Effect of the Monomer Cross-Linker Ratio on the Separation Selectivity of Monoclonal Antibody Subunits in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.


Filep C(1), Guttman A(1)(2).
Author information:
(1)Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, 98 Nagyerdei krt, Debrecen H-4032, Hungary.
(2)Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem u, Veszprem H-8200, Hungary.


With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.