A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation

Abstract Biotherapeutics, such as mAbs and fusion proteins, are a major and rapidly growing class of pharmaceuticals. Majority of the biopharmaceuticals are glycoproteins, wherein about 1 to 30% of their molecular weight (MW) are contributed by the glycans. Determination of MW of heavily glycosylated proteins, such as Fc‐fusion proteins, is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation influences the expected size of the glycoprotein, which leads to disproportionate MW estimation, in size‐dependent methods. Hence, in this study, we have demonstrated the advantages and limitations of four widely used MW estimation techniques for three proteins having varying levels of glycosylation. It was proven that glycosylation had least impact on MW determination by SEC‐MALS and SV‐AUC. However, MW estimation by LC‐MS and SDS‐PAGE was extensively hampered by the degree of glycosylation. It is, thus, essential to consider the structural characteristics of proteins while selecting a technique for determining their MW.