HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF CARBOHYDRATE MASS COMPOSITION IN GLYCOPROTEINS

A method has been developed which characterizes the carbohydrate portion of glycoproteins by its mass composition (mass carbohydrate per mass protein). This size-exclusion HPLC method uses both UV and refractive index detectors in series and requires only a single injection of intact glycoprotein. It has been shown that for calibration purposes any carbohydrate or protein which chromatographs well on the sizing system can be substituted for the carbohydrate or protein moiety of the glycoprotein of interest. The accuracy of this method was demonstrated for RNase B and fetuin. Mass compositions of various monoclonal IgMs were determined with an average intraday precision of approximately 6%; the limit of lot-to-lot resolution. Thus, the primary utility of this simple HPLC method is to identify substantial changes in the carbohydrate moiety among various glycoprotein preparations.