mFISH and FISH (mFISH/FISH) analysis Monoclonality evaluation of cell lines for biopharmaceutical production
Heterogeneity of cell lines has potential impact on the quality of cell-produced products (biopharmaceuticals) such as monoclonal antibodies. Therefore, regulatory authorities including FDA and EMA/CHAMP recommend more stringent control on the monoclonality of cell lines. Thus, new technological development has been expected to evaluate master cell banks (MCBs).
Sumika Chemical Analysis Service, Ltd. (SCAS) offers sophisticated technology for the monoclonality evaluation, called mFISH/FISH analysis. This is the combination analysis of FISH (Fluorescence in situ hybridization) and multicolor FISH analyses, and provides detailed information on the chromosome integrated transgenes. FDA introduced mFISH/FISH analysis in their presentation document in 2016 as an effective method for monoclonality evaluation of cell lines.
Features
Mammalian cells such as CHO cells and HEK293 cells are widely used for biopharmaceutical production and known to have unstable karyotypes. Chromosomal rearrangements during cell expansion cause variation of chromosomal composition, even in a population expanded from a single cell. Due to such a feature, it is difficult to map the transgene on chromosomes.
mFISH/FISH analysis can identify the transgene integration sites regardless of karyotypic instability as shown in the pictures below. The information on integration sites obtained by mFISH/FISH analysis allows us to evaluate the clonal status of a cell line.
Advantage of mFISH/FISH analysis
- Cell-by-cell analysis of transgene integration sites.
- Precise information provided on the chromosome of transgene integration.
- An effective method for monoclonality evaluation, recommended by FDA.
A: FISH image of the CHO metaphase chromosomes
B: Multicolor FISH image of the CHO metaphase chromosomes
C: Karyogram of B
D: Mapping of the FISH signals
Green arrows show the FISH signals.
A white arrow shows the chromosome with the FISH signals.
Other applications
mFISH/FISH analysis can be applied for various purposes throughout the whole process of cell bank construction, as below.
- Confirmation of transfection methods
- Screening for research cell banking
- Characterization of master cell banks (MCBs), working cell banks (WCBs) and end of production cell banks (EPCBs)
As presented above, mFISH/FISH analysis is again a valuable tool for the evaluation of transgene status during the cell line development for biopharmaceutical production.
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