Capillary gel electrophoresis for the analysis of heterodimer purity in a highly glycosylated, bispecific Fc fusion protein

Introduction Bispecific therapeutic fusion proteins require analytical methods capable of detecting and resolving process- and product-related impurities while remaining practical to implement and validate. Such methods are essential for supporting both process development and good manufacturing practice (GMP) -compliant testing. Methods A sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS) method was developed to separate structurally similar protein species based on differences in the number of N-linked glycosylation sites. The approach leverages glycosylation-dependent electrophoretic mobility to resolve heterodimeric and related molecular species. Results The CE-SDS method effectively separated bispecific protein variants that differed in glycosylation, enabling discrimination between the desired bispecific product and associated impurities. This was confirmed by comparing native and deglycosylated Fc-fusion protein. The method was successfully applied to inform process development decisions aimed at enriching the target bispecific protein and was demonstrated to be suitable for GMP release and stability testing by verifying ICH criteria for method validation. Discussion This analytical strategy provides a robust and broadly applicable means of separating heterodimeric and bispecific glycoproteins based on glycosylation differences. The method can be readily extended to other fusion proteins, provided sufficient glycosylation asymmetry exists between constituent polypeptide chains, making it a valuable tool for both development and manufacturing control.