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

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

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 Confidential

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!

“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

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?

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.

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

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 “$”

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

Consumables: Caps Type A Type E Type D Abgene Tape

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

Operating Principle – Perc Cassette Individual Reactor Detail

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

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

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

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

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

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

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.

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

Questions? Email: narayana_rao@pall.com

Thank You Confidential 23