Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies

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Samuel H Aeschlimann1, Christian Graf2, Dmytro Mayilo1

  • 1Novartis Institutes for BioMedical Research, Integrated Biologics Profiling Unit, CH-4002, Basel, Switzerland.

Biotechnology Journal
|February 23, 2019
PubMed

Insights

Targeted Locus Amplification (TLA) coupled with Next-Generation Sequencing (NGS) offers an efficient genomic screening method for Chinese hamster ovary (CHO) cell line development. This TLA/NGS approach robustly detects genetic variants and rearrangements, accelerating biotherapeutic protein production.

Area of Science:

  • Biotechnology
  • Genomics
  • Cell Line Development

Background:

  • Early analytical clone screening is crucial for selecting Chinese hamster ovary (CHO) cell lines with optimal stability and product quality for biotherapeutic protein production.
  • Traditional methods like mass spectrometry lack the throughput and speed required for efficient clone analytics.
  • Next-Generation Sequencing (NGS) presents a promising alternative for analyzing CHO clones.

Purpose of the Study:

  • To report an efficient NGS workflow using Targeted Locus Amplification (TLA) for genomic screening of antibody-expressing CHO clones.
  • To evaluate TLA/NGS for detecting single-nucleotide variants (SNVs) and transgenic rearrangements in CHO cell lines.
  • To assess the utility of TLA/NGS in identifying clone contaminations and genetically identical clones.

Main Methods:

  • Implementation of an efficient NGS workflow utilizing the Targeted Locus Amplification (TLA) strategy.
  • Targeted sequencing of the integrated transgenic DNA in CHO clones without requiring prior locus information.
  • Analysis of genomic DNA for single-nucleotide variants (SNVs) and transgenic DNA rearrangements.

Main Results:

  • TLA/NGS successfully identified CHO clones with high-level SNVs in the antibody gene.
  • The study demonstrated TLA/NGS's capability to detect rearrangements at the transgenic DNA level.
  • Detection limits for SNV calling were determined, and the potential for identifying clone contaminations was established.
  • TLA/NGS can also identify genetically identical clones, processing up to 24 CHO clones in under 7 workdays.

Conclusions:

  • TLA/NGS is a robust and efficient screening method for routine clone analytics in CHO cell line development.
  • This method enables targeted sequencing of integrated DNA, robustly detecting SNVs and rearrangements.
  • TLA/NGS accelerates the selection of high-quality CHO clones for biotherapeutic production.
Keywords:
Chinese hamster ovaryLC-MSclone selectionnext-generation sequencingtargeted locus amplification

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