Heparin Sepharose 6 Fast Flow
Contents
1. 8 Toclean in place exchange the buffer tank for wash buffer tank containing cleaning solution Packing from below To pack from the bottom carry out the same procedure for the connections and flow path via the bottom nozzle The column is now ready to equilibrate and test Note Itis also possible to use a slightly different packing method where the amount of medium is predetermined In this case the complete amount of medium is packed into the column causing compression of the bed When all medium has entered the column the pump is stopped the top nozzle is retracted the bottom mobile phase valve closed and the medium is allowed to decompress within the column p 9 SLURRY INLET OUTLET C MOBILE PHASE MOBILE PHASE D MOBILE PHASE B BUFFER WASTE SLURRY a WASTE SLURRY BUFFER Packing position The top nozzle is extended part of the way mid position into the column The bottom nozzle is fully retracted Slurry enters the column via the top nozzle and excess liquid exits via the bottom mobile phase outlet After packing the slurry lines are isolated from the mobile phase and can be cleaned independently from the rest of the column Running position The bottom and top nozzles are retracted Mobile phase enters the column directly into an annulus immediately behind the bed support The annulus is cut through at an angle to ensure that linear flow rate is kept2. 8 1000 72 XK 50 20 50 mm i d 8 1000 71 Prepacked columns HiTrap Heparin HP 1 ml 5x1 ml 7 0406 01 HiTrap Heparin HP 5 ml x5 ml 7 0407 01 HiPrep 16 10 Heparin FF 20 ml 7 5189 01 Large scale columns Data File CHROMAFLOW columns 8 1138 92 Data File BPG columns 8 1115 23 Data File BPG 450 columns 8 1060 59 Data File INdEX columns 8 1115 61 Data File Bio Process Stainless Steel columns 8 1121 08 p 20 Reference literature For general advice on lab scale use check the Affinity Chromatography Handbook from GE Healthcare Code no 18 1022 29 For general advice on optimization scaling up and other aspects relating to process chromatography we recommend Handbook of Process Chromatography A Guide to optimization scale up and validation Academic Press pp 188 214 1997 Sofer G and Hagel L p 21 p 22 www gehealthcare com protein purification www gehealthcare com GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Drop Design INdEX Sepharose HiTrap HiPrep BioProcess CHROMAFLOW Tricorn and BPG are trademarks
3. constant during distribution of the mobile phase across the bed Unpacking position In this position both bottom and top nozzles are fully extended into the column thereby exposing a third passage through which medium leaves the column Cleaning solution can be pumped through the nozzles and sprayed into the column In this way the column is easily and effectively cleaned without exposing the interior or the medium to the environment or without dismantling the column Figure 3 Principle of operation CHROMAFLOW columns p 10 Pressure packing BPG Columns BPG Columns are supplied with a movable adaptor They are packed by conventional pressure packing by pumping the packing solution through the chromatographic bed at a constant flow rate or back pressure 1 Pour some water or packing buffer into the column Make sure that there is no air trapped under the bottom bed support Leave about 2 cm of liquid in the column 2 Mix the packing buffer with the medium to form a 50 70 slurry sedimented bed volume slurry volume 0 5 0 7 Pour the slurry into the column Insert the adaptor and lower it to the surface of the slurry making sure no air is trapped under the adaptor Secure the adaptor in place 3 Seal the adaptor O ring and lower the adaptor a little into the slurry enough to fill the adaptor inlet with packing solution 4 Connect a pump anda pressure meter and start packing at the predetermined p
4. flush the hydraulic chamber E free of air and any residual medium a s Close C and open the elution inlet outlet B to allow trapped air in the adaptor bed support to escape DO Close B and open the elution inlet outlet D to start the packing applying a predefined constant hydraulic packing pressure When packing Sepharose 6 Fast Flow based media in an INdEX column to a bed p 13 so 10 1 m 12 di height of 15 cm the recommended hydraulic packing pressure is 1 5 bar for INdEX 100 and 0 8 bar for INdEX 200 When the adaptor has reached the surface of the settled bed continue to run the pump until the adaptor has been lowered 5 mm into the packed bed Close A and D and stop the pump Run the column with upward flow for a few minutes to remove residual air trapped in the adaptor The column is now ready for use To unpack the column connect the outlet from the pump to B and open C while keeping D closed This will cause the adaptor to rise from the bed surface B cq p Figure 4 Schematic representation of INdEX column with a 4 port 2 way valve mounted at the bottom outlet Evaluation of packing To check the quality of the packing and to monitor this during the working life of the column column efficiency should be tested directly after packing prior to re use and when separation performance i
5. starting material but one CIP cycle is generally recommended every 5 separation cycles Sanitization For inactivation of microbial contaminants equilibrate the column with buffer consisting of 0 1 M NaOH and 20 ethanol and allow to stand for 1 h Note Alternatively equilibrate with 70 ethanol and allow to stand for 12 h if working in an explosion proof environment Wash the column thoroughly with running buffer after sanitization Sterilization Autoclaving is the only recommended sterilization treatment Equilibrate the medium with 0 5 M NaCl pH 7 Dismantle the column and autoclave the medium at 120 C for 30 minutes Sterilize the column parts according to the instructions in the column manual Re assemble the column then pack and test it as recommended Storage Store Heparin Sepharose 6 Fast Flow at 4 to 30 C in 0 05 M sodium acetate containing 20 ethanol as preservative p 19 6 Ordering information Product Pack size Code No Heparin Sepharose 6 Fast Flow 50 ml 7 0998 01 Heparin Sepharose 6 Fast Flow 250 ml 7 0998 25 Heparin Sepharose 6 Fast Flow 1L 7 0998 03 Heparin Sepharose 6 Fast Flow 5L 7 0998 04 Related products Lab scale columns ricorn 5 20 column 5 mm i d 8 1163 08 ricorn 5 50 column 5 mm i d 8 1163 09 ricorn 10 20 column 10 mm i d 8 1163 13 ricorn 10 50 column 10 mm i d 8 1163 14 ricorn 10 100 column 10 mm i d 8 1163 15 XK 16 20 16 mm i d 8 8773 01 XK 26 20 26 mm i d
6. GE Healthcare Instructions 71 7190 00 AE Affinity media Heparin Sepharose 6 Fast Flow Heparin is a naturally occurring glycosaminoglycan which serves as an effective affinity binding and ion exchange ligand for a wide range of biomolecules including coagulation factors and other plasma proteins lipoproteins protein synthesis factors enzymes that act on nucleic acids and steroid receptors By coupling heparin to Sepharose 6 Fast Flow with a chemically optimized linkage GE Healthcare provides an excellent medium for both laboratory and process scale affinity purification 0 Sepharose Contents 1 Medium characteristics 2 Packing columns 3 Evaluation of packing 4 Operation 5 Maintenance 6 Ordering information p 2 14 17 18 20 1 Medium characteristics Heparin is a glycosaminoglycan consisting of alternating hexuronic D glucuronic or L iduronic and D glucosamine residues The polymer is heavily sulphated carrying sulphamino N sulphate groups at C 2 of the glucosamine units as well as ester sulphate O sulphate groups in various positions Figure 1 The heparin ligand used in Heparin Sepharose 6 Fast Flow is isolated from porcine intestinal mucosa and has a molecular weight distribution over the range 5 000 30 000 The base matrix Sepharose 6 Fast Flow consists of highly cross linked 6 agarose beads It provides the medium with high physical stability and excellent flow characteristics Heparin is l
7. acking flow rate or pressure Keep the flow rate or pressure constant during packing and check the pressure at the column inlet Never exceed the pressure limit for column or medium 5 When the bed has stabilized close the bottom valve and stop the pump The bed starts rising in the column Loosen the O ring and lower the adaptor to 0 5 1 0 cm above the bed surface 6 Seal the O ring start the pump and continue packing Repeat steps 5 and 6 until there is a maximum of 1 cm between bed surface and adaptor when the bed has stabilized Mark the bed height on the column tube 7 Close the bottom valve stop the pump disconnect the column inlet and without loosening the adaptor O ring push the adaptor down to approximately 3 mm below the mark on the column tube The packing solution will flush the adaptor inlet Remove any trapped air by pumping liquid from the bottom after the inlet tubing and the bottom valve have been properly filled p 11 Suction packing BPSS Columns BioProcess Stainless Steel Columns are supplied with fixed end pieces They are packed by suction i e by sucking packing solution through the chroma tographic bed at a constant flow rate 1 Fit a packing device on top of the column tube 2 Pour some water or packing buffer into the column Make sure that there is no air trapped under the bottom bed support Leave about 2 3 cm of liquid in the column 3 Mix the packing buffer with the medium to fo
8. aphy media e g for purification of coagulation factors or nucleic binding proteins Different substances may differ in their affinity for Heparin Sepharose 6 Fast Flow The binding capacity of a particular protein will depend upon parameters such as buffer composition pH flow rate and temperature Heparin Sepharose 6 Fast Flow might also function as a cation exchanger due to the negatively charged sulphate and carboxylate groups on the immobilized heparin ligand Acommonly used binding buffer for the purification of plasma proteins is 10 20 mM sodium citrate buffer pH 7 4 Since the heparin ligand acts as an affinity ligand in these cases it may be advisable to include low concentration of NaCl in order to eliminate unspecific ionic interactions In other applications 10 mN sodium phospate pH 7 0 or 20 mM Tris HCl pH 8 0 are often recommended as binding buffers Elution Elution is commonly performed by increasing the ionic strength of the buffer Elution using a continuous linear gradient or step gradient with NaCl KCI or NH SO up to 1 5 2 M is most frequently used p 17 5 Maintenance For best performance from Heparin Sepharose 6 Fast Flow over a long working life follow the procedures described below Cleaning In Place Cleaning in place CIP is a cleaning procedure that removes contaminants that may remain in the packed column after regeneration Regular CIP also prevents the build up of these contaminants in the med
9. ere L Bed height cm N number of theoretical plates V Peak elution distance W Peak width at half peak height V and W are in the same units p 15 To facilitate comparison of column performance the concept of reduced plate height is often used The reduced plate height is calculated HETP d where d is the diameter of the bead As a guideline a value of lt 3 is normally acceptable The peak should be symmetrical and the asymmetry factor as close as pos sible to 1 values between 0 8 1 5 are usually acceptable A change in the shape of the peak is usually the first indication of bed deterioration due to use Peak asymmetry factor calculation A b a where a 1st half peak width at 10 of peak height b 2nd half peak width at 10 of peak height Figure 5 shows a UV trace for acetone in a typical test chromatogram in which the HETP and A values are uv Column BPG 300 absorption Medium Sepharose 6 Fast Flow 4 Bed height 57 5 cm Bed volume 40 6 litres Eluent Distilled water Sample 1 05 litres 1 v v acetone Flow velocity 19 cm h V 18 7 W 0 9 HEPT 0 024 cm a 0 90 b 0 85 A 0 94 gt Volume Figure 5 UV trace for acetone in a typical test chromatogram showing the HETP and A value calculations p 16 4 Operation Binding Immobilized heparin has two main modes of interaction with proteins Heparin Sepharose 6 Fast Flow can be used as an affinity chromatogr
10. eservoir and fit the adaptor to the column LO With the adaptor inlet disconnected push down the adaptor approximately 2 mm into the bed allowing the packing solution to flush the adaptor inlet 1 C3 Connect the pump open the bottom outlet and continue packing The bed will be further compressed at this point and a space will be formed between the bed surface and the adaptor Close the bottom outlet Disconnect the column inlet and lower the adaptor approximately 2 mm into the bed Connect the pump The column is now ready to use 1 pa Packing large scale columns General packing recommendations Columns can be packed in different ways depending on the type of column and equipment used Always read and follow the relevant column instruction manual carefully p 6 Sepharose 6 Fast Flow based media are easy to pack since their rigidity allows the use of high flow rates see Figure 2 Four suitable types of packing methods are given e CHROMAFLOW packing e Pressure packing for columns with adaptors e Suction packing for large columns with fixed bed heights e Hydraulic pressure packing Linear flow rate Jon 700 600 500 4 400 300 4 200 100 Figure 2 Pressure flow rate curve for Sepharose 6 Fast Flow medium How well the column is packed will have a major effect on the result of the separation It is therefore very important to pack and test the column accor ding to the f
11. for bed volumes up to 0 55 ml at bed heights up o 2 8 cm e Tricorn 5 50 5 mm i d for bed volumes up to 1 14 ml at bed heights up o 5 8 cm e Tricorn 10 20 10 mm i d for bed volumes up to 2 2 ml at bed heights up o 2 8 cm e Tricorn 10 50 10 mm i d for bed volumes up to 4 56 ml at bed heights up o 5 8 cm p 4 Tricorn 10 100 10 mm i d for bed volumes up to 8 48 ml at bed heights up to 10 8 cm XK 16 20 16 mm i d for bed volumes up to 30 ml at bed heights up to 5cm XK 26 20 26 mm i d for bed volumes up to 80 ml at bed heights up to 5cm XK 50 20 50 mm i d for bed volumes up to 275 ml at bed heights up to 15cm Large scale columns BPG variable bed glass columns Inner diameters from 100 450 mm bed volumes from 2 4 131 litres bed height max 83 cm BioProcess Stainless Steel BPSS fixed bed columns Inner diameters from 400 1400 mm bed volumes from 12 1500 litres bed height 10 100 cm INdEX variable bed columns Inner diameters from 70 200 mm bed volumes up to 24 8 litres bed heights of max 79 cm CHROMAFLOW variable bed columns Inner diameters from 280 2000 mm Packing lab scale columns 1 2 Ww P Assemble the column and packing reservoir if necessary Remove air from the column dead spaces by flushing the end piece and adaptor with packing buffer Make sure no air has been trapped under the column bed support Close the column outlet leaving the bed suppor
12. ia bed and helps to maintain the capacity flow properties and general performance of Heparin Sepharose 6 Fast Flow Heparin Sepharose 6 Fast Flow withstands exposure to 0 1 M NaOH for long periods with no significant loss of binding capacity for antithrombin IIl When contamination is severe 0 5 M NaOH can be used however a decrease in functionality will be seen over time Figure 6 Other reagents in which the medium is stable include 8 M urea and 6 M guanidine hydrochloride 0 1 M NaOH m 0 5 MNaOH o 0 01 M HCI Binding capacity mg AT Wm drained gel T 1 o 50 100 150 Contact time hours Figure 6 Functional stability of Heparin Sepharose Fast Flow was tested in three different CIP studies CIP with 0 5 M NaOH can be used effectively over shorter periods p 18 Recommended cleaning in place procedures are summarized in Table 2 Always wash the column thoroughly with equilibration buffer after cleaning in place Table 2 CIP protocol For removal of time Wash with Column volumes Contact lonically bound proteins 2 M NaCl 0 5 10 15 min Precipitated or 0 1 M NaOH 4 1 2h denatured proteins or 6 M guanidine HCI approx 2 30 min 1h or 8 M urea approx 2 30 min 1h Hydrophobically 0 1 0 5 non ionic 4 1 2h bound proteins detergent A specific CIP protocol should be designed for each process according to the type of contaminants present The frequency of CIP depends of the nature and the condition of the
13. inked to the Sepharose matrix by reductive amination which is a very stable binding even in alkaline conditions The stability of this medium is limited only by the heparin itself The essential characteristics of Heparin Sepharose 6 Fast Flow are summarized in Table 1 Al B Figure 1 Heparin consists of alternating hexuronic acid A and D glucosamine residues B The hexuronic acid can be either D glucuronic acid top or its C 5 epimer L iduronic acid Ra H or SO Bes SO or COCH p 3 Table 1 Characteristics of Heparin Sepharose 6 Fast Flow Ligand density Average particle size Bead structure Flow rate Recommended pH working and long term short term cleaning in place Chemical stability 1 week 40 C 1 week 20 C Autoclavable Delivery and storage conditions approx 4 mg heparin ml drained medium 90 um 45 165 pm 6 highly cross linked spherical agarose 2300 cm h at 100 kPa XK 50 60 column bed height 25 cm eluent distilled water 20 C 4 12 4 13 0 01 M NaOH 0 05 M sodium acetate pH 4 0 4M NaCl 8 M urea 6 M guanidine hydrochloride 0 1 M NaOH 121 C for 20 minutes in distilled water Supplied in 0 05 M sodium acetate containing 20 ethanol 2 Packing columns Heparin Sepharose 6 Fast Flow is supplied pre swollen Decant the 20 ethanol solution and replace it with binding buffer before use Recommended columns Lab scale columns Tricorn 5 20 5 mm i d
14. is gradually built up The hydraulic pressure can be generated using a pump and a pressure relief valve When the adaptor reaches the surface of the settled medium it continues downwards under hydraulic pressure compressing the medium The extent to which the medium is compressed depends upon the pressure from the adaptor and the elasticity of the medium The quantity of medium required when packing Sepharose 6 Fast Flow based media by hydraulic pressure is approximately 1 2 litre sedimented medium per litre packed bed 1 Pour some water or packing solution into the column Make sure that there is no air trapped under the bottom bed support Leave about 2 cm of liquid in the column N Pour the 75 slurry into the column Fill the column with packing solution up to the top of the glass tube and mix the slurry Allow the medium to sediment to just below the bevel of the glass tube G see Figure 4 Gi Put the adaptor in a resting position against the bevel of the glass tube Avoid trapping air bubbles under the adaptor by slightly tilting the adaptor while mounting Lower the lid and secure it in place wn Connect a pump to the inlet of the hydraulic chamber A with a manometer and a pressure reliefe valve in line between the pump and the hydraulic chamber The manometer should be placed after the valve in the direction of the flow Open the hydraulic inlet A and the hydraulic outlet C Start the pump and
15. ng flow rate pressure is 70 100 of the maximum flow rate pressure The operational flow rate pressure should be lt 70 of the packing flow rate pressure Note For BPSS columns first pack the column by suction packing at a low flow rate Then determine the flow pressure characteristics as above by pumping buffer downwards through the column Packing CHROMAFLOW columns Procedure 1 Prepare the column for packing as described in the User Manual Packing from the top 1 Set the top nozzle to the pack position mid position 2 Fully retract the bottom nozzle run position D 3 Ensure that the top mobile phase is closed 4 Open the bottom mobile phase 5 Open Inlet C and start the packing pump Adjust the flow to achieve the required packing conditions for the selected medium Monitor column pressure and the outlet flow rate in order to record column packing parameters Remember to stir the medium slurry during packing to prevent it from settling 6 Continue pumping until the column is fully packed and the pump stalls due to build up of medium in its pipelines Turn off the packing pump 7 Fully retract the top nozzle to its run position Close Outlet C Open Inlet B from the water buffer tank and open Outlet D The pump should now be restarted to rinse the top slurry lines If the nozzle is full of liquid when in the packing position make sure that the waste slurry outlet is open before retracting the nozzle
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17. ollowing recommendations Begin the packing procedure by determining the optimal packing flow rate Guidelines are given below for determining the optimal packing flow rates for columns with adaptors and fixed bed heights Determining optimal packing flow rate The optimal packing flow rate is dependent on column size and type bed height packing solution and temperature The optimal packing flow rate must therefore be determined empirically for each individual system p 7 To determine the optimal packing flow rate proceed as follows 1 Calculate the exact amount of medium needed for the slurry this is especially important for columns with fixed bed heights The quantity of medium required per litre packed bed is approximately 1 15 litre sedimented medium 2 Prepare the column exactly as for column packing 3 Begin running the column at a low flow rate e g 30 of the expected max flow rate and record the flow rate and back pressure when the bed is packed and the pressure has stabilized 4 Increase the flow rate in small steps and record the flow rate and pressure at each step after the pressure has stabilized 5 Continue recording flow and pressure until the maximum flow rate has been reached i e when the flow rate levels off at a plateau indicating bed compression or when the pressure reaches the pressure specification of the column used 6 Plot pressure against flow rate as indicated in Figure 3 The optimal packi
18. rm a 50 slurry sedimented bed volume slurry 0 5 Pour the slurry into the column Stir gently to make sure it is homogeneous 4 Connect the column outlet valve to the suction side of a pump and start packing the bed by suction through the bed at the predetermined flow rate Keep the flow rate constant during packing 5 When the bed has stabilized the top of the bed should be just below the junction between the column and the packing device If when stabilized the level of the bed is incorrect add or remove slurry Always stir the slurry thoroughly before packing 6 Just before the last of the solution has entered the packed bed before the surface starts to dry close the valve at the column outlet stop the pump quickly remove the packing device and replace it with the lid Note This final operation should be completed as quickly as possible because the bed will expand when the flow stops 7 Start pumping buffer with upward flow through the column to remove any air bubbles trapped under the lid Hydraulic packing INdEX Columns INdEX Columns are supplied with a hydraulic function which allows an extremely simple rapid and reproducible packing procedure The medium is packed at the same time as the adaptor is lowered into position at the correct pressure p 12 The adaptor is pushed down by a constant hydraulic pressure forcing water or packing buffer through the slurry and compressing it so that a packed bed
19. s seen to deteriorate The best method of expressing the efficiency of a packed column is in terms of the height equivalent to a theoretical plate HETP and the asymmetry p 14 factor As These values are easily determined by applying a sample such as 1 v v acetone solution to the column Sodium chloride can also be used as a test substance Use a concentration of 2 0 M NaCl in water with 0 5 M NaCl in water as eluent The calculated plate number will vary depending on the test conditions and it should therefore be used as a reference value only It is also important that conditions and equipment are kept constant so that results are comparable Changes in solute solvent eluent sample volume flow rate liquid pathway temperature etc will influence the results For optimal results the sample volume should be at max 2 5 of the column volume and the flow velocity between 15 and 30 cm h If an acceptance limit is defined in relation to column performance the column plate number can be used as part of the acceptance criteria for column use Method for measuring HETP and A To avoid dilution of the sample apply it as close to the column inlet as possible Conditions Sample volume 2 5 of the bed volume Sample conc 1 0 v v acetone Flow velocity 15 cm h UV 280 nm 1 cm 0 1 AU Calculate HETP and As from the UV curve or conductivity curve if NaCl is used as sample as follows HETP L N N 5 54 V W 2 wh
20. t covered with packing buffer Resuspend medium stored in its container by shaking avoid stirring the sedimented medium Mix the packing buffer with the medium to form 50 70 slurry sedimented bed volume slurry volume 0 5 0 7 Pour the slurry into the column in a single continuous motion Pouring the slurry down a glass rod held against the column wall will minimize the introduction of air bubbles p 5 5 If using a packing reservoir immediately fill the remainder of the column and reservoir with packing buffer Mount the adaptor or lid of the packing reservoir and connect the column to a pump Avoid trapping air bubbles under the adaptor or in the inlet tubing a Open the bottom outlet of the column and set the pump to run at the desired flow rate Ideally Sepharose 6 Fast Flow based media are packed at a constant pressure of approximately 1 5 bar 0 15 MPa If the packing equipment does not include a pressure gauge use a packing flow velocity of approximately 500 cm h 10 cm bed height 25 C low viscosity buffer If the recommended pressure or flow rate cannot be obtained use the maximum flow rate the pump can deliver This should also give a reasonably well packed bed Note Do not exceed 75 of the packing flow velocity in subsequent chromatographic procedures using the same pump be When the bed has stabilized close the bottom outlet and stop the pump D If using a packing reservoir disconnect the r
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