Liquid Chromatography: Chapter 8. Glycans and Monosaccharides

Glycosylation is the most common form of posttranslational modification found on proteins and is critical for a wide range of functions. Many biopharmaceuticals are glycosylated, so it is imperative to identify the glycosylation attributes that are critical for drug safety and efficacy and to monitor these throughout the drug’s life cycle. Full structural analysis of glycans involves identifying both the sequence and the linkages of the monosaccharides in each glycan, as small changes can affect the activity or immunogenicity of the glycoprotein. A number of high-performance liquid-chromatography (HPLC) modes are available, including amide-bonded hydrophilic interaction (HILIC); reversed phase (RP); weak anion exchange (WAX), and porous graphitized carbon (PGC). Fluorescent labeling allows for quantitative detection at femtomole levels for glycan and monosaccharide analysis. These HPLC methods are easy to use, robust, and provide high-resolution, reproducible, quantitative data. The resolution and speed of analysis continue to improve with the availability of columns with sub-2-micron particles.