Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion chromatography-reverse phase liquid chromatography with UV detection

•Reverse phase column for SEC and RP separation of hydrophilic and hydrophobic macro molecules.•Use of phosphate buffer for sensitive detection of polysaccharide at UV 200 nm.•SEC-RPLC/UV method is simple and generic for analysis of free polysaccharides. The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (