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

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.

Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies

“…Of the total identified Rituximab glycans, ∼50% of the glycans were galactosylated, ∼1% of them were terminally sialylated, ∼1% of the glycans were hybrid, and ∼97% were core-fucosylated. A low level of sialylation was detected, which is a usual attribute observed with therapeutic mAbs …”

“…We note that high mannose and G4H3F (G0F) are the prominent glycoforms in Evolocumab. Sialylation was detected low, a common feature observed with therapeutic mAbs …”

“…A low level of sialylation was detected, which is a usual attribute observed with therapeutic mAbs. 38…”

Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines

“…The glycan composition is a determining factor in the interaction of antibodies with receptors and their effector functions [ 34 ]. Under our experimental conditions, it was possible to observe a higher percentage of fucosylated glycans, as expected, and a lower amount of antibodies with high mannose moieties [ 35 , 36 ]. On the other hand, although the method adopted has limitations, it was possible to identify differences between the mAbs, which may eventually be due to cultivation conditions [ 36 ].…”

“…Under our experimental conditions, it was possible to observe a higher percentage of fucosylated glycans, as expected, and a lower amount of antibodies with high mannose moieties [ 35 , 36 ]. On the other hand, although the method adopted has limitations, it was possible to identify differences between the mAbs, which may eventually be due to cultivation conditions [ 36 ]. Since the glycan composition strongly influences the binding of the antibody to the Fc receptor, further cell culture optimization and more refined analysis of the glycan composition of these antibodies need to be performed after the cloning of the pools.…”