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

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Emerging Trends in Plasma-free Manufacturing of
Recombinant Protein Therapeutics Expressed in
Mammalian Cells
Leopold Grillberger, Thomas R. Kreil, Sonia Nasr,
and Manfred Reiter

• Yiben Wang
• 11/16/11

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Recombinant Therapeutic Proteins -Microorganisms
-Plant cell cultures -Insect cell
lines -Mammalian cell lines -Transgenic
animals Over 165 biopharmaceutical products
globally -Majority are proteins
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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 -Unabl
e to perform necessary modifications
(glycosylation) needed for large, complex
proteins.
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Mammanlian cells -Used for large-scale
production of therapeutic proteins -Post-translat
ional 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
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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
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Additives -Derived from human or animal
sources -Blood -Milk -Bones -Hides -Tendons
-Hair -Skin -Pancreas
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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
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Threats of infectious diseases -Risk of using
human or animal component -Serum albumin and
gelatin stabilizers in formation
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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
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Transmissible spongiform encephalpathies
(TSEs) -Prions self-replicating infectious
proteins -Highly resistant -Physical/Chemica
l inactivation -Virus-removal methods cant
target -No detection method in plasma donors
early stages/pre-symptomatic of infection
-Bovine spongiform encephalpathies
(BSE) -Variant Creutxfeldt-Jacob disease (vCJD)
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• 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

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Response From Regulatory Agencies Physicians
Organizations
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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
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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
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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)
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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
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Methods Inactivation and Removal of
Viruses -Pasteurization -Vapor heating -Low
pH -Solvent/detergent treatment -Separation/puri
fication techniques -Ion-exchange -Immunogenic
ity chromatography -Nanofiltration
FDA The International Conference on
Harmonisation -Documents guiding the sourcing,
characterization, testing of raw materials, and
evaluating of therapeutic proteins for virus.
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Discussion
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Recombinant Therapeutic Proteins -Blood
factors -Anticoagulants -Growth
factors -Cytokines -Hormones -Vaccines -Therap
eutic enzymes -Monoclonal antibodies
Evolution in production -Enhanced
safety -Lower immunogenicity -Increased
half-life -Improved bioavailability -Alternative
routes of administration
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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.
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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
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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.
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Future -False sense of security -PF technology
prevention -Area of research -Different
culture, formulation, and storage conditions
-Physical stability of proteins