Protein pellet sample preparation for the analysis of N-glycans of therapeutic proteins.

Profiling of N-glycans of therapeutic proteins is a critical component in the control of biopharmaceutical products. N-Glycosylation, a common post-translational modification has been shown to impact the structure, function, stability, pharmacokinetics, and overall therapeutic efficacy of therapeutic proteins. Regulatory agencies around the world require detailed profiling of glycosylation to meet high product quality standards. The standard sample preparation for N-glycan analysis involves the enzymatic release of the glycan from the glycoprotein, fluorescent derivatization, and the time- and resource-consuming solid-phase extraction to remove excess fluorophore and reaction reagents before chromatographic glycan analysis. In this paper, we report a rapid sample preparation workflow that simplifies glycan purification after derivatization. The method uses the "protein pellet" approach to purify the fluorescently labeled glycan before analysis. The accuracy of the "protein pellet" method was established by comparing the results to the standard solid-phase extraction method that utilizes a polyamide stationary phase. The repeatability and linearity were also demonstrated.