SME Biotech Consulting Niche Outsourcing: When You Don’t Need It All Barry Rosenblatt, Ph.D. Cell Banking and Characterization
May 11, 2015
SME Biotech Consulting
Niche Outsourcing: When You Don’t Need It All
Barry Rosenblatt, Ph.D.
Cell Banking and Characterization
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Niche Outsourcing
Why outsource? Resource constraints Capital avoidance Core competency Virtual companies
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Niche Outsourcing
Areas to Outsource Preclinical/Toxicology Development
Cell line development Cell Banking
Manufacturing Upstream (cell culture) Downstream Formulation/Fill
Clinical
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Niche Outsourcing
Why Outsource Cell Banking? Performed under GMP Untested Cell Lines in Manufacturing Plant Controlled Cold (LN2, -70 Deg) Storage Characterization testing
Mycoplasma, Spiroplasma Sterility TEM Virus (Phage) Genetic Stability
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Niche Outsourcing
Where to go? Large CMO Smaller CMO CSO
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Cell Banking & Cell Line Characterization
What is needed for Banking? Development data
Media requirements Growth characteristics
Pre-bank Testing Mycoplasma Sterility
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Cell Banking
Batch Record development Performed in concert with the client
Pre-banks Research Cell Bank Ascension (seed) Bank
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Characterization of Continuous Cell Lines
What do the ICH guidelines and “Points to Consider” suggest?
Definitions of cell banks
Test descriptions
A typical cell testing approach
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ICH Guidelines And Points To Consider
Cell Lines History & genealogy Cell seed systems (banks) Culture medium Growth in vitro Time of sampling and testing Production and testing facilities
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Master Cell Bank (Definition)
The Master Cell Bank (MCB) is a quantity of cells derived from a single tissue and stored frozen at -70°C or below in aliquots, one or more of which would be used for the production of the Manufacturer’s Working Cell Bank (MWCB)
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Manufacturer’s Working Cell Bank
The Manufacturer’s Working Cell Bank (MWCB) is a quantity of cells derived from one or more of the ampoules of the cell seed and of uniform composition stored as -70°C or below in aliquots, one or more of which would be used for the production of a biological product.
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Extended Cell Bank
Cells obtained several generations past the stage at which crude product was harvested.
End of Production Cells or “Cells at the Limit of in vitro Cell Age Used for Production” are typically derived from the expansion of the WCB
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Cell Line History & Genealogy
Age, sex and species of the donor
Individual and family medical history
Culture history of the line including
methods used for the isolation of the
tissues from which the line was
derived, split ratio used in
passaging, media, etc.
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MCB & MWCB Storage
Stored in liquid or vapor phase of
liquid nitrogen
Documented location, identity, and
inventory of ampoules
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Culture Medium
Raw materials testing, i.e., serum adventitious agents
Accurate records on composition and sources
Testing for residual amount in final product
Absence of antibiotics
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Growth Characteristics In Vitro
Pattern and morphological appearance
stable Population doubling time (PDL) to post-
production
Determine PDL’s through senescence
Sampling of Beginning, Middle, and End Vials
are thawed for viability and recovery.
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Time of Sampling and Testing
Tests should be performed on cell suspensions (lysates)/fluids derived from the MWCB propagated to or beyond the level at which they are to be used in
production.
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Production and Testing Facilities
Absence of contamination with
transmissible agentsCross-contamination with other
cell lines
i.e., Good Manufacturing Practices
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Cell Line Characterization
Mammalian (general) Mycoplasma &
Sterility In Vivo adventitious
agent detection In Vitro adventitious
agent detection Karyology &
Isoenzymes Transmission Electron
Microscopy Reverse
Transcriptase Activity Tumorigenicity
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Cell Line Characterization
Bacterial (general) Purity Viability Presence of prophage Genetic stability
Copy number, restriction mapping Plasmid stability, retention of construct Retention of selectable markers
DNA sequencing
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Test Descriptions Tumorigenicity Testing In Vivo
Nude Mice Immunosuppressed newborn hamsters, mice
or rats Thymectomized and irradiated mice or rats
In Vitro Colony formation in soft agarose (Must be shown to be at least as sensitive as in
vivo)
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Test Descriptions
Karyology/Isoenzyme Gross abnormalities in chromosome number or
morphology Abnormalities intrinsic to original fetal material Exposure to chemical or physical mutagens Mislabeling and/or cross contamination
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Test Descriptions
Mycoplasma & Sterility Mycoplasma
Cultivable and non-cultivable
Aerobic and anaerobic
Culture and DNA fluorochrome methods
Sterility Bacteria and fungi
USP, 21 CFR 610.12
(1% of vials, 6 vials for Bacteriostasis/fungistasis)
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Test Descriptions
Presence of Viruses In vitro cell culture
Cell line being characterized or one from the
same species
A normal human embryo cell line
A monkey kidney cell line Minimum 14 day culture
Normal morphology
Hemadsorbing viruses
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Test Descriptions
Electron Microscopy Cells at or beyond production level TEM 100 sections at 50,000 X Pelleted cell supernatant, negative
staining Examine for viruses or other microbial
agents
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Test Description
In Vivo Safety Tests
Adult and suckling mice
Embryonated hen’s eggs
21 CFR 630.35
Simian cell lines may require Rabbits
Guinea pigs
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Test Descriptions
Specific Tests for Retroviruses Reverse transcriptase conventional
PBRT (PCR based reverse transcriptase)
Inoculation of RV-supporting cell lines with
supernatants from production cultures
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Murine Cell Line Characterization(Master Cell Bank)
In vitro (3 cell line, 14 day)
Mouse Antibody Production (MAP)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma
Reverse Transcriptase Sterility Extended XC Extended ERV Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional)
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Murine Cell Line Characterization(Manufacturer’s Working Cell Bank)
Sterility
Mycoplasma
In vitro ( 3 cell line, 14 day - optional)
Isoenzyme (optional)
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Murine Cell Line Characterization (End of Production Cells)
In vitro (3 cell line, 14 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma Reverse Transcriptase
Sterility Extended XC Extended ERV Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional)
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CHO Cell Line Characterization (Master Cell Bank)
Hamster Antibody Production (HAP)
In vitro (3 cell line, 14 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma Reverse
Transcriptase Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional)
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CHO Cell Line Characterization (Manufacturer’s Working Cell Bank)
Sterility
Mycoplasma
In vitro (3 cell line, 14 day - optional)
Isoenzymes (optional)
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CHO Cell Line Characterization (End of Production Cells)
In vitro (3 cell line, 14 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma
Reverse Transcriptase
Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional) Co-cultivation with
human cells
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Insect Cell Line Characterization (Master Cell Bank)
In vitro (3 cell line, 14 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Mycoplasma & Spiroplasma
Transmission Electron Microscopy
Reverse Transcriptase
Sterility Isoenzymes Bovine & Porcine
Viruses (optional)
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Insect Cell Line Characterization (Manufacturer’s Working Cell Bank)
Sterility Mycoplasma & Spiroplasma In vitro (3 cell line, 14 day - optional) Isoenzymes (optional)
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Human Cell Line Characterization (Master Cell Bank)
Human Virus (HIV 1 & 2, HTLV-1&2, Hepatitis A, B, C, Cytomegalovirus, EBV, Herpes 6, 7 & 8)
In vitro (3 cell line, 28 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma Reverse Transcriptase Sterility Isoenzymes &
Karyology Bovine & Porcine
Viruses (optional)
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Human Cell Line Characterization (Manufacturer’s Working Cell Bank)
Sterility
Mycoplasma
In vitro (3 cell line, 28 day - optional)
Isoenzyme (optional)
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Human Cell Line Characterization (End of Production Cells)
Human Virus (HIV 1 & 2, HTLV 1 & 2, Hepatitis A, B, C, Cytomegalovirus, EBV, Herpes 6, 7 & 8)
In vitro (3 cell line, 28 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma Reverse Transcriptase Sterility Isoenzymes &
Karyology Bovine & Porcine
Viruses (optional)
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Primate Cell Line Characterization (Master Cell Bank)
Virus (HIV 1 & 2, HTLV-1&2, SRV 1,2 & 3, SMRV, SIV, SV40, Foamy, B-virus, Simian Adenovirus, Simian Cytomegalovirus)
In vitro (3 cell line, 28 day) In vivo (adult & suckling mice, eggs, guinea pigs) Transmission Electron Microscopy Mycoplasma Reverse Transcriptase Sterility Isoenzymes & Karyology Bovine & Porcine Viruses (optional)
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Primate Cell Line Characterization (Manufacturer’s Working Cell Bank)
In vitro (3 cell line, 28 day) Mycoplasma Sterility Isoenzymes & Karyology
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Primate Cell Line Characterization (End of Production Cells)
Virus (HIV 1 & 2, HTLV-1&2, SRV 1,2 & 3, SMRV, SIV, SV40, Foamy, B-virus, Simian Cytomagalovirus, Simian Adenovirus)
In vitro (3 cell line, 28 day)
In vivo (adult & suckling mice, eggs, guinea pigs)
Transmission Electron Microscopy
Mycoplasma Reverse Transcriptase Sterility Isoenzymes &
Karyology Bovine & Porcine
Viruses (optional)
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E. coli Cell Characterization (Master Cell Bank)
Presence of Bacterial Virus (phage) Purity Restriction Map of Plasmid Plasmid Copy Number Plasmid Stability with & without
Antibiotics Retention of Expression Construct Sequencing of the Plasmid
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Conclusions
The major drivers for Niche outsourcing are no different than for full outsourcing
Choice of an appropriate site is linked to the scope of the Niche
Cell Banking is a prime candidate for Niche outsourcing due to the unique combined regulatory requirements of manufacturing and testing