Antibody Glycan Analysis

Antibody isotypes are glycosylated at highly conserved positions within their constant heavy chain regions. Glycosylation is an important factor for several effector functions such as complement activation and antibody activity. Variation in glycosylation can be attributed to several factors including the type of cell in which it is produced as well as processes including fermentation, purification, formulation or storage.
Mass spectrometry (MS) has gained widespread use in glycosylated protein analysis due to its high selectivity, sensitivity, and ability to analyze complex mixtures rapidly. The advent of soft ionization techniques such as ESI and MALDI has revolutionized the biomedical research field by providing new insights into the structural details on many levels for various important classes of biopharmaceuticals.

Glycan analysis of antibodies by MS is typically achieved by three main approaches.
  • Method 1 is conventional glycan analysis, using chemical or enzymatic treatment to liberate the glycans from the antibody, followed by derivatization prior to MS analysis.
  • Method 2 is intact antibody analysis which requires no sample pre-treatment before analysis.
  • Method 3 is analysis of glycopeptides derived by proteolytic digestion of the antibody.