RESUMO
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.
Assuntos
Indústria Farmacêutica/métodos , Escherichia coli/metabolismo , Proinsulina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Corpos de Inclusão/metabolismo , Proinsulina/biossíntese , Desnaturação Proteica , Dobramento de Proteína , Renaturação Proteica , Proteínas Recombinantes/biossíntese , Solubilidade , Compostos de Sulfidrila/metabolismoRESUMO
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.
Assuntos
Contaminação de Medicamentos/prevenção & controle , Insulina/química , Engenharia Genética , Humanos , Insulina/genética , Controle de Qualidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Padrões de ReferênciaRESUMO
Size-exclusion chromatography based on silica-diol sorbent was employed to analyze the recombinant proinsulin fusion protein obtained during the process of refolding and the following ion-exchange purification. The assay was qualified as a control method estimating its accuracy, precision, linearity, limit of detection, limits of quantitation, specificity, and robustness. The results show the reliability for the intended use.
Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Proinsulina/análise , Proteínas Recombinantes de Fusão/análise , Eletroforese em Gel de Poliacrilamida , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
One of the main stages of human recombinant insulin (HRI) production is the hormone purification by means of reversed phase high-performance liquid chromatography (RP HPLC). The optimization of this stage determines the increase of the total manufacturing yield. Therefore, the cost of the sorbent used in HPLC influences the cost of the manufacturing product, i.e. HRI substance. However, resolution between HRI and its admixtures decreases with time. The reason for this is the sorbent contamination with strongly adsorbed impurities (SAI) which are accumulated during elution. In the following research several methods for sorbent protection are studied. The opinion that SAI are mainly high-molecular weight compounds was examined using gel filtration. Different sorbent types were tested for the use in guard column. The results obtained were applied and improved at preparative level.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Insulina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Dióxido de Silício/química , Cromatografia em Gel , Humanos , Insulina/genética , Insulina/metabolismo , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.
Assuntos
Hipoglicemiantes/isolamento & purificação , Insulina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Insulina/análogos & derivadosRESUMO
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.