Monitoring of recombinant human insulin production by narrow-bore reversed-phase high-performance liquid chromatography, highperformance capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry

An analytical scheme for monitoring recombinant human insulin (rhI) product ion is suggested. The scheme includes high-performance separation micro-tec hniques (narrow-bore RP-HPLC, HPCE) based on different separation mechanism s and matrix-assisted laser desorption ionisation rime-of-flight RIS, and a llows one to obtain unambiguous information about purity and primary struct ure of all intermediates of the rhI production. The use of this scheme at a ll production steps provided optimisation of certain technological paramete rs [conditions for a fusion protein (FP) refolding, temperature and duratio n of the FP cleavage with trypsin, conditions for carboxypeptidase B digest ion of di-Arg(B31-B32)-insulin] and achievement of a high purity of the end -product. The proposed scheme may be used for solving various problems in m onitoring production of other recombinant proteins. (C) 2001 Elsevier Scien ce B.V. All rights reserved.