Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time...

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• Mammalian cell cultures are crucial for biotherapeutic production, requiring stable recombinant DNA.
• Immortalized cells accumulate point mutations, potentially altering recombinant gene expression and protein products.
• Accurate assessment of recombinant DNA integrity is vital for quality assurance in cell line development.

Purpose of the Study:

• To develop and validate a sensitive method for detecting low-frequency point mutations in recombinant DNA sequences.
• To quantify the prevalence and characteristics of mutations in engineered mammalian cell lines.
• To establish a quality assurance metric for recombinant gene fidelity in biomanufacturing.

Main Methods:

• Utilized Pacific Biosciences single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology.
• Developed a rDNA sequence analysis tool to process sequencing data from approximately 40,000 single recombinant DNA molecules.
• Applied statistical filtering to raw sequencing data for accurate mutation detection.

Main Results:

• The analytical method can detect single point mutations at a minimum frequency of 0.0042% (<1/24,000 bases).
• In a stable CHO transfectant pool, 28% of recombinant plasmid copies had at least one low-frequency (<0.3%) point mutation.
• Mutations were primarily in GC base pairs (85%), with no positional bias, and affected coding and non-coding DNA equally.

Conclusions:

• The developed SMRT™ CCS-based method offers high sensitivity for detecting low-frequency DNA mutations, surpassing traditional next-generation sequencing (NGS) and second-generation sequencing (SGS) platforms.
• This methodology provides a robust tool for assessing recombinant DNA fidelity during cell line development and biotherapeutic production.
• Understanding and quantifying these mutations are essential for ensuring the consistency and safety of biotherapeutics.