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Expanded Bed - Department of Molecular and Cellular Biology

1. 154 If the boundary conditions of a closed system are chosen then an analytical solution of eq 1 cannot be obtained and the equation has to be solved numerically 80 From eq 1 the broadening of a tracer pulse due to axial mixing may be estimated Figure 42 shows a set of curves for different degrees of mixing represented by differing Bodenstein number Bo The ideal plug flow would be characterised by infinitely high Bo Fig 42 A set of RTD curves for different degrees of axial mixing represented by differing Bodenstein number Bo The tanks in series model considers the column to exist from a cascade of ideal stirred tank reactors The larger the number of tanks in the cascade the closer the mixing behaviour is to ideal plug flow For the tanks in series model an analytical solution may also be found equation 5 describing E as a function of the number of tanks N 5 N N I exp N O E O Figure 43 presents a series of RTD curves for different values of N Again an infinitely high tank number represents ideal plug flow 155 Fig 43 A set of RTD curves for different degrees of axial mixing represented by differing values of N The dimensionless groups Pe and Bo as well as N may be determined by fitting the analytical solutions according to equations 2 and 5 to experimentally obtained RTD curves by non linear regression The coefficient of axial mixing Dax is o
2. Or reduce viscosity by further homogenization of the feed stock intracellular products or prevent possible release of nucleic acids through cell lysis extracellular products by on line dilution and by increasing osmolality of the diluent see pages 33 117 119 Use fresh cultures to prevent cell lysis extracellular products and release of nucleic acids Problem Cause Remedy Build up of particulates underneath the adaptor net High back pressure Sedimented bed height is too large Aggregation of biomass inside the column is trapped in the adaptor net Build up of particulates underneath the adaptor net Clogging of the bottom distribution system by nucleic acids in the feed stock Clogging of the bottom distribution system due to aggregation of biomass in the feed stock at low pH e g during cation exchange chromatography Clogging of the bottom distribution system due to agglomeration of cells in the feed stock Decrease sedimented bed height Nominal sedimented bed height is around 15 cm Replace the adaptor net with the elutriation sealing See above Treat the feed stock with nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99 and or prevent possible release of nucleic acids through cell lysis extracellular products by on line dilution and by increasing osmolality of the diluent see pages 33 117 119 and or
3. 2 0E 06 1 0E 06 0 0E 00 Feed 1 5L 11 8L 23 6L 35 4L 47 1L 60L Wash Eluate Particle Count 1 ml Fig 17 Total particle concentration in different fractions from purification of a monoclonal antibody on STREAMLINE rProtein A Reproduced with permission from Ref 64 33 Washing may also be performed with a buffer containing a viscosity enhancer such as glycerol which may reduce the number of bed volumes needed to clear the particulates from the bed A viscous wash solution follows the feedstock through the bed in a plug like manner increasing the efficiency of particulate removal Complete removal of particulate material by washing with one expanded bed volume of 25 50 v v glycerol has been reported by Chang and Chase 34 Even if the clarification efficiency of an expanded bed adsorption step is very high some interaction between cell cell debris material and adsorbent beads can be expected which retain small amounts of cells and or cell debris on the adsorbent Such particulates may be removed from the bed during regeneration for instance when running a high salt buffer through an ion exchanger or during cleaning between cycles using a well defined CIP protocol Cells retained on the adsorbent may be subjected to lysis during the washing stage Such cell lysis can be promoted by reduced ionic strength when wash buffer is introduced into the expanded bed Nucleic acids released due to cell lysis can cause signific
4. Expanded Bed Adsorption in Capture from Bacterial Fermentation Cultures This section describes adsorption from bacterial fermentation cultures It includes applications where the target molecule is accumulated intracellularly in soluble form or as inclusion bodies as well as where it is secreted into the cell culture broth Recovery of recombinant Annexin V from unclarified E coli homogenate by expanded bed anion exchange adsorption Expanded bed anion exchange adsorption has been used for pilot scale recovery of recombinant human placental annexin V from an Escherichia coli homogenate 28 Annexin V is an anticoagulant protein found in placenta Its molecular weight is approximately 34 kD and the isoelectric point is 4 9 It was cloned to be expressed intracellularly in E coli and was released from the harvested cells by three passages at 700 900 bar through a high pressure homogenizer This procedure effectively disrupted the cells and also reduced the viscosity caused by released nucleic acids Due to the tendency of the annexins to associate with membranous structures phospholipids a detergent Triton X 100 1 v v final concentration was added to the homogenate prior to purification The biomass dry weight of the homogenate was 3 6 Method scouting was performed using clarified feed material on STREAMLINE DEAE packed in an XK 16 column to a bed height of 10 cm When optimal conditions for feed conditioning adsorption washin
5. 1 Bicinchoninic acid trademark owned by Pierce 2 Specific activity represents the amount of therapeutic protein in mg per mg of total protein 89 Purification of a recombinant anti HIV Fab fragment from E coli homogenate by expanded bed cation exchange adsorption Expanded bed cation exchange adsorption on STREAMLINE SP was used in the capture step during purification of a recombinant anti HIV Fab fragment from an Escherichia coli homogenate 29 The work was performed by Pharmacia Biotech in collaboration with the Karolinska Hospital Stockholm Sweden and the Swedish Institute for Infectious Disease Control Stockholm Sweden with the purpose of producing Fab fragments active in neutralizing human immunodeficiency virus type 1 HIV 1 The Fab fragment was directed against the envelope protein gp120 of the HIV 1 virus The Fab fragment was expressed in the periplasm of E coli and was released from the harvested cells by 3 passages through a high pressure homogenizer at a pressure of 700 800 bar An endonuclease Benzonase Merck Nycomed Pharma A S was added to the buffer used during homogenization at a ratio of 10 ul per 4 litre of buffer to reduce the viscosity The biomass dry weight of the homogenate was 1 4 The isolelectric point of the Fab fragment was determined to be 10 3 Method scouting was performed on a small packed bed of STREAMLINE SP using clarified feed material Method optimization in expanded mode using crude
6. 79 Table 20 Chemicals and solvents that can be used with STREAMLINE columns Chemical agent Concentration Purpose Comments NaOH 1M CIP1 SIP2 Not recommended for STREAMLINE rProtein A and STREAMLINE Heparin NaOH 0 01 M Storage Not recommended for STREAMLINE rProtein A and STREAMLINE Heparin Ethanol 70 CIP SIP Ethanol 20 Storage lsopropanol 30 CIP NaCl 2M Regeneration CIP HCl 0 01 M CIP Not recommended for STREAMLINE SP STREAMLINE SP XL and STREAMLINE Heparin Acetic acid 25 CIP Recommended for STREAMLINE SP and STREAMLINE SP XL only for a contact time of up to 30 minutes Not recommended for STREAMLINE Heparin Guanidine HCl 6M CIP Urea 8M GIP Triton X 100 1 CIP Tween 1 CIP Glycerol 10 50 Wash 1 Cleaning in place 2 Sanitization in place 80 STREAMLINE systems Manual systems A portable system for operating STREAMLINE columns manually is available with two different tubing dimensions 6 mm i d tubing for STREAMLINE 50 and 10 mm i d tubing for STREAMLINE 200 The system contains valves tubing manifolds and an air trap permitting complete operation of the column including hydraulic control of adaptor movement and reverse flow All components are assembled on a 700 x 550 mm stainless steel frame The system is delivered with a stainless steel cover and handle which makes it easy to relocate without disassembling The valves in the system are manual diaphragm valves with integrated tubing T p
7. Applied BSA mg ml adsorbent Fig 6 Breakthrough curves for BSA on STREAMLINE DEAE in packed mode in an XK 16 column compared with expanded mode in a STREAMLINE 50 and STREAMLINE 200 column Work by Pharmacia Biotech Fig 7 compares the adsorption of lysozyme to STREAMLINE SP adsorbent in expanded mode with adsorption to SP Sepharose Fast Flow in packed mode using identical adsorption conditions SP Sepharose Fast Flow is a cation exchange medium for packed bed chromatography that is frequently applied for the initial capture of proteins A small difference in breakthrough capacity and steepness of the breakthrough curves can be observed The avarage particle size of STREAMLINE SP adsorbent is 200 pm compared to 90 pm for the SP Sepharose Fast Flow adsorbent which explains for the later breakthrough and steeper curve for SP Sepharose Fast Flow 1t _ Packed SP Sepharose Fast Flow 0 8 __ Expanded STREAMLINE SP 0 4 0 2 0 20 40 60 80 100 Applied lysozyme mg ml adsorbent Fig 7 Breakthrough curves for lysozyme on STREAMLINE SP and SP Sepharose Fast Flow at a flow velocity of 300 cm h Work by Pharmacia Biotech 12 Operating pressure Due to the high bed voidage void volume fraction of an expanded bed typically around 0 7 0 8 bed voidage of a packed bed is in the range 0 3 0 4 the restriction of flow is insignificant and consequently the operating pressures are extremely lo
8. Expanded bed adsorption on STREAMLINE SP was evaluated as an alternative to the cell harvesting and the initial cation exchange steps see Fig 38 Cell Culture Broth Dimers Harvest by TFF Cation Exchange Expanded Bed Adsorption 3 other chromatography 3 other chromatography steps steps Formulation Formulation Fig 38 Alternative purification paths for the recombinant human Nerve Growth Factor 114 The recombinant human Nerve Growth Factor rh NGF which was produced in CHO cells is a 26 kDa homodimer consisting primarily of a 118 amino acid residue monomer The isoelectric point is gt 9 Method scouting was performed on a 1 0 x 4 5 cm packed bed of STREAMLINE SP using clarified feed stock After having defined suitable conditions for binding and elution method optimization in expanded bed mode was performed on a STREAMLINE 25 column 25 mm i d The method was finally scaled up to a STREAMLINE 200 column 200 mm i d containing 3 litres of STREAMLINE SP adsorbent corresponding to a sedimented bed height of approximately 10 cm The method optimization studies revealed that by applying the feed at a temperature of 37 C it was possible to increase flow velocity and still achieve high binding capacity The optimum flow velocity was defined as 375 cm h which was the flow velocity applied during bed expansion equilibr
9. It enables more monitors to be connected and has a number of convenient programming features for method development and production These together with the intuitive user interface simplify production UNICORN also provides data integrity definable user access levels and extensive batch documentation which are important aspects of operating in GLP and GMP environments A UNICORN controlled system can be manually operated or fully automated through a user defined programming sequence For instance the system can be programmed to respond with lifting or lowering the adaptor flow stop flow reversal backflushing lowered flow rate etc when the adsorbent sensor STREAMLINE CD columns detects adsorbent particles under the adaptor net Each STREAMLINE system houses UNICORN hardware Up to four chromatographic systems can be connected to one PC running UNICORN control system software For more information about UNICORN contact your nearest Pharmacia Biotech office STREAMLINE production scale systems can also be supplied with a Programmable Logical Controller PLC as an alternative to UNICORN 85 7 Applications This Chapter shows how Expanded Bed Adsorption has been used successfully to capture target molecules from crude unclarified feed material The applications cover different types of feed material and illustrate a variety of separation techniques including ion exchange affinity and hydrophobic interaction chromatography
10. Number of theoretical plates 31 27 n d n d Breakthrough capacity for lysozyme mg lysozyme ml sedimented bed 85 n d 85 85 1 H expanded bed height when the adsorbent has been expanded and equlibrated with start buffer Ho sedimented bed height J2 The results indicate that the adsorbent could be reused for more than 50 cycles without compromising its function No effect on the tested parameters could be seen over 50 cycles SDS PAGE on collected fractions revealed no loss in chromatographic performance over the 50 cycles Purification of a recombinant Pseudomonas aeruginosa exotoxin A from unclarified E coli lysate by expanded bed anion exchange adsorption Expanded bed anion exchange adsorption has been used in the purification of a genetically modified recombinant Pseudomonas aeruginosa exotoxin A expressed in the periplasm of Escherichia coli 53 Gram quantities of inactivated Pseudomonas aeruginosa exotoxin A were needed to prepare several polysaccharide conjugated vaccines The inactivated exotoxin A serves as the carrier protein moiety that is covalently bound to the polysaccharide moiety The conjugate was to be used as a vaccine against certain pathogenic strains of methicillin resistant Staphylococcus aureus and Shigella The genetically modified inactivated exotoxin A was cloned into E coli and expressed as a soluble protein in the periplasm of the recombinant bacteria The harvested bacterial cells 4 5 kg were sus
11. The dynamic binding capacity of STREAMLINE SP XL and STREAMLINE Q XL is thus extremely high Typical dynamic binding capacities of STREAMLINE SP XL for lysozyme in a STREAMLINE 25 column at 15 cm sedimented bed height and flow velocities of 400 cm h are 190 220 mg ml adsorbent The high dynamic binding capacities obtained with STREAMLINE XL ion exchangers can improve throughput and productivity in different ways Speed can be increased by allowing a higher feed application flow rate for binding a certain amount of target protein to a certain amount of adsorbent Speed can also be increased by allowing a higher conductivity during adsorption of the target protein The crude feed may then not have to be diluted before application to the expanded bed This smaller volume reduces feed application time Productivity can also be improved by using a smaller amount of adsorbent for binding a certain amount of target protein i e reducing the scale of work 67 Table 12 compares dynamic binding capacities of STREAMLINE SP and STREAMLINE DEAE with STREAMLINE SP XL and STREAMLINE Q XL Table 12 Comparison of dynamic binding capacities of STREAMLINE SP DEAE and STREAMLINE SP Q XL for different molecules STREAMLINE SP STREAMLINE SP XL STREAMLINE DEAE STREAMLINE Q XL mg ml adsorbent mg ml adsorbent mg ml adsorbent mg ml adsorbent higG 12 60 s BSA 39 149 Ovalbumin 38 143 Lysozyme 78 213 Loading capacity at 10 breakt
12. The first new unit process operation in decades Bio Technol 11 1993 1059 McCormick D K Expanded Bed Adsorption A New Way for Industrial Recovery of Recombinant Proteins Poster presented at New Zealand Biotech Association Meeting Palmerston North New Zealand May 1993 Schmidt C et al Characterization of a Novel Adsorbent for Recovery of Proteins in Expanded Beds Poster presented at 6th European Congress on Biotechnology Florence Italy June 1993 K mpe S Hjorth R Nystrom L E Expanded Purification of Proteins using Purpose Designed Adsorbents 6th European Congress on Biotechnology Florence Italy June 1993 Volume III p WE 013 Chase H A Chang Y K Purification of Proteins from Crude Feedstock using STREAMLINE Expanded Bed Adsorption Presented at Thirteenth International Symposium on HPLC of Proteins Peptides and Polynucleotides San Francisco USA November 30 December 3 1993 Kampe S Barnfield Frej A K et al Analysis of some operating parameters of Novel Adsorbents for Recovery of Proteins in Expanded Beds Bioseparation 5 1995 217 223 Hjorth R Kampe S Carlsson M Development of operating conditions for protein purification using expanded bed techniques The effect of the degree of bed expansion on adsorption performance Biotech c amp Bioeng 49 1996 512 526 Chang Y K Chase H A Protein Recovery from E coli Homogenate using Expanded Bed Adsorption Chromatography Pr
13. abruptly increasing the flow rate from zero to 320 cm h thus causing extreme turbulence in the bed This procedure was repeated 140 times passing a total volume of 800 litres of water 4000 bed volumes through the bed over a period of 14 days Particles leaving the column during the expansion phase were collected and their volume was found to be less than 0 2 of the total The degree of expansion was unaffected Binding capacity The porosities of STREAMLINE base matrices and the coupling densities of the ligands attached provide high binding capacities for biological macromolecules This ensures high throughput and high productivity As in any type of adsorption the binding capacity for a specific target molecule depends not only on the inherent properties of the adsorbent and the target molecule but also on the type and extent of impurities in the crude feed applied to the column Binding capacity data for selected model proteins are given under the description of each STREAMLINE adsorbent STREAMLINE SP STREAMLINE DEAE Product characteristics STREAMLINE SP and STREAMLINE DEAE are ion exchange adsorbents for expanded bed mode Both are based on highly cross linked 6 agarose modified by including an inert quartz core to give the desired density BioProcess STREAMLINE SP is a strong cation exchanger The sulphonate groups maintain full protein binding capacity over the entire long term pH stability range of 4 13 STREAMLIN
14. during expansion equilibration and wash was 50 mM potassium phosphate pH 7 0 Elution was performed both in expanded and packed bed mode using a flow velocity of 360 and 90 cm h respectively The elution buffer was 100 mM sodium citrate pH 4 0 The eluate was collected in a vessel containing 1 M Tris HCl pH 8 0 for direct neutralization After elution the expanded bed was cleaned with five sedimented bed volumes of 2 M urea followed by five sedimented bed volumes of 1 M acetic acid Table 49 summarizes results from all three scales In one run on the STREAMLINE 25 column and in two runs on the STREAMLINE 50 column elution was performed in an expanded mode of operation which explains the lower concentration of antibody in the eluate The eluate from the different columns contained IgG at very high purity as determined by SDS PAGE Fractions of the feed applied the washing step and the final eluate were analysed for particle content with a Coulter Counter A more than 100 fold clarification was achieved in addition to the very high purity of the antibody and the significant volume reduction Comparing the curent capacity with the capacity during a previous purification of the IgG antibody by ion exchange expanded bed adsorption on STREAMLINE SP 35 shows a nearly 100 fold increase in capacity with STREAMLINE rProtein A The reduced capacity on the STREAMLINE SP was due to the high conductivity 13 mS cm in the undiluted feed applie
15. ethanol and finally adsorption buffer Five sedimented bed volumes of each solution were applied at a flow velocity of 50 cm h The breakthrough capacity for BSA and expansion characteristics were determined before cycle 1 and after cycles 1 5 and 10 Table 36 summarizes the results Table 36 Summary of results from a study on the re useability of STREAMLINE DEAE Start 1cycle 5cycles 10 cycles Breakthrough capacity for BSA mg BSA ml sedimented bed 58 7 59 2 58 2 58 6 Liquid velocity cm h to give 2x bed expansion in aqueous buffer at 20 C 200 205 196 203 103 The results indicated that the adsorbent could be reused for more than 10 cycles without compromising its function No effect on the tested parameters could be seen over 10 cycles It was also concluded that the resolution of proteins eluting from the adsorbent that had been regenerated by the CIP protocol was the same as that of the untreated adsorbent No evidence was found of any carry over or build up of contaminants as a result of reusing the adsorbent in multiple cycles of operation Recovery of glucose 6 phosphate dehydrogenase from S cerevisiae homogenate by expanded bed dye ligand affinity adsorption Purification of glucose 6 phosphate dehydrogenase G6PDH from crude unclarified homogenate of baker s yeast was performed by the use of expanded bed affinity adsorption to Procion Red H E7B immobilized onto STREAMLINE adsorbent 33 The start materi
16. recovered at high yield with a 15 fold concentration Table 46 Summary of results from the laboratory scale and pilot scale purification of humanized monoclonal IgG by expanded bed adsorption on STREAMLINE rProtein A STREAMLINE 25 STREAMLINE 200 run 1 run 2 Adsorbent L 0 075 0 075 4 7 Feed Volume L 1 5 1 5 93 Conc IgG mg ml 0 407 0 407 0 407 Amount IgG mg 611 611 37900 Eluate Volume L 0 0912 0 0912 6 134 Conc IgG mg ml 7 41 7 46 7 68 Amount IgG mg 676 677 47109 BSA ng mg IgG 8 6 25 10 Transferrin ng mg IgG lt 0 2 lt 0 4 lt 0 4 DNA pg mg IgG N A N A lt 0 26 Yield 111 111 124 Purification of a murine IgG antibody from hybridoma cell culture broth by expanded bed affinity adsorption A murine IgG monoclonal antibody was purified from a hybridoma cell culture by expanded bed adsorption on STREAMLINE rProtein A 73 A mouse hybridoma cell line producing an IgG monoclonal antibody was cultivated in a spinner system a 5 L bioreactor and a 100 L bioreactor The cells were cultivated on a serum free medium The cell density was 3 x 106 living cells ml The product titre was in the range 70 to 100 mg IgG L Method scouting was performed on a small packed bed of STREAMLINE rProtein A using clarified feed material After having defined optimal loading conditions pH ionic strength and flow velocity on the small packed bed laboratory scale expanded bed experiments were perform
17. use fresh cultures to prevent cell lysis extracellular products and release of nucleic acids Switch to anion exchange chromatography which allows a higher pH Remove large aggregates by an in line crude mesh filter Stir the feed stock during feed application to prevent cell agglomeration Remove large agglomerates with an in line crude mesh filter 137 Problem Cause Remedy Reduced flow rate Clogging of the expanded bed Channelling in the expanded bed 138 High back pressure Aggregate formation due to interaction of released nucleic acids and or negatively charged cells with positively charged adsorbent beads anion exchange adsorbents Clogging of the bottom distribution system by nucleic acids in the feed stock Clogging of the bottom distribution system due to aggregation of biomass in the feed stock at low pH e g during cation exchange chromatography See above Treat the feed stock with nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99 and or prevent possible release of nucleic acids through cell lysis extracellular products by on line dilution and by increasing osmolality of the diluent see pages 33 117 119 and or use fresh cultures to prevent cell lysis extracellular products and release of nucleic acids If problems are persistent try a cation exchange adsorbent instead Treat the feed stock with
18. 0 9 10 7 22 1 2 0 Mab Affinity Products Flow through 0 0 0 100 86 200 100 89 200 Mab Affinity Products Eluates 100 89 200 100 92 200 100 94 200 Table 52 shows the anticoagulant activity of immunopurified rhPC as percentage of immunopurified hPC derived from human plasma Immunopurified rhPC from unadsorbed effluents of STREAMLINE DEAE expanded bed adsorption showed no anticoagulant activity by APTT activated partial thromboplastin time assay Essentially no rhPC was obtained in unadsorbed effluents from loadings of untreated whey Immunopurified rhPC from NaCl eluate pools of STREAMLINE DEAE showed 43 58 and 75 APTT activity for untreated 2 mM Zn2 treated and 4 mM Zn treated loadings respectively It was concluded that the majority of background proteins and immature populations of rhPC were precipitated and pass through the column unadsorbed in the flow through fraction when the whey was treated with 4 mM Zn prior to loading onto the STREAMLINE DEAE expanded bed Hence metal dependant conformational changes associated with major milk proteins and target protein sub populations was used as an efficient tool for achieving highly selective expanded bed anion exchange adsorption of transgenic milk Table 52 Anticoagulant activity of immunoaffinity purified rhPC STREAMLINE DEAE products Metal present Sample Purity Activity by APTT hPC ref None STREAMLINE flow through fraction NA NA STREAMLINE eluate gt 95
19. 1997 219 227 Maurizi G Di Cioccio V Macchia G Boss P Bizzarri C Visconti U Boraschi D Tagliabue A Ruggiero P Direct Capture of Monoclonal Antibodies using a New rProtein A Matrix in Fluidized Bed Chromatography under Lysis Free Conditions Poster presented at Recovery of Biological Products VIII Tucson Arizona October 20 25 1996 L tkemeyer D Ameskamp N Tebbe H Wittler J Lehmann J Development of Immobilised Metal Affinity Chromatography of Proteins in Expanded Beds Poster presented at First International Conference on Expanded Bed Adsorption EBA 96 Cambridge UK December 1996 Paper no P5 8 Clemmit R H Ghose S Chase H A 151 INA 7 Sb 76 ae 78 To 80 81 152 The recovery of a Recombinant Therapeutic Protein from a High Cell Density Fermentation Process Using Expanded Bed Adsorption Chromatography Poster presented at First International Conference on Expanded Bed Adsorption EBA 96 Cambridge UK December 1996 Paper no P6 3 Binieda A Lewis C A Pearce Higgins M Purvis J A Expanded Bed Adsorption Chromatography A Case Study from the Real World Poster presented at First International Conference on Expanded Bed Adsorption EBA 96 Cambridge UK December 1996 Paper no P6 4 Purvis J A Binieda A Lewis C A Pearce Higgins M Varley P G Protein Separation from Transgenic Milk J Chem Tech Biotechnol 54 1994 110 Wrig
20. 2 69 units G6PDH ml 0 21 units GGPDH mg protein The biomass dry weight of the final 25 w v homogenate was 6 5 w w The viscosity was 5 0 mPa 4 C at a shear rate of 106 s 1 and the conductivity was 7 4 mS cm 4 C The 25 w v unclarified cell homogenate was applied on STREAMLINE DEAE adsorbent expanded and equilibrated with 50 mM sodium phosphate pH 6 0 in a STREAMLINE 50 column 50 mm i d The column contained 435 ml of sedimented adsorbent providing a sedimented bed height of 22 cm The total activity of G6PDH loaded onto the bed corresponded to 43 of the equilibrium capacity of the column The flow velocity during expansion equilibration was 196 cm h causing the bed to expand to a height of 44 5 cm The expanded bed height was maintained constant throughout feed application and wash by continuous adjustment of the flow velocity The position of the float of a rotameter positioned in the inlet to the bed was used to estimate the location of the top of the expanded bed Non adsorbed components were washed out from the expanded bed using 25 v v glycerol in 50 mM sodium phosphate pH 6 0 This wash solution having a viscosity similar to the feed stock allowed complete removal of residual particulate material from the bed by passage of a single expanded bed volume through the bed The glycerol solution was subsequently removed from the bed by 102 washing with buffer and the bed was converted to a packed configuration pri
21. 852 2811 8693 F 852 2811 5251 Asean Countries T 603 7353972 Malaysia F 603 735 4672 E mail pbasean po jaring my Australia Tollfree 1 800 252 265 T 61 3 9887 3909 F 61 3 9887 3912 E mail amradpb2 ozemail com au Hong Kong T 852 2811 8693 F 852 2811 5251 E mail phhkmkt hkstar com India T 91 44 434 0747 F 91 44 434 5537 E mail pharma giasmd01 vsnl net in Indonesia T 62 21 384 8884 F 62 21 384 9636 E mail hilabsci indo net id Malaysia T 60 3 703 1888 F 60 3 703 8047 E mail intersec po jaring my New Zealand Tollfree 008 733 893 T 64 9 638 7097 F 64 9 638 7098 E mail amradpbnz xtra co nz People s Republic of China T 852 2811 8693 F 825 2811 5270 Beijing T 86 10 6256 4308 F 86 10 6256 5603 Guangzhou T 86 20 8760 1566 F 86 20 8760 1566 Shanghai T 86 21 6267 4621 4656 F 86 21 6267 4611 Phillipines T 63 2 634 6571 F 63 2 635 4817 E mail secura mnl sequel net Republic of Korea T 822511 0801 F 822 511 3711 E mail phdi chollian dacom co kr Singapore T 65 250 3330 F 65 250 0003 E mail biolab singnet com sg Taiwan T 886 2 831 6021 F 886 2 831 5311 Thailand T 662 615 2130 F 662 271 4533 E mail becthai ksc7 th com Vietnam T 84 8 835 4652 F 84 8 835 2997 Japan Tokyo T 03 3492 9499 F 03 3492 9337 internet http www biotech pharmacia se 160
22. Place CIP of STREAMLINE rProtein A Poster presented at Cell Culture Engineering V San Diego California USA January 28 February 2 1996 Asplund M K mpe S Jagersten C Cleaning Sanitization and Storage Handbook of Process Chromatography A Guide to Optimization Scale up and Validation Academic Press London UK ISBN 0 12 654266 Hagel L Sofer G Metal Chelate Affinity Chromatography a new approach to protein purification Nature 258 1975 598 599 Porath J Carlsson J Olsson I et al Purification of Proteins by IMAC Trends in Biotechnol 3 1985 1 7 Sulkowski E Protein Interactions with Immobilized Transition Metal Ions Quantitative evaluations of variations in affinity and binding capacity Anal Biochem 191 1990 160 168 Hutchens T W Yip T T Proteoglycans structures and interaction Ann Rev Biochem 60 1991 443 475 Kjell n L Lindahl U STREAMLINE 25 column Data File 18 1112 02 Pharmacia Biotech AB Large scale recovery and purification of periplasmic recombinant protein from E coli using expanded bed adsorption chromatography followed by new ion exchange media J Biotechnol 48 1996 9 14 Johansson H J J gersten C Shiloach J 149 54 33 56 oye 58 59 60 61 62 63 64 150 Purification of Secreted Recombinant Proteins from Hansenula polymorpha by Fluidized Bed Adsorption Poster presented at First International Confer
23. Symposium Book No H00887 1993 Lindgren A Johansson S Nystr m L E Scale up Validation of Expanded Bed Adsorption Processes Poster presented at 6th European Congress on Biotechnology Florence Italy June 1993 Johansson S Lindgren A Nystrom L E 39 40 41 42 43 44 45 46 47 48 49 50 Jl a2 53 Expanded bed adsorption at production scale Scale up verification process example and sanitization of column and adsorbent Bioprocess Engineering 16 1997 57 63 Barnfield Frej A K Johansson H J Johansson S Leijon P Purification of Recombinant Human Retinoblastoma Protein at Canji Inc using STREAMLINE Expanded Bed Adsorption Downstream No 17 1994 Pharmacia Biotech AB European Patent Application EP 0 699 687 A2 1996 Noda M Sumi A Ohmura T Yokoyama K Purification of Proteins by Adsorption Chromatography in Expanded Beds Trends in Biotech 12 1994 296 303 Chase H A Physical Chemical Properties of STREAMLINE Ion Exchangers Poster presented at 7th European Congress on Biotechnology Nice France February 1995 Hansson K A Expanded Bed Adsorption Optimization of the Wash and Elution Steps on STREAMLINE Ion Exchangers Poster presented at 7th European Congress on Biotechnology Nice France February 1995 Carlsson M STREAMLINE SP STREAMLINE DEAE Cleaning in place Application Note 18 1115 27 Pharmacia Biotech AB Cleaning In
24. and the non ionic detergent Triton CF 10 66 Product availability STREAMLINE SP and STREAMLINE DEAE are supplied as suspensions in packs of 100 ml 300 ml 7 5 litres and 60 litres For larger quantities please contact your local Pharmacia Biotech office STREAMLINE SP is supplied in 20 ethanol containing 0 2 M sodium acetate STREAMLINE DEAE is supplied in 20 ethanol STREAMLINE Q XL Product characteristics BioProcess STREAMLINE SP XL 3 EXTREME LOAD STREAMLINE SP XL and STREAMLINE Q XL are two high capacity ion exchange adsorbents for expanded bed mode Their extremely high loading capacities increase the productivity of manufacturing operations STREAMLINE SP XL is a strong cation exchanger The sulphonate groups maintain full protein binding capacity over the entire long term pH stability range of 4 13 STREAMLINE Q XL is a strong anion exchanger The quaternary amine groups maintain full protein binding capacity over the entire long term pH stability range of 2 12 Both are based on highly cross linked 6 agarose modified by including an inert quartz core to give the desired density Long molecules of dextran are coupled to the agarose matrix and the strong Q and SP ion exchange groups are then attached to these dextrans chains through chemically stable ether bonds This will cause an increase in the effective interacting volume as well as in the steric availability of the ligands for the substance to be adsorbed
25. be a good complement to a NaOH based cleaning protocol About three sedimented bed volumes are applied at a flow velocity of approximately 100 cm h If the use of organic solvents are considered less attractive due to requirements for classified areas and explosion proof equipment a non ionic detergent may be an alternative Another alternative to an organic solvent may be a wash with hot water which is a technique frequently used in the dairy industry for lipid removal About 10 sedimented bed volumes of hot distilled water 60 95 C are applied at a flow velocity of 100 cm h A combined wash with 25 acetic acid 20 ethanol has sometimes proven to be an efficient cleaning protocol that can also be combined with an initial NaOH wash 39 Occasionally the presence of nucleic acids in the feed is the cause of fouling the adsorbent and in such a case treating the adsorbent with a nuclease e g Benzonase Merck Nycomed Pharma A S could restore performance Benzonase can be pumped into the bed and be left standing for some hours before washing it out Sometimes the delicate nature of the attached ligand prevents the use of harsh chemicals such as NaOH For instance protein ligands such as in STREAMLINE rProtein A will hydrolyse when exposed to high concentrations of NaOH which may limit the working life time of the medium Alternative cleaning agents that can be recommended in such cases are 6 M guanidine hydrochloride 6 M urea and 1 M aceti
26. bottom valve This gives an early indication on any pressure build up in the system Pressure can build up if the column nets are clogged or if adsorbent beads start packing against the adaptor net This can occasionally happen during application of a crude and viscous feed and may require periodic reversal of flow reduction of flow rate or reduction of viscosity of the feedstock Fig 20 shows a basic set up of a manual STREAMLINE system 45 1 OA so GS eat gl oo Lae oR Pressure monitor OoN P1 CP ada awe Double channet je Dae D A Tor ee A UV Cond pH P2 Single channet O 3 or 4 port Waste Fig 20 Schematic representation of a manual STREAMLINE system with two pumps Valve V1 changes the direction of flow from upward to downward when eluting bound substances from the sedimented bed It can also be used to reverse flow intermittently to prevent the column nets clogging or adsorbent beads building up under the adaptor end plate during feed application This valve may also be needed to dissolve possible plug formation in the bed prior to cleaning in place Valve V2 directs effluent to the product collection vessel during elution It also blocks the flow through the bed to allow the adaptor to be lifted in the column when Pump 1 pumps upward flow through the bed Valve V3 directs the flow of hydraulic liquid from the hydraulic chamber to waste when the adapt
27. chromatography on Q Sepharose High Performance When crude unclarified material was applied directly on STREAMLINE SP a purity of 90 92 with a recovery of 85 was achieved very close to that obtained by centrifugation filtration and S Sepharose High 101 Performance chromatography After subjecting the pooled fractions from STREAMLINE SP to anion exchange chromatography a final purity of 98 was reached identical to that obtained with the traditional route It was concluded that centrifugation filtration and cation exchange chromatography on S Sepharose High Performance could all be replaced by a single adsorption step on STREAMLINE SP Expanded Bed Adsorption in Capture from Yeast Fermentation Cultures This section contains applications of adsorption from yeast fermentation cultures including examples where the target molecule is accumulated intracellularly or secreted into the cell culture broth Recovery of glucose 6 phosphate dehydrogenase from S cerevisiae homogenate by expanded bed anion exchange adsorption Glucose 6 phosphate dehydrogenase G6PDH was purified from crude unclarified homogenate of bakers yeast by the use of expanded bed anion exchange adsorption 34 G6PDH an intracellular enzyme found in baker s yeast was released from the yeast cells by bead milling The resulting 50 w v wet weight homogenate was diluted 1 2 with 50 mM sodium phosphate pH 6 0 to a total protein concentration of 12 8 mg ml
28. from crude feedstock and for cleaning and sanitization Table 19 lists the principle components together with the Materials used in the construction of large scale STREAMLINE CD columns include electropolished stainless steel ASTM 316L column tube2 distributor plate net piping fittings and valves PTFE valves PP adsorbent sensor and EPDM gaskets and seals Table 20 lists chemicals and solvents that can be used with STREAMLINE columns 2 STREAMLINE CD columns can also be delivered with a transparent column tube manufactured from cast polymethylmethacrylate PMMA 78 Table 19 Materials of construction Designation STREAMLINE 25 STREAMLINE 50 STREAMLINE 200 Lid PEEKS PP4 SS ASTM 316L Column tube Borosilicate glass Borosilicate glass Borosilicate glass Top piece SS2 PP4 PP4 ASTM 316 End piece PEEK PP SS ASTM 316L Net top and bottom PP4 SS Ss SS1 ASTM 316 ASTM 316L ASTM 316L Adaptor distributor plate SS2 SS SS ASTM 316 ASTM 316 ASTM 316 Bottom distributor plate SS SS2 PVDF5 ASTM 316 ASTM 316 Adaptor PTFE PEEKS and PP4 SS2 SS O rings and gaskets SS2 ASTM 316 EPDM ASTM 316L EPDM NBR8 ASTM 316L EPDM 1 Stainless steel 2 Stainless steel electropolished 3 Polyetheretherketone 4 Polypropylene 5 Polyvinylidenefluoride 6 Polytetrafluoroethylene 7 Ethylenepropylenedimonomer 8 Nitrile rubber only in contact with hydraulic fluid
29. hibitane digluconate and 20 ethanol Allow to stand for 6 hours then wash with at least 5 bed volumes of sterile binding buffer Protocol 2 Equilibrate the bed with a solution consisting of 0 1 M acetic acid and 20 ethanol Allow to stand for 1 hour then wash with at least 5 bed volumes of sterile binding buffer Protocol 3 Equilibrate the bed with 70 ethanol Allow to stand for 12 hours then wash with at least 5 bed volumes of sterile binding buffer Note that specific regulations for classified areas and explosion proof equipment may apply when handling large volumes of organic solvents Storage We recommend storing STREAMLINE rProtein A in 20 ethanol Product availability STREAMLINE rProtein A is supplied as a suspension in 20 ethanol in packs of 75 ml 300 ml and 5 litres 1 Sodium N lauroylsarcosinate weak anionic detergent 2 The bed is equilibrated with 3 sedimented bed volumes and then left standing for 90 minutes 75 STREAMLINE columns Pharmacia Biotech manufactures a range of STREAMLINE columns specifically for use in expanded bed adsorption STREAMLINE columns are optimized to give stable expanded beds with STREAMLINE adsorbents from small scale optimization through pilot scale and up to final production Table 17 summarizes the STREAMLINE column range More detailed information is available in the User Manual supplied with each column Table 17 STREAMLINE columns available from Pharmacia Biotec
30. immobilized hybridoma cells The average cell concentration in the culture broth was 1 x 106 cells ml The product titre was in the range 14 to 50 mg IgG L Method scouting was performed on a 1 ml packed bed of STREAMLINE rProtein A using clarified feed material Optimum pH conditions for binding and elution were defined as pH 7 0 and pH 4 0 respectively Laboratory scale expanded bed experiments were performed on an XK 16 40 column Pharmacia Biotech a column designed for packed bed chromatography To improve flow distribution in expanded mode a 1 cm bed of non porous glass ballotini 3 mm diameter was packed at the bottom inlet The column contained 124 20 ml of STREAMLINE rProtein A adsorbent corresponding to a sedimented bed height of 10 cm Initial experiments with cell free hybridoma supernatant revealed that the capacity for the IgG antibody was 14 mg ml adsorbent The method was scaled up in two steps The first scale up step was run on a STREAMLINE 25 column 25 mm i d containing 50 ml of adsorbent corresponding to a sedimented bed height of 10 cm The second was run on a STREAMLINE 50 column 50 mm i d containing 150 ml of adsorbent which corresponds to a sedimented bed height of 7 6 cm Bed expansion equilibration feed application and wash were performed at an upward flow velocity of 360 cm h The crude cell containing hybridoma broth was applied directly onto the expanded bed after adjustment of pH The buffer used
31. ion exchange adsorbent would reduce capacity This situation may call for dilution before application to the expanded bed to achieve maximum loading capacity If conductivity is minimized at the end of the fermentation step dilution is unnecessary This results in less feed volume and shorter feed application time In an intracellular system conductivity of feed stock can be reduced by running the homogenization step in water or a dilute buffer The pH range defined during method scouting should also be verified in expanded bed mode since reduced pH in some systems may cause aggregation of biomass This aggregation can block the column distribution system causing poor flow distribution and an unstable bed Physical parameters are optimized in expanded bed mode since they relate to the hydrodynamic properties of the expanded bed Cell density and biomass content both affect viscosity which may reduce the maximum operational flow rate by over expanding the bed Temperature also affects the viscosity and hence the operational flow rate in the system Fig 12 shows the effect of temperature on the degree of expansion in a buffer system Increased temperature can improve binding kinetics as demonstrated in Fig 13 which shows breakthrough curves for BSA at two different temperatures Optimization experiments are usually carried out at room temperature but a broth taken directly from the fermentor may have a higher temperature This difference in
32. ixim 18 8207 03 1 4 PE 1x5m 19 0385 01 1 2 PE 1x5m 18 1015 10 6 mm PVC 1 x 30cm 18 0005 42 1x 75 cm 18 0005 43 1x 125 cm 18 0005 44 1x 150 cm 18 0005 45 1 x 200 cm 18 0005 47 10 mm PVC 1 x 30 cm 18 1012 85 1 x 40 cm 18 1012 86 1 x 90 cm 18 1012 62 1 x 140 cm 18 1012 63 1x 170 cm 18 1012 64 1 x 200 cm 18 1012 87 144 Peristaltic tubing 1 6 mm i d 3 2 mm i d 9 6 mm i d Pumps HiLoad Pump P 50 Watson Marlow 504 U RL peristaltic Watson Marlow 604 U R peristaltic UV monitor Flow cells Monitor UV 1 S 2 flow cell Industrial flow cell 6 mm i d Industrial flow cell 10 mm i d 280 nm Filter kit Miscellaneous GradiFrac includes 2 solenoid valves PSV 50 GradiFrac Rack Recorder REC 101 single channel operation Recorder REC 102 dual channel operation Plastic clamp 25 mm o d Gasket 6 mm i d Gasket 10 mm i d Blind flange 25 mm o d and packing Stop plug Flanging kit 120 V Flanging kit 220 V Contact your nearest Pharmacia Biotech office for ordering Peristaltic tubing can be supplied with moulded on 25 mm o d clamp connectors on request SN h ee eee N N l Ss SS 19 1992 01 44 2677 05 44 2686 01 18 1003 66 19 4840 02 18 1000 66 18 1000 65 19 2433 01 18 1993 01 18 1993 05 18 1001 42 18 1001 43 44 0508 05 44 0581 01 44 0581 02 18 1001 25 19 5170 01 18 4603 70 18 4603 71 1 To flange tubing 1 9 x 2 7 mm ends so that the tubing is retained insid
33. mg ml sedimented adsorbent Fig 14 Breakthrough curves for BSA on STREAMLINE DEAE from laboratory to production scale Work by Pharmacia Biotech 28 4 Method Optimization Feed application This section indicates what effect different feed stock variables may have on the behaviour of expanded bed adsorption and gives guidelines on suitable corrective actions to ensure stable expansion and consistent function with different types of feed material Viscosity When the crude feed is pumped onto the column using the same flow rate as was used for bed expansion and equilibration the expansion usually increases further due to the viscosity of the feed stock being higher than the viscosity of the equilibration expansion buffer Very high viscosities can have a negative impact on the stability of the expanded bed A moderate increase in viscosity does not effect bed stability but it can cause over expansion when using nominal flow velocities of around 300 cm h Such a high degree of expansion causes adsorbent beads to pack tightly against the adaptor net A packed zone of beads against the adaptor net acts as a depth filter and traps particulate material present in the crude feed eventually blocking flow through the column If particulates are seen building up against the adaptor net during feed application a periodic back flush helps remove them A switch to downward flow eliminates the build up after a few seconds When the build up h
34. more detail in Section 3 Experimental Design and Section 4 Method Optimization Adsorption characteristics The stability of expanded beds based on STREAMLINE adsorbents provides adsorption characteristics similar to those of packed bed chromatography As with packed bed chromatography the available binding capacity depends on the molecular weight of the target substance the binding strength to the ligands on the matrix flow velocity and other conditions of the process The absolute values for protein capacity given here are therefore only valid for specific proteins under defined flow velocities and process conditions Axial dispersion is usually an order of magnitude higher in an expanded bed compared with a packed bed However the adsorption characteristics of an expanded bed are very similar to a packed bed This is demonstrated in Fig 6 showing the breakthrough capacity for BSA on STREAMLINE DEAE adorbent in both expanded and packed bed mode Only small differences are seen in breakthrough capacity and slope of the breakthrough curve The same type of finding has been reported by Chase and Chang 20 using a similar test model with BSA and STREAMLINE DEAE adsorbent They concluded that breakthrough curves in packed and expanded modes were indistinguishable indicating that adsorption performance is approximately the same in both 11 1 0 O Packed XK 16 STREAMLINE 50 a _ STREAMLINE 200 00 20 40 60 80 100
35. necessary to elute the column in expanded mode In this case removal of aggregates from the bed by applying an efficient cleaning in place protocol is crucial to prevent build up from cycle to cycle Cell lysis As already discussed in Section 3 cell lysis is one of the main concerns when processing feed material from secretion systems since it usually releases nucleic acids lipids and other cell membrane components causing bed instability during processing and fouling of the adsorbent This makes it more difficult to restore performance between purification cycles In addition release of intracellular proteases can have a negative effect on the overall yield of active product Cell lysis may be of special concern in ion exchange applications since the culture broth has to be diluted and pH adjusted prior to loading of the feed stock Such Operations must be exercised with care since shifts in pH and or osmolality can accelerate cell lysis Dilution buffer to adjust pH and ionic strength should be added immediately before adsorption to minimize exposing cells to conditions that will promote cell lysis This is especially important when sample application time is long It will be even more important in large scale applications since scaling up usually increases holding times between the different operational steps Dilution just prior to adsorption can easily be achieved by applying on line dilution according to Fig 15 Ideally the diluent s
36. of 6400 ml of crude unclarified culture broth was applied to a STREAMLINE 50 column 50 mm i d containing 300 ml of expanded STREAMLINE SP corresponding to a sedimented bed height of 15 cm Following feed application unbound proteins and residual biomass were washed out from the bed in expanded mode The flow velocity used during expansion equilibration adsorption and wash was 300 cm h The buffer used during expansion equilibration and wash was 20 mM sodium citrate pH 3 5 The expansion equilibration with buffer at a flow velocity of 300 cm h resulted in a three fold expansion of the bed Desorption of aprotinin from the adsorbent was performed with downward flow in sedimented mode applying a two step elution procedure In the first step contaminating proteins were desorbed with 0 5 M NaCl in start buffer The second step employed 0 9 M NaCl in start buffer to desorb the aprotinin The matrix was finally regenerated with 2 M NaOH in sedimented mode The aprotinin containing eluate from the STREAMLINE column was subjected to further purification by reversed phase chromatography The eluate was supplemented with 0 1 v v of TFA and applied to a RP 18 Lichroprep HPLC column Merck Darmstadt at 2 cm min without further pre treatment Pure aprotinin was eluted by a gradient from 0 1 TFA in aqueous solution to 50 of 0 1 TFA in 40 v v isopropanol The aprotinin containing fractions were finally desalted by gel filtration on a Sephadex G 25 co
37. point where adsorbent beads start to pack against the adaptor net especially when a highly viscous feed is applied Increased sedimented bed height also gives increased cycle time Increasing residence time by decreasing flow velocity may have a negative effect on bed stability due to decreased expansion However it may be possible to decrease flow velocity without compromising the degree of expansion since the viscosity is usually significantly higher in the feed than in the equilibration buffer How significant the effect of this is depends on the resistance to mass transfer in the system It may be significant for a high molecular weight protein especially if the viscosity in the feed is high and slows down molecular diffusion This approach was taken by Chang and Chase 23 34 who noticed a 2 5 fold increase in dynamic binding capacity when applying a viscous feed containing lysozyme to STREAMLINE SP at a reduced flow velocity that was continously controlled to keep the degree of expansion constant twice the sedimented bed height during the entire process 80 60 40 QB mg ml adsorbent 20 0 10 20 30 40 Bed height cm Fig 19 Effect of sedimented bed height on breakthrough capacity QB for lysozyme on STREAMLINE SP Work by Pharmacia Biotech 44 5 Experimental Technique Chapter 5 examines the practical aspects of expanded bed adsorption It shows examples of different system set ups and describes t
38. released and cause high viscosity Cell lysis can also release significant amounts of lipids Agglomeration of cells can occur Cytoplasmic Cell debris high content of protein Lipid DNA and proteases are present Very thick feedstock which needs dilution Intact bacterial cells and endotoxins are present Cytoplasmic Cell debris high content of protein Lipid DNA and proteases are present Very thick feedstock which needs dilution Intact yeast cells are present Cytoplasmic Unusual location for product accumulation Periplasmic Cell debris high content of protein Lipid and proteases are present Thick feedstock which needs dilution DNA is present if cytoplasmic membrane is pierced Intact bacterial cells and endotoxin are present Periplasmic Not applicable to yeast cells Periplasmic Not applicable to mammalian cells Inclusion body Cell debris high content of protein Lipid and proteases are present Very diluted solutions after renaturation Intact bacterial cells DNA and endotoxin are present Precipitation of misfolded variants occurs in a time dependent manner Inclusion body Not applicable to yeast cells Inclusion body Not applicable to mammalian cells contaminant may also be present as charged particulates that can act as ion exchangers and adsorb proteins especially basic ones if the ionic strength of the homogenate is low This problem is ho
39. sedimented bed volumes of distilled water at 100 cm h 3 sedimented bed volumes of 30 isopropanol at 100 cm h 3 sedimented bed volumes of 25 acetic acid at 100 cm h and finally adsorption buffer until the pH and conductivity of the outlet stream were the same as the buffer 93 Fig 33 shows the expanded bed adsorption step on STREAMLINE DEAE A Column STREAMLINE 200 200 mm i d See Adsorbent STREAMLINE DEAE 4 7 litres 2 0 Feed 4 7 kg of cells were subjected to osmotic shock and suspended in a final volume of 180 litres 50 mM Tris buffer pH 7 4 before application onto the expanded bed Buffer A 50 mM Tris buffer pH 7 4 Buffer B 50 mM Tris 0 5 M NaCl pH 7 4 Flow velocity 400 cm h during feed application 100 cm h during elution and wash Heigh of expanded bed cm Sample application Washing Elution Buffer A Buffer B 100 200 260 Volume liters Fig 33 Capture of recombinant Pseudomonas aeruginosa exotoxin A on STREAMLINE DEAE Work by National Institute of Health Bethesda Maryland USA in collaboration with Pharmacia Biotech Uppsala Sweden Following the Capture step on STREAMLINE DEAE the material was further purified by two Intermediate Purification steps and a final Polishing step The first intermediate purification step was hydrophobic interaction chromatography HIC on Phenyl Sepharose 6 Fast Flow high sub packed in a BPG column Pharmacia Biotech This step removed a
40. temperature must be considered when basing decisions on results from small scale experiments It may be worth testing feed application at elevated temperatures since reduced viscosity and improved binding kinetics can allow a higher flow rate and thus shorter cycle times 19 Degree of Expansion Wa rere fiers i a az ta pees EN SS EAN SE aa 18 20 22 24 26 28 Temp Leverage C Fig 12 Degree of expansion with varying temperature C Co o Temperature of BSA 21 C 1 0 Temperature of BSA 36 28 C 0 4 0 2 0 20 40 60 80 100 Applied BSA mg ml adsorbent Fig 13 Breakthrough curves for BSA on STREAMLINE DEAE at different temperatures Work by Pharmacia Biotech The effect of critical parameters on the different stages of an expanded bed adsorption step is discussed in more detail under Feed characteristics and in Section 4 Method Optimization 20 Feed characteristics The most critical aspect of the design of an expanded bed adsorption process concerns interfacing of process conditions with the properties of the starting material This is important in any type of downstream process but is of particular importance in expanded bed adsorption since the interaction of the raw feed with an adsorbent is so much more complex than the traditional application of clarified pretreated feed to a packed bed of adsorbent Different starting materials affect bo
41. that reported in the literature The overall recovery of enzyme activity was greater than 80 of the activity originally found in the starting batch of skimmed milk 127 Table 50 Summary of results from the purification of equine lysozyme from skimmed milk Volume Total Total Specific Purification Yield ml protein activity activity factor mg U 106 U mg Skimmed milk 4990 42964 57 35 1335 1 0 100 STREAMLINE SP 560 4603 50 98 11075 8 3 88 9 Phenyl Sepharose 6 Fast Flow 2750 1547 46 88 26939 2 4 91 3 Purification of a recombinant protein from the milk of transgenic livestock by expanded bed anion exchange adsorption The active sub population of recombinant human protein C rhPC was purified from milk of transgenic pigs by expanded bed anion exchange adsorption on STREAMLINE DEAE 60 Milk is a relatively complex mixture containing serum passover proteins such as albumin broadly specific proteases and caseins The total protein content is 40 to 60 g protein L This complexity can make downstream processing difficult on a large scale Recombinant proteins have been harvested from g L levels in the milk of transgenic livestock using precipitation techniques in combination with chromatography For example multiple PEG precipitations in combination with ion exchange adsorption have been used to purify recombinant alpha 1 antitrypsin from the milk of transgenic sheep 76 and human protein C from the mi
42. the cleaning protocol should be applied at the end of each purification cycle Productivity Productivity in downstream processing is a complex issue which relates to all the different characteristics of both the feed material and the adsorbent In specific terms productivity is defined as the amount of product produced per adsorbent volume and time unit Productivity g litre h 1 40 Dynamic binding capacity The amount of product produced per adsorbent volume g litre relates to the dynamic binding capacity and yield of the target protein Dynamic binding capacity is a function of the flow velocity through the expanded bed and the uptake rate of the target protein under defined processing conditions i e pH conductivity viscosity etc Due to the impact on mass transfer resistance in the system dynamic binding capacity generally decreases with increase in flow velocity and with increase in viscosity of the feed The effect on mass transfer by viscosity is via the effect on the diffusion coefficient which decreases at increased viscosity The viscosity is usually of greater significance in expanded bed adsorption than in traditional packed bed chromatography due to high biomass content and presence of particulate material in crude unclarified feed materials Particulates and polyionic macromolecules in a raw feed may also affect dynamic binding capacity by interacting with binding sites in the adsorbent Dynamic binding capacity is de
43. to a packed bed by using conventional chromatographic adsorbents based on agarose in a column equipped with a purpose designed liquid distribution inlet giving a plug flow in the column The application of mixtures of proteins and cells onto these expanded beds showed the potential of the technique for recovery of proteins from particle containing feedstocks 9 10 11 The breakthrough capacity in such beds expanded by a factor of two was very similar to the breakthrough capacity in a packed bed However low flow velocities had to be applied to prevent the bed from expanding too much which resulted in a low overall productivity It was obvious from the experiments of Draeger and Chase that there was a need for particles with a higher sedimentation velocity to fully exploit the features of the expanded bed technology In 1992 the first results from such prototype adsorbents based on agarose were reported 12 13 14 15 Commercially available adsorbents based on amorphous silica have also been investigated as possible candidates for use in expanded beds 16 These adsorbents are denser than agarose based adsorbents but the smaller bead size enables this material to expand to the same degree as beds of agarose beads at comparable flow velocities A drawback of silica containing material is the limited stability at high pH values which makes it less suitable for biopharmaceutical production where high pH is frequently used for cleaning in pla
44. unclarified cell homogenate was performed with a STREAMLINE 50 column SO mm i d containing 300 ml adsorbent corresponding to a sedimented bed height of 15 cm The method was finally scaled up to pilot scale in a STREAMLINE 200 column 200 mm i d containing 4 7 litres of adsorbent The flow velocity during expansion equilibration adsorption and wash was 300 cm h which caused the bed to expand 2 8 times during expansion equilibration and 3 4 times during feed application The buffer used during expansion equilibration and wash was 50 mM sodium acetate pH 5 0 Desorption of the Fab fragment from the adsorbent was performed with downward flow in sedimented mode using 50 mM sodium acetate pH 5 0 containing 1 M NaCl The flow velocity during desorption was 100 cm h Fig 32 shows a chromatogram from the run on the STREAMLINE 50 column Table 25 summarizes the experiments at the two scales The same fermentation batch was used for both runs but homogenization was performed at different times 90 Table 25 Summary of results from lab scale and pilot scale expanded bed adsorption of recombinant anti HIV Fab fragment from unclarified E coli homogenate STREAMLINE 50 STREAMLINE 200 Volumes L Adsorbent 0 3 4 7 Homogenate 4 85 60 Eluate 0 50 6 0 Fab conc ug ml 6 8 in 62 5 out 23 in 122 5 out Yield 95 100 AUs50 am Column STREAMLINE 50 50 mm i d Adsorbent STREAMLINE SP 300 ml 2 0 Buffer A 50 mM sodium ace
45. up to the second heat treatment step using 1 ton of crude unclarified culture medium in each run The average yield after the heat treatment at 68 C for 30 minutes and the heat treatment with cysteine is 98 6 and 88 4 respectively The total yield of the four runs is 87 1 which is in good agreement with the results obtained at pilot scale on a STREAMLINE 50 column 50 mm i d It was concluded that the purity of rHSA obtained by this process heat treatment expanded bed adsorption is almost comparable to that of rHSA obtained by a conventional 5 step process filtration ultrafiltration heat treatment ultrafiltration cation exchange chromatography Thus the expanded bed process route reduces the number of steps from five to two which shortens the processing time and increases the yield by 30 Table 41 Summary of results from four production scale runs from the purification of rHSA Run No Step Volume rHSA Yield litres g 1 culture medium 922 5868 100 0 heat treatment 68 C 30 min 1900 5399 92 0 flow through 6000 eluate 200 heat treatment with cysteine 62 4840 82 5 2 culture medium 943 6246 100 0 heat treatment 68 C 30 min 1960 6351 101 7 flow through 6400 eluate 300 heat treatment with cysteine 61 5674 90 9 3 culture medium 937 6200 100 0 heat treatment 68 C 30 min 1877 6261 101 1 flow through 5777 462 7 4 eluate 200 heat treatment with cysteine 1 5594 90 2 4 cul
46. velocity of 100 cm h The protein was eluted with 8 bed volumes of a linear gradient from 0 to 0 5 M NaCl In total 73 of the initial exotoxin A was recovered with a specific activity of 0 1 mg toxin mg protein The total processing time for 4 5 kg cells is calculated to be 8 10 hours The protein was eluted from the expanded bed with a specific activity of 0 06 mg toxin mg protein The processing time was 2 5 hours Thus processing the crude unclarified feed material directly on an expanded bed adsorption column was 3 times faster than traditional processing using clarified feed on a packed chromatography column Although the specific activity of the exotoxin eluted from the expanded bed was lower the yield was slightly higher Table 29 compares the capture step peformed on the expanded bed with that on the packed bed Table 29 Comparing capture steps for processing of 4 5 kg E coli cells for the production of recombinant Pseudomonas aeruginosa exotoxin A DEAE Sepharose Fast Flow STREAMLINE DEAE Volumes L Cell lysate 90 180 Eluate 36 13 5 Time hrs Centrifugation 2 3 Clarification 2 3 Loading wash elution 3 2 5 Specific activity mg toxin mg protein 0 1 0 06 Yield 73 79 Packed column numbers are extrapolated values from a run on an XK 16 column with a packed bed volume of 21 ml Recovery of recombinant human Interleukin 8 from E coli inclusion bodies by expanded bed cation exchange adsorption
47. 0 2 0 06 Wash 1 1000 lt 1 lt 0 03 Wash 2 4200 55 1 7 Eluate 1050 2773 84 A number of analytical techniques were employed to demonstrate that the end product produced by the expanded bed route was of equivalent quality to the product produced by the established pilot scale process All results indicated that the products from the two different purification routes were indistinguishable in terms of purity identity and biological activity The use of this material in phase II clinical trials resulted in British Biotech being the first company to file an IND with expanded bed adsorption central to the purification process 113 Expanded Bed Adsorption in Capture from Mammalian Cell Cultures This section contains some applications on expanded bed adsorption from crude unclarified mammalian cell culture broth based on CHO cells myeloma cells and hybridoma cells Recovery of Nerve Growth Factor from CHO cell culture broth by expanded bed cation exchange adsorption Expanded bed adsorption on STREAMLINE SP was evaluated as an alternative capture step in the manufacturing of recombinant human Nerve Growth Factor 67 The work was performed by Genentech Inc So San Francisco CA USA in collaboration with Pharmacia Biotech The established process consisted of cell harvest by tangential flow filtration initial capture by packed bed cation exchange chromatography followed by three other chromatography steps and final product formulation
48. 0 cm h using a 2 0 mg ml solution of protein in 50 mM sodium phosphate buffer pH 7 5 lysozyme and 50 mM Tris HCl buffer pH 7 5 BSA Sedimented bed height was 15 cm Cleaning in place sanitization in place and sterilization The properties of the base matrix the derivatization chemistry used when attaching ligands and the inherent stability of the ligand groups result in very stable ion exchange media This high product stability allows exposure to harsh conditions such as 1 M NaOH for cleaning in place and sanitization Suitable cleaning in place protocols must be defined on a case by case basis depending on the nature of the feed applied to the expanded bed Chapter 4 Method Optimization contains general information about cleaning in place of STREAMLINE adsorbents The following protocols developed for STREAMLINE SP and STREAMLINE DEAE restore both hydrodynamic and chromatographic properties over a large number of purification cycles using different types of E coli homogenates 45 65 CIP procedure 1 0 5 M NaOH containing 1 M NaCl flow velocity 30 cm h contact time 4 hours distilled water flow velocity 100 cm h 3 sedimented bed volumes 30 isopropanol flow velocity 100 cm h 3 sedimented bed volumes 25 acetic acid flow velocity 100 cm h 3 sedimented bed volumes equilibration buffer flow velocity 100 cm h 5 10 sedimented bed volumes CIP procedure 2 0 5 M NaOH containing 1 M NaCl flow veloc
49. 26 Recovery IN O 120 100 80 60 40 20 0 31 36 38 43 44 48 41 39 37 49 50 56 54 61 66 67 74 75 77 78 Run numbers Average Recovery 103 5 22 5 n 20 Fig 31 Reproducibility study of recovery from a STREAMLINE SP used in STREAMLINE 50 column Eighty runs on the same column and adsorbent were performed with two E coli strains and four gene constructs Recovery was measured by RP HPLC Reproduced with permission from ref 58 The expanded bed adsorption step on STREAMLINE SP reduced total protein content 10 fold and resulted in a 3 fold increase in concentration of the target protein The purity was consistent between the two scales as determined by reversed phase HPLC Table 24 summarizes the efficiency of reduction of total protein endotoxin and DNA over the STREAMLINE SP step Since the endotoxin levels were low and consistent from batch to batch it was concluded that CIP with 0 5 M NaOH efficiently removed endotoxin from the column Table 24 Removal of total protein endotoxins and contaminating DNA during the expanded bed adsorption step on a STREAMLINE 50 column Protein concentration was determined by BCA endotoxin activity with the Limulus Amebocyte Lysate LAL kit Bio Whittaker and DNA with the Threshold system Molecular Devices Inc Total protein Specific Activity Endotoxins DNA mg EU ml ng ml Starting material 19 000 0 1 1 250 000 1 300 000 STREAMLINE SP eluate 2317 0 65 1 250 15
50. 43 2 mM Zn2 STREAMLINE flow through fraction gt 95 0 STREAMLINE eluate gt 95 58 4 mM Zn2 STREAMLINE flow through fraction gt 95 0 STREAMLINE eluate gt 95 75 130 8 Fault finding chart Start up Expansion Equilibration Problem Cause Remedy Beads stick to column wall when loading the adsorbent Expanded bed height H is lower than expected Adsorbent suspended in water Channelling in the expanded bed due to trapped air in the bottom distribution system Channelling in the expanded bed due to clogging of the bottom distribution system Channelling in the expanded bed due to the column not being in a vertical position Turbulence in the expanded bed due to fouling aggregation or infection of the adsorbent Suspend the adsorbent in starting buffer or in a salt solution e g 0 5 1 0 M NaCl Try to remove the air by pumping buffer at high flow velocity e g 300 500 cm h through the column using downward flow If the above does not help remove the adsorbent from the column Pump distilled water into the column through the bottom distri bution system and remove any trapped air using suction from above the adaptor net see page 53 Disassemble the column and clean the distributor plate and net see column User Manual Use a spirit level to adjust the vertical position of the column Clean and or sanitize the adsorbent see Cleaning in place page 38
51. 45 Chase H A Draeger M N Expanded bed adsorption of proteins using ion exchangers Separation Sci Technol 27 1992 2021 2039 Chase H A Draeger M N Adapting Chromatography for Initial Large Scale Protein Recovery ACS Conference Proceedings Series Harnessing Biotechnology for the 21st Century Ladisch M Bose A Eds 1992 pp 271 274 Hedman P Barnfield Frej A K Characterization of the Internal Flow Hydrodynamics in an Expanded Bed Adsorption Column Poster presented at Recovery of Biological Products VI Engineering Foundation Interlaken Switzerland September 1992 Wnukowski P Lindgren A Recovery of a Recombinant Protein from an E coli Homogenate using Expanded Bed Adsorption Poster presented at Recovery of Biological Products VI Engineering Foundation Interlaken Switzerland September 1992 Barnfield Frej A K Johansson S Hjorth R 15 16 ee 18 19 20 pale ZZ Ada 24 25 26 Hydrodynamic Stability of the Liquid Fluidized Bed of Small Particles An Experimental Study Poster presented at AIChE Annual Meeting Florida USA November 1992 Paper no 116dd Johansson B U Wnukowski P High performance liquid chromatography of amino acids peptides and proteins CX XIV Physical characterization of fluidized bed behaviour of chromatographic packing materials J Chromatogr 631 1993 115 124 Dasari G Prince I Hearn M T W Expanded Bed Adsorption
52. AMLINE CD column with an internal diameter of 600 mm followed by a second scale up step on a STREAMLINE CD with an internal diameter of 1200 mm Scalability was verified by consistency in yield as shown in Table 3 and by consistency of chromatographic performance as judged by the appearance of chromatographic curves and analytical gel filtration of collected product peaks RTD testing and breakthrough capacity determination gave further evidence of consistent performance at different scales see Fig 14 The discrepancy in wash volume between the two runs on the STREAMLINE 600 column is due to the fact that the adaptor was lowered to the bed surface earlier during the wash phase in run 2 compared with run 1 This change resulted in a significant reduction in consumption of wash buffer Table 3 Summary of process data from a scale up verification study Scale Sample Dry Load Wash Peak Yield volume weight g BSA litre volume volume L adsorbent SBV SBV STREAMLINE 25 run 1 0 74 4 71 19 7 12 1 1 90 run 2 0 75 4 83 19 9 11 1 2 87 STREAMLINE 200 48 4 82 20 4 16 1 9 88 STREAMLINE 600 run 1 420 4 87 19 9 17 2 2 87 run 2 440 4 48 20 8 11 2 1 92 STREAMLINE 1200 1640 5 60 20 4 12 2 3 88 SBV sedimented bed volumes C C 0 9 0 8 STREAMLINE 25 C 4 STREAMLINE 50 STREAMLINE 200 STREAMLINE 600 0 6 gt STREAMLINE 1200 0 7 0 5 0 4 0 3 0 2 0 1 0 0 Oo 10 20 30 40 50 60 70 80 90 100 Applied BSA
53. E DEAE is a weak anion exchanger The number of ligand groups that are charged varies with pH This adsorbent maintains consistently high capacities over the pH range of 2 9 More information about STREAMLINE SP and STREAMLINE DEAE including instructions for their use is available in Data File STREAMLINE SP STREAMLINE DEAE Code No 18 1111 73 Table 11 summarizes their characteristics 64 Table 11 Characteristics of STREAMLINE SP and STREAMLINE DEAE adsorbents Product STREAMLINE SP STREAMLINE DEAE Type of ion exchanger strong cation weak anion Total ionic capacity mmol ml gel 0 17 0 24 0 13 0 21 Particle size range um 100 300 100 300 Approx mean particle size um 200 200 Approx mean particle density g ml 1 2 1 2 Degree of expansion H H o at 300 cm h 2 3 2 3 OH stability long term 4 13 2 13 short term 3 14 2 14 Binding capacity mg ml gel lysozyme MW 14 500 gt 60 n d BSA MW 67 000 n d gt 40 Storage 0 2 M sodium acetate in 20 ethanol 20 ethanol Further information is available in Data File STREAMLINE SP STREAMLINE DEAE Code No 18 1111 73 n d not determined 1 Long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance Short term refers to the pH interval for regeneration and cleaning procedures 2 Breakthrough capacity determined in a STREAMLINE 50 column at a flow velocity of 30
54. E SP XL STREAMLINE Chelating and STREAMLINE Heparin For STREAMLINE rProtein A the approximate density of the adsorbent cake is 1 3 g ml Connecting the column Remove the adaptor and connect the column to the system via the column bottom valve as shown in Fig 20 Fill the column approximately 2 3 full with distilled water via the pump and bottom valve While pumping through the bottom valve suck out any air that may be trapped under the column end piece net with tubing connected to water suction or a peristaltic pump Move the tubing over the whole of the upper surface of the end piece net Leave about 5 cm of water in the column To drain adsorbent in a bucket connect one end of a piece of tubing containing a filter to water suction and use it to suck away the excess liquid the filter prevents loss of adsorbent Move the tubing very gently over the sedimented adsorbent surface Handle the drained adsorbent carefully to avoid damaging the adsorbent particles 53 Loading adsorbent Suspend the adsorbent in starting buffer to give an approximate 50 slurry Without allowing the adsorbent to sediment quickly pour the slurry into the column Wash out any remaining adsorbent from the container with buffer and pour this into the column Be careful not to trap air in the slurry Make sure that no aggregates of air adsorbent remain floating on the liquid surface Fill the column to the rim with distilled water Inserting the adaptor In
55. Expanded Bed Adsorption Principles and Methods Back to Collection 18 1124 26 Edition AB amersham pharmacia biotech 1 Introduction to Expanded Bed Adsorption This handbook introduces the principles of Expanded Bed Adsorption and serves as a practical guide to the use of STREAMLINE adsorbents and columns available from Pharmacia Biotech Critical operating parameters will be discussed as well as principles for method design and optimization that will ensure maximum exploitation of this unique unit operation The handbook is illustrated with examples of the different types of biological molecules which have been purified using Expanded Bed Adsorption The majority of biotechnology processes for producing pharmaceutical or diagnostic products involve the purification of proteins and peptides from a variety of sources Those include bacteria yeast and mammalian cell culture fluids or extracts from naturally occurring tissue Typically such purification schemes contain multiple unit operations including a number of chromatographic steps to ensure the safe removal of critical impurities and contaminants The type of product produced and its intended use will dictate the extent of purification needed Each step in the recovery process will affect the overall process economy by increasing operational cost and process time and also by causing loss in product yield Careful selection and combination of suitable unit operations du
56. Expanded bed cation exchange adsorption has been used to recover human interleukin 8 IL 8 expressed in E coli as inclusion bodies 31 Human IL 8 a pro inflammatory cytokine with a molecular weight of 8 3 kD and an isoelectric point of 9 was expressed in E coli both in a soluble form and as inclusion bodies To disrupt the cells and solubilize IL 8 the harvested cells were resuspended in a solution containing 6 M guanidine hydrochloride in 30 mM sodium phosphate pH 6 5 The amount of 6 M guanidine hydrochloride solution 95 added per gram cells wet weight paste was 2 3 ml The suspension was stirred at room temperature for approximately 3 hours IL 8 was then renatured by dilution with water in two steps The first dilution was performed with 3 volumes of water followed by stirring at room temperature for 30 minutes In the second step it was again diluted with 3 volumes of water and stirred at room temperature overnight The resulting unclarified debris and precipitate containing feed stock had a conductivity of 27 mS cm The pH was 6 6 Biomass content in the final feed stock was approximately 1 dry weight The crude unclarified feed stock was applied to a STREAMLINE 50 column 50 mm i d containing 300 ml STREAMLINE SP adsorbent corresponding to a sedimented bed height of 15 cm The flow velocity during expansion equilibration adsorption and wash was 300 cm h The buffer used during expansion equilibration and wash was 30 mM sodiu
57. High capacity strong cation exchanger STREAMLINE Q XL High capacity strong anion exchanger STREAMLINE rProtein A Affinity adsorbent for purification of monoclonal and polyclonal antibodies STREAMLINE Chelating Coupled iminodiacetic acid groups for immobilized metal affinity chromatography IMAC STREAMLINE Heparin Coupled heparin for purification of plasma proteins e g coagulation factors and other types of proteins 1 STREAMLINE Chelating and STREAMLINE Heparin are currently March 1997 available as CDM products see page 61 CDM Custom Designed Media 60 BioProcess Media BioProcess Media are a full range of separation media specially designed manufactured and supported to meet the demands of industrial scale biomolecule production BioProcess This symbol is your guarantee of e Secure long term supply of large batches of media e Comprehensive documentation and regulatory support to assist in process validation e Conformance to the ISO 9001 Quality system for reliable supply with high quality and high batch to batch consistency e High chemical stability to allow efficient cleaning and sanitization regimes e Scaleable performance from bench top to production hall e Compatible large scale columns and equipment Media in Chapter 6 that fulfill the above criteria are given the BioProcess Media symbol Custom Designed Media Custom Designed Media CDM meet the needs of specific industrial proce
58. MLINE 25 25 mm i d equipped with hydraulic adaptor 18 1110 50 STREAMLINE 25 25 mm i d equipped with manual adaptor 18 1110 51 STREAMLINE 50 50 mm i d equipped with hydraulic adaptor 18 1038 01 STREAMLINE 2001 200 mm i d equipped with hydraulic adaptor 18 1100 22 1 Stand must be ordered separately 18 1031 20 STREAMLINE CD For ordering contact your nearest Pharmacia Biotech office Systems Product Code No STREAMLINE manual portable system 6 mm i d 44 9431 01 STREAMLINE manual portable system 10 mm i d 44 9431 03 STREAMLINE pilot scale systems STREAMLINE production scale systems For ordering contact your nearest Pharmacia Biotech office Other components Item Qty pack Code No Valves SRV 3 1 19 2098 01 SRV 4 1 19 2099 01 L type 1 4 PP 1 19 0239 01 L type 6 mm SS 1 18 5757 01 L type 1 2 SS 1 18 1001 37 L type 10 mm SS 1 18 1012 56 4 way 1 4 PP 1 19 0240 01 4 way 6 mm SS 1 18 5758 01 4 way 1 2 SS 1 18 1001 36 4 way 10 mm SS 1 18 1012 57 Solenoid valve PSV 50 1 19 1994 01 Connectors M6 2 7 mm 5 18 4652 01 JACO 10 4 2 1 4 3 19 0273 01 JACO 10 8 6 1 2 5 18 6880 01 SRTC 3 2 19 2144 01 Unions 25 mm o d clamp to M6 2 18 1031 09 25 mm o d clamp to 1 4 threaded 2 18 0251 98 25 mm o d clamp to 1 2 threaded 2 18 1012 68 Tubing 1 9 x 2 7 mm PTFE unflanged 2xim 18 8210 01 1x2m 18 8207 01 1x25m 18 8207 02 1 9 x 2 7 mm PTFE S flanged
59. Media SOURCE 30Q Poster presented at Prep Tech 95 Rutherford New Jersey USA 1995 Johansson H J Shiloach J Jagersten C STREAMLINE Expanded Bed Adsorption for Recovery of Renatured Human Interleukin 8 from Escherichia coli Bioseparations 1997 In press Barnfield Frej A K Hammarstr m A Jones I Hjorth R Single step Recovery of a Secreted Recombinant Protein by Expanded Bed Adsorption Bio Technol 12 1994 285 288 Hansson M St hl S Hjorth R Uhl n M Moks T Development of an Expanded Bed Technique for an Affinity Purification of G6PDH from Unclarified Yeast Cell Homogenates Biotech amp Bioeng 48 1995 355 366 Chang Y K McCreath G E Chase H A Ion Exchange Purification of G6PDH from Unclarified Yeast Cell Homogenates using Expanded Bed Adsorption Biotech amp Bioeng 49 1996 204 216 Chang Y K Chase H A Purification of Monoclonal Antibodies from Whole Hybridoma Fermentation Broth by Fluidized Bed Adsorption Biotech Bioeng 45 1995 205 211 Thommes J et al Direct Capture of Recombinant Proteins from Animal Cell Culture Media using a Fluidized Bed Adsorber Animal Cell Technology Products for today prospects for tomorrow Griffiths B Spier R E Berthold W Eds Butterworth amp Heinemann Oxford 1994 pp 557 560 Erickson J C Finch J D Greene D C Scale up of Expanded Bed Adsorption Processes Poster presented at 7th BioProcess Engineering
60. NE Phenyl low sub prototype adsorbent 68 A 45 w v yeast suspension in 20 mM potassium phosphate pH 7 was homogenized in a high pressure homogenizer for 5 passages at 1 2 x 108 N m 2 The homogenate was diluted to a final total protein concentration of 10 mg ml and brought to an ammonium sulphate concentration of 0 78 M in potassium phosphate buffer pH 7 Before the expanded bed experiments on STREAMLINE Phenyl low sub method scouting was performed in packed bed mode using clarified feed material to investigate both anion exchange chromatography DEAE Sepharose Fast Flow and hydrophobic interaction chromatography Phenyl Sepharose Fast Flow low sub for their relative merits in capturing ADH The anion exchange medium exhibited low capacity and poor selectivity for ADH when applied to the column in 20 mM potassium phosphate pH 7 and was considered unsuitable for direct capture of the enzyme The hydrophobic interaction medium exhibited good capacity and high selectivity for ADH providing 93 yield and a purification factor of 7 6 when clarified feed was applied to a 5 ml packed bed of Phenyl Sepharose Fast Flow low sub The expanded bed adsorption was performed in a STREAMLINE 50 column 50 mm i d containing 300 ml of STREAMLINE Phenyl low sub corresponding to a sedimented bed height of 15 cm Expansion equilibration and wash were performed with 0 78 M ammonium sulphate in 20 mM potassium phosphate pH 7 using a flow vel
61. TREAMLINE 25 column Table 33 summarizes the experiments at both laboratory and pilot scale The difference in yield between the different scales is attributed to variation during assay of protein A concentration in the crude feed stock 99 A Column STREAMLINE 25 25 mm i d 280 Adsorbent STREAMLINE Q XL 75 ml Feed 2 L cell culture 0 4 mg rprotein A ml 2 L 10 mM Tris HCl pH 7 4 2 L distilled water plus MgCl added to a final concentration of 11 mM 10 ul Benzonase Buffer A 10 mM Tris HCI pH 7 4 10 mM MgCl Buffer B 10 mM Tris HCI pH 7 4 1 M NaCl Flow velocity 400 cm h during feed application and wash 100 cm h during elution 1 Sample application 5 2L Wash l Elution 400 cm h 100 cm h Fig 34 Chromatogram from laboratory scale expanded bed adsorption of recombinant protein A from unclarified E coli cell culture suspension using STREAMLINE Q XL in a STREAMLINE 25 column Work by Pharmacia Biotech Uppsala Sweden Table 33 Summary of results from expanded bed adsorption of recombinant protein A at laboratory and pilot scale STREAMLINE 25 STREAMLINE 200 Fermentor size L 10 100 Sample dilution 3x 3x Feed volume L 5 2 300 Yield 107 80 Total protein reduction 2 9 2 6 DNA reduction 12 13 Purification of human recombinant Interleukin 1 Receptor Antagonist from B subtilis fermentation broth by expanded bed cation exchange adsorption Human interleukin 1 recept
62. TREAMLINE Q XL in 20 ethanol During long term storage of STREAMLINE SP XL in unbuffered ethanol a gradual acidification of the storage solution may occur due to the acidic nature of the ligand We therefore recommend adding sodium acetate up to a concentration of 0 2 M As an alternative storage solution for both STREAMLINE SP XL and STREAMLINE Q XL we recommend 10 mM NaOH This is comparable to 20 ethanol from a bacteriostatic point of view Product availability STREAMLINE SP XL and STREAMLINE Q XL are supplied as suspensions in packs of 100 ml 300 ml and 7 5 litres For larger quantities please contact your local Pharmacia Biotech office STREAMLINE SP XL is supplied in 20 ethanol containing 0 2 M sodium acetate STREAMLINE Q XL is supplied in 20 ethanol 69 STREAMLINE Chelating Product characteristics STREAMLINE Chelating is an expanded bed adsorbent for immobilized metal affinity chromatography IMAC IMAC separates proteins and peptides on the basis of their affinity for metal ions immobilized by chelation 48 49 50 Certain amino acids e g histidine cysteine form complexes with these chelated metal ions around neutral pH It is mainly the histidine content of a protein that is responsible for binding which makes IMAC an excellent method for purifying recombinant proteins with poly histidine fusions as well as many natural proteins STREAMLINE Chelating may also be used as a weak cation exchanger due to th
63. There are two simple models that can mathematically describe RTD as a function of distinct parameters and quantify the amount of axial mixing within a column the dispersion model and the tanks in series model The dispersion model starts with a mass balance over a thin segment of an adsorbent column Considering convective and dispersive transport results in the well known equation 1 1 oc D d c Oc t a on a Under the boundary conditions of an open system an analytical solution may be obtained eq 2 which describes E t as a function of the dimensionless time t t and of a single variable the column Peclet number Pe which is defined according to equation 3 79 2 Pe Pe 1 E exp __ v4 T O 4 0 3 pe Dx Dax is the overall coefficient of axial mixing m s Pe is a dimensionless number that relates dispersed flow D to convective flow v L Increasing Pe then stands for decreased axial mixing As may be seen from the definition of Pe increasing bed length or flow velocity reduces the overall mixing within an adsorbent bed This dimensionless group has also been called Bodenstein number Bo A third dimensionless number representing axial mixing is the particle Peclet number Pe that contains the particle diameter d as the characteristic length It therefore characterises a specific adsorbent particle rather than the specific column set up 4 ved P D ax
64. Wnukowski 15 The investigation included three different sedimented bed heights ranging from 56 mm to 236 mm and flow velocities ranging from 100 to 300 cm h generating a degree of expansion from 1 7 to 3 times the sedimented bed height The general conclusion was that longitudinal mixing in the liquid phase was low and consistent indicating stable fluidization and a plug flow liquid profile through the bed i e a similar behaviour as in packed beds The axial dispersion coefficients were in the range 1 6 x 10 6 m sl Discrepancies in the results were observed for the lowest flow velocity which was explained by the bed not being purely random at low expansion ratios Instead large conglomerates of particles are formed together with channels and paravoids contributing to an unpredictable behaviour of the bed It was also concluded that for the same flow velocity the axial dispersion increased with the height of the bed an effect that can be explained by the plug flow being retarded at the wall of the column creating radial variations of the bed voidages across the cross section of the column Stability of expanded beds using STREAMLINE media was also verified by Wnukowski and Lindgren 13 by using a set up with a sedimented bed height of 225 mm expanded 3 2 times at a linear flow velocity of 300 cm h They reported an axial dispersion coefficient of 9 x 10 6 m2 s 1 Similarly Batt et al 25 reported an average axial dispersion coefficie
65. a G Lindgren A Barnfield Frej A K Leijon P Liten A Mayes T L et al Direct Capture of Nerve Growth Factor from CHO Cell Culture by EBA Presented at Recovery of Biological Products VIII Tucson Arizona October 20 25 1996 Abstr p 74 Beck J Liten A Viswanathan S Emery C Builder S A Comparative Engineering Study of the Use of Expanded Bed and Packed Bed Routes for the Recovery of Labile Proteins from Crude Feedstocks Proceeding Sth World Congress of Chemical Engineering July 1996 Vol 2 565 570 Smith M P Bulmer M Hjorth R Titchener Hooker N J STREAMLINE Chelating Characterization of a New Adsorbent for Expanded Bed Adsorption Poster presented at First International Conference on Expanded Bed Adsorption EBA 96 Cambridge UK December 1996 Paper no P5 7 Blomqvist I Lagerlund I Larsson L J Westergren H Shiloach J An approach to integrated antibody production Coupling of fluidized bed cultivation and fluidized bed adsorption Bioprocess Engineering 15 1996 21 29 Born C Th mmes J Biselli M Wandrey C Kula M R Polymer Shielded Dye Ligand Chromatography of Lactate Dehydrogenase from Porcine Muscle in an Expanded Bed System Bioseparations 6 1996 193 199 Garg N Yu I Mattiasson G Mattiasson B Purification of Human Recombinant Interleukin 1 Receptor Antagonist Proteins upon Bacillus subtilis Sporulation Protein Expression and Purification 9
66. a safety margin has to be applied to compensate for different sources of variability in the process that may affect binding capacity In practical terms this usually means that to avoid the risk for valuable product leaking off the column during feed application maximum loading should be in the range of 50 75 of the loading where the main breakthrough was defined in the breakthrough study 41 AU C Co Column STREAMLINE 50 i d 50 mm Adsorbent STREAMLINE SP 0 30 15 cm sedimented bed height Feed E coli homogenate containing a recombinant Fab fragment Buffer A 50 mM sodium acetate pH 5 0 Buffer B 50 mM sodium acetate 1 M NaCl pH 5 0 CIR 0 5 M NaOH 1 M NaCl 0 5 Sample loading __ _____ Washing gt Elution CIP 2 4 6 8 10 12 14 16 18 1 2 litres Fig 18 Determination of breakthrough capacity for a recombinant Fab fragment on STREAMLINE SP Work by Pharmacia Biotech Process time Process time is the time of a complete purification cycle i e the sum of the time for expansion equilibration feed application washing elution regeneration and cleaning in place The processing time is a function of volumetric flow rate and volume applied onto the column at each stage of the process The sample application stage often has the most significant impact on the processing time especially when the feed consists of an unprocessed diluted cell culture broth A long sample application time can also
67. aced by the elutriation sealing Cell agglomeration The formation of cell agglomerates in the feed material is a further point of concern when working with feed material from secretion systems This is more significant if the sample application time is long as when applying a dilute feed at maximum loading capacity This is because dead cells show an increased tendency to form agglomerates and cell viability in the feed decreases rapidly when the fermentation has been terminated and the feed prepared for application to the expanded bed column Cell agglomerates usually form in areas of stagnant liquid which makes it important to apply continuous stirring of the feed material throughout sample 32 application Once cell agglomerates have formed they serve as nuclei for the formation of larger aggregates If they enter the column they can partially block the inlet liquid flow distribution system which causes increased back pressure and channelling in the lower part of the bed versus the end of the sample application Large aggregates that cannot pass freely through the mesh screen of the column can be removed prior to application by using a simple in line crude mesh filter If aggregates enter the expanded bed they can interact with the adsorbent forming even larger aggregates that are difficult to remove from the bed during the wash phase If this occurs it can block the flow when the bed is to be eluted in the packed mode As a result it is
68. action revealed a series of proteins with isoelectric points below pH 8 The elution fraction only contained proteins with isoelectric points above pH 8 0 including lysozyme having an isoelectric point above 10 A mixture of proteins with different molecular masses was detected in this fraction by SDS PAGE The elution fraction contained only 10 of the original amount of protein but up to 89 of the lytic activity A280 nm 2 1 Elution 0 2 4 6 S o 2 Volume L Fig 41 Chromatogram showing the purification of lysozyme from skimmed equine milk on STREAMLINE SP in a STREAMLINE 50 column The speckled area represents the lysozyme containing fraction Reproduced with permission from reference 55 Following the Capture step on STREAMLINE SP the material was further purified by hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow The hydrophobic hydrophilic nature of equine milk lysozyme is modified depending on the presence or absence of Ca ions The lysozyme was adsorbed to the Phenyl Sepharose 6 Fast Flow column in the presence of an excess of EDTA 50 mM Tris HCl 1 mM EDTA pH 7 5 and desorbed with the same buffer when EDTA was replaced with Ca2 ions Table 50 summarizes the complete purification process The peak eluted from the Phenyl Sepharose 6 Fast Flow column contained a single protein according to isoelectric focusing and SDS PAGE The molecular mass was determined as 14 400 which is consistent with
69. activity in the cell culture broth during pH adjustment indicate that hybridoma cells are sensitive to such operations Consequently pH adjustment should be performed with great care with gentle stirring to prevent the formation of local extremes of pH Particle measurement in the flow through showed no increase of particles in the range 6 10 um dead cells and only a slight decrease of total cell amount in the range 10 60 um living cells No particles were found in the eluate Furthermore the DNA concentration and LDH activity were greatly reduced in the eluate from the expanded bed compared with the feed material Table 48 Detection of cell damage by measuring DNA concentration LDH activity and particle load DNA LDH activity Particle load ng ml nkat L 2 counts ml 6 10 um 10 50 um Cell culture broth pH 7 2 1917 1939 3 80 x 10 2 67 x 106 Feed pH 8 0 approx 1 litre 1926 3120 3 85 x 10 2 55 x 106 Passage total flow through and wash 2003 3000 4 00 x 10 2 52 x 108 Eluate 0 45 0 0 1 Detection limit 1 ng ml 2 Maximum LDH limit after destruction of all cells approx 15000 nkat L nkat nanokatal Purification of a murine IgG antibody from hybridoma cell culture broth by expanded bed affinity adsorption A murine IgG monoclonal antibody was purified from a hybridoma cell culture by expanded bed adsorption on STREAMLINE rProtein A 64 The monoclonal antibody was produced in a continuous culture using
70. adsorbent can even form a compact plug in the column when re expansion is attempted after elution in packed bed mode This can only be dissolved by repeated backflushing or stirring of the adsorbent in the column It is therefore vital to define efficient CIP protocols designed and optimized on a case by case basis to restore both hydrodynamic and chromatographic functionality between runs Such a protocol should be applied after each chromatographic run using upward flow through the column with the adaptor positioned at a level equivalent to twice the sedimented bed height The CIP procedure should be carried out immediately after the elution of the target protein If adsorbent is allowed to remain in the column overnight before cleaning it is usually more difficult to clean Before applying the first CIP solution the bed should be expanded and the adaptor lifted to twice the sedimented bed height by pumping elution buffer with upward flow through the bed at a flow velocity of 100 cm h The flow velocity during CIP should be moderate to allow a high contact time between adsorbent beads and the cleaning agent The volume of CIP solution should be reasonably large to allow efficient wash out of solubilized contaminants from the bed The efficiency of the CIP protocol should be verified by running repetitive purification cycles and testing several functional parameters such as degree of expansion number of theoretical plates in the expande
71. al was the same type of yeast homogenate as described in the previous application for recovery of G6PDH by expanded bed adsorption on STREAMLINE DEAE 34 G6PDH was released from yeast cells by bead milling cells suspended in 50 mM sodium phosphate pH 6 0 The resulting homogenate was diluted 1 2 with 50 mM sodium phosphate pH 6 0 to a total protein concentration of 12 3 mg ml 3 3 units G6PDH ml 0 27 units G6PDH mg protein The biomass dry weight of the final homogenate was 6 8 w w and the viscosity was 5 0 mPa The unclarified yeast homogenate was applied to a STREAMLINE 50 column 50 mm i d containing 420 ml STREAMLINE adsorbent coupled with Procion Red H E7B corresponding to a sedimented bed height of 21 cm The total activity of G6PDH loaded onto the bed corresponded to 23 of the estimated dynamic capacity of the column Prior to feed application the adsorbent was expanded and equilibrated with 50 mM sodium phosphate pH 6 0 at a flow velocity of 152 cm h causing the bed to expand to a height of 42 5 cm The expanded bed height was maintained constant throughout feed application and wash by continuous adjustment of the flow velocity to compensate for the increased viscosity of the homogenate Debris and non adsorbed components were washed out from the expanded bed using 25 v v glycerol in 50 mM sodium phosphate pH 6 0 A single expanded bed volume of the wash solution allowed complete removal of residual particulate material fr
72. and specific cleaning and saniti zation recommendations in Section 6 Product Guide 131 Problem Cause Remedy Expanded bed height Increased density of beads See above H is lower than due to fouling expected Decreased viscosity due to Check and control the increased temperature temperature if necessary Decreased flow velocity due Disassemble the column to clogging of the bottom and clean the distributor and or adaptor distribution plates and nets see column system User Manual Decreased flow velocity due Replace the net s to folding of the bottom and or adaptor net Decreased flow velocity due Remove and clean the to clogging in valves respective parts connectors tubing etc Decreased flow velocity due Replace pump tubing to worn pump tubing Follow the pump manufac turer s recommendations for pump use Most pump manufacturers recommend that the pump tubing is removed from the pump rotor when the pump is not in use to prolong tubing life Expanded bed height Increased viscosity due to Check and control the H is higher than decreased temperature temperature if necessary expected Low number of theoretical plates RTD test 152 Increased flow velocity due to high hydrostatic pressure from buffer tank Channelling in the expanded bed due to trapped air in the bottom distribution system Eliminate or decrease the difference in height between the liquid surface in the buffer tank and the s
73. ant aggregation and clogging owing to the glueing effect of nucleic acids forming networks of cells and adsorbent beads If not corrected during the washing stage wash volume time may increase due to channelling in the bed Other problems may also arise during later phases of the purification cycle such as high back pressure during elution in packed bed mode and increased particulate content in the final product pool If such effects are noted during washing a modified wash procedure containing Benzonase Merck Nycomed Pharma A S can be applied to degrade and remove nucleic acids from the expanded bed The following protocol is an example of a suitable wash procedure for removing aggregation caused by released nucleic acids The protocol may have to be further optimized according to the specific conditions in any particular application Wash the bed with 5 sedimented bed volumes of starting buffer upward flow 300 cm h Wash the bed with 2 sedimented bed volumes of starting buffer containing 2 mM MgCl and 50 pl 13000 units Benzonase per litre buffer upward flow 300 cm h Wash the bed with 8 10 sedimented bed volumes of starting buffer upward flow 300 cm h Elution When cells cell debris and other particulate material have been removed from the expanded bed during the wash stage the bed can be sedimented in the column and eluted in packed mode in exactly the same way as in packed bed chromatography Elution in pack
74. applied during feed application Hence the residence time can be increased by decreasing flow velocity or by increasing the bed height The effect of bed height on dynamic binding capacity is demonstrated in Fig 19 The early breakthrough observed at a sedimented bed height of 5 cm is partly due to the inherent instability i e back mixing in the lower part of an expanded bed but also due to a short residence time It can also be explained by a decrease in the number of theoretical plates mass transfer units which lowers the efficiency of the adsorption process When the sedimented bed height is increased to 10 cm the bed stabilizes which together with the increased number of mass transfer units causes a significant increase in dynamic binding capacity A further increase in sedimented bed height is accompanied by a further gradual increase in dynamic binding capacity as the capacity approaches the total available binding capacity of the bed A sedimented bed height of at least 10 cm is required to achieve stable expansion 43 A sedimented bed height of 15 cm is recommended as a starting point for method development to avoid the risk of bed instability and to assure a reasonable number of mass transfer units Increasing the sedimented bed height significantly above 15 cm in an attempt to further increase binding capacity may not be an attractive approach in expanded bed adsorption This is because it can cause the bed to expand up to the
75. aria antigen PfISS RESA An efficient and reproducible production purification scheme is of interest since ZZ M5 and related fusion proteins have been discussed as possible components in a blood stage malaria vaccine The rational behind the design of the fusion protein was to achieve a low combined isoelectric point to enable selective recovery using a DEAE anionic adsorbent at relatively low pH The isoelectric point of the fusion protein was 4 5 which allowed adsorption on the STREAMLINE DEAE adsorbent at pH 5 5 where most of the E coli proteins are not adsorbed The fusion protein was secreted into the culture medium in high yields to give a final concentration of approximately 550 mg L in the fermentation broth The cell density in the fermentation broth was 101 cfu ml and the dry weight was 37 g L The crude fermentation broth adjusted to pH 5 5 was loaded directly onto a 97 STREAMLINE 50 column 50 mm i d containing 200 ml STREAMLINE DEAE corresponding to a sedimented bed height of 10 cm The conductivity and viscosity of the broth was adjusted by online 1 1 mixing with loading buffer immediately before the broth entered the column Online dilution was applied to minimize cell lysis since release of negatively charged DNA might reduce the capacity of the anion exchange adsorbent and significantly increase the viscosity of the fermentation broth The flow velocity during expansion equilibration adsorption and wash was 200 cm h D
76. as been eliminated a switch back to upward flow quickly restabilizes the expanded bed Viscosity is more of a concern when working with intracellular systems compared to the more dilute broths resulting from systems where the target molecule is secreted to the culture medium The effect of feed stock viscosity and biomass content on expanded bed adsorption has been studied by Barnfield Frej et al 28 in an application to recover Annexin V from unclarified E coli homogenate Other host organisms or other experimental conditions may give different results but the data reported by Barnfield Frej et al can be useful as a general guide to the effect of feed stock characteristics on the hydrodynamic properties of an expanded bed To study the effect of biomass dry weight the cell suspensions were homogenized until the viscosity was less than 10 mPa s at a shear rate of 1 st see Table 4 29 Table 4 Example of biomass content and viscosities in E coli homogenates tested on a STREAMLINE DEAE adsorbent 15 cm sedimented bed height expanded in a STREAMLINE 50 column 5 cm i d 100 cm tube height applying a flow rate of 300 cm h 28 Biomass content Viscosity No of passages in homogenization Dry weight Wet weight At 1s shear At 50 s 1 shear At approx 700 bar rate mPa s rate mPa s 4 14 7 3 3 5 17 8 4 4 6 21 9 5 4 7 24 15 7 7 8 27 30 15 6 Trouble free expansion was achieved at biomass dry weights up to 5 At h
77. ated above is significantly shorter than the theoretical residence time hydrodynamic residence time calculated from the reactor volume and the applied flow rate it indicates insufficient fluidization and the formation of flow channels in the lower part of the bed causing early breakthrough of the buffer front Negative step input signal mark UV signal 0 Time Fig 10 UV signal recording during the test procedure for the determination of the number of theoretical plates Note Large dead volumes in a complete configuration with column pumps valves and tubing may cause low values for the number of theoretical plates The positive step signal from 0 to 100 is not recommended for evaluation as the reproducibility of the results is not as high as for the negative step signal Measurement of the theoretical plate number can also be done by injecting a volume of the tracer as a pulse 16 3 Experimental Design A complete downstream processing scheme will consist of different stages Fig 11 Each stage serves a specific purpose related to the state of the feed material the estimated final scale of operation processing time requirements and the amount of purification needed at that stage Each stage will be represented by one or several unit operations and may be referred to as Capture Intermediate Purification and Polishing Capture is the first critical stage in all bioproduct recovery processes It ma
78. ating are substituted with small synthetic ligands and can be exposed to 1 M NaOH In contrast biological ligands such as protein A and heparin are susceptible to hydrolysis at extremes of pH Care must therefore be taken when cleaning and sanitizing these media to avoid decomposing the attached ligands and significantly shortening their working life Specific information about pH stability is found in the description of each STREAMLINE adsorbent STREAMLINE adsorbents with the exception of STREAMLINE rProtein A can also be repeatedly sterilized by autoclaving e g at 121 C for 30 minutes Mechanical stability The mechanical stability of STREAMLINE adsorbents is very high This has been verified in both batch and expansion experiments with STREAMLINE ion exchangers 43 In the batch experiments a 50 slurry of the adsorbent was subjected to different types of treatment and the particle then size analysed Table 10 shows the results as the fraction of particles smaller than 125 pm Note that grinding effects such as those produced by magnetic stirrers should be avoided Table 10 Fragmentation of STREAMLINE adsorbent particles as a result of different types of physical treatment Treatment dn lt 125 um Untreated 9 Dried and rehydrated 9 Propeller mixer high speed 9 Peristaltic pump twice 14 Magnetic stirrer 30 minutes 62 63 In the expansion experiments the mechanical stability of the beads was tested by
79. ation feed application and wash The binding capacity for rh NGF was 10 mg ml adsorbent under those conditions The crude unclarified feed was applied directly onto the expanded bed after adjustment of pH to 5 7 The buffer used during expansion equilibration and wash was 25 mM MES NaMES 0 3 M sodium acetate pH 6 0 Elution was performed at a flow velocity of 100 cm h using downward flow in sedimented bed mode The elution buffer was 25 mM MES NaMES 1 M sodium acetate pH 6 0 After each purification cycle the column was subjected to cleaning in place using a solution consisting of 1 0 M NaOH and 0 5 M NaCl Figure 39 shows a chromatogram from a purification cycle on the STREAMLINE 25 column and Table 43 summarizes the results after scale up to the STREAMLINE 200 column Recovery of rhNGF ranged from 93 to 100 The purification factor was at least 11 fold The concentration factor was 30 50 fold The product pool from the STREAMLINE 200 column was further processed through the established process minus the cation exchange chromatography step The final product was compared with the product produced by the established process The product processed by expanded bed adsorption was equivalent to that processed using the existing process with respect to product quality Purity was determined by SDS PAGE RP HPLC and other assays that detect contaminant levels It was concluded that expanded bed adsorption met all the criteria for implementation in
80. ble in Data File STREAMLINE Chelating 1 Long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance Short term refers to the pH interval for regeneration and cleaning procedures 70 Cleaning in place sanitization in place and sterilization The properties of the base matrix the derivatization chemistry used when attaching the ligand and the inherent stability of the ligand group result in a very stable product This high stability allows exposure to harsh conditions such as 1 M NaOH for cleaning in place and sanitization Suitable cleaning in place protocols must be defined on a case by case basis depending on the nature of the feed applied to the expanded bed Chapter 4 Method Optimization contains general information about cleaning in place of STREAMLINE adsorbents The protocols previously described for STREAMLINE ion exchange adsorbents can also be applied for STREAMLINE Chelating STREAMLINE Chelating can be sanitized by washing the bed with 0 5 1 0 M NaOH for a contact time of 30 60 minutes This is an effective disinfectant treatment for vegetative bacteria yeast and moulds 47 STREAMLINE Chelating can be sterilized by autoclaving the adsorbent at 121 C for 30 minutes Storage We recommend storing STREAMLINE Chelating in 20 ethanol As an alternative we recommend 10 mM NaOH which is comparable to 20 ethanol fr
81. btained from the dispersion model Applying the tanks in series model yields N which is similar to the number of stages according to the HETP concept under non binding conditions Both models allow quantification of axial mixing in liquid fluidized beds from tracer pulse experiments A distribution curve like the RTD may be quantified by the moments of the distribution which are calculated according to equation 6 6 oO m Gy C t dt 0 C t is the concentration at the column outlet my stands for the area below the concentration time curve the total amount of tracer applied and is used to normalize the moments The first normalized moment p4 represents the mean residence time qt the second normalised moment p is used to calculate the variance of the distribution o2 as shown in equations 7 and 8 156 7 o0 fe CDd py T fedt 0 8 O Hi Mean and variance of the RTD may now be used to calculate the dimensionless groups Bo or Pe as well as N without needing to perform non linear regression 81 Under the boundary conditions of a closed system o2 is correlated to Bo Pe according to equation 9 Equation 10 is used to calculate the tank number N 9 2 1 exp B ai fan 10 03 If a correct experimental set up is chosen especially with regard to selecting a tracer and applying the pulse correctly then the methods described above can be used to describe overall mixing in a
82. c acid More detailed recommendations about suitable cleaning protocols can be found in the instructions accompanying each medium A logical approach to defining a suitable cleaning protocol can be as follows See also Section 5 Experimental Technique for detailed information on how to operate the column during CIP 1 Set up a small scale system and run a number of purification cycles without a CIP cycle in between until the point is reached where it is obvious that hydrodynamic and or chromatographic properties are compromized Sample load should be in the same percent range as in the final production method 2 Try to restore bed performance by cleaning with different types of agents Start with 0 5 1 0 M NaOH or a mixture of 0 5 M NaOH and 1 M NaCl using a fairly long contact time e g 4 hours If performance is not restored extend the exposure time to overnight treatment If performance is still not completely restored try other cleaning agents such as an organic solvent a detergent 25 acetic acid 20 ethanol a chaotropic agent etc 3 After one or several cleaning agents have been identified for their effectivness in recovering stability and performance of the expanded bed a suitable cleaning protocol is defined and applied in a scaled down version of the process to verify cleanability and repetitive use of the bed The cleaning protocol should be as simple as possible Preferably such a study is performed on new medium and
83. cation 56 Wash 1 When all the sample has been applied switch to wash buffer and use upward flow in expanded mode to remove loosely bound material and cells Use the same flow velocity as that used during feed application 2 While washing at this flow velocity lower the adaptor to just above the surface of the expanded bed see Fig 26 This speeds up the wash cycle and reduces the consumption of wash buffer This effect will be further enhanced by repeatedly adjusting the adaptor to keep it just above the surface of the expanded bed for the remainder of the wash If the feed stock contains a lot of particulates wait until most have been flushed out before lowering the adaptor Particulates can be trapped in the net if the adaptor is lowered too early or too quickly 3 Continue washing until the signal from the UV monitor returns to the base line 4 Switch to downward flow for a few seconds and then back to upward flow Repeat this procedure several times This back flush removes any particulates that might still be trapped in the distribution system 5 Turn off the pump and allow the bed to sediment 6 When the adsorbent has sedimented move the adaptor down towards the surface Stop when the edge of the adaptor net touches the bed Waste Fig 26 Schematic representation of valve positions and liquid flow in a manual STREAMLINE system during washing The figure shows valve positions when lowering the adapto
84. ce and sanitization in place procedures In 1993 Pharmacia Biotech introduced new types of chromatographic adsorbents and columns called STREAMLINE 17 18 products specially designed for Expanded Bed Adsorption STREAMLINE adsorbents and columns allow the formation of stable fluidized beds at high operating flow velocities The first media introduced were two ion exchangers STREAMLINE DEAE and STREAMLINE SP both developed from the highly biocompatible agarose base matrix by the inclusion of an inert crystalline quartz core material to provide the required density The defined particle size and density distribution of the STREAMLINE adsorbents together with the specially designed STREAMLINE columns yield expanded beds with well defined and consistent hydrodynamic properties and with adsorption characteristics similar to those of packed beds of standard chromatography media 19 20 21 22 23 The properties of Expanded Bed Adsorption make it the ultimate capture step for initial recovery of target proteins from crude feed stock The process steps of clarification concentration and initial purification can be combined into one unit operation providing increased process economy due to a decreased number of process steps increased yield shorter overall process time 24 reduced labour cost 25 and reduced running cost and capital expenditure 26 Expanded Bed Adsorption has also proved to be a versatile tool that can be applied on all
85. cedures are necessary to restore bed performance between runs Proper cleaning ensures repeated use of the adsorbent over a large number of purification cycles When the effects of the crude feed on the expanded bed have been examined the appropriate adjustments made and the flow rate set for feed application optimal loading of crude feed in expanded bed mode should be determined by performing a breakthrough capacity study This is similar to the breakthrough capacity studies for clarified feed previously performed in packed bed mode The various problems that may be encountered during the method optimization phase are discussed in more detail in Section 4 Method Optimization Process verification Verification of the optimized method for expanded bed mode can be carried out by scaling up to pilot scale using STREAMLINE 50 50 mm i d or STREAMLINE 200 200 mm i d columns providing sedimented bed volumes of up to 9 litres STREAMLINE 200 at a maximum sedimented bed height of 30 cm A nominal bed height of 15 cm gives a sedimented bed volume of 0 29 litre in a STREAMLINE 50 column and 4 7 litres in a STREAMLINE 200 column The principle for scale up is similar to that used in packed bed chromatography Scale up is performed by increasing the column diameter and maintaining the sedimented bed height flow velocity and expanded bed height This preserves both the hydrodynamic and chromatographic properties of the system Scalability and
86. commonly used source materials Successful processing by Expanded Bed Adsorption has been reported for E coli homogenate 14 24 27 29 40 57 58 63 74 E coli lysate 30 53 57 E coli inclusion bodies 31 secreted products from E coli 32 57 yeast cell homogenate 33 34 68 secreted products from yeast 41 54 56 69 75 whole hybridoma fermentation broth 35 64 70 73 myeloma cell culture 65 whole mammalian cell culture broth 25 36 66 67 milk 55 60 animal tissue extracts 71 and culture supernatant from a continuous fluidized bed bioreactor 61 Expanded Bed Adsorption by the use of STREAMLINE adsorbents and columns has also proven to be a scalable technique 37 39 66 that has found its way into the production halls of pharmaceutical manufacturers 40 41 A review of protein purification by adsorption chromatography in expanded beds has been published by Chase 42 2 Principles of Expanded Bed Adsorption Expanded bed adsorption is a single pass operation in which desired proteins are purified from crude particulate containing feed stock without the need for separate clarification concentration and initial purification The expansion of the adsorbent bed creates a distance between the adsorbent particals i e increased voidage void volume fraction in the bed which allows for unhindered passage of cells cell debris and other particulates during application of crude feed to the column Ba
87. cule by proteases released upon cell lysis extracellular products Elute in expanded bed mode see page 37 Remove the aggregates during cleaning in place Decrease flow velocity during elution Increase elution strength of the eluting buffer Change flow direction Pause the system stop the flow after having filled the bed with eluent buffer Continue with elution after a static incubation time of approximately one hour Apply a wash procedure containing nuclease e g Benzonase to degrade and remove nucleic acids from the bed prior to elution see page 36 Prevent cell lysis by on line dilution and by increasing the osmolality of the diluent see pages 33 117 119 Use fresh cultures to prevent extensive cell lysis 9 Ordering information Adsorbents Product Pack size Code No STREAMLINE SP 100 ml 17 0993 05 300 ml 17 0993 01 7 5L 17 0993 02 60 L 17 0993 03 STREAMLINE DEAE 100 ml 17 0994 05 300 ml 17 0994 01 TOL 17 0994 02 60 L 17 0994 03 STREAMLINE SP XL 100 ml 17 5076 05 300 ml 17 5076 01 75k 17 5076 02 STREAMLINE Q XL 100 ml 17 5075 05 300 ml 17 5075 01 7 5L 17 5075 02 STREAMLINE Chelating 300 ml 17 1280 01 75 1 17 1280 02 STREAMLINE Heparin 75 ml 17 1284 06 300 ml 17 1284 01 igen 17 1284 02 STREAMLINE rProtein A 75 ml 17 1281 01 300 ml 17 1281 02 5L 17 1281 03 Columns Product Code No XK 16 20 16 mm i d 18 8773 01 XK 26 20 26 mm i d 18 1000 72 STREA
88. d bed and breakthrough capacity Studies with different types of feed materials on different types of STREAMLINE adsorbents 29 31 33 34 45 46 58 have revealed that hydrodynamic and 38 chromatographic properties can be maintained over a large number of purification cycles if simple but efficient CIP protocols are performed between each run If the nature of the coupled ligand allows it an efficient CIP protocol would be based on 0 5 1 0 M NaOH as the main cleaning agent NaOH is the most widely accepted cleaning agent in downstream processing since it is a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pyrogens It is also a safe technique since it leaves no harmful traces in the column that can contaminate the product If the medium to be cleaned is an ion exchange medium the column should always be washed with a concentrated aqueous solution of a neutral salt e g 1 2 M NaCl before cleaning with NaOH Usually this is part of the regeneration at the end of the elution stage This removes most of the residual proteins and other contaminants attached to the medium by strong electrostatic interaction The NaOH wash will then remove irreversibly precipitated denatured substances and lipids In its simplest form the CIP protocol can be composed of a single wash with NaOH possibly adding 1 M NaCl to further increase the cleaning efficiency The flow velocity sh
89. d flow at the pre determined feed application flow velocity 4 Allow the bed to stabilize at this flow velocity for approximately 30 40 minutes The bed is stable when no further expansion can be observed and only small circular movements of the individual particles are visible see Fig 8 5 When the pH conductivity of the outlet stream is the same as the equilibration buffer recirculate the buffer by connecting the system outlet to the equilibration buffer container Continue until test sample or feed is applied to the bed 6 Check the stability of the expanded bed using the test principles outlined in Evaluation of bed stability in Chapter 2 P1 Waste Fig 24 Schematic representation of valve positions and liquid flow in a manual STREAMLINE system during expansion equilibration 55 Feed application 1 Position the adaptor 3 4 cm below the top of the column tube to allow for the bed expanding which usually occurs when feed is applied When the behaviour of the feed stock is better known the adaptor can be positioned 5 10 cm above the height to which the bed expands during feed stock application 2 When the expanded bed is stable and equilibrated with adsorption buffer switch to feed application as shown in Fig 25 Stir the feed stock continuously during application to preven
90. d to the adsorbent No such reduction in capacity is seen with the salt tolerant affinity adsorption on STREAMLINE rProtein A 125 Table 49 Summary of results from three different scales of expanded bed adsorption of a monoclonal IgG on STREAMLINE rProtein A XK 16 40 STREAMLINE 25 STREAMLINE 50 Run1 Run2 Run1 Run2 Run3 Runt Run2 Adsorbent volume ml 20 20 50 50 50 150 150 Feed volume L 10 10 20 20 20 60 60 Conc IgGo in feed mg L 18 9 13 8 34 50 50 40 29 Challenge mg lgG2 ml adsorbent 8 8 6 9 13 6 20 20 16 11 6 Conc IgGs in eluate mg L 2500 1200 11992 2084 2509 11972 9622 Concentration factor 132 87 35 42 50 30 33 Yield 126 87 95 78 84 83 82 1 Flow velocity during expansion equilibration feed application and wash was 420 cm h 2 Elution was performed in expanded mode using a flow velocity of 360 cm h Expanded Bed Adsorption in Capture from Milk This section contains two applications on expanded bed adsorption from milk one from skimmed equine milk and the other one from milk of transgenic livestock Purification of lysozyme from equine milk by expanded bed cation exchange adsorption Highly active pure lysozyme was purified from skimmed equine milk by expanded bed cation exchange adsorption on STREAMLINE SP 55 The purpose was to develop a method to prepare lysozyme from milk that was faster than existing methods In previously reported preparative techniques caseins are first removed by sa
91. de numbers Fraction collector Controller GradiFrac Manual valves Double channel SRV 4 Single channel SRV 3 Solenoid valves PSV 50 Pump P 50 Tubing 1 9 x 2 7 mm PTFE UV monitor Flow cell UV 1 S2 Connectors 2 7 mm o d M6 Recorder REC 101 102 Miscellaneous Rack Automated systems Small scale operation with AKTAexplorer STREAMLINE 25 column may be connected to AKTAexplorer chromatography system for completely automated method optimization and small scale processing Re configure the AKTAexplorer system as described below See Fig 22 for the complete system configuration 1 Disconnect the prefilter and the mixer 2 Connect Pump A directly to flow direction valve V7 position 7 50 3 Disconnect the sample loop and the sample pump from the injection valve V1 4 Connect Pump B directly to injection valve position 2 5 Connect injection valve position 3 to the hydraulic inlet of the STREAMLINE column 6 Install 1 0 mm 1 d tubing in the flow path after the column By pass the pH flow cell and the flow restrictor to reduce system back pressure in the column 7 Fit a stop plug in outlet valve V4 position 8 Setting the injection valve to position 2 and the outlet valve to position 8 opens the flow path from the hydraulic chamber of the column to W1 and blocks the column outlet This lifts the adaptor in the column when pump A pumps upward flow through the bed The adaptor is lowered by pumpin
92. ded bed adsorption unit operation are listed in Table 2 Table 2 The different phases of developing an expanded bed adsorption unit operation Method Method Process Production scouting optimization verification Purpose Screening of Optimization of Verification at pilot Production at full binding and binding elution scale scale elution conditions wash and CIP Production for using clarified using unclarified clinical trials material in material in packed bed mode expanded mode at small scale Column XK 16 or XK 26 STREAMLINE 25 STREAMLINE 50 STREAMLINE CD STREAMLINE 200 custom designed Sedimented bed 0 02 0 15 0 05 0 15 0 2 9 up to several volume litres hundred litres Method scouting Method scouting i e defining the most suitable STREAMLINE adsorbent and the optimal conditions for binding and elution is performed at small scale using clarified feed in packed bed mode Selection of adsorbent is based on the same principles as in packed bed chromatography The medium showing strongest binding to the target protein while binding as few as possible of the contaminating proteins i e the medium with the highest selectivity and or capacity for the protein of interest will be the medium of choice Regardless of the binding selectivity for the target protein STREAMLINE adsorbents are compatible with any type of feed material However when purifying proteins from mammalian cell cultu
93. dients position the beads at specific heights in the expanded bed depending on the sedimentation velocity of the individual adsorbent particles The smaller lighter particles move to positions at the top of the expanded bed the larger heavier particles to the bottom resulting in a stable uniform expansion In other words the beads find their ideal position in the column which is the reason for the low axial dispersion in expanded bed adsorption as illustrated in Fig 3 The column The column also has a significant impact on the formation of stable expanded beds STREAMLINE columns are equipped with a specially designed liquid distribution system to allow the formation of a stable expanded bed The need for a specially designed liquid distribution system for expanded beds derives from the low pressure drop over the expanded bed Usually the flow through a packed bed generates such a high pressure drop over the bed that it can assist the distributor in producing plug flow through the column Since the pressure drop over an expanded bed is much smaller the distributor in an expanded bed column must produce a plug flow itself Consequently it is necessary to build in an additional pressure drop into the distribution system Besides generating a pressure drop the distributor also has to direct the flow in a vertical direction only Any flow in a radial direction inside the bed will cause turbulence that propagates through the column Shear stre
94. ducts on page 61 More information about STREAMLINE Heparin including instructions for use is available in Data File STREAMLINE Heparin Table 15 summarizes the characteristics of STREAMLINE Heparin Table 15 Characteristics of STREAMLINE Heparin Ligand density 4 mg heparin ml gel Particle size range 100 300 um Approx mean particle size 200 um Approx mean particle density 1 2 g ml Degree of expansion H HO at 300 cm h 2 3 pH stability long term 4 12 short term 4 13 Storage 20 ethanol Further information is available in Data File STREAMLINE Heparin 1 The heparin used for immobilization is isolated from porcine intestinal mucosa and has a molecular weight distribution over the range 5000 30000 2 Long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance Short term refers to the pH interval for regeneration and cleaning procedures 72 Cleaning in place sanitization in place and sterilization The pH stability of STREAMLINE Heparin is limited by the susceptibility to hydrolysis of the heparin ligand at extremes of pH However up to 0 1 M NaOH can be used in cleaning and sanitization without significant loss of binding capacity Alternative treatments for removing strongly adsorbed precipitated or denatured proteins are washing with 6 M guanidine hydrochloride or 8 M urea Non ionic detergents at a conc
95. e negative charge of the deprotonated form of the carboxylic acid groups When used as an ion exchanger a significant part of the carboxylic acid groups lose their negative charge below pH 6 The total ionic capacity of STREAMLINE Chelating adsorbent used as a weak cation exchanger is expected to be 60 70 umole Nat ml gel which is lower than STREAMLINE ion exchange media Despite this STREAMLINE Chelating used as a cation exchanger may provide an alternative selectivity to the STREAMLINE ion exchange adsorbents STREAMLINE Chelating is based on highly cross linked 6 agarose which has been modified by including an inert quartz core to give the desired density It is substituted with iminodiacetic acid groups on spacer arms coupled to the STREAMLINE matrix via stable covalent linkages STREAMLINE Chelating is a Custom Designed Media CDM product See CDM products on page 61 More information about STREAMLINE Chelating including instructions for use is available in Data File STREAMLINE Chelating Table 14 summarizes the characteristics of STREAMLINE Chelating Table 14 Characteristics of STREAMLINE Chelating Chelating group Iminodiacetic acid Total capacity 40 umol Cu2t ml gel Particle size range 100 300 um Approx mean particle size 200 um Approx mean particle density 1 2 g ml Degree of expansion H HO at 300 cm h 2 3 OH stability long term 3 13 short term 2 14 Storage 20 ethanol Further information is availa
96. e objectives are e Stabilize the product e Remove proteases glycosidases etc e Remove solids e Remove cells e Remove water e Remove bulk quantities of proteins nucleic acids and carbohydrates e Prepare for further chromatography At the Capture stage high throughput i e capacity and speed is very important for processing large sample volumes keeping the scale of equipment as small as possible and giving the shortest possible cycle time Processing time is critical at this stage since fermentation broths and crude cell homogenates contain proteases and glycosidases that reduce product recovery and produce degradation products that may be difficult to remove later To prevent product degradation it is desirable to minimize the time the product is exposed to such enzyme activity Adsorption of the target molecule on a solid adsorbent decreases the likelihood of interaction between degradative enzymes and susceptible intramolecular bonds in the target molecule For this reason it is desirable to bind the target molecule as quickly as possible to an adsorbent This can be achieved with STREAMLINE expanded bed adsorption as crude feed can be applied directly to the adsorbent without prior clarification or concentration Expanded bed adsorption also increases productivity at the Capture stage due to the reduced overall processing time and increased yields that result from reducing the number of unit operations applied Furtherm
97. e the tubing connector M6 145 10 11 LZ 13 14 146 10 References Methods of Plasma Protein Fractionation Curling J M Ed Academic Press London UK 1980 pp 117 128 Brummelhuis H G J A novel ion exchange method for the isolation of streptomycin Chem Eng Prog 54 1958 49 52 Barthels C R Kleinman G Korzon N J Irish D B Development of a recovery process for novobiocin Biotechnol Bioeng 15 1973 533 549 Belter P A Cunningham EL Chen J W Fluidized bed adsorption for whole broth extraction Biotechnol Prog 6 1990 370 375 Gailliot F P Gleason C Wilson J J Zwarick J Batch fluidized ion exchange column for streams containing suspended particles J Chromatogr 201 1980 319 327 Buijs A Wesselingh J A Continuous affinity chromatography using a magnetically stabilized fluidized bed Biotechnol Prog 1 1985 95 103 Burns M A Graves D J The effects of magnetic stabilization on the structure and performance of fluidized beds Bioseparations 2 1991 217 230 Nixon L Koval C A Xu L Noble R D Slaff G S Liquid fluidized beds for protein purification I Chem Eng Symp Ser No 118 12 1 12 12 1990 Draeger M N Chase H A Liquid fluidized bed adsorption of proteins in the presence of cells Bioseparations 2 1991 67 80 Draeger M N Chase H A Affinity purification of proteins using expanded beds J Chromatogr 597 1992 129 1
98. e tubing sizes of 3 mm and 10 mm i d for STREAMLINE 50 and STREAMLINE 200 columns respectively The larger tubing size can also accommodate custom designed columns with flow rates of around 400 litre h Table 21 lists technical data for the pilot scale automated STREAMLINE systems STREAMLINE pilot scale system for method development and small scale production Table 21 Technical data for STREAMLINE pilot scale systems 3mm system 10 mm system Tubing diameter 3mm 10 mm Operating pressure 2 bar 2 bar Flow rate max 12 L h 180 L h Flow rate min 0 6 L h 9 L h Operating temperature 4 40 C 4 40 C STREAMLINE pilot scale systems are equipped with the same valves tubing and instruments used in BioProcess Systems supplied by Pharmacia Biotech for packed bed chromatography The valve is a pneumatically driven diaphragm valve with an integrated tubing T piece zero dead leg valve This design reduces the length of the flow path and minimizes the dead volume of the system thereby improving overall system performance and eliminating stagnant areas where microbial growth could occur Sanitary TC connections are used throughout the system including inlet outlet and column connections The hygienic status of this system has been verified in microbial challenge tests 82 Monitors for recording UV pH conductivity pressure and flow rate are included Extra monitors may be added to the system for recording these param
99. ecific adsorption of macromolecules and well documented industrial CIP protocols The reduction in porosity as a result of incorporating core material is insignificant for most applications Derivatization chemistry Ligands are coupled to the STREAMLINE adsorbent with epoxy chemistry which provides stable ether thioether linkages Fig 28 shows the structures of the coupled ligands ve R CH CH sof STREAMLINE SP N STREAMLINE SP XL CH ee ca ee ae tS STREAMLINE Q XL X CH CH 2 5 O CH CH N H STREAMLINE DEAE S C H CH COOH O R CHOH CH N STREAMLINE Chelating N CH COOH i 0 R CHOH CH NH CH 3 Heparin STREAMLINE Heparin S O R CHOH CH S ProtA STREAMLINE rProtein A N Fig 28 The structures of coupled ligands in STREAMLINE adsorbents 62 Chemical stability STREAMLINE adsorbents are stable in all commonly used aqueous buffers detergents organic solvents e g 70 ethanol 30 isopropanol and chaotropes or dissociating agents e g 6 M guanidine hydrochloride and 6 M urea commonly used to operate clean and sanitize chromatography columns in downstream processing Extremes of pH e g high concentrations of sodium hydroxide are often used for cleaning depyrogenation virus inactivation and sanitizing columns The pH stability of STREAMLINE adsorbents varies according to the stability of the ligands attached to the base matrix STREAMLINE ion exchangers and STREAMLINE Chel
100. ecular weight of 17 kD was expressed intracellularly in soluble form in E coli and was released from the harvested cells by high pressure homogenization at 1000 bar Expanded bed adsorption on STREAMLINE SP was the first step capture step in a sequence of three chromatographic steps to purify this protein which was intended to be used in clinical trials Method scouting was performed using clarified feed material on STREAMLINE SP packed in an XK 26 column to a bed height of 5 cm After having defined optimal running conditions unclarified feed stock was used for optimization in expanded mode on a STREAMLINE 50 column 50 mm i d containing 400 ml of adsorbent providing a sedimented bed height of 20 cm The method was finally scaled up to pilot scale in a STREAMLINE 200 column 200 mm i d containing 8 litres of adsorbent Seventy six litres of unclarified feed material containing 410 g of protein were applied to the STREAMLINE 200 column 88 Cleaning in place was performed after each purification cycle by exposing the adsorbent to 0 5 M NaOH for one hour without flow This solution was then replaced by 0 1 M NaOH or 20 ethanol for storage until the next run More than 80 capture cycles followed by CIP were successfully run on the same STREAMLINE SP adsorbent Recovery varied between 73 and 156 as measured by reversed phase HPLC Fig 31 Recovery for the same E coli strain and plasmid was 85 22 n 6 s D O 126 1
101. ed Bed Affinity Purification and Validation Poster presented at Cell Culture Engineering V San Diego California USA January 28 February 2 1996 Katinger H Schmatz C Lenz S Koller G Kreismayr G Klima G Unterluggauer F Katinger D Doblhoff Dier O Construction of a rProtein A Affinity Media for use in Expanded Bed Adsorption Chromatography Presented at 211th American Chemical Society National Meeting New Orleans Louisiana USA March 24 28 1996 Kennedy R M Large Scale Purification and Characterization of Recombinant Fibroblast Growth Factor Saporin Mitotoxin Protein Expression and Purification 8 1996 97 108 McDonald J R Ong M Shen C Parandoosh Z Sosnowski B Bussel S Houston L L Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds J Chromatogr A 752 1996 111 122 Thommes J Bader A Halfar M Karau A Kula M R 65 66 67 68 69 70 7a ee 73 74 Capture of a Humanized IgG4 Directly from the Fermenter Using STREAMLINE rProtein A Presented at Recovery of Biological Products VIII Tucson Arizona October 20 25 1996 Abstr p 16 J gersten C Johansson S Bonnerjea J Pardon R Expanded Bed Adsorption Chromatography Purification of a Monoclonal Antibody Presented at Recovery of Biological Products VIII Tucson Arizona October 20 25 1996 Abstr p 16 Zapat
102. ed harvested cell culture fluid in packed bed mode in a 1 x 10 cm column The conductivity in the cell culture fluid was approximately 15 mS cm To achieve good binding the cell culture fluid had to be diluted to around 6 mS cm before application to the STREAMLINE SP adsorbent Optimal binding was achieved at around pH 5 5 After the method scouting in packed bed mode the method was optimized in expanded mode using a STREAMLINE 25 column 25 mm i d The column contained 75 ml of STREAMLINE SP corresponding to a sedimented bed height of 15 cm The crude unclarified cell culture suspension was applied directly onto the STREAMLINE SP adsorbent using on line dilution to reduce conductivity immediately before the feed entered the column In the initial experiments plain water or starting buffer was used as diluent Cell viability in the cell culture was 66 which had been reduced to 20 at the time when the feed had passed through the column Cell viability was measured as the activity of lactate dehydrogenase LDH in the cell culture suspension The decrease in cell viability indicated that cells lysed as a consequence of changed osmotic pressure during dilution Extensive expansion of the bed during feed application gave further evidence for cell lysis causing increased viscosity due to release of nucleic acids Further experiments using 200 300 mM glucose as diluent confirmed that extensive cell lysis occured if the cells were not protected agains
103. ed mode reduces the volume of eluent needed and gives a more highly concentrated product pool It also decreases the risk of contaminating the product pool with trace amounts of cells and aggregates that may still be bound to the adsorbent beads and desorbed with the target protein during elution 36 Step wise elution is often preferred to continuous gradients since it allows the target protein to be eluted in a more concentrated form reduces buffer consumption and gives shorter cycle times Being a typical capture step separation from impurities in expanded bed adsorption is usually achieved by selective binding of the product which can simply be eluted from the column at high concentration with a single elution step The flow during elution can be directed either in the same direction as during sample application i e upwards or in the reverse direction i e downwards If only a small fraction of the adsorbent s capacity has been used resulting in adsorbed material being located predominately at the inlet of the bed reversed flow direction is likely to give a more concentrated product pool If the maximum capacity of the adsorbent has been used resulting in adsorbed material being located over the complete bed or predominantly at the bed outlet due to displacement effects elution by upward flow may be preferred The flow velocity during elution also affects the concentration of the product pool A lower flow velocity will give a smal
104. ed on a STREAMLINE 25 column 25 mm i d The STREAMLINE 25 column contained 75 ml of STREAMLINE rProtein A adsorbent corresponding to a sedimented bed height of 15 cm The column was operated in a semi automated system based on the programmable fraction collector GradiFrac and the piston pump P 50 Pharmacia Biotech 122 The method was finally scaled up to pilot scale using a STREAMLINE 200 column 200 mm i d containing 5 litres of STREAMLINE rProtein A corresponding to a sedimented bed height of 15 cm The STREAMLINE 200 column was operated by a peristaltic pump Bed expansion equilibration feed application and wash were performed at an upward flow velocity of 300 cm h The crude unclarified feed was applied directly onto the expanded bed after adjustment of pH The buffer used during expansion equilibration and wash was 50 mM Tris HCl pH 8 0 containing 150 mM NaCl Elution was performed at a flow velocity of 100 cm h using downward flow in sedimented bed mode The elution buffer was 50 mM phosphate 50 mM sodium citrate pH 4 5 containing 150 mM NaCl A concentrated basic buffer 0 5 M Tris HCl 150 mM NaCl pH 8 6 was introduced into the eluate to adjust pH to a more moderate level Table 47 summarizes results from both laboratory and pilot scales The results for the STREAMLINE 25 scale are average values from five purification cycles The procedure was not run at maximum loading capacity The capacity was 14 mg IgG ml adsorbent a
105. eic acids can be circumvented by treating the feed material with nuclease e g Benzonase which will degrade the nucleic acids into smaller fragments 46 l Benzonase is a genetically engineered endonuclease produced in E coli which is active on all forms of DNA and RNA According to information provided by the supplier Merck Nycomed Pharma A S the enzyme is produced under strict regulatory control to make it a suitable tool in industrial scale bioprocesses The enzyme is more than 90 pure free from viral contaminants and proteases Any impurities derive solely from the E coli host An ELISA kit is available for validating processes where Benzonase is used 31 Even if the concentration of nucleic acids in the feed is not extremely high and there is no sign of deteriorated hydrodynamic properties of the expanded bed they may still attach to the adsorbent beads by non specific interaction causing a slow build up which may effect binding characteristics after a number of purification cycles Such contamination may be removed from the column by washing with a few bed volumes of 1M NaOH 1M NaCl Complete removal may only be accomplished by treating the bed with a nuclease Another consequence of the presence of nucleic acids in the feed material is that they may effect the binding capacity and or the selectivity This is not solely related to nucleic acids but also to other types of polyionic macromolecules or highly charged insoluble mater
106. ell weight at harvest of 60 g L The molecular weight of the recombinant protein was 7 7 kDa and the isoelectric point was 4 5 Method optimization was performed on a STREAMLINE 50 column 50 mm i d containing 410 ml of STREAMLINE SP adsorbent which corresponds to a sedimented bed height of 21 cm Final production of phase II clinical material was performed on a STREAMLINE 200 column 200 mm i d containing STREAMLINE SP adsorbent providing the same sedimented bed height of 21 cm 111 The method development work included optimization of wash steps replacement of gradient with stepwise elution and optimization of flow velocity and column loading to maximize product recovery The effect of biomass dry weight on degree of expansion and bed stability was also examined At biomass dry weights greater than 40 g L the bed was found to expand to the top of the column when the feed was applied at a flow velocity of 200 cm h At a biomass dry weight of 95 g L product recovery decreased to 66 At biomass dry weights up to 65 g L recoveries were consistently greater than 82 A biomass dry weight of 40 g L was considered to offer the best balance in terms of feed stock dilution degree of bed expansion overall processing time and product recovery The crude unclarified feed was applied directly onto the expanded bed after dilution and adjustment of pH to 3 5 The flow velocity during feed application was 200 cm h The same flow velocity was appl
107. emoval of suspended cells after a wash with 4 litres of buffer 34 I2 25 5ra 5M D25 Volume L 100 1 SPpH5 5 10 N E DEAE pH 7 6 O DEAE pH 5 5 Column STREAMLINE 50 5 cm i d 1 m length Binding 30 mM NH4Ac pH 5 5 or washing buffers 30 mM Tris HCl pH 7 6 Elution buffers 0 1 0 25 or 0 5 M NaCl in binding buffer Flow rate 300 cm h at sample application and wash 100 cm h at elution 102 108 Sample 2 L E coli homogenate dry weight 3 4 104 Analysis The number of colony forming units cfu per mL was determined in the homogenate and in samples collected during the run Relative reduction of living bacteria 10e Fig 16 Relative reduction of living E coli cells during the wash and elution steps on STREAMLINE DEAE and SP at pH 5 5 and 7 6 Work by Pharmacia Biotech In an application on STREAMLINE rProtein A for purification of monoclonal antibodies 64 clarification efficiency was determined by particle analysis using a Coulter Counter Fig 17 A total of 60 litres of a whole hybridoma cell culture broth was applied to 150 ml of adsorbent Wash was performed with buffer until the UV signal returned to the base line It was shown that after an initial retardation all the particles contained in the feed left the column with the flow through fractions A more than 100 fold reduction of particles was detected in the eluate fraction 3 0E 06
108. en after extensive centrifugation of the feed stock precipitation continues and may finally block a packed chromatography bed When a non secreted product accumulates in the periplasmic compartment it can be released by disrupting the outer membrane without disturbing the cytoplasmic membrane Accumulation in the periplasmic space can thus reduce both the total volume of liquid to be processed and the amount of contamination from intracellular components However it is usually very difficult to release the product from the periplasmic space without piercing the cytoplasmic membrane and thereby releasing intracellular contaminants such as large fragments of nucleic acids which may significantly increase the viscosity of the feed stock 23 In traditional downstream processing the initial unit operations often include some type of pre treatment to remove contaminants such as lipids and DNA to reduce fouling the adsorbent and increase the working life of the column In expanded bed adsorption these problems are addressed by careful selection of an efficient cleaning in place protocol to apply after each purification cycle Cleaning in place procedures and other measures to reduce the effect of cells cell debris and contaminants on the chromatographic and hydrodynamic properties of the expanded bed will be further discussed in Section 4 Method Optimization Experimental strategy The different phases of developing an optimized STREAMLINE expan
109. en eluted from the column the CIP solution was recirculated through the bed for another 6 hours Table 44 summarizes the results from processing of 7324 litres of unclarified cell culture suspension on the STREAMLINE 1200 column The process resulted in complete removal of cellular mass and a five fold concentration of the antibody Furthermore 99 of the antibody was recovered Table 44 Summary of results from the recovery of a recombinant monoclonal antibody on STREAMLINE SP in a STREAMLINE 1200 column Volume L Adsorbent 170 Feed stock undiluted 7324 Feed stock diluted 17396 Wash 4807 Elution 1409 CIP 4800 Time hours Feed application 5 5 Wash 2 4 Elution 1 3 CIP 6 Cell density in feed stock cells ml 13 8 x 105 Cell viability in feed stock 52 Challenge mg antibody mg adsorbent 21 5 Concentration factor 5 Yield 99 Recovery of an antibody from CHO cell culture broth by expanded bed cation exchange adsorption A process has been described for expanded bed adsorption on STREAMLINE SP to capture an antibody from crude unclarified CHO cell culture broth 25 Two pilot runs were performed by applying unclarified serum free whole culture broth to a 50 mm inner diameter column containing 170 ml of STREAMLINE SP adsorbent corresponding to a sedimented bed height of 8 6 cm Prior to loading of 118 feed stock 25 mM MES buffer pH 5 4 was run through the column to equilibrate the adsorbent and gradually ex
110. ence on Expanded Bed Adsorption EBA 96 Cambridge UK December 1996 Paper no P6 5 Gellissen G Keup P Th mmes J Kula M R Simple two step procedure for the preparation of highly active pure equine milk lysozyme J Chromatogr 719 1996 327 331 Noppe W Hanssens I De Cuyper M Production of Two Aprotinin Variants in Hansenula polymorpha Process Biochemistry 31 1996 679 689 Zurek C Kubis E Keup P H rlein D Beunink J Th mmes J Kula M R Hollenberg C P Gellissen G A General Method for the Purification of Recombinant Proteins Expressed in E coli Poster presented at Prep 96 Washington USA May 1996 Daniels A I Bjork P Ljungl6f A Danielsson A Purification of a therapeutic recombinant protein using expanded bed adsorption chromatography Downstream No 23 1996 Pharmacia Biotech AB Ollivier M Bussone P Wallet J C Purity of Recombinant Protein A Used as a Ligand in New Affinity Chromatography Media Validation of Analytical Methods Poster presented at Pittcon 96 Chicago USA Isaksson K Hellberg U Moberg A Expanded Bed Purification of a Recombinant Protein from the Milk of Transgenic Livestock Presented at 211th American Chemical Society National Meeting New Orleans Louisiana USA March 24 28 1996 Degener A Belew M Velander W H Contract Manufacture of Clinical Grade CHO Recombinant Human Mab s Perfused Fluidized Bed Production Expand
111. ent of elution mode 37 Cleaning in place CIP The working life of chromatographic media has a significant impact on process economy in downstream processing A long working life means less frequent replacement of the media resulting in decreased running costs and increased overall process economy The working life of a STREAMLINE adsorbent is affected by the different types and amounts of contaminating material present in the feed stock applied to the column In any type of chromatography precipitated denatured or non specifically bound substances can block binding capacity and or interfere with the chromatographic selectivity of the adsorbent In expanded bed adsorption media are further challenged by the nature of the feed stock which may contain cells cell agglomerates cell debris and other membrane associated particulate material as well as a high content of lipids and nucleic acids released by cell lysis Such contamination can disturb the hydrodynamic properties of the bed by physical entrapment of large aggregates in the bed or by strong interaction causing aggregation of adsorbent beads The effect of such disturbances may be increased axial dispersion or even severe channelling in the bed often accompanied by a reduced degree of expansion Channelling and turbulence in the bed can also cause an increase in the volume of buffer needed before the UV signal returns to baseline during the wash stage In severe cases of fouling the
112. entration of 0 1 0 5 can be used to remove hydrophobically bound substances Chapter 4 Method Optimization contains general information about cleaning in place of STREAMLINE adsorbents STREAMLINE Heparin can be sanitized by washing the bed with a mixture of 0 1 M NaOH and 20 ethanol for a contact time of 1 hour An alternative procedure is to equilibrate the bed with 70 ethanol and allow it to stand for 12 hours Note that specific regulations for classified areas and explosion proof equipment may apply when handling large volumes of organic solvents STREAMLINE Heparin can be sterilized by autoclaving the adsorbent at 121 C for 30 minutes Storage We recommend storing STREAMLINE Heparin in 20 ethanol Product availability STREAMLINE Heparin is supplied as a suspension in 20 ethanol in packs of 75 ml 300 ml and 7 5 litres For larger quantities please contact your local Pharmacia Biotech office STREAMLINE rProtein A Product characteristics STREAMLINE rProtein A is an affinity adsorbent for purifying monoclonal and polyclonal antibodies 62 It is based on highly cross linked 4 agarose modified by including an inert metal alloy core to provide the required high density BioProcess The ligand is a recombinant protein A specially engineered by fusing a cysteine residue to the C terminus to favour an oriented coupling to the matrix by epoxy chemistry This coupling generates a stable thioether linkage between t
113. eriod of time without adverse effects on its subsequent chromatographic performance Short term refers to the pH interval for regeneration and cleaning procedures 2 Breakthrough capacity determined in a 4 4 ml packed bed at a flow velocity of 300 cm h using a 2 0 mg ml solution of protein in 50 mM Glycine HC l buffer pH 9 0 lysozyme and 50 mM Tris HCl buffer pH 7 5 BSA Bed height was 10 cm 68 Cleaning in place sanitization in place and sterilization The properties of the base matrix the derivatization chemistry used when attaching spacers and ligands and the inherent stability of the ligand groups result in very stable ion exchange media This high product stability allows exposure to harsh conditions such as 1 M NaOH for cleaning in place and sanitization Suitable cleaning in place protocols must be defined on a case by case basis depending on the nature of the feed applied to the expanded bed Chapter 4 Method Optimization contains general information about cleaning in place of STREAMLINE adsorbents The protocols previously described for STREAMLINE SP and STREAMLINE DEAE adsorbents can also be applied for STREAMLINE SP XL and STREAMLINE Q XL STREAMLINE SP XL and STREAMLINE Q XL can be sanitized by washing the bed with 0 5 1 0 M NaOH for a contact time of 30 60 minutes This is an effective disinfectant treatment for vegetative bacteria yeast and moulds 47 Storage We recommend storing STREAMLINE SP XL and S
114. es of STREAMLINE rProtein A Fig 40 shows chromatograms from laboratory scale and pilot scale runs on STREAMLINE 25 and STREAMLINE 200 respectively a Column STREAMLINE 25 25 mm i d Adsorbent STREAMLINE rProtein A Feed 1 5 litres of myeloma cell culture Buffer A 50 mM glycine 250 mM NaCl pH 8 Buffer B 100 mM glycine pH 3 0 A280 Flow velocity 300 cm h at equilibration feed application and wash 100 cm h at elution and CIP 80 160 Sample loading Washing Washingne so expanded mode packed mode Time min b Column STREAMLINE 200 200 mm i d Adsorbent STREAMLINE rProtein A Feed 93 litres of myeloma cell culture A Buffer A 50 mM glycine 250 mM NaCl pH 8 280 Buffer B 100 mM glycine pH 3 0 Flow velocity 300 cm h at equilibration feed application and wash 100 cm h at elution and CIP 80 endl e 160 Sample loading Washing Washing Elution expanded mode packed mode Time min Fig 40 Laboratory a and pilot scale b purification of a monoclonal IgG 4 antibody on STREAMLI NE rProtein A Work by Pharmacia Biotech in collaboration with Celltech Biologics Plc 121 Table 46 summarizes the results from two runs at laboratory scale on STREAMLINE 25 and the pilot scale run on STREAMLINE 200 The purity as determined by SDS PAGE was high and consistent between the different runs Very low levels of BSA transferrin and DNA were found in the eluate The antibody was
115. esented at 205th American Chemical Society National Meeting Denver Colorado USA April 1993 Paper no 61 Suding A Tomusiak M Expanded Bed Adsorption Process for Protein Recovery from Whole Mammalian Cell Culture Broth Bioseparations 5 1995 41 52 Batt B C Yabannavar V M Singh V Impact of Improved Chromatographic Media on Productivity and Process Design in Downstream Processing Poster presented at 12th Meeting of European Animal Cell Technology Wirtzburg Germany May 1993 Schmidt C Berglof J H Lindquist L O 147 L Zo 29 30 31 32 33 34 35 36 37 38 148 Pilot Scale Purification of Recombinant Annexin V using Expanded Bed Adsorption STREAMLINE and Hydrophobic Interaction Chromatography Butyl Sepharose 4 Fast Flow Poster presented at 6th European Congress on Biotechnology Florence Italy June 1993 Sk ld A Daniels I Barnfield Frej A K Pilot Scale Recovery of Recombinant Annexin V from Unclarified Escherichia coli Homogenate using Expanded Bed Adsorption Biotech amp Bioeng 44 1994 922 929 Barnfield Frej A K Hjorth R Hammarstr m A Purification of Recombinant Anti HIV Fab Fragment Expressed in Escherichia coli Poster presented at Recovery of Biological Products VII in San Diego California USA September 1994 Jagersten C et al Purification of a Recombinant Bacterial Exotoxin A by Expanded Bed Adsorption and a New Ion Exchange
116. esorption of the fusion protein was performed with downward flow in sedimented bed mode using 0 5 M NaCl The flow velocity during desorption was 100 cm h Following the initial purification step on STREAMLINE DEAE the fusion protein was further purified by affinity chromatography on IgG Sepharose utilizing the IgG affinity of the fusion partner ZZ Table 32 summarizes the results from both purification steps As determined by SDS PAGE the eluate from the expanded bed contained almost exclusively ZZ indicating the low amounts of extracellular E coli proteins that bind to an anion exchanger at pH 5 5 The second purification step on IgG Sepharose gave no further visual purification of ZZ M5 but was included as a polishing step to reduce the amount of DNA and endotoxin in the final product Considering an immunization dose of 50 100 pg protein the levels of DNA and endotoxin after the two step purification procedure were in the range acceptable by regulatory authorities 10 100 pg DNA dose and 10 EU mg product respectively The expanded bed adsorption also enabled efficient cell removal and a 16 fold volume reduction It was concluded by viable count measurements that 99 99 of the cells could be removed by washing the expanded bed with six bed volumes The whole expanded bed process was completed within four hours and the entire procedure from inoculation of the fermentor to the recovery of the purified product was completed within two working da
117. eters at additional locations in the liquid flow A three channel line recorder is available as an option for recording UV pH and conductivity All system pumps are peristaltic Table 22 lists materials of construction Table 22 Materials of construction for STREAMLINE pilot scale systems Component Material of construction Tubing PP Valves PP1 EPDM2 Air trap Glass Stainless steel ASTM 316L EPDM Pressure sensor Stainless steel ASTM 316L UV flow cell Stainless steel ASTM 316L quartz Conductivity flow cell and sensor Stainless steel ASTM 316L PEEKS OH flow cell and sensor PP glass Flow monitor cell Stainless steel ASTM 316L PFA4 1 Polypropylene 2 Ethylenepropylenedimonomer 3 Polyetheretherketone 4 Polyperfluoro alkoxy ethylene Perfluoro alkoxy alkane Production scale automated systems Large scale STREAMLINE systems are custom designed to match the requirements of large scale STREAMLINE CD columns These systems are based on the same design concept as the modular STREAMLINE systems used in process development and small scale production which facilitates scale up The main materials of construction are high alloy stainless steel polypropylene EPDM and glass Sanitary TC connections are used throughout the system including inlet outlet and column connections The hygienic design of STREAMLINE production scale systems has been verified by Sanitization studies A system was challenged with culture b
118. ewhat larger bed volume is needed for processing of a specific amount of feed In expanded bed adsorption the maximum flow velocity through the bed is limited by its effect on the degree of expansion and bed stability Nominal flow velocity with STREAMLINE media is around 300 cm h at room temperature giving a degree of expansion of around 3 times with normal aqueous based buffers Flow velocities significantly higher or lower than this can negativly effect the stability of the expanded bed Application of a viscous feed to the column further increases the degree of expansion If viscosity is significant a flow velocity of 300 cm h could be enough to cause the bed to expand up to the position of the adaptor forcing the beads up against the adaptor net Hence it will be difficult to inrease speed in an expanded bed system by increasing flow velocity However sample application time can be reduced by over sizing the column i e using a wider column with the same sedimented bed height This gives a higher volumetric flow rate at preserved flow velocity through the bed When processing time is not considered an important issue optimization could be focused on reducing the scale of work i e utilizing the available binding capacity of the adsorbent to its maximum The adsorption in an expanded bed is a process which is controlled by the residence time of the target protein in the column The residence time is the bed height divided by the flow velocity
119. f 4 C The resulting whey fraction was treated with 2 mM and 4 mM Zn This was applied to a STREAMLINE 50 column 50 mm i d without removing the precipitated material The column contained 300 ml of STREAMLINE DEAE adsorbent which corresponds to a sedimented bed height of 15 cm The flow velocity during expansion equilibration adsorption and wash was 300 cm h The buffer used during expansion equilibration and wash was 25 mM Tris HCl pH 7 2 Desorption was performed with downward flow in sedimented mode using a three step elution procedure The following buffers were used in sequence 1 125 mM NaCl in 25 mM Tris HCl pH 7 2 2 250 mM NaCl in 25 mM Tris HCl pH 7 2 3 500 mM NaCl in 25 mM Tris HCl pH 7 2 After elution the bed was regenerated with 2 M NaCl and subjected to a cleaning in place CIP procedure by washing with a solution of 0 5 M NaOH and 1 M NaCl Following the Capture step on STREAMLINE DEAE the rhPC containing fraction was further purified by immunoaffinity chromatography Table 51 summarizes the complete purification process Processing whey without addition of Zn2 ions resulted in about 2 of the rhPC and 45 of the total whey proteins passing through the bed without being adsorbed Treatment with 2 mM Zn ions resulted in the elution of 27 of the rhPC and 56 of the total whey proteins in the unbound fraction The loading of whey treated with 4 mM Zn ions resulted in 51 of the rhPC and 85 of the total w
120. ffers specific for the type of chromatographic principle applied This regeneration removes the more strongly bound proteins which are not removed during the elution phase 0 Sedimented 1 Equilibration 2 Sample appl 3 Washing 4 Elution 5 Regeneration adsorbent expanded expanded expanded packed bed packed bed Fig 1 Schematic presentation of the steps of expanded bed adsorption Finally a cleaning in place procedure is applied to remove non specifically bound precipitated or denaturated substances from the bed and restore it to its original performance During this phase a moderate upward flow is used with the column adaptor positioned at approximately twice the sedimented bed height Stable fluidization Expanded bed adsorption is based on controlled stable fluidization thus combining the hydrodynamic properties of a fluidized bed with the chromatographic properties of a packed bed The fluidization allows particulate matter to pass through the bed unhindered The expanded bed principle i e the formation of stable fluidization with a minimum of back mixing channelling and turbulence in the bed allows the formation of several mass transfer units or several theoretical plates in the expanded bed mimicing the performance of a traditional packed chromatography column Results from studies of the hydrodynamic properties of expanded beds based on STREAMLINE media were reported by Johansson and
121. fined by determining the breakthrough capacity for the target protein in expanded bed mode using frontal analysis This is done at the end of the method optimization phase when the effect of the crude feed on the hydrodynamic and chromatographic performance of the expanded bed has been carefully examined and all necessary corrections have been made to ensure consistent functionality and robustness This work also includes the final decision on what will be the most suitable flow rate to apply during feed application in expanded mode The technique is the same as that applied during the method scouting phase using clarified feed in packed bed mode The process feed is continously applied to the column at the defined flow velocity until breakthrough of the target protein can be detected in the column effluent A breakthrough curve from expanded bed adsorption of a recombinant Fab fragment on STREAMLINE SP is shown in Fig 18 The breakthrough profile of the Fab fragment was determined by running an ELISA on single fractions collected during feed application and wash The concentration of the Fab fragment in single fractions C was plotted in the chromatogram in relation to the concentration of the Fab fragment in the feed applied to the column C In this specific application the main breakthrough occurred when approximately 8 litres of feed had been applied to the column When defining a suitable maximum loading capacity in subsequent production runs
122. g and elution had been defined the method was optimized on a small expanded bed of STREAMLINE DEAE using crude unclarified feed material to define optimal running conditions The column used during optimization was a STREAMLINE 50 column with an internal diameter of 50 mm The sedimented bed height was 15 cm The method was finally scaled up to pilot scale in a STREAMLINE 200 column with an internal diameter of 200 mm 86 Bed expansion equilibration feed application and wash were performed at an upward flow velocity of 300 cm h The buffer used during expansion equilibration and wash was 30 mM ammonium acetate pH 5 5 Elution was performed at 100 cm h using downward flow in sedimented bed mode The elution buffer was 30 mM ammonium acetate containing 250 mM NaCl pH 5 5 Cleaning in place was performed after each purification cycle using upward flow with the adaptor positioned at twice the sedimented bed height The cleaning protocol was 0 5 M NaOH containing 1 M NaCl at a low flow velocity giving a contact time of at least 4 hours 3 sedimented bed volumes of distilled water at 100 cm h 3 sedimented bed volumes of 30 isopropanol at 100 cm h 3 sedimented bed volumes of 25 acetic acid at 100 cm h and finally adsorption buffer until the pH and conductivity of the outlet stream were the same as the buffer Figure 30 and Table 23 summarize the experiments The yield of annexin V was approximately 95 as determined by an anticoagulant acti
123. g hydraulic liquid via pump B into the hydraulic chamber Open the flow path from pump B to the hydraulic inlet by switching the injection valve to position 1 G5 Flow Direction Valve V7 G3 eer G4 wh T ANG G10 Go Mixer G1 G2 Injection Valve V1 ee SIL Pump Pump AA aoe Gon vv S a B RUY Sample Pump 5 W3 Boe E E all Poa RE G16 y cio QD F A1 A2 BI B2 aoe aa ee Uh Ea y U G17 g Fraction Collector A2 B1 B2 Buffer Valve V6 Sample Valve V5 G7 auie Valve V4 Gia ae nag Ate aA Column Valve V3 A11 A12 A13 A14 A15 A16 A17 A18 51 2 53 54 55 56 S7 S8 Fe F3 F4 F5 F6 F7 F8 Fig 22 Schematic representation of KTAexplorer system reconfigured for expanded bed adsorption chromatography using a STREAMLINE 25 column Small scale operation with BioPilot System The STREAMLINE 25 column can also be connected to BioPilot System for completely automated control of expanded bed adsorption chromatography at small scale Re configure BioPilot System as described below See Fig 23 for the complete system configuration 51 1 Disconnect Superloop from Valve 1 position 2 It is not required 2 Connect tubing between Valve 1 position 2 and the column hydraulic inlet 3 Disconnect the tubing from Valve 1 position 1 and connect it to a SRTC 3 connector 4 Connect tubing between the SRTC 3 connector and Valve 1 position 1 5 6 Connect tubing between outlet W1 and the
124. g where they cause severe fouling during microfiltration Hybridoma cells are generally considered to be particularly sensitive to shear forces resulting from vigorous agitation or sparging In contrast CHO cells have relatively high resistance to shear rates and a good tolerance to changes in osmotic pressure The use of expanded bed adsorption reduces the amount of cell lysis that occurs as compared with traditional centrifugation and cross flow filtration unit operations since the cells are maintained in a freely flowing low shear environment during the entire capture step Nevertheless it is important to actively prevent cell lysis during processing for instance by avoiding exposure to osmotic pressure shocks during dilution of the feed stock and by minimizing the sample application time Non secreted products sometimes accumulate intracellularly as inclusion bodies which are precipitated protein aggregates that result from over expression of heterologous genes Inclusion bodies are generally insoluble and recovery of the biologically active protein requires denaturation by exposure to high concentration of chaotropic salts such as guanidine hydrochloride or dissociants such as urea The subsequent renaturation by dilution provides very large feedstock volumes Expanded bed adsorption can be advantageous since precipitation of misfolded variants increases with time which usually causes problems for traditional packed bed chromatography Ev
125. h Column Application area STREAMLINE 25 Optimization work at laboratory scale STREAMLINE 50 Optimization and verification work at pilot scale STREAMLINE 200 Verification work at pilot scale and small scale production STREAMLINE CD Custom designed columns for full scale production Column design The unique design of the liquid distributor at the base of STREAMLINE columns ensures the formation of stable expanded beds The distributor has been optimized for each column size to provide stable fluidization with STREAMLINE adsorbents at flow velocities between 200 and 500 cm h using water based buffers as mobile phase STREAMLINE columns are also equipped with a movable adaptor operated by a hydraulic drive This allows the height of the expanded bed to be altered during the different stages of an expanded bed adsorption cycle The adaptor is lowered by pumping liquid into the hydraulic compartment above the adaptor plate It is raised by pumping liquid upwards through the column while allowing the hydraulic liquid to flush out of the hydraulic compartment The adaptor is kept at its upper position for bed expansion and feed application and is lowered to the surface of the sedimented bed for desorption in packed bed mode It can also be lowered to the surface of the expanded bed for the wash cycle after feed application This speeds up the wash cycle and considerably reduces the consumption of wash buffer Large scale STREAMLINE CD colum
126. h at room temperature giving a degree of expansion of around 3 fold with normal aqueous based buffers Extend equilibration time to one hour or more Increase sedimented bed height Nominal sedimented bed height is around 15 cm Minimum recommended sedimented bed height is 10 cm 133 Problem Cause Remedy High back pressure Fuzzy bed surface Channelling in the lower part of the expanded bed 134 Clogging of the bottom and or adaptor distribution system Folding of the bottom and or adaptor net Clogging in valves connectors tubing etc Presence of adsorbent fines in the expanded bed Unsufficient equilibration following buffer exchange Trapped air in the bottom distribution system Clogging of the bottom distribution system Disassemble the column and clean the distributor plates and nets see column User Manual Replace the net s Remove and clean the respective parts Remove fines by elutriation see column User Manual Extend equilibration time after buffer exchange Try to remove the air by pumping buffer at high flow velocity e g 300 500 cm h through the column using downward flow If the above does not help remove the adsorbent from the column Pump distilled water into the column through the bottom distri bution system and remove any trapped air using suction from above the adaptor net see page 53 Disassemble the column and clean
127. have a negative effect on productivity by decreasing the yield of active product due to cell lysis releasing proteases and or glycosidases The height of the expanded bed also influences processing time In principle a wide short bed gives shorter processing time compared to a narrow tall bed This is because processing a certain volume of liquid through a certain volume of adsorbent using a specified flow velocity is faster if the bed is wide and short since the processing flow velocity corresponds to a higher volumetric flow rate through the system When applying adsorption chromatography in preparative mode the general guideline is therefore to keep the bed height as short as possible to reduce process time 42 Optimizing throughput Throughput i e the amount of feed that can be applied per volume of adsorbent and time unit is a function of capacity and speed of the purification process As in any type of chromatography optimization of one of these parameters can only be realized at the expense of the other The characteristics of the feed and the anticipated final scale of operation form the basis for the balance between capacity and speed in any particular application High speed may be required to reduce sample application time particularly if cell lysis occurs releasing destructive nucleic acids proteases glycosidases etc In practical terms it may be important to apply high volumetric flow rates even if this means that a som
128. he bed collapsed to close to the sedimented bed height These results seems to agree with results reported by Chang and Chase 34 using STREAMLINE DEAE in a STREAMLINE 50 column for purification of glucose 6 phosphate dehydrogenase from unclarified yeast cell homogenates They concluded that a biomass dry weight of 7 could be readily processed with no sign of bed instability if the flow rate was decreased to prevent excessive expansion of the bed 30 When high biomass content and high viscosity cause frequent build up of adsorbent against the adaptor net the flow rate should be reduced or the viscosity of the feed stock decreased to reduce bed expansion during feed application Reduction of viscosity can easily be accomplished by diluting the feed stock with buffer or water When the target protein is accumulated intracellularly the viscosity may be reduced by further homogenization of the feed stock After a few runs through a high pressure homogenizer the viscosity is usually in the order of 5 mPa s Nucleic acids High viscosity can also be related to a high content of nucleic acids in the feed stock Treatment of the feed stock with a nuclease e g Benzonase can give the desired decrease of viscosity 28 In an intracellular system reduction of viscosity by nucleic acid degradation using a nuclease is more efficient if the nuclease is added to the cell suspension prior to rather than after he homogenization This treatment e
129. he ligand and the STREAMLINE matrix The oriented coupling provides high binding capacities for IgG due to the enhanced interaction between protein A and the F region of the antibody 73 The recombinant protein A is produced in E coli and purified by a multi step chromatographic procedure before being coupled to the base matrix 59 The purification of protein A does not involve the use of coupled IgG or any other protein The purified recombinant protein A is tested according to established specifications before being released for the manufacture of STREAMLINE rProtein A More information about STREAMLINE rProtein A including instructions for use is available in Data File STREAMLINE rProtein A Code No 18 1115 67 and in the Instructions included in the pack Table 16 summarizes the characteristics of STREAMLINE rProtein A Table 16 Characteristics of STREAMLINE rProtein A Ligand Recombinant protein A E coli Ligand density 6 mg protein A ml gel Particle size 80 165 um Approx mean particle density 1 3 g ml Degree of expansion H HO at 300 cm h 2 3 OH stability long term 3 10 short term 2_11 Binding capacity Total 50 mg human IgG ml gel Dynamic 20 mg human IgG ml gel Storage 20 ethanol Further information is available in Data File STREAMLINE rProtein A Code No 18 1115 67 1 Long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its sub
130. heir operation in detail System configurations This section describes how to set up a system for expanded bed adsorption It covers system configurations and selecting components for both manual and automatic modes The instructions for start up and system operation refer to a manual or semi automated system as outlined in figures 20 and 21 Manual systems A STREAMLINE column is easily set up for manual operation It requires two pumps manual valves and UV conductivity and pH monitors One of the pumps controls the adaptor movement by pumping hydraulic liquid into the hydraulic chamber of the STREAMLINE column The other pumps liquid through the column The most suitable type of pump for expanded bed adsorption is a peristaltic pump The advantage of peristaltic pumping is that the crude feed does not contaminate the pump making stripping and cleaning unnecessary The pumping action is also reasonably gentle which minimizes cell lysis when pumping whole cell culture broth The limited tolerance to back pressure during peristaltic pumping is not a problem since expanded bed adsorption systems generate only low pressure One double channel valve is needed to reverse flow through the column A number of single channel valves are needed to select buffer or feed at the inlet side collect product at the outlet side by pass the column and control adaptor movement by the hydraulic pump A pressure monitor can be installed before the column
131. hey proteins passing through the expanded bed unadsorbed The 250 mM and 500 mM NaCl step eluates were pooled for subsequent immunoaffinity chromatography The purification factors of the NaCl step eluates ranged from less than 1 to 8 29 of the original whey protein and 84 of the rhPC was contained in the pooled eluates from untreated whey loading The 2 mM Zn2 treated whey loading gave an eluate pool of 23 of the original total whey protein and 66 of the rhPC The 4 mM Zn treated whey loading gave an eluate pool of 18 of the original total whey protein and 41 of the rhPC The rhPC yield from the immunoaffinity step was 90 to 94 for salt eluate pools and about 86 to 89 for unadsorbed effluent from the STREAMLINE DEAE column All immunoaffinity products were greater than about 95 pure as judged by SDS PAGE A purification factor of about 200 was achieved by combination of STREAMLINE DEAE expanded bed adsorption and immunoaffinity chromatography 129 Table 51 Summary of results from the purification of rhPC from milk of transgenic pigs No Zinc 2 mM Zinc 4 mM Zinc Step Total Yield Purifi Total Yield Purifi Total Yield Purifi protein cation protein cation protein cation factor factor factor Feed 100 100 1 100 100 1 100 100 1 Flow through 44 9 2 0 05 56 2 26 8 0 5 85 51 1 0 6 Elution 1 14 7 4 5 0 3 14 0 1 5 0 1 6 5 0 5 0 1 Elution 2 10 7 83 1 7 8 73 50 6 7 0 7 19 2 7 Elution 3 18 1 1 3 0 1 16 0 14 5
132. hould have a high osmolality to prevent cell lysis due to increased osmotic pressure The osmolality of a cell culture is approximately 300 mOsm kg An example of a suitable diluent is a solution of 200 mM D glucose in water giving an osmolality of approximately 200 mOsm kg Fermentor Loading on expanded bed Elution Buffer tank Fig 15 Schematic diagram of process flow during adsorption with on line dilution of the culture broth 33 Wash In any type of adsorption chromatography the washing stage removes non bound and weakly bound soluble contaminants from the chromatographic bed In expanded bed adsorption washing also removes remaining particulate material from the bed Since expanded bed adsorption combines clarification concentration and initial purification the particulate removal efficiency is a critical functional parameter for the optimal utilization of the technique Washing is performed by pumping starting buffer through the expanded bed with upward flow until the UV signal from the column effluent returns to close to the base line This requires approximately 5 20 sedimented bed volumes of buffer which will also ensure an almost complete removal of particulate material from the column The required wash volume depends on the type of feed material used Feed material based on secretion systems e g hybridoma cell cultures requires smaller wash volumes for complete particulate removal The flow rate at s
133. hrough determined in a STREAMLINE 25 column at a flow velocity of 400 cm h for STREAMLINE SP Q XL and 300 cm h for STREAMLINE SP DEAE using a 2 mg ml solution of protein in 50 mM Tris HCl pH 7 5 BSA and ovalbumin 50 mM Glycine pH 9 0 lysozyme and 50 mM sodium acetate pH 5 0 hIgG Sedimented bed height was 15 cm Work by Pharmacia Biotech More information about STREAMLINE SP XL and STREAMLINE Q XL including instructions for their use is available in Data File STREAMLINE SP XL STREAMLINE Q XL Code No 18 1123 81 Table 13 summarizes their characteristics Table 13 Characteristics of STREAMLINE SP XL and STREAMLINE Q XL adsorbents Product STREAMLINE SP XL STREAMLINE Q XL Type of ion exchanger strong cation strong anion Total ionic capacity mmol ml gel 0 18 0 24 0 23 0 33 Particle size range um 100 300 100 300 Approx mean particle size um 200 200 Approx mean particle density g ml 1 2 1 2 Degree of expansion H HO at 300 cm h 2 3 2 3 pH stability long term 4 13 2 12 short term 3 14 2 14 Recommended working flow velocity cm h 300 500 300 500 Binding capacity mg ml gel lysozyme MW 14 500 gt 140 n d BSA MW 67 000 n d gt 110 Storage 0 2 M sodium acetate in 20 ethanol 20 ethanol Further information is available in Data File STREAMLINE SP XL STREAMLINE Q XL Code No 18 1123 81 n d not determined 1 Long term refers to the pH interval where the gel is stable over a long p
134. ht G Binieda A Udell M A scalable method for the purification of recombinant human protein C from the milk of transgenic swine Adv Bioproc Eng 1994 501 507 Drohan W N Wilkins T D Latimer E Zhou D Velander W Lee T K Lubon H Estimating plate heights in stacked membrane chromatography by flow reversal J Chromatogr A 702 1995 69 80 Roper D K Lightfoot E N Review of liquid mixing in packed bed biological reactors Biotechnol Progr 4 1988 134 148 Swaine D E Daugulis A J Analysis of boundary conditions in the axial dispersion model by application of numerical laplace inversion Chem Eng Sci 46 1991 2567 2571 Seidel Morgenstern A Chemical Reaction Engineering Wiley amp Sons New York 1977 Levenspiel O APPENDIX AXIAL MIXING IN LIQUID FLUIDIZED BEDS The main difference between adsorption in packed beds and in fluidized beds is the mobility of the adsorbent particles within the fluidized bed Therefore the traditional limiting factors of protein adsorption to porous matrices have to be extended by mixing in the solid phase which arises from particle movement during fluidization These limiting factors are equilibrium kinetics of the protein ligand interaction mixing in the liquid phase fluid side transport and particle side transport Up to now no data are available on particle mixing in beds of fluidized adsorbents However it can be suspected that the particle movement
135. ial such as phospholipids polysaccharides cell debris or whole cells Nucleic acids which carry negative charges can bind to anion exchangers and block capacity but they may also form complexes with the target protein in cation exchange applications where the target protein is positively charged This may cause loss in product yield since neutral protein nucleic acid complexes show weak binding to the medium and are eluted in the flow through fraction This type of interaction between nucleic acid and contaminating proteins in the feed material also affects selectivity during adsorption The net effect of these interactions will depend on which adsorbent is used the working pH and the profile of contaminating proteins in the feed material Aggregation of biomass Cells and cell debris from different hosts often tend to aggregate at low pH If this effect is severe it blocks the column inlet distribution system It is therefore important to test at an early stage that the conditions selected during the method scouting phase are compatible with the unclarified feed stock When problems occur they are usually associated with the low pH used during cation exchange chromatography Problems can thus be circumvented by applying anion exchange chromatography instead Occasionally debris may aggregate inside the expanded bed during feed application eventually blocking the column adaptor net To prevent this type of problem the adaptor net can be repl
136. ieces zero dead leg valve a design which reduces the system dead volume and eliminates stagnant areas where microbial growth could occur The system is supplied with four inlets for sample buffers and CIP solution and one extra outlet to collect product The connections in the system including inlets outlets and column connections are all sanitary 25 mm o d clamp connections f ap m ee se CE Li 7 E Manual STREAMLINE system Pumps one to pump liquid through the column and one for the hydraulic drive monitors UV pH or conductivity and tubings are not included and must be ordered separately Extra tubing is needed to connect tanks to the system inlet and outlet valves to connect the column inlet outlet and hydraulic inlet to connect monitors between the flow reversal valve and the system outlet valve to connect the pumps inlet and outlet and to connect the hydraulic liquid container to the system to dispose of hydraulic liquid when lifting the adaptor See Table 7 to select suitable pumps tubings monitors etc Materials of construction are polypropylene tubing manifolds valves EPDM valves gaskets air trap stainless steel ASTM 316L air trap 4 port 2 way valve PTFE 4 port 2 way valve and glass air trap 81 Pilot scale automated systems Automated STREAMLINE systems are available in two sizes suitable for method development and small scale production They hav
137. ied during expansion equilibration prior to feed application and during the wash in expanded mode after feed application The buffer used during expansion equilibration and wash in expanded mode was 50 mM acetate buffer pH 3 5 containing 250 mM NaCl The degeree of expansion before feed application was typically 2 3 2 4 times the height of the sedimented bed After the initial wash out of residual cells and particulates in expanded mode the bed was sedimented and washed with upward flow in two separate steps In the first step wash 1 the bed was washed with purified water to facilitate buffer exchange In the second step wash 2 the bed was washed with 50 mM phosphate buffer pH 6 containing 50 mM NaCl to selectively remove bound contaminants prior to product elution Elution was performed at a flow velocity of 200 cm h using upward flow in sedimented bed mode Fig 37 shows a typical chromatogram from a run on the STREAMLINE 50 column The chromatograms from the phase II production runs looked identical A280 2 1 8 1 6 1 4 12 i 0 8 0 6 0 4 0 2 0 50 100 150 200 250 300 350 400 Time min Feed application Cell Removal Wash 1 Wash 2 Elution Fig 37 Chromatogram from a typical process run on the STREAMLINE 50 column Reproduced with permission from ref 75a 112 After each purification cycle the column was subjected to cleaning in place using a solution of 0 5 M NaOH and 1 0 M NaCl The cleaning solution was
138. igher dry weights the bed expanded to the top of the column and caused a build up of adsorbent beads against the adaptor net At dry weights of 7 and above it was not possible to reduce viscosity to below 10 mPa s by multiple passages through the homogenizer but it was still possible to use dry weights of 7 8 if the flow direction was reversed periodically to prevent build up against the adaptor net No evidence of decreased bed stability e g channelling in the expanded bed could be detected at dry weights up to 8 Dry weights higher than 8 resulted in channelling in the expanded bed and poor recovery of the target protein At a dry weight of 9 2 the expanded bed collapsed to close to the sedimented bed height due to heavy channelling Barnfield Frej et al also studied the effect of viscosity while keeping the biomass dry weight constant at approximately 3 4 The viscosity was varied by passing the feed stock three times through the homogenizer at pressures ranging from 300 to 950 bar Trouble free expansion was achieved at viscosities up to 10 mPa s At viscosities above 10 mPa s the bed expanded to the top of the column requiring periodic reversal of flow direction to prevent build up against the adaptor net It was possible to use viscosities up to 50 mPas without evidence of channelling in the bed Feed stocks with viscosities above 50 mPa s gave rise to channelling and poor recovery of the target protein At viscosities of 500 mPa s t
139. initially pumped through the sedimented bed for 30 minutes using downward flow The flow velocity was 100 150 cm h Then the flow direction was reversed for 30 minutes with the adaptor in its uppermost position The bed was then left in contact with the cleaning solution for 1 hour method development with the STREAMLINE 50 column or overnight phase II production with the STREAMLINE 200 column No effect on the dynamic binding capacity of the STREAMLINE SP could be detected for adsorbent that had undergone 12 process and CIP cycles Table 42 summarizes the results from a typical process run on the STREAMLINE 50 column using a feed stock with a biomass dry weight of 30 g L The product was recovered with a yield of 84 and a 6 fold concentration The majority of the protein contaminants were separated from the product as determined by RP HPLC Potentially detrimental proteases were also selectively removed from the product stream This was verified by comparing mass spectra of the feed stock wash 2 and eluate incubated for 1 week at 25 C with controls stored at 70 C The mass spectra showed high intrinsic stability for the product in the eluate while proteolytic degradation of the product in the feed stock and wash 2 was clearly evident Table 42 Typical results from a process run on the STREAMLINE 50 column Volumes Total product Yield ml mg Fermentation 6500 Adsorbent 410 Feed stock 18500 3300 100 Flow through 2450
140. ioindfr eu pharmacia com Germany T 0761 490 30 F 0761 4903 405 E mail serve bioindde eu pharmacia com Great Britain T 01727 814000 F 01727 814 001 E mail serve bioindgb eu pharmacia com Italy T 02 273 221 F 02 273 022 12 E mail serve bioindit eu pharmacia com Austria Netherlands T 0165 580 400 F 0165 580 401 E mail serve bioindni eu pharmacia com Norway T 63892310 F 63 8923 15 E mail serve bioindno eu pharmacia com Portugal T 01 424 9200 F 01 424 9299 Spain T 93 589 07 01 F 93 589 34 74 E mail serve bioindes eu pharmacia com Sweden T 08 623 85 00 F 08 623 00 69 E mail serve bioindse eu pharmacia com Switzerland T 01 802 8150 F 01 802 8151 E mail serve bioindch eu pharmacia com Regional office Pharmacia Biotech Export Vienna T 43 1 982 3826 F 43 1 985 8327 E mail rfeike phexport vie co at CIS amp NIS T international 7 503 956 1137 Moscow domestic 7 095 956 1137 F international 7 503 232 0250 domestic 7 095 232 0250 Czech Republic T 02 205 11 392 F 02 205 11 392 Hungary T 01 1747 584 F 01 1757 819 Israel T 972 3 535 15 05 ext 131 Gamidor Ltd F 972 3 534 65 73 Poland T 022 651 63 33 F 022 651 75 57 Ukraine T 044 543 19 71 F 044 418 10 76 Regional office Greece Africa and Middle East T 30 1 960 0687 F 30 1 960 0693 E mail eliask eexi gr Athens internet http www biotech pharmacia se 159 Asia Pacific Main office Hong Kong T
141. ity 30 cm h contact time 4 hours distilled water room temp flow velocity 100 cm h 3 sedimented bed volumes distilled water 85 95 C flow velocity 100 cm h 10 sedimented bed volumes 25 acetic acid 20 ethanol flow velocity 100 cm h 1 sedimented bed volume equilibration buffer flow velocity 100 cm h 5 10 sedimented bed volumes CIP procedure 3 1 w v DARACLEAN 8471 flow velocity 30 cm h contact time 4 hours equilibration buffer flow velocity 100 cm h 5 10 sedimented bed volumes Procedures 1 and 2 also provide a good sanitization effect Washing with 0 5 1 0 M NaOH for a contact time of 30 60 minutes is an effective disinfectant treatment for vegetative bacteria yeast and moulds 47 STREAMLINE SP and STREAMLINE DEAE can be sterilized by autoclaving the adsorbent at 121 C for 30 minutes Storage We recommend storing STREAMLINE DEAE and STREAMLINE SP in 20 ethanol During long term storage of STREAMLINE SP in unbuffered ethanol a gradual acidification of the storage solution may occur due to the acidic nature of the ligand We therefore recommend adding sodium acetate up to a concentration of 0 2 M As an alternative storage solution for both STREAMLINE SP and STREAMLINE DEAE we recommend 10 mM NaOH This is comparable to 20 ethanol from a bacteriostatic point of view 1 DARACLEAN Grace Dearborn Ltd is a commercially available cleaning agent containing caustic soda alkaline salts
142. ked bed adsorption was insufficient to completely clarify the feed stock Table 39 Variation in yield of ADH as a function of load volume and number of CIP cycles One CIP cycle was performed after each adsorption cycle Cumulative volume loaded ADH Yield ADH Yield bed volumes Expanded Bed Packed Bed 1 95 85 3 114 71 5 105 58 107 Purification of recombinant Aprotinin Variants from H polymorpha fermentation broth by expanded bed cation exchange adsorption Expanded bed adsorption on STREAMLINE SP was used in the capture step during purification of two recombinant DesPro 2 aprotinin variants from Hansenula polymorpha fermentation broth 56 Aprotinin is a bovine pancreatic trypsin inhibitor with a molecular weight of 6 5 kDa and an isoelectric point of 10 5 Aprotinin inhibits a range of proteases and has excellent potential as a therapeutic and diagnostic compound DNA sequences coding for the two aprotinin variants were expressed in the methylotropic yeast Hansenula polymorpha The coding sequences were fused to the KEX2 recognition site of the S cerevisiae derived Mfal preproleader sequence which causes secretion of the recombinant aprotinin variants into the culture broth The culture broth was prepared for expanded bed adsorption by dilution 1 1 with deionized water to a conductivity of 25 mS cm and by adjustment of pH to 3 5 The diluted culture broth had a biomass content of 5 dry weight A total
143. lar caused by air under the and liquid channels e g movements of the distributor plate or caused by the column particles no back mixing pulsation from the pump not being level or mixing of the particles Fig 8 Visual patterns of movement of adsorbent beads in an expanded bed 13 Visual inspection alone however does not give the complete picture of the flow distribution across the entire column cross section Bed stability should be evaluated by more accurate techniques such as measuring the degree of expansion and number of theoretical plates before each run Measuring the degree of expansion Measuring the degree of expansion is a quick and useful measure of bed stability although less accurate than determining the number of theoretical plates The degree of expansion is determined from the ratio of expanded bed height to sedimented bed height H HO as defined in Fig 9 If the degree of expansion differs from the expected value it may indicate an unstable bed Absolute values for the degree of expansion can only be compared if the buffer system liquid density and viscosity and temperature are constant between runs A significant decrease in the degree of expansion may indicate poor stability or channelling due to trapped air under the distributor plate infection or fouling of the adsorbent the column not being in a vertical position or a blocked distributor plate t t m t t Sedimented bed height Expa
144. le up to final production is performed in STREAMLINE CD custom designed columns These columns are designed with a distribution system that ensures the same distribution of flow and the same stability of the expanded bed as the laboratory scale and pilot scale columns used during method optimization Consistent hydrodynamic and chromatographic performance has been verified in columns with inner diameters up to 1200 mm providing sedimented bed volumes of more than 150 litres at a sedimented bed height of 15 cm Processing data from such a verification study 39 are shown in Table 3 The feed material used in this study was based on BSA spiked into a suspension of baker s yeast The concentration of yeast in the feed was 4 6 dry weight Expansion equilibration sample application and wash were performed at a flow velocity of 300 cm h using 20 mM Tris pH 7 5 Elution was performed by a single step procedure using a solution of 1 0 M NaCl in equilibration buffer Elution was performed in packed bed mode using downward flow at a flow velocity of 100 cm h The adsorbent used was STREAMLINE DEAE and the amount of adsorbent used at each scale corresponded to a sedimented bed height of 15 cm The method 27 development work was performed at laboratory scale on a STREAMLINE 25 column 25 mm i d The established process was then verified in a pilot scale set up using a STREAMLINE 200 column 200 mm i d before scaling up to production on a STRE
145. ler elution volume The optimal flow velocity for eluting proteins from STREAMLINE adsorbents is in the range of 50 150 cm h considering the time for elution and volume of the collected product pool If large aggregates are formed in the bed during application of the feed stock it can be difficult to remove them all during the wash stage If such aggregates are still present in the bed at start of elution it might be necessary to elute the column in expanded mode to avoid excessive back pressure in the column The aggregates subsequently have to be removed from the bed during the cleaning in place stage The concentration effect will also be substantial when eluting in expanded bed mode which is demonstrated in Table 5 showing data from elution of lysozyme from STREAMLINE SP 44 Table 5 The effect of elution mode on the volume of eluted fraction of lysozyme Lysozyme was loaded on STREAMLINE SP at 20 of the adsorbents total capacity and eluted with a step elution procedure using 1 M NaCl in binding buffer Elution flow rate was 100 cm h Elution mode Eluted volume Ratio eluted volume ml sedimented bed volume Sedimented downward flow 308 0 99 Sedimented upward flow 350 1 12 Expanded upward flow 426 1 36 Elution in sedimented mode with downward flow gave the smallest elution volume Elution in expanded mode increased the elution volume by approximately 40 The symmetry of the eluted peaks was virtually identical independ
146. liquid fluidised bed to decide whether the bed will provide efficient adsorption from a dispersion point of view or whether bed stability has to be improved prior to the adsorption process As a general rule of thumb Bo gt 40 or N gt 20 may be regarded as sufficient to ensure that the adsorption process is not limited by liquid mixing 157 Trademarks The following designations are trademarks owned by Pharmacia Biotech AB STREAMLINE EXTREME LOAD FineLINE Sephadex Sepharose Mono Q SOURCE GradiFrac AKTA BioPilot BioProcess UNICORN 158 Order from Head Office Pharmacia Biotech AB Bj rkgatan 30 S 751 82 Uppsala Sweden Tel 46 0 18 16 50 00 Fax 46 0 18 16 64 05 North America USA T 1 800 526 3593 between 8 30 am and 8 00 pm EST F 1 800 FAX 3593 Canada T 1 800 463 5800 between 8 30 am and 6 00 pm EST F 1 800 567 1008 Central amp South America Brazil T 55 11 872 6833 F 55 11 873 0464 E mail pharmaciabrasil originet com br Europe Main European office Freiburg Germany T 49 0 761 45190 F 49 0 761 4903 159 T 01 68 66 250 F 01 68 79 03 E mail serve bioindat eu pharmacia com Belgium T 32721469 F 3272 16 37 E mail serve bioindni eu pharmacia com Denmark T 4814 1000 F 4814 1006 E mail serve bioinddk eu pharmacia com Finland T 3589 8520 7400 F 358 9 8531 933 E mail serve bioindfi eu pharmacia com France T 01 69 35 67 00 F 0169 41 96 77 E mail serve b
147. lizes negative charges on nucleic acids present in the feed This prevents the nucleic acids from binding to the cationic ligands of the adsorbent which will otherwise reduce binding capacity for the target protein Method scouting was performed in a packed bed of STREAMLINE Q XL using clarified feed material Optimal conditions for binding wash and elution were defined The breakthrough capacity as determined in the packed bed experiments was found to be approximately 15 mg recombinant protein A per ml adsorbent Method optimization in expanded mode was performed on a STREAMLINE 25 column 25 mm i d The column contained 75 ml of STREAMLINE Q XL which corresponds to a sedimented bed height of 15 cm The method was finally scaled up to pilot scale in a STREAMLINE 200 column 200 mm i d Bed expansion equilibration feed application and wash were performed at an upward linear flow velocity of 400 cm h The buffer used during expansion equilibration and wash was 10 mM Tris HCl pH 7 4 containing 10 mM MgCl Elution was performed at 100 cm h using downward flow in sedimented mode The elution buffer was 10 mM Tris HCl pH 7 4 containing 1 M NaCl After elution the bed was subjected to cleaning in place by washing with a solution of 0 5 M NaOH and 1 M NaCl The cleaning solution was applied with upward flow with the adaptor positioned at twice the sedimented bed height Figure 34 shows a chromatogram from a laboratory scale run in a S
148. lk of transgenic swine 77 It has often been necessary to apply affinity chromatography to separate desired protein species from inactive recombinant protein sub populations Addition of PEG the use of filtration or centrifugation to remove cells and protein precipitates and subsequent immunoaffinity chromatography all add significantly to the costs of producing therapeutic proteins from transgenic milk Downstream processing of rhPC from milk of transgenic pigs was simplified by an initial selective precipitation of a and b caseins with low concentrations of Zn ions The whey containing precipitated proteins was applied directly to an expanded bed of STREAMLINE DEAE The precipitated proteins passed through unhindered and eluted in the flow through fraction while the rhPC was adsorbed onto the expanded bed The addition of Zn ions also increased the selectivity of the adsorption process due to reaction of Zn2 ions with immature inactive populations of rhPC This specific interaction causes conformational changes within these populations which allow them to pass through the bed unadsorbed while the active population of rhPC binds to the adsorbent 128 Immediately after collecting the milk from transgenic lactating sows it was diluted with 200 mM EDTA pH 7 0 in a 1 1 ratio This solubilizes the caseins which exist as micelles The milk EDTA mixture was defatted by centrifugation at 4500g for approximately 90 minutes at a temperature o
149. lt CS X on off Adaptor down lt Z Z off on Adaptor down on on Upward flow 47 Table 7 Components recommended for a manual STREAMLINE system at different scales of operation See Ordering Information for descriptions pack sizes and code numbers Component STREAMLINE 25 STREAMLINE 50 STREAMLINE 200 Valves Double channel Single channel SRV 4 SRV 3 4 way 1 4 i d PP 4 way 6 mm i d SS2 L type 1 4 i d PP1 L type 6 mm i d SS2 4 way 1 2 i d SS 4 way 10 mm i d SS2 L type 1 2 i d SS L type 10 mm i d SS2 Tubing 1 9 x 2 7 mm PTFE 1 4 i d PE 1 2 i d Pe 6 mm i d PVC2 10 mm i d PVC2 Pumps Watson Marlow Watson Marlow Watson Marlow 504 U RL 504 U RL 604 U R Rotation speed Rotation speed Rotation speed 220 rpm 220 rpm 165 rom Peristaltic tubing 3 1 6 mm i d 3 2 mm i d 9 6 mm i d Connectors 2 7 mm o d M6 1 4 i d JACO 10 4 2 1 1 2 i d JACO 10 8 6 valve connectors valve connectors Unions 25 mm o d clamp 25 mm o d clamp to 25 mm o d clamp to to M6 4 1 4 threaded 1 2 threaded 5 UV monitor Flow cell UV 1 S2 UV 1 Industrial UV 1 Industrial 6 mm i d 2 10 mm i d 2 Recorder REC 102 REC 102 REC 102 Miscellaneous Stop plug Blind flange and packing Blind flange and packing Plastic clamp 25 mm Gasket 6 mm i d Plastic clamp 25 mm Gasket 6 mm i d Pla
150. lt or acid precipitation This is followed by dialyzation and lyophilization steps and finally at least two chromatographic steps These manipulations are time consuming and can result in significant inactivation of the enzyme Therefore direct capture of lysozyme from skimmed milk by expanded bed adsorption was evaluated as an alternative process Equine milk was defatted at 4 C by centrifugation at 7500 g for 10 minutes About two volumes of 60 mM Tris HCl buffer were added to 1 6 litres of the milk to give a final Tris HCl concentration of 20 mM The pH was adjusted to 8 0 with HCl The defatted and diluted milk was applied to a STREAMLINE 50 column 50 mm i d containing 300 ml STREAMLINE SP corresponding to a sedimented bed height of 15 cm The flow velocity during expansion equilibration adsorption and wash was 300 cm h The buffer used during expansion equilibration and wash was 126 20 mM Tris HCl 0 02 NaN3 pH 8 0 Desorption was performed with downward flow in sedimented mode using 20 mM Tris HCl 0 02 NaN3 pH 8 0 containing 1 M NaCl Figure 41 shows a chromatogram from the purification of skimmed milk on STREAMLINE SP in a STREAMLINE 50 column The chromatogram showed two fractions the flow through fraction and the fraction eluted by the desorption buffer The flowthrough fraction was slightly translucent probably as a result of light scattering caused by casein micelles Isoelectric focusing of samples from this fr
151. lumn and freeze dried Table 40 summarizes the results of the complete purification procedure 108 Table 40 Summary of results from purification of aprotinin Purification step Aprotinin concentration Purification Yield mg litre factor Crude starting material 202 1 100 STREAMLINE SP 1412 3 0 76 RP 18 HPLC 719 5 45 36 Sephadex G 25 543 5 5 35 The purification step on the STREAMLINE SP adsorbent resulted in a 7 fold concentration and a 3 8 fold purification of aprotinin at a yield of 76 Efficient removal of particulate material during the expanded bed adsorption step allowed direct application of the eluted material onto the RP HPLC column without pre treatment The separation of incorrectly processed aprotinin was accomplished in the RP HPLC step HPLC SDS PAGE and N terminal sequencing confirmed the fidelity and homogeneity of the isolated aprotinin Process for purification of recombinant human serum albumin from P pastoris fermentation broth by expanded bed cation exchange adsorption Expanded bed adsorption on STREAMLINE SP is used in the large scale production of recombinant human serum albumin rHSA 41 In the process rHSA is expressed in the yeast Pichia pastoris and secreted into the culture medium At the end of fermentation the culture medium including host cells is heat treated to inactivate proteases originating from the host The heat treatment is followed by a two fold dilution with distilled wa
152. m 58 1 Switch valve V1 to upward flow Switch valve V3 to open the flow path from the hydraulic chamber to waste Set valve V2 to the closed position to block the flow through the bed 2 Lift the adaptor by pumping elution buffer with pump 1 at a flow rate of 100 cm h 3 When the adaptor reaches a level equivalent to twice the sedimented bed height stop the adaptor by setting valve V2 to the waste position and valve V3 to its closed position 4 If there is any tendency for plug formation in the bed at this stage reverse flow to dissolve the plugs Switch valve V1 to downward flow and return to upward flow after a few seconds Repeat this procedure until the plugs have been broken up 5 Continue cleaning the bed by pumping cleaning in place solution with upward flow through the column according the pre defined cleaning in place protocol Maintenance Storage To store STREAMLINE adsorbent in the column pump at least 5 sedimented bed volumes of storage solution through the sedimented bed at a flow rate of 100 cm h using upward flow Specific recommendations about storage solutions for different types of STREAMLINE adsorbents are included in the instructions accompanying each medium Position the adaptor on top of the sedimented bed Remove the lid from the column and suck out any liquid remaining in the space above the adaptor Add 20 ethanol before replacing the lid Elutriation Even if STREAMLINE adsorbents show high
153. m phosphate pH 6 5 Desorption of IL 8 from the adsorbent was performed with downward flow in sedimented mode using 30 mM sodium phosphate pH 6 5 containing 0 5 M NaCl The flow velocity during desorption was 100 cm h Table 30 summarizes the adsorption step on STREAMLINE SP An 11 fold concentration of IL 8 was achieved The yield of IL 8 in the eluate as determined by FEIA was 97 and the purification factor was 4 8 Table 30 Summary of results from expanded bed adsorption of recombinant IL 8 from unclarified E coli feed stock Volume Total protein IL 8 Yield ml mg mg Fermentation 6700 Feed stock 12080 40 s b v 3745 870 100 Flow through and wash 13800 46 s b v 2990 30 3 Eluate 1075 3 6 s b v 750 840 97 1 5 b v sedimented bed volumes After each purification cycle on the STREAMLINE SP adsorbent the column was subjected to a cleaning in place procedure using upward flow with the adaptor positioned at twice the sedimented bed height The cleaning protocol consisted of 1 w v DARACLEAN 8471 at a flow velocity of 30 cm h giving a contact time of 4 hours followed by 4 sedimented bed volumes of adsorption buffer at a flow velocity of 100 cm h 1 DARACLEAN Grace Dearborn Ltd is a commercially available cleaning agent containing caustic soda alkaline salts and the non ionic detergent Triton CF 10 96 To verify the function of the adsorbent after repeated use the STREAMLINE 50 column was
154. manufacturing 115 10 000 Total Protein mg ml 1000 O NGF mg ml A Turbidity nephalose Protein mg ml 100 Turbidity nephalose 10 O 60 120 180 240 300 360 420 465 495 555 Load Wash ROO Time min Fig 39 Expanded bed adsorption of rhNGF on STREAMLINE SP in a STREAMLINE 25 column Work by Genentech Inc So San Francisco CA USA in collaboration with Pharmacia Biotech Table 43 Summary of results from the recovery of rhNGF on STREAMLINE SP ina STREAMLINE 200 column Adsorbent volume 3L Feed stock volume 400 L Cell viability 52 Packed cell volume 2 7 Degree of expansion maximum 5 4 Concentration factor 30 Purification factor 11 Yield 100 Cycle time 6 hours 116 Recovery of a recombinant monoclonal antibody from CHO cell culture broth by expanded bed cation exchange adsorption at large scale Expanded bed adsorption on STREAMLINE SP was used to capture a recombinant monoclonal antibody from crude unclarified CHO cell culture suspension at 12000 litres scale 66 The work was performed by Genentech Inc So San Francisco CA USA in collaboration with Pharmacia Biotech The recombinant monoclonal antibody was produced in CHO cells at an expression level of 500 600 mg L It had an isoelectric point of 9 1 and therefore bound efficiently to a cation exchange resin at acidic pH values Optimal binding and elution conditions were defined using clarifi
155. mechanical stability 43 elutriation may sometimes be needed to prevent the accumulation of adsorbent fines generated by repeated operation or handling of the adsorbent These fines are washed out from the column adsorbent using an elutriation procedure where the adaptor net has been replaced with an elutriation sealing This procedure is described in the Instruction Manual accompanying each STREAMLINE column Replacing the adsorbent Resuspend the adsorbent in buffer Handle the sedimented adsorbent carefully to avoid damage by shear forces Either pour out the adsorbent slurry from the column or siphon it off 59 6 Product Guide Pharmacia Biotech supplies a complete range of STREAMLINE products from method development at laboratory scale up to full scale routine production This Chapter describes the different STREAMLINE adsorbents columns and systems Basic product characteristics and technical data are included For information about pack sizes and code numbers see Ordering Information STREAMLINE adsorbents Pharmacia Biotech manufactures a range of STREAMLINE adsorbents for ion exchange and affinity expanded bed adsorption Table 9 summarizes this product range All adsorbents are available in both laboratory pack sizes and bulk quantities Table 9 STREAMLINE adsorbents available from Pharmacia Biotech STREAMLINE SP Strong cation exchanger STREAMLINE DEAE Weak anion exchanger STREAMLINE SP XL
156. mentations and by subjecting the adsorbent to different types of shear force 43 Particles made only of organic material have limited density and would need to have very large diameters for the high sedimentation velocity required Such large particle diameters result in long diffusional path lengths which cause considerable mass transfer resistance counteracting productivity STREAMLINE adsorbents are therefore based on a composite particle containing an inert core material that is denser than organic materials Such particles can be designed so that their sedimentation velocity is high also at a reasonable particle size STREAMLINE adsorbents exhibit a Gaussian like distribution of particle size and particle density which is illustrated in Fig 2 work from Pharmacia Biotech 43 Sedimented d D gel volume Fig 2 Distribution of beads of a STREAMLINE ion exchanger expanded 2 5 times with water at a flow velocity of 300 cm h in a STREAMLINE 50 column 50 mm i d Work by Pharmacia Biotech Fluidized bed with mixing Expanded bed adsorption t t Fig 3 Comparison of particle movement in a fluidized bed with extensive mixing with particle movement in an expanded bed At the stable fluidization of an expanded bed only small circular movements of the adsorbent beads can be seen This particle polydispersity is an important design factor contributing to the stability of the expanded bed The size and density gra
157. n hydraulics Position the adaptor at a height corresponding to approximately four times the height of the sedimented bed to allow for bed expansion Set valve V3 to its closed position see Fig 24 and valve V2 to its waste position Filling the system Set the column bottom valve V4 to the by pass position Start pump 1 and fill all tubing with the correct buffer by switching the appropriate valves on the inlet side Fill the sample inlet tubing with starting buffer Make sure that all air has been removed from tubings and valves Increase the flow rate in the system to force out any remaining air bubbles While pumping equilibration buffer through the system put the column in line by switching valve V4 to direct the flow to the bottom column inlet 54 System operation Expansion Equilibration 1 Check that the stand base is mounted vertically Use a spirit level to check that the column is vertical Adjust if necessary and secure the stand feet or wheels Note A vertical column is crucial for optimal results 2 Mark the height of the sedimented bed on the column tube Before the first run the bed must be expanded and sedimented in the column to be able to determine the correct sedimented bed height The sedimented bed height is used to determine the degree of expansion when expansion and equilibration is completed 3 Position the valves according to Fig 24 Start pumping equilibration buffer through the column with upwar
158. nables fewer passages through a high pressure homogenizer to reach a viscosity suitable for expanded bed adsorption The effect of nucleic acids may be particularly severe if they originate from lysis of cells in an extracellular expression system or from a pierced cytoplasmic membrane during release of product accumulated in the periplasmic compartment These two cases release larger fragments of nucleic acids than does release from an intracellular expression system by normal application of high pressure homogenization for cell disintegration The nucleic acids released from lysed cells may not have a severe effect on the viscosity of the feed but are more likely to cause problems related to aggregation clogging and fouling of the adsorbent They may cause significant clogging of the inlet liquid distribution system of the column generating increased back pressure and uneven flow distribution over the column cross section An uneven flow distribution in turn causes channelling in the expanded bed and reduced expansion during sample application If the effect is less severe it may only be seen as a moderate increase in back pressure and some slight channelling in the lower part of the expanded bed Sometimes nucleic acids that have entered the bed may cause formation of large aggregates in the expanded bed which may be difficult to remove from the bed during feed application and the subsequent wash phase Problems with clogging due to released nucl
159. nded bed flow rate 0 cm h flow rate 300 cm h Degree of expansion 5 A 2 5 at 300 cm h 0 Fig 9 Definition of the degree of expansion Number of theoretical plates The Residence Time Distribution RTD test is a tracer stimulus method that can be used to assess the degree of longitudinal axial mixing dispersion in the expanded bed by defining the number of theoretical plates A dilute acetone solution is used as a tracer input into the fluid entering the column The UV absorbance of the acetone is measured in the exit stream from the column The number of theoretical plates are calculated from the mean residence time of the tracer in the column and the variance of the tracer output signal representing the standard band broadening of a sample zone The RTD test is a simple but efficient tool for function testing 14 complete systems If used to test systems before feed application the risk of wasting valuable feed is reduced considerably The test should be performed with the buffer and flow rate that are to be used during process operation Note that when using a small tracer molecule such as acetone with a porous adsorbent such as STREAMLINE media the measurement of RTD is a function of tracer permeation in the matrix pores in addition to the actual dispersion in the liquid phase A description of the test procedure and calculations used to determine the number of theoretical plates when performing the test on a negative
160. ng upward flow with the adaptor positioned at twice the sedimented bed height The cleaning protocol consisted of 0 5 M NaOH containing 1 M NaCl at a flow velocity of 30 cm h giving a contact time of 4 hours 3 sedimented bed volumes of distilled water room temperature at 100 cm h 10 sedimented bed volumes of distilled water 85 95 C at 100 cm h 1 sedimented bed volume of 25 acetic acid 20 ethanol at 100 cm h and finally 10 sedimented bed volumes of adsorption buffer at 100 cm h To verify the function of the adsorbent after repeated use the STREAMLINE 50 column was subjected to 50 subsequent purification cycles each cycle followed by the CIP protocol described above The feed material used in this study was prepared by releasing the Fab fragment from the cells by osmotic shock by sucrose instead of high pressure homogenization An endonuclease Benzonase Merck Nycomed Pharma A S was added to the lysate to reduce viscosity The degree of expansion was determined before each cycle The number of theoretical plates of the expanded bed was determined before cycle 1 and after cycle 20 Breakthrough capacity for lysozyme was determined before cycle 1 and after cycles 30 and 50 The breakthrough capacity was determined in expanded bed mode Table 27 summarizes the results Table 27 Summary of results from a study on the re useability of STREAMLINE SP Start 20 cycles 30 cycles 50 cycles Degree of expansion H Hp 3 2 3 2 3 1 3 2
161. ns these contributions are usually not isolated in fluidized bed adsorption The result without these considerations will still be sufficient to judge whether a fluidized bed is stabilized so that it will allow efficient adsorption However it may be helpful to consider these different sources of mixing when trouble shooting cases of increased mixing within the adsorbent bed The simplest way of describing the overall mixing in a liquid fluidized bed is via the residence time distribution RTD of fluid elements RTD describes the probability distribution of a fluid element spending a certain time t in the column A wide RTD represents a situation where gross mixing of liquid elements leads to a broad range 153 of possible times t that a fluid element can spend in the column If we consider the application to a column of an infinitely narrow pulse of a suitable tracer that does not interact with the adsorbent ideal plug flow would demand that the pulse travels unchanged through the bed so that each tracer molecule has precisely the same residence time t This residence time is calculated from the ratio of bed length to flow velocity L v If one or more of the factors discussed above causes axial mixing then some elements of the pulse will be retarded and have increased t Other parts will travel faster and show reduced t Thus a variety of t is obtained which is characterised by the residence time distribution function E t
162. ns which are manufactured from stainless steel can be supplied with an adsorbent sensor The adsorbent sensor is mounted under the screen on the adaptor see Fig 29 and consists of a transmitter and a receiver The transmitter sends out an ultrasonic signal and the receiver is adjusted to detect the signal in buffer solutions and fermentation broths containing cells etc but not in solutions where adsorbent particles are present The adsorbent sensor is 1 STREAMLINE CD columns can also be delivered with a transparent column tube manufactured from cast polymethylmethacrylate PMMA 76 used to control the position of the adaptor during the run It can also be used to back flush the column if it detects build up of adsorbent particles under the adaptor net during feed stock application STREAMLINE CD columns are also supplied with an adaptor position indicator which provides continuous information about the position of the adaptor during the entire process STREAMLINE columns comply with hygienic requirements of process development and production i e from laboratory to manufacturing scales The hygienic design of small scale columns such as STREAMLINE 25 as well as large scale STREAMLINE CD columns has been verified by sanitization studies The columns were challenged with culture broths of yeast and bacteria to mimic a real expanded bed adsorption process 39 52 Following a predefined sanitization in place protocol the adsorbent the h
163. nt is an anion exchanger try a cation exchanger instead if the problems persist Prevent cell lysis by on line dilution and by increasing osmolality of the diluent see pages 33 117 119 Use fresh cultures to prevent extensive cell lysis 139 Problem Cause Remedy Turbulent flow pattern in the expanded bed Poor adsorption of target molecule low recovery or low capacity 140 Aggregation and clogging of the adsorbent due to formation of cell agglomerates and cell adsorbent aggregates in the expanded bed Decreased efficiency of the adsorption process due to channelling and turbulent flow pattern in the expanded bed Decreased efficiency of the adsorption process due to short residence time Binding capacity blocked anion exchange adsorbents by nucleic acids and other polyanionic macromolecules present in the feed stock Reduced charge on target molecule cation exchange applications due to complexing with nucleic acids and other polyanionic macromolecules in the feed stock Stir the feed stock during feed application to prevent the formation of cell agglomerates which can enter the bed and form large aggregates of cells and adsorbent beads See above Increase residence time by decreasing flow rate or by increasing sedimented bed height see page 43 Change to a cation exchange adsorbent Or add Mg ions to the feed stock to form complexes with the nega
164. nt of 1 86 x 10 6 m s t in a series of experiments on STREAMLINE SP using a flow velocity of 135 cm h and a sedimented bed height of 8 6 cm Thommes et al 64 reported results from hydrodynamic studies with a small scale column using a sedimented bed height of 6 cm They concluded that a certain minimum flow velocity is required for development of a stable fluidized bed Axial mixing was significantly lower at the highest flow velocity compared with the lowest corresponding to a decrease in Bodenstein number from 33 to 11 when the flow velocity was decreased from 375 cm h to 200 cm h in this specific experimental system The axial dispersion coefficients were in the range 6 9 x 10 m s1 Hence the same order of magnitude of axial mixing has been reported by different investigators demonstrating that stable fluidization can be achieved with STREAMLINE adsorbents Sedimented bed height and linear flow velocity are critical process parameters that may have a significant impact on the performance of an expanded bed The importance of sedimented bed height flow velocity and other critical processing parameters including the effect of physico chemical properties of the crude feed stream will be discussed in more detail in Section 3 Experimental Design and Section 4 Method Optimization Design features The adsorbent Tailoring the chromatographic characteristics of an adsorbent for use in expanded bed adsorption includes careful control of
165. nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99 and or prevent release of nucleic acids through cell lysis extracellular products by on line dilution and by increasing osmolality of the diluent see pages 33 117 119 Switch to anion exchange chromatography which allows a higher pH Remove large aggregates with an in line crude mesh filter Problem Cause Remedy Channelling in the expanded bed Turbulent flow pattern in the expanded bed Clogging of the bottom distribution system due to agglomeration of cells in the feed stock Instability caused by high biomass content and high viscosity of the feed stock Aggregation clogging and fouling of the adsorbent due to nucleic acids present in the feed stock Aggregation clogging and fouling of the adsorbent due to cell lysis extracellular products which releases nucleic acids lipids and other cell membrane components into the feed stock Stir the feed stock during feed application to prevent cell agglomeration Remove large agglomerates with an in line crude mesh filter Reduce viscosity by diluting feed stock with buffer or water or reduce viscosity by treating the feed stock with nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99 Treat the feed stock with nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99 If the adsorbe
166. ocity of 200 cm h The same flow velocity was used during application of the crude unclarified yeast homogenate to the expanded bed which resulted in a degree of expansion of around 3 2 Desorption of the enzyme from the adsorbent was performed with downward flow in sedimented mode by step elution from 0 78 M ammonium sulphate to 0 M ammonium sulphate in start buffer Fig 35 shows the expanded bed adsorption step on STREAMLINE Phenyl low sub 106 Total Protein mg ml 1 o 0 1000 2000 3000 4000 5000 Retention Volume ml Fig 35 Purification of ADH from yeast homogenate by expanded bed adsorption on STREAMLINE Phenyl low sub represents step elution from 0 78 M ammonium sulphate to 0 M ammonium sulphate Reproduced with permission from ref 68 The expanded bed adsorption on STREAMLINE Phenyl low sub was compared with packed bed adsorption using Phenyl Sepharose Fast Flow low sub packed in an XK 50 40 column 50 mm i d to a final bed height of 15 cm Table 39 presents the results from successive loadings onto both the expanded and the packed bed in terms of ADH eluted as percent yield of total loaded In the expanded bed route 95 of the total ADH loaded was recovered compared with 85 for the packed bed The packed bed fouled more rapidly than the expanded bed for the same volume of material processed indicating that the high speed centrifugation 38 000 g for 60 minutes employed prior to pac
167. olumns connected to the system Valve V2 directs the column effluent to the fraction collector It also blocks the flow through the bed to allow the adaptor to be lifted when pump 1 pumps upward flow through the bed Valve V3 directs the flow of hydraulic liquid from the hydraulic chamber to waste when the adaptor is lifted It also opens the flow path from pump 2 to the hydraulic chamber of the column when the adaptor is lowered Valve V3 should be closed during normal upward or downward flow through the bed see Fig 21 49 Alternatively it can be left in its waste position to allow for upward movement of the adaptor in case of build up of pressure drop over the adaptor net The fourth port of the valve has to be blocked Valve V3 and pump 2 are optional They are needed to operate a STREAMLINE 25 column supplied with a hydraulic adaptor but are not required if the STREAMLINE 25 column is supplied with a manual adaptor Valve V4 by passes columns connected to the system Valves VS and V6 are used to select individual columns connected to the system Table 6 shows valve positions and pump modes used to direct flow and control adaptor movement Table 8 lists components suitable for assembling a complete semi automated system based on STREAMLINE 25 and GradiFrac Table 8 Components for a semi automated STREAMLINE system based on a STREAMLINE 25 column and GradiFrac See Ordering Information for descriptions pack sizes and co
168. om a bacteriostatic point of view Product availability STREAMLINE Chelating is supplied as a suspension in 20 ethanol in packs of 300 ml and 7 5 litres For larger quantities please contact your local Pharmacia Biotech office 71 STREAMLINE Heparin Product characteristics STREAMLINE Heparin is an expanded bed affinity adsorbent for affinity chromatography using immobilized heparin Heparin is a highly sulphated glycosaminoglycan that binds a wide range of biomolecules 51 Among the protein classes successfully purified on immobilized heparin are enzymes mast cell proteases lipoprotein lipase coagulation enzymes superoxide dismutase serine protease inhibitors antithrombin III protease nexins growth factors fibroblast growth factor Schwann cell growth factor endothelial cell growth factor extracellular matrix proteins fibronectin vitronectin laminin thrombospondin collagens nucleic acid binding proteins initiation factors elongation factors restriction endonucleases DNA ligase DNA and RNA polymerases hormone receptors oestrogen and androgen receptors and lipoproteins STREAMLINE Heparin is based on highly cross linked 6 agarose modified by including an inert quartz core to give the desired density Heparin is immobilized to the STREAMLINE matrix by reductive amination giving a stable coupling even under very alkaline conditions STREAMLINE Heparin is a Custom Designed Media CDM product See CDM pro
169. om the bed After the glycerol wash the bed was allowed to settle and 50 mM sodium phosphate pH 6 0 was used to wash out the glycerol from the bed at a flow velocity of 152 cm h The enzyme was desorbed from the bed by a multiple step elution procedure using downward flow at a flow velocity of 152 cm h The following elution steps were applied 1 50 mM triethanolamine HCl pH 8 0 2 100 mM NaCl in 50 mM triethanolamine HCl pH 8 0 3 10 mM NADP in 50 mM triethanolamine HCl pH 8 0 and 4 2 M NaCl in 50 mM triethanolamine HCl pH 8 0 104 G6PDH was recovered from the unclarified homogenate with a yield of 99 and an average purification factor of 103 No particulate material was found in the eluted enzyme as judged by turbidometric and microscopic analysis The process time for the complete purification cycle was 3 hours 40 min equilibration 30 min feed application 60 min wash 50 min elution Table 37 summarizes the experimental results Table 37 Purification of G6PDH from yeast cell homogenate using expanded bed dye ligand affinity adsorption Purification Volume Liquid Total Total Specific Purification Yield of step ml velocity activity protein activity factor G6PDH cm h U mg U mg Homogenate 830 2732 10309 0 265 1 0 100 Flow through 830 152 53 6 4814 Wash 440 53 90 3 3987 Eluate 1 625 152 7 639 Eluate 2 500 152 0 227 Eluate 3 450 152 2698 99 27 25 103 98 8 El
170. or antagonist IL 1ra and IL ra mutants expressed in Bacillus subtilis have been purified by expanded bed adsorption on STREAMLINE SP 72 IL 1ra is the antagonist member of the interleukin 1 IL 1 family It exerts its activity by competing with IL 1 for binding to its receptors The inhibitory activity of IL 1ra on the effect of IL 1 makes it a candidate for therapeutic use in a number of pathologies in which IL 1 activity is involved 100 IL 1ra was expressed in B subtilis in endocellular form and released into the culture medium by starvation induced sporulation a process which allows spontaneous release of the recombinant protein without need for a specific cell disruption step The sporulation was induced within the fermentor by simultaneous starvation and temperature shift IL 1ra was purified from the culture medium by two alternative routes The first was traditional processing of clarified feed material by packed bed ion exchange chromatography the second was direct adsorption of crude unclarified feed material on an expanded bed of STREAMLINE SP The complete scheme of the traditional route consisted of centrifugation 20 000 g for 1 hour at 4 C filtration initial purification on a S Sepharose High Performance column 3 5 x 10 cm and final purification on a Q Sepharose High Performance column 3 5 x 10 cm or Mono Q HR 10 10 The complete scheme of the alternative route consisted of direct capture of the unclarified cult
171. or is lifted It also opens the flow path from Pump 2 to the hydraulic chamber of the column when the adaptor is lowered Valve V3 is closed during normal use with upward or downward flow through the bed see Fig 24 Alternatively valve V3 can be positioned to open the flow path from the hydraulic chamber to waste This will allow the adaptor to move upwards in case the adaptor net is clogged and thus prevent pressure build up in the system This is useful if no pressure monitor is connected to the column inlet If valve V3 is a 4 port valve the fourth port must be blocked 46 Valve V4 is a column bottom valve used to by pass the column when filling the system at the inlet side The bottom valve also allows a column containing sedimented adsorbent to be connected or disconnected without being drained Table 6 shows valve positions and pump modes used to direct flow and control adaptor movement Table 7 lists recommended equipment for assembling complete manual systems based on STREAMLINE 25 STREAMLINE 50 and STREAMLINE 200 columns Table 6 Pump mode and valve positions for directing flow and controlling adaptor movement in a manual semi manual STREAMLINE system as outlined in Fig 20 and Fig 21 Operation Valve 1 Valve 2 Valve 3 Pump 1 Pump 2 double channel single channel single channel 4 port 3 or 4 port 3 or 4 port Upward flow OS 2N 6 on off Downward flow OA Z L on off Adaptor up
172. or to elution The enzyme was desorbed from the bed by a three step elution scheme using a flow velocity of 200 cm h The first step was elution by 0 05 M NaCl 50 mM sodium phosphate pH 6 0 the second was elution by 0 15 M NaCl 50 mM sodium phosphate pH 6 0 and the third step was 1 0 M NaCl 50 mM sodium phosphate pH 6 0 Table 35 summarizes the experimental results The enzyme was recovered with a yield of 98 in the second elution step with 0 15 M NaCl The purification factor was 12 The process time for the complete purification cycle was 3 3 hours equilibration 40 min feed application 30 min wash 60 min elution 70 min Table 35 Purification of G6PDH from yeast cell homogenate using expanded bed anion exchange adsorption Purification step Volume Liquid Total Total Specific Purification Yield of ml velocity activity protein activity factor G6PDH cm h U mg U mg Homogenate 1068 2873 13670 0 21 1 0 100 Flow through 1068 196 66 4 273 0 14 Wash 550 66 122 5 4102 0 17 Eluate 1 1300 200 42 258 1 46 Eluate 2 2100 200 2819 1125 2 51 12 0 98 1 Eluate 3 900 200 6 917 0 2 Total recovery 100 0 100 0 s To verify the function of the adsorbent after repeated use STREAMLINE DEAE was subjected to 10 cycles of feed application each followed by a wash with 25 v v glycerol solution and a cleaning in place procedure consisting of 1 0 M NaCl 1 0 M NaOH 1 0 M NaCl 70
173. ore process economy benefits from the reduction of costs associated with labour consumables maintenance and capital expenditure when comparing a single expanded bed adsorption step with a multiple step approach based on the traditional sequence of centrifugation filtration and packed bed chromatography Critical parameters The critical parameters in expanded bed adsorption can be divided into chemical parameters and physical parameters Chemical parameters are the parameters related to the selectivity and capacity of the separation process and include pH ionic strength types of ions and buffers used The influence on separation performance of these parameters is virtually the same in expanded bed adsorption as in traditional packed bed chromatography 18 Physical parameters are the parameters related to the hydrodynamics and stability of a homogeneous fluidization in the expanded bed Some physical parameters are related to the broth composition e g cell density biomass content and viscosity Others are related to operating conditions such as temperature flow velocity and bed height Chemical parameters are optimized during method scouting in packed bed mode as described under Experimental strategy Some of the chemical parameters such as pH and conductivity are worth investigating thoroughly to optimize interfacing fermentation and expanded bed adsorption For example high conductivity feed stock applied directly to an
174. ould be low e g 30 50 cm h and the volume applied should be large enough to allow a minimum contact time of 4 hours If this does not restore the performance of the adsorbent an even longer contact time should be tried A combination of long contact time and a moderate consumption of cleaning solution can be applied by first directing the main peak of the material eluted by the CIP solution to waste and then recirculating the CIP solution on the column for the remainder of the cleaning time which can then be extended to overnight exposure or beyond Even if the adsorbent shows a grainy appearance and poor expansion due to heavy channelling and thus poor contact with the cleaning liquid it slowly improves during the wash phase if the contact time is long enough If aggregation of the bed is severe resulting in the formation of a compact plug in the column this must be eliminated by periodic back flushing before starting the cleaning This is done by intermittently changing the flow direction in the bed for short periods applying a high flow velocity e g up to 2500 cm h to mechanically break up the plug and clumps If the performance of the adsorbent cannot be restored by a wash with NaOH or NaOH NaCl alone solvent or detergent based cleaning methods should be used in conjunction with NaOH after having washed out the NaOH from the column with distilled water A polar organic solvent such as 30 isopropanol or 20 70 ethanol can
175. pand the bed to a final height of 2 4 times its sedimented height The culture broth was diluted three fold on line by a separate buffer feed to reduce the ionic strength and pH to a level suitable for adsorption to the STREAMLINE SP adsorbent On line dilution was applied to minimize exposure of cells to reduced pH and osmolality which could cause cell lysis The combined flow velocity of the two feeds was such that approximately the same 2 4 times expansion of the bed was maintained throughout adsorption This corresponded to an average flow velocity of 135 cm h through the bed in the first run In the second run the required average flow velocity to generate the same degree of expansion was 144 cm h The cell culture broths were stored for 2 and 3 days respectively before processing which reduced the cell load by partial sedimentation The cell concentration in the undiluted culture broth was 5 x 104 cells per ml at the start of the process Following adsorption the expanded bed was washed with approximately 24 sedimented bed volumes of equilibration buffer to remove unadsorbed protein and any remaining cells from the bed Following the wash the bed was allowed to sediment and the antibody was eluted with a linear gradient from 40 to 400 mM NaCl in 25 mM MES buffer pH 5 4 In the second run the pH of the elution buffer was increased to 6 4 to tighten the product peak and reduce the number of fractions required to recover the antibody Af
176. pended in 2 volumes of 20 sucrose in 50 mM Tris buffer pH 7 4 containing 1 mM EDTA The exotoxin A was released by osmotic schock after dilution with 18 volumes of 50 mM Tris buffer pH 7 4 An endonuclease Benzonase Merck Nycomed Pharma A S was added at a ratio of 0 2 pl 75 U per gram cells and the suspension was diluted with 18 volumes of the Tris buffer The endonuclease was needed to reduce the viscosity of the cell extract The concentration of dry cell mass in the cell extract was 6 g L The unclarified cell extract was applied to STREAMLINE DEAE adsorbent expanded and equilibrated with 50 mM Tris buffer pH 7 4 in a STREAMLINE 200 column 200 mm i d The column contained 4 7 litres of adsorbent providing a sedimented bed height of 15 cm The adsorbent expanded 5 times during the adsorption phase After loading the adsorbent was washed in expanded mode with 40 litres 8 5 sedimented bed volumes of buffer Elution was performed with downward flow in sedimented bed mode using a flow velocity of 100 cm h The buffer used during expansion equilibration and wash was 50 mM Tris buffer pH 7 4 The elution buffer was 50 mM Tris buffer pH 7 4 containing 0 5 M sodium chloride Cleaning in place was performed after each purification cycle using upward flow with the adaptor positioned at twice the sedimented bed height The cleaning protocol was 0 5 M NaOH containing 1 M NaCl at a low flow rate giving a contact time of at least 4 hours 3
177. r while maintaining upward flow through the expanded bed The adaptor is lowered by pumping hydraulic liquid from pump 2 into the hydraulic compartment of the column 57 Elution 1 Switch valve V1 to downward flow and start pumping 1 2 sedimented bed volumes of wash buffer through the sedimented bed at a flow velocity of approximately 100 cm h 2 Switch to elution buffer and continue pumping in the same direction to elute the target protein from the sedimented bed Pump at a flow velocity of 50 100 cm h see Fis 27 3 Collect eluted fractions as indicated by the UV monitor 4 Turn off the pump and immediately start cleaning in place CIP Hyacraulic iquid aN Pump 2 Buffer 1 Pa Buffer 2 E ii Me a CS Pressure monitor Me P1 Ty Ooo bo UV Cond pH Waste Fig 27 Schematic representation of valve positions and liquid flow in a manual STREAMLINE system during elution Cleaning in place Design and optimize the cleaning in place protocol according to the properties of the adsorbent ligand and the nature of the feed material applied The general operating principles of cleaning in place an expanded bed adsorption column are described below More detailed information about designing cleaning in place protocols is given under Cleaning in place in Chapter 4 Method Optimization Specific recommendations for different types of STREAMLINE adsorbents are included in the instructions accompanying each mediu
178. ractions 1 395 64 4 556 8 0 116 column strip effluent 0 7 9 4 530 0 0177 2 whole broth 36 7 43 270 0 0275 diluted feed 108 2 14 90 0 0238 flow through 108 0 38 70 2 5 84 x 10 3 pooled eluate fractions 0 765 294 9 1473 5 0 200 column strip effluent 0 7 1 18 410 2 88 x 103 The operating cost for the expanded bed adsorption process was compared with an alternative process consisting of a microfiltration step and an ultrafiltration step Labour costs filter adsorbent costs and reagents cost were calculated for processing of 250 L of whole cell culture broth The labour cost was found to be four times lower for the expanded bed process Assuming one set of filters would be sufficient for 100 filtration batches and the given adsorbent volume would be sufficient for only 10 batches the calculated filter cost was still twice as high as the adsorbent cost The overall operating cost for the expanded bed process was estimated to be 3 6 times less than the filtration process Furthermore it was concluded that the expanded bed process offers further advantages due to the partial purification achieved and the higher recovery compared with the alternative process Purification of a humanized IgG antibody from myeloma cell culture broth by expanded bed affinity adsorption A humanized IgG monoclonal antibody was purified from a myeloma cell culture by expanded bed adsorption on STREAMLINE rProtein A 65 The work was performed by Pharmacia Biotech Upp
179. re systems by ion exchange chromatography STREAMLINE SP is recommened as the first 24 choice This is because of the high density of negatively charged glycoproteins on the surface of mammalian cells These may interact with the positively charged surface of an anion exchanger such as STREAMLINE DEAE The effect of this will be more severe above pH 7 Suitable columns are XK 16 or XK 26 columns providing sedimented bed volumes of up to 0 15 litres XK 26 at a maximum sedimented bed height of 30 cm Nominal bed height is 15 cm which gives a sedimented bed volume of 0 03 litres in an XK 16 column and 0 08 litres in an XK 26 column The flow velocity during method scouting should be similar to the flow velocity to be used during the subsequent experiments in expanded mode The nominal flow velocity for STREAMLINE expanded bed adsorption is 300 cm h This may need adjustment during optimization depending on the properties of the feed stock A small amount of clarified feedstock is applied to the packed bed at various binding conditions to define those that provide the optimal selectivity and capacity for the target protein Elution can be performed step wise or by applying a gradient Linear gradients are applied in the initial experiments to reveal the relative binding of the target molecule versus the contaminants This information can be used to optimize selectivity for the target molecule i e to avoid binding less strongly bound contaminant
180. ring the design phase may reduce the number of steps needed To design in the fewest possible processing steps thus offers the most efficient way of reaching high process economy in the overall production process The initial purification of the target molecule has traditionally been addressed by adsorption chromatography using a conventional packed bed of adsorbent This necessitates clarification of the crude feed before application to the chromatography column The standard techniques used for removal of cells and or cell debris have been centrifugation and microfiltration The efficiency of a centrifugation step depends on particle size density difference between the particles and the surrounding liquid and viscosity of the feed stock When handling small cells such as E coli or cell homogenates small particle size and high viscosity reduce the feed capacity during centrifugation and sometimes make it difficult to obtain a completely particle free liquid To obtain a particle free solution that can be further purified by traditional packed bed chromatography centrifugation is usually combined with microfiltration However microfiltration also has its drawbacks Although microfiltration yields cell free solutions the flux of liquid per unit membrane area is often dramatically decreased during the filtration process Fouling of the microfiltration membranes is another problem that significantly adds to the Operational cost The combined
181. roths of yeast and bacteria to mimic a real expanded bed adsorption process 39 Following a predefined sanitization in place protocol a number of critical test points in the system were all free from vegetative organisms Fig 29 shows the configuration of a STREAMLINE production scale system designed for the operation of a STREAMLINE 600 column custom designed column with an internal diameter of 600 mm For more information about custom designed STREAMLINE systems contact your nearest Pharmacia Biotech office 83 A production scale STREAMLINE system and column Product Product Waste Feedstock Inlets e g buffers eq and OH AT cleaning Ra saniti zation y fluids RA moe O To V O Ri Main pump P2 Hydraulic pump Cleaning Hydraulic F Flow meter 2 sanitization fluid V Vent valve 5 fluid CV Check valve B Back pressure valve AT Air trap 2 AS Air sensor 2 S Steam trap 3 P Pressure sensor 3 C Conductivity meter D Zero dead leg valve 2 UV UV monitor pH pH meter 1 Temperature sensor 4 AdS Adsorbent sensor APS Adaptor position sensor Fig 29 STREAMLINE column and system for production scale operation 84 System control STREAMLINE pilot scale systems can be controlled by the Process Controller C 3 or by the UNICORN multipurpose controller STREAMLINE production scale systems are controlled by UNICORN or other commercially available control systems UNICORN adds flexibility
182. s It can also be used to define the step wise elution to be used in the final expanded bed When selectivity has been optimized the maximum dynamic binding capacity is determined by performing breakthrough capacity tests using the previously determined binding conditions The breakthrough capacity determined at this stage will give a good indication of the breakthrough capacity in the final process in the expanded bed as has been discussed previously in Section 2 Method optimization Method optimization for the expanded mode is performed on small scale using crude unclarified feed A suitable column is the STREAMLINE 25 25 mm i d which provides a sedimented bed volume of up to 0 15 litres at a maximum sedimented bed height of 30 cm A nominal bed height of 15 cm gives a sedimented bed volume of 0 074 litres in a STREAMLINE 25 column The purpose of the method optimization in expanded mode is to examine the effects of the crude feed on the stability of the expanded bed and on the chromatographic performance If necessary adjustments are made to achieve stable bed expansion with the highest possible recovery purity and throughput During method optimization the process should be challenged by applying a sample load close to the maximum sample load as defined in the breakthrough study performed at the method scouting phase Challenging the process gives an 25 identification of critical process parameters and reveals what cleaning pro
183. s defined by frontal analysis of breakthrough during method scouting on the small packed bed column The purity of the eluted antibody was higher than 90 as determined by SDS PAGE No contaminating albumin initial concentration 500 mg ml remained in the eluate Table 47 Summary of results from laboratory scale and pilot scale purification of a monoclonal IgG by expanded bed adsorption on STREAMLINE rProtein A STREAMLINE 25 STREAMLINE 200 Adsorbent volume L 0 075 5 Feed volume L 94 3 Conc IgG in feed mg ml 0 09 0 031 Amount IgG applied mg 90 2 93 Conc IgG in eluate mg ml 1 7 0 66 Concentration factor 15 21 Yield 95 100 90 6 Process time minutes 100 115 During the laboratory scale runs on the STREAMLINE 25 column the extent of cell damage caused by the P 50 piston pump and by passage through the expanded bed was investigated Concentration of DNA activity of lactate dehydrogenase LDH and the particle load in the flow through were used as indicators on cell damage Table 48 summarizes the results from those studies 123 Despite the sensitivity of hybridoma cells to shear force neither the high flow velocity with the piston pump nor the passage of the cells through the expanded bed caused any detectable cell damage No increase in DNA concentration or LDH activity was found in the flow through and wash fraction compared with the sample applied to the expanded bed The increase in LDH
184. sala Sweden in collaboration with Celltech Biologics Plc UK Crude unclarified cell culture broth from the fermentor was applied directly onto a STREAMLINE column containing STREAMLINE rProtein A at an amount corresponding to a sedimented bed height of 15 cm The temperature of the cell culture broth was 37 C at the time of feed application Prior to feed application the bed was expanded and equilibrated with 50 mM glycine glycinate pH 8 120 containing 250 mM NaCl The flow velocity during expansion equilibration and feed application was 300 cm h After the sample had been applied the expanded bed was washed with starting buffer until the signal from the UV monitor returned to baseline This was followed by an additional 10 sedimented bed volumes of starting buffer Elution was performed with downward flow in sedimented mode at a flow velocity of 100 cm h The elution buffer was 0 1 M glycine pH 3 0 After elution the bed was subjected to cleaning in place by passing 2 sedimented bed volumes of 6 M guanidine hydrochloride through the bed using a flow velocity of 100 cm h in expanded bed mode Two subsequent runs as described above were performed at laboratory scale by applying 1 5 litres of cell culture broth to a STREAMLINE 25 column 25 mm i d containing 75 ml of STREAMLINE rProtein A The process was then scaled up to pilot scale by applying 93 litres of cell culture broth to a STREAMLINE 200 200 mm i d containing 4 7 litr
185. scale up In any type of chromatographic process a successful scale up to full production can only be achieved by designing in scalability during the method development stage Designing in scalability has to do with the careful selection of suitable media buffers chemicals columns and system hardware and with building robustness into the process at the early stages of method optimization To assure scalability and robustness sources of variation have to be defined characterized tested and ideally eliminated Sources of variation that cannot be eliminated must be carefully controlled by setting specifications for the upper and lower limits of all critical process parameters Safety margins have to be built into the process control parameters based on challenge tests performed at the upper and lower limits of normal variations The most critical sources of variation in expanded bed adsorption are related to the feed material e g variations in product concentration concentration of contaminating proteins biomass content viscosity conductivity cell lysis nucleic acids etc Other sources of variation are related to the process 26 conditions e g raw materials buffer preparation equipment personnel etc Time may also be a critical source of variation since holding times between different steps feed application time etc can vary especially with scale up When scalability is considered from the start and built into the process d
186. sequent chromatographic performance Short term refers to the pH interval for regeneration and cleaning procedures 2 Breakthrough capacity in packed and expanded bed at a flow velocity of 300 cm h Sedimented bed height 15 cm Cleaning in place and sanitization in place The pH stability of STREAMLINE rProtein A is limited by the susceptibility to hydrolysis of the protein A ligand at high pH values This excludes using high concentrations of NaOH for cleaning and sanitization The following alternative cleaning in place protocol has therefore been developed for STREAMLINE rProtein A It has been verified to restore both hydrodynamic and chromatographic performance over several purification cycles 46 74 2 M NaCl and 1 mM NaOH in 20 ethanol flow rate 100 cm h contact time 120 minutes 5 Sarcosyl 20 mM EDTA 0 1 M NaCl in 20 mM sodium phosphate pH 7 0 100 cm h contact time 90 minutes 50 mM acetic acid in 20 ethanol to remove the detergent 300 cm h contact time 20 minutes Other cleaning agents that can be recommended to remove strongly adsorbed precipitated or denatured proteins are 6 M guanidine hydrochloride 6 M urea and 1 M acetic acid Chapter 4 Method Optimization contains general information about cleaning in place of STREAMLINE adsorbents The following alternative protocols can be recommended to sanitize STREAMLINE rProtein A Protocol 1 Equilibrate the bed with a solution consisting of 2
187. sert the assembled adaptor into the column at an angle so that one side of the adaptor net is in the water filled column Without trapping air under the net carefully put the adaptor into a vertical position Slowly push the adaptor down until the gasket on the adaptor net is submerged in water This ensures that the gasket forms a tight seal with the glass tube Fill the space above the adaptor with distilled water so that the hydraulic drive can function Push down the lid and secure it in place For the STREAMLINE 25 column insert the adaptor into the column without force so that the adaptor O ring rests on top of the glass tube Fill the space above the adaptor with distilled water so that the hydraulic drive can function Push down the lid and secure it in place Switch valve V3 to open the flow path from pump 2 to the hydraulic chamber of the column Set valve V2 to waste position to open the column outlet Slowly move the adaptor down by pumping hydraulic liquid from the hydraulic pump into the hydraulic chamber of the column Stop the hydraulic pump when the adaptor has been lowered a few centimeters Switch valve V3 to open the flow path from the hydraulic chamber to waste Set valve V2 to the closed position to block the flow path through the bed Lift the adaptor by pumping buffer with upward flow from pump 1 to remove any air trapped in the hydraulic chamber If necessary repeat this procedure until all air has been removed from the colum
188. sic principles of operation The principle of expanded bed adsorption is shown in Fig 1 STREAMLINE adsorbent is expanded and equilibrated by applying an upward liquid flow to the column A stable fluidized bed is formed when the adsorbent particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward liquid flow velocity The column adaptor is positioned in the upper part of the column during this phase Crude unclarified feed is applied to the expanded bed with the same upward flow as used during expansion and equilibration Target proteins are bound to the adsorbent while cell debris cells particulates and contaminants pass through unhindered Weakly bound material such as residual cells cell debris and other type of particulate material is washed out from the expanded bed using upward liquid flow When all weakly retained material has been washed out from the bed the liquid flow is stopped and the adsorbent particles quickly settle in the column The column adaptor is then lowered to the surface of the sedimented bed Flow is reversed and the captured proteins are eluted from the sedimented bed using suitable buffer conditions The eluate contains the target protein increased in concentration clarified partly purified and ready for further purification by packed bed chromatography After elution the bed is regenerated by washing it with downward flow in sedimented bed mode using bu
189. ss associated with flow constrictions also requires consideration when designing the liquid distributor Shear stress should be kept to a minimum to reduce the risk of molecular degradation Another function of the distribution system is to prevent the adsorbent from leaving the column This is usually accomplished by a net mounted on that side of the distributor which is facing the adsorbent The net must have a mesh size that allows particulate material to pass through and yet at the same time confine the adsorbent to the column The distributor must also have a sanitary design which means that it should be free from stagnant zones where cells cell debris can accumulate More information about STREAMLINE columns is found in Section 6 Product Guide Characteristics of expanded beds Bed expansion Fluidization occurs when particles are pushed upwards in a column at a velocity corresponding to their sedimentation velocity The degree to which a bed expands i e how far up in the column a particular bead is transported is controlled by the size and the density of the adsorbent beads the linear flow velocity of the mobile phase and the viscosity of the mobile phase The size and density of STREAMLINE adsorbents beads have been defined to allow optimal expansion at flow velocities that will ensure high productivity of the purification system STREAMLINE adsorbents expand about 2 to 3 times in normal buffer solutions at room temperature a
190. ss separations when chromatography media from our standard product range are not suitable Custom Designed Media comprise media for both packed bed chromatography and expanded bed adsorption They can be made to BioProcess Media specifications if required The Custom Designed Media group at Pharmacia Biotech works in close collaboration with the customer to design manufacture test and deliver media for specialized separation requirements Several CDM products are also available to the general market Media first produced as Custom Designed Media have often proven so successful that they have subsequently been introduced as BioProcess grade Pharmacia Biotech products Custom Designed Media in Chapter 6 are given the CDM symbol Product availability Contact your nearest Pharmacia Biotech office for further details of CDM products and services 61 Base matrices The base matrix in STREAMLINE media is a highly cross linked beaded agarose derivative based on 6 or 4 agarose This has been modified by including an inert core to provide the required density for stable expansion at high flow rates The macrostructure of the agarose is composed of polysaccharide chains arranged in bundles that are further strengthened by inter chain cross linking The resulting macroporous structure combines high physical and chemical stability with good capacities for large target molecules Agarose based matrices are also well appreciated for their low non sp
191. step input signal is given below 1 When the bed is fully expanded at the test flow rate mark the expanded bed height on the column and continue pumping buffer 2 Lower the adaptor so there is about 0 5 to 1 cm between the net and the expanded bed surface 3 Start the recorder and UV monitor When the baseline is stable change to buffer acetone mixture 0 25 v v and wait for the positive step input UV signal Fig 10 4 Change back to buffer when the UV signal is stable at maximum absorbance 100 Mark this change on the recorder paper mark in Fig 10 5 Wait for the negative step input UV signal and allow the signal to stabilize at the baseline level 0 6 Calculate the number of theoretical plates N from the negative input UV signal N t2 o2 where t mean residence time o standard deviation t is the distance from the mark in step 4 of the test procedure to 50 of the maximum UV signal Fig 10 o is measured as half the distance between the points 15 85 and 84 15 of the maximum UV signal Fig 10 15 A difference of more than 20 in the number of theoretical plates between two runs performed under identical test conditions indicates that the bed is not stable A reasonably good value for N is within the range 25 30 at a sedimented bed height of 15 cm using a nominal flow velocity of 300 cm h This corresponds to a plate number of 170 200 N m If the mean residence time as calcul
192. stic clamp 25 mm Gasket 10 mm i d 1 Threaded connections 2 25 mm o d clamp connections 3 Peristaltic tubing ordered from Pharmacia Biotech can be supplied with moulded on 25 mm o d clamp connectors as an option if sanitary connections are required 4 For connection to peristaltic tubing supplied with 25 mm o d clamp connectors 5 For connection of PE tubing threaded polyethylene tubing to column peristaltic tubing and UV flow cell 48 Semi automated systems A small scale semi automated system can be set up with the STREAMLINE 25 column and GradiFrac a fraction collector with built in control of flow rate three switch valves and recorder start and stop Fig 21 shows a schematic representation of this configuration The system shown in Fig 21 contains one STREAMLINE 25 column and one extra column for traditional packed bed chromatography More columns can be connected if more valves are added to the system Hydraulic iquid i Pump 2 P 50 Buffer B Buffer A W C A AA 4 Pump 1 P 50 Waste IN Sample V4 Waste Product Fig 21 Schematic representation of a semi automatic STREAMLINE system based on the STREAMLINE 25 column and GradiFrac fraction collector Pump 1 and the PSV 50 valves are controlled by a programmed sequence in the GradiFrac controller Valves V1 V2 V3 V4 V5 and V6 are manual valves Valve V1 changes flow direction through the c
193. subjected to 50 subsequent purification cycles each followed by the CIP protocol described above The degree of expansion was determined before each cycle and breakthrough capacity for lysozyme was determined before cycle 1 and after cycles 25 and 50 Breakthrough capacity was determined in expanded bed mode Table 31 summarizes the results Table 31 Summary of results from the study on the re useability of STREAMLINE SP Start 25 cycles 50 cycles Degree of expansion H Ho 2 8 2 4 2 5 Breakthrough capacity for lysozyme mg lysozyme ml sedimented bed 69 71 74 l H expanded bed height when the adsorbent has been expanded and equlibrated with start buffer Ho sedimented bed height The results indicated that the adsorbent could be reused for more than 50 cycles without compromising its function No effect on the tested parameters could be seen over 50 cycles SDS PAGE on collected fractions revealed no loss in chromatographic performance over the 50 cycles Purification of a secreted recombinant protein from E coli fermentation broth by expanded bed anion exchange adsorption Expanded bed anion exchange adsorption on STREAMLINE DEAE was used in the purification of a recombinant fusion protein directly from crude Escherichia coli fermentation broth 32 The fusion protein consisted of two synthetic IgG binding domains ZZ derived from staphylococcal protein A and a repeat structure M5 from the central repeat region of the mal
194. substantial part of the UV absorbing material that could interfere with the following steps The second intermediate purification step was anion exchange chromatography on SOURCE 30Q packed in a FineLINE 100 column This step removed the majority of the remaining contaminants The polishing step was HIC on SOURCE 15 PHE The four step procedure starting with crude unclarified lysate resulted in a pure protein according to PAGE and RPC analysis with an overall recovery of 51 Table 28 summarizes the purification process Table 28 Purification table for the complete process Values are extrapolated from actual runs except for STREAMLINE DEAE which was performed at the scale given in the table Purification step Volume Total protein Exotoxin A Step recovery litres gram gram Bacterial extract 180 351 0 10 8 STREAMLINE DEAE 13 5 140 0 8 54 79 Phenyl Sepharose Fast Flow high sub 11 4 41 0 6 60 17 SOURCE 30Q 30 2 12 6 6 04 91 SOURCE 15PHE 12 2 n d 5 5 91 1 Activity was determined with a radial immunodiffusion assay using Goat anti exotoxin A antibodies List USA 94 The Capture step using expanded bed adsorption with crude unclarified feed material was compared with traditional processing using clarified feed material on a packed bed of chromatography media In the packed bed process the cell lysate was centrifuged clarified and applied on a packed bed of the anion exchanger DEAE Sepharose Fast Flow at a flow
195. t aggregates forming 3 Continuously monitor the level of the expanded bed The degree of expansion usually increases during this phase due to the increase in viscosity from the crude feed 4 Periodically back flush to clear the adaptor net if particulates build up underneath it due to increased bed expansion Switch valve V1 to downward flow to disrupt this build up Return to upward flow after a few seconds when the build up has been cleared The expanded bed quickly re stabilizes Decrease expansion by reducing the flow velocity if build up is observed frequently If high biomass content and high viscosity cause bed instability and channelling decrease viscosity by diluting the feed stock Refer to Feed application in Chapter 4 Method Optimization for more details about the effect of feed material on bed stability and performance Valve V3 can be put in its waste position during feed application and wash especially if there is no pressure monitor connected to the system This will allow the adaptor to move upwards in case of build up of pressure drop over the adaptor net Buffer 2 a m p ra O D Z D Oo UV Cond pH Waste Fig 25 Schematic representation of valve positions and liquid flow in a manual STREAMLINE system during feed appli
196. t change in osmotic pressure during dilution Using 200 300 mM glucose as diluent gave higher cell viability lower degree of expansion higher product yield and also made it easier to restore bed expansion characteristics by cleaning in place After method optimization in the STREAMLINE 25 column the method was scaled up 2200 fold allowing the purification of recombinant monoclonal antibody from 12000 litres of cell culture suspension Using on line dilution with 200 mM glucose unclarified cell culture suspension was applied directly onto a STREAMLINE 1200 column customized column with 1200 mm i d after adjustment of pH to 5 4 The column contained 170 litres of STREAMLINE SP corresponding to a sedimented bed height of 15 cm 117 The buffer used during expansion equilibration and wash was 20 mM MES pH 5 4 The flow velocity during expansion equilibration feed application and wash was 300 cm h Elution was performed at a flow velocity of 100 cm h using downward flow in sedimented bed mode The elution buffer was 0 25 M tetramethyl ammonium chloride TMAC 1 M NaCl After the purification cycle had been completed the column was immediately subjected to cleaning in place using a solution of 0 5 M NaOH and 1 M NaCl The solution was pumped through the bed with upward flow at a flow velocity of 100 cm h The adaptor was positioned at three times the sedimented bed height After approximately two hours when the main contaminant peak had be
197. t flow velocities ranging from 200 to 400 cm h These are considerably higher flow velocities than can be applied with unmodified agarose adsorbents to achieve the same degree of expansion which is illustrated in Fig 4 Degree of expansion H H 0 100 200 300 400 500 Flow velocity cm h71 Fig 4 Relative expansion at different flow velocities of STREAMLINE adsorbents and an agarose matrix with the same particle size 100 300 pm and agarose content 6 but without inert core material O Work by Pharmacia Biotech 10 Mi 100 mM acetate buffer pH 5 MO 25 v v glycerol A 32 v v glycerol Bed height cm Liquid Velocity cm h Fig 5 Expanded bed height of STREAMLINE SP sedimented bed height 10 cm with varying flow rate in a glycerol buffer system in a STREAMLINE 50 column Reproduced with permission from ref 23 Note that absolute values for the degree of expansion will vary with working temperature and the buffer system used liquid density and viscosity The effect of increased viscosity of the buffer system is an increased degree of expansion This is an important consideration during application of a crude and viscous feed material The effect of increased viscosity of the mobile phase on bed expansion has been studied by Chang and Chase 23 and is illustrated in Fig 5 The effect of viscosity and its implication in an expanded bed adsorption experiment will be discussed in
198. tart of the wash cycle should be the same as during feed application The wash volume can be minimized by lowering the adaptor at start of the wash and keeping it just above the surface of the expanded bed for the remainder of the wash cycle 39 In large scale STREAMLINE columns an adsorbent sensor is available for automatic lowering of the adaptor The efficiency of washing out particulate material from a STREAMLINE DEAE and STREAMLINE SP column is demonstrated in Fig 16 44 E coli homogenates containing approximately 10 cfu ml were applied to 250 ml media The relative reduction of living cells was approximately 105 after washing with buffer for a volume corresponding to 20 sedimented bed volumes which is in the same range as when using traditional clarification techniques A slight increase in the number of living cells was observed in the eluate from STREAMLINE DEAE when the NaCl concentration in the eluate was increased This is due to the fact that E coli cells are negatively charged and bind to the positively charged adsorbent surface Similar results have been reported by Hansson et al 32 and Batt et al 25 Hansson et al applied 8 litres of crude E coli fermentor broth to 200 ml STREAMLINE DEAE adsorbent and reported a 4 log reduction of viable cell count after a wash with six expanded bed volumes of buffer Batt et al applied 26 litres of crude CHO culture broth to 170 ml STREAMLINE SP adsorbent and reported an almost complete r
199. tate pH 5 0 Buffer B 50 mM sodium acetate 1 M NaCl pH 5 0 Flow velocity 300 cm h during feed application and wash 100 cm h during elution reversed flow Volume ml Fig 32 Capture of recombinant anti HIV Fab fragment on STREAMLINE SP Work by Pharmacia Biotech Uppsala Sweden Following Capture on STREAMLINE SP the Fab fragment was further purified by Intermediate Purification on Phenyl Sepharose 6 Fast Flow high sub and by a Polishing step on SOURCE 15S This three step procedure starting with crude unclarified homogenate resulted in a pure Fab fragment according to SDS PAGE It was also found to have retained immunological activity towards the surface protein gp120 of HIV 1 as determined by ELISA and retained neutralizing activity towards HIV 1 The neutralizing activity was measured as the Fab fragment s ability to inhibit the HIV 1 infection of T cells in in vitro cell cultures Table 26 summarizes the complete purification process 91 Table 26 Summary of results from the three step procedure for the purification of recombinant anti HIV Fab fragment Purification step Volume Fab conc Step recovery litres ug ml Bacterial homogenate 4 85 6 8 STREAMLINE SP 0 5 62 5 95 Phenyl Sepharose 6 Fast Flow high sub 0 026 122 5 96 SOURCE 15S 0 005 940 56 After each purification cycle on the STREAMLINE SP adsorbent the column was subjected to a cleaning in place procedure usi
200. ter elution the column was stripped with 1 M NaCl 25 mM MES buffer to remove any protein still adsorbed Finally the adsorbent was cleaned with 1 M NaOH followed by 1 M NaCl 3 bed volumes each then distilled water to bring the pH to neutral Table 45 summarizes the results from the two pilot runs The antibody elution profile in the first run exhibited a broad peak with a long tail The pooled fractions contained 69 4 of the antibody in the culture broth Another 5 1 was found in the column strip effluent which means that the overall antibody recovery was 74 5 In the second run the antibody peak was much sharper due to the increased pH of the elution buffer Only 0 4 of the antibody was found in the column strip effluent Due to the higher antibody load in the second run breakthrough of antibody was observed in the flow through but nearly 100 of the adsorbed antibody was recovered 119 A 19 fold reduction of volume and a 6 fold increase in antibody purity was achieved in the first run In the second run the product volume was 47 times less than the whole broth and the purification factor was 7 3 Table 45 Summary of results from two pilot runs of expanded bed adsorption on STREAMLINE SP Run Step Volume Antibody Total protein mg antibody No litres conc mg L conc per mg mg L total protein 1 whole broth 26 4 98 260 0 0192 diluted feed 78 1 44 100 0 0144 flow through 78 0 053 87 5 6 06 x 104 pooled eluate f
201. ter to reduce conductivity and adjustment of pH to 4 5 The process was scaled up to about 1000 litres of culture medium giving a final volume of diluted unclarified culture medium of about 2000 litres The diluted unclarified feed stock was applied to a STREAMLINE CD column with an inner diameter of 1000 mm containing 150 litres of STREAMLINE SP adsorbent corresponding to a sedimented bed height of 19 cm The adsorbent was expanded and equilibrated with 50 mM acetate buffer pH 4 5 containing 50 mM sodium chloride prior to feed application The feed was applied at a flow velocity of 100 to 250 cm h under continuous stirring to prevent cell agglomeration After feed application the expanded bed was washed with 50 mM acetate buffer pH 4 5 containing 50 mM sodium chloride and subsequently the rHSA was desorbed from the adsorbent in sedimented mode using a downward flow velocity of 50 to 100 cm h The elution buffer was 100 mM phosphate buffer pH 9 containing 300 mM sodium chloride 109 The rHSA obtained from the expanded bed adsorption step is further purified by a combination of chromatography steps in packed bed configuration Prior to this purification the rHSA containing fraction from the STREAMLINE SP column is again heat treated in the presence of a reducing agent and stabilizers to reduce the degree of colouring of rHSA and to accelerate the conversion of dimer to monomer Table 41 summarizes the outcome of four production scale runs
202. th the chromatographic and hydrodynamic performance of an expanded bed in completely different ways The content of a feed stock depends on the source material and its handling In the case of a recombinant system it also depends on the location of the accumulated product in the producing organism A good understanding of the characteristics of different source materials and the results of their handling processing is helpful to process development and optimization Table 1 lists some of the common characteristics of recombinant feed stocks used to produce pharmaceuticals and other bioproducts A significant effect on the performance of expanded bed adsorption is whether the target molecule is secreted from the production organism into the culture medium or if it is accumulated intracellularly in the producing organism Secretion systems generate dilute low viscosity feed stock that contains rather low amounts of protein and intracellular contaminants thus providing favourable conditions for downstream processing Intracellular systems on the other hand generate feed stocks rich in intracellular contaminants and cell wall cell membrane constituants Along with the nutrient broth these contaminants pose a greater challenge during the optimization phase of expanded bed adsorption Much of the nutrient broth and associated contamination can be removed prior to cell lysis by thorough washing of the cells but such steps introduce additional costs
203. the distributor plate and net see column User Manual Problem Cause Remedy Channelling in the lower part of the expanded bed Large circular movements and liquid channels in the expanded bed Turbulent flow pattern in the expanded bed Pulsations from the pump Column not in a vertical position Fouling aggregation or infection of the adsorbent Change to a pump giving less pulsation or install a pulse dampener between the pump and the column or change to a smaller pump tubing diameter that will allow the pump to be run at a higher speed Use a spirit level to adjust the vertical position of the column Clean and or sanitize the adsorbent see Cleaning in place page 38 and specific cleaning and sanitization recommenda tions in Section 6 Product Guide 135 Feed application Problem Cause Remedy Build up of Over expansion due to high Periodically back flush to particulates viscosity or high particle clear the adaptor net underneath the adaptor net 136 content of the feed stock high cell density high bio mass content high content of nucleic acids low temperature see page 56 If build up is frequent reduce the flow velocity or reduce viscosity by diluting feed stock with buffer or water or reduce viscosity by treating the feed stock with nuclease e g Benzonase to degrade nucleic acids see pages 31 90 93 99
204. the sedimentation velocity of the adsorbent beads The sedimentation velocity is proportional to the density difference between the adsorbent and the surrounding fluid multiplied by the square of the adsorbent particle diameter To achieve the high throughput required in industrial applications of adsorption chromatography flow velocities must be high throughout the complete purification cycle The first results reported from expanded bed adsorption using conventional chromatographic adsorbents based on agarose 8 revealed an obvious need for particles with higher sedimentation velocity to allow the operation of expanded beds at high flow velocities without the beads being carried out of the column by the lifting liquid flow STREAMLINE adsorbents are based on agarose a material proven to work well for industrial scale chromatography The macroporous structure of the highly cross linked agarose matrices combines good binding capacities for large molecules such as proteins with high chemical and mechanical stability High mechanical stability is an important property of a matrix to be used in expanded bed mode to reduce the effects of attrition when particles are moving freely in the expanded bed The modified agarose matrix used in the manufacture of STREAMLINE adsorbents is less brittle than inorganic material such as some glass or ceramic materials The mechanical stability of STREAMLINE adsorbents has been verified by repeated expansions and sedi
205. third port on the SRTC 3 connector Direct tubing from outlet W2 to the container for hydraulic liquid 7 Fit a stop plug in Valve 6 position 8 8 Remove the filter from the Prefilter 6000 between pump A and the mixer The flow path from pump A to the expanded bed will be open whenever Valve 1 is in positions 1 or 3 When Valve 1 is in position 3 the flow path from the hydraulic chamber of the column to the hydraulic liquid container will also be open Setting Valve 1 to position 3 and Valve 6 to position 8 blocks the column outlet and lifts the adaptor by upward flow through the column The adaptor is lowered by pumping hydraulic liquid via pump C into the hydraulic chamber To open the flow path from pump C to the hydraulic inlet valve 1 should be in position 1 ERE E g ees VALVE 5 AV say Nes bape N aair SYNE VALVE 4 FREFILTER EJ e3 PUMP A PORT 4 MIXER F SRTC 3 COLUMN BYPASS ET PREFILTER PUMP B ae ERIS SUPERLOOP PORT 4 E PREFILTER AIR SENSOR VALVE 3 PUMP Ai Buffer A A2 Sample B1 Buffer B C1 Hydraulic liquid Fig 23 Schematic representation of a BioPilot system reconfigured for expanded bed adsorption chromatography using a STREAMLINE 25 column 52 Pilot scale production scale Pharmacia Biotech supplies both manual and fully automated STREAMLINE systems in a range of scales Systems based on STREAMLINE 50 and STREAMLINE 200 col
206. tively charged phosphate groups of the nucleic acids see page 99 Change to an anion exchange adsorbent or add Mg ions to the feed stock to form complexes with the negatively charged phosphate groups of the nucleic acids Problem Cause Remedy Poor adsorption of target molecule low recovery or low capacity Poor binding due to high conductivity in the feed stock ion exchange adsorbents Wash Dilute the feed stock to a conductivity below 5 mS cm Or minimize conductivity in the culture broth at the end of the fermentation process Or try the high capacity ion exchangers STREAMLINE Q XL or STREAMLINE SP XL Problem Cause Remedy Increased wash volume time Channelling in the expanded bed due to aggregation caused by nucleic acids being released during lysis of cells retarded on the adsorbent beads Apply a wash procedure containing nuclease e g Benzonase to degrade and remove nucleic acids from the bed see page 36 141 Elution Problem Cause Remedy High back pressure Large product volume Precipitation in the eluate Low yield 142 Formation of large aggregates during application of feed stock that are not removed during the washing stage Excessive zone spreading during elution Elution by low pH causing precipitation of nucleic acids present in the bed at the start of elution Degradation of target mole
207. to the process The main source of contaminants in feed where the target molecule is located within the host cell is the complex cell membrane that has to be disrupted to release the target molecule Bacterial and yeast cell walls have a high polysaccharide content that can nucleate into larger structures that foul solid surfaces Proteins and phospholipids are other integral parts of such cell walls that will be released upon cell disintegration Bacterial cell walls are particularly rich in phospholipids lipopolysaccharides peptidoglycans lipoproteins and other types of large molecules that are associated with the outer membrane of a bacterial cell These contaminants may complicate downstream processing by fouling the chromatographic adsorbent This type of 21 Table 1 Characteristics of feed stocks according to the location of the product in the recombinant organism E coli Yeast Mammalian cells Secreted Dilute low viscosity feed containing low amounts of protein Proteases bacterial cells and endotoxins are pre sent Cell lysis often occurs with handling and at low pH DNA can be released and cause high viscosity Secreted Dilute low viscosity feed containing low amounts of protein Proteases and yeast cells are present Secreted Dilute low viscosity feed containing low amounts of protein Proteases and mammalian cells are present Cell lysis often occurs with handling and at low pH DNA can be
208. ture medium 916 6845 100 0 heat treatment 68 C 30 min 1885 6818 99 6 flow through 5885 2 eluate 300 heat treatment with cysteine 111 5804 84 8 110 Purification of a recombinant protein pharmaceutical from P pastoris fermentation broth by expanded bed cation exchange adsorption Expanded bed adsorption on STREAMLINE SP was evaluated by scientists at British Biotech plc UK as an alternative capture step in the manufacturing of a therapeutic protein for Phase II clinical studies 75a b Material for phase I clinical studies was initially produced by a small pilot scale process starting with a fermentation volume of 60 litres This process could not be scaled up for manufacturing the amounts required for the phase II clinical studies Expanded bed adsorption on STREAMLINE SP was evaluated as an alternative to the clarification concentration and initial cation exchange step in the established pilot scale process see Fig 36 Subsequent purification steps remained unchanged Phase Phase Il Fermentation 60 L Fermentation 450 L Centrifugation Concentration Conditioning Conditioning Diafiltration pH adjustment Cation exchange Expanded Bed Adsorption Fig 36 Alternative purification routes for the recombinant protein pharmaceutical The product was secreted by the yeast Pichia pastoris with a biomass dry c
209. uate 4 500 152 121 521 2 Total recovery 104 99 8 z 7 To verify the function of the adsorbent after repeated use the Procion Red H E7B coupled STREAMLINE adsorbent was subjected to 10 cycles of feed application each followed by a wash with 25 v v glycerol solution and a cleaning in place procedure using a cocktail containing 0 5 M NaOH and 4 M urea in 60 v v ethanol Five sedimented bed volumes of the cocktail were applied at a flow velocity of 50 cm h The breakthrough capacity for lysozyme and expansion characteristics were determined before cycle 1 and after cycles 1 5 and 10 Table 38 summarizes the results The results indicated that the adsorbent could be reused for more than 10 cycles without compromising its function No effect on the tested parameters could be seen Table 38 Summary of results from a study on the re useability of Procion Red H E7B coupled STREAMLINE adsorbent Start 1cycle 5 cycles 10 cycles Breakthrough capacity for lysozyme mg lysozyme ml sedimented bed 21 2 20 3 22 1 20 4 Liquid velocity cm h to give 2x bed expansion in aqueous buffer at 4 C 153 154 153 152 105 Recovery of alcohol dehydrogenase ADH from S cerevisiae homogenate by expanded bed hydrophobic interaction adsorption The intracellular enzyme alcohol dehydrogenase ADH was purified from crude unclarified homogenate of baker s yeast by the use of expanded bed hydrophobic interaction adsorption on STREAMLI
210. umns are suitable for method development and small scale production Full scale production systems based on STREAMLINE CD custom designed columns have a capacity of several hundred litres sedimented bed volume See Chapter 6 Product Guide for more information about large scale STREAMLINE columns and systems Start up This section describes the preparation required prior to an expanded bed adsorption separation Detailed information about assembling and operating STREAMLINE columns and components is given in the individual column Instruction Manuals Sampling the adsorbent Each STREAMLINE adsorbent contains particles with a wide range of bead size and density When taking a sample of adsorbent from the container take great care to ensure that the beads represent this range in both size and density Gently shake the container until the adsorbent is completely suspended Then immediately pour all the adsorbent into a glass filter funnel porosity 3 or a bucket depending on the amount Allow the adsorbent to drain cut out a triangular slice of the cake and weigh it Calculate the amount of adsorbent needed for a specific height of sedimented bed in the STREAMLINE column Amount of drained adsorbent g density of adsorbent cake g ml x sedimented bed height cm x cross sectional area of the column cm2 The approximate density of the adsorbent cake is 1 2 g ml for STREAMLINE DEAE STREAMLINE SP STREAMLINE Q XL STREAMLIN
211. ure medium onto STREAMLINE SP followed by final purification on Mono Q In the expanded bed route the crude feed material was applied to a STREAMLINE 50 column 50 mm i d containing 300 ml of STREAMLINE SP adsorbent corresponding to a sedimented bed height of 15 cm The flow velocity during expansion equilibration adsorption and wash was 300 cm h The buffer used during expansion equilibration and wash was 25 mM MES NaOH 1 mM EDTA pH 6 25 Desorption of IL 1ra from the adsorbent was performed with downward flow in sedimented mode using a linear gradient of NaCl from 0 to 0 5 M in 25 mM MES NaOH 1 mM EDTA pH 6 25 The flow velocity during elution was 100 cm h Fractions containing IL 1ra were pooled and subjected to final purification on a Mono Q HR 10 10 column Table 34 summarizes the complete purification sequence by both the traditional route and the expanded bed route Table 34 Summary of results from purification of recombinant IL 1ra DoB 0039 by two alternative routes Purification step Traditional route Expanded bed route Purity Recovery Purity Recovery Crude starting material 14 100 14 100 Centrifugation n d n d Filtration 14 96 z Cation exchange 94 84 90 92 85 Anion exchange 98 74 98 73 A final purity of 98 and an overall recovery of 74 was achieved when clarified start material was purified by cation exchange chromatography on S Sepharose High Performance followed by anion exchange
212. uring the method development work actual scaling up is usually a straightforward process The strategy is to maintain all the parameters that are related to the chromatographic and hydrodynamic performance such as sedimented bed height expanded bed height flow velocity sample load volume of adsorbent and volume of process buffers expressed in terms of the number of sedimented adsorbent bed volumes Some system factors may affect performance after scale up and may call for fine tuning of the process such as adjustment of equilibration volume wash volume and elution volume A major concern when scaling up an expanded bed adsorption step is the column especially the inlet and outlet liquid distribution systems The most critical design parameters are the number of inlets and the extent of the pressure drop generated by the distribution system A certain pressure drop has to be built into the distribution system for formation of plug flow A large industrial column requires a higher pressure drop and a greater number of inlets than a small laboratory scale column These two parameters have to be adjusted with the dimension of the column Other important design parameters are the chemical resistance of the wetted material and the hygienic design High chemical resistance allows the use of harsh chemicals during cleaning in place procedures A hygienic design eliminates stagnant zones in the column where cells and cell debris can be trapped Sca
213. use of centrifugation and filtration often results in long process times or the use of comparatively large units causing significant capital expenditure and recurrent costs for equipment maintenance It also results in significant product loss due to product degradation Hence direct adsorption from crude feed stocks potentially offers significant reduction of time and cost compared to traditional processes An alternative to traditional clarification and packed bed chromatography is adsorption to a resin in a stirred tank This technique can be used to advantage when recovering the target substance from a large volume of crude feed In packed bed mode this would require a long sample application time and initial removal of particulate material to prevent clogging of the bed This method has for instance been used for many years on a commercial scale for the isolation of plasma coagulation Factor IX with DEAE Sephadex 1 However the well mixed batch adsorption process is a single stage adsorption procedure that requires more adsorbent to achieve the same degree of adsorption as in a multi stage multi plate process such as packed bed chromatography Hence a multi plate process represents a more efficient use of the resin which reduces the cost of the process Adsorption of the target molecule to an adsorbent in a fluidized bed also eliminates the need for particulate removal Fluidized beds have been used in industry for many years for the reco
214. very of antibiotics including batch processing techniques for recovery of streptomycin 2 and semi continuous systems for novobiocin 3 A method has also been published describing the successful capture of immunomycin from a Streptomyces culture at large scale 4 In the fully fluidized bed channelling turbulence and backmixing is extensive constituting at the most a single equilibrium stage i e showing characteristics very similar to a batch process in a stirred tank The single equilibrium stage in a fluidized bed decreases the efficiency of the adsorption process with low recoveries re cycling needed inefficient washing procedures and increased processing time Several attempts have been made to stabilize fluidized beds to accomplish a multi stage fluidized bed reactor with separation characteristics similar to packed bed chromatography The first approach used segmentation of the bed by insertion of a number of plates with suitably sized holes into the adsorption column 5 In another approach magnetic adsorbent particles and a magnetic field over the fluidized bed column were used to stabilize the bed 6 7 A substantial stabilization of the bed was achieved using magnetic adsorbents but the experiments were carried out at small laboratory scale and scaling up requires complicated and expensive equipment 2 Draeger and Chase 8 were able to create a stable fluidized expanded bed with chromatographic characteristics similar
215. vity assay and reversed phase HPLC No annexin V was found in the flow through or wash Scanning SDS PAGE gels showed an increase in the annexin V content from 9 in the clarified homogenate to 20 in the eluate Table 23 Summary of results from lab scale and pilot scale expanded bed adsorption of recombinant annexin V from an unclarified E coli homogenate STREAMLINE 50 STREAMLINE 200 Volumes L Fermentation 3 4 50 Homogenate 1 7 26 5 Adsorbent 0 3 4 7 Wash 4 5 15 bed vol 71 15 bed vol Eluate 0 6 2 bed vol 10 2 1 bed vol Time min Column set up bed expansion and equilibration 60 60 Feed stock application 17 17 Wash 46 45 Elution 18 19 TOTAL 2 h21 min 2 h21 min Yield gt 95 gt 95 87 Application of homogenate Wash Elution A 280 nm 2 0 1 0 Anticoagulant activity a 0 0 10 20 30 40 50 60 70 80 90 100 Time min Fig 30 Chromatogram from pilot scale expanded bed adsorption of recombinant annexin V from unclarified E coli homogenate using a STREAMLINE 200 column with STREAMLINE DEAE adsorbent Work from Pharmacia Biotech Uppsala Sweden Purification of a therapeutic recombinant protein from unclarified E coli homogenate by expanded bed cation exchange adsorption The use of expanded bed cation exchange adsorption to purify a recombinant protein from Escherichia coli homogenate has been reported by Rh ne Poulenc Rorer GENCELL France 58 The protein which had a mol
216. w during the whole operating cycle Operating pressure is normally below 0 5 bar 50 kPa Evaluation of bed stability Mastering the hydrodynamics of the expanded bed is critical for the performance of an expanded bed adsorption operation The hydrodynamics of a stable expanded bed run under well defined process conditions are characterized by a high degree of reproducibility which allows the use of simple and efficient test principles to verify the stability i e functionality of the expanded bed before the feed is applied to the column The same type of test principles used to verify functionality of a packed chromatography column are used in expanded bed adsorption Visual inspection When working with laboratory and pilot scale columns with glass tubes visual inspection of movements in the expanded bed can be performed before feed application starts The bed is stable when only small circulatory movements of the adsorbent beads are observed Other movements may indicate turbulent flow or channelling which leads to inefficient adsorption Large circular movements of beads in the upper part of the bed usually indicate that the column is not in a vertical position Channelling in the lower part of the bed usually indicates air under the distributor plate or a partially clogged distribution system These visual patterns are illustrated in Fig 8 A stable expanded bed Liquid channels e g Large circular movements with small circu
217. wever not specifically related to expanded bed adsorption and should be addressed when selecting conditions for cell disruption The main concern when processing a feed based on a secretion system would be to maintain intact cells thereby avoiding the release of cell membrane components and intracellular contaminants such as DNA lipids and intracellular proteins that may 22 foul the adsorbent or block the inlet distribution system of the column Release of intracellular proteases is a further concern since it will have a negative impact on the recovery of biologically active material Animal cells lack a cell wall which makes them more sensitive to shearing forces than microbial cells The mammalian cell membrane is composed mainly of proteins and lipids It is particularly rich in lipids composing a central layer covered by protein layers and a thin mucopolysaccharide layer on the outside surface Due to the high membrane content of mammalian cells lysis can complicate the downstream process by causing extensive lipid fouling of the adsorbent Another consequence of cell lysis is the release of large fragments of nucleic acids which can cause a significant increase in the viscosity of the feedstock or disturb the flow due to clogging the column inlet distribution system Nucleic acids may also bind to cells and adsorbent causing aggregation in the expanded bed These types of contamination also lead to problems in traditional processin
218. will also lead to increased fluid phase mixing A short theoretical description of mixing phenomena is presented below Some methods of correct quantification of mixing in liquid fluidized beds are also explained In general the expression axial mixing summarises all possible deviations from a plug flow movement of fluid elements in a chromatographic bed irrespective whether it is fluidized or packed In a more precise picture three different contributions to overall mixing are found as has been pointed out by Roper and Lightfoot for adsorptive membranes 78 Their considerations are also valid in this case Firstly micro scale fluid phase mixing which may be caused by mechanical obstructions to regular flow recirculation eddies etc as well as by diffusion comprises the so called convective dispersion A second contribution is the presence of different flow paths through the adsorbent bed which results in a broad distribution of residence times of fluid elements In the case of liquid fluidized beds channels at the column inlet or zones of preferential passage through the bed are examples of these phenomena The third effect comes from extra column mixing e g in large tubing volumes mixing plates at column inlets or in detectors These effects are very important in short adsorbent beds as the ratio of extra column residence time to the residence time in the bed is unfavourable To get an overall picture of mixing by simple mea
219. y include a number of different unit operations such as cell harvesting product release feed stock clarification concentration and initial purification For highest possible productivity and process economy of the overall process the ultimate goal will be to reach the targets for purity and yield in as few steps as possible and with the simplest possible design Expanded bed adsorption technology with STREAMLINE is specifically designed to address the problems related to the beginning of the downstream sequence and may serve as the ultimate Capture step since it combines clarification concentration and initial purification into one single operation Stages in downstream processing Bioreactor ee ee ae ee ae yen ee eee eee Chromatography Steps Cell Separation Cell Disruption Clarified Culture Cell Debris Medium Removal Product Recovery and Concentration Capture Column Based Separation Intermediate Other Separation Purification Operation Polishing Purified Bulk Drug Substance lt q _ bulsse90 1d wea1sumoq Fig 11 Schematic diagram of a bioproduct recovery process showing the different stages in downstream processing 17 Strategic considerations The overall purpose of the Capture stage is to rapidly isolate the target molecule from critical contaminants remove particulate matter concentrate and transfer to an environment which conserves potency activity Some typical Captur
220. ydraulic liquid the column eluate and a number of critical test points in the column were all free from vegetative organisms STREAMLINE columns with inner diameters of 1000 25 and 600 mm Table 18 lists the technical and operating characteristics of STREAMLINE columns For more information about STREAMLINE CD columns contact your nearest Pharmacia Biotech office 77 Table 18 Technical and operating characteristics of STREAMLINE columns for expanded bed adsorption Property STREAMLINE 25 STREAMLINE 50 STREAMLINE 200 Inner diameter 25 mm 50 mm 200 mm Tube height 1000 mm 1000 mm 950 mm Max height during 2300 mm 2300 mm 2400 mm operation Foot print of column stand Sedimented bed height min max Sedimented adsorbent volume min max Max operating pressure Recommended flow rates expanded mode min expanded mode max sedimented mode elution 400 mm x 400 mm 100 kPa 1 bar 1 L h 200 cm h 2 5 L h 500 cm h 0 25 0 75 L h 50 150 cm h 300 mm x 300 mm 10 cm 30 cm 0 2L 0 6L 100 kPa 1 bar 4 L h 200 cm h 10 L h 500 cm h 1 3 L h 50 150 cm h 600 mm x 600 mm 100 kPa 1 bar 63 L h 200 cm h 157 L h 500 cm h 16 47 L h 50 150 cm h 1 Column with stand and adaptor in uppermost position materials of construction Column materials The materials used in STREAMLINE columns are compatible with chemicals and solvents commonly used to recover proteins
221. ys Table 32 Summary of results from the two step procedure for the purification of the recombinant fusion protein ZZ MS Purification step Volume ZZ M5 DNA Endotoxin Step recovery litres gram pg mg EU mg Fermentation 8 40 s b v 4 4 n d n d STREAMLINE DEAE Wash 6 30 s b v Eluate 0 5 2 5 s b v 4 1 60 000 12 000 93 IgG Sepharose 0 5 4 1 700 10 100 1 5 b v sedimented bed volumes 98 Recovery of recombinant protein A from E coli fermentation broth by expanded bed adsorption on the high capacity anion exchange adsorbent STREAMLINE Q XL Expanded bed adsorption on STREAMLINE Q XL was used to recover a recombinant protein A directly from crude unclarified Escherichia coli fermentation broth Work by Pharmacia Biotech Uppsala Sweden The recombinant protein A was expressed in E coli at an expression level of 0 4 mg ml cell culture suspension It had a molecular weight of 34 kDa and the isolelectric point was 4 5 The cell culture suspension was diluted to a final conductivity of 5 3 mS cm using one part distilled water and one part 10 mM Tris HCl buffer pH 7 4 Benzonase Merck Nycomed Pharma A S 5 pl L and MgCl 11 mM was added and the cell culture suspension was then allowed to stand under gentle stirring for approximately 60 minutes at room temperature before application to the expanded bed Benzonase is an endonuclease added to reduce viscosity by degrading released nucleic acids Mg2 neutra
222. ystem outlet Alternatively install a restrictor in the line between the buffer tank and the system inlet Try to remove the air by pumping buffer at high flow velocity e g 300 500 cm h through the column using downward flow Problem Cause Remedy Low number of theoretical plates RTD test Channelling in the expanded bed due to clogging of the bottom distribution system Channelling in the expanded bed due to the column not being in a vertical position Turbulence in the expanded bed due to fouling aggregation or infection of the adsorbent Instability in the expanded bed due to low flow velocity giving a low degree of expansion Instability in the expanded bed due to unsufficient equilibration Instability in the expanded bed due to the sedimented bed height being too short If the above does not help remove the adsorbent from the column Pump distilled water into the column through the bottom distri bution system and remove any trapped air using suction from above the adaptor net see page 53 Disassemble the column and clean the distributor plate and net see column User Manual Use a spirit level to adjust the vertical position of the column Clean and or sanitize the adsorbent see Cleaning in place page 38 and speci fic cleaning and sanitization recommendations in Section 6 Product Guide Increase flow velocity Nominal flow velocity is around 300 cm

Expanded Bed - Department of Molecular and Cellular Biology

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