Online Hydrophilic Interaction Chromatography (HILIC) EnhancedTop-Down Mass Spectrometry Characterization of the SARS-CoV-2Spike Receptor-Binding Domain

SARS-CoV-2 cellular infection is mediated by theheavily glycosylated spike protein. Recombinant versions of the spikeprotein and the receptor-binding domain (RBD) are necessary forseropositivity assays and can potentially serve as vaccines against viralinfection. RBD plays key roles in the spike protein's structure andfunction, and thus, comprehensive characterization of recombinantRBD is critically important for biopharmaceutical applications. Liquidchromatography coupled to mass spectrometry has been widely usedto characterize post-translational modifications in proteins, includingglycosylation. Most studies of RBDs were performed at the proteolyticpeptide (bottom-up proteomics) or released glycan level because ofthe technical challenges in resolving highly heterogeneous glycans atthe intact protein level. Herein, we evaluated several online separationtechniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2)capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) toseparate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS).Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differsignificantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g.,cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by usingglycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facilecomparison of biosimilars in quality control applications.