The drug loading clearly increases in steps of two, linked to the two accessible cysteine amino acids when a disulfide bridge is reduced. Discussion Here, we describe a method to minutely characterize in the undamaged protein level micro-heterogeneity originating from complex N-glycosylation, truncations and chemically- or biologically-induced modifications on mAbs. The method offered here will aid in the comprehensive analytical and practical characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies Keywords: native mass spectrometry, monoclonal antibodies, glycosylation, antibody-drug conjugates, biopharmaceuticals, biosimilars, protein micro-heterogeneity, proteoforms Introduction Controlling MS402 and understanding the protein micro-heterogeneity of monoclonal antibodies (mAbs) both in a qualitative and quantitative manner represents one of the main focuses in the development and manufacturing of this class of therapeutics. Post-translational modification (PTM) on mAbs needs to be minutely characterized because it may affect antibody structure, efficacy, and potency, and its potential antigenicity or immunogenicity. The most widespread PTM occurring on mAbs is usually N-glycosylation. The nature of the glycan chains influences Fc-effector function and serum half-life.1,2 In particular, the lack of core fucosylation enhances antibody dependent cellular cytotoxicity (ADCC),3,4 while the presence of (2C6)-linked sialic acids MS402 (values, which so far has been little explored on ion traps or Orbitraps. In this work, we aim for the detailed characterization of highly complex micro-heterogeneity, including glycosylation profiles, on intact native mAbs. We use the recently described Orbitrap Exactive Plus (ThermoFisher Scientific) that has been modified to perform native MS.24 We show that this instrument is capable, through its high sensitivity, mass accuracy and resolving power, of providing baseline separation of the different proteoforms on intact half- (~75 kDa) and full-mAbs (~150 kDa). The analysis by native MS around the Orbitrap at the intact protein level provides a number of advantages. Most importantly, a single highly resolved profile of all protein micro-heterogeneity MS402 could be obtained within Adamts4 a few minutes using a few femto-mole of sample, making it a time- and cost-efficient tool for routine analysis. Very little sample preparation is required, as the direct injection into the mass spectrometer excludes the need for a chromatographic step prior to MS analysis. Additionally, differences in the chemical nature of the glycan chains do not substantially affect the ionization efficiency of the intact protein, allowing the relative quantification of all proteoforms/glycoforms, including highly sialylated glycans. The detailed qualitative and quantitative profiles we observe reveal, in some cases, more than 30 different proteoforms of a single mAb,25 extending the depth of structural characterization usually obtained by current technologies. Results Benchmarking the performance of native MS using an Orbitrap mass analyzer in the characterization of protein micro-heterogeneity of intact full-length mAbs To test the performance and demonstrate the versatility of the new analysis workflow, we selected three different samples: (1) a full-length (150 kDa), hinge deleted, IgG4 that exists in equilibrium with its half-antibody (75 kDa); (2) IgG4 mutants exhibiting highly complex glycosylation profiles; and (3) an IgG1 antibody-drug conjugate (ADC). We benchmarked our approach evaluating the glycosylation profile on a wild-type, hinge-deleted IgG4 antibody (hingeIgG4). The deletion of the hinge region excludes intermolecular disulfide bonds between the two heavy chains, making the dimerization of the two half-antibodies occur solely through non-covalent interactions.26 The full native mass spectrum of the hingeIgG4WT antibody is shown in Determine?1A. Notably, as described earlier27 this spectrum can be generated in a matter of a few minutes, consuming just a few femtomoles of sample. The native MS spectrum provides a glimpse of the equilibrium, caused by the deletion of the hinge region, that exists between the MS402 half- and MS402 full-antibody in answer.