Recombinant human insulin. V. Optimization of the reversed-phase high-performance liquid chromatographic separation.
The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.
Klyushnichenko
V E
VE
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Yakimov
S A
SA
Arutyunyan
A M
AM
Ivanov
A E
AE
Maltsev
K V
KV
Wulfson
A N
AN
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D011384:Proinsulin / Q000302:isolation & purification; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D007328:Insulin; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C018037:insulin, desamido (A21)-; D011384:Proinsulin