Multiple-parallel-protease digestion coupled with high-resolution mass spectrometry: An approach towards comprehensive peptide mapping of therapeutic mAbs
IR@IMTECH: CSIR-Institute of Microbial Technology, Chandigarh
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| Title |
Multiple-parallel-protease digestion coupled with high-resolution mass spectrometry: An approach towards comprehensive peptide mapping of therapeutic mAbs
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| Creator |
Pradhan, Gauri
Sneha, J.M. Sonwane, Babasaheb P Santhakumari, B Rao, Alka Kulkarni, Mahesh |
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| Subject |
QR Microbiology
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| Description |
Therapeutic monoclonal antibodies (mAbs) are structurally large and complex molecules. To be safe and efficacious, a biosimilar mAb must show high similarity to its reference product in Critical Quality Attributes (CQA). mAbs are highly sensitive to protein expression, production, manufacturing, supply chain, and storage conditions. All these factors make biosimilar mAbs intrinsically susceptible for variability during production. Accordingly, several lots of references and tests are required to establish the biosimilarity of a test mAb. The primary structure is a CQA of a mAb affecting its safety and efficacy. Here, we apply peptide mapping as an analytical method to decipher the primary structure and associated modifications for a quick quality assessment of TrastuzumAb and RituximAb innovator and biosimilar. A multiple-parallel-protease digestion strategy followed by high-resolution mass spectrometric analysis consistently achieved 100% sequence coverage along with reliable detection of post-translational modifications. Additionally, the use of supporting methods such as intact mass analysis and circular dichroism helped us to decipher the primary and higher order structures of these mAbs. We identify discernible variations in the profile of the innovator and biosimilar mAbs and validate the method for quick yet deep comparability analysis of the primary structure of biosimilar mAbs sold in the market.
Significance: Peptide mapping using bottom-up approach is one of the most common methods for the characterization of therapeutic monoclonal antibodies. Herein, we describe a multi-parallel-protease digestion strategy using a combination of five different proteases followed by high-resolution mass spectrometric analysis with TrastuzumAb and RituximAb as an example. This resulted in a comprehensive identification of peptides with increased reliability and identification of different PTMs. Additional supporting orthogonal methods like intact mass and higher-order structure analysis helped evaluate broader conformational properties.
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| Publisher |
Elsevier Science
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| Date |
2021-02-10
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| Type |
Article
PeerReviewed |
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| Relation |
https://www.sciencedirect.com/science/article/abs/pii/S1874391920304218
http://crdd.osdd.net/open/2649/ |
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| Identifier |
Pradhan, Gauri and Sneha, J.M. and Sonwane, Babasaheb P and Santhakumari, B and Rao, Alka and Kulkarni, Mahesh (2021) Multiple-parallel-protease digestion coupled with high-resolution mass spectrometry: An approach towards comprehensive peptide mapping of therapeutic mAbs. Journal of proteomics, 232. ISSN 1876-7737
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