凝胶排阻色谱-多角度激光散射法测定破伤风类毒素相对分子质量分布

doi:10.19894/j.issn.1000-0518.200374

摘要/Abstract

摘要： 为更好地表征精制破伤风类毒素(精制TT)相对分子质量分布的情况,利用凝胶排阻色谱与多角度激光散射仪联用法(SEC-MALS法)对精制破伤风类毒素分子质量及其分布进行了初步探索。通过优化实验参数,确定了最佳实验条件:流动相为0.2 mol/L磷酸盐缓冲液-1%异丙醇(pH=7.0)(PBS),样品浓度为2.0 mg/mL,柱温为25 ℃,流速为0.5 mL/min。与聚丙烯酰胺凝胶电泳法相比,SEC-MALS法能够提供准确的精制TT单体分子质量测量值。此外,SEC-MALS法能够得到质均相对分子质量M_w、数均相对分子质量M_n以及二者的比值——多分散性M_w/M_n等重要物理参数,可以更加准确地反映精制TT溶液中单聚体、二聚体、多聚体分布状态,可以量化分子大小批间差异情况,有利于评价精制TT生产过程中的工艺调整或变化对蛋白单体收率的影响,当作为多糖蛋白结合疫苗常用的载体蛋白时,有利于以准确可量化的质量参数参与结合物的关键工艺控制。

关键词: 破伤风类毒素, 相对分子质量分布, 凝胶排阻色谱与多角度激光散射仪联用法

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

中图分类号:

O657

马庆华, 朱向国, 郑佳, 刘刚, 杜琳. 凝胶排阻色谱-多角度激光散射法测定破伤风类毒素相对分子质量分布[J]. 应用化学, 2021, 38(6): 739-742.

MA Qing-Hua, ZHU Xiang-Guo, ZHENG Jia, LIU Gang, DU Lin. Relative Molecular Mass Distribution of Tetanus Toxoid Determined by Gel Exclusion Chromatography and Multi Angle Laser Scattering[J]. Chinese Journal of Applied Chemistry, 2021, 38(6): 739-742.

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