Comprehensive n-glycosylation characterization of therapeutic antibodies by capillary electrophoresis and mass spectrometry

S3.3 Glycomics
Location (hall): 
Start/end time: 
Monday, July 1, 2019 - 17:15 to 17:30

Andras Guttman1, Marton Szigeti2, Laszlo Hajba2

1Sciex, San Diego, United States, 2Research Institute for Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary

Comprehensive characterization of the carbohydrate moiety of protein pharmaceuticals is of high importance, especially when glycosylation changes can impact the biological effect of the drug. Well over half of these new generation drugs are monoclonal antibodies, where the linked carbohydrates not only affect their physicochemical properties and stability, but also their receptor binding activity, circulating half-life and very importantly immunogenicity. State of the art bioanalytical techniques are crucial for N-glycosylation characterization of therapeutic antibodies for the biopharmaceutical industry, especially in clone selection, process development and lot release. N glycosylation analysis, however, represents a very challenging bioanalytical task as glycans are very complex groups of molecules. The lack of chromophore / fluorophore moieties and, in many instances, easily ionizable groups usually require derivatization of carbohydrates before their analysis by high performance bioanalytical techniques. Full structural elucidation of glycans1 (e.g., sequencing) utilizes consecutive enzymatic digestions by sugar and linkage specific exoglycosidases, followed by capillary electrophoresis analysis of the digests. 

This presentation will cover the state of the art of liquid phase separation methods for structural elucidation of protein glycosylation, mostly focusing on capillary electrophoresis and its combination with mass spectrometry (CE-MS and CESI-MS). Particular attention will be paid to the comparative interpretation of CE and LC results in case of the analysis of monoclonal antibody therapeutics (both innovative products and biosimilars).2 Comprehensive glycosylation characterization of this recently emerging class of very successful new generation drugs will be discussed in respect to quality by design with the main goal to demonstrate structural and functional equivalence of the products focusing on the analysis of core fucosylation, possible presence of alpha-gal residues and N-glycolylneuraminic acid content. Assisted by the emerging field of glycoinformatics, the identity of glycan structures can be readily assigned relative to their electrophoretic migration time based glucose unit (GU) values. With the aid of such database capillary electrophoresis offers a reliable, precise and traceable glycan structural elucidation method for biotherapeutics and other glycoproteins of biomedical importance.

  1. M. Szigeti and A. Guttman, Scientific Reports, 7 (2017) 11663.
  2. L. Hajba, A. Szekrenyes, B. Borza, A. Guttman, Drug Discovery Today, 23 (2018) 616.