Quantitative High-Throughput and High-Sensitivity Glycoprofile Analysis of Therapeutic Glycoproteins Using HILIC-FLD...

Protein glycosylation is recognized as a Critical Quality Attribute for the biological and therapeutic activity of many recombinant proteins. Therefore, glycosylation should be monitored rigorously to ensure the desired quality, safety, and potency of monoclonal antibodies and other therapeutic glycoproteins. However, glycans are highly heterogeneous structures in proteins, and this poses a challenge for glycoprofile analysis. Hydrophilic interaction liquid chromatography with fluorescence detection has been recognized as a standard method for separation and quantification of released N-glycans after conventional 2-aminobenzamide labeling, but the sample preparation procedure is time-consuming with low sensitivity and poor reproducibility. Here we present a streamlined 96-well plate format platform for high-throughput and high-sensitivity glycan profiling. By taking advantage of rapid glycoprotein denaturation, enzymatic deglycosylation, highly sensitive fluorescent derivation, and on-matrix glycan cleanup, the reaction time has been shortened significantly to approximately 10 min. A detailed workflow including two-dimensional chromatography fractionation, exoglycosidase sequential digestion, mass spectroscopy glycan mass confirmation, and data interpretation is described. Potential pitfalls and precautions to be taken to ensure data accuracy are also discussed in this chapter.