Low level sequence variant analysis of recombinant proteins: an optimized approach

Area of Science:

• Biopharmaceutical Development
• Analytical Chemistry
• Molecular Biology

Background:

• Sequence variants in recombinant biopharmaceuticals can unpredictably affect clinical safety and efficacy.
• Sensitive analysis of these variants is critical during bioprocess development.

Purpose of the Study:

• To present a two-stage analytical approach for sensitive detection and quantitation of aberrant sequence variants in recombinant proteins.
• To enable rapid comparison of candidate production cell lines and in-depth analysis of selected clones.

Main Methods:

• A two-stage analytical strategy: initial multi-clone comparison followed by in-depth variant identification and quantitation.
• Differential analysis for sensitive batch-to-batch comparison of recombinant proteins.
• Detection of single and multiple amino acid substitutions, including those in post-translational modifications (e.g., glycopeptides) and structural isomers.

Main Results:

• The approach successfully detected sequence variants down to sub-percentage levels in two case studies.
• Identified variants included single amino acid substitutions (e.g., Thr → Asn), truncated/elongated variants, and double substitutions.
• Correlated a Thr → Asn variant to a DNA mutation and a Phe → Tyr variant to amino acid limitation in the fermentation medium.

Conclusions:

• The presented two-stage analytical approach is suitable for sensitive and rapid detection of sequence variants in recombinant proteins.
• This method aids in identifying critical sequence variants impacting biopharmaceutical quality.
• The findings support robust bioprocess development and quality control for biopharmaceuticals.