Overview of Protein Therapeutics

1

Contents

Introduction1

Production 2

Delivery33

Future Direction44

It is currently estimated that there are 25,000–40,000 different

genes in the human genome, viewed from the perspective of

disease mechanisms, as disease may result

when any one of these proteins contains

mutations or other abnormalities, so it

gives a tremendous opportunity for

Protein therapeutics to alleviate

these disease.

What is Protein therapeutics?

Why protein therapeutics?

Proteins cannot be mimicked by simple chemical compounds.

There is often less potential for protein therapeutics to interfere

with normal biological processes and cause adverse effects.

It is often well tolerated and are less likely to elicit immune responses.

Provide effective replacement treatment without the need for gene therapy

Time of protein therapeutics may be faster

A STAR IS BORN Pre-1986

1986–1991 MORE BIOTECHNOLOGY SUCCESSES

BIOTECHNOLOGY INDUSTRY1992–1999

BIOTECHNOLOGY IMPROVEMENT2002 and beyond

History and Development

The Evolution of Protein Therapeutics : A Timeline

1953 First accurate model of DNA suggested

1982 Human insulin, created using recombinant DNA technology

1986 Interferon alfa and muromonab-CD3 approved

1997 First whole chimeric antibody, rituximab, and first humanized antibody, daclizumab, approved

1993 CBER's Office of Therapeutics Research and Review (OTRR) formed

2002 Market for biotechnology products represents approximately $30 billion of $400 billion in yearly worldwide pharmaceutical sales

2006 An inhaled form of insulin (Exubera) approved, expanding protein products into a new dosage form.

Group I: protein therapeutics with enzymatic or regulatory activity

Group II : protein therapeutics with special targeting activity

Group III : protein vaccines

Group IV : protein diagnostics

Classification of protein therapeutics

Protein therapeutics replacing a protein that is deficient or abnormal (Group Ia)*

Protein therapeutics augmenting an existing pathway (Group Ib)*

Protein therapeutics providing a novel function or activity (Group Ic)

Protein therapeutics that interfere with a molecule or organism (Group II a)*

Protein therapeutics that deliver other compounds or proteins (Group II b)

Protein vaccines (Group III )*

Protein diagnostics (Group IV )

Protein Therapeutics also have disadvantages that may limit

their more widespread acceptance, include low oral and

transdermal bioavailability, moreover,

injections must be given frequently

because the half-lives of proteins

are short.

Remaining Disadvantages

Manufacturing of Recombinant Protein Therapeutics

-Microorganisms-Plant cell cultures

-Insect cell lines-Mammalian cell lines-Transgenic animals

Types of Cell Factories:

Recombinant proteins – a platform for developing more advanced products:

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

Initial Production:Established microbial expression systems using bacteria or yeast.

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

Mammalian cells:

Used for large-scale production of therapeutic proteins-Post-translational modifications-Proteins – natural form-60-70% of all recombinant therapeutic proteins produced in mammalian cells, Chinese Hamster Ovary (CHO).

CHO:Ease of manipulationProven safety profile in humansSimilar glycosylation patterns

Alternative, non-mammalian cell system:Advances in modulating the glycosylation patterns in certain yeast strains

-Pechia. 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

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:

•Development• Selection of a cell line that can yield high protein

output in serum-free medium•Upstream processing

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

•Downstream processing• Purification without the addition of other plasma

proteins•Final formulation

• Formulation without animal-derived additives•Testing

• Assure safety of product

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.

“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 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