Characterization of Cell bank and Seed bank By Juree Charoenteeraboon

Source of contamination in Vaccine Raw materials Animal cell substrate Original source adventitious agents Serum, trypsin,…. (cultivation) Seed or other cell substrate (virus, bacteria, yeast,…) Contamination Excipients Production process Container

Terminology Cell bank: MCB: Master cell bank WCB: Working cell bank a collection of appropriate containers whose contents are of uniform composition, stored under defined conditions. Each container represents an aliquot of a single pool of cells MCB: Master cell bank A collection of cells of uniform composition derived from a single source WCB: Working cell bank Derived from one or more vials from MCB and used for production

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Cell bank Sources (human, animal and insect origin) Category Primary cell culture (PCC) Cell derived directly from animal, Chick Embryonic eggs Diploid cells (DCL): Derived from Primary cell culture to be a cell line with a finite in vitro lifespan paired (euploid) chromosomes and structurally identical to those of the derived species Continuous cell line (CCL): a cell line with an apparently unlimited capacity for population doubling. referred to as “immortal” Stem cell lines (SCL) A predominant stem cell population retaining the capacity to produce progenitors of differentiated cell

(Primary cell culture) DCLs (Diploid cell lines) Passage Isolation PCCs (Primary cell culture) Passage CCLs (Continuous cell lines/immortal cell)

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PCCs Majority : viral contamination Advantages comparatively easy to prepare using simple media and bovine serum. generally possess a broad sensitivity to a variety of viruses Disadvantages Contamination by infectious agents is a higher risk than with DCLs and CCLs The quality and viral sensitivity of cultures obtained from different animals are variable. PCCs cannot be tested as extensively as DCLs or CCLs.

DCLs human (e.g. WI-38, MRC-5) and monkey (i.e. FRhL-2) origin a finite capacity for serial propagation remain alive and metabolically active but may show morphological and biochemical changes non-tumorigenic; display diploid cytogenetic characteristics with a low frequency of chromosomal abnormalities

DCLs Advantages well characterized and standardized. a cryopreserved cell bank system that allows for consistency and reproducibility not tumorigenic

DCLs Disadvantages not easy to use in large-scale production more fastidious nutritional requirements than other cell substrates. difficultly adapt to serum-free growth. more difficult to transfect and engineer than CCLs

CCLs serial subcultivation of PCC of animal tumor (HeLa cells) Transform normal cells with oncogenic virus or viral sequence serial subcultivation of a primary cell population derived from normal tissue that having an apparently indefinite lifespan (Vero, BHK-21, CHO, MDCK, Hi5) Hybridoma cell line (myeloma cell and B cell)

CCLs Advantages Disadvantages well characterized and standardized a cryopreserved cell bank system that allows for consistency and reproducibility Grow more easy than DCLs and easily adapt to serum-free growth Can grow on micro-carrier (production scale) Disadvantages May express endogenous viruses and some are tumorigenic in immunosuppressed animal model Theoretical risks identified by nucleic acid, transforming protein and virus

SCLs Advantages well characterized and standardized a cryopreserved cell bank system that allows for consistency and reproducibility Some may be adapt to grow in suspension in bioreactor may produce unique proteins of potential importance as biotherapeutics Have the potential to generate cells and tissue-like structures that permitted the in vitro unculturable agents

SCLs Disadvantages Laborious May produce growth proteins with undefined effects on adults Require complex media (TSE risk) A little experience use SCLs in manufacture biological product

Conclusion of Special consideration PCCs Adventitious agents: SPF, Clean DCLs Determine the Karyotype of new DC strain for Identity and character Contamination from other cell lines Genetic stability monitoring throughout production CCLs Identity, Contamination, Homogenicity Tumorigenicity and Oncogenicity SCLs Ethic Periodic phenotype confrimation; Absence of non-diploid cells Sustained pluripotent capacity

Potential risk and risk mitigation Viruses and other transmissible agents Cellular nucleic acid Growth-promoting protein

ที่มา Annex 3

ที่มา Annex 3

Characterization of cell bank Recommendations Identity Stability Sterility Viability Growth characteristics Homogeneity (consistency of viability and growth characteristic in each vial) Tumorigenicity Oncogenetics Cytogenetics Microbial agents (adventitious and contamination)

Identity Karyology (chromosome) Isoenzyme analysis human cell DNA profiling (RFLP, PCR,…) Human leukocyte antigen (HLA) typing Cell line for rProtein: vector integrity Recombinant cell: Expression plasmid copy number, insertions, deletions, verification of protein-coding sequences, protein-production level,….

Stability Once very 5 years for cryopreserve Genetic stability Global or target gene expression Proteomic or metabolic profile Phenotype makers Viability Copy number of construction mRNA and protein level (rProtein)

Viability Viability test Membrane integrity: Metabolic activity: Lactate Dehydrogenase leakage Metabolic activity: MTT, WST, … DNA replication : Count cell

Growth Characteristics Well understood Viability, morphology, cell-doubling time Plating efficiency …… Correlate to Homogeneity

Tumorigenicity CCLs: Assay some contains oncogene or virus to be immortal cell Classified as tumorigenic: BHK-21, CHO, Hela Assay In vivo: Inject 107 cells (IM or SC) into immunocompromized animal CCLs or New DCL Nodule growing metastases (microscopic: liver, heart, spleen and lymph node)

Oncogenicity CCL, SCL Animal model Tumorigenicity positive New born nude mice, newborn hamsters and newborn rats A cellular DNA Observe at least 4 months

Cytogenetics DCL, SCL, CCL DCL: count of stained chromosomes CCLs: Marker chromosome Support genetic stability

Testing for adventitious agents In vivo tests adults mice, sucking mouse, guinea pigs, rabbits, embryonated chicken eggs, antibody production test In vitro tests for viruses Cell culture Safety test (cytopathic effect) Transmission Electron Microscopy (TEM) Biochemical test In vitro tests for Non viral agents Mycoplasma/Spiroplasma (PCR, ELISA, Cultivation) Bacterial and Fungal sterility Mycobacteria testing

Characterization of Seed bank Identity Stability Sterility/contamination Viability Adventitious agents Others Non-virulent test Tumorigenicity and Oncogenicity

Characterization of Seed bank Identity Phenotype: Morphology Microscopy; Bright field microscope (fungi/bacteria) or TEM Genotype: DNA Sequencing, PCR Recombinant protein : marker Biochemical properties Immunological assay

Biological Raw materials Adventitious agent Serum Trypsin Animal acids Other Biological Reagent

References Annex 1. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks Annex 3: Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications ICH Topic Q 5 D Quality of Biotechnological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products http://www.bioreliance.com/eg/services/biopharmaceutical-services/cell-line-characterization/cell-line-authentication John Geigert. The challege of CMC regulatory Compliance for Biopharamceuticals. 2004. Kluwer Academic/Plenum Publisher. New York.