Abstract: In order to better characterize the relative molecular mass distribution of purified tetanus toxoid (purified TT), the relative molecular mass and distribution of purified TT were preliminarily explored by size exclusion chromatography and multi angle laser scattering (SEC-MALS). By optimizing the experimental parameters, the optimal conditions were determined as follows: the mobile phase is 0.2 mol/L phosphate buffer solution-1% isopropanol (pH=7.0), the sample concentration is 2.0 mg/mL, the column temperature is 25 ℃, and the flow rate is 0.5 mL/min. Compared with polyacrylamide gel electrophoresis, SEC-MALLS method can provide accurate measurement of relative molecular of TT mass. SEC-MALS method can obtain important physical parameters such as mass-average molecular mass (M_w), number-average relative molecular mass (M_n) and their ratio (M_w/M_n), which can more accurately reflect the distribution state of monomers, dimers and polymers in refined TT solution, and quantify the difference of molecular size between batches, which is conducive to the evaluation of industrial performance in refined TT production process. The influence of technology adjustment or change on the yield of protein monomer is beneficial to the carrier protein commonly used in polysaccharide protein conjugate vaccine, and to participate in the key technology of conjugate with accurate and quantifiable quality parameters.

Key words: Tetanus Toxoid, Relative molecular mass distribution, Size exclusion chromatography and multi angle laser scatterometer