Cellulose functionalized magnetic beads for high throughput glycosylation analysis in biotherapeutic modalities

The increasing demand for biotherapeutics has necessitated the evaluation of their critical quality attributes, one of which is glycosylation, an essential post-translational modification found on many biological molecules. In particular, the purification of N-glycans after their release from the proteins and derivatization is important in ensuring the removal of the deglycosylated protein, excess labelling reagents and salts for subsequent analysis. However, current methods of N-glycans purification are either expensive, laborious, time-consuming or not catered for high throughput analysis. To overcome these constraints, we developed a high throughput purification method for fluorescent derivatized N-glycans using cellulose functionalized magnetic beads (CMBs). We compared the method with two current purification methods, hydrophilic interaction chromatography solid phase extraction (HILIC-SPE) and gel filtration using human serum IgG (hsIgG) and bovine fetuin and assessed their reproducibility. The CMB method yielded highly similar glycan profiles to the two methods with very good precision. We then assessed the compatibility of the method to purify N-glycans derivatized with different fluorescent labels ( Rapi Fluor MS, 2-aminobenzamide and procainamide). We also applied the methodology to analyse N-glycans in the biotherapeutic protein, recombinant alpha-1-antitrypsin (rAAT) which was modified with higher sialylation content. Importantly, the method successfully captured the differences in the glycan profiles between the modified and unmodified rAAT. Finally, we automated the method together with the digestion and labelling protocol onto a robotic liquid handler for high throughput glycosylation analysis. The versatility of the CMB method, together with its affordability and robustness, may provide an alternative workflow for the high throughput analysis of glycans in biotherapeutic modalities.