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

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.

Overview: Single-Use BioReactors (SUBR)

Disposable Bioreactor Bag Bioreactor System

SUBR

Sparger with multiple discs

Air and CO2

Historically at KBI

SUBR

Sparger with one disc size

Open Pipe

CO2 and O2

Historically at KBI Air, 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).

6 5

8

7

1 2

3

4 SUBR

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

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.

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

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.

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 .

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

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

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

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.

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.

SUBR

Sparger with drilled holes

Open Pipe

OR Air 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

SUBR

Sparger with drilled holes

Open Pipe

OR Air

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).

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.

O2

pH

Air

Base

CO2

SUBR

Open Pipe

20 micron sparger

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.

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

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.

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

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

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

• 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

Thanks

Questions??