Estudo do equilibrio liquido-liquido, da partição de insulina e da pre-purificação da proteina de fusão precursora da insulina humana em sistemas aquosos bifasicos do tipo PEG/Sal / Study of liquid-liquid equilibrium, the partition of insulin and pre-purification of the fusion protein precursor of human insulin in aqueous biphasic systems of the type PEG / salt

AUTOR(ES)
DATA DE PUBLICAÇÃO

2003

RESUMO

In this work the primary purification of a fusion protein precursor of human insulin and produced by recombinant E. coli has been studied using aqueous two-phase systems. The fusion protein was provided by the pharmaceutical company Biobrás S.A (Montes Claros/Brazil) and was dissolved in 8M area. Partitioning essays of fusion protein were investigated in the poly(ethylene glycol) (PEG)/potassium phosphate and poly(ethylene glycol)/sodium citrate aqueous two-phase systems at 25°C. Two 32 experimental design were done to evaluate the effects of PEG molecular weight and the polymer/salt concentration ratio on the purification factor and on the recovery of the fusion protein in the top phase. The studied levels were 1500, 3350 and 8000 for PEG molecular weight and 1,6; 1,636 and 1,67 for the polymerlsaIt concentration ratio. The partitioning experiments were performed at pH 9 and 12. An analytical method for the fusion protein was developed using Immobilized Metal Affinity Chromatography and the Bradford method. Solids on the interface top were observed in all cases examined The fusion protein partitioned between the PEG-rich phase and the solids on interface. Contaminating proteins were eliminated to some extent, which resulted in an almost 2-fold fusion protein purification with good recoveries. Phase diagrams of the system poly(ethylene glycol)1 sodium citrate/water were determined for three different molecular weights of PEG ( 600, 1500, 3000) at 25°C. At least four tie-lines of each system were measured. The experimental results were correlated by applying a model for the excess Gibbs energy. This model is the Virial equation used by Pitzer for salt solutions, but concentrations are expressed by surface fraction. It is called Virial Equation with Relative Surface fractions (VERS). All phase equilibrium calculations were performed using this model and minimizing the Gibbs energy of the feed under the constraint that both phases are electroneutral. Agreement between the experimental and calculated results within the experimental uncertainty was obtained. The average percents of deviation between experimental and calculated values using VERS model for all components were 1,16%, 1,05% and 1,19% for the systems PEG6001 Na3Cit/H2O, PEGl500/ Na3Cit/H2O PEG30001 Na3Cít/ H2O respectively. Human and porcine insulin partitioning was investigated in the system PEG/sodium citrate/water for three pHs (4,5; 7 and 9,5) at 25°C. Human and porcine insulin showed great affinity for the PEG-rich phase and the partition coefficients were higher than 10. The human insulin partition coefficient is practically independent of the PEG molecular mass. However, there is a tendency for the porcine insulin partition coefficient to increase with the PEG molecular mass at pH 7 and 9,5, attaining values- above 50. The porcine insulin partition coefficient was predicted in the system PEG/sodium citrate/water at 25°C using VERS model and good results were obtained for the system at pH 7

ASSUNTO(S)

polietileno liquid-liquid equilibrium modeling equilibrio liquido-liquido modelagem insulin polyethylene insulina

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