Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid-chromatography coupled to mass spectrometry (LCMS) has been widely used to characterize post-translational modifications in proteins including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogenous glycans at the intact protein level. Herein, we evaluated several online separation techniques: 1. C2 reverse-phase liquid chromatography (RPLC), 2. capillary zone electrophoresis (CZE), and 3. acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down MS. Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation, N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogenous glycoproteins for facile comparison of biosimilars in quality control applications.