Chemical Engineering and Processing 56 (2012) 29–33 Contents lists available at SciVerse ScienceDirect Chemical Engineering and Processing: Process Intensification jo u rn al hom epage: www.elsevier.com/locate/cep Determination of crystal growth rate for porcine insulin crystallization with CO 2 as a volatile acidifying agent Gisele A.M. Hirata, André Bernardo, Everson A. Miranda ∗ LEBp – Laboratório de Engenharia de Bioprocessos, Departamento de Engenharia de Materiais e de Bioprocessos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, UNICAMP, Av. Albert Einstein, 500, CEP 13083-852 – Campinas, SP, Brazil a r t i c l e i n f o Article history: Received 15 February 2011 Received in revised form 8 February 2012 Accepted 8 March 2012 Available online 16 March 2012 Keywords: Crystallization CO2 Growth crystal Insulin Protein a b s t r a c t Crystallization is controlled by two steps that determine the quality and the final size of the product, nucleation and growth, which are functions of supersaturation. Recently, Hirata et al. [1] crystallized insulin using CO 2 as a volatile acid to impose supersaturation on the system. The objective of the present work was to determine the growth kinetics of insulin crystallization in 50 mM NaHCO 3 solution with 0.4 mM ZnCl 2 in a CO 2 atmosphere at 15 ◦ C, adjusting the parameters of the equation G = k g × S g to the experimental data. The solubility of insulin in the NaHCO 3 /CO 2 /ZnCl 2 system at 15 ◦ C was determined as a function of pH in the range of 6.30–7.34. The crystal growth data allowed determination of the growth order “g” (g = 2.9). Although protein crystallization has some features that differ from the crystallization of less complex molecules, the apparent growth kinetics of insulin were successfully analyzed here with the same empirical methods used for small molecules, which can easily be scaled up for industrial applications to achieve specific size and purity, the goals of industrial crystallization. The method used in this work is a useful tool for describing and simplifying optimization of industrial protein crystallization processes. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Crystallization is a separation and purification technique gov- erned by two main steps, nucleation and growth, which are caused by a change in system parameters (pH, temperature, solution com- position, etc.). Nucleation results from the aggregation of molecules in the development of the first crystals [2]. After the formation of nuclei and the subsequent creation of crystals, growth – the addition of monomers to the crystal surface – occurs. Nucleation and growth control essential characteristics of a product such as crystal habit and structure (a compound can have different crys- talline structures, which are different products since each has a distinct dissolution rate, stability, solubility, etc. [3,4]), crystal size distribution, and physical properties. Knowledge of super- saturation level (the driving force of the process) is crucial for determination of the kinetic parameters of nucleation and crystal growth rates [5]. The production of optimum crystals is influenced by other factors in addition to the thermodynamics and kinetics of the crystallization process. The hydrodynamics of the system, type of stirrer, shear stress, etc. have strong consequences on the crystal size distribution and crystal form and size. These character- istics are of paramount importance to the final product since, for ∗ Corresponding author. Tel.: +55 19 3521 3918; fax: +55 19 3521 3890. E-mail address: everson@feq.unicamp.br (E.A. Miranda). example, size and form are directly related to the dissolution behav- ior, important information in the case of many pharmaceutical products. Isoelectric precipitation and crystallization of proteins are pro- cesses usually conducted with acids such as H 2 SO 4 and HCl used for pH adjustment to the isoelectric point. The use of volatile elec- trolytes is an alternative to these processes, since extreme local pH values – which can denature fragile biomolecules such as proteins – are avoided. Another important advantage of volatile electrolytes is that they can be removed from the system relatively easily by reducing pressure and increasing temperature and recycled in the process [6]. Several authors have used CO 2 as a volatile acid to pre- cipitate proteins from complex mixtures such as milk and soybean extracts [6–9]. Moreover, Tashima et al. [10] recently reported the precipitation of a single protein using CO 2 . They studied the isoelec- tric precipitation of porcine insulin with CO 2 in NaHCO 3 solutions under different conditions (temperature and pH) with preserva- tion of the biological activity of the molecule. They also developed a thermodynamic model to correlate experimental solubility data. Hirata et al. [1] were the first to report the crystallization of a sin- gle protein using CO 2 as acidifying agent (porcine insulin in the presence of zinc). Along this same line, the aim of this study was to determine the growth kinetics for the crystallization of insulin with CO 2 through determination of protein solubility and crystal- lization runs. Growth rates were measured to get basic information about the supersaturation and the growth order for the system studied, information required for better understanding of the 0255-2701/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2012.03.001