More than two thirds of recombinant biopharmaceutical products on the market are glycoproteins, and nearly every stage of their manufacture is carefully monitored and regulated to ensure consistency in quality, safety, and effectiveness. Consequently, international regulatory agencies require use of state-of-the-art glycan analyses methods to help ensure the successful development and commercialization of effective and safe glycosylated biotherapeutics. To address this need, Waters offers a variety of robust, reproducible, complementary, information-rich analytical methods for this application.
HILIC for Large Biomolecules An alternative, high resolution separation technology
Hydrophillic Interaction Chromatography (HILIC) has been widely used to separate small polar compounds, yet its application to large biomolecules, other than released glycans, has been surprisingly limited. With the widepore Waters ACQUITY UPLC Glycoprotein BEH Amide 300A 1.7μm column and some new ideas on separation methods, it is now possible to use HILIC to glean previously unattainable information from intact proteins (with or without glycosylation), protein fragments, and complex, released glycans.
Glycoprotein Column Chemistry
The ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Column allows scientists to obtain novel yet complementary glycan related information of biotherapeutic proteins at the intact glycoprotein, glycoprotein subunit, or glycopeptide level. ◾Optimized wide-pore, HILIC stationary phase for resolving glycoforms from intact or digested glycoproteins ◾Generation of domain specific glycan linkages with or without MS ◾Elucidation of site specific glycan occupancy ◾High resolution glycopeptide mapping without limitations due to peptide/glycan size or composition ◾Improved resolution in separations of large, released N-glycans (EPO, Factor IX) ◾QC tested with Waters Glycoprotein Performance Test Standard
The figure to the left shows the separation of the Glycoprotein Performance Test Standard(RNase A + RNase B glycoforms) using an ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm, 2.1 x 150 mm Column. Fluorescence detection at Ex 280 nm and Em 320 nm and a column temperature of 45 ˚C.
Intact Glycoprotein Analyses
The ACQUITY UPLC Glycoprotein BEH Amide 300Å, 1.7 µm Columns separate individual intact protein glycoforms as well as deliver information about glycan occupancy. Using a high organic solvent concentration with TFA ion pairing and elevated temperature, one is able to successfully enhance the solubility of intact glycoproteins for this HILIC-based, gradient separation. ◾Measure glycan occupancy of an intact therapeutic mAb ◾Relative abundance of aglycosylated forms (-2 and -1 N-glycans) can be monitored by fluorescence ◾Widepore phase facilitates the development of previously unimagined separations