Monitoring of recombinant human insulin production by narrow-bore reversed-phase high-performance liquid chromatography, high-performance capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry.
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.
Sergeev
N V
NV
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow. sergeev@ibch.ru
Gloukhova
N S
NS
Nazimov
I V
IV
Gulyaev
V A
VA
Shvets
S V
SV
Donetsky
I A
IA
Miroshnikov
A I
AI
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000032:analysis* / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D007328:Insulin; D011993:Recombinant Fusion Proteins; D014357:Trypsin