Suspension Culture raav-modified GS -/- CHO Cell Line

Transcription

1 Suspension Culture raav-modified GS -/- CHO Cell Line CATALOG NO. HD-BIOP3 1.0 Product Description/Overview The raav-modified GS -/- CHO (Chinese Hamster Ovary) cell line HD-BIOP1 was created using Horizon s proprietary recombinant Adeno-Associated Virus (raav) technology. These cells were adapted to suspension growth in animal component free, chemically defined media and designated HD-BIOP3. raav homologous recombination vectors are more than 1000 times more efficient at gene-targeting than plasmid based methods. Using homologous recombination based gene targeting does not induce double strand breaks or activate the error prone DNA repair pathways, allowing genetic alteration to base pair resolution. Glutamine synthetase (GS) is one of the most commonly used selectable markers in the biopharmaceutical industry. The GS pathway converts glutamate into glutamine. Cells that lack the GS enzyme must be grown in media supplemented with glutamine to survive. Methionine Sulphoximine (MSX) inhibits the GS pathway. In cells that have a functional GS gene, MSX can be used to suppress endogenous GS activity. MSX can also be used to select producing clones. However, MSX is a toxic chemical, and cannot be used in drug production. An MSX-free process is advantageous, and to do that, a GS knock-out cell line is needed. The GS -/- cells were generated by removal of exon 6 of the Glutamine Synthetase gene in CHO K1 cells. These knockout cells have been adapted to suspension growth in commercially available, animal component free media. This biomanufacturing ready cell line can be used in MSX-free selection processes for the generation of high producing recombinant cell lines.

2 1.0 PRODUCT DESCRIPTION/OVERVIEW PRECAUTIONS AND DISCLAIMER STORAGE AND STABILITY CONTENTS BACKGROUND: GS -/- CELL LINE REQUIRED CELL CULTURE REAGENTS EQUIPMENT/MATERIALS NEEDED PART I: MEDIA PREPARATION FOR HD-BIOP3 CELLS PURPOSE REAGENTS AND EQUIPMENT PROCEDURE The following procedure is for the preparation of 1 L of growth media The following procedure is for the preparation of 1 L of selection media The following procedure is for the preparation of 1 L of transfection media for use in PEI mediated transfection PART II: STOCK CULTURE INITIATION / THAWING OF GS -/- CELLS PURPOSE REAGENTS AND EQUIPMENT PROCEDURE PART III: STOCK MAINTENANCE / SUBCULTURING OF GS -/- CELLS PURPOSE REAGENTS AND EQUIPMENT PROCEDURE (TO BE PERFORMED EVERY 3-4 DAYS) PART IV: CELL BANKING OF HD-BIOP3 CELLS PURPOSE REAGENTS AND EQUIPMENT PROCEDURE PART V: TRANSFECTION AND SELECTION PURPOSE REAGENTS AND EQUIPMENT PROCEDURE Electroporation PEI mediated transfection Selection Clonal dilution PART VI: SMALL SCALE FED-BATCH CULTURING PURPOSE REAGENTS AND EQUIPMENT PROCEDURE

3 Day 0 Set up Feed Screening Assay sampling Assay termination PART VII: BIOREACTOR CULTURE

4 2.0 Precautions and Disclaimer Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. 3.0 Storage and Stability Store the cells in the vapor phase of liquid nitrogen immediately upon arrival. 3.1 Contents Component Catalog Number Quantity GS -/- cell line HD-BIOP3 1 Vial of cells with >1x10 7 cells/ml 4.0 Background: GS -/- cell line The GS -/- cell line was generated using Horizon s raav technology. The starting cell line was ECACC CHO K1. The cell line was transfected with an raav vector containing homology arms targeting the sequences flanking exon 6 of the CHO GS gene. This is the exon that codes for the substrate binding domain of the GS enzyme, therefore deletion of this exon results in non-functional protein. The transfected pool was cultured under selection for two weeks before being single cell cloned, followed by sequencing to confirm deletion at the target site. The second allele was targeted using the same strategy. Selection cassettes were removed by cre-mediated excision. Several single cell clones were isolated that contained biallelic knockout mutations at the GS locus. After extensive characterization of the clones, one clone was identified as having more robust characteristics than the others. The clone ID for this GS -/- cell line is 2C9-1A5 which was given the catalogue number HD-BIOP1. This clone was adapted to suspension growth in commercially available, chemically defined, animal component free media and was defined as HD-BIOP3. Figure 1. This is a schematic representation of the targeting strategy for removal of exon 6 from the CHO GS gene, which codes for the substrate binding domain of the protein. 4

5 5.0 Required Cell Culture Reagents RECOMMENDED CULTURING CONDITIONS Cell culture reagents Manufacturer Cat. No. GS -/- cells Horizon Discovery HD-BIOP3 CD FortiCHO TM media Life Technologies A CD CHO media Life Technologies DMSO Sigma-Aldrich D2438 L-Glutamine or GlutaMAX Life Technologies Opti-MEM I Reduced Serum Media Life Technologies Freestyle TM MAX Reagent Life Technologies Albucult Sigma A mg HT Supplement Life Technologies Fatty acid supplement Sigma F7050 Synthetic Cholesterol Sigma S5442 Anti-clumping agent Life Technologies AE 5.1 Equipment/materials needed 100% ethanol 100% isopropanol 15 ml sterile conical tube 384 well microtiter plate, tissue culture treated 50 ml sterile conical tube 6 well microtiter plate, tissue culture treated 70% ethanol 75cm 2 vented culture flask 96 well microtiter plate, tissue culture treated Amaxa 4D-Nucleofector Automated cell counter or hemocytometer Biosafety cabinet Centrifuge CO 2 incubator, humidified (5% CO 2, 37 C) Cryovial labels (must be LN 2 resistant) Erlenmeyer flasks 5

6 Freezer (-20 C) Freezer buddies with isopropanol or controlled rate freezer LN 2 freezer LN 2 freezer boxes Micropipetters and sterile tips Refrigerator SF Cell Line 4D-Nucleofector X Kit L Shaking CO2 incubator (5% CO2, 37 o C) Sterile cryovials Sterile 1.5 ml tubes Sterile filter: 1000 ml capacity Sterile pipettes Ultra cold freezer (-80 C) Water bath at 37 C NOTE: The following procedures should be performed only by personnel trained to: Work with bio hazardous materials Use Universal Precautions Use aseptic technique NOTE: All cell culture and media handling in these protocols must be carried out in a sterile biosafety cabinet. 6

7 6.0 Part I: Media Preparation for HD-BIOP3 Cells 6.1 Purpose This protocol describes procedures for creating growth media for use with HD-BIOP3 cells. 6.2 Reagents and equipment 70% ethanol Biosafety cabinet CD FortiCHO TM media CD CHO TM media Freezer (-20 C) L-Glutamine or GlutaMAX Albucult HT supplement Fatty acid supplement Synthetic Cholesterol Anti-clumping agent Refrigerator Sterile filter (1000 ml capacity) Sterile pipettes Water bath at 37 C 6.3 Procedure The following procedure is for the preparation of 1 L of growth media i) Thaw L-Glutamine if required in a 37 C water bath until completely thawed. ii) Add Glutamine to CD FortiCHO TM media to a final concentration of 4 mm iii) Filter media through 1000 ml capacity 0.22 µm filter. iv) Mark the date on the media bottle and store media in fridge until needed. v) Discard unused media after one month The following procedure is for the preparation of 1 L of selection media i) Filter CD CHO TM media through 1000 ml capacity 0.22 µm filter ii) Mark the date on the media bottle and store media in fridge until needed. iii) Discard unused media after one month The following procedure is for the preparation of 1 L of transfection media for use in lipidbased transfection i) Thaw L-Glutamine if required in a 37 C water bath until completely thawed. ii) Add Glutamine to CD CHO TM media to a final concentration of 4 mm iii) Filter media through 1000 ml capacity 0.22 µm filter. iv) Mark the date on the media bottle and store media in fridge until needed. 7

8 v) Discard unused media after one month. RECOMMENDED CULTURING CONDITIONS The following procedure is for the preparation of 1 L of limiting dilution media i) Thaw reagents as required in a 37 C water bath until completely thawed. ii) Add excipients (table below) to CD FortiCHO medium according to the values given. iii) Filter media through 1000 ml capacity 0.22 µm filter. iv) Mark the date on the media bottle and store media in fridge until needed. v) Use media on day of manufacture. Cell culture reagents Original concentration Final Concentration Volume (ml) CD FortiCHO medium N/A 1X L-Glutamine 200 mm 4 mm 20 GlutaMAX 200 mm 2 mm 10 Albucult 100 mg/ml 1 mg/ml 10 HT Supplement 100X 1X 10 Fatty acid supplement 4000X 1X 0.25 Synthetic Cholesterol 500X 1X 2 Anti-clumping agent 1000X 1X 1 8

9 7.0 Part II: Stock Culture Initiation / Thawing of GS -/- Cells 7.1 Purpose This protocol describes procedures for the stock initiation of GS -/- cells. 7.2 Reagents and Equipment 125 ml Erlenmeyer flask 15 ml sterile conical tube 70% ethanol Automated cell counter or hemocytometer Biosafety cabinet Cell culture growth media (prepared as per section 6.3.1) Centrifuge CO 2 incubator, humidified (5% CO 2, 37 C) Frozen vial of HD-BIOP3 cells Sterile pipettes Water bath at 37 C 7.3 Procedure i) Adjust incubator settings to 37 C and 5% CO 2 with shaking set to 125 rpm and ensure there is water for humidity control (~85%). ii) Warm growth media to room temperature or to 37 C in a water bath. iii) Obtain sterile 15 ml conical centrifuge tube. iv) Transfer 10 ml of sterile growth media to the 15 ml conical tube. v) Transfer 20 ml of sterile growth media to the 125 ml Erlenmeyer flask. vi) Obtain frozen vial from LN 2 freezer. vii) Immediately thaw vial in 37 C water bath until just thawed. viii) Spray the vial with a copious amount of 70% ethanol and place in the biosafety cabinet. ix) Transfer cells from the cryovial to the 15 ml conical centrifuge tube containing fresh media. x) Centrifuge at 220 rcf for 5min at room temperature to pellet the cells. xi) Carefully aspirate off the supernatant without disturbing the cell pellet. xii) Add 10 ml fresh growth media to the conical centrifuge tube and gently resuspend the cell pellet by pipetting. xiii) Seed cells in the 125 ml Erlenmeyer flask containing 20 ml growth media 9

10 8.0 Part III: Stock Maintenance / Subculturing of GS -/- Cells 8.1 Purpose This protocol describes procedures for stock maintenance/scale-up of HD-BIOP3 cells. 8.2 Reagents and Equipment 70% ethanol Automated cell counter or hemocytometer Biosafety cabinet Cell culture growth media (prepared as per section 6.3.1) CO 2 incubator (5% CO 2, 37 C) Erlenmeyer Flasks Sterile pipettes Water bath at 37 C 8.3 Procedure (to be performed every 3-4 days) i) Verify that the incubator is set to 37 C and 5% CO 2 with shaking set to 125 rpm and has water for humidity control (~85%). ii) Warm growth media to room temperature or to 37 C in a water bath. iii) Remove stock cell culture(s) from incubator. Place the flask in the Biosafety cabinet and swirl to ensure that all the cells are suspended. iv) Aseptically remove a cell culture sample and count by trypan blue exclusion using a hemocytometer or an automated cell counter. v) Take note of viable cell density (VCD) and viability (%V). VCD should be within logarithmic phase of growth, and %V should be 90%. Cells should not be allowed to exceed a density of 6x10 6 cells/ml during sub-culturing vi) Calculate cell volume needed to obtain x 10 5 cells/ml. Use the following working volumes for the appropriately sized culture vessel (see table on p11) vii) Aseptically transfer calculated volume of stock culture to an appropriately sized Erlenmeyer flask with prewarmed media. viii) For cell culture scale up, repeat protocol steps 2 7 until desired numbers of cells needed for medium/feed screen are achieved 10

11 Corning Polycarbonate Erlenmeyer Flasks Capacity Recommended Working Volume* Approx. Height (A) Approx. Width (B) 125 ml ml 114 mm 66 mm 250 ml mL 138 mm 83 mm 500 ml ml 178 mm 101 mm 1 L ml 207 mm 128 mm 2 L ml 291 mm 163 mm 3 L ml 255 mm 233 mm * Baffled flasks may allow higher working volumes or low shaking speeds; recommend testing for optimum culture parameters. 11

12 9.0 Part IV: Cell Banking of HD-BIOP3 Cells 9.1 Purpose This protocol details procedures for establishing a working cell bank of GS -/- cells. 9.2 Reagents and Equipment 100% ethanol 100% isopropanol 15 ml sterile conical tubes 50 ml sterile conical tubes Automated cell counter or hemocytometer Cell culture growth media (prepared as per section 6.3.1) Centrifuge Cryovial labels (must be LN 2 resistant) DMSO Ultra Cold Freezer (-80 C) Freezer buddies or controlled rate freezer GS -/- stock cell culture LN 2 freezer LN 2 freezer boxes Sterile cryovials Sterile pipettes 9.3 Procedure i) If using freezer buddies, fill with fresh 100% isopropanol. ii) Label cryovials. iii) Prepare freezing media: growth media + 7.5% DMSO. iv) Aseptically remove a cell culture sample from the culture flask and count by trypan blue exclusion using a hemocytometer or an automated cell counter. Do not proceed if cell viability is less than 90%. v) Calculate the volume of cell stock and freezing media needed to obtain 1x10 7 viable cells per cryovial (1 ml volume). vi) Note: Once cell preparation is initiated, work must proceed quickly to get vials into freezer. It is recommended that the total time from removing cells from the stock culture to placing freezer buddies in the 80 C freezer take no more than 30 minutes. vii) Aseptically transfer calculated volume of stock culture to an appropriately sized conical centrifuge tube. viii) Centrifuge at 220 rcf for 5 min at room temperature. ix) Carefully aspirate off the supernatant without disturbing the cell pellet. x) Gently resuspend the cells with calculated volume of freezing media. xi) Mix thoroughly by pipetting. xii) Immediately aliquot cell suspension into labeled cryovials. Cap tightly. xiii) Transfer the vials to prepared freezer buddies and store in -80 C freezer, or transfer vials to a controlled rate freezer for overnight freezing. xiv) Transfer vials to LN 2 freezer within hours of freezing. 12

13 10.0 Part V: Transfection and Selection 10.1 Purpose This protocol describes the procedure for transfecting cells with plasmid using electroporation or lipid based methods. These parameters serve as a guide, and may need to be further optimized. Following transfection, cells are selected for those expressing sufficient amounts of Glutamine Synthetase to not be reliant on exogenous glutamine for viability Reagents and Equipment Automated cell counter or hemocytometer Biosafety cabinet Cell culture growth media (prepared as per section 6.3.1) Cell culture selection media (prepared as per section 6.3.2) Centrifuge Erlenmeyer Flasks 75cm 2 vented culture flasks GS -/- stock cell culture Plasmid DNA (Linearized, filter sterilized through a 0.22 µm filter) Shaking CO 2 incubator (5% CO 2, 37 C) Sterile pipettes For electroporation only: 6 well microtiter plate, tissue culture treated SF Cell Line 4D-Nucleofector X Kit L Amaxa 4D-Nucleofector For lipid-based transfection only Transfection media prepared as per section FreeStyle TM MAX Reagent (for lipid-based transfection) Opti-MEM I Reduced Serum Media Sterile conical tubes Sterile 1.5 ml tubes For clonal dilution 384 well microtiter plates, tissue culture treated Cell culture limiting dilution media (prepared as per section 6.3.4) 13

14 10.3 Procedure Electroporation using Amaxa 4D-Nucleofector and 100 μl Nucleocuvette -Lonza Two days before transfection i) Subculture the cells two days prior to electroporation to ensure log phase growth at time of transfection (e.g. seed at 3 x 10 5 cells/ml 48h before transfection) On the day of transfection: ii) Verify that the incubator is set to 37 C and 5% CO 2 and has water for humidity control (~85%). iii) Warm growth media with no antibiotics (prepared as per section 6.3.1) to room temperature or to 37 C in a water bath. iv) Aseptically remove a cell culture sample and count by trypan blue exclusion using a hemocytometer or an automated cell counter. Do not proceed if cell viability is less than 90%. For each transfection condition: v) Add 1 ml of pre-warm culture media in each well of a 6-well plate vi) Centrifuge 6 x 10 6 cells (5 minutes, 220 rcf) at room temperature vii) Prepare 600 µl of Nucleofector solution by mixing 492 µl of SF Cell line solution with 108 µl of Supplement viii) Resuspend 6 x 10 6 cells pellet in 600 µl of Nucleofector solution ix) Add 12 µg of plasmid DNA into 600 µl of transfection reaction (final concentration ~ 20 µg/ml) x) Transfer 100 µl of the transfection reaction containing cells and DNA into one Nucleocuvette (6 total) xi) Tap the cuvette to remove bubbles and place the Nucleocuvette into 4D-Nucleofection device xii) Perform Nucleofection using the program DT-133 xiii) Remove the cuvette and incubate for 10 minutes at room temperature xiv) Resuspend transfected cells in 500 µl pre-warmed media per nucleocuvette. Avoid repeated aspiration of the sample xv) Transfer transfected cells from each nucleocuvette into individual well of a 6-well plate containing 1 ml of prewarm culture media (Final volume ~1.6 ml) xvi) Incubate the cells in a humidified incubator at 37 C and 5% CO 2 until further analysis. xvii) Proceed to section Lipid-based transfection This protocol describes the transfection of CHO cells in 30ml of culture medium per transfection condition (plasmid/control). Depending on transfection efficiency a variety of transfection reagent:dna ratios should be tested using the same steps. 2-3 days before transfection: i) Subculture the cells at 1.3 x 10 5 cells/ml in growth media. Cells should be greater than 90% confluence and should have been in culture for more than 3 passages. The day before transfection: i) Prepare and pre-warm transfection media, remove antibiotics if previously included. ii) Aseptically remove a cell culture sample from the culture flask and count by trypan blue exclusion using a 14

15 hemocytometer or an automated cell counter. Do not proceed if cell viability is less than 90%. iii) Transfer enough cells for a final density of 6 x 10 5 cells/ml in 22 ml per transfection condition. iv) Harvest cells by centrifugation (5 min, 100 rcf), resuspend pellet in transfection media and transfer to appropriately-sized culture flask/s. v) Incubate cells overnight in a humidified incubator at 37 C and 5% CO 2 with shaking set to 125 rpm. On the day of transfection: i) Pre-warm transfection media, bring all other reagents to room temperature. ii) Dilute 37.5 µl FreeStyle TM MAX Reagent in 600 µl Opti-MEM I Reduced Serum Media per transfection condition. If preparing multiple transfections, prepare a mastermix in a sterile conical tube. iii) In a sterile 1.5 ml tube, dilute 37.5 µg of linearized plasmid DNA in 600 µl Opti-MEM I Reduced Serum Media per transfection condition (this media must not contain anti-foam agents such as Pluronic F-68 or any antibiotics). Prepare a mock-transfection condition by omitting DNA from 1 tube. iv) Immediately add diluted FreeStyle TM MAX Reagent to diluted DNA solution, keeping the pipette tip just below the meniscus. Incubate for at least 10 minutes at room temperature but not more than 20 minutes. v) Whilst the transfection mix is incubating, aseptically remove a cell culture sample from the culture flask and count by trypan blue exclusion using a hemocytometer or an automated cell counter. Do not proceed if cell viability is less than 95%. The cell density should be in the region of x 10 6 cells/ml. vi) Aliquot 30ml cells at 1 x 10 6 cells/ml into 1 vented 125 ml culture flask per transfection condition. vii) Aseptically transfer 1.2 ml FreeStyle TM MAX Reagent /DNA transfection mix to each cell flask and swirl to homogenize the culture. viii) Incubate in a humidified incubator at 37 C and 5% CO 2 with shaking set to 125 rpm for 48 hours. ix) Proceed to section Selection i) After 48 hours viability should be assessed through aseptic removal of a cell culture sample and counting by trypan blue exclusion using a hemocytometer or an automated cell counter. ii) Centrifuge 20 x 10 6 cells (5 min, 100 rcf) and resuspend in 40 ml selection media. iii) Split cells across 2 x 75cm 2 vented culture flasks per transfection condition. iv) Incubate in a humidified static incubator at 37 C and 5% CO 2. v) Every 5 days, count cells and replace selection media (remove selection media, centrifuge at 100 rcf for 5 min and resuspend cells in 20 ml fresh selection media per flask). For cell counts scrape the bottom of the flask and take a sample for counting prior to media removal. vi) Once cells have begun to recover, cryofreeze cells and proceed to clonal dilution Clonal dilution The day before clonal dilution: i) Subculture the cells one day prior to single cell dilution to ensure log phase growth at time of clonal dilution (e.g. seed at 1.3 x 10 5 cells/ml 24h before clonal dilution). Subculturing the cells 48h or 72h prior clonal dilution greatly reduces clone survival rate. On the day of clonal dilution ii) Generate an intermediate working dilution by mixing 1 x 10 4 cells in 1 ml of clonal dilution media 15

16 iii) Dilute previous working dilution 1000 times in clonal dilution media to obtained a cell density of 10 cells/ml iv) Dispense 50 µl of cell suspension into individual wells (0.5 cell/well) of as many 384 well plates as are required to obtain a suitable number of clones for productivity analysis v) Incubate plate up to days without changing medium vi) Observe for growth of isolated clones vii) Subculture cells as described in section 8 Part VI: Small Scale Fed-Batch culturing 10.4 Purpose This protocol details procedures for fed-batch culturing of HD-BIOP3 in Erlenmeyer flasks Reagents and equipment 70% ethanol Automated cell counter or hemocytometer Biosafety cabinet Cell culture growth media (prepared as per section 6.3.1) CO 2 incubator (5% CO 2, 37 C) Erlenmeyer Flasks Sterile pipettes Water bath at 37 C 10.6 Procedure Day 0 Set up i) Verify that the incubator is set to 37 C, 5% CO 2, and has water for humidity control (~85%). ii) Warm growth media to room temperature or to 37 C in a water bath. iii) Remove stock cell culture(s) from incubator. Place the flask in the Biosafety cabinet and swirl to ensure that all the cells are suspended. iv) Aseptically remove a cell culture sample and count by trypan blue exclusion using a hemocytometer or an automated cell counter. v) Take note of viable cell density (VCD) and viability (%V). VCD should be within logarithmic phase of growth, and %V should be 90%. vi) Calculate cell volume needed to obtain 0.8 x 10 6 cells/ml vii) Aseptically transfer calculated volume of stock culture to an appropriately sized Erlenmeyer flask with prewarmed media viii) Place assay tubes into incubator. A Day 0 count is optional Feed Screening i) It is recommended that the culture should be fed glucose so that is not limiting the performance of media/feeds in the screening experiment ii) As a starting condition, on Day 4, Day 6 and Day 8, add 5-10% feed. Feed addition optimization can be performed based on the strategies in the table on p16. iii) Add glucose to maintain the levels at 4 g/l 16

17 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Option % 5.00% 5.00% Option % 5.00% 5.00% Option % 5.00% 5.00% Option % 7.50% 7.50% Option % 7.50% 7.50% Option % 10.00% 10.00% Option % 10.00% 10.00% Option % 10.00% 10.00% Assay sampling i) To be performed daily from Day 1 to Day 14 ii) Triturate the cell suspension before aseptically removing a cell culture sample and counting by trypan blue exclusion using a hemocytometer or an automated cell counter. iii) Record viable cell density (VCD) and viability (%V). iv) On days 3, 6, 9, 12 and 14, collect ml of spent media for productivity analysis and transfer to an eppendorf tube. Return cell cultures to the incubator without disturbing the remainder of the culture. v) Centrifuge at 15,000 rcf for 5 minutes to pellet cells and debris. vi) Transfer supernatant into new sample collection tube. vii) Freeze sample immediately at -20 C Assay termination i) Repeat assay sampling steps until day 14 or viability is <60%. ii) After collection of all samples on the final day of the assay, terminate the cultures. iii) Submit spent media samples for productivity quantification. 17

18 11.0 Part VII: Bioreactor culture RECOMMENDED CULTURING CONDITIONS NOTE: Although process parameters may be significantly different when using different bioreactor systems the below parameters can be used as a starting point for optimization. Parameters Temperature Suggested Settings 37 o C with no temperature shift CO2 5.00% ph ph 6.9 +/- 0.2 with no ph shift DO 50.00% RPM Calculated by N=(1.3/D2)1/3 constant P/V = 1.3, N is propeller rotation per min, D is the tip diameter of a down flow marine impeller design i) Remove the appropriate number of vials from your Working Cell Bank to thaw directly into the media as described in section 7. ii) Subculture as described until desired volume and biomass has accumulated to enable inoculation of the bioreactor at 0.8 x 10 6 cells/ml. iii) One day before the cells reach inoculation density, warm the growth medium to 37 C and polarize the DO probe. Pre-warm medium at 37 C for 24 hours in the bioreactor to stabilize ph and DO2 level. iv) Inoculate bioreactor by seeding cells directly into medium at a starting cell density of 0.8 x 10 6 cells/ml. v) Monitor glucose levels and feed cultures by aseptic addition of sterile glucose to 4g/L and of sterile glutamine to 3 mm. vi) Feed supplementation should be administered by addition of 5-10% of starting volume of medium on days 4, 6 and 8 post inoculation. vii) Starting at day 0, collect 5-10 ml of spent media for productivity analysis and cell counts as described in viii) Centrifuge productivity samples at 15,000 rcf for 5 minutes to pellet cells and debris. Transfer supernatant into new sample collection tube. Freeze sample immediately at -20 C. ix) Repeat steps VCD and productivity Assay sampling until day 14 or %V is <60%. x) After collection of all samples on the final day of the assay, terminate the cultures. xi) Submit spent media samples for productivity quantification. 18