Nicheoutsourcing

SME Biotech Consulting

Niche Outsourcing: When You Don’t Need It All

Barry Rosenblatt, Ph.D.

Cell Banking and Characterization

Niche Outsourcing

Why outsource? Resource constraints Capital avoidance Core competency Virtual companies

Niche Outsourcing

Areas to Outsource Preclinical/Toxicology Development

Cell line development Cell Banking

Manufacturing Upstream (cell culture) Downstream Formulation/Fill

Clinical

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

Niche Outsourcing

Where to go? Large CMO Smaller CMO CSO

Cell Banking & Cell Line Characterization

What is needed for Banking? Development data

Media requirements Growth characteristics

Pre-bank Testing Mycoplasma Sterility

Cell Banking

Batch Record development Performed in concert with the client

Pre-banks Research Cell Bank Ascension (seed) Bank

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

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 Manufacturer’s Working Cell Bank (MWCB)

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.

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

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 & MWCB Storage

Stored in liquid or vapor phase of

liquid nitrogen

Documented location, identity, and

inventory of ampoules

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

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.

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.

Production and Testing Facilities

Absence of contamination with

transmissible agentsCross-contamination with other

cell lines

i.e., Good Manufacturing Practices

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

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

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

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

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)

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

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

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 Descriptions

Specific Tests for Retroviruses Reverse transcriptase conventional

PBRT (PCR based reverse transcriptase)

Inoculation of RV-supporting cell lines with

supernatants from production cultures

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)

Murine Cell Line Characterization(Manufacturer’s Working Cell Bank)

Sterility

Mycoplasma

In vitro ( 3 cell line, 14 day - optional)

Isoenzyme (optional)

Murine Cell Line Characterization (End of Production Cells)

Mycoplasma Reverse Transcriptase

Sterility Extended XC Extended ERV Extended S+L- Karyology &

Viruses (optional)

CHO Cell Line Characterization (Master Cell Bank)

Hamster Antibody Production (HAP)

Mycoplasma Reverse

Transcriptase Extended S+L- Karyology &

Viruses (optional)

CHO Cell Line Characterization (Manufacturer’s Working Cell Bank)

Sterility

Mycoplasma

In vitro (3 cell line, 14 day - optional)

Isoenzymes (optional)

CHO Cell Line Characterization (End of Production Cells)

Mycoplasma

Reverse Transcriptase

Extended S+L- Karyology &

Viruses (optional) Co-cultivation with

human cells

Insect Cell Line Characterization (Master Cell Bank)

Mycoplasma & Spiroplasma

Reverse Transcriptase

Sterility Isoenzymes Bovine & Porcine

Viruses (optional)

Insect Cell Line Characterization (Manufacturer’s Working Cell Bank)

Sterility Mycoplasma & Spiroplasma In vitro (3 cell line, 14 day - optional) Isoenzymes (optional)

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)

Mycoplasma Reverse Transcriptase Sterility Isoenzymes &

Karyology Bovine & Porcine

Viruses (optional)

Human Cell Line Characterization (Manufacturer’s Working Cell Bank)

Sterility

Mycoplasma

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

Isoenzyme (optional)

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)

Viruses (optional)

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)

Primate Cell Line Characterization (Manufacturer’s Working Cell Bank)

In vitro (3 cell line, 28 day) Mycoplasma Sterility Isoenzymes & Karyology

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)

Viruses (optional)

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

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

Nicheoutsourcing

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