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

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 hydrophilic interaction chromatography (HILIC) columns optimized for protein separation. These monoliths allow lower TFA usage and support effective glycoprotein analysis.

The scientists then applied their monolithic HILIC-mass spectrometry (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 reversed-phase liquid chromatography (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, to learn more about their research (1). Here, they explain how careful control of polymerization conditions, pore structure, and ion-pairing chemistry enabled a breakthrough in intact glycoprotein separation and characterization.

For the first part of the interview, click here.

How does HILIC improve glycoform separation, and what role do additives like trifluoroacetic acid (TFA) play in this process?

HILIC is a method of choice for the analysis of many polar analytes and has demonstrated high structural selectivity in the characterization of glycoproteins at various level (2). With respect to small molecule analysis, where a high % of ACN is used (for example, 95%), sometimes using isocratic elution, a lower amount of ACN is used for subunit or intact protein analysis, typically using gradient elution with a relatively large amount of water used (for example, 20 to 40%). Due to the high-water content, we suggest that no partitioning occurs, but only polar interactions (such as hydrogen bonding and ion exchange) take place. Since glycans are highly hydrophilic components, subtle structural differences, such as the number or size of the overall glycan structure, lead to differential retention. Glycoforms with higher hydrophilicity in HILIC are retained longer (and eluted later), allowing for resolution that, in the case of proteins like ribonuclease B, can be at the single glycan level, which RPLC cannot achieve (co-elution of glycoforms).

In HILIC analysis of glycoproteins, amide-based stationary phases are used, and TFA is employed as an ion-pairing reagent to mask charged sites on the antibody, thereby minimizing secondary interactions that can broaden peaks. This ensures that hydrophilicity, rather than electrostatic effects, primarily drives separation. In our study, 0.1% TFA was necessary to achieve narrow peak widths, while lower concentrations, such as 0.02% (v/v), produced broader peaks (3). For smaller glycoproteins, lower TFA levels may be sufficient due to their reduced charge density (4).

What advantages do poly(acrylamide-co-N,N-methylene-bis(acrylamide)) monolithic stationary phases offer over conventional silica-based stationary phases for intact glycoprotein analysis?

Poly(acrylamide) monolithic columns offer two principal advantages over conventional, particle-packed silica-based stationary phases for the HILIC analysis of intact glycoproteins like mAbs: i) a non-charged surface chemistry, and ii) unique morphology.

Our materials are polymer-based monoliths, as opposed to silica-based particles functionalized by an immobilized amide chemical selector. Therefore, on our material, there are no secondary ionic interactions common to silica columns (silanol activity). This reduces nonspecific adsorption from charged groups (basic AA) of the large mAbs, leading to sharper peaks, significantly improved glycoform resolution, and better compatibility with mass spectrometry (lower TFA amounts are needed).

The continuous, porous monolithic structure provides high permeability and facilitates the diffusion of mAb, enabling highly efficient separation compared to particle-packed beds.

What challenges arise in separating intact monoclonal anibody (mAb) glycoforms, given their large molecular size and the relatively small mass differences between glycoforms?

The most significant challenge is the small difference in mass and chemistry introduced by the glycans. A single monosaccharide difference, such as a galactose residue (~162 Da), represents less than 0.1% of the total ∼150 kDa mAb molecular weight. This makes chromatographic resolution exceptionally difficult.

The mAb's structure contains ∼approximately 1300 amino acids, each with chemical groups that can cause multiple interactions (for example, ionic and hydrophilic) with the stationary phase. These dominant, non-specific interactions from the protein backbone mask the subtle, desired retention differences caused by the glycans, necessitating the use of ion-pair agents.

Finally, the mAb's large size results in slow mass transfer, which poses challenges in terms of column efficiency. In our group, to overcome this, we developed columns with good efficiency for large protein separations and applied (very) shallow gradients (0.2% B/min) to chromatographically resolve the minute elution differences (Figure 1).

Figure 1: Base peak chromatogram (BPC) with extracted ion chromatogram (EIC) traces of separated glycoforms of (A) trastuzumab and (B) nivolumab. Each EIC characterized a specific glycoform: EIC 1 – G0F, EIC 2 – G1F, EIC 3 – G0F/G0F, EIC 4 – G0F/G1F, EIC 5 – G1F/G1F, EIC 6 G1F/G2F, EIC 7 – G2F/G2F. From reference (3). 

How did optimizing the polymerization conditions, such as varying dimethyl sulfoxide (DMSO) content, improve the performance of acrylamide monolithic HILIC columns for intact mAb glycoform analysis?

Optimizing the DMSO-to-octanol ratio, was essential to improve the performance of acrylamide monolithic HILIC columns for intact mAb glycoform analysis. In our work, the porogen composition was systematically varied to control the pore size distribution and morphology of the polymeric stationary phase. DMSO served as a "good" solvent (dissolving the monomers), while octanol, a "poor" solvent, promoted the formation of optimal flow-through pores by inducing controlled polymer precipitation.

We suggest that increasing the DMSO content relative to octanol enhances column performance by increasing the effective surface area, resulting in improved retention and resolution (3). The increased DMSO ratio led to a monolithic structure with a higher accessible surface area for mAb. In HILIC, this larger surface area promotes interaction between the hydrophilic analytes (mAb glycoforms) and the stationary phase, resulting in higher retention.The optimized pore structure also facilitated more efficient mass transfer for the large mAb molecules. This resulted in narrower peak widths and, consequently, significantly higher resolution and selectivity for the closely related intact mAb glycoforms.

This successful optimization was crucial, enabling the first demonstrated LC-MS method capable of resolving intact mAb glycoforms.

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. Gargano, A. F. G.; Haselberg, R.; Somsen, G. W. Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry for the Characterization of Glycoproteins at the Glycan, Peptide, Subunit, and Intact Level; in Carbohydrate Analysis by Modern Liquid Phase Separation Techniques, Elsevier; 2021, p. 209–278. DOI: 10.1016/B978-0-12-821447-3.00018-4.
3. 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, 13569–13576. DOI: 10.1021/acs.analchem.5c02033.
4. Passamonti. M.; Zhai, Z.; Moreschini, M.; Wilson, J. W.; Zhou, M; Schoenmakers, P. J. et al. Influence of Ion-Pairing Reagents on the Separation of Intact Glycoproteins Using Hydrophilic-Interaction Liquid Chromatography-High-Resolution Mass Spectrometry. J. Chromatogr. A 2023, 1688, 463721. DOI: 10.1016/j.chroma.2022.463721