RESUMO
The optimization of the separation of recombinant human insulin (rhI) and recombinant human proinsulin (rhP) by free-solution capillary zone electrophoresis in uncoated fused-silica capillaries with ionic and zwitterionic buffers was carried out. The relationship between the selectivity and pH of the buffer was established. The effects of pH and ionic strength of the buffer on protein adsorption on the capillary walls and Taylor diffusion was investigated. The separation of rhI and rhP with an efficiency of 200,000 theoretical plates was achieved using 20 mM Na2HPO4-NaOH buffer (pH 11.2). The proposed method allows the simultaneously determination of rhP and some rhI degradation products (which are also included in pharmacopoeias) in pharmacopoeially limited amounts in rhI.
Assuntos
Proinsulina/isolamento & purificação , Soluções Tampão , Eletroforese , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Proinsulina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
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.
Assuntos
Insulina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Insulina/análogos & derivados , Insulina/química , Focalização Isoelétrica , Proinsulina/isolamento & purificação , Compostos de Amônio Quaternário/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e., size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography), to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.
