Emerging Trends in Plasma-free Manufacturing of Recombinant Protein Therapeutics Expressed

in Mammalian Cells

Yiben Wang11/16/11

Leopold Grillberger, Thomas R. Kreil, Sonia Nasr, and Manfred Reiter

Recombinant Therapeutic Proteins:

-Microorganisms-Plant cell cultures

-Insect cell lines-Mammalian cell lines-Transgenic animals

Over 165 biopharmaceutical products – globally-Majority are proteins

Recombinant protein benefit – a platform for developing more advanced products:

-Enhanced safety-Lower immunogenicity-Increased half-life-Improved bioavailability

Production:

-Established microbial expression systems using bacteria or yeast.

Problem:

-Unable to perform necessary modifications (glycosylation) – needed for large, complex proteins.

Mammanlian cells:

-Used for large-scale production of therapeutic proteins-Post-translational modifications-Proteins – natural form-60-70% of all recombinant therapeutic proteins –

mammalian cells, Chinese hamster ovary (CHO).

CHO:

-Ease of manipulation-Proven safety profile in humans-Similar glycosylation patterns

Alternative, non-mammalian cell system:

-Advances in modulating the glycosylation patterns in certain yeast strains

-P. pastoris

Hemophilia A:

-X-linked coagulation disorder-Mutations in the coagulation factor VIII (FVIII) gene.

FVIII replacement therapy:

-Plasma-derived purified FVIII concentrates (1970s)-Recombinant FVIII concentrates (1992)-Animal and human plasma free recombinant FVIII (2003)

-Eliminated the risk of blood-borne infections during therapy

Additives:

-Derived from human or animal sources:-Blood-Milk-Bones-Hides-Tendons-Hair-Skin-Pancreas

Serum:

-Production of therapeutic proteins on a commercial scale

-Main threat – serum-derived proteins

-Risk of pathogen transmission-Viral outbreaks-Mad cow disease

-High protein content and variability-Increase in immunogenicity

Threats of infectious diseases:

-Risk of using human or animal component-Serum: albumin and gelatin – stabilizers in formation

Risks:

Amplified:-Multiple steps in manufacturing-Repeated administrations

Virus transmission:-Blood-borne infectious agents

-long-lasting, silent carrier states – no noticeable symptoms; highly infectious blood and plasma

-Solvent/detergent and nanofiltration – not 100% efficient

Transmissible spongiform encephalpathies (TSEs):

-Prions – self-replicating infectious proteins-Highly resistant

-Physical/Chemical inactivation-Virus-removal methods can’t target

-No detection method in plasma donors – early stages/pre-symptomatic of infection

-Bovine spongiform encephalpathies (BSE)-Variant Creutxfeldt-Jacob disease (vCJD)

Plasma-free production process:

1.Development1. Selection of a cell line that can yield high protein

output in serum-free medium2.Upstream processing

1. Production of protein that is stable in animal-free cell culture medium

3.Downstream processing1. Purification without the addition of other plasma

proteins4.Final formulation

1. Formulation without animal-derived additives5.Testing

1. Assure safety of product

Response From Regulatory Agencies & Physicians’

Organizations

Measures to assure product safety:-Controlling the source-Test raw material-Implement virus-inactivation and removal-Test end products

BSE outbreak:

-Strict requirements regarding bovine-derived materials’ country of origin-1998 – expansion of restricted countries

-BSE known to exist-Department of Agriculture

Center for Biologics Evaluation and Research (CBER)

-Manufacturers - products:-Cell culture history-Isolation-Media-Identity and pathogen testing of cell lines

Politics:

-Safety regulations -Donor screening policies

US Centers for Disease Control and Prevention (CDC):

-Single greatest risk of transfusion-transmitted viral infections

-Failure of screening – infected donors – pre-seroconversion phase of infection

More sensitive tests:-PCR-based nucleic acid amplification testing (NAT)

-Minipool NAT-Single donor testing (ID NAT)

NAT:

-Shorten the lag time – no detection of infection-HIV: 22 days 12 days-HCV: 70 days 14 days-No complete elimination of lag time

Pathogens:-HBV-HCV-HIV-1 and HIV-2-HTLV-I and HTLV-II-Syphilis-WNV

Methods – Inactivation and Removal of Viruses:-Pasteurization-Vapor heating-Low pH-Solvent/detergent treatment-Separation/purification techniques

-Ion-exchange-Immunogenicity chromatography

-Nanofiltration

FDA & The International Conference on Harmonisation:-Documents guiding the sourcing, characterization,

testing of raw materials, and evaluating of therapeutic proteins for virus.

Discussion

Recombinant Therapeutic Proteins:

-Blood factors-Anticoagulants-Growth factors-Cytokines-Hormones-Vaccines-Therapeutic enzymes-Monoclonal antibodies

Evolution in production:-Enhanced safety-Lower immunogenicity-Increased half-life-Improved bioavailability-Alternative routes of

administration

“The risk of pathogen transmission through the use of human- or animal-

derived raw materials in the manufacture of pharmaceuticals was the major driver

behind the development of PF technology.”

Erythropoesis-stimulating agents:

-Manage anemia – chronic kidney disease-Good example of evolution

-Introduced in 1980s – blood-derived-A recombinant product-Longer half-life-Conversion to an HAS-free formulation-Conversion to serum-free formulation-PF, PEGylated recombinant – longer half life

Complete Elimination of Risk of Transmission:

Recombinant Therapeutic Proteins:

-Production: cell lines free of human- or animal-derived proteins-Processing: strict pathogen removal and/or inactivation-Testing: lipid- and non-lipid-enveloped viruses-Packaging: in absence of human- or animal-derived proteins

Average cost for developing a biopharmaceutical product exceeding $1 billion.

Future:

-False sense of security-PF technology

– prevention

-Area of research: -Different culture, formulation, and

storage conditions -Physical stability of proteins