INTRODUCTION

Reliable analysis of the molar masses of biomacromolecules is essential for control of the manufacturing process and product release. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) is a technique for characterizing macromolecules by providing the molar mass and polydispersity for all molecules in solution and does not require molecular weight calibration standards. In chromatography mode, when a light scattering instrument is coupled to a fractionation technique, as well as concentration detectors [e.g., UV, refractive index (RI)], absolute molar mass and size distributions can be directly measured. A laser beam is directed at eluting molecules, and the resulting scattered light is directly proportional to the product of the molar mass and the concentration. This chapter describes a validated SEC-MALS method to determine the molar mass of polysaccharides used for the manufacture of human vaccines. Note that the procedure presented here may be adapted to activated and/or derivatized polysaccharides and monovalent protein-polysaccharide conjugates with some minor modifications depending on sample properties (e.g., functional groups, or solution composition differences between samples and mobile phases).