Overview of Protein
Therapeutics
1
Contents
1
Introduction
2
Production
3
Delivery
4
Future Direction
What is Protein therapeutics?
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.
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
History and Development
2002 and beyond
1992–1999
1986–1991
Pre-1986
BIOTECHNOLOGY IMPROVEMENT
BIOTECHNOLOGY INDUSTRY
MORE BIOTECHNOLOGY SUCCESSES
A STAR IS BORN
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
1993
CBER's Office of Therapeutics Research and Review (OTRR) formed
1997
First whole chimeric antibody, rituximab, and first humanized
antibody, daclizumab, approved
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.
Classification of protein therapeutics
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
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 )
Remaining Disadvantages
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.
Manufacturing of Recombinant
Protein Therapeutics
Types of Cell Factories:
-Microorganisms
-Plant cell cultures
-Insect cell lines
-Mammalian cell lines
-Transgenic animals
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 manipulation
Proven safety profile in humans
Similar 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 – preseroconversion 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 animalderived raw materials in the
manufacture of pharmaceuticals was
the major driver behind the
development of PF technology.”
Erythropoesis-stimulating agents:
Manage anemia – chronic kidney diseaseGood 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 animalderived 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