In-Depth Glycan Characterization of Therapeutic Glycoproteins by Stepwise PGC SPE and LC-MS/MS

Glycosylation of biologics, an important factor in pharmacological functions such as efficacy, safety, and biological activity, is easily affected by subtle changes in the cellular environment. Therefore, comprehensive and in-depth glycan characterization of therapeutic glycoproteins should be performed to ensure product quality and process consistency, but it is analytically challenging due to glycan microheterogeneity occurring in the glycan biosynthesis pathway. LC-based chromatographic separation combined with mass spectrometry (MS) has been widely used as a prominent tool for the qualitative and quantitative analysis of glycosylation of therapeutic glycoproteins. However, prior to LC/MS analysis, glycans are selectively captured and fractionated by solid-phase extraction (SPE) utilizing physicochemical characteristics for comprehensive characterization of a wide range of glycan heterogeneity on glycoengineered therapeutic proteins. In particular, porous graphitized carbon (PGC) SPE has been employed as a useful technique for the fractionation of native glycans having different sizes and polarities. Here, we describe a systematic method for comprehensive glycan characterization of therapeutic proteins using stepwise PGC SPE and LC/MS.