FRhK-4 cells, an adherent mammalian cell line, was scaled from a roller bottle into a bench-top perfusion bioreactor system using microcarriers.

1. SCALE UP OF FRhK-4 CELL GROWTH FROM ROLLER
BOTTLE TO BENCH-TOP PERFUSION SYSTEM
Shriram Kaliannan Chandramohan, Carolyn Lanzkron, Joseph Philipp, Logan Trimble
Nathaniel Swanson, Jean-François P. Hamel
(Contact info: Dr. Hamel- jhamel@mit.edu )
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, USA
Objective
Conclusions
• This study focused on the development of a scalable
process for expanding FRhK-4 cells. Cells were initially
grown in the T-flask and roller bottle, and expanded to a
Cytodex-1 microcarrier suspension culture in a bioreactor.
• The 2-L Sartorius Biostat-B univessel was set up to operate
continuously, in the perfusion mode, using a settling tube.
• Glucose, measured online by biochemical analysis
(BioPAT® Trace), was the limiting nutrient and triggered
change in the perfusion rate.
• Successful perfusion experiment was performed in the
bioreactor with 2 g/L of Cytodex-1 at 1.5 L without
generating aggregates or foam.
Objectives Approach Findings
Achieving/ Attaining adequate
Cell attachment
Agitation profiles
Continuous vs. intermittent agitation
Standard concentration vs Concentrated medium (2x)
Intermittent agitation facilitated adequate cell
attachment within 24 h after inoculation
Concentrated medium did not facilitate cell
attachment
Maximizing cell concentration
Monitoring cell growth kinetics
Repeated batch vs perfusion method
Intermittent agitation profile (IAP)
Microcarrier Cytodex-1 (2-4 g/L)
Direct vs, indirect bead-to-bead transfer
Online biochemical analyzer connected to the bioreactor to
enable real time nutrient/metabolite level monitoring
Maintaining uniform microcarrier suspension
Choosing bioreactor platform- tested various configuration
of impellers (Pitched, Rushton, Paddle)
Modification in mode of operation
Medium considerations
-Exchange in medium triggered when glucose <1g/L
-Use of powered and ready-to- use (RTU) media
Mitigate shear stress
Perfusion method facilitated effective cell growth
Settling of microcarriers during medium change
did not affect cell viability
-Indirect bead-to-bead transfer provided 2.2
times more cell yield than direct bead to bead
transfer.
-Direct bead-to-bead transfer suffered aggregate
formation even with intermittent agitation profile
Allows control over:
-Maintain glucose concentration >1g/L
-Minimize lactate accumulation
Dual impeller configuration
(Rushton with pitched blade in up-flow
orientation) allowed uniform suspension
Rocking bioreactor prevented microcarriers from
settling down
4.2 times more cell yield with medium exchange
Powdered medium can be used interchangeably
with liquid medium
Minimizing aggregate formation
Between reactor parts
Modification in reactor design
In culture
-Evaluated different impeller configurations (Rushton,
Pitched blade, Paddle)
-Varied agitation rate in response to settling microcarriers
Increased distance between vessel wall and
probes and avoid overcrowding vessel with
internal parts
Dual impeller
(Rushton with pitched-blade in up flow
orientation) ensured uniform suspension
Rocking the reactor broke up aggregates and
killed foam
Addressing foaming issues
Preventive approach
Chemical
Evaluated different configurations of aeration systems
(ring, micro, open-tube spargers)
Reactive approach
Mechanical
-Use of antifoam C, Pluronic F-68
-Reduce serum concentration in medium
Open-tube sparger was superior to ring and
micro sparger because of reduced foam
formation.
Rocking the bioreactor killed foam while
maintaining uniform microcarrier suspension
Optimizing Cell Detachment
conditions
Trypsinization
Evaluated the effect of trypsin concentration on indirect
bead-to-bead transfer
Contamination issues/ Bioreactor handling
Optical vs. polarographic probes to prevent contamination
issues
Effective cell removal was achieved
with both 25% and 50% trypsin (w/v)
Optical probe can be disconnected to facilitate
trypsinization in indirect bead-to-bead transfer
Spargers
Impellers
Microcarriers
Cell Yield (cell number/ surface area):
• With the perfusion mode, over the course of the year-long project the cell
yield improved 15 fold.
• Compared to the roller bottle up to 63% cell yield was successfully
obtained in the bioreactor
Bioreactor design:
• Optimization of bioprocesses is highly important: Compare glass bench-
top bioreactor vs. single-use bioreactor
Bead-to-Bead (B2B) transfer:
• Direct B2B transfer more attractive than indirect B2B transfer for scale up
process
• Direct B2B transfer poses more challenges: more studies are needed to
optimize conditions
Scale up:
The optimized design and conditions can be used a basis for scale up
operations
Acknowledgements
For providing resources in support of this project:
Biokit, Spain
Sartorius Stedim, USA
Nova Biomedical Corporation, USA
And, Jayni Jatin Shah for assistance with making the poster.
Presented at :
BPI Conference
Boston, MA
October 26-29, 2015
DC
A B
Cytodex-1TM surface microcarriers (GE healthcare Life Sciences) 2 g/L, images
acquired at 200x magnification. Fig A: Microcarriers at 0 h , Fig. B: Microcarriers
at 166 h.
Aggregate formation. Fig C: 100x magnification, Microcarriers at 355 h. Fig. D:
aggregate in the vessel due to foam formation