Molecular Diagnostics Services, Inc.
A Contract Research Organization

Typical Mammalian Cell Bank Characterization Program

All tests are to be 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:

Recommended tests for the MCB are listed below. 

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)
    • HPV16, Human Papilloma Virus
    • HPV18, Human Papilloma Virus
    • HCMV, Human Cytomegalovirus
    • EBV (HHV 4, Human Herpes Virus, Epstein-Barr)
    • 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

For more Info:

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

Additional testing recommended by MDS:

Other Potential adventitious agents:

Recommended tests for the Working Cell Bank
  1.  Identification & Characterization of Cultured Cells by Analysis of 4 Isoenzymes. electrophoresis analysis to indicate consistency with in the banks.
  2. Total viable cell/vial
  3. Cell viability post bank thaw
  4. In vivo:  Extended in vivo adventitious agents assay using embryonated hens’ eggs, suckling mice, adult mice, and guinea pigs
  5. Sterility Testing by Direct Inoculation Method
  6. Test for Mycobacterium sp Culture Medium Method
  7. Test for Mycobacterium sp  by PCR Method
  8.  Direct Culture and DNA Staining Test for the Detection of Mycoplasmas
  9. Endotoxin (LAL)

Recommended tests for the END OF PRODUCTION CELL BANK

In vivo tumorigenicity (120 or 210 days , WHO  guideline (TRS 878 Annexure 1, (Appendix 1, Page 87,and page 43; and  CBER guidelines 2010, page 35),  10 animals per group) the sacrifice of the animals shall be done at the end of observation period with periodical palpation for nodule formation at the site of injection and visceral organs.

In vivo Oncogenicity (120 days, FDA Guidance 2010)

TEM: Transmission Electron Microscopic Examination of Cell Cultures (200 Cell profiles)

You may select using new born nude mice (preferred by CBER) or athymic mice  as test system (Appendix 2; WHO TRS 878)

Purity

Basic Microbiology

Note:  Isoenzyme Electrophoresis for both MCB and WCB & 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.

Additional services


SMALL SCALE GMP PRODUCTION SERVICES

CELL BANK RELEASE TESTING SERVICES

IMMUNOGENICITY TESTING SERVICES

ELISPOT for spleen, lymph node or PBMC
Th-1 and/or Th-2 cytokine expression: IL2, IL4, IFVγ
Intracellular cytokine staining FACS (spleenor lymph node or PBMC), i.e.:
IL2, IL4, IFVγ
Cytokine release by ELISA (spleen, lymph nodes or PBMC) i.e.:
IL2, IL4, IFVγ
T-cell stimulation/proliferation assays (spleen, lymph node or PBMC)
T cell subset: i.e. CD4, CD8, NK cells, macrophages, dendritic cells, etc
Mixed leukocyte response (MLR) against allogeneic leukocytes
T-cell co-cultured with dendritic or other Antigen Presenting Cells (APC) cells:

  1. Delayed-type hypersensitivity response to sheep RBCs or other such as chicken RBCs
  2. Skin Rash
  3. Lymphadenopathy
  4. Urticaria (Hives)
  5. IgE

Host resistance challenge models (endpoints)

  1. Syngeneic tumor cells: MethA fibrosarcoma (growth and regression); B16 melanoma (lung burden)
  2. Bacterial models: Listeria monocytogenes (mortality); Streptococcus species (mortality)
  3. Viral models: Cytomegalovirus (viral load in salivary gland, lung, kidney, spleen, and liver)
  4. T-cells co-cultured with dendritic cells.
  5. Delayed-type hypersensitivity response to sheep RBCs or other such as chicken RBCs
  6. Skin rash
  7. Lymphadenopathy
  8. Urticaria (hives)
  9. IgE

Immunopathology

  1. Complete blood count, differential white blood cell count
  2. Lymphoid organ weights; body weight and temperature
  3. Histopathology, gross and microscopic (spleen, thymus, lymph nodes, Peyer's patches and bone marrow)

Immunoefficacy of Recombinant Vaccine

MDS offers custom monoclonal and polyclonal antibody development for recombinant vaccine. This approach involves the direct immunization of the host animal with plasmid DNA encoding the client's protein of interest. The immunized host then produces the encoded protein and raises antibodies. Genetic immunization is perfectly suited to producing antibodies when a protein is difficult to express, purify or when a gene has been obtained, but the protein itself is unknown.

Advantages:

  1. Large-scale synthesis and purification of proteins are no longer necessary.
  2. Antibodies are directed only against the encoded protein.
  3. Unwanted antibodies to contaminants found in purified protein preparations are eliminated.
  4. Plasmid DNA also acts as an adjuvant to stimulate immune responses.
  5. These antibodies can be used in many applications, including, but not limited to, purifying proteins from cells or tissues, identifying proteins in prognostic and diagnostic assays, etc.
  6. No additional charge when used with either monoclonal or polyclonal antibody development programs.

Our Technical Support Staff is available to assist you with the:

Validation Assays:
Standard method development and validation includes testing for accuracy, precision, specificity, detection limit and quantitation limit. Method optimization and matrix-interference testing is also available.

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