INTRODUCTION
The success of a robust bioprocess relies on the ability to accurately and effectively control critical process parameters. In the field of
single-use bioreactors (SUB) these evaluations still remain rare and proper control strategies are not clearly outlined. As a result, this study was
aimed to provide a rigorous understanding towards developing methods for optimal O2 supply, lower dCO2 built up and establishing a pH control
strategy in large scale SUB.
As a first step, mass transfer capabilities were studied using the static gassing out method for determining the volumetric mass transfer
coefficient (kLa) for O2 by varying the size and number of microspargers in the SUB. To evaluate the effects and possible interactions of several
critical factors such as agitation rate, sparge rate and working volumes, design of experiments with full factorial design was conducted. The
response surfaces with second degree polynomial were used to select the optimal controlled parameters with efficient kLas for the cell culture
studies. The kLa’s were determined across different working volumes, agitation and O2 sparge rates.
The air sparge for CO2 stripping in SUB was evaluated via a drilled holes in a sparger and a pipe. Both the strategies, drilled holes in a
sparger or in a pipe, are efficient ways to remove CO2 from the SUB. However, the process pH increases upon removal of CO2 and the rate of
increase in pH was observed to be dependent on the strategy used for CO2 removal. Thus, in order to establish a better control on this pH
deviation in SUB, the PID parameters in the pH-PID loop were tuned and CO2 cascade feedback strategies (microspargers vs drilled holes) were
also evaluated and optimized for the cell culture studies.
Following the optimization of the aforementioned physical characteristics in the SUB, two CHO cell line based 200L fed-batch processes,
one with minimal and the other with improved control strategies, were conducted in SUB. This study successfully demonstrated the advantages of
improved pO2, pH and pCO2 control in a SUB system.
Establishing improved O2 supply, lower dCO2 built up and pH control in large scale single-use bioreactors.
Shahid Rameez, Sigma S. Mostafa and Abhinav A. Shukla KBI Biopharma, 1101 Hamlin Road, Durham, NC 27704
LARGE SCALE SINGLE-USE BIOREACTORS (SUB)
OPTIMAL SUPPLY FOR O2
CONCLUSION
Our aim was to develop a robust O2 supply, dCO2 removal and pH control strategy for large-scale fed-batch processes in
SUB. The dissolved O2 concentration in a suspension of cell culture depends on the rate of O2 transfer from the gas phase
to the liquid, rate at which O2 is transported into the cells, and on O2 uptake by the cells for growth, maintenance and
production. The gas–liquid mass transfer was evaluated with different size sparger discs. A significant impact on supply of
O2 was observed changing the size and number of sparger discs.
For CO2 removal, our approach was to first conduct stripping rate studies using spargers and pipes with drilled holes to
determine rate of removal of CO2 from SUB. Supply of CO2 on demand to control deviations in process pH was evaluated
using the microspargers. In addition, the PID parameters in the pH-PID loop were tuned from default manufacturer’s
settings to establish a faster response on these pH deviations. Finally using the best strategy for the proof of concept run at
200-L scale we demonstrated reduction in dCO2 built up during a CHO cell culture process producing an monoclonal
antibody.
O2 transfer is often the rate-limiting step in the aerobic bioprocess due to the low solubility of O2 in the cell culture medium. The measurement
and/or prediction of the volumetric mass transfer coefficient, (kLa), is a crucial step in the design and scale-up of SUBs. This work aimed at
measurement of kLas using different size and number of sparger discs to provide a better knowledge about the selection, design, scale-up in SUBs.
SUBs are widely used in mammalian cell culture processes and are
rapidly replacing conventional stirred tank bioreactors. SUBs have
impellers like conventional bioreactors, however, with different designs
and sizes. Moreover, the impellers are integrated into the plastic bag.
The plastic bag and the integrated impeller are pre-sterilized.
Prior to onset of cell culture process the sterilized bag is mounted
in the bioreactor vessel and the impeller is connected, mechanically or
magnetically, to a driver connected to a motor.
SUB enhance the biological and process safety during
manufacturing by reducing cleaning and sterilization demands and risk
of cross contamination.
To be representative of the process, troubleshooting experiments were carried in a salt solution to outline a control strategy.
The physical properties of this salt solution more closely mimic the physical properties of the cell culture medium. The solution
was controlled at pH 6.85 and a high CO2 was artificially built up in the SUB. While using drilled holes for removal of CO2
in SUB we evaluated two strategies to attenuate pH deviations caused by CO2 removal.
Strategy 1: CO2 on demand was passed through the micro-sparger instead of pipe with drilled holes. Hence, better mass transfer
for CO2 and thus better control on the pH deviation.
Strategy 2: During the stripping for CO2, pH-PID parameters were tuned for faster response to gain better control on deviation
in pH from process pH setpoint.
Although, the results from such studies are from a salt buffer solution instead of medium with cells, these experiments provide
for a platform to design a control strategy which is eventually employed in a cell culture process.
Results: The kLa is dependent on the O2 transfer from the gas sparge rates to the liquid, working liquid volumes
in the SUB and the stirrer speeds (agitation rate). Figures on the right show experimental and predicted kLa
values, under different operational conditions, using full factorial DOE based design in a 200L SUB . Working
volume and gas flow rates were observed as significant factors in regulating kLas. kLas decreased when working
at lower gas flow rates as a consequence of the O2 transport limitation.
Results:
Changing the Size and number of sparger discs can result in significant impact on supply of O2
CO2 STRIPPING IN SUB 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 and pH-dependent enzymatic reactions in the cell. 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 SUBs these evaluations remain rare and proper stripping strategies are not clearly outlined.
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.
CO2 stripping causes the pH in the system to
rise over time. The stripping rate and control on
process pH deviations will depend on type of
stripping strategy employed (drilled holes in
sparger/pipe).
Case Study: 200L Cell Culture run, 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.
Results: Supplying the CO2 on demand through the microsparger
established better control on the pH deviation in the salt solution.
Furthermore, tuning the pH-PID parameters increased the
response to control pH deviation within dead band of the pH set
point in the solution.
Results: Employing aforementioned gassing strategies in a 200L
CHO cell culture process producing a monoclonal antibody,
approximately 40% reduction in dCO2 built up was achieved.
O2 SUPPLY CONSIDERATIONS
In SUBs 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 benchmark during the design, operation and scale-up for O2 delivery.
Implementation of aforementioned strategy in a 200L CHO cell culture process
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