SME Biotech Consulting Regulatory Considerations in Upstream Process Development Barry Rosenblatt, PhD
May 11, 2015
SME Biotech Consulting
Accelerating Your Success:Regulatory Considerations in Upstream Process Development
Barry Rosenblatt, PhD
SME Biotech Consulting
Target Identificati
onCandidateSelection
Candidate Optimizatio
nPre-IND Evaluation and Clinical Trials
Time
Con
c t1/2
TargetValidation
Biotech Process of Creating a Drug
SME Biotech Consulting
Characterization of Continuous Cell Lines
What do the ICH/EU guidelines and “Points to Consider” suggest?
Definitions of cell banks
Test descriptions
A typical cell testing approach
SME Biotech Consulting
Guideline Documents• ICH:
– VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN Q5A(Rl)
– DERIVATION AND CHARACTERISATION OF CELL SUBSTRATES USED FOR PRODUCTION OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS Q5D
• FDA:– Points to Consider in the Characterization of Cell Lines Used to
Produce Biologicals (1993)
• EU:– GUIDELINE ON VIRUS SAFETY EVALUATION OF
BIOTECHNOLOGICALINVESTIGATIONAL MEDICINAL PRODUCTS Doc. Ref. EMEA/CHMP/BWP/398498/2005 (FEB 2009)
SME Biotech Consulting
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
SME Biotech Consulting
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 Working Cell Bank (WCB)
SME Biotech Consulting
Working Cell Bank (Definition)
The Working Cell Bank (WCB) 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.
SME Biotech Consulting
End of Production or Cells at In-Vitro Limit
Cells obtained at or several generations past the stage at which crude product was harvested.
End of Production Cells (EoP)or “Cells at the Limit of in vitro Cell Age Used for Production” (CAL) are typically derived from the expansion of the WCB
SME Biotech Consulting
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.
SME Biotech Consulting
MCB & WCB Storage
Stored in liquid or vapor phase of liquid
nitrogen
Documented location, identity, and
inventory of ampoules
Stored in multiple locations/sites (minimum
of 2)
SME Biotech Consulting
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
SME Biotech Consulting
Growth Characteristics In Vitro
Stable Pattern and morphological
appearance
Population doubling time (PDL) to post-
production
Determine PDL’s through senescence
Determine number of generations
SME Biotech Consulting
Time of Sampling and Testing
Tests should be performed on cell suspensions (lysates)/fluids derived from the WCB propagated to or beyond the level at which they are to be used in production.
SME Biotech Consulting
Production and Testing Facilities
Absence of contamination with
transmissible agents
Cross-contamination with other cell lines
i.e., Good Manufacturing Practices 21CFR parts 210-211
ICH Q7
SME Biotech Consulting
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
SME Biotech Consulting
Test Description
Mycoplasma & Sterility
• Mycoplasma
– Cultivable and non-cultivable
– Aerobic and anaerobic
– Culture and DNA fluorochrome methods
• Sterility
– Bacteria and fungi
– USP, 21 CFR 610.12, EP 2.6.1
SME Biotech Consulting
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
SME Biotech Consulting
Test Description
Presence of Viruses
• In vitro cell culture (Cell Line dependant)
– 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
SME Biotech Consulting
Test Description
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
SME Biotech Consulting
Test Description
Electron Microscopy TEM sections at 50,000 X Pelleted cell supernatant Examine for viruses or other microbial agents
SME Biotech Consulting
Test Description
Specific Tests for Retroviruses
Reverse transcriptase conventional
PBRT (PCR based reverse transcriptase)
Inoculation of RV-supporting cell lines with
supernatants from production cultures
SME Biotech Consulting
Test Description
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)
SME Biotech Consulting
Murine Cell Line Characterization(Master Cell Bank)
In vitro (3 cell line, 28 day)
Mouse Antibody Production (MAP)
In vivo (adult & suckling mice, eggs)
Transmission Electron Microscopy
Mycoplasma
Reverse Transcriptase
Sterility Extended XC Extended ERV Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional)
SME Biotech Consulting
Murine Cell Line Characterization(Working Cell Bank)
Sterility
Mycoplasma
In vitro ( 3 cell line, 28 day - optional)
Isoenzyme (optional)
SME Biotech Consulting
Murine Cell Line Characterization (End of Production Cells)
In vitro (3 cell line, 28 day)
In vivo (adult & suckling mice, eggs)
Transmission Electron Microscopy
Mycoplasma Reverse Transcriptase
Sterility Extended XC Extended ERV Extended S+L- Karyology &
Isoenzymes Bovine & Porcine
Viruses (optional)
SME Biotech Consulting
ICH Summary Virus TestingTable 1: Virus Tests to Be Performed Once at Various Cell Levels MCB WCBa Cells at the limitb
Tests for Retroviruses and Other Endogenous Viruses
Infectivity + - +
Electron microscopyc +c - +c
Reverse transcriptased +d - +d
Other virus-specific testse as appropriatee - as appropriatee
Tests for Non-endogenous or Adventitious Viruses
In vitro Assays + -f +
In vivo Assays + -f +
Antibody production testsg +g - -
Other virus-specific testsh +h - -
a. See text - Section III.A.2. b. Cells at the limit: cells at the limit of in vitro cell age used for production (See text - Section III.A.3). c. May also detect other agents. d. Not necessary if positive by retrovirus infectivity test. e. As appropriate for cell lines which are known to have been infected by such agents. f. For the first WCB, this test should be performed on cells at the limit of in vitro cell age, generated from that WCB; for WCBs subsequent to the first WCB, a single in vitro and in vivo test can be done either directly on the WCB or on cells at the limit of in vitro cell age. g. e.g., MAP, RAP, HAP - Usually applicable for rodent cell lines. h. e.g., tests for cell lines derived from human, non-human primate or other cell lines as appropriate.
SME Biotech Consulting
EU Guidance (Cell Harvest)
In vitro testing
Tests for infectiousretroviruses*
In vivo testing*
CHO Yes, all bulks§
No No
NS0 and Sp2/0
Yes, all bulks§
Yes, once for given scale
No
All other cell lines
Yes, all bulks§
Yes, once for given scale
Yes, once for given scale
* Where possible, test material should contain cells or cellular fragments in order to detect cell associated viruses. For perfusion cell cultures, manufacturers should determine and justify the mostappropriate stage at which to derive samples containing cells for testing. It is also acceptable toderive test material from cells that have been cultured beyond the scale used to generate the batch of product; in these circumstances, the approach taken should be justified. Testing for infectiousretroviruses may be omitted when more sensitive tests have shown negative results.§ Quantification of retroviruses or retroviral-like particles need only be performed for the first threebulks for a specific stage of development (or less, if less than three bulks are prepared).
SME Biotech Consulting
The Cycle
SME Biotech Consulting
Comparability & Setting Specifications
DevelopDevelop
ApplyApply
AnalyzeAnalyze
Specs?Specs?
SME Biotech Consulting
Comparability & Setting Specifications
Phase 0 Phase 1 Phase 2 Phase 3
DevelopDevelop
ApplyApply
AnalyzeAnalyze
SpecsSpecs??
Comparability?Comparability?Comparability?Comparability?
SME Biotech Consulting
Conclusions
Cell Line optimization can occur at multiple points during development
Media optimization goes hand-in-hand with cell line optimization
Customized media may require customized testing
Re-testing is required for every new cell line (Establishment of bank)
Comparability testing should be planned in advance of cell line/process changes