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 Confidential

End-to-End Disposable Facility for Cell Culture manufacturing

Disposable Seed, Production, & Harvest

Shake Flasks (20 mL – 2000 mL)

Wave Bags (2L – 25L)

Bioreactors (40L – 2000L)

Depth Filters (Sized based on need)

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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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

Sparger Selection

6

• 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

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

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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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)

Viability

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• 4 GMP runs at 200L scale show minimal variability

• Data from KBI and Client match well (not shown)

Titer

11

• 4 GMP runs at 200L scale show minimal variability

• Data from KBI and Client match well (not shown)

Titer

12

• 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

Fin

al

Tite

r

Client KBI

Source

Onew ay Analysis of Final Titer By Source

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

Max

dC

O2

Client KBI

Source

Onew ay Analysis of Max dCO2 By Source

Product Quality

14

• KBI’s product quality data within range provided by client

60

65

70

75

80

85

90

95

100

G0

F

Client KBI

Source

Onew ay Analysis of G0F By Source

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

Titer

16

• 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

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

Upstream Process Development

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Shake Flask vs. 3L Bioreactor

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Titer

Different Feed & Supplement Combinations

3L Glass Bioreactor Characterization

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kL

a kLa

• Characterization of small scale bioreactors is a necessity

• Per cell oxygen uptake rate was determined at this scale

Comparability Across Scale – 3L, 15L, 200L, and 2000L

Viable Cell Density

• VCD data matches across scale

Viability

22

• Viability data matches across scale

Comparability Across Scale – 3L, 15L, 200L, and 2000L

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• Titer data matches across scale

Titer

Comparability Across Scale – 3L, 15L, 200L, and 2000L

Product Quality

24

• KBI’s product quality data within range provided by client

25

30

35

40

45

50

55

60

G0

F

Client KBI

Source

Onew ay Analysis of G0F By Source

Harvest Clarification

• Current platform: depth filtration

• Alternate platforms being evaluated: Disposable Centrifuges

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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)

Resis

tan

ce (

psi/L

MH

)

Depth Filtration Evaluation

• Resistance across different depth filters studied

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

Min

imu

m A

rea

(m

2)

Sterile Filter

Secondary Filter

Primary Filter

• Comparison was made of the total minimum area used for the depth and

sterile filters

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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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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Depth Filtration Evaluation

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Turbidity vs. Throughput

0 50 100 150 200 250 300

Throughput (Volume/Surface Area)

Tu

rbid

ity

• Turbidity post depth filter was studied

0.22 μm Polishing Filter Study: Vmax Throughput vs. Time

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0 5 10 15 20 25 30

Time (min)

Th

rou

gh

pu

t (l

/m 2

)

.

post filter 1

post filter 2

post filter 3

Polishing Filter Evaluation

• Throughput across polishing filters varied based on load material

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