Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development
| Autor: | Christina S. Alves, Chongfeng Xu, Yizhou Zhou, Baohong Zhang, Justin Crisafulli, Eric Marshall, Patrick Cullen, Fergal Casey, Thomas M. Carlile, Chao Sun, Dongdong Lin, Joost Groot |
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| Rok vydání: | 2021 |
| Předmět: |
Transgene
Clone (cell biology) Robustness (evolution) High-Throughput Nucleotide Sequencing General Medicine Computational biology Biology Amplicon Applied Microbiology and Biotechnology DNA sequencing Cell Line Benchmarking Cell culture Mutation False positive paradox Molecular Medicine Humans Transgenes Throughput (business) |
| Zdroj: | Biotechnology journalREFERENCES. 16(8) |
| ISSN: | 1860-7314 |
| Popis: | 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. This article is protected by copyright. All rights reserved. |
| Databáze: | OpenAIRE |
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