Bio-manufacturing inmammalian cells – challengesand opportunities

Prof. Nigel Jenkins, National Institute for BioprocessingResearch & Training, Ireland (NIBRT)

Nigel.Jenkins@nibrt.ie ++353 87 266 8984

http:/www.nibrt.ie

Antibody Structure

Slide 3/33

Structure of Mouse IgG2aIgG Structure

Variable Region(Fab or F(ab)2)

Variable Region(Fab or F(ab)2)

disulfide bonds

Constant Region(Fc)

Monoclonal Antibodies on the Market

• Most antibodies under development are fully humanized.

Name Company Indication Approval TypeOrthoclone J&J Used in transplant rejection 1986 MouseReoPro Centocor (J&J) Cardiovascular stents 1994 ChimericRituxan Biogen-IDEC Lymphoma & Leukemia 1997 ChimericSimulect Novartis Used in transplant rejection 1998 ChimericRemicade Centocor (J&J) Rheumatoid Arthritis & other

Autoimmune diseases1998 Chimeric

Erbitux Imclone-BMS Colorectal & Neck Cancer 2004 ChimericZenapax PDL BioPharma Used in transplant rejection 1997 HumanizedSynagis Medimmune (Astra-

Zeneca)RSV infections 1998 Humanized

Herceptin Genentech Breast Cancer 1998 HumanizedRaptiva Genentech Psoriasis 2003 HumanizedXolair Genentech Allergies & Asthma 2003 HumanizedAvastin Genentech Colon & Lung Cancer 2004 HumanizedHumira Abbott Rheumatoid Arthritis 2002 TNF-Fc Fusion

protein

Orencia Bristol-Myers-Sqibb Rheumatoid Arthritis 2006 CTLA4-FcFusion protein

Mylotag Wyeth Used in transplant rejection 2000 Antibody-Cytotoxin

Protein Synthesis

Protein Folding & Assembly

Post-Translational Modifications

Types:

Misfolding & Aggregation

Variable Glycosylation (cell & process-specific)

Methionine Oxidation, Asparagine Deamidation

Proteolysis

Effects

Half life in vivo

Efficacy

Immunogenicity

Drug stability (shelf life)

Cell Culture (Upstream Bioprocessing, USP)

Fermentation (300L-20,000L) is the culturing of cells to producevaluable product.

Wave-Type Disposable Bioreactors (<550L)

Protein Purification (Downstream Processing, DSP)

• Cost of a Protein A column can be up to $2 million

Multiple sprayports give evenresin bed

Relative Sizes of Viruses and Bacteria

Units: microns (µm)

= x10³ nanometers (nm)

Animal cell = 12-14 μm,

2x size of this slide

Virus Filtration using Viresolve NormalFlow Parvovirus (NFP) filters

NFP is capable of removing viruses as small as Parvovirus: (0.018 μm).

But cannot handle high loads and small pore sizes build up backpressure & slow flow rates

Therefore NFP filtration is used near the end of purification.

Fill-Finish Line

• Most protein formulations are either: Liquid, Frozen liquid, orLyophilized (freeze-dried) protein.

• Excipients are used to stabilize the protein for up to 2 years.

The Finished Drug Product

Most MAbs are administered intravenously (iv) or sub-cutaneously (sc)

Protein Damage can occur at every Process Step

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ProteinSynthesis &

Secretion

BioreactorConditions

ProteinPurification

17 log VirusInactivation

Formulation& Storage up

to 2 Years

Low pH,detergents, high

salt

Buffers,excipients,containers

Productivity,secretion

machinery,robustness

Agitation, mediacomponents, cell

viability, light

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Mammalian Cells for BiopharmaceuticalProduction

Advantages

Product is secreted

Relatively simple purification

CHO close to human protein modifications

Productivity 2-10 g/L

Disadvantages

High costs: $250/g

Slow growth (doubling time 20-28h)

Need to eliminate viruses & other contaminants

Proteins are fragile

National Institute forBioprocessing Research and

Training (NIBRT)

http://www.nibrt.ie

Education and Training

Education solutions include: Masters degrees in Biopharmaceutical Engineering and Biopharmaceutical Science.

Training solutions include: Bespoke accredited, training delivered in house for Industry. Technician training delivered in association with FAS. Bioprocessing seminars and stand-alone training

Research: NIBRT Strategy

NIBRT’s research strategy is focused on the design, development and optimization of bioprocesses for the safe and

economic manufacture of biopharmaceuticals.

PostTranslationalModification

Upstreamprocessing

ProductAnalytics

DownstreamprocessingFormulation

Cell Lines

Product

Prof JenkinsProf Rudd

Prof RuddProfAl-RubeaiProf Marison

ProfMarisonProf Al-Rubeai

To be hiredProf Jenkins

MAb

NIBRT Building

The NIBRT Building will be 5,700m2 (approx 58,000 ft2) including 6 researchlabs and an upstream pilot plant suite up to 150L capacity.

The NIBRT Building is scheduled to be complete by Q4 2009 and willprovide a holistic solution to the research and training needs of thebiopharma industry.

The Building will be located on the UCD campus, Dublin, Ireland.

Summary

Monoclonal antibodies are one of the fastest growing sectors of thePharma industry, with sales of >$20 billion by next year.

Bioprocessing operations for MAbs are more complex than forchemical compounds, however the drop-out rates in late-stageclinical trials are low.

NIBRT offers training, research and facility solutions to all aspectsof BioPharma processes.