Identifying low‐level sequence variants via next generation sequencing to aid stable CHO cell line screening

Abstract

Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here, for the first time, we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify, and eliminate cell lines containing low-level point mutations in recombinant coding sequence. Among the top eleven mAb producers chosen from transfectant, clone or subclone stages, three of the cell lines contained either missense or nonsense point mutations at a low level of less than 2%. Subsequent LC/MS/MS characterization detected ∼3% sequence variants with an amino acid change from Ser to Leu at residue 117 in the heavy chain of transfectants 11 and 27. This substitution is consistent with the RNAseq finding of a C/T mutation located at 407 base pair (TCA→TTA) in the heavy chain coding sequence. Here we demonstrate that RNAseq is a rapid and highly sensitive method to identify low-level genetic mutation de novo corresponding to the amino acid substitution that elicits sequence variant(s). Its implementation in CLD constitutes an early and effective step in identifying desired CHO expression cell lines.

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

“…Deviations or variants from the primary protein sequence or higher‐order structure could have an impact on efficacy or safety of the final therapeutic proteins and should be avoided . Such protein sequence variants mostly originate from DNA mutations or erroneous DNA replication and mistranslations during protein synthesis . Often, sequence variants have been first identified at the protein level using analytical methods such as charge‐ or hydrophobicity‐based separation methods and mass spectrometry.…”

“…Peptide mapping with liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and protein subunit LC–MS methods are most commonly used to receive information on location, identity, and relative quantification of sequence variants at the amino acid level with high sensitivity . These techniques, however, are still labor‐intense and low throughput, especially when detection of low‐level sequence variants is required . Novel next‐generation sequencing (NGS) techniques have revolutionized the gene and genome analysis lately in terms of speed, costs, throughput, and sensitivity and are increasingly applied in biopharmaceutical research and development .…”

“…These reports used in both cases messenger RNA (mRNA) as a source of sequencing (whole transcriptome RNA sequencing and Amplicon sequencing). Therefore, the integrity of recombinant DNA outside of open reading frames was not assessed . NGS techniques can also be applied for whole‐genome analysis and capture‐based targeted sequencing.…”

Utilization of sequence variants as biomarkers to analyze population dynamics in cloned cell lines

“…Detection of low level SVs has occurred more frequently due to improvements in sequencing (Pekin et al, 2011;Zhang et al, 2015b) and analytical instrumentation specifically in mass spectrometry and related software (Brady et al, 2015;Harris et al, 1993;Zeck et al, 2012;Zhang et al, 2013). In terms of protein analytics, there is much higher sensitivity and selectivity for product characterization and low level SV detection (Feeney et al, 2013;Wan et al, 1999).…”

“…Sanger sequencing (Casales et al, 2010) or, more recently, next generation sequencing (NGS) have been used to detect genetic heterogeneity with high accuracy (Zhang et al, 2015b). Upon further investigation, the corresponding mutations in the genomic DNA and RNA of the gene-of-interest were confirmed.…”