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Proving Monoclonality: What Are Your Options?

In recent years proof of clonality has become a focus for the FDA and other regulatory authorities. Many companies have received comments back on this topic during the IND review process of their biological products. Although not explicitly stated in any regulatory documents, the development of clonal cell banks is referenced in ICH Q5D and EMA/CHMP. Clonality is thought to minimize the heterogeneity of cell banks and thus allow for consistent manufacture of a product. The absence of a clonal cell line could potentially result in the growth of undesired cell populations during manufacturing process changes, causing changes in the final product and disruptions in product supply while troubleshooting is performed.

The simplest approach to provide high probability of a clonal cell bank is documentation of deposition of a single cell into a microtiter well, by either cell sorting (FACS) or images taken at the earliest possible stage during cell line development. For this purpose, sophisticated cell sorting, imaging, and cell printing instruments have now become available to companies performing these tests (e.g., ClonePix).

While the above approaches are applicable to newly generated production cell lines, they fail in the case of established cell lines that were subcloned and isolated before this requirement was in place. Complementary technologies in addition to the above mentioned single cell documentation approaches are available for this purpose. These include Southern blot techniques to detect the presence of a recombinant gene in specified genomic segments, junction PCR or targeted locus amplification (TLA) to identify the precise genome integration site, and fluorescence in situ hybridization (potentially in combination with chromosome painting) to detect the chromosomal location of the recombinant gene.

The previous techniques focus on providing data that a unique and single integration event took place, statistically implying that all cells with that specific integration site and sequence were derived from a single clone. In most cases, it is unlikely that multiple cells integrated the gene of interest at precisely the same location. However, in the case of targeted integration protocols, these approaches for the support of monclonality would fail. Also, for those using CHO cells it is important to take into consideration that the genome of CHO cells is constantly rearranging at a high rate due to the rapid division rate of these cells in culture. This relates to both the occurrence of genomic rearrangements, such as translocations and SNPs, and changes in the epigenome and the transcriptome which affects the behavior of cells. While a subclone may behave in a specific way, if re-subcloned, the properties of the new subclones may again diverge while still maintaining the same genome integration site. This makes protocols looking at integration sites alone unreliable for monoclonality determination. Therefore, for a more definitive identification of subclonality and cell lineage (identification of the maternal subclone or host cell line), more sophisticated tools are required that analyze the genome in more detail. Next generation sequencing (NGS) of the entire genome is a powerful tool and is becoming the method of choice in providing evidence of monoclonality for cell banks.

The use of NGS, also known as whole genome sequencing (WGS), can answer the following questions with a high level of statistical significance:

  • Is the cell line a subclone or a pool of cells?
  • Is the subclone indeed a sibling of the presumed host cell line?
  • How many integration sites are present and what are the precise locations of plasmid integration?
  • Is the sequence of the product gene intact or are there rare mutants?
  • Are the required regulatory elements such as UTRs and promoters present?
  • How many copies of the product gene are integrated?

With the use of NGS, companies can answer many questions at once instead of running multiple tests in support of the monoclonality of a cell bank. The partnership between Charles River and PathoQuest offers companies cell banking and cell line characterization along with a complete NGS service for genetic characterization, including insertion site mapping and sequencing, short tandem repeat (STR) sequencing, TLA sequencing, and whole genome sequencing.

If you would like to learn more about next generation sequencing and its various applications, contact us at [email protected].