In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Abstract

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies

Evolution of Orbitrap Mass Spectrometry Instrumentation

“…To better meet the requirements necessary for the analysis of intact antibodies, antibodydrug conjugates, large protein complexes, and native proteins, an extended mass range Orbitrap instrument was developed with improved sensitivity to these analytes (47)(48)(49)(50). These mass spectrometers have been optimized for improved transmission of higher mass-to-charge-ratio ions, enabling detection up to m/z 20,000.…”

Native Mass Spectrometry: What is in the Name?

“…With increased sensitivity, analyzer resolution, and enhanced desolvation, the achievable resolving power on proteins and protein assemblies using the Orbitrap mass analyzer increased substantially in comparison with traditional Q-ToF instruments, especially in the higher m / z range ( m / z  > 4000). This increased resolving power has enabled small differences in proteoforms, such as glycosylation on antibodies [53, 54] and phosphorylation on proteins and protein complexes [55, 56], to be more readily resolved. One striking example is the analysis of erythropoietin, whereby 236 glycan proteoforms were separated by m / z and identified [57].…”

Boundaries of Mass Resolution in Native Mass Spectrometry

“…However, owing to the high resolving power of the Orbitrap, this instrument can potentially open up new avenues in native MS analysis. The Orbitrap Exactive Plus EMR, first reported in 2012 [33], was already proven to be an important advance for the field since it is able to resolve small mass shifts even in complex spectra enabling, for example, the identification and quantification of complex glycosylation profiles of antibody mixtures [23,51].…”