ABSTRACT
Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.
Subject(s)
Drug Industry/methods , Escherichia coli/metabolism , Proinsulin/isolation & purification , Recombinant Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Humans , Inclusion Bodies/metabolism , Proinsulin/biosynthesis , Protein Denaturation , Protein Folding , Protein Renaturation , Recombinant Proteins/biosynthesis , Solubility , Sulfhydryl Compounds/metabolismABSTRACT
A method for monitoring the manufacture of genetically engineered human insulin by HPLC was developed. The method was validated by the estimation of its linearity, correctness, accuracy, specificity, and stability; the limits of detection and quantitative assessment were also determined. It was proven that HPLC analysis enables reliable and reproducible results to be obtained and can be used for monitoring insulin manufacture.
Subject(s)
Drug Contamination/prevention & control , Insulin/chemistry , Genetic Engineering , Humans , Insulin/genetics , Quality Control , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Reference StandardsABSTRACT
Studies of replacement therapy of diabetes mellitus resulted in introduction of series of forms of insulin and new insulin analogs which exhibit better control of blood glucose level. The present paper deals with basic tendencies in this field.
