Download Characterization of Cell bank and Seed bank

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
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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
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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
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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
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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
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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.