Sergeev N V, Gloukhova N S, Nazimov I V, Gulyaev V A, Shvets S V, Donetsky I A, Miroshnikov A I
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
J Chromatogr A. 2001 Jan 12;907(1-2):131-44. doi: 10.1016/s0021-9673(00)01016-5.
An analytical scheme for monitoring recombinant human insulin (rhI) production is suggested. The scheme includes high-performance separation micro-techniques (narrow-bore RP-HPLC, HPCE) based on different separation mechanisms and matrix-assisted laser desorption ionisation time-of-flight MS, and allows one to obtain unambiguous information about purity and primary structure of all intermediates of the rhI production. The use of this scheme at all production steps provided optimisation of certain technological parameters [conditions for a fusion protein (FP) refolding, temperature and duration of the FP cleavage with trypsin, conditions for carboxypeptidase B digestion of di-ArgB31-B32-insulin] and achievement of a high purity of the end-product. The proposed scheme may be used for solving various problems in monitoring production of other recombinant proteins.
提出了一种监测重组人胰岛素（rhI）生产的分析方案。该方案包括基于不同分离机制的高效分离微技术（窄孔反相高效液相色谱法、高效毛细管电泳法）和基质辅助激光解吸电离飞行时间质谱，能够获取有关rhI生产所有中间体纯度和一级结构的明确信息。在所有生产步骤中使用该方案可优化某些工艺参数[融合蛋白（FP）复性条件、用胰蛋白酶切割FP的温度和持续时间、二精氨酸B31 - B32 -胰岛素的羧肽酶B消化条件]并实现最终产品的高纯度。所提出的方案可用于解决监测其他重组蛋白生产中的各种问题。
