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

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.