Niket Bubna, Cameron T. Phillips, Sigma S. Mostafa and AbhinavA. Shukla. KBI Biopharma, Durham, NC 253rd ACS National Meeting & Exposition April 2-6, 2017 • San Francisco, CA #acsSanFran • www.acs.org/SanFran2017

1. Scalability of cell culture processes in single-use bioreactors using different CHO cells line variants
Chinese Hamster Ovary (CHO) cells have been used for clinical and commercial manufacturing of
biopharmaceutical products for decades. Several CHO cell line variants that achieve high titers
are now being cultured in single-use bioreactors (SUBs), but data for scalability of cell culture
processes in single-use bioreactors is limited. Rapid development of early-stage cell culture and
purification processes is essential to ensure production of FIH bulk drug substance within a
reasonable timeframe. A platform process development paradigm combined with our single-use
bioreactor platform for manufacturing provides the flexibility to scale-up and manufacture multiple
molecules with quick turnaround while maintaining high plant capacity. Several case studies
showing comparison of process performance across development (glass and ambr15) and
manufacturing scales (100 L to 2000 L) are provided. Key factors like power input and volumetric
gas flow rates were maintained across all different processes, thereby establishing a platform for
scale-up into single-use bioreactors. This analysis confirmed that our scale-up platform using 200
and 2000 liter SUBs provides robust performance using a variety of CHO cell lines and cell culture
processes.
Abstract
Niket Bubna, Cameron T. Phillips, Sigma S. Mostafa and Abhinav A. Shukla
KBI Biopharma, Durham, NC
References
Case Study II – CHO-K1 Cell Line Variant
Conclusions
Acknowledgments
We would like to thank Sigma and Abhinav for their support and guidance; and express our
gratitude to Lynwel Cunanan, James Hamlin, Bryan Howarth, Shaunak Uplekar and Jingshu Zhu
for their help.
Several challenges were faced while developing a cell culture
process for this cell line: Doubling time was >30 hours and the
cell line was observed to grow poorly upon exposure to single-
use bioreactor bags. The product was also known be prone to
clipping. Given the amount of drug substance needed to initiate
clinical trials for this molecule, a 600 L-scale bioreactor volume
was necessary. In order to meet the harvest volume requirement,
a seed bioreactor-stage with 2 passages within the same vessel
was developed. Passage culture was diluted with fresh basal
medium and supplements while maintaining culture temperature
above 33 deg C (above). Inoculum was then transferred to the
production bioreactor bag to conduct a 12-14 day fed-batch
process (right).
Vial
Inoculum
Expansion in
Shake Flasks
Inoculum
Expansion in
Wave Cellbag
Inoculum
Expansion in
XDR-200 SUB
Production
Run in XDR-
2000 SUB
Parameter XDR-50 SUB XDR-200 SUB XDR-2000 SUB
Impeller Diameter 0.2159 m 0.2159 m 0.4191 m
Impeller Power Number 1.50 1.15 0.72
Pitched-blade Impeller 3 blades at 40° 3 blades at 40° 4 blades at 40°
Turn-down Ratio 2.2:1 5:1 5:1
Aspect Ratio 1.5:1 1.5:1 1.5:1
Parameter Xcellerex Single-Use Bioreactor Stainless Steel Bioreactor
Sterilization Pre-sterilized Bags (γ irradiated) CIP and SIP
Culture Vessel Single-use LDPE Bag Stainless Steel Vessel
Agitator Bottom-mounted pitched-blade
Top/Bottom mounted impellers
(with or without baffles)
Gas Supply
Sparge discs
(with or without open-pipe)
Ring sparger, Microsparger, etc.
Sample Port Welding sterile bags SIP
Even though each CHO cell line variant requires unique culture conditions and expresses different
biopharmaceutical products, cell culture processes developed in small-scale bioreactors are being
successfully scaled-up using a platform approach for a variety of CHO cell variants. Given the
consistent performance observed using this scale-up platform, process development studies can
be focused on achieving product quality attributes or titer requirements. Several different
strategies have been employed test robustness of this scale-up platform using single-use
bioreactors and in each case the processes have been found to be scalable. This platform using
the Xcellerex single-use bioreactors will be used for commercial manufacturing in the future.
Chinese Hamster Ovary (CHO) cell lines are widely used in biopharmaceutical processing to
produce monoclonal antibodies and therapeutic proteins. Although clinical or commercial production
occurs in large-scale bioreactors, process development and optimization is performed at much
smaller scales, in bioreactors ranging from milliliters (microbioreactors) to a few liters (bench-scale
bioreactors). Several factors, including bioreactor geometry, agitation rate and gassing strategy must
be considered when increasing scale of operation.
Several different strategies can be used for scale-up:
• Maintain bioreactor geometry, P/V, and VVM across scales – Used at KBI Biopharma to develop
scalable processes.
• Maintaining bioreactor geometry, kLa, and superficial gas flow – This method prioritizes oxygen
transfer over mixing, using constant kLa to determine the agitation speed. A similar method
replaces superficial gas velocity with constant tip speed or shear rate ensuring the cells are
subjected to the same shear stress at all scales.
𝑃𝑃
𝑉𝑉
=
𝑁𝑁𝑝𝑝 𝑁𝑁3
𝐷𝐷𝑖𝑖
5
𝜌𝜌
𝑉𝑉
𝑉𝑉𝑉𝑉𝑉𝑉 =
𝑄𝑄𝐺𝐺
𝑉𝑉
where,
Di = Impeller diameter [m]
N = Agitation speed [s-1]
Np = Impeller power number [-]
P = Power [W]
S = Cross-sectional area [m2]
V = Volume [L]
where,
QG = gas flow rate [m3/s]
V = Volume [L]
VVM = Vessel volumes per minute [min-1]
Clarification
using POD
Depth Filters
Typical Cell Culture Process Flow for 2000 L-Scale cGMP Manufacturing at KBI Biopharma
• Dreher, T. et al. (2014) Design space definition for a stirred single‐use bioreactor family from 50
to 2000 L scale. Eng. Life Sci.14, 304-310
• Eibl, D. et al. (2011) Single-Use Bioreactors – An Overview. In Single-Use Technology in
Biopharmaceutical Manufacture (Eibl, D. and Eibl, R., ed.), pp. 33-48, John Wiley & Sons
• Shukla, A. and Gottschalk U. (2013) Single-use disposable technologies for biopharmaceutical
manufacturing. Trends Biotechnol. 31, 147-154
• Yang, J. et al. (2007) Fed‐batch bioreactor process scale‐up from 3‐L to 2,500‐L scale for
monoclonal antibody production from cell culture. Biotechnol. Bioeng. 98, 141-154
253rd ACS National Meeting & Exposition
April 2-6, 2017 • San Francisco, CA
#acsSanFran • www.acs.org/SanFran2017
Case Study III – CHO-S Cell Line
Scaling up CHO Cell Line Variants for Clinical Manufacturing
Case Study I – CHO-DG44 Cell Line
0
5
10
15
20
25
30
2013-14 2015 2016
New Products Mfg Batches
Project Type of Molecule Cell Line Type Cell Line Vendor Basal Medium Peak VCC (1^6 cells/mL)
A Non-mAb CHO-DG44 A / B OptiCHO / Dynamis 10
B mAb CHO-S A / B Dynamis 25-30
C Non-mAb CHO-S A / B Dynamis 25-30
D mAb CHO-K1 C ProCHO 5 6-8
E-H 4 x mAbs CHO-S A / B Dynamis 21
I mAb CHO-DG44 D BalanCD Growth A 30-40
J mAb CHO-GS E FortiCHO 7-15
K mAb CHO-GS F CD CHO 12-15
L Non-mAb CHO-S G CD OptiCHO 7-8
M mAb CHO variant H BalanCD Growth A 30
O mAb CHO-GS Client Proprietary Client Proprietary 7-10
P mAb CHO-GS F OptiCHO / FortiCHO 12-15
Q mAb CHO variant H BalanCD Growth A 30-40
R mAb CHO-K1 I OptiCHO / PowerCHO-GS 10-11
S mAb CHO-DG44 J ExCell CHO 13-20
T-W 4 x Non-mAbs CHO-DG44 A / B OptiCHO 10
X Bispecific mAb CHO variant H BalanCD Growth A 25
Y mAb CHO-K1 B ProCHO 5 6-10
Z Non-mAb CHO-DG44 D ExCell ACF CHO 17-19
AA mAb CHO variant K OptiCHO 12
AB-AE 4 x Bispecific mAbs CHO variant H BalanCD Growth A 30-40
80
82
84
86
88
90
92
94
96
98
100
0
1
2
3
4
5
6
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
CellViability
ViableCellCount
Days
Mfg Run 1-VCC
Mfg Run 2-VCC
Mfg Run 3-VCC
Mfg Run 1-Viability
Mfg Run 2-Viability
Mfg Run 3-Viability
50 L
200 L
Transfer to Production
N-2 N-1
ViableCellCount
Days
3 L-Scale 3 L-Scale 600 L-Scale 600 L-Scale
Titer
Time (Days)
3 L-Scale 3 L-Scale 600 L-Scale 600 L-Scale
Gene synthesis & vector
construction
Stable Pools
Cloning
Analytical Method
Dev.
Analytical Method
Qual.
MCB Prep
cGMP Run
BDS Disposition
IND Submission
Upstream Platform
Assessment / Supply
Downstream Platform
Assessment / Supply
Clone
Selection*
Demo Run
Tech Transfer
Pre-form Dev.
Formulation Dev.
Viral Clearance
Top pool
Top clone
Cell Line Development
Cell Culture Process Development
Overlapping Activities (CLD-PD-Mfg)
Stable Pool
HCCF Supply Run
(50 L SUB)
Clone Selection
Platform Process
Assessment
Process Confirmation
(50 L SUB)
Scale-up
(200 L SUB)
Top 3 Clones
Top
Clone
SUB performance evaluation
Wave or Seed bioreactor
performance evaluation
This case study illustrates an expedited
model of process development, where
gene synthesis to release of the first batch
of bulk drug substance purified from a
2000 L-scale cGMP bioreactor run was
achieved in less than 14 months. Pre-
determined medium-feed combination and
process parameters were used to reduce
the need for optimization. Several tests
were carried out in single-use bags to
ensure scalability and compatibility with
the cell culture process. Key goal was
speed and consistent process
performance, which was successfully
achieved for four monoclonal antibodies
within 16 months.
ViableCellCount
Days
2000 L-Scale 200 L-Scale 50 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
ProductConcentration
Days
2000 L-Scale 200 L-Scale 50 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
Glucose
Days
2000 L-Scale 200 L-Scale 50 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
Lactate
Days
2000 L-Scale 200 L-Scale 50 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
Media Equilibration
N-2 Passage
N-1 Passage
Temperature PV (deg C) Temperature SP (deg C) Vessel Weight (kg)
ViableCellCount
Days
2000 L GMP Run #1
200 L-Scale
15 L-Scale
3 L-Scale
DoublingTime
RCB Shake Flasks (Control)
MCB Shake Flasks (Control)
Film 1
Film 2
Film 3
Film 4
Film 5
Clinical Manufacturing Batches (Manufacturing Plant in Durham, NC)
List of CHO Cell Line Variants and Molecules Manufactured
Comparison of Key Parameters between Xcellerex SUBs
Comparison of Key Parameters between Xcellerex SUBs & Stainless Steel Bioreactors
Scale-up Considerations for Late-stage Process Development
Limited studies were performed for
early-stage cell culture process
development for this cell line. A platform
process was implemented to select the
Top Stable Clone and generate Tox
material. Initial attempt to scale-up in
single-use bioreactors showed poor cell
growth, but bioreactor bag pre-treatment
and nutrient addition improved
performance. So far, 10 clinical
manufacturing batches have been
successfully filled. This clinical
manufacturing process is now being
developed and optimized for
commercial manufacturing. The key
objectives for this late-stage
development is to improve specific
productivity and pH control in the
bioreactor culture; and to ensure single-
use storage containers and bioreactor
bag films are appropriately selected to
overcome impact on cell growth and cell
viability. Shown here are results of
functional tests for several Wave
Cellbags and Bioprocess container
films.
ViableCellCount
Days
Control (T=1)
Control (T=2)
Control (T=4)
Film 1 (T=1)
Fim 1 (T=2)
Film 1 (T=3)
Film 1 (T=4)
Film 2 (T=1)
Film 2 (T=2)
Film 2 (T=3)
Film 2 (T=4)
Titer
Days
2000 L GMP Run #1
200 L-Scale
15 L-Scale
3 L-Scale
Comparison of cell growth in two single-use storage container (BPC) films
Comparison of cell growth in Wave Cellbag films.
Error bars represent standard deviation (n=3 to 5)
Early-stage development studies showed poor cell growth in initial attempts to scale-
up in single-use bioreactor bags
Gene to IND in 12-14 Months
Strategies for expedited process development and clinical manufacturing for mAbs
Manufacturing a Low Titer Recombinant Protein at 600 L-Scale
Temperature control and cell growth during the N-2 and N-1 seed bioreactor passages in the same bioreactor bag to generate enough inoculum
Comparison of results from the production
bioreactor runs at 3 L and 600 L-scale