Immunoglobulin G Glycoprofiles are Unaffected by Common Bottom-Up Sample Processing.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Immunoglobulin G (IgG) glycosylation is a critical post-translational modification impacting IgG function.
• It serves as a biomarker and a critical quality attribute for antibody therapeutics.
• Accurate IgG glycosylation analysis relies on stable glycopeptide profiles during sample processing.

Purpose of the Study:

• To investigate the stability of IgG glycosylation during common sample preparation steps.
• To identify potential sources of instability, particularly acid hydrolysis of sialic acids.
• To assess the impact of acid denaturation, vacuum concentration, and storage on IgG glycosylation.

Main Methods:

• Analysis of intravenous IgG (IVIg) under various stress conditions.
• Evaluation of deviations from a reference method for IgG glycosylation analysis.
• Measurement of key glycosylation features: sialylation, galactosylation, bisection, and fucosylation.

Main Results:

• Most glycosylation features (sialylation, galactosylation, bisection, fucosylation) remained stable across tested conditions.
• Prolonged exposure to acidic conditions at 37°C led to a significant decrease in sialylation.
• Subtle changes in galactosylation were observed under prolonged acidic conditions.

Conclusions:

• Standard sample preparation methods for bottom-up glycoproteomics are generally suitable for IgG analysis.
• Avoidance of prolonged or intense heating in acidic solutions is recommended to preserve sialylation.
• The study provides crucial insights into maintaining IgG glycosylation integrity during analysis.