02995nas a2200265   4500008004100000022001400041245015900055210006900214260000800283300001100291490000800302520207900310653001302389653000802402653001402410653003802424653001602462100001902478700001802497700002702515700002102542700001402563700002502577856012702602       2021                        eng d  a1874-391900aMultiple-parallel-protease digestion coupled with high-resolution mass spectrometry: an approach towards comprehensive peptide mapping of therapeutic mAbs0 aMultipleparallelprotease digestion coupled with highresolution m  cFEB  a1040530 v2323 a<p>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.</p>
10aLC-MS/MS10aPTM10aRituximAb10aTherapeutic monoclonal antibodies10aTrastuzumAb1 aPradhan, Gauri1 aSneha, J., M.1 aSonwane, Babasaheb, P.1 aSanthakumari, B.1 aRao, Alka1 aKulkarni, Mahesh, J.  uhttps://library.ncl.res.in/content/multiple-parallel-protease-digestion-coupled-high-resolution-mass-spectrometry-approach