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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
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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
0.1
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Prod
uct C
once
ntra
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at H
arve
st (m
g/L)
New to Technology >2 Years Experience
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Analysis of Variance for Experienced FirmsProfessor Matt Croughan, Keck Graduate Institute
y = -0.0804x + 50.266R2 = 0.0005
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Perc
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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
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Fed-batch cell culture in 1990
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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
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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
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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
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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%
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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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VC
D (
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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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0 1 2 3 4 5 6 7 8 9 10 11
% V
iabi
lity
Days
% ViabilityControl 125:125 mg/L Cellastim:Lacromin
250 mg/L Cellastim 500 mg/L Cellastim
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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
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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
6.66.8
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pH
Days
pH vs. Time125:125 pH Control pH
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Glucose Trends EXP4 BioreactorsControl 250 mg/L Cellastim
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Con
cent
rati
on (
g/L)
Day
Lactate Trends EXP4 BioreactorsControl 250 mg/L Cellastim
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pH Trends EXP4 BioreactorsControl 250 mg/L Cellastim
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Osm
olal
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OsmolalityTrends EXP4 BioreactorsControl 250 mg/L Cellastim
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Purification Step Using GE-ÄKTApilot
Column Equilibration
Sample Loading/ Flow-Through Collection Column Wash Eluted
Fractions
Column Re-
equilibration
a-IL-8
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SDS-PAGE
1 2 3 4 5 6 7 8 9 10
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
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SDS-PAGE
1 2 3 4 5 6 7 8 9 10
25kD
75kD
50kD
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
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IgG Standard Curve
y = 197.13e1.28x
R2 = 0.9936
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OD 450nm
Tite
r (ng
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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
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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