Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies
Non-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rea...
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| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2024.2420805 |
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| author | Andrew Kleinberg Yuan Mao Ning Li |
| author_facet | Andrew Kleinberg Yuan Mao Ning Li |
| author_sort | Andrew Kleinberg |
| collection | DOAJ |
| description | Non-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rearrangement of disulfide connections (disulfide scrambling), thus generating connectivity profiles that do not accurately represent the protein being analyzed. Common misconceptions (e.g. avoiding basic-pH digestion to prevent disulfide scrambling) have led to the development of alternative reagents and conditions that can alleviate this issue, but yield problematic digestion profiles. Herein, we systematically and comprehensively examine the primary considerations for accurate non-reduced peptide mapping, and provide effective, practical solutions to minimize undesired behavior while still yielding high-quality digests. Additionally, we present a method that exploits intentional disulfide scrambling as a reference tool to demonstrate the robustness of our proposed strategies. We also introduce maleimide as a cysteine-alkylating reagent and demonstrate its benefits over industry-leading analogs such as N-ethylmaleimide in terms of compatibility with regulatory reports. |
| format | Article |
| id | doaj-art-d63c0735261e40d287f7b79802a4f2ca |
| institution | DOAJ |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj-art-d63c0735261e40d287f7b79802a4f2ca2025-08-20T03:22:19ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2024.2420805Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodiesAndrew Kleinberg0Yuan Mao1Ning Li2Department of Analytical Chemistry, Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USADepartment of Analytical Chemistry, Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USADepartment of Analytical Chemistry, Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USANon-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rearrangement of disulfide connections (disulfide scrambling), thus generating connectivity profiles that do not accurately represent the protein being analyzed. Common misconceptions (e.g. avoiding basic-pH digestion to prevent disulfide scrambling) have led to the development of alternative reagents and conditions that can alleviate this issue, but yield problematic digestion profiles. Herein, we systematically and comprehensively examine the primary considerations for accurate non-reduced peptide mapping, and provide effective, practical solutions to minimize undesired behavior while still yielding high-quality digests. Additionally, we present a method that exploits intentional disulfide scrambling as a reference tool to demonstrate the robustness of our proposed strategies. We also introduce maleimide as a cysteine-alkylating reagent and demonstrate its benefits over industry-leading analogs such as N-ethylmaleimide in terms of compatibility with regulatory reports.https://www.tandfonline.com/doi/10.1080/19420862.2024.2420805Acidic digestioncysteine alkylationdisulfide scramblingnon-reduced peptide mappingthiol-disulfide exchangeβ-elimination |
| spellingShingle | Andrew Kleinberg Yuan Mao Ning Li Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies mAbs Acidic digestion cysteine alkylation disulfide scrambling non-reduced peptide mapping thiol-disulfide exchange β-elimination |
| title | Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies |
| title_full | Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies |
| title_fullStr | Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies |
| title_full_unstemmed | Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies |
| title_short | Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies |
| title_sort | practical solutions for overcoming artificial disulfide scrambling in the non reduced peptide mapping characterization of monoclonal antibodies |
| topic | Acidic digestion cysteine alkylation disulfide scrambling non-reduced peptide mapping thiol-disulfide exchange β-elimination |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2024.2420805 |
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