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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: – 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 www.atcc.org 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 DCLs (Diploid cell lines) Passage Isolation PCCs (Primary cell culture) Passage CCLs (Continuous cell lines/immortal cell) http://nas-sites.org/stemcells/stem-cellbasics/download-stem-cell-figures/ 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 – 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: • 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: – 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 – Tumorigenicity positive • Animal model – 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/cellline-characterization/cell-line-authentication John Geigert. The challege of CMC regulatory Compliance for Biopharamceuticals. 2004. Kluwer Academic/Plenum Publisher. New York.