title: Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry.
abstract: The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.
authors.affiliation: 1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands. [3].
authors.forename: Sara
authors.initials: S
authors.lastname: Rosati
authors.affiliation: 1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands. [3].
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: 1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands.
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.affiliation: 1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D004867:Equipment Design; D006031:Glycosylation; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D011499:Protein Processing, Post-Translational
chemical_list: D000911:Antibodies, Monoclonal