Typical Mammalian Cell Line

All tests are conducted and documented in compliance with Good Laboratory Practices, 21 CFR 58 and in accordance with the EU or US Pharmacopoeia and EMEA or FDA guidelines such as:

• Good Laboratory Practices for Non-Clinical Laboratory Studies, 21 CFR 58. US Food and Drug Administration.
• U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research. Points to Consider in the Characterization of Cell Lines used to Produce Biologicals (1993).
• Guidance for industry: characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious diseases: February, 2010. 
• ICH Harmonized Tripartite Guideline: Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin; Q5a(R1): 23 September 1999.
• International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q5A/Q5B.
• ICH Guidance, Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products, (63 FR 50244; September 21, 1998).

Recommended tests for the Master Cell Bank  
Identity Testing:

1. Isoenzyme electrophoresis to indicate consistency with human dermal fibroblast control (see below)
2. Karyology analysis (karyotyping) of cell lines (50 metaphases) to confirm diploid status
3. DNA fingerprinting / genetic profiling (STR) to demonstrate comparability to a reference cell line
4. Total viable cells/vial
5. Cell viability (post bank thaw)

Purity:

1. In vitro:  as recommended by FDA, PTC:  28-days including cell cultures, hemagglutination (HA) and hemadsorption (HAD) tests
2. In vivo:  extended in vivo adventitious agents assay using embryonated hens’ eggs, suckling mice, adult mice, and guinea pigs
3. In vitro assay: detection of porcine viral contaminants using PPK indicator cells according to 9CFR
4. Detection of reverse transcriptase activity by F-PERT assay
5. Retrovirus 293 Co-cult (5 Passages, FPERT end-point)
6. PCR detection of simian viruses (SV40 &SV-5)
7. PCR for human and animal pathogens.  Endpoint or QPCR panel for T. pallidum (if applicable) and the following viruses:

• Human Immunodeficiency Virus Type 1 (HIV I) DNA
• Human Immunodeficiency Virus Type 1 (HIV I) RNA
• Human Immunodeficiency Virus Type 2 (HIV II) DNA
• Human Immunodeficiency Virus Type 2 (HIV II) RNA
• HTLVI (DNA)
• HTLVI (RNA)
• HTLVII (DNA)
• HTLVII (RNA)
• Hepatitis B Virus (HBV)
• Hepatitis C Virus (HCV)
• Hepatitis A Virus (HAV)
• Human Papilloma Virus 16 (HPV16)
• Human Papilloma Virus 18 (HPV18)
• Human Cytomegalovirus, (HCMV)
• Epstein Barr Virus (EBV) (HHV 4, Human Herpes Virus)
• Parvovirus-B19
• Bovine Herpes Virus I (BHV I)
• Bovine Herpes Virus IV(BHV IV)
• Porcine Circovirus-1 and 2 (B/PCV1)
• Swine Hepatitis E Virus (HEV)
• Mycobacterium sp

Testing for animal viruses according to 9 CFR 113.46, 113.47 and 113.53, using cell culture monolayers:

• Testing serum alone for bovine virus diarrhea virus (BVDV1) and bovine rotavirus (BRV)
• Testing trypsin alone for porcine parvovirus (PPV)
• Testing product for both porcine and bovine viruses

Additional testing recommended by MDS:

• Basic Microbiology
• Bacteriostasis/Fungistasis
• Sterility (Direct Inoculation Method (EP, USP, JP and 21 CFR)
• Endotoxin (LAL)

Testing for other potential adventitious agents to be considered:

• Mycoplasma PCR
• Direct culture and DNA staining test for the detection of Mycoplasmas

Recommended tests for the Working Cell Bank
Identification and characterization of cultured cells by analysis of 4 isoenzymes. Electrophoresis analysis to indicate consistency within the banks.
Total viable cells/vial
Cell viability post bank thaw
Purity:

1. In vitro:  as recommended by FDA, PTC:  28-days including cell cultures, hemagglutination (HA) and hemadsorption (HAD) tests
2. In vivo:  extended in vivo adventitious agents assay using embryonated hens’ eggs, suckling mice, adult mice, and guinea pigs

Sterility testing by direct inoculation method
Test for Mycobacterium sp culture medium method
Test for Mycobacterium sp  by PCR
Direct culture and DNA staining test for the detection of Mycoplasmas
Endotoxin(LAL)
Recommended tests for the End of Production Cell Bank
In vivo tumorigenicity (120 or 210 days) WHO guidelines (TRS 878 Annexure 1) and CBER guidelines
In vivo oncogenicity (120 days, FDA Guidance 2010)
TEM: Transmission electron microscopic examination of cell cultures (200 cell profiles)
Identity Testing

• Isoenzyme electrophoresis to indicate consistency with WCB & MCB
• Karyology analysis (karyotyping) of cell lines (50 metaphases) to confirm diploid status
• DNA fingerprinting/genetic profiling (STR) to demonstrate comparability to a reference cell line
• Total viable cells/vial
• Cell viability (post bank thaw)

Purity

• In vitro:  28 days including cell cultures, hemagglutination (HA) and hemadsorption (HAD) tests (performed on MCB and WCB) but we are talking about EPCB
• In vivo: Extended in vivo adventitious agents assay using embryonated hens’ eggs, suckling mice, adult mice, and guinea pig

Basic Microbiology

• Bacteriostasis/Fungistasis
• Sterility (direct inoculation method
• Test for Mycobacterium sp by direct culture and DNA staining
• Endotoxin (LAL)
• Mycoplasma PCR
• Transmission electron microscopic examination of cell cultures (200 cell profiles)

Note: Isoenzyme Electrophoresis for MCB, WCB and EPCB
From the banding patterns of the enzymes used, a “process of elimination” is employed to determine the species identity of the cell line. Standard isoenzyme analyses include determination of the isoenzyme gel electrophoresis banding pattern for at least three of the following enzymes.

• Glucose-6-phosphate dehydrogenase (G6PD)
• Lactate dehydrogenase (LD)
• Malate dehydrogenase (MD)
• Nucleoside phosphorylase (NP)
• Aspartate aminotransferase (AST)
• Peptidase B (PepB)
• G-Protein coupled receptors (GPCR)
• cAMP phosphodiesterase Assay
• Protein kinase (PK)