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Effect of temperature and pH on the crystallization of insulin with carbon dioxide as the acidifying agent

Grant number: 11/50281-0
Support type:Regular Research Grants
Duration: May 01, 2011 - April 30, 2012
Field of knowledge:Engineering - Chemical Engineering - Industrial Operations and Equipment for Chemical Engineering
Principal Investigator:Everson Alves Miranda
Grantee:Everson Alves Miranda
Home Institution: Faculdade de Engenharia Química (FEQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil


In isoeletric precipitation, an important technique for the precipitation and crystallization of proteins, the pH is adjusted to or close to the pi of the protein. In this condition, the solubility of the molecule is reduced and the formation of the second (solid) phase occurs. Since a large proportion of proteins has an acidic pi, it is very common the use of mineral acids like sulfuric or phosphoric acids in these processes. Studies on the use of volatile electrolytes such as CO2 in protein precipitation showed that these agents are a promising alternative to conventional acids. Volatile electrolytes dissolve and dissociate in water resulting in ions whose concentrations are dependent on the temperature and pressure of the system, what facilitates their removal from system. However, until recently, there was no report of the use of volatile electrolyte in protein crystallization. Hirata et al. (2010) were the first work to describe the use of a volatile acid (CO2) in the crystallization of a protein (porcine insulin). Rhombohedral crystals - the Gommon shape for porcine insulin crystals that contain zinc in their structure - were obtained after adjustment of pH, initial concentrations of insulin and zinc to specific conditions. The solubility curve for insulin in the NaHCO3/CO2/ZnCl2 system at 15°C as a function of pH in the range of 6.0 to 8.0 and the crystal growth kinetics as function of supersaturation were determined. In the same line of this work of Hirata et al. (2011), the objective of this project is to verify the effect of temperature (5, 15 and 25 °C), pH (6.0 a 7.0) and initial protein concentration (1.0, 1.5, and 2,0 mg/ml) in the crystal growth rate, particle size distribution, and crystal shape. Temperatura, pH, and initial protein concentration are important process variables in crystallization that have impact in the purity and crystal habit of the product and are also key in the development of large scale industrial processes. (AU)