Protein Spray-Freeze Drying. Effect of Atomization Conditions on Particle Size and Stability
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Purpose. To investigate the effect of atomization conditions on particle size and stability of spray-freeze dried protein.
Methods. Atomization variables were explored for excipient-free (no zinc added) and zinc-complexed bovine serum albumin (BSA). Particle size was measured by laser diffraction light scattering following sonication in organic solvent containing poly(lactide-co-glycolide) (PLG). Powder surface area was determined from the N2 vapor sorption isotherm. Size-exclusion chromatography (SEC) was used to assess decrease in percent protein monomer. Fourier-transform infrared (FTIR) spectroscopy was employed to estimate protein secondary structure. PLG microspheres were made using a non-aqueous, cryogenic process and release of spray-freeze dried BSA was assessed in vitro.
Results. The most significant atomization parameter affecting particle size was the mass flow ratio (mass of atomization N2 relative to that for liquid feed). Particle size was inversely related to specific surface area and the amount of protein aggregates formed. Zinc-complexation reduced the specific surface area and stabilized the protein against aggregation. FTIR data indicated perturbations in secondary structure upon spray-freeze drying for both excipient-free and zinc-complexed protein.
Conclusions. Upon sonication, spray-freeze dried protein powders exhibited friability, or susceptibility towards disintegration. For excipient-free protein, conditions where the mass flow ratio was > ∼0.3 yielded sub-micron powders with relatively large specific surface areas. Reduced particle size was also linked to a decrease in the percentage of protein monomer upon drying. This effect was ameliorated by zinc-complexation, via a mechanism involving reduction in specific surface area of the powder rather than stabilization of secondary structure. Reduction of protein particle size was beneficial in reducing the initial release (burst) of the protein encapsulated in PLG microspheres.
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- 1.S. P. Schwendeman, M. Cardamone, M. R. Brandon, A. M. Klibanov, and R. Langer. Stability of proteins and their delivery from biodegradable polymer microspheres. In S. Cohen and H. Bernstein (eds.), Microparticulate systems for the delivery of proteins and vaccines, Marcel Dekker, New York, 1996 pp. 1-49.Google Scholar
- 2.O. L. Johnson, W. Jaworowicz, J. L. Cleland, L. Bailey, M. Charnis, E. Duenas, C. Wu, D. Shepard, S. Magil, T. Last, A. J. S. Jones, and S. D. Putney. The stabilization of human growth hormone into biodegradable microspheres. Pharm. Res. 14:730-735 (1997).Google Scholar
- 3.J. H. Crowe, J. F. Carpenter, L. M. Crowe, and T. J. Anchordoquy. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27:219-231 (1990).Google Scholar
- 4.J. F. Carpenter, S. J. Prestrelski, and T. Arakawa. Separation of freezing and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies. Arch. Biochem. Biophys. 303:456-464 (1993).Google Scholar
- 5.S. J. Prestrelski, T. Arakawa, and J. F. Carpenter. 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 (1993).Google Scholar
- 6.Y.-F. Maa, P.-A. Nguyen, T. Sweeney, S. J. Shire, and C. C. Hsu. Protein inhalation powders. Spray drying vs. spray freeze drying. Pharm. Res. 16:249-254 (1999).Google Scholar
- 7.S. Brunauer, P. H. Emmett, and E. Teller. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60:309-319 (1938).Google Scholar
- 8.K. Griebenow and A. M. Klibanov. On protein denaturation in aqueous-organic mixtures but not in pure organic solvents. J. Am. Chem. Soc. 118:11695-11700 (1996).Google Scholar
- 9.H. R. Costantino, K. G. Carrasquillo, R. A. Cordero, M. Mumenthaler, C. C. Hsu, and K. Griebenow. The effect of excipients on the structure and stability of lyophilized recombinant human growth hormone (rhGH). J. Pharm. Sci. 87:1412-1420 (1998).Google Scholar
- 10.K. G. Carrasquillo, R. A. Cordero, S. Ho, J. Franquiz, and K. Griebenow. Structure-guided encapsulation of bovine serum albumin in poly(DL-lactic-co-glycolic) acid. Pharm. Pharmacol. Commun. 4:563-571 (1998).Google Scholar
- 11.K. G. Carrasquillo, H. R. Costantino, R. A. Cordero, C. C. Hsu, and K. Griebenow. On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer. J. Pharm. Sci. 2:166-173 (1999).Google Scholar
- 12.H. R. Costantino, T. H. Nguyen, and C. C. Hsu. Fourier transform infrared spectroscopy demonstrates that lyophilization alters the secondary structure of recombinant human growth hormone. Pharm. Sci. 2:229-232 (1996).Google Scholar
- 13.P. Herbert, K. Murphy, O. Johnson, N. Dong, W. Jaworowicz, M. A. Tracy, J. L. Cleland, and S. D. Putney. A large-scale process to produce microencapsulated proteins. Pharm. Res. 15:357-361 (1998).Google Scholar
- 14.W. D. Rhine, D. S. T. Hsieh, and R. Langer. Polymers for sustained macromolecule release: procedures to fabricate reproducible delivery systems and control release kinetics. J. Pharm. Sci. 69:265-270 (1980).Google Scholar
- 15.R. A. Siegel and R. Langer. A new monte-carlo approach to diffusion in constricted porous geometries. J. Coll. I. Sc. 109:426-440 (1986).Google Scholar
- 16.J. A. Dean, Ed., Lange's Handbook of Chemistry, McGraw-Hill Book Company, New York, 1985.Google Scholar
- 17.Y.-F. Maa, P.-A. Nguyen, and S. W. Hsu. Spray-drying of air-liquid interface sensitive recombinant human growth hormone. J. Pharm. Sci. 87:152-159 (1998).Google Scholar
- 18.M. J. Pikal, S. Shah, D. Senior, and J. E. Lang. Physical chemistry of freeze-drying: measurement of sublimation rates for frozen aqueous solutions by a microbalance technique. J. Pharm. Sci. 72: 635-650 (1983).Google Scholar
- 19.B. M. Eckhart, J. Q. Oeswein, and T. A. Bewley. Effect of freezing on aggregation of human growth hormone. Pharm. Res. 11: 1360-1364 (1991).Google Scholar
- 20.H. Willemer. Measurements of temperature, ice evaporation rates and residual moisture contents in freeze-drying. Dev. Biol. Stand. 74:123-134 (1992).Google Scholar
- 21.J. A. House and J. C. Mariner. Stabilization of rinderpest vaccine by modification of the lyophilization process. Dev. Biol. Standard 87:235-244 (1996).Google Scholar
- 22.K. Masters. Spray Drying Handbook, John Wiley and Sons, New York, 1991.Google Scholar
- 23.C. C. Hsu, H. M. Nguyen, D. A. Yeung, D. A. Brooks, G. S. Koe, T. A. Bewley, and R. Pearlman. Surface denaturation at solid-void interface. A possible pathway by which opalescent particle form during the storage of lyophilized tissue-type plasminogen activator at high temperatures. Pharm. Res. 12:69-77 (1995).Google Scholar
- 24.B. Y. Chang, B. S. Kendrick, and J. F. Carpenter. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J. Pharm. Sci. 85:1325-1330 (1996).Google Scholar
- 25.G. B. Strambini and E. Gabellieri. Proteins in frozen solutions. Evidence of ice-induced partial folding. Biophys. J. 70:971-976 (1996).Google Scholar
- 26.K. Fu, K. Griebenow, L. Hsieh, A. M. Klibanov, and R. Langer. FTIR characterization of proteins encapsulated within PLGA microspheres. J. Control. Release 58:357-366 (1999).Google Scholar
- 27.H. R. Costantino, L. Shieh, A. M. Klibanov, and R. Langer. Heterogeneity of serum albumin with respect to solid-state aggregation via thiol-disulfide interchange-Implications for sustained release from polymers. J. Control. Release 44:255-261 (1997).Google Scholar
- 28.R. M. C. Dawson, D. C. Elliot, W. H. Elliot, and K. M. Jones, Eds. Data for Biochemical Research, Oxford University Press, New York, 1994.Google Scholar
- 29.H. R. Costantino, K. Griebenow, P. Mishra, R. Langer, and A. M. Klibanov. Fourier-transform infrared (FTIR) spectroscopic investigation of protein stability in the lyophilized form. Biochim. Biophys. Acta 1253:69-74 (1995).Google Scholar
- 30.V. E. Bychkova, O. B. Ptitsyn. The molten globule in vitro and in vivo. Chemtracts Biochem. Mol. Biol. 4:133-163 (1993).Google Scholar