Multiple Options for Proving Monoclonality

In recent years, proof of clonality has become a focus of attention for regulatory agencies. Although not explicitly stated in any regulatory documents, the development of cloned cell banks is cited in the ICH Q5D and EMA/CHMP. Clonality is thought to minimize cell bank heterogeneity, resulting in the consistent production of products. The absence of clonal cell lines may result in the growth of unwanted cell populations during changes in the manufacturing process, leading to changes in the final product and interruption of product supply when troubleshooting is performed.

Clonality Evaluation by Southern Blotting Analysis

• Currently, Southern blot analysis is the gold standard technique for clonality molecular studies, at least when fresh tissue is available. Such studies typically focus on the rearrangement of IG and TCR gene fragments and utilize the combinatorial libraries observed in these genes. In particular, Southern blotting can be used to detect gene rearrangements based on changes in the distance between restriction enzyme cleavage sites in genomic DNA.
• Despite the high reliability of Southern blot analysis, there are several inherent drawbacks, including significant demands in terms of time and technical skill, the need for large quantities of high-quality DNA, and limited sensitivity.

Clonality Evaluation by PCR Analysis

• In recent years, PCR-based analyses have gradually replaced other techniques for routine clonality workups. PCR is fast, accurate, and requires only small amounts of nucleic acid as a template. In addition, PCR can also amplify partially degraded DNA.
• Currently, two different types of PCR analysis, qualitative and quantitative, are used for clonality assessments. Regardless of the approach, the detection of IG/TCR gene rearrangements using PCR-based techniques requires prior, precise knowledge of the rearranged gene segments to design appropriate primers at opposite sides of the junctional regions.

Clonality Evaluation by NGS Analysis

Since the complete sequencing of the first human genome, there has been a rapid development of technologies to facilitate DNA analysis using NGS. NGS can be applied to either the whole genome or to selected DNA regions. NGS studies are based on the fragmentation of target DNA probes, with subsequent amplification and sequencing of the obtained DNA fragments. Despite the promise of these approaches, the translation of these innovative technologies into routine diagnostic practice will be complex and difficult.

Clonality Evaluation by FISH Analysis

• Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed in the early 1980s. FISH uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope.
• Creative Bioarray provides the genetic characterization of (CHO) producer cell lines by FISH, which offers information on transgene integrity and integration sites. We offer Clonality Analysis Service (FISH) for your scientific research as follows, probe design and synthesis, chromosome spread preparation, FISH on chromosome spreads imaging, and data analysis.
• In addition, please browse our related products and services in the table below.

Reference

1. Gazzola A, et al. (2014). "The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies." Ther Adv Hematol. 5 (2), 35-47.