Micro Reactor Technologies - Improved productivity and efficiency of cell culture process development

Making the World Safer, Greener, Better

Making the World Safer, Greener, Better

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AgendaAgenda

•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

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Traditional Cell Line Selection and Process Optimization Traditional Cell Line Selection and Process Optimization

UncontrolledUncontrolled Processes:

Early and Late in Development

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

Controlled Processes: Late in Development

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Why New Tools?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!

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“Losers” in Shake Flasks but … “Winners” in Bioreactors“Losers” in Shake Flasks but … “Winners” in Bioreactors

Industrial data

• All CHO clones had low titres in shake flasks• Best of low titer clones progressed to minireactors• Titer improvement when clones were run controlled

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Pro

du

ctivity (mg

/ml)

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What is the Significance of Not Running Controlled?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?

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Next Generation: “High-throughput” Micro BioreactorsNext 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.

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High-throughput and Scalable Micro-24 MicrobioreactorHigh-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

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High-throughput and Scalable Micro-24 MicrobioreactorHigh-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 “$”

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Consumables: CassettesConsumables: 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

Baffle cassette Perc cassette REG2 cassette

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Consumables: CapsConsumables: Caps

Type A Type E

Type DAbgene

Tape

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Operating Principle – REG2 Individual Reactor DetailOperating Principle – REG2 Individual Reactor Detail

CO2, Buffer (NaBicarb) - required for cell culture CO2, NH3 - required for microbes

Options: Air, O2, N2, up to 3 gases in total

pH 470 nm and DO 505 nmby luminescence quenching

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Operating Principle – Perc Cassette Individual Reactor DetailOperating Principle – Perc Cassette Individual Reactor Detail

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Micro-24 MicroReactor – FEATURESMicro-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

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Screen clones in a controlledenvironment

Identify clonesthat respond

to feed conditionsearlier

Identify operating ranges earlier

Generate moreyield data and

downstream processchallenges 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

ApplicationsApplications

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

Control SoftwareControl Software

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• 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!!!

Micro-24 Microbioreactor : OrganismsMicro-24 Microbioreactor : Organisms

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Screening: ParametersScreening: Parameters

Micro-scale to industrial scale fermentation : implementation of scale-down andDOE combination for process modelisation and improvement of industrial scalevaccine processChaudet.Nicolas1, Lintot.Emilie2, Jourdat.Catherine1, Barbirato.Fabien2, Guillaume.Jean-Marc11Sanofi Pasteur, Marcy l’Etoile, France, Bioprocess Research and Development Upstream, 2 Sanofi Pasteur, Marcy l’Etoile, France, Manufacturing Technology

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Micro-24 and Feed DevelopmentMicro-24 and Feed Development

Feed response would not have been identified in Shake Flasks

Micro-24 results are similar to 3 L bioreactor resultsProof of concept: Screen for feed response and develop

feeds and media for bioreactor processes.

Industrial Data

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Micro-24 and 2L Reactor: Viability and Cell Count ComparisonMicro-24 and 2L Reactor: Viability and Cell Count Comparison

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.

• Good comparison between the Micro-24 and 2L bioreactors• No compromise on the product quantity or quality issues

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Scalable Bioreactors for Process OptimizationScalable 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

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Questions?Questions?

Email: narayana_rao@pall.comEmail: narayana_rao@pall.com

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