Detecting low level sequence variants in recombinant monoclonal antibodies

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Recombinant monoclonal antibodies are crucial therapeutics, but low-level protein sequence variants can impact efficacy and safety.
• Accurate detection of amino acid substitutions is essential for quality control in antibody production.

Purpose of the Study:

• To develop and validate a systematic analytical approach for detecting and identifying low-level protein sequence variants in recombinant monoclonal antibodies.
• To demonstrate the application of this method in early and late-stage antibody projects for clone selection.

Main Methods:

• Combined tryptic and chymotryptic peptide mapping with Mascot Error Tolerant Search (ETS).
• Optimized reversed-phase HPLC separation, UV detection, and mass spectral acquisition parameters.
• Utilized model samples with known low levels of recombinant antibodies for method optimization.

Main Results:

• Successfully detected and identified low-level amino acid substitutions (e.g., M83R at 5%, P274T at 42%, L413Q at 0.3%) in recombinant antibodies.
• Demonstrated rapid screening capability for early-stage clone selection, identifying variants in CHO cell clones.
• Applied the method to identify a low-level variant in a late-stage antibody production candidate.

Conclusions:

• The developed peptide mapping and ETS approach is effective for sensitive detection and identification of protein sequence variants in monoclonal antibodies.
• This systematic analysis offers practical applications for clone selection and quality control in the biotechnology industry.
• The method has potential for detecting polymorphisms in endogenous proteins.