Merck Moving Beyond Passwords: FIDO Paris Seminar.pptx
Bio manufacturing summit croughan et al jan 2010
1. Optimizing Overall Manufacturing
System Performance through the
Use of Animal-free Cell Culture
Supplements
KiriLynn Svay, Jeff Rosenbloom,
Delyan Rusev, and Matt Croughan
Amgen Bioprocessing Center
Keck Graduate Institute, Claremont, CA
BioManufacturing Summit, January 27, 2010
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 Experience
Product 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. Analysis of Variance for Experienced Firms
Professor M Croughan, Keck Graduate Institute
att
100.0
Percent 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.
5. “..It” flows down hill
Consequences of
10g/L
Cell
Culture
10g/L
• Cell density (volume)
• Cell debris
• Product Mass
• HMW
Purification
•Drug
Product
Development
•From Jon Coffman
7. Cell culture strategies to optimize
overall 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
8. 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 Lipids
Vitamins
Hormones
◦ Buffering agent
◦ Detoxifying agent
◦ Protectant from shear
9. 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
10.
11. 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%
12. 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 greatest
VCD (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
13. 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
90
VCD (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
14. 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 decreased
sp. 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 1
pH
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
19. SDS-PAGE
Coomassie Blue Staining
1 2 3 4 5 6 7 8 9 10
75kD
50kD
25kD
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
20. SDS-PAGE
Silver Staining
1 2 3 4 5 6 7 8 9 10
75kD
50kD
25kD
1 Protein Marker
2 Supernatant: 250 mg/L Cellastim
3 Flow – through fraction (time point:10-11min) of #2 run through Protein A Column
4 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim
5 Waste Fraction (time point: 59-60min) of #2 run through Protein A Column
6 Supernatant from Protein A Column after column disassembling
7 Supernatant: 125/125 mg/L Cellastim/Lacromin
8 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/L
9
Cellastim/Lacromin
10 Waste Fraction (time point: 47-48min) of # 7 run through Protein A Column
22. 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
