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

Sumit K Singh,Kelvin H Lee

doi:10.3389/fbioe.2021.805788

Abstract

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.

Highlights

Monoclonal antibodies are the fastest-growing modality in the biopharmaceutical industry with application towards treating many diseases including cancer (Grilo and Mantalaris, 2019)
The glycan analysis of the VRC01 using the standard library search method resulted in the identification of 53 confirmed N-glycans, out of which 44 were assigned based on both Glucose Units (GU) values and accurate mass, while the remaining nine glycan species were annotated based on GU values alone (Figure 1)
The percentage calculated is the sum of individual normalized amounts (Table 1) for each glycan species contributing to the overall abundance of a given attribute

Summary

Introduction

Monoclonal antibodies (mAbs) are the fastest-growing modality in the biopharmaceutical industry with application towards treating many diseases including cancer (Grilo and Mantalaris, 2019). Glycan Screening Method for mAbs. The most common type of glycosylation observed in mAbs is N-glycosylation. The overwhelming diversity of possible glycan types on a mAb necessitates analytical methods that can characterize the mAb glycosylation at different levels of protein architecture (Jensen et al, 2012; Reusch et al, 2015a; Reusch et al, 2015b). These include analysis at the level of intact proteins, protein subunits, peptides, and released glycans often utilizing liquid chromatography with mass spectrometry and/or fluorescence detection (Kaur, 2021). Other approaches include capillary electrophoresis-mass spectrometry (CE-MS), capillary electrophoresis-laser induced fluorescence detection (LIF), highperformance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and/or nuclear magnetic resonance (NMR) spectroscopy (Duivelshof et al, 2019)

Methods

Results

Discussion

Conclusion