Aeschlimann, Samuel, Graf, Christian, Mayilo, Dmytro, Munschy, Helene, Urda, Lorena, Kappes, Nora, Simonis, Marieke, Splinter, Erik, van Min, Max and Laux, Holger (2019) Shortened abstract for Enhanced CHO clone screening using Next-Generation Sequencing and Targeted Locus Amplification publication. Enhanced CHO clone screening: Applying Targeted Locus Amplification and Next-Generation Sequencing technologies during cell line development.

Abstract

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next-generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single-nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high-level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.

Item Type:	Article
Keywords:	Chinese hamster ovary, Clone selection, LC-MS, Next Generation Sequencing, Targeted Locus Amplification
Date Deposited:	26 Jun 2019 00:45
Last Modified:	26 Jun 2019 00:45
URI:	https://oak.novartis.com/id/eprint/39649