Antibody Glycotyping: PNGase Released Glycomic Analysis of IgG Without Purification

Antibody glycosylation is a critical quality attribute (CQA) in disease research and quality control of therapeutic antibodies. This study presents a streamlined approach for the direct analysis of N-glycans released from IgG by PNGase F using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without any prior purification. By utilizing a 1,5-diaminonaphthalene (DAN)/2,5-dihydroxybenzoic acid (DHB) matrix, multiple glycan-derived peaks were selectively detected directly from the protein-rich digestion mixture, demonstrating the superior glycan selectivity of the DAN/DHB system. Furthermore, systematic dilution of the digest was found to significantly enhance the signal-to-noise ratio, enabling the detection of sialylated glycans while simultaneously suppressing the formation of undesirable DAN adducts. Comparative evaluation of three matrices—DAN/DHB, DHB/0.1% TFA, and DHB—across varying IgG concentrations revealed that the DAN/DHB matrix offers the highest sensitivity and robustness for unpurified samples. Although there are some limitations for quantitative analysis, especially for minor and sialylated glycoform, this novel, purification-free methodology provides a rapid and easily automatable platform for IgG glycan profiling.