Optimizing Overall Manufacturing System Performance through the Use of Animal-free Cell Culture Supplements

BioManufacturing Summit, January 27, 2010

KiriLynn Svay, Jeff Rosenbloom, Delyan Rusev, and Matt Croughan

Amgen Bioprocessing CenterKeck Graduate Institute, Claremont, CA

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

y = 0.082e0.3477x

R2 = 0.9593

y = 9.941e0.205x

R2 = 0.7939

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New to Technology >2 Years Experience

Analysis of Variance for Experienced FirmsProfessor Matt Croughan, Keck Graduate Institute

y = -0.0804x + 50.266R2 = 0.0005

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“..It” flows down hill

Consequences of 10g/L

• Cell density (volume)• Cell debris• Product Mass• HMW

Cell Culture 10g/L

Purification •Drug Product Development

•From Jon Coffman

Fed-batch cell culture in 1990

Cell culture strategies to optimize overall system performance

Consistent and sufficient titers◦ Every process, every timeShorter culture duration◦ Higher vol. productivity, lower contamination riskReduced cell death◦ Lower degradative enzyme levels◦ Lower HCP and debris loads downstreamImproved downstream processing◦ Higher yields or fewer/simpler stepsImproved product quality and/or stability

Recombinant Human Serum Albumin (rHSA) made in an animal free production host by InVitria (www.InVitria.com)Roles of Albumin in Cell Culture:◦ Binding and transport mechanism

Supply of LipidsVitaminsHormones

◦ Buffering agent◦ Detoxifying agent◦ Protectant from shear

Recombinant Lactoferrin is a naturally occurring iron-binding protein that was made in an animal free production 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

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%

Batch Shake Flask Results

Cells supplemented with 250 mg/L Cellastim or 500 mg/L Cellastim reached the greatest cell density in batch shake flasks

This data was very similar to the InVitria studies using the same conditions

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Viable Cell DensityControl 125:125 mg/L Cellastim:Lacromin

250 mg/L Cellastim 500 mg/L Cellastim

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% ViabilityControl 125:125 mg/L Cellastim:Lacromin

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Bioreactor Density TrendsSimilar trends appeared in the fed batch bioreactors

•Better growth than un-supplemented cells•Slower decline rate at end of culture

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Bioreactor Viable Cell Densities and % Viability125:125 mg/L Cellastim:Lacromin VCD Control VCD

125:125 mg/L Cellastim:Lacromin %VIA Control %VIA

Specific Glucose and Lactate Trends

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Specific Glucose Consumption in Fed Batch Bioreactors

Control 125:125 mg/L Cellastim:Lacromin

In fed batch bioreactors there was decreased sp. glucose consumption and decreased sp. lactate production from cells supplemented with Cellastim and Lacromin

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Specific Lactate Production in Fed Batch Bioreactors

Control 125:125 mg/L Cellastim:Lacromin

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pH vs. Time125:125 pH Control pH

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Glucose Trends EXP4 BioreactorsControl 250 mg/L Cellastim

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Lactate Trends EXP4 BioreactorsControl 250 mg/L Cellastim

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pH Trends EXP4 BioreactorsControl 250 mg/L Cellastim

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OsmolalityTrends EXP4 BioreactorsControl 250 mg/L Cellastim

Purification Step Using GE-ÄKTApilot

Column Equilibration

Sample Loading/ Flow-Through Collection Column Wash Eluted

Fractions

Column Re-

equilibration

a-IL-8

SDS-PAGE

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Coomassie Blue Staining

25kD

75kD

50kD

1 Protein Marker2 Supernatant: 125/125 mg/L Cellastim/Lacromin

3 Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L Cellastim/Lacromin

4 UF Purified a-IL-8 of #3 product5 Supernatant: 250 mg/L Cellastim6 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim7 UF Purified a-IL-8 of #6 product8 Supernatant: 500 mg/L Cellastim9 Purified a-IL-8 by Protein A Column from supernatant containing 500 mg/L Cellastim

10 UF Purified a-IL-8 of #9 product

SDS-PAGE

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Silver Staining

1 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 disassembling 7 Supernatant: 125/125 mg/L Cellastim/Lacromin8 Flow – through fraction (time point:10-11min) of # 7 run through Protein A Column

9 Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L Cellastim/Lacromin

10 Waste Fraction (time point: 47-48min) of # 7 run through Protein A Column

IgG Standard Curve

y = 197.13e1.28x

R2 = 0.9936

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SampleAmount of IgG loaded

(milligrams)

Amount of IgG recovered

(milligrams)% Recovered

CONTROL

0 mg/L Cellastim

0 mg/L Lacromin

784 328 41.8

125mg/L Cellastim

125 mg/L Lacromin619 321 51.9

250 mg/L Cellastim 537 315 58.7

500mg/L Cellastim 449 313 69.7

ELISA

Summary

Optimize overall system performanceCellastim/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 throughReduced non-specific losses of MAb