Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies.

ABSTRACT

Peptide mapping is an important tool used to confirm that the correct sequence has been expressed for a protein and to evaluate protein post-translational modifications (PTMs) that may arise during the production, processing, or storage of protein drugs. Our new orally administered drug (Ab-1), a single-domain antibody, is highly stable and resistant to proteolysis. Analysis via the commonly used tryptic mapping method did not generate sufficient sequence coverage. Alternative methods were needed to study the Ab-1 drug substance (75 mg/mL) and drug product (3 mg/mL). To meet these analytical needs, we developed two new peptide mapping methods using lysyl endopeptidase (Lys-C) digestion. These newly developed protein digestion protocols do not require desalting/buffer-exchange steps, thereby reducing sample preparation time and improving method robustness. Additionally, the protein digestion is performed under neutral pH with methionine acting as a scavenger to minimize artifacts, such as deamidation and oxidation, which are induced during sample preparation. Further, the method for low-concentration samples performs comparably to the method for high-concentration samples. Both methods provide 100% sequence coverage for Ab-1, and, therefore, enable comprehensive characterization for its product quality attribute (PQA) assessment. Both methods can be used to study other antibody formats.