Enhanced Intact Glycoform Characterization of Monoclonal Antibodies Using Polyacrylamide Monolithic HILIC-MS: Part I

Monoclonal antibodies (mAbs) contain N-linked glycans that influence pharmacokinetics and efficacy, making glycoform profiling critical for therapeutic development. Intact-level characterization provides valuable insights into glycoform combinations while minimizing sample preparation. However, conventional methods like reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) have a difficult time separating glycoforms due to limited selectivity, and other techniques, such as ion-exchange or capillary electrophoresis, are less effective for neutral glycoforms. Hydrophilic interaction chromatography (HILIC) offers improved resolution of intact glycoforms by leveraging glycan hydrophilicity. Using neutral amide-based stationary phases and additives like trifluoroacetic acid (TFA), HILIC enables high-resolution separation.

In a study conducted at the van 't Hoff Institute for Molecular Sciences at the University of Amsterdam (the Netherlands), researchers developed poly(acrylamide-co-N,N-methylene-bis(acrylamide) monolithic HILIC columns optimized for protein separation. These monoliths allow lower TFA usage and support effective glycoprotein analysis. The scientists then applied their monolithic HILIC-MS platform to separate and characterize intact glycoforms of large proteins, including IgG mAbs (~150 kDa). Given the small relative mass differences of glycans, the team optimized polymerization conditions to enhance pore morphology and separation performance. They validated the method on five IgG mAbs and compared results to RPLC-MS, demonstrating improved glycoform resolution. LCGC International spoke to Annika van der Zon and Andrea Gargano, both from the van 't Hoff Institute and corresponding authors of the paper resulting from this research, to learn more about their research (1).

Why is glycosylation profiling of monoclonal antibodies (mAbs) critical for therapeutic applications? How can changes in glycosylation impact efficacy?
Glycosylation is the enzymatic attachment of carbohydrate chains (glycans) to proteins, a critical post-translational modification (PTM) that influences the structural and functional properties and overall therapeutic efficacy of mAbs (Figure 1). Comprehensive glycosylation profiling of mAbs is essential in biopharmaceutical development and quality control. The specific glycan structures present on the antibody surface directly affect its pharmacodynamic and pharmacokinetic properties, as well as its safety and efficacy in clinical use. Changes in the Fc glycan structure have a profound impact on the antibody's interactions with the immune system. For example, antibodies lacking fucose residues (afucosylated antibodies) have enhanced binding affinity to the FcγRIIIa receptor on natural killer (NK) cells, leading to improved antibody-dependent cellular cytotoxicity (ADCC) and stronger tumor cell killing activity a characteristic very important in cancer therapy. Conversely, antibodies with a higher degree of sialylation tend to display reduced ADCC activity but increased anti-inflammatory properties, which is a desirable feature in the treatment of autoimmune diseases, like rheumatoid arthritis.

Figure 1: Schematic representation of an IgG antibody highlighting post-translational modifications (PTMs).

Can you explain the advantages and limitations of the different multilevel approaches (released glycans, peptide, subunit, and intact levels) used for glycoprotein characterization?
The characterization of a glycoprotein, like a mAb requires a multilevel approach to achieve a complete picture of its glycoforms (the molecular variants arising from different glycan structures) (Figure 2). The four primary levels of analysis each offer unique information, trading off between specificity (glycan detail) and context (glycoform combination).

Figure 2: Illustration of commonly used digestion approaches used to characterize glycoforms of mAbs. From reference (2).

What makes intact-level analysis particularly valuable for glycoform profiling compared to other levels of characterization?
In our view, the unique contribution of intact-level analysis (analyzing the full ∼150 kDa mAb) is its ability to directly determine the glycoform combination (or glycopairing) across the two heavy chains (Figure 2).

While released glycan and peptide-level analyses provide superior structural detail (for example,specific linkages and precise composition), they lose the connectivity information. They only tell you the type of glycans present, not how they are distributed (paired) on the final therapeutic molecule. Intact analysis provides the context that the other, more detailed approaches cannot.

IgGs have typically two N-glycosylation sites located in the Fc region (N-glycan, Asn297); the overall conformation and spatial arrangement of the Fc region is responsible for the activity (such as antibody-dependent cellular cytotoxicity [ADCC]), which depends on the combined presence of the two Fc glycans. For instance, an antibody could have a highly fucosylated glycan on one chain and an afucosylated glycan on the other. Only intact-level analysis can easily confirm this heterogeneity and distinguish it from a fully afucosylated or fully fucosylated molecule.

Why does RPLC have limited selectivity for glycoforms, and what are the consequences of this limitation in mAb analysis?
In RPLC, separation relies on differences in hydrophobicity. The mAb backbone is a large, highly hydrophobic biopolymer. The hundreds of hydrophobic amino acid residues of the protein structure determine its retention time in RPLC. The glycans have a relatively small molecular weight compared to the mAb backbone and are hydrophilic. Their presence slightly reduces the overall hydrophobicity of the protein; however, the minute hydrophobic difference introduced by subtle changes between glycoforms (such as, for example, adding one galactose, a ∼162 Da change) is insufficient to overcome the massive hydrophobic forces exerted by the ∼150 kDa protein backbone. As a result, RPLC cannot resolve the subtle differences in hydrophobicity between different glycoforms, causing them to co-elute in a single, broad peak.

Join us on Wednesday for part two of this interview, as Annika van der Zon and Andrea Gargano explain how careful control of polymerization conditions, pore structure, and ion-pairing chemistry enabled a breakthrough in intact glycoprotein separation and characterization.

References

1. van der Zon, A. A. M.; Hana, L. N.; Husein, H.; Holmark, T.;Zhai. Z.; Gargano, A. F. G Hydrophilic Interaction Chromatography HRMS with Acrylamide Monolithic Columns: A Novel Approach for Intact Antibody Glycoform Characterization. Anal. Chem. 2025,97 (25), 13569-13576. DOI: 10.1021/acs.analchem.5c02033
2. van der Zon, A. A.M.; Verduin, J.; van den Hurk, R. S.; Gargano, A. F. G.; Pirok, B. W. J. Sample Transformation in Online Separations: How Chemical Conversion Advances Analytical Technology. Chem. Comm. 2024, 60, 36–50. DOI: 10.1039/D3CC03599A.