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

Immunoglobulin
G (IgG) glycosylation is a key post-translational
modification in regulating IgG function. It is therefore a prominent
target for biomarker discovery and a critical quality attribute of
antibody-based biopharmaceuticals. A common approach for IgG glycosylation
analysis is the measurement of tryptic glycopeptides. Glycosylation
stability during sample processing is a key prerequisite for an accurate
and robust analysis yet has hitherto hardly been studied. Especially,
acid hydrolysis of sialic acids may be a source for instability. Therefore,
we investigated acid denaturation, centrifugal vacuum concentration,
and glycopeptide storage regarding changes in the IgG glycosylation
profile. Intravenous IgG was analyzed employing imaginable deviations
from a reference method and stress conditions. All glycosylation features
sialylation, galactosylation, bisection, and fucosylationremained
unchanged for most conditions. Only with prolonged exposure to acidic
conditions at 37 °C, sialylation decreased significantly and
subtle changes occurred for galactosylation. Consequently, provided
that long or intense heating in acidic solutions is avoided, sample
preparation for bottom-up glycoproteomics does not introduce conceivable
biases.