Cell Line Characterization and Authentication Methods

Cell line characterization is performed for Master Cell Banks (MCB), Working Cell Banks (WCB), and End-of-Production Cells (EPC) or cells at the limit (CAL) of in vitro cell age used for production. According to current regulations, the following aspects must be considered:

  • The origin and history of the cell line cellular morphology and growth characteristics
  • Cellular identity
  • Purity of the cell lines (i.e., absence of contaminating cells, microbial contaminations, and contaminations by adventitious viruses)
  • Tumorigenicity and oncogenicity
  • Stability

Cell Line Authentication Package Recommendations

Considerations include species of the cell line (e.g., hamster, human, chicken, E. coli, P. pastoris) and its intended use (e.g., recombinant protein production, vaccine production, gene therapy). Furthermore, documentation of the source of the cell line and its development history, as well as the biological properties of the cells, determines which testing should be performed. When executing the assays, the guidelines recommend using testing procedures that are validated to show their suitability for the intended purpose.


Using NGS to Characterize Cell Lines Used for Production of Biologics: Verifying Clonality and Identity

Microscopic image of DNA.

View this webinar replay to learn about the use of the latest sequencing technologies for the characterization of your cell lines.

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Microbial & Mammalian Cell Line Authentication Methods

Validated methods for mammalian and microbial cell line characterization are available at our sites in both the United States and Europe. References to guidelines concerning cell line characterization can be found in our regulatory library.

The tables below list recommended testing plans for the characterization of general mammalian and microbial cell lines used to produce a recombinant protein, along with a specific example for CHO cells.

  • Mammalian Cell Line Characterization
    Mammalian Cell line Characterization Panel1 MCB WCB EPC/CAL
    Microbial Contamination Sterility X X X
    Mycoplasma X X X
    Mycobacterium X    
    Spiroplasma X    
    Cell Line Identity DNA Barcoding X X X
    DNA fingerprinting/STR X   X
    Karyology X   X
    Genetic stability DNA sequencing X   Optional
    Gene copy number by qPCR/STR X   X
    Virus Testing Retroviruses: Reverse transcriptase and retroviral infectivity assays X   X
    In vitro adventitious viruses X X X
    In vivo adventitious viruses X   X
    Test for specific viruses such as:
    • Bovine viruses
    • Porcine viruses
    • Human viruses
    • Minute virus of mice
    X    
    In vivo biosafety: Mouse, hamster, and rat antibody production assays (MAP, RAP, and HAP) X    
    Transmission electron microscopy X   X
    Tumorigenicity/Oncogenicity     X

    1Novel molecular methods based on next generation sequencing (NGS) technology can also be offered to supplement the test program.

  • Microbial Cell Line Characterization
    Microbial Cell Line Characterization Panel MCB WCB
    Bacteriophage detection X X
    Purity X X
    Identification X X
    Viability X X
    Copy number X X
    DNA Sequencing X X
    Retention of selectable markers X X
    Retention of recombinant construct X X
    Restriction Endonuclease Analysis X X
  • CHO Cell Line Characterization
    CHO Cell line Characterization Panel MCB WCB EPC/CAL
    Microbial Contamination Sterility X X X
    Mycoplasma X X X
    Cell Line Identity DNA Barcoding X X X
    Genetic stability DNA sequencing X   X
    Gene copy number X   X
    Restriction endonuclease analysis X   X
    Virus Testing Endogenous and non-endogenous retroviruses: reverse transcriptase and retroviral infectivity assays X   X
    In vitro adventitious viruses X   X
    In vivo adventitious viruses (suckling and adult mice, embryonated eggs) X   X
    Test for specific viruses such as:
    • Bovine viruses
    • Porcine viruses
    • Minute virus of mice
    X    
    In vivo biosafety: Mouse and hamster antibody production assays (MAP and HAP) X    
    Transmission electron microscopy X   X

     

With all of these things to consider, it is important to determine not only the most effective, but also the most relevant and efficient cell line authentication plan for you.

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Frequently Asked Questions (FAQs) for Cell Line Characterization: