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Bio manufacturing summit croughan et al jan 2010

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Bio manufacturing summit croughan et al jan 2010

  1. 1. Optimizing Overall ManufacturingSystem Performance through theUse of Animal-free Cell CultureSupplementsKiriLynn Svay, Jeff Rosenbloom,Delyan Rusev, and Matt CroughanAmgen Bioprocessing CenterKeck Graduate Institute, Claremont, CABioManufacturing Summit, January 27, 2010
  2. 2. 25 Years of Progress in Clinical Protein Production from Recombinant CHO Cells in Batch and Fedbatch Culture Professor Matt Croughan, Keck Graduate Institute, Claremont, CA 100000 New to Technology >2 Years ExperienceProduct Concentration at Harvest (mg/L) 10000 0.205x y = 9.941e 2 R = 0.7939 1000 100 10 0.3477x y = 0.082e 2 R = 0.9593 1 0.1 0 5 10 15 20 25 30 Years Since 1980
  3. 3. Analysis of Variance for Experienced Firms Professor M Croughan, Keck Graduate Institute att 100.0Percent CV for Product Concentration at Harvest 90.0 80.0 y = -0.0804x + 50.266 70.0 2 R = 0.0005 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0 5 10 15 20 25 30 Years Since 1980
  4. 4. “..It” flows down hill Consequences of 10g/L Cell Culture 10g/L • Cell density (volume) • Cell debris • Product Mass • HMWPurification •Drug Product Development •From Jon Coffman
  5. 5. Fed-batch cell culture in 1990
  6. 6. Cell culture strategies to optimizeoverall system performance Consistent and sufficient titers ◦ Every process, every time Shorter culture duration ◦ Higher vol. productivity, lower contamination risk Reduced cell death ◦ Lower degradative enzyme levels ◦ Lower HCP and debris loads downstream Improved downstream processing ◦ Higher yields or fewer/simpler steps Improved product quality and/or stability
  7. 7. Recombinant Human Serum Albumin (rHSA) made inan animal free production host by InVitria(www.InVitria.com)Roles of Albumin in Cell Culture:◦ Binding and transport mechanism Supply of Lipids Vitamins Hormones◦ Buffering agent◦ Detoxifying agent◦ Protectant from shear
  8. 8. Recombinant Lactoferrin is a naturally occurring iron-binding protein that was made in an animal freeproduction system by InVitria (www.InVitria.com)Advantages of Lactoferrin in Cell Culture◦ Can be used as a growth factor◦ Protects against oxidation due to Fe3+ ions◦ Microbial deterrent◦ Iron transport to cells
  9. 9. Impact of Cellastim/Lacromin Supplements on CHO Cell Culture in CD medium % Improvement, Supplemented/Control (data from five separate experiments) Peak viable cell density (VCD) ◦ Average: 28%, Range: 2 - 47% Peak titer (product concentration) ◦ Average: 40%, Range: 2 – 88% Volumetric productivity at peak titer ◦ Average: 69%, Range: 3 – 162%
  10. 10. Batch Shake Flask Results Viable Cell Density Control 125:125 mg/L Cellastim:Lacromin 250 mg/L Cellastim 500 mg/L Cellastim Cells supplemented with 250 8 mg/L Cellastim or 500 mg/L Cellastim reached the greatestVCD (million cells/mL) 7 6 5 cell density in batch shake 4 flasks 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 Days % Viability Control 125:125 mg/L Cellastim:Lacromin 250 mg/L Cellastim 500 mg/L Cellastim 100 This data was very 90 80 similar to the InVitria 70 % Viability 60 studies using the same 50 40 conditions 30 20 0 1 2 3 4 5 6 7 8 9 10 11 Days
  11. 11. Bioreactor Density Trends Similar trends appeared in the fed batch bioreactors •Better growth than un-supplemented cells •Slower decline rate at end of culture Bioreactor Viable Cell Densities and % Viability 125:125 mg/L Cellastim:Lacromin VCD Control VCD 125:125 mg/L Cellastim:Lacromin %VIA Control %VIA 12 100 95 10 90VCD (million cells/mL) 85 8 80 %Viability 6 75 70 4 65 60 2 55 0 50 0 1 2 3 4 5 6 7 8 9 10 11 Days
  12. 12. Specific Glucose and Lactate Trends Specific Glucose Consumption in Fed Batch Bioreactors In fed batch bioreactors there Control 125:125 mg/L Cellastim:Lacromin was decreased sp. glucose 0.6 consumption and decreasedsp. Glucose Consumption 0.5 sp. lactate production from (ng/cell*day) 0.4 cells supplemented with 0.3 Cellastim and Lacromin 0.2 0.1 0 0-Prefeed Post Feed - 7 7-10 Specific Lactate Production in Fed Batch Bioreactors pH vs.Time Control 125:125 mg/L Cellastim:Lacromin sp. Lactate Production (ng/cell*day) 125:125 pH Control pH 1.4 7.6 1.2 7.4 7.2 1pH 7 0.8 6.8 0.6 6.6 0.4 0 1 2 3 4 5 6 7 8 9 10 11 0.2 Days 0 0-Prefeed Post Feed - 7 7-10
  13. 13. Glucose Trends EXP4 Bioreactors Control 250 mg/L Cellastim 10.00 9.00 8.00Concentration (g/L) 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 2 4 6 8 10 12 14 16 18 Day Lactate Trends EXP4 Bioreactors Control 250 mg/L Cellastim 4.50 4.00 Concentration (g/L) 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0 2 4 6 8 10 12 14 16 18 Day
  14. 14. pH Trends EXP4 Bioreactors Control 250 mg/L Cellastim 7.15 7.10 7.05 7.00 6.95pH 6.90 6.85 6.80 6.75 6.70 0 2 4 6 8 10 12 14 16 18 Day Osmolality Trends EXP4 Bioreactors Control 250 mg/L Cellastim 500.00 450.00 Osmolality (mOsm) 400.00 350.00 300.00 250.00 200.00 0 2 4 6 8 10 12 14 16 18 Day
  15. 15. Purification Step Using GE-ÄKTApilot a-IL-8 Column Column Sample Loading/ Eluted Column Wash Re-Equilibration Flow-Through Collection Fractions equilibration
  16. 16. SDS-PAGE Coomassie Blue Staining 1 2 3 4 5 6 7 8 9 1075kD50kD25kD 1 Protein Marker 2 Supernatant: 125/125 mg/L Cellastim/Lacromin Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L 3 Cellastim/Lacromin 4 UF Purified a-IL-8 of #3 product 5 Supernatant: 250 mg/L Cellastim 6 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim 7 UF Purified a-IL-8 of #6 product 8 Supernatant: 500 mg/L Cellastim 9 Purified a-IL-8 by Protein A Column from supernatant containing 500 mg/L Cellastim 10 UF Purified a-IL-8 of #9 product
  17. 17. SDS-PAGE Silver Staining 1 2 3 4 5 6 7 8 9 1075kD50kD25kD1 Protein Marker2 Supernatant: 250 mg/L Cellastim3 Flow – through fraction (time point:10-11min) of #2 run through Protein A Column4 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim5 Waste Fraction (time point: 59-60min) of #2 run through Protein A Column6 Supernatant from Protein A Column after column disassembling7 Supernatant: 125/125 mg/L Cellastim/Lacromin8 Flow – through fraction (time point:10-11min) of # 7 run through Protein A Column Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L9 Cellastim/Lacromin10 Waste Fraction (time point: 47-48min) of # 7 run through Protein A Column
  18. 18. ELISA IgG Standard Curve 1200 1000 800 Titer (ng/mL) y = 197.13e1.28x R2 = 0.9936 600 400 200 0 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 OD 450nm Amount of IgG loaded Amount of IgG recovered Sample % Recovered (milligrams) (milligrams) CONTROL 0 mg/L Cellastim 784 328 41.8 0 mg/L Lacromin125mg/L Cellastim 619 321 51.9125 mg/L Lacromin250 mg/L Cellastim 537 315 58.7500mg/L Cellastim 449 313 69.7
  19. 19. Summary Optimize overall system performance Cellastim/Lacromin supplementation ◦ Higher product concentration (titers) ◦ Higher specific productivity ◦ More efficient glucose metabolism ◦ Reduced cell death ◦ Higher consistency ◦ Equal or improved yields at protein A capture Supplements in flow through Reduced non-specific losses of MAb

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