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Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion
Deanna C Schuchmann1, Weiying Hou1, Joshua Creahan1
1Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 875 Chesterfield Parkway West, Chesterfield, MO 63017, United States.
Abstract:
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 (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 μg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0-108.5 %.
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