1. Micro Reactor Technologies - Improved productivity and efficiency of cell culture process development
Making the World Safer, Greener, Better

2. Traditional cell line and process optimization
Agenda
Traditional cell line and process optimization
Next generation screening methods
Micro-24 Microbioreactor: What is it?
Applications
Advantages
Next generation cell line and process optimization
Implementing the “High-throughput” Micro-24 Bioreactor

3. Traditional Cell Line Selection and Process Optimization
Uncontrolled Processes:
Early and Late in Development
Controlled Processes:
Late in Development
Uncontrolled
Shake Flasks (SF)
Benefits:
Large number of clones
No bioreactor resources needed
Concerns:
Screening, selection, initial process optimization is uncontrolled
Commercial process will be controlled
SF are not the standard for scale-up to pilot scale and beyond
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4. Value to your organization!
Why New Tools?
Shake Flasks are not always scalable
“system” does not transfer to bioreactors
Cells LIKE bioreactors more than shake flasks
Higher density cultures in mammalian platforms
No online data
Cost of NOT having the “right” data and screens?
QbD and Regulatory
Robust Process development
Evolving: Where does the process actually start?
Value to your organization!

5. “Losers” in Shake Flasks but … “Winners” in Bioreactors
Productivity (mg/ml)
All CHO clones had low titres in shake flasks
Best of low titer clones progressed to minireactors
Titer improvement when clones were run controlled
Industrial data
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6. What is the Significance of Not Running Controlled?
Additional screening necessary.
Why screen the same way?
Increase demand on process optimization’s scarce resources
Bioreactors, operators, scientist
Increase development time
Does one really have the best commercial cell line and process at the end of the day?

7. Next Generation: “High-throughput” Micro Bioreactors
Uncontrolled
Controlled Processes: Early and Late in Development
Large number of clones and conditions can be screened controlled without using valuable 3L bioreactor resources
Learn more about process earlier using high-throughput 10 mL bioreactors.
Increase the likelihood of selecting and developing the best clone and process for commercialization.

8. High-throughput and Scalable Micro-24 Microbioreactor
Small scale and controlled bioreactor system
24 individually monitored and controlled reactors pH
Ammonia (Microbial)
Sodium Bicarbonate (Mammalian)
Dissolved Oxygen
Temperature
Controllable agitation

9. High-throughput and Scalable Micro-24 Microbioreactor
Data acquisition in real time
MRT is amenable to studying factors/ levels and replicates that total 24
Multiple factor studies uncover interactions
And, it’s “scalable”
Saves on time and “$”

10. Consumables: Cassettes
Baffle cassette
Perc cassette
REG2 cassette
3 Cassette Types
REG2 and Baffled- Gases are bubbled into cassette
Perc Plate: Gases are placed in the overlay
10 mL Total Volume per well/ ”reactor”
Pre-sterilized

11. Consumables: Caps
Type A
Type E
Type D
Abgene Tape

12. Operating Principle – REG2 Individual Reactor Detail
pH 470 nm and DO 505 nm
by luminescence quenching
CO2, Buffer (NaBicarb) - required for cell culture
CO2, NH3 - required for microbes
Options: Air, O2, N2, up to 3 gases in total 12

13. Operating Principle – Perc Cassette Individual Reactor Detail

14. Micro-24 MicroReactor – FEATURES
INDEPENDENT CONTROL OF pH
Operating range: 4.0 – 10.0,
Accuracy is ± 0.2 pH units
INDEPENDENT CONTROL OF DO
Range and accuracy ±5% at 0% saturation, 10% at 100% saturation
INDEPENDENT CONTROL OF TEMPERATURE
Operating range: 18 – 45OC
AGITATION
Operating Range: 0 – 800 rpm 14

15. Applications Reduce development time
Screen clones in a controlled environment
Generate more yield data and downstream process challenges earlier
Identify operating
ranges earlier
Identify clones that respond
to feed conditions earlier
More thoroughly
investigate design space
Reduce development time
Improve chance of finding the best cell line
Reduce burden on process optimization team and equipment

16. Control Software
Individual set points for temp, pH, DO in each of the 24 reactors
Real-time monitoring & control of temp, pH, DO and gas fluxes 16

17. Micro-24 Microbioreactor : Organisms
Escherichia coli
Pseudomonas fluorescens
Saccharomyces cerevisiae
Pichia pastoris
Bacillus subtilis
Shewanella oneidensis MR-1
Aspergillus nidulans
Trichoderma reesei
Aspergillus oryzae
Chinese Hamster Ovary (CHO) Cells
Hybridoma
Carrier-based tissue engineering
Cell-free expression systems
Your Favourite Organisms!!! 17

18. Screening: Parameters
Micro-scale to industrial scale fermentation : implementation of scale-down and
DOE combination for process modelisation and improvement of industrial scale
vaccine process
Chaudet.Nicolas1, Lintot.Emilie2, Jourdat.Catherine1, Barbirato.Fabien2, Guillaume.Jean-Marc1
1Sanofi Pasteur, Marcy l’Etoile, France, Bioprocess Research and Development Upstream, 2 Sanofi Pasteur, Marcy l’Etoile, France, Manufacturing Technology

19. Micro-24 and Feed Development
Feed response would not have been identified in Shake Flasks
Micro-24 results are similar to 3 L bioreactor results
Proof of concept: Screen for feed response and develop feeds and media for bioreactor processes.
Industrial Data

20. Micro-24 and 2L Reactor: Viability and Cell Count Comparison
Good comparison between the Micro-24 and 2L bioreactors
No compromise on the product quantity or quality issues
Chen, Aaron, Rajesh Chitta, et al.. Twenty-Four Well Plate Miniature Bioreactor System as a Scale-Down Model for Cell Culture Process Development. Biotechnology and Engineering 2009;102(1) :

21. Scalable Bioreactors for Process Optimization
Screening clones and processes controlled
Identify operating ranges earlier
Identify clones that respond to feed earlier in process
Develop “bioreactor” media and feeds
Generate more yield data and identify downstream process challenges earlier
Reduce development time
Improve likelihood of finding the best cell line
Reduce team and equipment burdens
Ideal for quality by design (QbD)
Enhance technology transfer

22. Questions?

23. Thank You
Confidential 23