HILIC Glycopeptide Mapping with a Wide-Pore Amide Stationary Phase

Importance of the Topic

Glycopeptide mapping plays a critical role in characterizing protein modifications such as glycosylation in biopharmaceuticals
Conventional reversed phase methods often struggle to resolve hydrophilic glycopeptides with high selectivity
High resolution hydrophilic interaction chromatography with a wide pore amide phase offers complementary and orthogonal separations to unlock detailed glycoform information

Objectives and Study Overview

This study demonstrates the use of a wide pore Glycoprotein BEH Amide 300 Angstrom 1.7 micron column for high resolution HILIC glycopeptide separations
Comparisons are made with reversed phase peptide mapping and two commercial amide columns to benchmark performance
Applications include mapping of trastuzumab and cetuximab glycopeptides and lot to lot profiling, using UV, fluorescence and MS detection

Methodology and Instrumentation

Sample preparation involved non reduced Lys C digests for trastuzumab and an IgG1K reference, and a combined Lys C tryptic digest for cetuximab
HILIC separations were performed on an ACQUITY UPLC H Class Bio System with Glycoprotein BEH Amide 300 Angstrom 1.7 micron column
Mobile phases included 0.1 percent TFA in water and acetonitrile, with sample dilution in acetonitrile dimethyl sulfoxide mixtures
Detection modes comprised UV absorbance, fluorescence at Ex 280 Em 320 nanometer and ESI MS on a SYNAPT G2 S HDMS system with MassLynx and UNIFI software

Main Results and Discussion

Reversed phase mapping of trastuzumab Lys C digest resolved peptides broadly but compressed glycopeptides into a narrow window
HILIC on the wide pore amide column class separated non glycosylated peptides from glycopeptides and significantly improved glycoform selectivity
MS enabled identification of biantennary, sialylated and minor glycoforms on the 29 residue Fc peptide K16, matching known released glycan profiles
Lot to lot HILIC UV separations of K16 from two trastuzumab batches revealed differences in terminal galactosylation consistent with released glycan studies
Cetuximab HILIC glycopeptide mapping detected over 25 high abundance glycoforms across Fc peptide T22 and Fab peptide T8, including immunogenic epitopes
Fluorescence based peak capacity measurements showed the Glycoprotein BEH Amide column achieved 72.8 versus 37.1 and 51.9 for two competitor amide phases

Benefits and Practical Applications

Wide pore amide HILIC provides orthogonal selectivity to reversed phase enabling enhanced characterization of hydrophilic glycopeptides
MS compatibility allows detailed glycoform assignment and detection of minor species without extensive sample cleanup
Optical detection options support high throughput quality control for lot to lot comparability and monitoring glycosylation profiles

Future Trends and Possibilities

Integration of HILIC glycopeptide mapping with subunit level analyses will further enhance site specific glycosylation insights
Advances in ion mobility and high resolution MS promise deeper profiling of low abundance glycoforms and diagnostic oligosaccharides
Automation of sample preparation and column conditioning may support expanded use in regulated environments and biosimilar development

Conclusions

Wide pore Glycoprotein BEH Amide HILIC offers exceptional resolving power for glycopeptide mapping complementing reversed phase methods
Enhanced peak capacity and MS compatibility provide rich data on glycoform microheterogeneity across multiple domains of monoclonal antibodies
This approach supports detailed structural characterization and robust quality control of glycoprotein therapeutics

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