Routine o-glycan characterization in nutritional supplements — a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation

Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pI of the different CGMP glycoforms was evaluated with pI internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency.