Abstract
Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35°C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35°C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.
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Arakawa, T., Prestrelski, S. J., Kenney, W. C., and Carpenter, J. F., Factors affecting short-term and long-term stabilities of proteins.Adv. Drug Delivery Rev., 10,1–28 (1993).
Benet, L. Z., Kroetz, D. L., and Sheiner, L B., The dynamics of drug absorption distribution and elimination.In The pharmacological Basis of Therapeutics; G. A. Goodman, R. H. Gilman,Eds., 9th ed. MaGraw-Hill, New York pp 3–27, (1996).
Carpenter, J. F. and Crowe, J. H., An infrared spectroscopic study of the interactions of carbohydrates with dried proteins.Biochem., 28, 3916–3922 (1989).
Chang, B. S., Beauvais, R. M., Dong, A., and Carpenter, J. F., Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation. Arch.Biochem. Biophys., 331, 249–58 (1996).
Chi, E. Y, Krishnan, S., Kendrick, B. S., Chang, B. S., Carpenter, J. F., and Randolph, T. W., Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor.Protein Sci., 12,903–13(2003).
Chung, S. I., Lewis, M. S., and Folk, J. E., Relationships of the catalytic properties of human plasma and platelet transglu-taminases (activated blood coagulation factor XIII) to their subunit structures.J. Biol. Chem., 249, 940–950 (1974).
Cleland, J. L., Lam, X., Kendrick B., Yang J., Yang T. H., Overcashier D., Brooks, D., Hsu, C., and Carpenter, J. F., A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody.J. Pharm. Sci., 90, 310–321 (2001).
Cochrance Injuries Group Albumin Reviwers, Human albumin administration in critically ill patients: systemicreview of randomisedcontrolled trials.BMJ., 317,235–240 (1998).
Dong, A. C., Huang, P., and Caughey, W. S., Protein secondary structure in water from second-derivative amide I infrared spectra.Biochemistry, 29, 3303–3308 (1990).
Dong, A., Prestrelski, S. J., Allison, S. D., and Carpenter, J. F., Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation.J. Pharm. Sci., 84, 415–424(1995).
Duddu, S. P. and Dal Monte, P. R., Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody.Pharm. Res., 14, 591–595(1997).
Franks, F., Freeze-drying: from empiricism to predictability.Cryoletters, 11, 93–100 (1990).
Han, Y., Ulrich, M. A., and Cutler, J. E.,Candida albicans mannan extract-protein conjugates induce a protective immune response against experimental candidiasis.J. Infect. Dis., 179,1477–1484(1999).
He, X. M. and Carter, D. C., Atomic structure and chemistry of human serum albumin.Nature, 358, 209–215 (1992).
Izutsu, K., Yoshioka, S., and Kojima, S., Physical stability and protein stability of freeze-dried cakes during storage at elevated temperatures.Pharm. Res., 11, 995–999 (1994).
Johnson, R. E., Kirchhoff, C. F., and Gaud, H. T., Mannitol-sucrose mixture-versatile formulations for protein lyophilization.J. Pharm. Sci., 91, 914–922 (2002).
Kim, Y. S., Jones, L. S., Dong, A., Kendrick B. S., Chang, B. S., Manning, M. C., Randolph, T. W., and Carpenter, J. F., Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins.Protein Sci., 12, 1252–1261 (2003).
Kragh-Hansen, U., Molecular aspects of ligand binding to serum albumin.Pharmacol. Rev., 33,17–53 (1981).
Krishnan, S., Chi, E. Y., Webb, J. N., Chang, B. S., Shan, D., Goldenberg, M., Manning, M. C., Randolph, T. W., and Carpenter, J. F., Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.Biochemistry, 41, 6422–31 (2002).
Kreilgaard, L., Frokjaer, S., Flink, J., Randolph, T. W., and Carpenter, J. F., Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid.Arch. Biochem. Biophy, 360, 121–134(1998).
Laemmli, U. K., Cleavage and structural proteins during the assembly of the head of bacteriophage T4.Nature, 227, 680–685(1970).
MacAulay, J., Thompson, K., Kiberd, B. A., Barnes, D. C., and Peltekian, K. M., Serum creatinine in patients with advanced liver disease is of limited value for identification of moderate renal dysfunction: are the equations for estimating renal function better?Can. J. Gastroenterol., 20, 521–526 (2006).
Matsushita S., Chuang V. T., Kanazawa M., Tanase S, Kawai K., Maruyama T., Suenaga A., and Otagiri M., Recombinant human serum albumin dimmer has high blood circulation activity and low vascular permeability in comparison with native human serum albumin.Pharm. Res., 23, 882–891 (2006).
Offringa, M., Excess mortality after human albumin administration in critically ill patients.BMJ., 317,223–224 (1998).
Patro, S.Y., Freund, E., and Chang, B.S., Protein formulation and fill-finish operations.Biotechnol. Annu. Rev., 8,55–84. (Review) (2002).
Peters, T., All about albumin: Biochemistry, Genetics and Medical Applications, Academic Press. San Diego, CA (1996).
Prestrelski, S. J., Arakawa, T., and Carpenter, J. F., Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy.Arch. Biochem. Biophys., 303,465–473 (1993a).
Prestrelski, S. J., Pikal, K. A., and Arakwa, T., Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy.Pharm. Res., 12, 1250–1259 (1995).
Prestrelski, S. J., Tedeschi, N., Arakawa, T., and Carpenter, J. F., Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers.Biophys. J., 65, 661–671 (1993b).
Roy, M. L., Pikal, M. J., Rickard, E. C., and Maloney, A. M., The effects of formulation and moisture on the stability of a freeze-dried monoclonal antibody-vinca conjugate: a test of the WLF glass transition theory.Dev. Biol. Stand, 74, 323- 339(1992).
Seefeldt, M. B., Kim, Y. S, Tolley, K. P., Seely, J., Carpenter, J. F, and Randolph, T.W., High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist-thermodynamics, kinetics, and application to accelerated formulation studies.Protein Sci., 14,2258–66 (2005).
Slade, L. and Levine, H. A., Food polymer science approach to structure-property relationships in aqueous food systems: non-equilibrium behavior of carbohydrate-water systems.Adv. Exp. Med. Biol., 302, 29–101 (1991).
Takakura, N., Wakabayashi, H., Yamauchi, K., and Takase, M., Influences of orally administered lactoferrin on IFN-γ and IL-10 production by intestinal intraepithelial lymphocytes ’ and mesenteric lymph-node cells.Biochem. Cell Biol., 84, 363–368 (2006).
Wi, S., Pancoka, P., and Keiderling, T. A., Polarizing microscopy of eyespot of chlamydomonas: in situ observation of its location, orientation and multiplication.Biospectroscopy 4, 92–99(1998).
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Han, Y., Jin, BS., Lee, SB. et al. Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage. Arch Pharm Res 30, 1124–1131 (2007). https://doi.org/10.1007/BF02980247
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DOI: https://doi.org/10.1007/BF02980247