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 50mM NaHCO 3 solution with 0.4mM 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.56 2933Hirata, G.A.M., Bernardo, A., Miranda, E.A., Crystallization of porcine insulin with carbon dioxide as acidifying agent (2010) Powder Technol., 197, pp. 54-57Chernov, A.A.J., Protein crystals and their growth (2003) Struct. Biol., 142, pp. 3-21Garcia, E., Veesle, S., Boistelle, R., Hoff, C., Crystallization and dissolution of pharmaceutical compounds: an experimental approach (1999) J. Cryst. Growth, pp. 1360-1364Yu Shekunov, B., York, P., Crystallization processes in pharmaceutical technology and drug delivery design (2000) J. Cryst. Growth, 211, pp. 122-136Cheng, Y.C., Lobo, R.F., Sandler, S.I., Lenhoff, A.M., Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions (2005) Biotechnol. Bioeng., 94, pp. 177-188Khorshid, N., Hossain, M.M., Farid, M., Precipitation of food protein using high pressure carbon dioxide (2007) J. Food Eng., 79, pp. 1214-1220Jordan, P.J., Lay, K., Ngan, N., Rodley, G.F., Casein precipitation using high pressure CO 2 (1987) New Zeal. J. Dairy Sci., 22, pp. 247-256Hofland, G.W., de Rijke, A., Thiering, R., van der Wielen, L.A.M., Witkamp, G.J., Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid (2000) J. Chromatogr. B, 743, pp. 357-368Thiering, R., Hofland, G., Foster, N., Witkamp, G.J., van der Wielen, L.A.M., Fractionation of soybean proteins with pressurized carbon dioxide as a volatile electrolyte (2001) Biotechnol. Bioeng., 73, pp. 1-11Tashima, A.K., Ottens, M., van der Wielen, L.A.M., Cyntra, D.E., Pauli, J.R., Pessôa Filho, P.A., Miranda, E.A., Precipitation of porcine insulin with carbon dioxide (2009) Biotechnol. Bioeng., 103, pp. 909-919Crowfoot, D., X-ray single crystal photographs of insulin (1935) Nature, 135, pp. 591-592Dodson, E.J., Dodson, G.G., Lewitova, A., Sabesan, M., Zinc-free cubic pig insulin: crystallization and structure determination (1978) J. Mol. Biol., 125, pp. 387-396Ladisch, M.R., (2001) Bioseparations Engineering: Principles, Practice, and Economics, , Wiley-Interscience, New YorkSakabe, N., Sakabe, K., Sasaki, K., X-ray studies of water structure in 2 Zn insulin crystal (1985) J. Bioscience., 8, pp. 45-55Schlichtkrull, J., Insulin crystals. II. Shape of rhombohedral Zn-insulin crystals in relation to species and crystallization media (1956) Acta Chem. Scand., 10, pp. 1459-1464Wintersteiner, O., Abramson, H.A., The isoelectric point of insulin: electrical properties of adsorbed and crystalline insulin (1933) J. Biol. Chem., 99, pp. 741-753Yip, C.M., Defelippis, M.R., Frank, B.H., Brader, M.L., Ward, M.D., Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly (1998) Biophys. J., 75, pp. 1172-1179Norrman, M., Schluckebier, G., Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH (2007) BMC Struct. Biol., 7, pp. 1-14Chausmer, A.B., Zinc, insulin and diabetes (1998) JACN, 17, pp. 109-115Brown, L.R., Glucose-mediated insulin delivery from implantable polymers (2002) Polymeric Biomaterials, pp. 1101-1116. , Marcel Dekker, New York, S. Dumitriu (Ed.)Schlichtkrull, J., Insulin crystals. IV. The nucleation and growth of insulin crystals (1957) Acta Chem. Scand., 11, pp. 439-460Schlichtkrull, J., The growth of insulin crystals (1957) Acta Chem. Scand., 11, pp. 1248-1256Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal. Biochem., 72, pp. 248-254Myerson, A.S., (2002) Handbook of Industrial Crystallization, , Butterworth-Heinemann, WoburnAsherie, N., Protein crystallization and phase diagrams (2004) Methods, 34, pp. 266-272McPherson, A., (1999) Crystallization of biological macromolecules, , Cold Spring Harbor, New YorkPusey, M.L., Snyder, R.S., Naumann, R., Protein crystal growth: growth kinetics for tetragonal lysozyme crystals (1986) J. Biol. Chem., 261, pp. 6524-6529Mersmann, A., Kind, M., Chemical engineering aspects of precipitation from solution (1988) Chem. Eng. Technol., 11, pp. 264-276Chen, Y.H., Kim, K.J., Kim, S.H., A study on crystallization kinetics of pentaerythritol in a batch cooling crystallizer (2005) Chem. Eng. Sci., 60, pp. 4791-4802Chen, P.C., Kou, K.L., Tai, H.K., Jin, S.L., Lye, C.L., Lin, C.Y., Removal of carbon dioxide by reactive crystallization in a scrubber - kinetics of barium carbonate crystals (2002) J. Cryst. Growth, pp. 2166-2171Rosenberger, F., Inorganic and protein crystal growth - Similarities and differences (1986) J. Cryst. Growth, 76, pp. 618-636