# Hydrophilic Interaction Chromatography HRMS with Acrylamide Monolithic Columns: A Novel Approach for Intact Antibody Glycoform Characterization

## TL;DR

A new HILIC-MS method using acrylamide monolithic columns improves the analysis of intact antibody glycoforms, enabling detection of low-abundance variants.

## Contribution

Development of acrylamide monolith HILIC-MS for intact mAb glycoform characterization, enabling detection of low-abundance glycoforms.

## Key findings

- Increasing DMSO content in the polymerization mixture improved retention and reduced peak widths in HILIC columns.
- The optimized HILIC-MS method achieved baseline separation of glycoforms like G0F vs G0F/G0F in trastuzumab.
- Acrylamide-monolith HILIC-MS detected low-abundance glycoforms (e.g., single G0F and M5/M5) not detectable by RPLC-MS.

## Abstract

Glycosylation significantly
impacts the pharmacokinetics and efficacy
of monoclonal antibody (mAb) biotherapeutics. Characterizing mAbs’
glycoform profiles is crucial for optimizing therapeutic outcomes,
and intact antibody analysis provides key information about the glycoform
combinations present. While state-of-the-art RPLC-MS methods are commonly
used for intact mAb analysis, they lack the selectivity to resolve
glycoforms and, therefore, may not detect lower-abundance glycoforms.
In contrast, HILIC methods have demonstrated good resolving power
for middle-up mAb glycoform analysis. However, to date, no application
of HILIC has been described to characterize mAb glycoforms at the
intact level. This study describes the development of acrylamide monoliths
for HILIC-MS intact mAb glycoprofiling. We studied how the porogen
composition (octanol and DMSO ratio) in the polymerization mixture
affects the column permeability and separation performance. Our findings
indicated that increasing the DMSO content increased retention and
decreased the peak widths. The optimized HILIC-MS method was applied
to analyze five reference intact mAbs (IgG1 and IgG4). The method demonstrated glycoform selectivity at the intact
protein level, achieving baseline separations between single and double
Fc glycosylation (e.g., for trastuzumab, resolution (Rs) of 3.62 for
G0F vs G0F/G0F) and partial separations between glycoforms differing
by one glycan unit (e.g., for trastuzumab, Rs of 1.06 between G0F/G0F
and G0F/G1F). Compared to state-of-the-art RPLC-MS, acrylamide-monolith
HILIC-MS enabled the measurement of low-abundance glycoforms (e.g.,
single G0F and M5/M5). The selectivity and sensitivity (ng of sample
injection) of this method open opportunities for studies of IgG heterogeneity
in bioanalytical applications.