Benchmarking and optimization of a high‐throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development. Issue 8 (3rd June 2021)

• Record Type: Journal Article
• Authors: Groot, Joost; Zhou, Yizhou; Marshall, Eric; Cullen, Patrick; Carlile, Thomas; Lin, Dongdong; Xu, Chong‐Feng; Crisafulli, Justin; Sun, Chao; Casey, Fergal; Zhang, Baohong; Alves, Christina
• Is Part Of: Biotechnology journal. Volume 16:Issue 8(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-06-03
• Subjects: bioprocess engineering -- cell line development -- CHO cells -- next‐generation sequencing -- sequence variant analysis; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202000548
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: In recent years, High‐Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non‐mutated antibody clone to generate spike‐in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false‐positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low‐level true mutations. Abstract : Biotherapeutic protein producing cell line clones are screened for mutations in their biotherapeutic transgenes using a targeted RNA‐sequencing assay. We report on improvements in assay computational analysis and evaluated the performance of 16 different preparation protocols using an RNA mutation spike‐in series. The assay updates furthered robustness and speed and included new protocols that reduced background mutation error levels; thus, improving the ability to identify low‐level true mutations.