Dr. Rodney Banwort walks through how Emory Pharma analyzes glycans on monoclonal antibodies. The process uses PNGase F to release glycans, then labels them with 2-AB or procainamide and runs them through UPLC-MS. Glycan patterns matter because they affect drug activity, immunogenicity, and whether biosimilars are truly equivalent.
Chapters
Hello. Today you will learn about glycan analysis, why it's important and how it's done. My name is Dr. Rodney Banwort and I am a principal scientist here at Emory Pharma. Prior to Emory, I did LC Ms. Analysis of biologics for 20 years at places like Alcon, Novartis and Pfizer. A simple glycosylation on a monoclonal antibody is illustrated in the figure. Mabs are glycosylated at a specific arginine residue on the heavy chain. There are two heavy chains in the MAB and both are glycosylated. The end release and labeled. Glycan determination is a detailed procedure that is useful throughout the whole product development cycle of biopharmaceuticals. The glycan profile of therapeutic proteins and mabs can affect the protein's activity and function and its immunogenicity. The profile will change depending upon the health of the expression cell and is therefore monitored for lot to lot variability. The glycan profile is also important for biosimilarity. The profile is routinely elucidated in comparability and characterization studies as well. Indeed, the consistency of drug production is supported by the consistency of the glycan profile. Even though this is not the most sensitive technique. Glycan information is often obtained from LC Ms. Mass analyses. In the case of the NISMAB, the information on the glycosylations G0F, G1F and G2F is demonstrated. Subunit and peptide mapping analyses also revealed glycosylation information. Some of the major glycans are illustrated on this slide. They contain N acetylglucosamine, mannose, fucose, galactose and sialic acid. This slide only illustrates a few of the glycans that are possible. In this slide I am presenting the protocol we use at Emory Pharma to release and label the glycans for the mabs and proteins we study Study in the workflow, the protein or MAB sample is first treated with the enzyme PNGSF at 37 degrees for two hours. The enzyme cleaves the glycans from the asparagine residue in the protein and creates a mixture of both the protein and the released oligosaccharides. The next step isolates the saccharides from the protein mixture using solid phase extraction. I prefer to use the Hillock phase in this step. After elution, the glycans are derived by lyophilization and are derivatized with compounds such as 2ab or procainamide. Prior to LCMS analysis. On this slide we have illustrated the labeling reaction of the saccharide. Chromatographic parameters that I prefer are presented here. The two AB glycan libraries are commercially available and are injected into the same UPLC Ms. System as the samples. Because the vendor has identified the main peaks in the library, the glycan assignments in the sample are simplified via retention time comparison. Fluorescence detection is very sensitive and is recommended for glycan analysis. In this slide I have presented the LC Ms. Chromatograms of the 2ab labeled Glycan Library on top and and the results from our case study on the NISMAB on the bottom. In the NIST MAB we identified over 25 glycans. In conclusion, the N release and glycan labeling analysis is the most sensitive and accurate method for characterizing the glycan profile of proteins and maps. Since the glycan profile can affect the activity and function of the map, its importance cannot be neglected. This analysis is useful from early development up through to commercialization and is included in comparability and characterization studies. While our work utilizes a modern high field orbitrap mass spectrometer, the method can be performed with a fluorescence detector, significantly decreasing the cost of the analysis but not the sensitivity. Thank you and please contact us@emorypharma.com with any further questions and or inquiries. Sa.
