Improving Standard N-Glycan Sample Preparation with Manual Automation using Microchromatography to Improve Efficiency, Accuracy, and Reproducibility

The search for more efficient tools that streamline the sample preparation process for characterization of N-Glycans continues to be at the forefront in the field of glycomics. The goal is to more effectively streamline the sample preparation and data analysis to allow for a deeper understanding of the multiple structures and functions that result from glycosylation. Adding sugars to proteins in the process of forming glycoproteins can be complex. Abnormal glycosylation, congenital disorders of glycosylation (CDG), can occur and be linked to a number of diseases. Better understanding of this structure and function relationship can assist with understanding how these diseases occur and thus developing disease biomarkers. Monitoring bioprocessing of monoclonal antibodies (Mabs) is another area where product glycosylation is important. Eliminating manufacturing variability from batch-to-batch is critical, but the traditional laboratory sample preparation approach can often take too long. Traditionally, sample preparation for N-Glycans takes more than three days and often involves specific laboratory technique, not including the time for sample analysis. The transition to manual automation tools combined with microchromatography reduces sample preparation to less than 2.5 hours, while providing the added confidence that N-Glycan sample preparation and the sample data are reliable. Human Immunoglobulin G (hIgG) was mixed with bovine fetuin prior to microchromatography purification and subequent sample preparation, labeleing and analysis using HPLC with fluorescence detection. Results indicate that manual automation tools show improved accuracy and reproducibility between samples while allowing for more effective and more efficient characterization of N-Glycans to be performed.