Agilent Biopharma Workflow Solutions Virtual Symposium: Advances in Glycan and Sialic Acid Analysis

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.

Monoclonal antibodies are heterogeneous molecules that include glycosylation variants, carboxyl truncation, and deamidation that require extensive analytical characterization. These molecules are one of the fastest growing classes in the biopharmaceutical industry. This presentation will discuss a complete workflow for intact monoclonal antibody analysis with the use of the 6545XT AdvanceBio LC/Q-TOF platform and automatic data processing with MassHunter BioConfirm software.

This talk will explore HILIC chromatography methods for the analysis of released N-glycans labeled with InstantPC or 2-AB dyes. Best practices for robust methods will be introduced. Method development strategies for resolving critical pairs will be discussed including gradient and mobile phase optimization.

N-Glycans on biotherapeutics can affect immunogenicity, pharmacokinetics and pharmacodynamics, making characterization of N-glycans essential in the development process. Here we present rapid N-glycan sample preparation and analysis workflows for biotherapeutics, including released N-glycan labeling with InstantPC and 2-AB for characterization by liquid chromatography (LC) with fluorescence (FLD) and mass spec (MS) detection. In addition, we will discuss approaches to identify different sialylated N-glycan species as well as quantitating the sialic acid content of biotherapeutic glycoproteins using plate-based and LC/FLD/MS approaches.