Download Emerging Trends in Plasma-free Manufacturing of Recombinant

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
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. Development
1. Selection of a cell line that can yield high protein
output in serum-free medium
2. Upstream processing
1. Production of protein that is stable in animal-free cell
culture medium
3. Downstream processing
1. Purification without the addition of other plasma
proteins
4. Final formulation
1. Formulation without animal-derived additives
5. 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 – 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.
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 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 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 animalderived 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