Increasing adoption of single-use technologies for bioprocessing along with higher titers from cell culture bioreactor processes has allowed clinical and even commercial manufacturing to be successfully performed in 2000 L-scale single-use bioreactors. Several biopharmaceutical manufacturers have successfully adopted single-use bioreactors for production. However, information about process scalability from glass bioreactors to 2000 L single-use bioreactors for different types of CHO cell lines is not widely available. Here we provide an overview of the key differences between single-use and conventional stainless steel bioreactors, and highlight factors that are employed while scaling-up from small-scale glass bioreactors to 2000 L-scale single-use bioreactors. Several case studies focusing on process performance across scales into single-use bioreactors are provided. This analysis confirms that the 2000 L-scale single-use bioreactorsystem can be robustly employed for biopharmaceutical manufacturing.

1. Single-use bioreactors (SUBs) are being increasingly utilized for biopharmaceutical manufacturing.
Minimal requirements for cleaning, higher plant capacity due to quick turnaround and flexibility of
manufacturing multiple products without extensive changeover are key attributes that have led to
rapid adoption of disposable technology for cell culture manufacturing.
Xcellerex XDR SUBs ranging from 50 L to 2000 L have been proven as successful replacement for
stainless steel stirred-tank bioreactors. Xcellerex SUBs feature a cylindrical bag that uses a
magnetically coupled, bottom-driven impeller with easy installation prior to culturing cells. The
impeller is positioned at an angle, 15° off-centered at the bottom of the bag. Bioreactor bags with
pre-installed disc spargers (micro and macrospargers) or open-pipe spragers can be used
depending on the process. Sparge discs in Xcellerex SUB bags are fabricated on the impeller
assembly.
Stainless steel bioreactors are typically used with a
top-mounted stirrer shaft installed with one or more
impellers depending on the vessel size and process
requirement. Stainless steel bioreactor tanks are also
known to use baffles to prevent vortex while mixing
the culture. Impellers in Xcellerex SUBs are
positioned off-center which prevent vortex formation
and thus does not require baffles for proper mixing. Stainless steel bioreactors can be used with a
variety of impeller types like Rushton, marine or pitched-blade impellers whereas Xcellerex SUB
bags include 3-4 pitched-blade impellers at a 40° pitch. SUB bags are pre-sterilized by gamma
irradiation and can be typically used to remove sterile by welding sterile sample containers whereas
steam sterilization is required for stainless steel vessels increasing the ease of using SUBs.
Scalability of a Single-use Bioreactor Platform for Biopharmaceutical Manufacturing
Increasing adoption of single-use technologies for bioprocessing along with higher titers from cell
culture bioreactor processes has allowed clinical and even commercial manufacturing to be
successfully performed in 2000 L-scale single-use bioreactors. Several biopharmaceutical
manufacturers have successfully adopted single-use bioreactors for production. However,
information about process scalability from glass bioreactors to 2000 L single-use bioreactors for
different types of CHO cell lines is not widely available. Here we provide an overview of the key
differences between single-use and conventional stainless steel bioreactors, and highlight factors
that are employed while scaling-up from small-scale glass bioreactors to 2000 L-scale single-use
bioreactors. Several case studies focusing on process performance across scales into single-use
bioreactors are provided. This analysis confirms that the 2000 L-scale single-use bioreactor
system can be robustly employed for biopharmaceutical manufacturing.
Abstract
Niket Bubna, Cameron T. Phillips, Sigma S. Mostafa and Abhinav A. Shukla
KBI Biopharma, Durham, NC
Single-use and Stainless Steel Bioreactors
Factors for Scale-up to 2000 L-Scale Bioreactors
References
Case Studies Across Bioreactor Scales
Conclusions
Acknowledgments
Case Study I – CHO-K1 Cell Line
Case Study II – CHO-S Cell Line
Case Study III – CHO-DG44 Cell Lines (Cell Line A and B)
We would like to thank Sigma and Abhinav for their support and guidance; and express our gratitude
to Brian Baker, Lynwel Cunanan, Bryan Howarth, Kathryn Olson, Shaunak Uplekar and Jingshu Zhu
for their help.
Comparison between two CHO-DG44 cell lines is shown above (Cell Line A and B from the same
vendor). This comparison demonstrates a case where variable cell culture performance was
observed in Cell Line A during the first attempt to scale-up in a 200 L-scale SUB bag (red line), but
not in Cell Line B. Four process modifications (no change to raw materials or consumables) were
implemented to the Cell Line A bioreactor process, resulting in improved cell growth and
productivity. The same changed were applied to Cell Line B ensuring seamless scale-up from 3 L
to 200 L-scale.
An early-stage cell culture
process was transferred from a
client. This process first tested
at 15 L-scale showed
comparable cell culture
performance, but required a
modification to the day of
harvest to achieve consistent
product quality results. A
strategy to initiate harvest
depending on the % acidic
species was successfully
implemented at 50 L and 2000
L-scale.
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. Platform
cell culture and purification processes
were successfully applied to optimize the
final process using a stable pool of cells.
Process performance was confirmed in 3
L-scale glass bioreactor using Top 3
Clones before scaling up in 200 L and
2000 L-scale SUBs using the Top
Candidate Clone.
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
Rushton Impeller Marine Impeller Pitched-blade Impellers
(including sparge discs)
Stainless Steel Bioreactors Xcellerex Single-Use Bioreactors
Cell culture processes developed in small-scale glass bioreactors were successfully scaled to
single-use bioreactors ranging from 50 L to 2000 L for several CHO cell lines. Scale-up to larger
bioreactor volumes was implemented by maintaining power input per volume and VVM across
different scales. Results in four CHO cell lines variants show that cell growth, metabolite levels and
productivity can be reproduced in large-scale SUBs. We have also developed process steps to
overcome suboptimal cell growth in single-use bioreactors as observed in Cell Line A (Case Study
IV). Based on the scalability of this single-use bioreactor platform, it will be used for commercial
manufacturing in the near 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.
Agitation Rate
• Homogenous mixing is important to provide uniform nutrient, oxygen concentration, pH and
temperature around the cell – improper mixing will develop gradients and result in suboptimal
performance
• Limit to maximum mixing in mammalian cells due to cell physiology – lack of cell wall and cell
diameter – leads to increased sensitivity to shear forces
• Power input per volume is commonly used to normalize the agitation speed based on the power
provided to the impeller. This method is used for scale-up of most mammalian cell culture
processes at KBI Biopharma.
Gassing Strategy
•Important variables include sparge type, sparge hole size and gas flow rates
•A ratio of volumetric gas flow to liquid volume, vessel volumes per minute (VVM), can also be used
to scale up gas flow rates. This method is arguably better since it depends on the actual liquid
volume in the bioreactor instead of an area which will not change. This allows one to operate at
multiple volumes in one vessel while changing flow rates based off the volume.
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. This ensures 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 [L/min]
V = Volume [L]
VVM = Vessel volumes per minute [min-1]
Vial
Inoculum
Expansion in
Shake Flasks
Inoculum
Expansion in
Wave Cellbag
Inoculum
Expansion in
XDR-200 SUB
Production
Run in XDR-
2000 SUB
Clarification
using POD
Depth Filters
Typical Cell Culture Process Flow for 2000 L-Scale cGMP Manufacturing at KBI Biopharma
ViableCellCount
Days
2000 L-Scale 2000 L-Scale 50 L-Scale
15 L-Scale 3 L-Scale 3 L-Scale
Glucose
Days
2000 L-Scale 2000 L-Scale 50 L-Scale
15 L-Scale 3 L-Scale 3 L-Scale
Ammonia
Days
2000 L-Scale 2000 L-Scale 50 L-Scale
15 L-Scale 3 L-Scale 3 L-Scale
ProductConcentration
Days
2000 L-Scale 2000 L-Scale 50 L-Scale
15 L-Scale 3 L-Scale 3 L-Scale
%AcidicSpecies
Days
50 L-Scale 2000 L-Scale 2000 L-Scale
Lactate
Days
2000 L-Scale 2000 L-Scale 50 L-Scale
15 L-Scale 3 L-Scale 3 L-Scale
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
ViableCellCount
Days
100 L-Scale 100 L-Scale 100 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
ProductConcentration
Days
100 L-Scale 100 L-Scale 100 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
Glucose
Days
100 L-Scale 100 L-Scale 100 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
Lactate
Days
100 L-Scale 100 L-Scale 100 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
ViableCellCount
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
ProductConcentration
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
Glucose
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
CellViability
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
Lactate
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
Ammonia
Days
200 L-Scale 200 L-Scale 200 L-Scale
200 L-Scale 15 L-Scale 3 L-Scale
3 L-Scale
Cell Line A Cell Line B
1. Shukla, A. and Gottschalk U. (2013) Single-use disposable technologies for biopharmaceutical manufacturing.
Trends Biotechnol. 31, 147-154
2. 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
3. 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
4. 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
Case Study IV – CHO/dhFr- (Client’s Proprietary Cell Line)
Rapid process development (4
months) using the Top Clone resulted
in a robust process with 2.5-fold
increase in bioreactor titer. Top Clone
was utilized for a feed screening
experiment and optimization of pH
control range. Final process from 3 L-
glass bioreactor was scaled to 200 L
single-use bioreactor prior to process
transfer for clinical manufacturing at
2000 L-scale. Cell growth, productivity
and metabolite levels were similar in
all three scales.
ViableCellCount
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale
Glucose
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale
CellViability
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale
ProductConcentration
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale
Lactate
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale
Ammonia
Days
2000 L-Scale 2000 L-Scale 2000 L-Scale
2000 L-Scale 2000 L-Scale 200 L-Scale
3 L-Scale 3 L-Scale 3 L-Scale
3 L-Scale 3 L-Scale