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Expanding the Analytical Toolbox: Developing New Peptide Map Methods to Fully Characterize the Proteolysis-resistant Single-domain Antibody New Modality
Liu, Y.D.; Beardsley, M.I.; Yang, F. Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies. Pharmaceuticals2023, 16, 1327.
Liu, Y.D.; Beardsley, M.I.; Yang, F. Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies. Pharmaceuticals 2023, 16, 1327.
Liu, Y.D.; Beardsley, M.I.; Yang, F. Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies. Pharmaceuticals2023, 16, 1327.
Liu, Y.D.; Beardsley, M.I.; Yang, F. Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies. Pharmaceuticals 2023, 16, 1327.
Abstract
Peptide mapping is an important tool to confirm the correct sequence expressed for a protein and to evaluate protein post-translational modifications (PTMs) that may arise during 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 by the commonly used tryptic map 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 analytical needs, we developed two new peptide map methods with 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 was 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 attributes (PQA) assessment. Both methods can be used to study other antibody modalities.
Keywords
Biopharmaceuticals; drug substances; sample preparation; mass spectrometry
Subject
Chemistry and Materials Science, Analytical Chemistry
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
