title: Genetic mutation analysis at early stages of cell line development using next generation sequencing.
abstract: A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next-Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high-throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813-817, 2016.
authors.affiliation: Cell Culture Development, Biogen, Cambridge, MA.
authors.forename: Chapman
authors.initials: C
authors.lastname: Wright
authors.affiliation: Computational Biology and Genomics, Biogen, Cambridge, MA.
authors.forename: Joost
authors.initials: J
authors.lastname: Groot
authors.affiliation: Cell Culture Development, Biogen, Cambridge, MA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Swahn
authors.affiliation: Computational Biology and Genomics, Biogen, Cambridge, MA.
authors.forename: Helen
authors.initials: H
authors.lastname: McLaughlin
authors.affiliation: Computational Biology and Genomics, Biogen, Cambridge, MA.
authors.forename: Mei
authors.initials: M
authors.lastname: Liu
authors.affiliation: Analytical Development, Biogen, Cambridge, MA.
authors.forename: Chongfeng
authors.initials: C
authors.lastname: Xu
authors.affiliation: Computational Biology and Genomics, Biogen, Cambridge, MA.
authors.forename: Chao
authors.initials: C
authors.lastname: Sun
authors.affiliation: Computational Biology and Genomics, Biogen, Cambridge, MA.
authors.forename: Eric
authors.initials: E
authors.lastname: Zheng
authors.affiliation: Cell Culture Development, Biogen, Cambridge, MA.
authors.forename: Scott
authors.initials: S
authors.lastname: Estes
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D019295:Computational Biology; D003412:Cricetulus; D059014:High-Throughput Nucleotide Sequencing; D013058:Mass Spectrometry; D009154:Mutation; D017422:Sequence Analysis, DNA
keywords: CHO; amplicon sequencing; early stage cell line development; mutation detection