This document discusses the scale-up and technology transfer from non-disposable to fully disposable systems for cell culture manufacturing. It describes KBI's strategy for characterization of disposable bioreactors and development of comparable processes. Case studies demonstrate comparable performance across scales from 3L to 2000L bioreactors. Key factors like sparger selection and dual gas supply were optimized to control dissolved CO2. Process data like cell density, viability and titer matched the client's data. Depth filtration and disposable centrifuge options were evaluated for harvest clarification.

1. A Case Study of Scale-up and Tech
Transfer from Non-Disposable to Fully
Disposable Systems
Sigma S. Mostafa, Ph.D., Director, Upstream Development
Abhinav Shukla, Ph.D., Vice President , Process Development

2. 2Confidential
End-to-End Disposable Facility for
Cell Culture manufacturing

3. Disposable Seed, Production, & Harvest
Shake Flasks (20 mL – 2000 mL) Wave Bags (2L – 25L)
Bioreactors (40L – 2000L)Depth Filters (Sized based on need)

4. Strategy for Tech Transfer to
Disposable Bioreactors
• Detailed characterization of disposable bioreactors
• Development of in-house scalability data
• Determination of a robust scaling factor (e.g. Power per volume)
• Delivery of a comparable process through choosing appropriate
aeration and agitation strategy
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5. Characterization of Bioreactors
• Response surface DOE for Mass Transfer Coefficient (kLa)
• Salt solution with ionic strength similar to cell culture
• Sparging Rate, Agitation Rate, and Working Volumes
are key factors
200L Disposable Bioreactor with 20um Sparger

6. Sparger Selection
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• Vendors offer multiple options for spargers:
2 μm, 20 μm, 0.5 mm, 1.0 mm
• Multiple disks of same porosity is available
• A dual sparger system: 1) micro and/or macro
sparger disks for oxygen; 2) wand (drilled pipe)
for air is available
• Each of these options will lead to different
mixing characteristics

7. Impact of Probes
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• Depending on agitation rate, impeller
orientation, and working volume, probe
location may impact probe reading
• Disposable probes preferable; however,
accuracy is still under question

8. Case Study - I
• Tech Transfer project (200L)
• At scale data available from other bioreactor
systems
• Shear damage a concern; as a result relatively
low agitation is used
• Sparger disk with 2 μm porosity used
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9. Viable Cell Density
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• 4 GMP runs at 200L scale show minimal variability
• Data from KBI and Client match well (not shown)

10. Viability
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• 4 GMP runs at 200L scale show minimal variability
• Data from KBI and Client match well (not shown)

11. Titer
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• 4 GMP runs at 200L scale show minimal variability
• Data from KBI and Client match well (not shown)

12. Titer
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• Titer data at KBI was similar to client’s data
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2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
FinalTiter
Client KBI
Source
Onew ay Analysis of Final Titer By Source

13. Dissolved CO2
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• KBI dCO2 high due to use of a microsparger and low agitation rate
50
70
90
110
130
150
170
190
MaxdCO2
Client KBI
Source
Onew ay Analysis of Max dCO2 By Source

14. Product Quality
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• KBI’s product quality data within range provided by client
60
65
70
75
80
85
90
95
100
G0F
Client KBI
Source
Onew ay Analysis of G0F By Source

15. Case Study - II
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• Tech Transfer project (200L, 2000L)
• Intermediate scale data available
• Dissolved CO2 a concern
• Dual sparging system used –
i) Wand with drilled holes for air supply (for CO2 removal)
ii) Four 20 μm sparger disks used for O2 supply

16. Titer
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• VCC, viability within ranges of
client data (not shown)
• Titer within range of client
data
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Client KBI
Source
Onew ay Analysis of Final Titer By Source

17. Case Study - III
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• Process Development project
• Data generated in 3L and 10L glass
bioreactors, and 200L and 2000L bioreactors
• Oxygen uptake rate (per cell) was high; cells
were sensitive to high dissolved CO2
• Antifoam addition was not an option

18. Upstream Process Development
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19. Shake Flask vs. 3L Bioreactor
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Titer
Different Feed & Supplement Combinations

20. 3L Glass Bioreactor Characterization
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kLa
kLa
• Characterization of small scale bioreactors is a necessity
• Per cell oxygen uptake rate was determined at this scale

21. Comparability Across Scale –
3L, 15L, 200L, and 2000L
Viable Cell Density
• VCD data matches across scale

22. Viability
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• Viability data matches across scale
Comparability Across Scale –
3L, 15L, 200L, and 2000L

23. 23
• Titer data matches across scale
Titer
Comparability Across Scale –
3L, 15L, 200L, and 2000L

24. Product Quality
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• KBI’s product quality data within range provided by client
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30
35
40
45
50
55
60
G0F
Client KBI
Source
Onew ay Analysis of G0F By Source

25. Harvest Clarification
• Current platform:
depth filtration
• Alternate platforms
being evaluated:
Disposable Centrifuges
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26. Resistance vs. Throughput Chart
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0.00
0.05
0.10
0.15
0.20
0.25
0 50 100 150 200 250 300
Throughput (L/m2)
Resistance(psi/LMH)
Depth Filtration Evaluation
• Resistance across different depth filters studied

27. Depth Filter Evaluation
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Minimum Area Comparison
0.0
0.5
1.0
1.5
2.0
2.5
3.0
A1->B1->C A1->B2->C A2->C
MinimumArea(m2)
Sterile Filter
Secondary Filter
Primary Filter
• Comparison was made of the total minimum area used for the depth and
sterile filters

28. Conclusions
• End-to-end disposable facility shortens change
over duration and eliminates cross
contamination risk
• A rigorous scientific approach in tech transfer
from non-disposable to disposable leads to
consistent success in the disposable platform.
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29. Questions ?
Contact Information:
Sigma S. Mostafa, Ph.D.
Director, Process Development
KBI Biopharma Inc.
1101 Hamlin Road
Durham, NC 27704
smostafa@kbibiopharma.com
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30. Depth Filtration Evaluation
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Turbidity vs. Throughput
0 50 100 150 200 250 300
Throughput (Volume/Surface Area)
Turbidity
• Turbidity post depth filter was studied

31. 0.22 μm Polishing Filter Study:
Vmax Throughput vs. Time
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0 5 10 15 20 25 30
Time (min)
Throughput(l/m2
).
post filter 1
post filter 2
post filter 3
Polishing Filter Evaluation
• Throughput across polishing filters varied based on load material

32. www.kbibiopharma.com
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