Making the World Safer, Greener, Better

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

2. 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 Controlled Processes: Late in Development Uncontrolled Processes: Early and 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 Confidential

4. Why New Tools? • Shake Flasks are not always scalable • “system” does not transfer to bioreactors • Cells LIKEbioreactors 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 Confidential

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. Baffle cassette Perc cassette REG2 cassette Consumables: Cassettes • 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. Abgene Tape Type A Type E Type D Consumables: Caps

12. Operating Principle – REG2 Individual Reactor Detail pH 470 nm and DO505 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

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

15. Applications • Reduce development time • Improve chance of finding the best cell line • Reduce burden on process optimization team and equipment Screen clones in a controlledenvironment Generate moreyield data anddownstream processchallenges earlier Identify operating ranges earlier Identify clonesthat respond to feed conditionsearlier More thoroughly investigatedesign space

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

17. Escherichia coli Pseudomonas fluorescens Saccharomyces cerevisiae Pichiapastoris Bacillus subtilis Shewanellaoneidensis MR-1 Aspergillusnidulans Trichodermareesei Aspergillusoryzae Micro-24 Microbioreactor : Organisms Chinese Hamster Ovary (CHO) Cells Hybridoma Carrier-based tissue engineering Cell-free expression systems Your Favourite Organisms!!!

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) : 148-160.

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? Email: narayana_rao@pall.com

23. Confidential Thank You