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Multi-6™ User Manual - Biovest Hollow Fiber
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1. Page 66 of 68 Mult 6 User Manual 700466 000 Rev B Blovest Intematior Multi 6 Producin Metabolic Data Record Production Batch Cell tine Product Teaio e ee EE T heda aeda Day lores pose OK ERE EUR EE voie volume Harvest Proa Comments and N Date Time pat fareto marat matat marhe manr iage agoes volume cone Pump Speeds Page of Mult 6 User Manual Page 67 of 69 700486 000 Rev B Blovest international Multi 6 Producin Metabolic Data Record Production Batch Cell tine Product Teaio eee EE T Day rete ease FE JE UR PR Voume oine rarest rod Comments and N Date Time pat fareto marat matat marhe manr Aguas agoes volume cone Pump Speeds Page of Page 68 of 68 Mult 6 User Manual 700866 000 Rev B Blovest international Biovest is the leader in large scale perfusion technology We provide a range of hollow fiber systems for a variety of production needs CRS EAA E mel ere 61 5 55 525 24 58 5 gt 55 24 B7H 2 916 GHB PETE Mult 6 User Manual Page 69 of 69 700466 000 Rev B
2. Changing Media and Outflow containers must be done aseptically and several times during the run Properly configuring these containers will enable their convenient and aseptic usage When using media bags of carboys we suggest they include at least 24 61 em of tubing to simplify their aseptic usage Most media suppliers offer media bags with a pre defined length of tubing that often is long to meet this need When using carboys the user is responsible for including this length of tubing on the ported cap assembly Having length of tubing connected to these containers allows their er connections to reach intoa laminar flow hood for aseptic connection and disconnection to the Mult 5 This length of tubing allows carboys and media bags to remain outside ofthe laminar low hood easing their use and minimizing items in the hood Experienced technicians develop the ability to quickly and effectively connect and change Media and Outflow containers outside ofa laminar flow hood Inexperienced technicians should perform these tasks in a hood Important Points See the Important Points of Moving the Mult 6 into the laminar flow hood Whenever using a laminar flow hood while connecting or changing Media and Outflow containers Minimize the amount of time the circulation pump is not running Like one s heart the circulation pump maintains oxygenation and pH control Minimize the amount of time the Multis out of the
3. Important Points At the start of the run use a small volume 1 5 L of basal medium in the Media Container Do not fill the container even if it can hold more media Circulating too large a volume can cause undesirable media dilution effects that may cause slow no growth after inoculation of the cells As the culture expands and the basal medium needs to be changed replace the container with new containers that have increasing volumes of basal medium see Figure 22 After the first several changes of the Media Container subsequent new containers will be connected at their full volume to supply enough nutrient Note the indicated days and the depicted volumes in Figure 22 are meant to be illustrative and only a guideline After Day 2 4 Day 3 5 Day 4 7 Day 7 and on Fill Flush Add Media Add Media Add Media Change Media o Day 1 2 Figure 22 Page 50 of 69 Muit 6 User Manual 700466 000 Rev B Blovest international Media Rate The Media Pump provides a slow flow rate of fresh basal medium into the IC to primarily control nutrients such as glucose glutamine and other small metabolites The Media Pump has a secondary affect on pH control but pH control is primarily controlled by other means The Media Pump has no affect on oxygenation which is controlled by the Circulation Pump Unlike the three other pumps whose speeds require litle management the speed of the Media Pump should be closely managed to e
4. nutrients and wastes The pores havea range of molecular weight cut off mwco from approximately 10 39 KDa These pores allow only small molecules to freely move across the membrane Figure 3 Mult 6 User Manual Page 7of 69 700466 000 Rev B Blovest international Bioreactor Fluid Dynamics Two critical functions for cell growth and production are the supply of fresh nutrients and the removal of waste products This essential exchange occurs across the hollow fiber membrane within the bioreactor see Figure 4 The bioreactor contains thousands of hollow fibers which in a simple sense function as a single membrane The membrane has pores with a mwco range from approximately 10 39 KDa This mweo allows the exchange diffusion of basal media nutrients and Os and metabolic wastes and COs This muco range does not allow passage across the fiber membrane of the cells and most added growth supplements and secreted proteins The benefits of the fibers semi permeabilty ae Secreted product is concentrated and isnot diluted regardless of the volume of feed media used The large volume of feed media consumed during the run is product free and discarded as waste Therefore supernatant volumes for purification remain very small Nutrient concentrations can be manually controlled to either stimulate growth of the culture or secretion of product The six bioreactors allow up to six cell lines to be cultured si
5. Gas Exchange Cartridge act as the lungs to create respiration for the culture and provide oxygenation and pH control As cells secrete protein it is concentrated within a fluid circuit EC that is separate from the feed media and metabolic waste IO Concentrated product is harvested and fresh growth supplements ve edded insmall volumes using oying PATET surrounding With these general concepts in mind the detailed information on the following pages provides a better understanding of how and why the Mult works fibers to separate ICS from ECS Hollow Fiber Technology at each end The core of all hollow fiber based mammalian cell culture cross section systems is the hollow fiber bioreactor The bioreactor is a clear plastic cylindrical housing containing several thousand hollow fibers that are attached at each end of the cylinder see Figure 2 Hollow fibers contained within a cylinder create two separate fluid volumes within the bioreactor The combined volumes within the fibers is called the Intracapllary Space or ICS The volume surrounding the fibers is called the Extracapillary Space or ECS 8 de im S Figure 2 The ICS and ECS are connected to one another only via the small pores within the hollow fiber membrane which are visible in Figure 3 The fibers provide a substrate upon and between which the cells grow The permeability of the fiber membrane permits the exchange of
6. Innovation Leader since 1981 Blovest international Copyright Notice 2011 Biovest International Ine All rights reserved No part of this document may be reproduced in any form without the prior written consent of Biovest Intemational Inc Biovest International Inc makes no warranties with respect to this documentation and disclaims any implied warranties of merchantability and fitness for a particular purpose Information in this document is subject to change without notice Biovest International Inc assumes no responsibility for any errors that may appear in this document Trademark Acknowledgements Mult 6 is a trademark of Biovest International Inc Masterflex L S and Easy Load are registered trademarks of Cole Parmer Instrument Company Luer and Luer Lok ate trademarks of Becton Dickinson and Co Tygon is a registered trademark of Norton Performance Plastics PharMed is a registered trademark of Saint Gobain BD Cell is a trademark of Becton Dickinson Inc CLEARLINK is a trademark of Baxter International Inc GlucCell is a trademark of Cesco Bioengineering Co Ltd YSI 2700 Select is a trademark of YSI Ine Document Number 700466 000 WARNING Operating the Multi6 requires the use of pump motors and pump heads It is important to refer to the user manuals provided by the manufacturer of the pump motor and pump head to ensure they are used in a safe and proper manner CAUTION Wherever a cautio
7. TO Stainless steel mounting hardware for bwo L S Easy Load Il pump heads part number EW 77200 02 Need one gt Outflow Tubing Circulation and Gassing Pump System The setup pictured in Figure 8 of one motor driving two pump heads is preferable to drive gassing flow through the GEX and circulate medium through the Intracapillary Circuit However other setups will work The suggested and pictured items to purchase are a Masterflex Ley Circulation Tubing Pump driveand wo Makete pump heads I Masterfte L S digital drive 90 260 VAC gt Gassing Tubing part number EW47523 90 Need one T9 Mastrtex L S Rasy Load It Figure 8 pump head for precision tubing fixed occlusion stainless steel rotor part number EW 77200 60 Need one 9 Masterftex L S Fasy Load II pump head for high performance precision tubing fixed occlusion stainless steel rotor part number EW 77200 42 Need one Stainless steel mounting hardware for two L S Easy Load Il pump heads part number EW 77200 02 Need one Suggestion The four pump heads appear to be similar but are not It may be helpful to label the EW 77200 62 pump head Circulation Pump to reduce the chance of using the pump heads incorrectly The remaining three identical EW 77200 60 pump heads can be labeled Media Pump Outflow Pump and Gassing Pump Subsequent references to these pump heads will use this terminology See the Specifications section for further infor
8. B Blovest international Placing Multi 6 into the CO incubator The Multi6 operates within the incubator forthe Additionally the incubator s air CO gas mixture same reasons as flask cultures The incubator allows pH control and provides oxygenation of the provides the Multi6 with temperature contral cell culture medium Procedure 1D Position the Nut 6 inside the incubator Go to this section fr space requirements T The IC Sample line can remain inside the incubator 1D Route the pump tubing out of the incubator and secure it to prevent it from being pinched when incubator door is closed 1D Place the Media Motor and Circulation Motor as close to the incubator as possible O Load the tubing into the Media Motor See how to load the Media Motor for details O Load the tubing into the Circulation Motor See how to load the Ci 1D Open the Media and Outflow clamps 9 Open all ventilation filter clamps if present on the media and outflow containers ulation Motor for details O When the pumps are tumed on confirm the fluids and gas are pumped in the correct directions 1D The Media and Outflow pumps should rotate at the same rpm The Circulation and Gassing pumps should rotate at the same rpm Although each pair of pump heads will turn at the same Tate as one another the larger tubing will have a higher flow rate which is necessary 9 Set the Media and Circulation motors to the desired speeds See Media Ra
9. Media Reservoir traps the supplements within the IC so they will not reach the cell culture Because itis unnecessary to use growth supplements or expensive serum free media for the large volume of IC media consumed during the run Multi6 greatly reduces overall media costs versus other ir vitro cell culture methods Note some applications use protein free medium which means the IC and EC media are identical Because there are no large molecular weight additives in protein free media secreted protein is the only protein present in the bioreactor Harvest Port This location is primarily used to harvest supernatant and at the beginning of the run to inject the inoculum It also may be used to periodically collect small volumes for product analysis Mult 6 User Manual Page 13 of 69 700466 000 Rev B Blovest international Equipment and Supplies Req red Equipment Media and Outflow Pump System The setup pictured in Figure 7 of one motor os driving two pump heads is preferable to deliver media and collect outflow but other motors will Media Tubin work see Quick Start Guide The suggested and 9 pictured items to purchase are a Masterflex pump drive and two identical Masterlex pump heads O Mastertex L S digital drive 90 260 VAC part number EW 07523 80 Need one TO Mastertlex L S Easy Load II pump head for Figure 7 precision tubing fixed occlusion stainless steel rotor Part number EW 77200 60 Need two
10. Reservoir T Tum on the Media Motor for continuous mode operation to rotate counterclockwise at a rate of 1000 mL hour for size 14 pump tubing 76 rpm Confirm media is being pumped out of the Media Container and toward the Multi 6 If you se air bubbles rising in the Media Container stop the Media Motor Either the media tubing is loaded in the wrong direction in the Media Pump or the Media Motors rotation is reversed Fx the problem then restart the Media Motor T Allow the Media Motor to operate until the level of medium in the Media Reservoir is approximately half full TOT aea disinfection caution about how to do this safely Tighten the Media Reservoir 1 When the Media Reservoir is half full medium should automatically begin to be pumped out of the reservoir via the Outflow Pump Confirm media exits the reservoir via the top mounted Outflow tubing line see Figure 16 medium does not exit the Media Reservoir via the Outflow Pump when the reservoir is approximately half full either the outflow tubing is loaded in the wrong direction in the Outflow Pamp the Outflow Pump is not closed or the pump s rotation is reversed Stop the Media Motor i the problem then restart the Media Motor T Once Outflow medium has begun tobe pumped from the Media Reservoir turn on the Circulation Motor for continuous mode operation to rotate counterclockwise ata rate of 10 ml min for size 36 pump tubing 21 rpm Confirm medium is b
11. a higher than oxygenation and pH control only It does not affect necessary circulation rate is not detrimental metabolite concentrations which are controlled by However too slow a circulation rate can cause the Media Pump rate While oxygenation and pH anoxia and be dramatically detrimental are important for a healthy culture it is easy to determine the necessary circulation rate to use The circulation rate is quite fast mL min while the media and outflow rates both mL hour are slow Suggested circulation rates to use during the run are provided in the following table However if DO is being monitored these data can be used to adjust the circulation rate Maintain the DO concentration Because the cell culture is on the opposite side of from an IC Sample above 130 mmHg O gt the hollow fiber membrane from the circulating Setting the Circulation Motor Speed The suggested Masterflex motor directly displays the pump rate in mL min but the correct pump head size must be selected to achieve the displayed pump rate The Circulation Motor always will be sot for a size 36 pump segment Even though the Gassing Pump is also driven by the Circulation motor do not select its size 17 pump segment on the motor s keypad Ifyou are not using the suggested Masterflex motor as the Circulation Motor and it has a simple theostat surrounded by a dial with numbers to indicate relative speeds it does not directly display the pum
12. allow the aseptic exchange fair 1D Separately place the bottle or carboy ported cap assembly cap tubing fittings and ventilation filter in autoclavable packaging Autoclave the item using standard dry goods cycle parameters T After the bottle or carboy and ported cap assembly are sterile bring them to the laminar flow hood where the Mult 6 was prepared Disinfect the ouside of their packaging before placing them into the laminar flow hood 1 Remove the standard cap from the Media Container and aseptically fil it with sterile basal medium prepared separately per manufacturer s instructions 1 Remove the ported cap assembly from the sterile packaging Ty Install the ported cap assembly on to the bottle or carboy It can be difficult to aseptically guide the ported cap assembly s long sipper tubing into the bottle or carboy It is a good idea to practice this and develop a method before needing to do it aseptically for an actual Multi 6 production 9 Tighten the ported cap assembly on to the bottle or carboy When using pre filled media bags for the Fill amp Flush procedure review Figure 18 9 Place the media bag on a cart in front of the laminar flaw hood 9 Disinfect the media bag s tubing line that terminates with preferably a male luer fitting the one closed with a female luer plug Do not yet remove the small bag that typically is covering the Iuer fitting 1 Extend the disinfected tubing line into the laminar flow hood
13. and filling the bioreactor The growing culture frequently affects pH and needs ever greater amounts of fresh media ete Sampling frequency during the growth phase is often performed every one to two days During the production phase the cell number is relatively static which leads to a fairly stable pH and media consumption rate Therefore during the production phase sampling frequency can be lower than during the growth phase When the same cell line is cultured repeatedly sample data and experience from these runs can be used to create a general production strategy This strategy can then be repeated with reduced sampling requirements throughout the run IC samples are used to monitor the culture s pH and metabolic activity in order to know when to change the Media Rate EC samples are used to monitor the product secretion rate in order to optimize the harvesting frequency and volume removed Important Points Monitoring pH and metabolites is optional If the basal cell culture medium contains phenol red visually determining pH may be sufficient but perhaps not ideal Glucose can be measured using a ghucometer See the section Basal Media Changes for further information or contact Biovest s Account Services for further information If run to run consistency or optimal production yield is desired monitoring pH and metabolites should be performed Monitoring dissolved oxygen concentration is also optional and rare
14. assembly and several fest of the connected tubing Place the bottle inside the hood Disinfoct the replacement bottle and place it inside the hood See Connecting amp Changing Media and Outflow Containers for further instructions Option an experienced technician can change containers while the Multi remains in the COs incubator but more risk is involved To change the Media or Outflow container when using carboys or media bags leave the Mul ge 3 s outside the hood on the cart Locate the male female luer junction that connects the Muti to the relevant Media or Outflow container Disinfect the male female luer junction and tiwo to three feet of tubing on each side ofthe junction Extend the sterile tubing segments and luer junction into the hood Disinfect the tubing and fittings of the replacement carboy or media bag and extend them into the hood See Connecting amp Changing Media and Outflow Containers for further instructions Option an experienced technician can change containers while the Mult remains in the COs incubator but more risk is involved O Return to the instructions that commonly require removing the Multi from the COs incubator Inoculation Change Media and Outflow containers Initial Factor Addition Harvesting Supernatant and Adding Factor Page 36 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Connecting amp Changing Media amp Outflow Containers
15. cell culture The Media Pump and Outflow Pump can be off for short periods of time without affecting the culture While in growth or production phases Disinfect the Multi6 and miscellaneous supplies that enter the laminar flaw hood and be very careful about aseptic handling while working in the hood The Muti 6 is durable and can operate for months so preventing contamination allows it to be used for a long time ringes are used to remove medium from the IC Sample port The number and type of assays that will be performed determine the size of syringe and sample volume that are necessary Production Metabolic Data Record Metabolic data from sampling assays can be logged in the provided table Monitor pH metabolites and dissolved oxygen DO of the cell culture medium Procedure 1D Perform a two point calibration ofthe pH meter first or calibrate the DO meter first Then proceed with removing the sample for analysis This sequence minimizes 1 the time between withdrawing the sample and measuring its pH and or DO and 2 the drift of the sample s pH and or dissolved oxygen concentration 1 Measure the sample s pH and DO first as they can change rapidly Then measure the sample s metabolite concentration s if sampling will be done while the Multi6 is in the CO incubator go to the next step If sampling will be done with the Multi 6 in the laminar flow haod go to that section below 1D Allow all four pumps to continue pu
16. either direction Ensure itis rotating counterclockwise Some other motors rotate in only one direction DD Stop the motor and open both pump heads Once the direction of rotation of the motors is determined you will know how to orient the four pump segments in their respective pump heads This ensures they pump liquids and gas in the correct directions Page 22 of 68 Multi 6 User Manual 700466 000 Rev B Blovest international Loading Mult 6 s Media and Outflow Tubing in the Media Pump and Outflow Pump 9 Locate the Media Tubing s size 14 pump segment which is the yellow tubing marked 06508 14 PharMed This pump segment creates the flow of fresh basal cell culture medium from the Media Container into the flowppath of the Mult D Locate the Outflow Tubing s size 16 pump segment which is the yellow tubing marked 06508 16 PharMoi This pump segment creates the flow of spent basal cell culture medium out of the flowpath of the Multi6 and into the Outflow Container T There is one directional arrow label near each of these pump segments to indicate the required direction of fuid flow Remove the tape holding the media tubing in a coil Load the media pump segment into its pump head in the manner shown in Figure 14 Close the pump head DF Remove the tape holding the outflow tubing in a coil Load the outflow pump segment into its pump head in the manner shown in Figure 14 Close the pump head a NOTE PharMed tubing is speciall
17. flow 9 Remove the tape holding the circulation tubing in a coil Load the circulation pump segment into its pump head in the manner shown in Figure 15 lose the pump head J Remove the tape holding the gassing tubing in a coi Load the gassing pump segment into its pump head in the manner shown in Figure 15 Close the pump head NOTE PharMed tubing is specially made to withstand the mechanical rigor of the pump head Its Not necessary to shift new portions of the PharMed tubing into the pump head throughout the run CAUTION Do NOT load the off white silicone tubing in the pump head because it quickly will be damaged causing cell culture medium to leak Circulation Pump mounted in back Gassing Pump mounted in front These tubing ends are from the Multi 6 Ifthe motor turns clockwise ore beth pump segment the oppodte Artele ws shun This verlation iter does not iea ett bu eohevad wher Nat jeeni is Bases are hay purged Figure 15 age 24 of 8 ui Usor Manual 700466 000 Rev B Blovest international Flushing the Muti T Open the Media Clamp Mukti s Media Clamp and the Media Containers clamp if present 1 Open the Outtiow Clamp Mult 6s Outflow Clamp and the Outflow Container s clamp if present Inspect the Mul for pinched kinked or obstructed tubing Dy Media Tubing 1 Outils Tubing D Circulation Tubing T Gassing Tubing O Ic Circuit Tubing the loop from Media Reservoir to GEX to si bioreactors to Media
18. incubator to minimize temperature changes to the cell culture Disinfect the Muli bottles of fresh media and miscellaneous supplies that enter the laminar flow hood and be very careful about aseptic handling while working in the hood The Multi6 can operate for months so preventing contamination allows it to be used for a long time Procedure to connect the Media and Outflow containers T The first connection of these containers occurs as part of Fill amp Flush when the Mult is in the laminar flow hood Leave it in the hood to simplify the aseptic connection of the containers D For subsequent connections of containers the safest method is to return the Multi to a location near the laminar flow hood The MUli 6 itself does not need to be placed inside the hood to aseptically connect containers just its long Media and Outflow tubing lines Q Follow the instructions for Moving the Multi into the laminar fow hood Mult 6 User Manual 700466 000 Rev B Page 47 of 69 Blovest international Biovest s Cap Assembly amp Glass Bottle For 1 connections of Media amp Outflow containers disinfect the outside of the new bottle assemblies to be connected For2 and subsequent connections disinfect the outside of the new bottle assemblies and the bottle assemblies already connected to the Multi 6 s Media and Outflow tubing Place the bottle assemblies into the laminar flow hood T Connect the Media Container to
19. per week until the maximum yield is achieved Note maximum product yield may not correlate to highest product concentration Page 56 of 68 Multi 6 User Manual 700466 000 Rev B Blovest international Procedure for Adding Factor and Harvesting Supernatant CAUTION adding Factor and harvesting supernatant must always be done aseptically However itis possible to perform this procedure while the Mult remains in the COs incubator This procedure should be done in a laminar flow hood to minimize the risk of contamination Ty Prepare six syringes containing the desired volume of pre warmed Factor We suggest 20 mL to start and an increasing Factor volume as the culture expands Also draw about 3 mL of sterile air into the syringe 9 Gather another six empty syringes that are large enough to collect the desired volume of Supernatant This volume usualy is the same volume as Factor being injected To perform this procedure inthe laminar flow hood proceed to the first step notin a laminar flow hood set it on a bench or cart next to the COs incubator and close the doors to prevent changing its temperature and COs concentration Skip the next step Refer to Moving the Mult 6 into the laminar flow hood to begin this procedure Disinfect the Mult 6 especally the six EC Factor and six Harvest tubing lines and luer fittings Be careful to not spray alcohol on the polycarbonate plastic of the bioreactors Disinfect the EC F
20. problematic todo safely when using a media bag see caution note below Procedure T Connect the Muti 6 s Media Line to the Media Container s OUT port or tubing line as previously described TG Connect the Multi6 SOutflow Line to the IN port or tubing line of the same Media Container G Ensure a ventilation filter is connected to the Media Container CAUTION Be careful when using a media bag to simultaneously deliver media and collect outflow 1 The opening inside the media bag that leads to the media tubing MUST remain in contact with the liquid medium to allow it to be pumped out of the bag Otherwise air is pumped out of the bag 2 The opening inside the media bag that leads to the ventilation filter MUST NOT contact the medium or medium will ow into the ventilation tubing and wet the filter which will cause the bag to over inflate and cause a rupture somewhere Page 44 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Sampling Sampling is optional but suggested in order to monitor pH and respond to the nutrient needs of the large number of cells that the Mult can support approximately 10 10 per bioreactor Sampling does not require a lot of time yet can provide the following benefits stable pl maintenance improved product yield economical use of media and successful culture expansion Sampling typically is done more often during the growth phase of the run when the culture is expanding
21. the Mutts to the CO incubator Refer to Placing the MUl6 into the COs incubator 1 Ensure the Media and Outflow clamps are open before restarting the pumps O Set the Media Motor to 50 mL hour 4 rpm G Set the Circulation Motor to 250 m min 52 rpm for approximately the first week T Allow the system to run NOTE Luer connections can be made with the Multi 6 inside the COh incubator if aseptic technique is used For more precaution against contamination antibiotics can be added to the medium CAUTION When disinfecting the Multi 6 use caution to ensure disinfecting chemicals such as Isopropanol do not enter the gas side of the GEX Periodic Tasks Monitor the Multi 6 throughout the run on a daily periodic basis to determine when the listed tasks should be done Monitoring media is done preferably by sampling media and performing assays off line Ifthe inoculum number was 2 x 10 cells the first thing you likely will have to do is increase the Media pump rate on day 2 Day 0 is the day of inoculation I the inoculum number was higher the culture might need the Media pump rate increase on day 1 Additionally you periodically will need to T increase Media Rate 1D increase Circulation Rate DD change Media amp Outflow containers DD sample IC media to monitor pH and metabolites DD sample EC media to monitor product concentration DD add fresh Factor amp remove harvest Mult 6 User Manual Page 33 of 69 700466 000 Rev
22. the Outflow Container only has a capacity of 10 L we suggest that you decrease the Media Motor s speed to 375 mi hour 28 rpm to collect only 9 L This will prevent the potential of the Outflow Container over filling Upon returning to the lab increase the Media rate to 400 mL hour 30 rpm until the Outflow Container has 10 L of flush medium The Circulation Motors speed does not need to be reduced in this scenario CAUTION Fill amp Flush results in the Multi 6 being non cytotoxic This result is based on both the volume of media flushed and the 24 hour duration Increasing the Media Pump rate and decreasing the Fill amp e Flush duration may not result in a non cytotoxie flowpath T Once 10 Lof flush medium has been collected the Multi 6 is non cytotaxe TD Reduce the Media Motor s speed to 25 mL hour 2 rpm NOTE IF PBS was used as the 10 Aush volume after the 24 hour flashing period it must be removed and replaced with the chosen basal cell culture medium Replace the PBS container with a Media Container and collet at least 24 L of medium into the Outflow Container O Fill amp Flush is complete The Mult 6 is ready to begin the Pre Inoculation Phase ly to begi CAUTION Maintain the 25 mL hour media rate until the inoculation occurs This rate of media consumption even in the absence of cells to consume its nutrients offsets the loss of water volume that happens from the GEX functions of controllin
23. to calculate the provided equation to determine the necessary NaHCO to use Mult 6 User Manual 700466 000 Rev B Page 27 of 69 Blovest international D After adjusting CO wait 30 60 minutes for pH to equilibrate at the new value Wait a much longer time after adjusting NaHCO because medium exchange via the Media Pump occurs slowly which slows the equilibration of pH to its new value Remove another IC Sample recheck pH and make another adjustment if necessary pl pi i I Repeat the preceding step until pH is at the desired value Calculated Unknown Standard Calculation Spreadsheet Calculation Parameter pH PH 638 tog BOSNAHCO 4CO ROUNDISUM 638LOG103053 C2 82 2 co 3053 NaHCO lantilog pH 638 ROUND PRODUCT C230 53 10MA2 638 2 NaHCO 6 Os anti pH 438 1 3053 ROUNDKPRODUCT BE104 A2638 30 532 Table 1 a ee NaHCO Calculated Calculated Calculated i ie s L pH COs NaHCO x y z a b e Suggestion 1 Create a spreadsheet with these six columns 2 Copy amp paste or enter the Spreadsheet Calculations of Table 1 into their corresponding cells in Table 2 Usage enter any two of the three values x y or z to determine the unknown parameter Table 2 Adding growth supplements DD Adding growth supplements is covered in the next section Injecting complete medium into the cell sde of the bioreactor fr the first time Page 28
24. 6 production pH Concentration of glucose and or lactate and other metabolites notably glutamine for some cell lines Use mid log phase pH and metabolite information determined above for the Mult culture If possible regulate pH The easiest method may be adjusting the incubator s CO Increase Media Rate to maintain this glucose lactate or glutamine concentration Maintaining these parameters during the growth phase of the Muli 6 culture shorten post inoculation lag phase and minimize the amount of time until the bioreactor becomes confluent Once the Multi bioreactor is confluent continued growth isn t the main goal A somewhat lower PH lower glucose concentration higher lactate etc discourage continued culture expansion and result in greater protein secretion To achieve this goal do not further increase Media Rate once the bioreactor is confluent Media and Outflow container changes continue to occur when necessary A Cell Line Characterization protocol is provided to facilitate this worthwhile effort Evaluate several basal media Remember that RPMI is generally not the best cell culture medium for use in the Mult Harvest Optimization To optimize harvest strategy maximize yield increase the frequency and or volume of EC perfusion adding Factor and harvesting supernatant until you no longer see an increase in protein production Although rare the secretion rate of the protein of interest c
25. 69 700466 000 Rev B i qa Mesane Media Container with Male Luer Connectors e Media Container with Female Luer Connectors See Parts List for male luer adapter Media Container unused Either type of Media Container s male luer connects to Multi 6 s Media line s female luer Figure 18 Page 40 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international T Connect the Outflow Container to Mults s Outflow Line Biovest s Cap Assembly amp Glass Bottle Refer to Figure 19 Loosen the male luer plug in the eap s IN port of the empty bottle For 1 connection of Outflow Container discard the sterility protector covering the male luer taper of the Multh6 s Outflow Line For 2 and subsequent connections of Outflow Container remove the male luer taper of the Multi 6 5 Outflow Line from the caps IN port of the full outflow bottle Remove the luer plug in the IN port of the empty bottle s eap Press the exposed male luer taper of the Outflow Line into the empty bottle cap s IN port until itis tight Mult 6 User Manual 700466 000 Rev B Carboys or Media Bags Refer to Figure 20 Loosen the female hier that plugs the male luer in the Outflow Containers collection tubing of the empty Outflow Container For 1 connection of Outflow Container discard the male Iuer from the Mult 6 s Outflow Line s female luer port For 2 and subsequent connections of Outflow Con
26. Do not place the entre bag in the hood T Connect the Mult 6 s Media line to the chosen Media Container Refer to Figures 17 and 18 T Connect the Multi 6 s Outflow line to the chosen Outflow Container Refer to Figures 19 and 20 C Now that the sterile connections have been made the Mul 6 may be taken out of the laminar flow hood It may be easiest to do this by placing everything on a cart T Move the Muti into the CO incubator Refer to Placing the Multi into the CO incubator CAUTION regarding disinfecting the Multi 6 Spraying the translucent polypropylene plastic connectors with isopropanol or ethanol to disinfect them is OK However do not directly spray alcohols on to the clear polycarbonate plastic such as the bioreactors to prevent the chance this material will form stress cracks due to the alcohol s rapid evaporative cooling Instead use these alcohols to wet a sterile gauze and wipe the polycarbonate clean or use a sterile prop pad to wipe the area Mult 6 User Manual Page 21 of 69 700466 000 Rev B Blovest international Setup the Media and Outflow pump 9 Place ane pump motor the Media Pump and Outflow Pump both EW 77200 60 model on a bench next to the CO incubator TD Mount both pump heads on the pump motor as described in the operating manuals provided by their manufacturer Mount the Media Pump on the motor and the Outflow Pump on the Media Pump see Figure 14 Note although this motor is use
27. Loading the Media Tubing and Outflow Tubing in their pumps 3 Loading the Circulation Tubing and Gassing Tubing in their pumps Flushing the Mult 85 Pre inocullation Phase2Z Adjusting pH of the cell culture medium 7 Adding growth supplements28 Miscellaneous Pre inoculation tasks 4 Injecting Factor into the cell side of the bioreactor for the first time 30 Multi 6 User Manual Page Sot 69 700466 000 Rev B Blovest Intemational Inoculation 32 Periodic Tasks83 Placing Mult into the CO incubator Moving Mult into the laminar flow hood Connecting amp Changing Media amp Outflow Containers82 Alternative Method to Connect the Media and Outflow Containers Samplingl5 Monitor pH metabolites and dissolved oxygen of the cell culture medium Basal Medium Changest8 Media Container Change ProcedurctS Outflow Container Change Proceduret Alternative Change Basal Medium Method Media Rate Use Metabolite Analysis to Determine Media Rates Metabolite Assay Options 2 Setting the Media Motor Speed83 Outflow Rates Gassing Rate Circulation Rates Setting the Circulation Motor Speed SS Harvesting Supernatant and Adding PactorSs Procedure for Adding Factor and Harvesting SupernatantS7 Optimization Tips TroubleshootingSS Specifications Parts List Items Available From Biowest Carboy Components63 Media in Bags and Empty Bags Cell Line Characterization Worksheet Mult 6 Production Metabolic Data RecordAZ Biovest
28. Multi 65 Media Line Biovest s Cap Assembly amp Glass Bottle Refer to Figure 17 Loosen the male luer plug in the cap s OUT port of the full bottle For 1 connection of Media Container discard the sterility protector covering the male luer taper of the Multi6 s Media Line For 20 and subsequent connections of Media Container remove the male luer taper of the Multi 6 s Media Line from the cap s OUT port of the empty media bottle Remove the uer plug in the OUT port of the full media botle s cap Press the exposed male luer taper of the Media Line into the full media bottle cap s OUT port until it is tight Disinfect the following items depending on which type of container is in use vg items depending pe Carboys or Media Bags For 1 connections of Media amp Outflow containers disinfect the tubing lines connected to the carboys or media bags For 2 and subsequent connections disinfect the tubing lines connected to the new carbays or media bags and the tubing connected to the carboys or media bags already connected to the Mult 6 Media and Outflow tubing Also disinfect the portion of the Multi 6 s Media and Outflow tubing that enters the hood Extend the disinfected tubing lines into the laminar flow hood Do not place the earboy s or media bags into the hood Carboys or Media Bags Refer to Figure 18 Loosen the female her that plugs the male luer in the Media Containers deliv
29. See Setting the Media Motor Speed below for instructions Metabolite Assay Options There are several options for measuring metabolites Costs for the equipment and reagents or consumables greatly varies These options are provided as examples We do not sell support or endorse any specifie option GlucCell glucometer specially designed to measure glucose in cell culture media Diabetic Glucometers a review of two models Glucose and Lactate assay kits biowision com bioassaysys com trinitybiotech com YSL 2700 SELECT bench top instrument measures metabolites such as glucose lactate and glutamine Page 52 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Setting the Media Motor Speed The suggested Masterflex motor directly displays the pump rate in mL min but the correct pump head size must be selected to achieve the displayed pump rate The Media Motor always will be sot fora size 14 pump segment Even though the Outflow Pump is also driven by the Media motor do not select its size 16 pump segment on the motor s keypad See the next page regarding the Outflow Rate Refer to the Masterflex operating manual to program the Media Motor for continuous mode operation Ifyou are not using the suggested Masterflex motor as the Media Motor and it has a simple rheostat surrounded by a dial with numbers to indicate relative speeds it does not directly display the pump rate in mL min In this case you must use a st
30. a is removed from the Mult 6 at the required rate Mount the Outflow Pump and Media Pump to the same motor to achieve this goal Important Notice The Outflow Pump routinely pumps some air and some media This is normal However the flow of air into the Outflow Container requires it to have a ventilation filter that absolutely must not become wet Secure it in a manner that outflow media will not reach this ventilation filter A wet filter would stop ventilation over pressurize the Outflow Container and cause a rupture and media leak Gassing Rate The Gassing Pump creates the gassing rate The gassing rate in combination with the gas exchange cartridge GEX provides oxygenation and pH control While oxygenation and pH control are important for a healthy culture it is easy to set the correct gassing rate Simply ensure the Gassing Pump s rpm is equal to the Circulation Pump s rpm see next page to ensure sufficient flow through the GEX The GEX has a very large surface area and will ensure oxygen is not limiting and pH control is adequate Connect the Gassing Pump and Circulation Pump to the same motor to achieve this goal Page 54 of 68 Mult 6 User Manual 700466 000 Rev B Blovest International Circulation Rate The Circulation Pump creates the circulation rate medium the culture experiences no shear stress within the IC Circuit Circulation rate affects from the circulation low This means
31. a male luer plug The caps third ventilation port must connect ta sterilizing grade hydrophobic filter 1 Male and 2 Female Luer Connectors Carboys amp Caps suppliers of ported carboy caps include a selection of hose fitting diameters Again Y inch diameter tubing is lange enough However carboy cap port fittings often are either or Vainch in size inch ports are too large Vinch ports are OK T Carboys that will deliver fresh basal medium to the Multi6 s Media line require a male luer on the carboy cap s external tubing line that connects to the caps internal tubing line that reaches to the bottom of the carboy see Figure 18 The second liquid port can be closed with a ler plug The cap third ventilation port must connect to a sterilizing grade hydrophobic filter T Carboys that will collect spent basal medium from the Mut 6 s Outflow line require a male Incr on the carboy cap s extemal tubing line that connects to the cop s internal tubing line is short and does not reach the bottom of the carboy see Figure 20 The second liquid port can e closed with a luer plug The cap s third ventilation port must connect to a sterilizing grade hydrophobic filter Mult 6 User Manual Page 17 of 69 700466 000 Rev B Blovest international When using rigid containers such as bottles or carboys with a ported cap to deliver Media or collect Outflow the ported cap MUST include a sterilizing grade ventilation filter to allow the chan
32. actor Port and Harvest Port of one bioreactor If Clearlink connectors are in use they also must be disinfected before connecting a syringe Connect a filled syringe to the EC Factor Port Connect an empty syringe to the Harvest Port Open the EC Factor and Harvest port lamps Inject Factor at the same rate as pulling supernatant into the Harvest syringe Close the Harvest Port clamp Inject enough of the air that is inside the Factor syringe to push air past the Factor Port clamp but da not push air all the way into the bioreactor Close the Factor Port clamp oo oooo0oo0o0 O ag Replace the Harvest syringe with a new syringe that is the size needed to harvest supernatant the next time It remains connected until it is needed to harvest supernatant the next time Note if this procedure is being done ina laminar flow hood draw a few mLs of sterile air into the replacement syringe Connect the syringe to the Harvest Port Open the Harvest Port clamp Inject air past the Harvest Port clamp but do not push aie all the way into the bioreactor Close the Harvest Port clamp CAUTION At all times be careful to not introduce air bubbles into the ECS If this happens remove the air bubbles using a syringe connected to the EC Factor or Harvest port as necessary and if possible 1D Leave the empty Factor syringe connected until itis used to inject Factor the next time OF Refer to Placing the Mult into the COs incubator if this procedu
33. an be up or down regulated by the concentration of the protein itself Adjust the harvest strategy accordingly For protein free applications more frequent harvesting can minimize the potential for degradation or alteration of the secreted protein due to background culture lysis in the bioreactor When using serum supplementation decrease its concentration in steps once the bioreactor is full High density cultures confluent bioreactors often yield more product with decreased concentrations of FBS with the added benefit of a purer crude supernatant Page 58 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Troubleshooting Points Low pli In static culture allow a flask to go into the death phase Determine pH of the medium just prior to the decline in viability Do not let the medium in Multi 6 to decrease to this pH Refer to Adjusting pH for details about the options to control pH Confluent bioreactors usually metabolize high amounts of glucose into lactate The resulting lactate concentration an acid source decreases pH The COs concentration in the incubator another acid soutce also decreases pH If the incubator only contains the Mult6 reduce the ncubator s CO concentration to balance the culture s lactate production until pH of the medium in the Mult rises to the desired value Ifreducing the incubator s CO concentration isn t possible adjust the concentration of Sodium b
34. and correlate to increased product secretion There are two general ways to determine when to change the media rate metabolite analysis and visual observation These methods are discussed in detail below See Optimization Tips amp Troubleshooting and Cell Line Characterization Worksheet for further guidelines on setting the media rate and understanding a cell line s nutrient requirements Use Metabolite Analysis to Determine Media Rate Oo Refer to Moving the Mult 6 into the laminar flow hood to begin the Sampling procedure Alternatively leave the Mult 6 in the COs incubator and perform Sampling without moving it is Oo a Refer to Sampling to remove a sample of IC medium for off line analysis of the critical metabolite s Refer to Placing the Multi 6 into the CO incubator if Sampling was performed in the hood If Coll Line Characterization already was completed refer to its resultant target cancentration s af the important metabolite s Monitor metabolite concentration s as an indicator s for when to increase the media rate If cell line characterization has not been done yet use a target of 50 residual metabolite concentration until a cell line characterization can be done Mult 6 User Manual 700466 000 Rev B Page 51 of 69 Blovest Intemational TU Hthe assayed metabolite concentration s is below the target value s increase the Media Pump speed by approximately 25 50 when the metabolite concent
35. as good as the standard method isa toensure optimal consistent balanced nutrient simple method to setup and maintain particularly delivery during the run when using bottles and carboy containers Important Points The standard method of connecting Media and Outflow uses two containers The alternative method uses just one container see Figure 21 Media Lre Outfow Line The altemative method requires the Media Outflow Container to have a ventilation filter whether bottles carboys or media bagsarebeing Vertiaton The Media Outflow Container must have three ports or tubing ines Shown An IN port or tubing line to connect to the Muli 6s Media Line Pit An OUT port or tubing line to connect to the Mul 6 SOutflow Line A third port or tubing line to connect a ventilation filter Advantages of using this altemative method Simple setup when using media bottles or carboys Figure 21 Uses less bench floorspace Simpler to mave the Mult and container to the laminar flow hood when necessary Nowony about Media amp Outflow containers becoming empty or full Disadvantages of using this alternative method Can lead to confusing results if a nutrient is being consumed but not monitored This can cause growth phase problems confuse Media Rate adjustment requirements and create other problems Suggestion BD Cell MAb Media is designed for long term use between changes Media can become to spent or drive pH very low Can be
36. avable ane each Usage Connect two male luer connectors together when necessary 3319 0008terile Cover Tubing with a sterilizing grade porous plug Autoclavable ane each Usage Install on to fittings to keep them sterile after autoclaving 1092 006Female luer tubing fitting Polypropylene Autoclavable one each Usage Install into 3 16 tubing 100211 006Male ler tubing fitting Polypropylene Autoclavable one each Usage Install into 3 16 tubing 401183 000Cearlink Connector Sterile kit of fifteen individually packaged connectors Usage connect to the BC Factor Harvest and IC Sample ports to facilitate their rapid use outside of a laminar flow hood while reducing the risk of contamination Mult 6 User Manual Page 61 of 69 700466 000 Rev B Blovest International aps T Note 1825 000 fiter is nat a included with a a aoe ke 103247 000 Male Luer Adapter 5199 000 0017 000 A 600023 000 Medis Cap B 102360 003 Red Lock Ring Female Luer Adapter 10L Glass Bottle B 600022000 Outflow Cap 401183000 100211006 1092 005 Cleariink Connector Male Luer Tubing Fitting Female Luer Tubing Fitting 100553500 3319 000 White Ratchet Clamp Sterile Cover 02u Fiter with Female Luer e E 600100072 Sterile 72 Tubing Extension shown outside of sterile bag e a a a aen p s Senile arefing to simplify septal connecting the center Luer adapters 1 Collect all tems as shou above 2 press the sterte covers ful on to the Luer ng ba
37. d Mult 6 User Manual Page 47 of 69 700466 000 Rev B Blovest international Basal Medium Changes Small scale cell culture methods such as static culture and small hollow fiber products use a simple but time consuming media change procedure pour out their spent media and refill them with fresh media And this occurs often daily Mult 6 isa comparatively large scale system Unlike small scale products it uses Media and Outflow containers to significantly simplify the process of supplying adequate basal media during its many weeks of operation However connecting these containers to ease media handling is not the only necessity It also is important to increase the basal medium delivery rate as the culture expands Even when using high glucose basal media which we recommend the consumption rate can rise to 6 L day or higher because the Mult 6 supports a very high number of cells The maximum consumption rate depends on basal medium formulation and varies by cell line Changing basal medium when using the Muli6 involves two related but distinct tasks Media Container Changes Media Pump Rate Changes Media Container Changes are done whenever the Media Container is nearly empty The procedure to change the basal medium container is described below Media Pump Rate Changes generally are increases of the pump rate to deliver basal media nutrients ever faster to match the culture s expansion rat
38. d to pump Media and Outflow it will be referred to as the Media Motor to distinguish it from the Circulation Motor Briefly operate the Media Motor without tubing in either pump head in continuous mode operation in a counterclockwise rotation at a rate of 1000 mL hour for size 14 pump tubing 76 rpm While the motor is turning confirm both pump heads are rotating counterclockwise Note the suggested pump motor can rotate in either direction Ensure iti rotating counterclockwise Some other motors rotate in only one direction DD Stop the motor and open both pump heads Setup the Circulation and Gassing pump 9 Place another pump motor the Circulation Pump and Gassing Pump CP is EW 77200 62 GP is EW 77200 60 on a bench next to the COs incubator O Mount both pump heads on the pump motor as described in the operating manuals provided by their manufacturer Mount the Circulation Pump on the motor and the Gassing Pump on the Circulation Pump see Figure 15 Note although this motor is used to drive Circulation and Gassing it will be referred to as the Circulation Motor to distinguish it from the Media Motor DD Briefly operate the Circulation Motor without tubing in either pump head in continuous mode operation in a counterclockwise rotation at a rate of 100 mL minute for size 36 pump tubing 21 rpm While the motor is turning confirm both pump heads are rotating counterclockwise Note the suggested pump motor ean rotate in
39. duct Over time product concentration increases and becomes the primary reason that complete media and supernatant volumes are exchanged When serum is being used as the growth supplement many researchers observe that serum concentration can be decreased in steps with a corresponding increase in product secretion rate To accomplish this harvest a larger volume of supernatant than the volume of Factor that is injected Important Points Remember that the First Addition of Factor should occur before Inoculation Doing so allows the cells to move from static culture to hollow fiber culture with as little change to their environment as possible Pre warm complete media before injection into the ECS to prevent the culture experiencing a temperature shock Experimenting with various protocols frequency and exchanged volume for Factor addition and supernatant collection can yield greater product secretion rates General guidelines for adding Factor and harvesting supernatant until a specific protocol is developed Perform this three times a week Beginning exchange volumes should be 20 mL As the culture expands increase the frequency and or increase the exchange volume up toa maximum of 50 mL Check product concentration 5 days after each increase of the exchanged volume Calculate total product yield concentration x volume Continue increasing the exchange volume approximately once
40. e Increasing the media rate is the analog of changing madia in small scale products There are two general ways to determine when to change the media rate visual observation and metabolite analysis See Media Rate for further information about knowing when to increase the rate and how to doit See Optimization Tips amp Troubleshooting and Cell Line Characterization Worksheet for further guidelines on setting the media rate and understanding a cell line s nutrient requirements Important Point tis unnecessary to warm basal medium prior to use because it remains at room temperature during use The CO incubator warms it quickly enough as it enters the incubator Media Container Change Procedure Container oo Oo Oee Page 48 of 68 Perform the following procedure before the Media Container becomes empty Itis safer but more involved to perform this procedure in the laminar flow hood Refer to Moving the Mult 6 into the laminar flow hood to begin the media change procedure Alternatively leave the Mul 6 in the COs incubator and perform this procedure without moving it Refer to Connecting amp Changing Media amp Outflow Containers to replace the nearly empty Media Refer to Placing the Multi 6 into the CO incubator if this procedure was performed in the hood Record each new Media Container s volume of added basal media to the metabolic data record Mult 6 User Manual 700866 000 Rev B Blovest Intematio
41. eing pumped through the Circulation Pump as shown on the directional fow labels near the circulation pump segment If you see medium flowing backwards either the circulation tubing is loaded in the wrong direction in the Circulation Pump or the pumps rotation is reversed Pix the problem then restart the Circulation Motor The orientation of the gassing pump segment also should be checked to ensure it was loaded inthe correct direction Figure 16 Mult 6 User Manual Page 25 of 69 700466 000 Rev B Blovest International 1D Allow the Circulation Pump to operate slowly until no significant amount of air bubbles is being pumped along with the media through the IC Circuit Observe the lowpath for any leaks Refer to the CAUTION statement above if a leak occurs 9 the CP quickly drains the Media Reservoir and pumps air into the IC Circuit stop the pump until the Media Reservoir again is half full Then restart the CP O Once the Circulation Pump is pumping only medium the Media Reservoir is half full and no leaks are seen increase the Circulation Motor s speed to 5k mL min 104 rpm Ty Reduce the Media Motor s speed to 400 mL hour 30 rpm Reconfirm there are no media leaks If not allow the Circulation Motor and Media Motor to operate unattended for another 24 hours to perfuse 10 L of media through the Mult 6 CAUTION Peristaltic pumps have a pump rate accuracy of about 410 If you leave the pumps running overnight and
42. epeat the previous nine steps for the remaining five bioreactors DD Wait sic hours for growth supplements to disperse before inoculating 9 The Muti is ready for the next procedure Inoculation Proceed to the inoculation procedure only after pH and temperature are at the desired values NOTE media are injected into the bioreactor s lower EC Factor Port and removed from the upper Harvest Port to prevent leaving air bubbles in the ECS Prevent injecting air into the ECS NOTE experiments using six different growth supplements or various concentrations of growth supplements may be performed because the ECS of each bioreactor is segregated Mult 6 User Manual Page 31 of 69 700466 000 Rev B Blovest international Inoculation Inoculation is the result of scaling up cells using inoculation can lead to extended lag phase and conventional cell culture methods Passaging of the continue to have a long term impact on the hollow flask culture during scale up should be performed fiber culture consistently when the culture is in mid log phase growth Maintain this passaging routine for several passages before beginning the inoculation procedure below This passaging routine generally achieves maximum cell viability Care should be taken to ensure maximum viability of the inoculum Poor viability at the time of Prepare 2 10 viable cells per bioreactor The inoculum culture should have been maintained in mid log phase of grow
43. ers are too small to be practical with most cell lines Partially fil larger bottles or carboys to start the run and Completely fill them later when the media consumption rate is high enough Or use small media bags to start the run and switch to large bags when necessary Container Reuse the decision to reuse media and outflow containers is determined by user preference time and effort required for cleaning and re sterilzation and how the basal media is purchased Bottles and carboys usually are reused Bags usually are single use disposables If media is made from powder it can be filter sterilized into any container type Tfthe large volumes of media are purchased in liquid format the practical l container is media bags because media suppliers sell large volumes of media in media bags Once the media is consumed from media bags the resulting empty bags are discarded but they could then be used to collect Outflow media once and then would be discarded mi N Media Outflow Media Outflow Gutfow Container Container Container Container Container Figure 9 Figure 10 Figure 11 Page 16 of 68 Mult User Manual 700466 000 Rev B Blovest international Media and Outflow Container Connection Method Regardless of which container option is used it must include luer connectors because the Mut 6 includes ler connectors as its tubing connection method Female and male hier connectors are shown in Figure 12 Media Bags suppliers
44. erum to the IC circuit the Media Reservoir before inoculation at the same concentration as the EC In these eases supplementation of the IC circuit is often only necessary for the first seven to fourteen days After that time switching directly to purely basal medium usually results in an ongoing viable culture Increase the cell number in the inoculum from 2 10 up to 5 x 10 viable cells Small inoculum volumes lead to high inoculum density This can negatively impact some cell lines throughout the Multi6 production Even if the cells are not negatively impacted low inoculum volumes can lead to poor distribution of the inoculum in the bioreactor which can lead to poorer growth and filling of the bioreactor NS0 GS cell ines likely require cholesterol supplementation to the ECS particularly when using low concentrations of FBS Mult 6 User Manual Page 59 of 69 700466 000 Rev B Blovest Intemational Specifications Flowpath Volumes 7 IC Circuit 490 mL EC Volume 50 mL per bioreactor Peristaltic Pumps Pump sMasterflexFlow Rate Functionlfubing SizeinLirevolution Media Pump140 22 Outflow Pumpl60 80 Gassing Pumpl72 8 Circulation Pump368 8 Space Widtht25 s1sem Depth 93 em Height25 318 em Weight Ibs 09 kg Page 60 of 68 Multi 6 User Manual 700466 000 Rev B Blovest Intemational Parts List Items available from Biovest International Illustrations are on the next page 10309 2051 Mul
45. ery tubing of the full Media Container For 1 connection of Media Container discard the male uer from the Mult 6 s Media Line s female luer port For 2 and subsequent connections of Media Container loosen the male female luer connection of the empty Media Container connected to the Muti 65 Media Line Remove and discard the necessary items to be able to connect the male luer of the full Media Container to the female luer of the Multi 6 s Media Line CAUTION When using Media bags the opening inside the media bag that leads to the media tubing MUST remain in contact with the liquid medium to allow it to be pumped out of the bag If this opening inside the bag loses contact with the medium the Media Pump will create a vacuum Dy Instructions that describe how to connect Outflow Containers are after the following two pages Page 38 of 68 Mult 6 User Manual 700866 000 Rev B Blovest international Biovest s proprietary ready for use Cap Assembly Soe Pars List for ordering information Notes 1 Rigid media bottles require a sterilizing grade Ventilation fitert 2 OUT port leads to the caps internal sipper tubing that reaches the bottom of the bottle The bottle cap unused IN port remains closed with 2 male luer plug Media Container Multi 6 s Media line s male luer taper fitting connects to the Media Container Cap s OUT port Figure 17 Mult 6 User Manual Page 39 of
46. ev B Blovest international Overview The Mult 6 name derives from the multiple uses of its six hollow fiber bioreactors These uses include for example simultaneously culturing six established cell lines to produce six products producing six fold amounts of one product by culturing one cell line in all bioreactors evaluating multiple media formulations for optimal performance determining the highest secretor among six clones for further development ete Mult 6 is a low cost hollow fiber cell culture system capable of producing small to large quantities of highly concentrated monoclonal antibodies and other secreted proteins Multi is much simpler to use than conventional static culture methods Ascites production and competing hollow fiber products from other suppliers in order to produce an equivalent amount of material Mult allows the user to focus on science rather than protein production LE necessary development in the Multi can be scaled up to a range of larger hollow fiber systems provided by Biovest International Inc Mult 6 is supplied fully assembled sterile and ready for immediate use Simply fill and lush its Nowpath with basal medium to begin run startup Multi is single use to eliminate time consuming cleaning amp sterilization steps necessary with other in vitro cell culture equipment or the use and sacrifice of mice in Ascites production Mult 6 six bioreactor cartridges can be used to culture both
47. g pH and oxygenating the cell culture media Operating the Multi 6 for long periods of time at 37 C at a 0 mL hour Media rate can concentrate the circulating media components Page 26 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Pre Inoculation Phase There are two primary tasks to accomplish during the Pre Inoculation Phase Adjust pH Add growth supplements Adjusting pH of the medium in the Multi 6 to the PH value that coincides with mid log phase growth For information about determining pH set points see Cell Line Characterization Worksheet Adding growth supplements is necessary because Pill amp Flush is done using basal cell culture medium Add growth supplements to the bioreactors ECS before inoculation for the particular cell line in use can shorten the growth phase of the culture Monitoring pH during the run helps you prevent it from falling too far and know when to change the Media Rate Don t rush to inoculate the cells until until growth supplements have been added and pH is stable and appropriate for the cell line being used Doing so can dramatically reduce inoculum viability and increase lag phase Adjusting pH of the cell culture medium 1D Determine the desired pH see Cell Line Characterization Worksheet Determining pH occurs prior to using the Mult 9 Continue operating the Circulation Motor s speed at 500 mL min 104 rpm to allow pH to stabil
48. ging liquid volume in the bottle to be accommodated by air entering or exiting the bottle If the ventilation filter becomes wet the container will pressurize or experience vacuum These filters can be reused re sterlized but this practice is not recommended CAUTION The bottle cap s ventilation filter MUST NOT become wet to prevent a hazard to personnel and equipment Contact Biovest International for additional technical and purchasing information about specific container options configurations and requirements The Parts List section near the end of the manual includes some of the options that are available from Blovest Optional Equipment pH meter Glucose or lactate assay kit or glucometer Glutamine assay kit useful if the cell line doesn t use glucose as the main energy source Dissolved oxygen meter unnecessary for routine productions using the Mult 6 Page 18 of 68 Multi 6 User Manual 700466 000 Rev B Blovest Intemational Unpacking amp Setup The unpacking and setup steps ensure the product remains sterile and is leak free and ready for use It is important to ensure the clamps are open on arrival and that the fittings are not loose T Remove the sterile sealed packages containing the Multi 6 from the shipping carton T Disinfect the outside of the sealed packages and put them into a laminar flow hood T Remove the Muti from its sealed package and discard the white foam
49. h the following dimensions 3 16 internal diameter with a wall thickness of 3 32 or 14 4 Cut two 24 lengths for the liquid n and out ports 5 Cuta 3 length for the ventilation port A short length prevents tubing kinks from blocking ventilation Filter 024 Autoclavable vent filter available from Biovest 1825 000 Fittings 1 Because the MUlt 6 has female luer fittings on the Media and Outflow tubing lines the earboy s liquid in and out ports should terminate in male luer fittings 2 Male and female luer fittings and sterility covers are EE available from Biovest see details of the previous page 3 Ifa female luer becomes necessary for some unanticipated reason connecta female luer adapter 5199 000 on previous page to either the in or out port s male luer fitting Mult 6 User Manual Page 63 of 69 700466 000 Rev B Blovest international Items available from other suppliers Suggested items are illustrated below along with an example supplier names Use this information to source equivalent items from your preferred supplier While Biovest does not endorse any specific supplier or option we can assist you with questions about suitability of an item for use with the Multi 6 Media in Bags and Empty Bags All major suppliers of basal media provide bulk media in bags Each supplier uses bag designs of different sizes number of tubing lines length of tubing lines types of tubing connectors etc Bags are eithe
50. he concentration of low molecular weight metabolites such as glucose or lactate Dissolved oxygen or other low molecular weight components also may be assayed from these media samples Sampling and testing IC media is optional but can help you to determine when and by how much to change the MP speed to support the expanding cell culture Bioreactor Lumen The internal volume of the semi permeable hollow fibers within the bioreactors is part of the IC and is referred to as the Intracapillary Space ICS see Figure 2 The ICS is on the non cell side non product side of the hollow fiber membrane The hollow fiber internal volume and membrane permeability provide the ability to deliver low molecular weight nutrients and 2 to the cells 2 tollect metabolic waste products and CO gt from the cells Mult 6 User Manual Page 11 of 69 700466 000 Rev B Blovest international Extracapillary Circuit EC The Extracapillary Circuit is the fluid volume of the components and tubing that connect to the cell side of the hollow fiber membrane within each bioreactor and is shown in yellow in Figure 6 The EC enables two primary tasks adding Factor growth supplemented medium and harvesting supernatant Adding Factor enables the culture to receive necessary supplements to maintain high viability culture expansion and product secretion Harvesting Supernatant enables the technician to collect the secreted product EC Perfusion is the comb
51. henever you move the Mult6 ensure the tubing lines stay below the height of the Multi 6 to prevent their volume from draining into and overfilling the Media Reservoir T Piace the Mult 6 and Media and Outflow containers on a cart 1D Move the cart to the laminar fow hood GF you will inject fresh Factor into the ECS or withdraw harvest supernatant from the ECS Disinfect the Muti 6 especially the six EC Factor and six Harvest tubing lines and luer fittings and place it inside the incubator Be careful to not spray alcohol on the polycarbonate plastic of the bioreactors See Harvesting Supernatant and Adding Factor for farther instructions T To withdraw an IC Sample to analyze metabolites ar pH Disinfect the IC Sample line and extend it into the laminar ow hood The Mult6 Media Container and Outflow Container can remain on the cart When using a Clearlink on the IC Sample port it also must be disinfected before connecting a syringe Soe Sampling for further instructions Option an experienced technician can safely perform Sampling while the Mul 6 remains in the COs incubator but more risk is involved Using Clearlink connectors can reduce this risk Mult 6 User Manual Page 35 of 69 700466 000 Rev B Blovest Intemational T To change the Media or Outflow container when using Biovest s Cap Assembly and glass bottles leave the Mult 6 outside the hood on the cart Disinfect the connected Media or Outflow bottle cap
52. hollow fiber cell culture productions Complete Media EC Factor Complete media is cell culture medium containing high molecular weight typically growth supplements O Add the necessary amount of growth supplement FBS for example into the basal medium of choice T Alternative complete media a proprietary serum free medium formulation also works well in place of serum supplemented media When using serum free media as the complete media using classic basal medium for the IC Media often works well and reduces media costs For further information contact Biovest s Account Services DD Routine static cell culture equipment and supplies to produce a scale up inoculum of 2x10 viable cells per bioreactor Q Sterile syringes of various sizes such as 1 mL 5 mL 30 mL and 60 mL You may find other sizes are useful too Syringes are used to inject Factor and the inoculum Syringes also are used to remove IC medium samples for pH and metabolite analyses and EC medium to collect supernatant There are various methods and equipment for analyzing these samples The chosen methods and equipment determine the necessary sample sizes Mult 6 User Manual Page 15 of 69 700466 000 Rev B Blovest international Media and Outflow Container Options The Multi6 does not include Media and Outflow containers These containers must be purchased and prepared separately There are options for the type size and reusability of these containers D Contai
53. icarbonate in the basal medium to achieve the desired pH When the purchased media already contains NaHCO one option to increase the buffer concentration is to make a sterile solution suggest 75 of NaHCO and add it to the Media Container as necessary to achieve and maintain the desired pH When using separate Media and Outflow containers increasing Media Rate beyond the rate that is necessary to supply enough nutrients will dilute lactate and increase pH When pumping Media and Outflow from and to one container see the Alternate Method remember that lactate will increase and cause pH to decrease until the container is changed Do not be excessively frugal with media usage when using this method Adjusting the Sodium bicarbonate concentration for high lactate production rates may be necessary Using other buffers such as HEPES up to 15 mM can be effective but is generally not recommended However mixing pH buffers can complicate pH control when using an incubator that contains an amount of CO Poor Growth Long Lag Phase Same cell lines are sensitive to the concentration of certain uncharacterized low molecular weight components in serum These components are not formulated in the basal medium Therefore these components dialyze away from the ECS and their concentration becomes limiting to the cell culture This results in very slow initial growth or inoculum death To prevent this problem the simple solution is to add s
54. ined effect of adding Factor and harvesting supernatant The functions of the components of the Extracapillary Circuit are Bioreactor Extracapillary Space The extracapillary space ECS within the bioreactor consists of the space surrounding the hollow fibers and is separated from the lumen IC by the porous hollow fiber membrane see Figure 6 Within the ECS are the cells high molecular weight growth factors and cell secreted proteins They are to lange to pass through these pores and are retained within the ECS Note the ECS volumes of the six bioreactors are segregated from one another to prevent the cultures and products from mixing x ist Harvest Port Six Media Bioreactor Reservoir Manifold X sR EC Factor Port xmm Circulation eee le Semple Port a E Pump erer Gas Exchan a Cre imp EC Circuit Figure 6 Pago 120f60 Muti 6 User Manual 700466 000 Rev B Blovest international EC Factor Port This location is primarily used to inject fresh complete media each time supernatant is harvested Complete media is either basal medium supplemented with growth factor such as FBS or it isa defined serum free media In either case complete media contains large molecular weight supplements too large to fit through the fiber pores For this reason complete media must be added directly to the ECS in order for the supplements to reach the cell culture Adding complete media to the
55. ion through 48 hours of declining viability Set up this number of flasks plus one for a celltree control When recording sampling data an the following page sacrifice a flask for each sampling which will eliminate the necessity for aseptic technique Sepersion ature Seed __ spinner flasks or __T flasks with 0 1 x 10 cells ml Collect a minimum sample volume daily and record data on the back of his sheet Data required for recommended characterization are as follows PH viability total cell density and glucose lactate and product concentrations Additional metabolite data may be useful Collect data for an additional 48 hours after viability begins to fall Graph these data against time and determine the following results Pests Maximum product concentration Maximum viable density x10 cells ml Doubling time at midog phase Baseline Setpoints Use the setpoints below as a baseline strategy for entering process control parameters for the Multi production run Growth Phase Setpoinis Production Phase Setpoints values recorded at values recorded at maximum maximum growth rate product concentration pH Glucose cone mg d Lactate conc mg dL Mult 6 User Manual Page 65 of 69 700466 000 Rev B Blovest intematio Call Line Characteriza5n Data Sheet Celi tine Product houe leorelnne pors fest pesen zm pensi fovea aa roast ca Conor Flask
56. it into the laminar fow hood Note it is not necessary to place the entire Mult 6 the media outflow circulation and gassing lines and media and outflow containers into the hood DD Hithis isthe first use of the IC Sample port Loosen the male luer plug connected to the IC Sample port Removea 3mL syringe from its sterile packaging Remove and discard the male luer plug and connect the 3 ml syringe 1D Open the IC Sample clamp 1D Fill the 3 mL syringe to flush the IC Sample line DD Close the IC Sample clamp 1D Loosen the 3 ml syringe 9 Remove a 20 mL syringe from its sterile packaging T Remove and discard the 3 mL syringe T Connect the 20 mL syringe CAUTION Sample volumes greater than 20 mL may drain liquid volume from the bioreactor and result in the accumulation of air If larger sample volumes are necessary for the desired assay s proceed with caution 1 Open the IC Sample clamp 1D Fill the 20 mL syringe DD Close the IC Sample clamp 1D Remove a3 mL syringe from its sterile packaging 9 Remove and save the 20 mL syringe T Connect the new 3 mL syringe to the IC Sample port 1D Follow the instructions for Placing the Multi6 into the COs incubator 1D Open the Media and Outflow container clamps 1D Tumon the Media and Circulation motors to their former speeds O Assay the sample s pH and metabolite and DO concentrations 1D Discard the sample DD Record the data in the Metabolic Data Recor
57. ize as quickly as possible in the following steps 9 Remove an IC Sample according to the Sampling instructions TD CO Adjustment If pH of the medium is too low decrease the COs of the COs incubator If pH of the medium is too high increase the CO of the COs incubator Adjusting pH is more easily done by changing COs than the following alternative method NaHCO adjustment and is practical when the CO incubator contains no other cultures Using the information in Table 1 and Table 2 on the following page do either of the following to determine the necessary COs to use to achieve the desired pH enter the desired pH and the current concentration of Sodium bicarbonate NaHCO in the suggested spreadsheet use a calculator to calculate the provided equation NaHCO Adjustment Alternatively if pH is too low or high leave the iCO unchanged and instead adjust the concentration of Sodium bicarbonate NaHICO in the basal medium Adjusting NaHCO cannot always easly be done depending on the type of basal medium you purchased This method may be more practical than changing COs especially when the COs incubator is shared with other cultures Using the information in Table 1 and Table 2 on the following page do either of the following to determine the necessary NaHCO concentration to achieve the desired pH enter the desired pH and the COs incubator s current COs in the suggested spreadsheet use calculator
58. l bovine serum Alternatively various media vendors chemically defined serum free medium can be used instead of serum supplemented media Regardless of what is boing used for Factor what distinguishes it from basal medium is the presence of high molecular weight components that enhance the growth of the culture or secretion of the desired protein Because hollow fiber technology inherently uses a semi permeable membrane that has a very low supplement that is necessary compared to conventional cell culture methods Whereas basal medium flows through the IC circuit non cellside and readily exchanges with the EC space cellside and is metabolized by the culture Factor must he directly injected into the EC space to achieve delivery of the high molecular weight components to the culture It is important that the first injection of Factor occur at least several hours to one day before inoculation to allow the supplements to fully mix within the molecular weight cut off the high molecular weight supplements in Factor are mostly too large to pass through the hollow fibers pores This is a distinct benefit and greatly reduces the amount of ECS Do not rely on the inoculation procedure to serve as the first injection of Factor Important Paints Perform the following steps for the first injection of growth supplements during the P Phase These steps also will be performed each time supernatant is harvested fro
59. lt 6 User Manual Page 19 of 69 700466 000 Rev B Blovest international Fill amp Flush Hollow fibers are stored with a wetting agent to maintain their physical integrity This wetting agent must be removed before inoculation is performed The wetting agent is removed by Bushing the Mut with ten liters of sterile basal cell culture medium Using cell culture medium is preferable because it saves time and aseptic manipulations An alternative to using cell culture medium is to flush with 10 L of tx sterile PBS Using PBS may be preferred if an expensive cell culture medium will be used during the run However while using PBS may save some money compared with using cell culture medium using PBS adds time and complication to run startup The PBS must be prepared sterilized and flushed out of the Multi6 using the chosen basal cell culture medium anyway These tasks take time and increase the number of aseptic manipulations that must be performed correctly Do not skip the flushing procedure because the wetting agent is cytotoxic After ten liters of fush media have have been collected the Mult 5 will be non cytotoxic Fill amp Flush can be performed in the laminar flow hood ar in the COs incubator The flush volume rapidly circulates through the fibers via the Circulation Pump While the medium circulates 10 Lof medium are perfused by using the Media Pump and Outflow Pump Operate these pumps until the ten liters
60. ly necessary However DO data may be collected from the same sample of medium used to assay pH and metabolites when these data need to be monitored All sampling must be done aseptically even though fluids are only being removed form the flowpath Sampling may be done while the Muli 6 remains in the CO incubator or after it is moved into a laminar flow hood Sampling while it s in the incubator often has not resulted in contamination However you may prefer to perform sampling with the Multi in the hood to minimize the risk of contaminating it For more precaution against contamination antibiotics can be added to the media You may prefer to extend the IC Sample tubing line outside of the COs incubator and close its doors Amedium sample can be withdrawn without stopping the pumps or moving everything to the laminar flow hood or disturbing the incubator s temperature or CO concentration When dane put the tubing back into the incubator to keep it out of the way Whenever sampling is done in a laminar flow hood minimize the amount of time the circulation pump is not running Like your heart the circulation pump maintains oxygenation and pH control Oxygen concentration and pH rapidly decrease when the CP is off particularly with high cell numbers Mult 6 User Manual 700466 000 Rev B Page 45 of 69 Blovest international Minimize the amount of time the Mult 6 is out of the incubator to minimize temperature changes to the
61. m the bioreactor t is safest to perform the following steps with the Mult in a laminar flow hood because of the numerous connections of syringes involved However this procedure may be performed with the Muti in the CO incubator Follow standard procedures for disinfecting items prior to entering the hood and handling items within the hood Procedure O Prepare six 60 ml syringes with 50 mL of pre warmed Factor C Gather another six empty 60 mL syringes Q Follow the instructions to move the Multi 6 into the laminar flow hood 1 Disinfect the Muti6 especially the six EC Factor and six Harvest tubing lines and luer fittings Be careful to not spray alcohol on the polycarbonate plastic of the bioreactors T Place the Mult inside the laminar fow hood D Disinfect the EC Factor Port and Harvest Port of one bioreactor If Clearlink connectors are in use on these parts they also must be disinfected before connecting a syringe T Connect a filled syringe to the EC Factor Port 9 Connect an empty 60 mL syringe to the Harvest Port 1 Open the EC Factor and Harvest port clamps Dy Inject Factor at the same rate as removing basal medium into the empty syringe Inoculation Mult 6 User Manual 700466 000 Rev B Page 30 of 68 Blovest international DD Close the EC Factor and Harvest port clamps 9 Replace the filled syringe on the Harvest Port with a new 3 ml syringe 1D Discard the filled syringe from the previous step 1D R
62. mation about the fluids that are pumped Page 14 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Space in a Humidified 37 C CO Incubator 1D Width 125 31 8cm O Depth 75 19 2 em 1D Height 125 31 8 em NOTE the motor and pump head suggestions are relevant to operating a single Multi amp Tf multiple Muki 65 are simultaneously in use there are other motor and pump head options that may be preferable Part numbers for Masterflex items were current as of the writing of this user manual Purchase Masterflex items from your preferred supplier Regi red Supplies Basal cell culture medium IC Media D Suggest 45 g L Glucose 6 mM Glutamine 3 6 g L NaHCO and no other pH buffering agent DMEM FI2 1 1 works well for murine hybridomas and CHO cell lines O Medium consumption can be difficult to predict The Multi 6 supports approximately 2 5 x 10 cells per each of the six bioreactors They naturally will consume a lot of basal medium Depending on cell line stability the run will last weeks or months so prepare to have enough basal medium T Alternative basal media media such as BD Cell MAb Media are consumed in significantly smaller volumes than classic basal medium formulations Although these proprietary media have a higher per liter cost the benefits are very infrequent media changes which minimize handling time and contamination risk 1D RPMs generally not recommended for use in
63. ml in size 1D Open the Harvest clamp 1D Open the EC Factor clamp 1D Inject the inoculum into the top port while simultaneously withdrawing medium at the same rate into the empty syringe at the bottom port DD Close the Harvest Port lamp TD Slowly inject the medium collected in the syringe at the bottom port back into the bioreactor Note the fluid will be filtering through the hollow fiber membranes so itis normal to feel some pressure on the syringe and it will take a few moments to be complete Page 22 of 68 Mult User Manual 700466 000 Rev B Blovest international 1D Close the EC Factor clamp O Replace the empty syringe connected to the EC Factor Port with an new 3 mL syringe O Replace the empty inoculum syringe connected to the Harvest Port with the 3 ml syringe of fresh medium T Open the Harvest clamp TO Inject the 3 mil fresh medium into the top port T Close the Harvest clamp O Replace the empty syringe connected to the Harvest Port with a new 3 ml syringe TO Optional some cell lines are benefitted by adding the saved conditioned medium from the second inoculation step into the bioreactor via the EC Factor Part over the next few days Other call ines grow well without this extra effort so the remaining supernatant from step three above can be discarded When used the conditioned medium is used until it is gone in place of Factor T Repeat the preceding steps for the remaining five bioreactors Return
64. mping at their current rates Turning off the Media and Circulation motors is NOT necessary 1D Disinfect the IC Sample port If a Clearlink is in use it also must be disinfected before connecting a syringe O this is the first use of the IC Sample port Loosen the male luer plug connected to the IC Sample port Remove a mL syringe from its sterile packaging Remove and discard the male luer plug and connect the 3 mL syringe T Open the IC Sample clamp 1D Fill the 3 mL syringe to flush the IC Sample line DD Close the IC Sample clamp DD Loosen the 3 ml syringe 9 Remove a 20 mL syringe from its sterile packaging 1D Remove and discard the 3 mL syringe 1 Connect the 20 mL syringe 1D Open the IC Sample clamp 9 Fill the 20 mL syringe DD Close the IC Sample clamp 9 Remove a3 mL syringe from its sterile packaging Page 46 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international DD Remove and save the 20 mL syringe 1D Connect the new 3 mL syringe to the IC Sample port This syringe will function as a sterile port cover until the next sampling It will then be used to flush the IC Sample line Assay the sample s pH metabolite and DO concentrations xy the sample pi O Discard the sample and record the data sampling will be done with the Multis in the laminar fow hood go to the next step 1 Follow the instructions for Moving the MURS into the laminar fow hood 1D Disinfect the IC Sample line and extend
65. multaneously Their secreted products will not mix as long as their molecular weight sizes are larger than the retention of the fibers pore size Note most but not all secreted proteins are larger than the pore size Harvest Complete Medium O ne of thousands of semi permeable hollow fiber membranes Cells grow to tissue density in ECS Basal Medium Nutrients amp Oxygen Figure 4 Diffusion driven exchange of low molecular weight components is assisted by the circulation pump as it forces basal medium through the inside of the hollow fibers As basal medium enters the inside of the fibers there is a slightly higher pressure in the ICS than ECS This pressure difference forces medium through the pores of the hollow fiber and into the ECS near the proximal end entrance of the bioreactor carrying with it basal medium nutrients and Os As medium flows inside the ICS and seeps into the ECS the pressure within the ICS continues to drop Page 8 of 69 Mult User Manual 700466 000 Rev B Blovest international Near the distal end of the bioreactor there is a slightly higher pressure in the ECS than ICS This pressure difference forces medium through the pores of the hollow fiber and back into the ICS carrying with it the low molecular weight metabolic wastes and COs from the cell culture Due to the low of medium along the length of the ECS growth supplements such as FBS and secreted proteins can be at higher concentratio
66. n statement is used the documentation needs to be consulted in order to find out the nature of the potential hazard and any action which may need to be taken Failure to do so may result in injury or damage to the system NOTE NI equipment is for indoor use only Biovest International Inc 8500 Evergreen Boulevard Minneapolis MN 55433 USA Telephone 763 786 0302 Toll free n the US 800 3 Telefax 763 786 0915 Email accountservices biovestcom m2 Page 2 of 69 Mult 6 User Manual 700466 000 Rev B Blovest Intemational Table of Contents About this Manual 5 Supports Ordering Information Overviews Hollow Fiber Technology Bioreactor Fluid Dynamics6 Intracapillary Circuit IC Media ReservoirlD Media Pump and Media Container Outflow Pump and Outflow ContainerLO Circulation Pump Gassing Pump Gas Exchange Cartridge IC Sample Porth Bioreactor Lument Extracapillary Circuit ECW Bioreactor Extracapillary Spacel2 EC Factor Port Harvest Portl3 Equipment and Supplies Required Equipments Media and Outflow Pump SystemLd Circulation and Gassing Pump SystemtA Space in a Humidified 37 C CO Incubatort S Required Supplies5 Basal cell culture medium IC Media 5 Complete Media EC Pactor 15 Media and Outflow Container Options 6 Media and Outflow Container Connection Method7 Optional Equipment Unpacking amp Setupl Fill amp Flush Preparing for the Fill amp Flush Procedure 20
67. n the at proximal end This process is known as the Starling Effect Intracapillary Circuit IC The Intracapillary Circuit is the fluid cicuit ofthe components and tubing that connect to the non cell side non product side of the hollow fiber membrane within the bioreactor and is shown in red in Figure The IC enables two primary tasks basal medium circulation and basal medium perfusion Basal M irculation enables pH control and oxygenation via the coordinated high speed operation of the Circulation Pump and Gassing Pump Basal Medium Perfusion enables control of the levels of nutrients and metabolic waste products such as glucose glutamine lactate and ammonia via the coordinated low speed operation of the Media Pump and Outflow Pump x ist Harvest Port Six Media Bioreactor Reservoir Manifold x Sige EC Factor Port a N Circuation LI pC Sample Port cs aS Pump T 2 Gas Exchange Cartridge nu IC Circuit Figure 5 Multi 6 User Manual Page Got 69 700466 000 Rev B Blovest international The functions of the major components of the Intracapillary Circuit are Media Reservoir The Media Reservoir enables media circulation and media exchange Additionally the Media Reservoir has a constant volume of warm media that acts to warm the fresh incoming basal medium Media Pump and Media Container The Media Pump MP provides a slow flow rate of fresh basal medium into
68. nal Outflow Container Change Procedure Perform the following procedure before the Outflow Container becomes full Itis safer but more involved to perform this procedure in the laminar flow hood Refer to Moving the Mult 6 into the laminar flow hood to begin the outflow change procedure Alternatively leave the Multi 6 in the CO incubator and perform this procedure without moving it e o DD Refer to Connecting amp Changing Media amp Outflow Containers to replace the nearly full Outflow Container Oo Refer to Placing the Multi 6 into the COs incubator if this procedure was performed in the hood Mult 6 User Manual Page 49 of 69 700466 000 Rev B Blovest international Alternative Change Basal Medium Method Use this alternative method to change basal periodically change the container with a new medium when you also are using the Alternative container filled with fresh basal medium The Method to Connect Media and Outflow Containers frequency to change the container varies by cell In this alternative setup basal medium circulates line and increases as the culture expands nto and out of the Multi 6 from one container so pk heal neers This method still involves monitoring metabolite concentration s adjusting the media rate and However over time nutrient levels decrease and changing the container See Basal Medium Changes metabolic wastes inerease In response to this for further information about these tasks
69. ner Type examples include media bags with integral tubing lines Figure 9 glass bottles with ported caps Figure 10 carboys with ported caps Figure 11 plastic bottles with ported caps not shown and others Biovest sells some container items that are convenient for use with the Muli6 See the Parts List for details The advantages of media bags are they are available either filled with media or are empty to collect media They re simple to use are sterile and ready for use and disposable Media bottles and carboys are inexpensive and can be reused They are easy to use but require time and effort to be cleaned assembled and re sterilized for each use Plus preparing fresh basal media to add into them requires additional time and effort TD Container Size two key factors influence what the appropriate Media Container size is Media consumption rate higher media consumption rates mean larger container sizes are the practical choice to minimize the frequency of changing containers to minimize labor hours and contamination risk Length of time the basal media in the container remains at room temperature use a container volume that can be consumed over 10 14 days Nutrients generally will not degrade during this amount of time at room temperature Start the run using small volumes 5 10 liters Switch to large volumes approximately 2 3 weeks later for the remainder of the run 10 20 liters or more Containers smaller than 5 lit
70. nsure it is running fast enough but not too fast This ensures the culture is not limited by too litle nutrient nor is media being wasted by being pumped too fast Basal media is pumped out of the flowpath by the Outflow Pump as fast as it is pumped into the Fowpath by the Media Pump See Outflow Rate on the following page This Basal Medium Perfusion allows technicians to control nutrients at either the optimal concentration for the growth or production phases of the run The growth phase is the period fof time before the bioreactor is filled with cells Production phase begins when the bioreactor is essentially filled with cells The media rate should be increased frequently during the growth phase to ensure the increasing cell mass is supported by increasing nutrient delivery IF the media rate does not increase along with the cell mass nutrient levels of the media circulating in the IC Cireuit become depleted causing the culture s growth rate to stagnate and ultimately its viability to decline Once the bioreactor is filled with cells the media rate should not be further increased because the media rate that coincided with filling the bioreactor is the rate that is needed to maintain that amount of cell mass It is common for nutrient concentrations to somewhat decrease from this time forward for lactate and other metabolic waste products to increase somewhat and for pH to decrease somewhat These changes usually occur slowly
71. of 68 Multi 6 User Manual 700466 000 Rev B Blovest international Miscellaneous Pre inoculation tasks TD Reminder check the volumes in the Media Container and Outflow Container Change the containers if they are nearly empty and full respectively See Connecting amp Changing Media amp Outflow Containers for the necessary instructions TD How molecular weight supplements are necessary add them now and be prepared to add them throughout the run if necessary These supplements can be additional nutrients selection agents antibiotics ete Low molecular weight supplements can be added via the IC Sample line using a syringe but preferably are formulated into the basal medium Follow the same steps as when Sampling simply inject the solution rather than removing a sample Because these supplements are small they will cross the hallow fiber membrane Therefore add enough supplement for the combined IC EC volume approximately 790 mL T The Mutt is ready for the next procedure injecting Factor into the cell side of the bioreactor for the first ime T Once pH is at the desired valus reduce the circulation rate to 100 mL min 21 rpm See Setting the Circulation Pump Speed for further information Mult 6 User Manual Page 29 of 69 700466 000 Rev B Blovest international Injecting Factor into the cell side of the bioreactor for the first time Factor is generally a mixture of basal medium and growth supplements eg feta
72. of filled and empty media bags offer a selection of bags with various numbers of tabing lines diameters of tubing and connector types TO Media bags filled with media will noed minimally one tubing line with a male luer connector Other connector types are unnecessary AW tubing diameter is preferable No vent filter is necessary for media bags delivering media to the Mul The male luer of the media bag connects to the female luer of the Mul 6 s Media line Figure 12 see Figure 18 TO Media bags that are empty and used to collect Outflow media need minimally two preferably 4 diameter tubing lines that terminate with preferably one male luer each although one male luer and one female luer will work see Figure 20 Bottles amp Caps glass bottles with the GLAS threaded opening are readily available in Sand 10 L sizes They re also available but uncommon in 15 and 20 Lszes Because these bottles have the same threaded opening they accommodate Biovest s cap assembly specifically designed for them TO Bottles that will deliver fresh basal medium use the cap s OUT port to connect to the Mult 6 s Media line s male er taper ee Figure 17 The cap s IN port is closed with a male Iwer plug The cop s third ventilation port must connect to a sterilizing grade hydrophobic fier TO Bottles that will collect spent basal medium use the caps IN port to connect to the Mult6 s Outflow line s male luer taper see Figure 19 The caps OUT port is closed with
73. of medium are collected The collected Aush volume in the Outflow Container is aseptically removed and discarded Preparing for the Fill amp Flush Procedure tis unnecessary to pre warm the media used for Fill d Flush Prepare either of the following two options as an Outflow Container to collect the flush media Review Figures 19 and 20 and the assembly information in the Parts List 9 Autoclave sterilize an empty 10 L bottle or carboy or larger with a ported cap assembly loosely placed on the bottle or carboy Tighten the cap after the container cools to room temperature 1D Toan empty sterile 10 L or larger outflow bag connect a sterile ventilation filter one similar to 1825 000 see Parts List to preferably the outflow bag s male luer connector or alternatively the outflow bag s female luer connector using an adapter Important remember that this outflow bag needs a remaining male luer connector to connect to the Muli 6 s Outflow line Page 20 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international When using a bottle or catboy as a Media Container for the flush medium review Figures 17 and 18 and the assembly information in Parts List T Autoclave sterilize the bottle or carboy with a standard non ported cap Ensure the bottle or Carboy cap is not tight during the autoclave cycle to allow air to exit and enter the container Cover the cap and Media Container s cap and neck with wrapping to
74. opwatch and count motor revolutions for one minute Adjust the rheostat until you determine where the following revolutions per minute are achieved During pre inoculation set the media rate to 25 mL min 2 rpm Continue this media rate for the first day or two after inoculation One to several days after inoculation the culture will come out of lag phase and begin to expand When this occurs begin to increase the media rate every one to two days by 25 to 100 If glucose and or lactate are being assayed use these data instead to determine when and by how much to increase the media rate Suggested minimum and likely maximum media rates that will be necessary during the run are Motor Media Rate Media Rate Comments Revolutions mL min mL hr per Minute rpm Dep OsimL min 264m he Offsets GEX loss of water vapor that would undesirably concentrate nutrients opm 1342mL min 8052mL hr Likely maximum media rate necessary Calculation size 14 pump tubing 022 mL per pump head revolution Mult 6 User Manual Page 53 of 69 700466 000 Rev B Blovest international Outflow Rate The Outflow Pump creates the required flow rate of consumed basal medium out of the flowpath to maintain the correct fluid volume in the Muli The outflow rate is important but it is easy to set the correct outflow rate Simply ensure the Outflow Pump s rpm is equal to the Media Pump s rpm to ensure medi
75. oxygenation due to the function of the gas exchange cartridge The Circulation Pump CP is a self priming peristaltic pump The CP flow rate creates no shear or stress on the cell culture because this flow fs through the non cell side ofthe hollow fiber membrane Gassing Pump The Gassing Pump GP pulls a source of gases through the Gas Exchange Cartridge The source of the gases is generally the CO incubator generally a high percentage of CO Under some circumstances the source may be room air a low percentage of COs The GP also isa peristaltic pump Page 10 of 68 Mult 6 User Manual 700466 000 Rev B Blovest Intemational Gas Exchange Cartridge The Gas Exchange Cartridge GEX oxygenates the circulating cell culture medium The GEX also decreases or increases pH of the circulating cell culture medium When CO incubator gases flow through the GEX the cell culture medium is oxygenated and pH decreases When room air flows through the GEX the cell culture medium is oxygenated and pH The GEX is a membrane based device that separates two compartments the gas side and the cell culture medium side Gas blue in the figure flows on one side of the membrane while culture medium flows on the other side This membrane is permeable to gas transfer allowing bubble free exchange of COs and oxygen IC Sample Port The IC Sample Port allows the technician to remove a small volume of basal medium to monitor pH or t
76. p rate in ml min In this case you must use a stopwatch and count motor revolutions for one minute Adjust the rheostat until you determine where the following revolutions per minute are achieved see following table Suggested circulation rates to use during the run are Motor Revolutions per Minute Circulation Rate Run Day pm mL min 21 rpm 100 mL min Preinoculation also Fill amp Flush initially during leak testing 52 rpm 250 mL min Day 0 inoculation day Day 7 10t rpm 500 mL min Day7 Day 14 also majority of Fill amp Flush 208 rpm 1 000 mL min Day 14 and on Calculation size 36 pump tubing 4 8 mL per pump head revolution Mult 6 User Manual Page 55 of 69 700486 000 Rev B Blovest international Harvesting Supernatant and Adding Factor Many times during the course ofthe production run fresh complete media Factor is needed by the culture and supernatant is removed to collect product Factor and supernatant are exchanged in equal volumes to maintain the fuid volume in the bioreactor and the concentration of Factor This combined EC Perfusion frequency and or volume should increase as the culture expands Once the culture has come out of lag phase and has been growing for two to three days the culture likely will benefit from additional Factor Remember to remove an equal volume of supernatant to maintain a constant concentration of Factor despite the initially low pro
77. r 2D pillow or 3D in shape 2D bags work well with the Mult Empty media bags often referred to as bioprocess containers are available from media suppliers and other companies such as www appliedbpe com wwwadvancedscientifics com and www sartorius stedlim com Tubing 1 W internal diameter tubing is preferable but is OK 2 Media bags require only one tubing line 3 Outflow bags require two tubing lines Filter Connect to the Outflow bag a 0 2p autoclavable vent filter available from Biovest 1825 000 Fittings 1 Because the Multi 6 has female ler fittings in its Media and Outflow tubing lines Media and Outflow bags preferably should terminate in male luer fittings 2 Only luer connectors are necessary Other types ane unnecessary 3 Bags more commonly have female luer fittings In this case to use a Media or Outflow bag s Female ler connect a male luer adapter see 103247000 and 102360 003 that then connects to the Multi 6 Page 64 of 68 Multi 6 User Manual 700466 000 Rev B Blovest international Cell Line CharacterizaBn Worksheet cal re Date Cones Medium tots Supplements Serum other Determine the minimum sample volume required for measuring pH and metabolite and product concentrations Use this value to calculate the volume and number of flasks required for characterization Suggestion when using T flasks determine the number of days that will transpire from inoculat
78. ration s is somewhat below the target 50 75 when the metabolite concentration s is significantly below the target See Setting the Media Motor Speed below for instructions T Once the cultures mass in the bioreactors appears to be at equilibrium don t further increase the Media Pump speed because the run should shift from the growth to the production phase Pumping media faster may result in higher nutrient consumption but usually does not provide higher product yield Use Visual Observations to Determine Media Rate Visual observations are an easy but not the best method to assess the basal medium s condition Relying on visual observation can lead to poor and inconsistent results However this method is adequate if you do not need to optimize production If optimizing production is desired monitoring metabolite concentration s ensures better and consistent results which can be especially useful if the process eventually will scale up to larger hollow fiber production systems Visual observations require that the cell culture medium contains phenol red Visual observations require that the cell culture utilizes glucose as the primary energy source Do not use this method when glutamine is the culture s primary energy source 1D Increase the Media Pump speed depending on the amount of color change to the medium 25 50 when the discoloration is low to moderate 50 75 when the discoloration is moderate to high
79. rbed ends 3 Conect the iver tings together Pace ech amamah Rs own tronpatertatodoable pacta 5 Autoclave the tems using standard dry goods ce parameters 6 The fittings and tubing provide a large area for your fingers to grasp without Touching ipe center Luer adapters during their Connection othe MU Media and Outflow containers etc Page 62 of 69 Mult 6 User Manual 700466 000 Rev B Blovest Intemational Items available from other suppliers Suggested items are illustrated below along with an example supplier s name and ordering part number Use this information to source equivalent items from your preferred supplier While Biovest does not endorse any specific supplier or option we can assist you with questions about suitability ofan item for use with the Mult 6 Carboy Components 1 two 3219 000 wwwnalgenelabware com 2 Two 10210 006 Polycarbonate Carboys 10L 2251 0020201 2251 0050 Polypropylene Carboys OL 2250 0020201 2250 0030801 225040130 1 Three 100211 006 2Two 100553000 h Carboy Cap 83B Three V Ports Polypropylene S Two pieces of 2 2162 0831 fts al of the carboys listed above remse Tubing 4 One piece ota 1 Remove the tubing pieces that are provided on the caps ae external ports They are too short 5 One 1825 000 2 Donot use Vi internal diameter tubing It will not fit tightly on the V external ports al 3 Purchase platinum cured silicone tubing or C lex tubing wit
80. re was performed in the hood Log the volumes of collected supernatant and injected Factor into the metabolic data record 8 pe i Mult 6 User Manual Page 57 of 69 700466 000 Rev B Blovest International Optimization Tips amp Troubleshooting Optimization is optional Generalized protocols and inerease yield while reducing media often successfully yield a good amount of protein consumption When new cells lines routinely are being cultured once or twice only optimization in a sense is finding a general protocol that works with various similar cell lines even if it s not ideal for any one line specifically When a cell line is cultured several times then it is possible to optimize the process Following are areas to consider if optimization is pertinent to your application It may be helpful to discuss your goals and cell lines with Biovest s Account Services for assistance with optimization See the Cell Line Characterization worksheet a the end of this user manual Optimization Tips This tip is often overlooked and often significant Consistently passage the T flask roller bottle spinner scale up culture to maintain mid log phase growth Inconsistent scale up conditions can lead to long term differences in the Multi 6 culture Determine the conditions that correspond to mid log phase growth in static culture and use this information in developing the culturing strategy for the growth phase of the Mult
81. s larger seale equipment Page 4 of 69 Mult 6 User Manual 700466 000 Rev B Blovest Intemational About this Manual This User Manual will guide you through the concepts and proper steps to effectively use the Mult 6 including set up inoculation and operation There ae several hyperlinks within this User Manual Excluding graphics hyperlinks are indicated by blue text tems in the Table of Contents are hyperlinks to the corresponding section in the manual The footers on each page are hyperlinks back to the Table of Contents Other hyperlinks are provided to speed navigation between specific topics within the manual Blue text in figures are not hyperlinks This user manual applies to using the Mult 6 part number 60309 205 see Ordering Information below Support 1 you have any technical questions after reading this User Manual please contact Account Services for assistance Account Services alsa provides customer service for placing orders Ordering Information Multi 6 the complete pre assembled pre sterilized single use hollow fiber cultureware Its ordering part number is 600309 206 Other potentially necessary items are described in the Parts List We offer many other items not in the Parts List that may simplify using the Multi 6 or make using it more adapted to the layout of your laboratory or equipment Contact Biovest s Account Services for assistance Mult 6 User Manual Page Sof 69 700466 000 R
82. suspension and anchorage dependent cells to very high cell densities The six bioreactors are connected in parallel to ensure they receive equal nutrient and oxygen delivery Multi6 is maintained in a COs incubator to control pH and temperature see Figure 1 Basal media and Outflow containers and the two pump motors remain outside the CO incubator The inclusion of basal media and outflow containers and pumps allow long term unattended operation that greatly reduces technician support time and the risk of contamination compared to other cell culture methods Multi 6 Design Overview Mutt maintains cells inan optimal environment one that mimics the mammalian body In 1 fact we use the analogy of a Be nn body to describe how the Multi 6 i functions Cells are grown in the EC space surrounding the outside of the hollow fibers the capillaries le Fior port of the bioreactor The culture is crga ee Tip eror supported by the other 5 Pa parn jd Components Cell culture Eie Stem fones tu the nie cee iC Circut ofthe hollow fibers to carry Inside fresh nutrients and oxygen to EC Circuit CO2 Incubator the cells while carrying away cell F waste products such as lactate Figure 1 Page 6 of 69 Mult 6 User Manual 700466 000 Rev B Blovest international Ics ammonia and CO The Circulation Pump acts as the heart to circulate medium through the IC Circuit The Gassing Pump and
83. tainer loosen the male female Iuer connection of the filled Outflow Container connected to the Muli 6 Outflow Line Remove and discard the necessary items to be able to connect the male luer of the empty Outflow Container to the female luer of the Multi 6 SOutflovy Line Page 41 of 69 Biovest s proprietary ready for use Cap Assembly Seo Parts List for ordering information Notes 1 Rigid media bottles require asterilzing grade entation ster 2 IN port leads tothe cap internal short tubing unseen in illustration that does not reach the bottom ofthe bome 3 The bottle cap s unused port remains cased with a male luer plug Outfiow Container Multi 6 s Outflow line s male luer taper fitting connects to the Outflow Container Cap s IN port Figure 19 Page 42 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Outflow Container with Male Luer Connectors outflow Container with Female Luer Connectors See Parts Uist for male Luer adapter Outiow Container Either type of Outflow Container s male luer connects to Multi 6 s Outflow tubing s female luer Figure 20 Mult 6 User Manual Page 43 of 69 700466 000 Rev B Blovest International Alternative Method to Connect the Media and Outflow Containers Connecting the Media and Outflow containersas The following alternative connection method described in the previous section is the best method while not
84. te and Circulation Rate for further information CAUTION Whenever you move the Multi 6 ensure the tubing lines stay below the height of the Mult 6 to prevent their volume from draining into and overfilling the Media Reservoir NOTE The Media Container and Outflow Container remains at room temperature See Media Container Size for further information Page 24 of 68 Mult 6 User Manual 700466 000 Rev B Blovest international Moving Multi 6 into the laminar flow hood Mult 6 is retuned to the laminar flow hood for With experience these tasks can be performed Sampling Inoculation Changing Media amp Outflow without removing it from the incubator but the containers injecting Factor into the ECS and risk of contamination is higher than when done in withdrawing harvest supernatant from the ECS the laminar low hood Important Points Minimize the amount of time the Multi is out of the CO incubator to minimize temperature changes to the cell culture Minimize the amount of time the circulation pump is not running Like one s heart the circulation pump maintains oxygenation and pH control Oxygen concentration and pH rapidly decrease when the Circulation Pump is off particularly with high cell numbers Procedure DD Stop the Media Motor and Circulation Motor DD Close the Media and Outflow clamps O Open the four pump heads 1D Remove the pump tubing from the pump heads CAUTION W
85. th for several passages and havea viability greater than 40 Ifa significantly igher number of cells are inoculated a higher initial Media pump rate of basal medium may be beneficial Important Points Perform the following steps in a laminar flow hood and follow standard procedures for disinfecting items entering the hood and handling items within the hood The following procedure describes how to inoculate a bioreactor The same steps are used whether six cell lines will be inoculated into the six bioreactors or fewer cell lines will be used to inoculate the bioreactors Ech bioreactor should be inoculated with 2 108 viable cells until data are available that suggest a smaller or larger inoculum is preferable Prepare the scale up culture volume s according to the numberof cell ines to be inoculated Procedure DD Concentrate the scale up culture via centrifugation using routine methods and supplies 1D Decant the supernatant into a sterile container DD Resuspend the cell pellet in 15 mL of conditioned medium and keep the remaining supernatant sterile O Draw the 15 mL inoculum into a syringe 1D Prepare a 3 mL syringe with fresh medium 1D Disinfect the Multi 6 and bring it into the laminar flow hood 9 Disinfect the EC Factor and Harvest ports of one bioreactor T Remove the syringe from the Harvest Port and connect the inoculum syringe 9 Remove the syringe from the EC Factor Port and connect an empty syringe 15
86. the IC The MP rate is quite slow at the time of inoculation and incteases as the culture expands The technician adjusts MP speed based on color change of the medium or preferably from analysis of metabolites from an IC medium sample removed aseptically from the flowpath Regulating the MP speed allows the technician to increase media delivery and control glucose glutamine lactate and other metabolite concentrations witha simple pump speed adjustment rather than performing time intensive manual emptying and filling of media bottles The MP is a peristaltic positive displacement pump that is self priming The Media Container can be either a large volume vented bottle or carboy or it can be a large volume non vented flexible bag Such containers allow many days of unattended operation to minimize technician handling time and contamination risk Outflow Pump and Outflow Container The Outflow Pump OP removes consumed basal medium from the Media Reservoir at the same rate as the Media Pump adds it into the reservoir The OP also is a self priming peristaltic pump The Outflow Container can be either a large volume vented bottle or carbay or it can be a large volume vented flexible bag Such containers allow many days of unattended operation to minimize technician handling time and contamination risk Circulation Pump The Circulation Pump provides a high fow rate of medium through the IC to provide sufficient pH control and
87. ti 6 Completely assembled and sterile product 1825 000Steriizing grade ventilation filter 50 pack 600017 000101 Glass Bottle 4 pack 600022 000Delivery cap assembly for 10L glass bottle pack requires separate purchase of 1825 000 600023 000Receiving cap assembly for 10L glass bottle 4 pack requires separate purchase of 1825 000 400100 072Sterile tubing extension line 72 183 em long one each Male luer lock on one to connect to Multi 6 600309 205 Mult 6 Female luer on other end to connect to syringes or Media and Outflow containers Suggested Uses 1 Connect one end to IC Sample Line Connect other end to syringes Allows extended IC Sample Line to reach into laminar flow hood when necessary for aseptic sample removal 2 Connect to one end to Multi 65 Media Line or Outflow Line Connect other end to Media or Outflow container Allows extended Media or Outflow Line to reach into laminar flow hood Change container aseptically in laminar fow hood without moving Multi from incubator 102360 008Red lock ring Nylon Rotating Snap fi mount Auteclavable one each Usage hand press twa on to one 103247000 Converts both ends of 103247 000 toa locking male luer connector 103247 000Male luer adapter Polypropylene Snap fit mount Autoclavable one each Usage connect a Media or Outflow container that has only a female luer to the female uer of the Mult 6 Media Line or Outflow Line 5199 000Female luer adapter Polypropylene Autocl
88. wrapping and blue tape T Set the four coils of tubing next to the Multi6 as is shown in Figure 13 Place the IC Sample line not shown in figure 13 where it can be inspected Ensure the following plastic pinch clamps are open Contact Biovest s Account Services department if a clamp is closed 1D Media Clamp 1D Outflow Clamp O Gassing Clamp 1D IcSample Clamp EC Factor Clamps there are six near red luer fittings Q Harvest Clamps there are six near green luer fittings Ensure all luer fittings are not loose Do not over tighten them or they can become difficult to open when necessary 9 Media Line 1D Outflow Line O 1C Sample Line TEC Factor Lines the six red luer fittings DD Harvest Lines the six green luer fittings Close the following clamps 1D Six Harvest Clamps 1D Six EC Factor Clamps 1 1C Sample Clamp Dy Suggested Replace 13 male luer fittings and sterile covers six each for EC Factor amp Harvest and one for IC Sample with Clearlink connectors Clearlink reduces but does not eliminate the risk o contamination when connecting syringes or containers to these ports outside of a laminar flow hood The advantages of their use also include reduced handling time and complication throughout the run Refer to the insert sheet provided with the Clearlink connectors for usage information 9 Leave the Mult 6 in the laminar flow hood Itis ready for the Fill amp Flush procedure Figure 13 Mu
89. y made to withstand the mechanical rigor of the pump head Its Not necessary to shift new portions of the PharMed tubing into the pump head throughout the run CAUTION Do NOT load the off white silicone tubing in the pump head because it quickly will be damaged causing cell culture medium to leak Media Pump mounted in back Outflow Pump mounted in front Media toward p al Media Tubing _ Siow tom es Outflow from Ifthe mator tums clockwise orient both Multi 6 pump segments in the oppasite direction be shown NA Tubing not shown at actual lengths Multi 6 re Figure 14 Mult 6 User Manual Page 23 of 69 700466 000 Rev B Blovest International Loading Mult 6 s Circulation and Gassing Tubing in the Circulation Pump and Gassing Pump 9 Locate the Circulation Tubing s size 36 pump segment which is the yellow tubing marked 06508 36 PhaarMed This pump segment creates the circulating flow of cell culture medium through the IC Circuit of the Mult DD Locate the Gassing Tubing s size 17 pump segment which is the yellow tubing marked 06508 17 hard This pump segment creates the flow of incubator gases through the gas exchanger of the Mult for pH control and oxygenation T There are two directional arrow labels near each end of the Circulation pump segment and one directional arrow label near the Gassing pump segment These labels indicate the required direction of luid gas
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