Identifying low-level sequence variants via next generation sequencing to aid stable CHO cell line screening
Related Experiment Video
Updated: Apr 11, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Sheng Zhang1, Lisa Bartkowiak1, Bernard Nabiswa1
1Process Sciences Cell Culture, Abbvie Bioresearch Center, 100 Research Drive, Worcester, MA, 01605.
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.
Insights
Transcriptome sequencing (RNAseq) rapidly detects low-level mutations in Chinese hamster ovary (CHO) cell lines during biotherapeutic development. This method identifies sequence variants early, ensuring product safety and efficacy in monoclonal antibody production.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is critical for product safety and efficacy.
- Sequence variants, unintended amino acid substitutions, must be monitored during cell line development (CLD).
- Current methods may not efficiently detect low-level genetic alterations.
Purpose of the Study:
- To report the first application of transcriptome sequencing (RNAseq) for detecting low-level point mutations in recombinant coding sequences during monoclonal antibody (mAb) CLD.
- To evaluate RNAseq's sensitivity and speed in identifying sequence variants.
- To demonstrate RNAseq's utility in eliminating undesirable CHO cell lines early in development.
Main Methods:
- Application of RNAseq to eleven top Chinese hamster ovary (CHO) cell line producers at various development stages (transfectant, clone, subclone).
- Analysis of RNAseq data to detect point mutations in recombinant coding sequences.
- Liquid chromatography-tandem mass spectrometry (LC/MS/MS) for characterization of sequence variants.
Main Results:
- Three out of eleven cell lines showed low-level point mutations (missense or nonsense) below 2% via RNAseq.
- LC/MS/MS confirmed approximately 3% sequence variants with a Ser to Leu substitution in the heavy chain of specific transfectants.
- The identified amino acid substitution correlated with a C/T mutation detected by RNAseq in the heavy chain coding sequence.
Conclusions:
- RNAseq is a rapid and highly sensitive method for de novo identification of low-level genetic mutations causing amino acid substitutions.
- Implementing RNAseq in CLD provides an early and effective strategy for selecting desired CHO expression cell lines.
- This approach enhances the assurance of product safety and efficacy by minimizing developmental delays caused by undetected sequence variants.
More Related Videos
09:34Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
09:33Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens
Published on: August 25, 2023
