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Monoclonal Antibody Production: Building the Platform. Andrew Clutterbuck Eden Biodesign Ltd.

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(1)

Monoclonal Antibody Production:

Building the Platform

Andrew Clutterbuck

Eden Biodesign Ltd.

(2)

Questions

Questions are encouraged throughout the presentation and can be asked by using

p y g

the email address provided within your

webcast viewer.

(3)

Eden Biodesign

“Designing and developing valuable biopharmaceutical

medicines by the application of good science from day one”

g

medicines by the application of good science from day one”

(4)

Eden Biodesign

‰ CMO offering:

‰ Expression system development

g

‰ Expression system development

‰ Process and analytical development

‰ Cell banking

‰ cGMP production services p

‰ Consultancy services in CMC issues,

regulatory support, cGMP training, technical trouble shooting and clinical trial supply

trouble shooting and clinical trial supply logistics

‰ World class microbial, mammalian and viral process development and cGMP facilities process development and cGMP facilities located in Liverpool, UK

‰ US subsidiary located in Research Triangle Park NC

Park, NC

(5)

Presentation overview

‰ Ch ll f i tib d f t

‰ Challenges facing antibody manufacturers

‰ An overview of Antibody purification

‰ Case study into the development of a purification methodology for the clinical purification methodology for the clinical production of an IgG 4 monoclonal

antibody

(6)

Challenges in Antibody Production

‰ High dosing requirements:

g y

‰ High dosing requirements:

‰ Large amounts of product required

‰ Continuity of supply

‰ Increasing titres:

‰ Typical: 2 – 5 g/L

‰ Column and resin limitations

‰ Column and resin limitations

‰ Low viability on harvest:

‰ Clarification issues

‰ Clarification issues

‰ Increased contaminant levels

(7)

Challenges in Antibody Production g y

‰ High throughput processing:

‰ High throughput processing:

‰ Increased capacity resin

‰ High flow rates

‰ Disposable technology:

‰ Pre-packed columns

‰ Disposable membrane adsorbtion

‰ Disposable membrane adsorbtion technology

‰ Other options:

‰ Other options:

‰ Cation exchange primary capture

‰ Simulated moving bed

‰ T h t ti

‰ Two phase extraction

(8)

Typical mAb Purification Process

Fermentation H t & l ifi ti

yp

Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(9)

Harvest & Clarification

Fermentation H t & l ifi ti Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(10)

Harvest & Clarification

‰ Direct depth filtration

‰ Ideally suited for volumes (< 1000 L)

‰ Disposable options

‰ Centrifugation → Depth filtration g p

‰ Most practical for large volumes (> 1000 L)

‰ High shear

‰ Difficult to develop and control

‰ Equipment and cleaning validation requirements

‰ High operating costs

‰ Microfiltration

‰ Suited at all scales

‰ Equipment and cleaning validation requirements

‰ High buffer and utility consumption

(11)

Protein A Chromatography

Fermentation H t & l ifi ti

g y

Harvest & clarification Protein A chromatography

Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(12)

Protein A Chromatography g y

‰ Industry standard

‰ Industry standard

‰ Robust

‰ Efficient

‰ Efficient

‰High purity in singe step

‰Volume reduction

‰Volume reduction

‰ High initial capital outlay

‰ Column limitations

‰Binding capacity

‰H d

‰Hardware

(13)

Low pH Viral Inactivation

Fermentation H t & l ifi ti

p

Harvest & clarification

Protein A chromatography Low pH viral inactivationp

Cation exchange chromatography Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(14)

Low pH Viral Inactivation

‰ Inactivation of enveloped viruses

p

‰ Inactivation of enveloped viruses

‰ Cheap, simple and effective

‰ Minimal hardware requirements

‰ Precipitation of host cell proteins

‰ Precipitation of host cell proteins

‰ Product stability

‰ Product stability

‰ Aggregation

‰ Precipitation

‰ Precipitation

(15)

Cation Exchange Chromatography

Fermentation H t & l ifi ti

g g p y

Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(16)

Cation Exchange Chromatography

‰ Intermediate purification:

g g p y

‰ Intermediate purification:

‰Aggregate removal

‰Host cell protein reduction

‰Host cell protein reduction

‰Leached Protein A reduction

‰ Packed bed chromatography

‰ Packed bed chromatography

‰ Bind and elute mode

‰ Possible viral reduction step

(17)

Anion Exchange Chromatography

Fermentation H t & l ifi ti

g g p y

Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(18)

Anion Exchange Chromatography g g p y

‰ Polishing purification:

‰ Polishing purification:

‰Residual host cell protein removal

‰Endotoxin reduction

‰ Flowthrough mode:

‰Disposable membrane technology

‰Disposable membrane technology

‰ Viral reduction

(19)

Viral Reduction Filtration

Fermentation H t & l ifi ti Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(20)

Viral Reduction Filtration

‰ Nano-filtration

‰ Nano filtration

‰100 nm Pre-filter → 20 nm Final filter

‰ Small non enveloped viruses

‰ Small non-enveloped viruses

‰ Disposable capsule format

(21)

Concentration & Buffer Exchange

Fermentation H t & l ifi ti

g

Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(22)

Concentration & Buffer Exchange

‰ Ult filt ti / di filt ti

g

‰ Ultrafiltration / diafiltration

‰Crossflow filtration

‰ Volume reduction

‰ Buffer exchange

‰ Buffer exchange

‰ Various options

(23)

Bioburden Reduction Filtration

Fermentation H t & l ifi ti Harvest & clarification

Protein A chromatography Low pH viral inactivationp Cation exchange chromatography

Anion exchange chromatography

Concentration & buffer exchange Viral reduction filtration

Bioburden reduction

(24)

Bioburden Reduction Filtration

‰ 0 22 µm filtration

‰ 0.22 µm filtration

‰ Pre-filter → Final filter

‰ Disposable capsules

‰ Bioburden reduction

‰ Bioburden reduction

‰Patient safety

‰Reduce microbial spoilage p g

(25)

Monoclonal antibody purification:

Monoclonal antibody purification:

Case Study

(26)

Case Study: Process Development

‰ IgG 4

y p

‰ IgG 4

‰CHO cell line:

‰Enabling cell expression

‰Enabling cell expression technology

‰Stirred tank Bioreactor

‰Chemically defined media

‰ Target = 0 5 g/L expression

‰ Target = 0.5 g/L expression

(27)

Case Study: Process Development

‰ Depth filtration

y p

‰ Depth filtration

‰Gross cellular debris removal

‰Direct bioreactor clarification

‰Fully disposable

‰ Normal flow filtration

‰ Normal flow filtration

‰0.22 µm filtration

‰Bioburden reduction

‰Fine particulate removal

‰Disposable capsule format

(28)

Protein A – Resin Screening g

MabSelect A: GE Healthcare ProSep A: Millipore

MabCapture A: Applied Biosystems

MabCapture A: Applied Biosystems

(29)

Cation Exchange resin screening:

Capto S GE HealthCare

Run 1 – pH 5.5 Run 2 – pH 5.0 Run 3 – pH 4.5

Capto S – GE HealthCare

(30)

Cation Exchange resin screening:

GigaCap S TosoH GigaCap S – TosoH

Run 1 – pH 5.5 Run 2 – pH 5.0 Run 3 – pH 4.5

(31)

Cation Exchange resin screening:

Poros HS Applied Biosystems Poros HS – Applied Biosystems

Run 1 – pH 5.5 Run 2 – pH 5.0 Run 3 – pH 4.5

(32)

Anion Exchange resin screening

‰ Packed bed:

g g

‰ Packed bed:

‰Poros HQ – Applied Biosystems

‰Capto Q – GE Healthcare p

‰ Membrane adsorbtion:

‰Sartobind Q Sartorius

‰Sartobind Q – Sartorius

‰Adsep – Natrix Separations

‰Chromasorb – Millipore p

(33)

Case Study: 2 L Bioreactor y

‰ 2 L bioreactor

‰ 2 L bioreactor

‰ PowerCHO growth media

‰F d t t li d

‰Feed strategy applied

‰ 0.5 g/L expression

‰ 2 x 10 6 cells per mL (on harvest)

‰ < 50 % i bilit ( h t)

‰ < 50 % viability (on harvest)

(34)

Protein A: MabCapture A Applied Bios stems

Applied Biosystems

(35)

Cation Exchange: Poros HS Applied Bios stems

Applied Biosystems

(36)

Anion Exchange: Poros HQ Applied Bios stems

Applied Biosystems

(37)

Analytical Results: SDS-PAGE y

(38)

Analytical Results: SDS-PAGE y

(39)

Analytical Results: Size exclusion y

(40)

Analytical Results

Analysis Technique Result

Endotoxin LAL < 0.5 EU/mL

DNA qPCR 16 pg/mL

Purity (SEC) SEC 100 %

Purity (SEC) SEC 100 %

Host cell protein ELISA In development

Recovery ELISA In development

(41)

Questions

Questions are encouraged throughout the presentation and can be asked by using

p y g

the email address provided within your

webcast viewer.

(42)

Thank You

Title:

Thank You.

Title:

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