1. Characterization of Cell bank and Seed bankBy
Juree Charoenteeraboon

2. 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

3. 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

5. 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

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

8. 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.

9. 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

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

11. 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

12. 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)

13. 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

14. 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

15. 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

16. 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

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

18. ที่มา Annex 3

19. ที่มา Annex 3

20. 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)

21. 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,….

22. 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)

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

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

25. 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)

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

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

30. 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

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

34. 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

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

39. 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
John Geigert. The challege of CMC regulatory Compliance for Biopharamceuticals Kluwer Academic/Plenum Publisher. New York.