scaling down bioreactor process development · scaling down bioreactor process development:...

Scaling Down Bioreactor Process Development:

Comparison of Microbioreactor and Bench Scale Solutions.

Richard Lugg. Scientist I, MedImmune.European Laboratory Robotics Interest Group: High Throughput Bioprocess Development. 22 June 2011

2

Challenges Facing the Industry

� We are in an industry constantly under pressure to deliver high quality products:-

� Aiming for high titre manufacturing processes.

� Through efficient use of resources in a timely manner.

� Scaled down bioreactors can be one method of achieving this goal.

3

Typical Preclinical Project Plan.

VectorsParental cell lines

Clonal cell lines Phenotypic Cell Line Stability

Lead Clone Selected

SuspensionShake/T Flask50 Cell Lines

Static 24 Well Plate

200-300 Cell Lines

Bioreactor Platform Process

6-8 Cell Lines

Static 24 Well Plate

200-300 Cell Lines

SuspensionShake Flask

50 Cell Lines

Suspension Shake Flask

6-8 Cell Lines

Bioreactor Process Development

Bioreactor Process

Optimisation1 Cell Line

Multiple Bioreactors / Flasks

Process Lock

4

Typical Preclinical Project Plan.



Lead Clone Selected


Shaking plate(suspension)

24 Well Plate200-300 Cell Lines


6-8 Cell Lines




50 Cell Lines


6-8 Cell Lines


Bioreactor Process



Process Lock

5

Screening for the Best Cell Lines.

� Early cell line screening in static/suspension plates.

� Best clones evaluated in shake flasks.

� Only the final few (<10) are taken forward to bioreactors.

� In an ideal world every cell line would be evaluated in a bioreactor.

100s

10s

<10

6

Improving Quality of Data

Quality of Data

Ease of Handling

?

7

New Technologies for Screening Cells

� New technologies can now offer better scale down models of our cell culture and bioreactor processes.

Scale-down Bioreactor System

�Micro-24™ - Pall

�SimCell™ – Seahorse Bioscience

�ambr™ - TAP Biosystems

Fed Shake Flask

Shaken 24 well Plates (semi-automated)*Static 96 well Plate

Possible new approachesOld technology

*Silk et al. Biotechnology Letters (2010)

8

ambr™ - TAP Biosystems

� TAP Biosystems saw a need for a scaled down bioreactor.

� Approached MedImmune for our input in development of the system.

� Gave us scope to try another system that could be tailored to our requirements.

9

ambr™ Background

� ambr™ microbioreactor.

� Automated.� Liquid handling deck.

� Cell culture manipulation.> Inoculation.> Feeding.> Sampling.

� Disposable stirred bioreactor vessels.

� 24 or 48 vessels can be run at same time.

� 7mL-15mL working volume.

� Bioreactor control. � pH.

� Temperature.

� Dissolved oxygen.

� Stirrer speed. TAP Biosystems.

10

Liquid Handling Deck

� 2 Cell culture stations (24 vessel format).

� 12 vessels per culture station.

� 1 temperature setting for each cell culture station.

� 1 stirrer speed setting for each cell culture station.

� 2 deck for pipette tips.

� 4 decks for reagents and sampling lab ware.

Liquid handling arm Vessels (24 total)

Tips for feed addition / sampling

Inoculum, medium, feeds or

sample cups

11

Disposable Vessel.

pH and O2sensors

Impeller

Open pipe sparger

Inlet / outlet for fluids

• Gamma irradiated for sterility.• Presens fluorescence sensors for pH and O2 sensors.• 3 Gasses can be added through the sparger. • Each vessel can have different pH, dissolved oxygen set points. • Separate feeding regimes.• Measurements taken every 90 seconds.

12

� Considerations

�This technology requires a dedicated laminar airflow hood.

�Some infrastructure for gases.

�User handling required during run to replace tips, offline analysis etc.

�Flexibility.

�Simple to use.

Extra Considerations When Using ambr™

13

=Does ?

14

Vessel to Vessel Reproducibility.

� Batch over grow experiment ran using 24 vessels in batch with the same condition.

� Conditions.> pH, DO, temperature control.> No feed/glucose additions made.

� Off Line Analysis.> Cell counts were made using a Vi-CELL® (Beckman Coulter).> pH compared with using a blood gas analyser (Radiometer).

15

ambr Batch Overgrow Data.

Key:

Ambr : Orange and Red

Mean in Black Solid

3SD from mean in Black Dotted

� Good consistency between vessels was observed.

� Dotted lines show 3 standard deviations (SD) from the mean of the data.

� Data falls within the dotted lines.

� No differences between culture stations.

� No corner affects.

� No back row Vs front row affects.

0

20

40

60

80

100

120

0 2 4 6 8 10 12

Days

Via

ble

Cel

ls/m

L

Viable cell profile.

16

ambr Batch Over Grow Data.

� pH was controlled within range (pH SP ± 0.10).

� pCO2 as expected for batch data.

6.90

7.00

7.10

7.20

7.30

0 2 4 6 8 10 12

Days

pH

Low

er L

imit

p

H S

P

U

pper

Lim

it

0

20

40

60

80

100

120

140

0 2 4 6 8 10 12

DayspC

O2

(mm

Hg)

Off line pH Profile. Off line pCO2 Profile.

17

ambr Batch Over Grow Summary.

� Good vessel to vessel comparability.

� No positional effects.

� Front to back.

� Side to side.

� Culture station to culture station.

� pH well controlled for all vessels but it is slightly different in the microbioreactor in comparison to a benchtop bioreactor.

18

pH control strategy.

6.60

6.80

7.00

7.20

7.40

0 2 4 6 8 10 12 14 16

pH

� In ambr pH is controlled by addition of carbon dioxide or a base solution as for a benchtop bioreactor.

� There are subtle differences in the control of pH:

� Base additions are discontinuous with ambr where a benchtop bioreactor base addition is continuous.

� You have to determine a target value for the base addition to bring the pH back up to (see example below).

Example:

Here the base additions are made on days 5 and 7 to target a pH of 6.90 (half way between the SP and lower limit)

pH SP

Upper Limit

Lower Limit

19

Further Optimisation of Process.

� Gassing.

� Ballast flow rates can be optimised for process.

� Move to use same gases as benchtop bioreactor.

� Manual nature of the glucose measurements using the hand held meter became laborious.

� Implemented a YSI2700 with 24 sample turntable.

� pH control.

� pH profiles showed similar trends but base addition optimised toachieve similar profile to benchtop at the lower limit.

� Lower target base additions sets for fine tuning of pH the control.

20

Clone Ranking Experiment

� 24 vessels used to examine the ranking of 6 different clones using fed batch method.

� Each clone was evaluated in duplicate.

� 2 different gas flow rates.

� Controls.> Stirrer speed increased during run based on O2 demand.> DO, temperature control kept constant.> pH with dead band used. > Bolus nutrient and glucose feed additions made.

� Off Line Analysis.> Cell counts were made using a Vi-CELL® (Beckman Coulter).> Offline pH was measured using a blood gas analyser (Radiometer).> A YSI2700 instrument was used to measure glucose and lactate.

� Data was compared to that generated in benchtop bioreactor (DASgip).

21

ambr Vs Benchtop Bioreactor Data.

0

5

10

15

20

25

30

35

40

45

0 2 4 6 8 10 12 14 16

Days

Via

ble

Cel

ls /

mL

(e6)

A1 A19 A7 A13 D1

6.60

6.65

6.70

6.75

6.80

6.85

6.90

6.95

7.00

7.05

7.10

0 2 4 6 8 10 12 14 16

Days

pH

A1 A19 A7 A13 D1

pH SP

Upper limit

Lower limit

Viable Cell Number Profile. Off line pH Profile.

� Red line shows benchtop bioreactor data and blue line shows ambr data.

� 1 cell line shown as all clones were similar in their profiles.

� Viable cell count good reproducibility between ambr vessels difference towards end of run due to using different Vicell for ambr and benchtop bioreactor data.

� pH profile is very similar between ambr and benchtop bioreactor.

22

ambr Vs Benchtop Bioreactor pCO 2 Profile.

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

0 2 4 6 8 1 0 1 2 1 4 1 6

D a y s

pCO

2

A 1 A 1 9 A 7 A 1 3 D 1

pCO

2

Shows similar drop in pCO2 between ambr and benchtop bioreactorover time course.

The separate pCO2 profiles in ambr due to the different ballast flow rates.

23

Glucose and Lactate Profiles.

General trends in the ambr are comparable to the benchtop bioreactor.

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16

Days

Glu

cose

g/L

A1 A19 A7 A13 D1

0

1

2

3

4

5

0 2 4 6 8 10 12 14 16

Days

Lact

ate

g/L

A1 A19 A7 A13 D1

Glucose Profiles. Lactate Profiles.

24

Clone Ranking: Summary

� Generally cells grow better in the ambr than in benchtop bioreactors.

� Higher IVC and higher maximum viable cell density reached.

� Specific productivity (pg/cell/day) was similar between ambr and benchtop bioreactors.

� Titre is usually higher in the ambr compared with the bench top bioreactor vessels due to higher IVC.

� How does the clone ranking look?

25

Clone Ranking on Titre.

1 2

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

End

of R

un T

itre

� Final harvest titre of top 2 cell lines ranked in the same order by ambr and benchtop bioreactor.� Titres > 3.5g/L.

� Final harvest titre of other 4 cell lines in a different order.� Small data set for these cell lines at benchtop scale makes it more difficult to

differentiate between the different clones.

� Need to run more benchtop bioreactors to verify ranking order.

1 2 3 4 5 6

26

Clone Ranking Conclusions.

� Good vessel to vessel reproducibility in the ambr system.

� Very tight data set generated for the batch and fed batch ranking studies.

� Good process control in ambr comparable to benchtop systems.

� Allows for process optimisation (pH, DO, etc).

� Clone ranking in microbioreactor compared to benchtop bioreactor systems is ok.

� We need to build further data sets. > Parallel experiments using both benchtop bioreactors and ambr

needed.> Potential to push bioreactor screening earlier in our preclinical

project plan.

27

Typical Preclinical Project Plan



Lead Clone Selected





6-8 Cell Lines




50 Cell Lines


6-8 Cell Lines


Bioreactor Process



Process Lock

28

Future Preclinical Project Plan?



Lead Clone Selected




6-8 Cell Lines




50 Cell Lines


6-8 Cell Lines


Bioreactor Process



Process Lock

Bioreactor Platform Process?

Bioreactor Platform Process?

29

� Efficient use of resources is important.

� Microbioreactor systems as alternative to shake flasks/benchtop bioreactors.

� This technology can be applied earlier in our process than traditional bioreactors.

� Allowing for screening multiple cell lines in a manufacturing environment.

� Further process development and optimisation on a limited panel of cell lines for higher titre manufacturing process.

� Bioreactor quality data with shake flask resource.

� Better decision making during clone screening.

Summary

30

Efficient use of resources.

6 Benchtop bioreactors. 24 microbioreactors.

31

Acknowledgments

� ambr ™� Gareth Lewis, Alison Mason, Rahul Pradhan, Diane Hatton and Ray Field.

� Early Stage Bioreactor Team and Cell Sciences Group at MedImmune.

� From TAP Biosystems.> Richard Wales, Neil Bargh, Kenneth Lee, Dave Savage.

scaling down bioreactor process development · scaling down bioreactor process development:...

Documents