Unambiguous identification and quantification of galactose-α-1,3-galactose as a critical quality attribute with mass spectrometry and exoglycosidases.

Alpha-galactosylation, galactose-α-1,3-galactose (α-Gal), is always regarded as a critical quality attribute due to its potential to provoke immunogenic responses in patients. Consequently, monitoring alpha-galactosylation in therapeutic proteins is essential, but current analytical techniques fall short in terms of identification sensitivity and quantification accuracy. Specifically, the released glycan assay by hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry, the gold standard for glycan separation/identification, faces challenges due to ambiguities with isomeric glycan structures. To address these challenges, we developed a comprehensive analytical method that enhances both the identification sensitivity and quantification accuracy for α-Gal. We developed an integrated workflow that combines an advanced mass spectrometry technique - parallel reaction monitoring triple-stage mass spectrometry - with exoglycosidase sample treatment. This approach generates structurally specific signature ions, enhances identification sensitivity, enables the separation of α-Gal from its isomers, and improves quantification accuracy. By employing this more sensitive analytical approach without ambiguity in assignment for common glycan structures found in monoclonal antibodies, the safety of therapeutic proteins can be better assured, effectively minimizing the risk of α-Gal-induced immunogenicity.