Abstract
Choline dihydrogen phosphate (CDHP) is a low melting point organic salt reported to increase the thermal stability of model proteins. The purpose of the current work was to investigate the effect of CDHP on recombinant human interleukin-2 (rhIL-2) functional and structural integrity, a therapeutic protein used for treating advanced melanoma. Structural integrity and biological activity of rhIL-2 formulated in CDHP was measured below and above thermal midpoint unfolding temperature (T m) of the protein. Potential biocompatibility was assessed by exposing splenocytes and the B16F10 cell line to CDHP at various concentrations and conditions of pH. Formulation of rhIL-2 in an aqueous 680 mM CDHP pH 7.4 solution preserved rhIL-2 binding activity when the solution was heated to 23.3 °C above T m. CDHP solutions (≤80 mM), formulated with 0.33% (w/v) NaHCO3 to maintain pH ≥ 7.2, exhibited no cytotoxic activity toward primary splenocytes or B16F10 cells cultures. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30 mM CDHP aqueous solution with NaHCO3 (pH ≥ 7.2) compared to controls without CDHP. Choline DHP increases rhIL-2 thermal stability in the absence of inherent CDHP cytotoxicity. While increased T m is associated with a diminished rhIL-2 biological activity, the protein retains binding ability.
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Arakawa, T., R. Bhat, and S. N. Timasheff. Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system. Biochemistry 29(7):1914–1923, 1990.
Arakawa, T., J. S. Philo, and Y. Kita. Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin. Biosci. Biotechnol. Biochem. 65(6):1321–1327, 2001.
Byrne, N., L. M. Wang, J. P. Belieres, and C. A. Angell. Reversible folding-unfolding, aggregation protection, and multi-year stabilization, in high concentration protein solutions, using ionic liquids. Chem. Commun. (Camb.) (26):2714–2716, 2007.
Constatinescu, D., C. Herrmann, and H. Weingartner. Patterns of protein unfolding and protein aggregation in ionic liquids. Phys. Chem. Chem. Phys. 12(8):1756–1763, 2010.
Cooper, A., C. M. Johnson, J. H. Lakey, and M. Nollmann. Heat does not come in different colours: entropy–enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions. Biophys. Chem. 93(2–3):215–230, 2001.
Den Otter, W., J. J. Jacobs, J. J. Battermann, G. J. Hordijk, Z. Krastev, E. V. Moiseeva, R. J. Stewart, P. G. Ziekman, and J. W. Koten. Local therapy of cancer with free IL-2. Cancer Immunol. Immunother. 57(7):931–950, 2008.
Foureau, D. M., I. H. McKillop, C. P. Jones, A. Amin, R. L. White, and J. C. Salo. Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+T cell expansion in an immunocompetent mouse model. Cancer Immunol. Immunother. 60(9):1347–1356, 2011.
Fujita, K., M. Forsyth, D. R. MacFarlane, R. W. Reid, and G. D. Elliott. Unexpected improvement in stability and utility of cytochrome c by solution in biocompatible ionic liquids. Biotechnol. Bioeng. 94(6):1209–1213, 2006.
Fujita, K., D. R. MacFarlane, and M. Forsyth. Protein solubilising and stabilising ionic liquids. Chem. Commun. (Camb.) (38):4804–4806, 2005.
Fujita, K., D. R. MacFarlane, M. Forsyth, M. Yoshizawa-Fujita, K. Murata, N. Nakamura, and H. Ohno. Solubility and stability of cytochrome c in hydrated ionic liquids: effect of oxo acid residues and kosmotropicity. Biomacromolecules 8(7):2080–2086, 2007.
Geigert, J., N. Solli, P. Woehleke, and S. Vemuri. Development and shelf-life determination of recombinant interleukin-2 (proleukin). Pharm. Biotechnol. 5:249–262, 1993.
Gillis, S., and K. A. Smith. Long term culture of tumour-specific cytotoxic T cells. Nature 268(5616):154–156, 1977.
Hashimoto, T., Z. He, W. Y. Ma, P. C. Schmid, A. M. Bode, C. S. Yang, and Z. Dong. Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells. Cancer Res. 64(9):3344–3349, 2004.
Hicks, R. W., and S. C. Becker. An overview of intravenous-related medication administration errors as reported to MEDMARX, a national medication error-reporting program. J. Infus. Nurs. 29(1):20–27, 2006.
Hofmeister, F. About the science of the effect of salts. Arch. Exp. Pathol. Pharmacol. 24:247–260, 1888.
Kaushik, J. K., and R. Bhat. A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions. Protein Sci. 8(1):222–233, 1999.
Komsa-Penkova, R., R. Koynova, G. Kostov, and B. G. Tenchov. Thermal stability of calf skin collagen type I in salt solutions. Biochim. Biophys. Acta 1297(2):171–181, 1996.
Kuwahara, T., S. Asanami, and S. Kubo. Experimental infusion phlebitis: tolerance osmolality of peripheral venous endothelial cells. Nutrition 14(6):496–501, 1998.
Ma, A., R. Koka, and P. Burkett. Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis. Annu. Rev. Immunol. 24:657–679, 2006.
Macfarlane, D. R., R. Vijayaraghavan, H. N. Ha, A. Izgorodin, K. D. Weaver, and G. D. Elliott. Ionic liquid “buffers”-pH control in ionic liquid systems. Chem. Commun. (Camb.) 46(41):7703–7705, 2010.
Maclean, D. S., Q. Qian, and C. R. Middaugh. Stabilization of proteins by low molecular weight multi-ions. J. Pharm. Sci. 91(10):2220–2229, 2002.
Malek, T. R., and I. Castro. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 33(2):153–165, 2010.
Mealey, R. H., M. H. Littke, S. R. Leib, W. C. Davis, and T. C. McGuire. Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy. Vet. Immunol. Immunopathol. 121(1–2):8–22, 2008.
Miki, Y., K. Kakuyama, and K. Soda. Protein stability; optimization of electrostatic contributions by partially neutralizing surface ionic charges. Biosystems 44(1):69–77, 1997.
Minami, Y., T. Kono, T. Miyazaki, and T. Taniguchi. The IL-2 receptor complex: its structure, function, and target genes. Annu. Rev. Immunol. 11:245–268, 1993.
Miquel, K., A. Pradines, F. Terce, S. Selmi, and G. Favre. Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells. J. Biol. Chem. 273(40):26179–26186, 1998.
Nony, P., P. Girard, S. Chabaud, L. Hessel, C. Thebault, and J. P. Boissel. Impact of osmolality on burning sensations during and immediately after intramuscular injection of 0.5 ml of vaccine suspensions in healthy adults. Vaccine 19(27):3645–3651, 2001.
Olteanu, A., C. N. Patel, M. M. Dedmon, S. Kennedy, M. W. Linhoff, C. M. Minder, P. R. Potts, M. Deshmukh, and G. J. Pielak. Stability and apoptotic activity of recombinant human cytochrome c. Biochem. Biophys. Res. Commun. 312(3):733–740, 2003.
Proctor, V. A., and F. E. Cunningham. The chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Crit. Rev. Food Sci. Nutr. 26(4):359–395, 1988.
Ru, M. T., J. S. Dordick, J. A. Reimer, and D. S. Clark. Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content. Biotechnol. Bioeng. 63(2):233–241, 1999.
Shaker, M. A., and H. M. Younes. Interleukin-2: evaluation of routes of administration and current delivery systems in cancer therapy. J. Pharm. Sci. 98(7):2268–2298, 2009.
Sparano, J. A., R. I. Fisher, M. Sunderland, K. Margolin, M. L. Ernest, M. Sznol, M. B. Atkins, J. P. Dutcher, K. C. Micetich, G. R. Weiss, et al. Randomized phase III trial of treatment with high-dose interleukin-2 either alone or in combination with interferon alfa-2a in patients with advanced melanoma. J. Clin. Oncol. 11(10):1969–1977, 1993.
Tadeo, X., B. Lopez-Mendez, D. Castano, T. Trigueros, and O. Millet. Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation. Biophys. J. 97(9):2595–2603, 2009.
Tadeo, X., M. Pons, and O. Millet. Influence of the Hofmeister anions on protein stability as studied by thermal denaturation and chemical shift perturbation. Biochemistry 46(3):917–923, 2007.
Takeda, K., S. Akagi, S. Takahashi, A. Onishi, H. Hanada, and C. A. Pinkert. Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture. Cloning Stem Cells 4(3):223–229, 2002.
Viguier, M., F. Lemaitre, O. Verola, M. S. Cho, G. Gorochov, L. Dubertret, H. Bachelez, P. Kourilsky, and L. Ferradini. Foxp3 expressing CD4+CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J. Immunol. 173(2):1444–1453, 2004.
Vijayaraghavan, R., A. Izgorodin, V. Ganesh, M. Surianarayanan, and D. R. MacFarlane. Long-term structural and chemical stability of DNA in hydrated ionic liquids. Angew. Chem. Int. Ed. Engl. 49(9):1631–1633, 2010.
Vogt, G., S. Woell, and P. Argos. Protein thermal stability, hydrogen bonds, and ion pairs. J. Mol. Biol. 269(4):631–643, 1997.
Vonhippel, P. H., and K. Y. Wong. Neutral salts: the generality of their effects on the stability of macromolecular conformations. Science 145(3632):577–580, 1964.
Vrikkis, R. M., K. J. Fraser, K. Fujita, D. R. Macfarlane, and G. D. Elliott. Biocompatible ionic liquids: a new approach for stabilizing proteins in liquid formulation. J. Biomech. Eng. 131(7):074514, 2009.
Waldmann, T. A., S. Dubois, and Y. Tagaya. Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 14(2):105–110, 2001.
Weaver, K. D., H. J. Kim, J. Sun, D. R. MacFarlane, and G. D. Elliott. Cytotoxicity and biocompatibility of a family of choline phosphate ionic liquids designed for pharmaceutical applications. Green Chem. 12:507–513, 2010.
Weaver, K. D., R. M. Vrikkis, M. P. Van Vorst, J. Trullinger, R. Vijayaraghavan, D. M. Foureau, I. H. McKillop, D. R. Macfarlane, J. K. Krueger, and G. D. Elliott. Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid. Phys. Chem. Chem. Phys. 14(2):790–801, 2011.
West, W. H., K. W. Tauer, J. R. Yannelli, G. D. Marshall, D. W. Orr, G. B. Thurman, and R. K. Oldham. Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N. Engl. J. Med. 316(15):898–905, 1987.
Yamasaki, M., H. Yano, and K. Aoki. Differential scanning calorimetric studies on bovine serum albumin: I. Effects of pH and ionic strength. Int. J. Biol. Macromol. 12(4):263–268, 1990.
Yamasaki, M., H. Yano, and K. Aoki. Differential scanning calorimetric studies on bovine serum albumin: II. Effects of neutral salts and urea. Int. J. Biol. Macromol. 13(6):322–328, 1991.
Zhang, Y., and P. S. Cremer. Interactions between macromolecules and ions: the Hofmeister series. Curr. Opin. Chem. Biol. 10(6):658–663, 2006.
Zhao, H., O. Olubajo, Z. Song, A. L. Sims, T. E. Person, R. A. Lawal, and L. A. Holley. Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions. Bioorg. Chem. 34(1):15–25, 2006.
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This work was supported in part by NIH grant 1R21EB00740401A2 to GDE and DRM, and by an UNCC-CMC partnership grant to GDE and DMF.
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Associate Editor Joseph Le Doux oversaw the review of this article.
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Foureau, D.M., Vrikkis, R.M., Jones, C.P. et al. In Vitro Assessment of Choline Dihydrogen Phosphate (CDHP) as a Vehicle for Recombinant Human Interleukin-2 (rhIL-2). Cel. Mol. Bioeng. 5, 390–401 (2012). https://doi.org/10.1007/s12195-012-0243-x
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DOI: https://doi.org/10.1007/s12195-012-0243-x