A presentation by KBI Process Development Scientist Shahid Rameez, Ph.D. demonstrating the ability to accurately and effectively monitor and control critical process parameters in a large single-use bioreactor.

1. Establishing improved O2 supply, lower
dCO2 built up and pH control in large scale
Single-Use BioReactors (SUBR)
Shahid Rameez, Ph.D.
Scientist, Process Development.
245th ACS National Meeting & Exposition, 2013, New Orleans, Louisiana

2. The ability to accurately and effectively monitor/control
critical process parameters in a Bioprocess.
Successful Bioprocessing
Towards Robust Cell Culture Processes
Technology Development at KBI
Volumetric mass transfer coefficient (Kla)
Evaluating Microspargers (2 and 20 micron)
at different agitation and flow rates of O2.
CO2 stripping:
Demonstrating CO2 stripping via
Open pipe or drilled holes in sparger.
Establishing a better control with
tuning PID controllers.
In Single-Use Bioreactors these evaluations remain rare
and proper control strategies are not clearly outlined.

3. Overview: Single-Use BioReactors (SUBR)
Disposable Bioreactor Bag
Bioreactor System

4. SUBR
Sparger with multiple discs
Air and CO2
Historically at KBI
SUBR
Sparger with one disc size
Open Pipe
CO2 and O2
Historically at KBIAir, O2 and CO2
Historically at KBI
Multi-Sparger Bag Custom Bag with a Wand
Two types of bag designs: (1) Sparger with multiple discs of different sizes, (2) Sparger and an
open pipe (2mm drilled holes).

5. 65
8
7
1
2
3
4SUBR
Multiple sparger
1 and 5 = 20 micron disks.
2 and 6 = 2 micron disks.
3 and 7 = 2 micron disks with 5 – 1mm drilled holes.
4 and 8 = 2 micron disks with 5 – 0.5mm drilled holes.
Multi-Sparger Bag

6. SUBR
Sparger
Custom Bag with a Wand
Open Pipe
200L
4 - 20 micron disks.
2000L
8 - 20 micron disks.
200L
10 - 2mm drilled holes.
2000L
26 - 2mm drilled holes.

7. 4 - 20 micron disks. 2 - 20 micron disks.
1 - 2 micron disks.
2 - 2 micron disks.
PART 1:
O2 supply (kLa) in SUBR
Various combinations (Size + Number) of sparger discs for supply of O2

8. Example: O2 supply (kLa) in 200L SUBR using 20 micron spargers
Response Surface DOE for determination of kLa
Physical Properties
(For Example: Viscosity,
Density, Interfacial tension)
Geometric Properties
(For Example: Size of
Bioreactor and Impellers)
Energy Dissipation
(Mixing time, Bubble
Diameter)
Cell Culture Knowledge
(Shear Sensitivity, Cell
growth of the culture, etc.
Operational Requirements
(For Example: Agitation, Gas
flow velocities, Working Volumes)
Effective O2 supply to support the Process
Understanding of SUBR design along-with knowledge specific to cell line and product of
interest is required when determining the effective O2 supply.

9. 200L SUBR gassing with 20 micron disks
Note: Change in pH and its effect on kLa have to determined too. For this
study, with change in pH (by 1 unit or more), there was not significant
change in the values of kLa.
• Thus, based on the response surfaces, the optimal range
for operating parameters was determined for the process.
• Agitation in the tested study range was found to have
minimal impact on the kLa .
• Working volumes and the O2 sparge rates were found to
have significant impact on the kLa .

10. Case Study: Gassing strategies with different micron disks
• Changing the Size and number of
sparger discs can result in significant
impact on supply of O2

11.  The dissolved oxygen (dO2 ) concentration in a suspension of cell
culture depends on:
- Rate of O2 transfer from the gas phase to the liquid.
- Rate at which O2 is transported into the cells.
- O2 uptake by the cells for growth, maintenance/production.
 The gas–liquid mass transfer is strongly depended on the
hydrodynamic conditions in the bioreactors. These conditions are a
function of energy dissipation.
O2 Supply Considerations

12.  In SUBRs high values of mass transfer rates and excellent mixing
can be achieved with proper O2 delivery strategy.
 Many factors such as agitation, type and number of spargers , gas
flow rates etc, have to be evaluated in detail in order to achieve
optimal O2 supply.
 The correct measurement and/or prediction of (kLa), serves as
crucial step in the design, operation and scale-up for O2 delivery in
SUBRs.
O2 Supply Considerations

13. PART 2:
CO2 stripping in SUBR for a Cell Culture Process.
 One of the recurrent issues that is observed in industrial
mammalian cell culture especially in large-scale bioreactors is
accumulation of dissolved CO2 (dCO2) .
 The impact of dCO2 on cell culture has been studied in detail. It
has been shown to have effect on:
- Cell growth rate, specific productivity, decrease in cell density.
- Decrease in glucose, lactate, and glutamine specific metabolite rates.
- Changes in pH-dependent enzymatic reactions in the cell.

14. PART 2:
CO2 stripping in SUBR for a Cell Culture Process.
 In small-scale bioreactors majority of dCO2 is stripped via
surface aeration. However, in large-scale bioreactors, the liquid
surface-to-volume ratio decreases and thus other strategies for
dCO2 removal have to be designed.
 In SUBRs these evaluations remain rare and proper control
strategies are not clearly outlined.

15. SUBR
Sparger with drilled holes
Open Pipe
ORAir
Air
Both excessive stripping or accumulation of dCO2 are detrimental to
cell growth thus an optimal level of dCO2 has to be maintained for
cell culture. This optimal value will vary with cell line and product of
interest.
SUBRs at KBI
Stripping of CO2 via Spargers
Disks with 1mm or 0.5mm drilled holes
Stripping of CO2 via Open Pipe
Open pipe with 1mm drilled holes

16. SUBR
Sparger with drilled holes
Open Pipe
ORAir
Figure describes the pH curve over time as Air is passed through
the Open pipe or Sparger. CO2 removal causes the pH in the
system to rise over time.
Air
Note: The stripping rate and thus control on process pH drifts will
depend on type of stripping strategy employed (Sparger/Open pipe).

17. Case Study: 200L Cell Culture runs, where high CO2 built up was
expected due to addition of a basic feed which led to addition for high
amounts of CO2 to control pH increase in the culture.
Problem: The CO2 removal which could be achieved was minimal. The
pH started to increase rapidly as soon as Air was introduced in the
culture to achieve CO2 removal.

18. O2
pH
Air
Base
CO2
SUBR
Open Pipe
20 micron sparger

19. O2
pH
Air
Base
CO2
SUBR
Open Pipe
20 micron sparger
We employed two Strategies:
Strategy 1: CO2 in the cascade loop was passed through the micro-
sparger instead of open pipe. Thus, better mass transfer for CO2 and
thus better control on the pH drifts.

20. O2
pH
Air
Base
CO2
SUBR
Open Pipe
20 micron sparger
Strategy 2: The PID parameters are tuned to gain better control on the
drifts in pH.
PID

21. Results: CO2 in the cascade loop passed through the microsparger established better
control on the pH drift. In addition the tuned PID parameters were able to control
drift of pH within tight dead band of the pH set point.

22. Results: Comparison between two-200L cell culture runs (≈150L Working
Volumes) in SUBR, with/without gassing strategies employed.
≈ 40% reduction in dCO2 built up was observed when employing the
discussed gassing strategies.
Basic Feed Additions

23.  A rigorous understanding towards developing methods for
optimal O2 supply, lower dCO2 built up and establishing a pH
control strategy in large scale Single-Use Bioreactors.
 Flexibility to optimize critical physical process parameters (pH,
Dissolved Oxygen, low CO2 built up) enables to develop robust
cell culture processes.
Conclusions

24.  Robust large-scale process development excellence reduces
costs/timelines and can play a major factor in eventual commercial
cell culture development.
 Overall, these studies at KBI were aimed to provide a better
knowledge in selection, design and scale-up for development of
large scale cell culture processes in Single-Use Bioreactors.
Conclusions

25. • Joe McMahon President and CEO
• Abhinav Shukla, Ph.D. VP, Process Development and Manufacturing
• Sigma Mostafa, Ph.D. Director, Process Development
 Process Development Team at KBI
Acknowledgements

26. Thanks
Questions??