Development and validation of a label-free HPLC-CAD method to determine total sialic acid in therapeutic proteins

Sialylation plays a crucial role in influencing the pharmacokinetic, chemical, physical, and immunogenic properties of therapeutic proteins and is commonly tracked through total sialic acid analysis. In this study, we developed a novel label-free HPLC method to monitor total sialic acid levels in protein drug products. The method utilizes a replaceable, pre-column C18 cartridge that removes protein in-line, preventing protein interference with sialic acid and contamination of the analytical column and enabling direct sample injection. The optimized mixed-mode hydrophilic interaction-ion exchange liquid chromatography (HILIC-IEX) separation condition includes a volatile mobile phase for charged aerosol detection (CAD) of sialic acids. The method was validated to demonstrate satisfactory specificity, linearity (R > 0.999), precision (RSD between 0.4 % and 2.1 %), sensitivity (QL < 25 ng), and accuracy (recovery between 93 % and 102 %). It was successfully applied to determine total sialic acids in a model protein drug product, demonstrating promising potential as an accurate, robust, efficient, and cost-effective approach for sialylation evaluation.