Accelerating Usp

SME Biotech Consulting

Accelerating Your Success:Regulatory Considerations in Upstream Process Development

Barry Rosenblatt, PhD


Target Identificati

onCandidateSelection

Candidate Optimizatio

nPre-IND Evaluation and Clinical Trials

Time

Con

c t1/2

TargetValidation

Biotech Process of Creating a Drug


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


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)


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


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)


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.


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


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.


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)


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


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


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.


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


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


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


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


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


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


Test Description

Electron Microscopy TEM sections at 50,000 X Pelleted cell supernatant Examine for viruses or other microbial agents


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


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)


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)


Murine Cell Line Characterization(Working Cell Bank)

Sterility

Mycoplasma

In vitro ( 3 cell line, 28 day - optional)

Isoenzyme (optional)


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)


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.


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§



* 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).


The Cycle


Comparability & Setting Specifications

DevelopDevelop

ApplyApply

AnalyzeAnalyze

Specs?Specs?


Comparability & Setting Specifications

Phase 0 Phase 1 Phase 2 Phase 3

DevelopDevelop

ApplyApply

AnalyzeAnalyze

SpecsSpecs??

Comparability?Comparability?Comparability?Comparability?


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