Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Spectroscopy

Background:

• Glycans, attached oligosaccharides, significantly influence protein and lipid functions, including drug efficacy and immunogenicity.
• Traditional glycosylation analysis methods are often destructive, time-consuming, and sensitive to experimental conditions.
• Existing high-sensitivity IR spectroscopy methods had limited frequency scanning ranges, hindering comprehensive glycan analysis.

Purpose of the Study:

• To develop a rapid, nondestructive method for quantifying glycans and determining their monosaccharide composition using quantum cascade laser (QCL) infrared (IR) spectroscopy.
• To extend the frequency scanning range of QCL-IR spectroscopy for simultaneous analysis of glycan and protein vibrational fingerprints.
• To validate the method by comparing QCL-IR spectroscopy results with intact mass spectrometry for glycoproteins.

Main Methods:

• Implementation of a mirror-based double-pass acousto-optic modulator (AOM) in the solvent absorption compensation (SAC) and double-beam modulation (DBM) scheme to extend the IR frequency range.
• Acquisition of IR spectra for six glycoproteins and two nonglycosylated proteins in solution.
• Comparison of IR absorbance ratios (glycan ring band to protein amide band) with glycan-to-protein backbone mass ratios obtained from intact mass spectrometry.
• Application of multivariate analysis to determine monosaccharide composition and improve glycan-protein mass ratio prediction.

Main Results:

• The extended frequency range (970 to 1840 cm^-1) enabled simultaneous observation of monosaccharide ring bands and protein amide bands.
• A linear correlation was observed between the IR absorbance ratios and the glycan-to-protein backbone mass ratios for glycoproteins.
• Monosaccharide compositions derived from multivariate analysis aligned with existing databases, and improved the predictability of glycan-protein mass ratio.

Conclusions:

• The developed QCL-IR spectroscopy method is a rapid, sensitive, and nondestructive tool for quantifying glycosylation and monosaccharide composition.
• This technique can be a standard for monitoring batch-to-batch comparability in biopharmaceutical manufacturing.
• The method holds potential for analyzing glycosylation in new glycoproteins and other glycosylated biological systems.