Study of the isoelectric precipitation of porcine insulin in aqueous solutions with carbon dioxide

Author(s)

Alexandre Keiji Tashima

• Document type: Doctoral Thesis
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química
• Defense date: 04-10-2007

Examining board members

Carlos Eduardo Calmanovici; Paulo de Tarso Vieira e Rosa; Martin Aznar; Theo Guenter Kieckbusch

• Advisor: Everson Alves Miranda

Abstract

Precipitation is a technique frequently employed in downstream process for the recovery of proteins from aqueous solutions. One example of such technique is the isoelectric precipitation, which consists of the adjustment of solution pH to the value where the protein has zero net charge, the isoelectric point, pI. Commonly, mineral acids and bases are used for pH adjustment and to induce protein precipitation. However, in order to obtain high purity proteins and avoid environmental pollution, an additional process is needed to neutralize and remove residual salts. It is also necessary to take into account the fact that local pH extremes can cause denaturation of the targeted protein. Recent works have pointed the use of volatile electrolytes as promising alternatives to the precipitating agents conventionally utilized in protein recovery. Volatile electrolytes are obtained by dissolution of gases as carbon dioxide in aqueous solutions. In this process, the volatile electrolyte dissociates into ions, whose concentrations depend on system pressure and temperature. Biomolecules of pharmaceutical interest as insulin, for instance, may be potentially recovered by isoelectric precipitation process with carbon dioxide. Thus, in this project an experimental and theoretical study of the isoelectric precipitation of porcine insulin with carbon dioxide was conducted, in order to evaluate the influence of parameters such as temperature, pressure and concentrations of electrolytes and protein over the precipitation process. Kinetics of pH variation due to acidification of the insulin solutions was determined, as well as protein precipitation kinetics. The use of sodium bicarbonate as buffer agent with carbonic acid produced in solution allowed the equilibrium studies be conducted without the need of any additional component to control system pH. Solubility data of porcine insulin were obtained in the temperature range of 5 to 25oC and up to CO2 pressures of 16 bar. These data were correlated by a thermodynamic model in which the protein in solution was considered as an electrolyte, which allowed an analysis of electrostatic force effects over protein solubility (AU)