Analyzing mAb Glycosylation at Multiple Levels Using the 6545XT AdvanceBio LC/Q-TOF

Monoclonal antibodies (mAbs) and their derivatives represent a very important class of biopharmaceutical molecules with a wide range of applications. With the dramatic increase in approved mAb products and mAb product sales over the recent years, there is increased need for comprehensive analytical characterization capabilities. mAbs are heterogeneous molecules by nature which are composed of various types of sequences, modifications and structural variants. Protein glycosylation is one of the major post-translational modifications (PTMs) of a mAb that plays an important role in many biological processes. The distribution and composition of the glycans bound to the mAb molecules can have an effect on therapeutic efficacy and immunogenicity, consistent glycosylation-associated quality control of therapeutic monoclonal antibodies (mAbs) has become a high priority in pharmaceutical bioprocessing.

 

There are typically four levels of LC/MS workflows for Glycan/Glycoforms in characterization: level 1 and level 2 focus on the analysis of glycoforms on the intact and reduced mAb molecules. Level 3 is the analysis of glycopeptide generated from proteolytical digestion of mAbs, commonly part of the peptide sequence mapping workflow. Level 4 is the characterization of glycans that have been released by enzymatic cleavage or other mechanism.

 

We present here the complete N-glycan quantitation workflow solutions using liquid chromatography (LC) with fluorescence detection (FLD) or mass spectrometry (MS) for four levels of biotherapeutic analysis: intact mass, mAb subunits, glycopeptides, and released glycan. These solutions dramatically improve not only productivity by allowing convenient sample preparation and streamlined data acquisition, but also accuracy in data analysis.