Pharmaceutical Research

, Volume 8, Issue 4, pp 427–436 | Cite as

The Effects of Formulation Variables on the Stability of Freeze-Dried Human Growth Hormone

  • Michael J. Pikal
  • Karen M. Dellerman
  • Michael L. Roy
  • Ralph M. Riggin
Article

Abstract

Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the “shelf-life” stability of the freeze-dried product. This research presents the results of a formulation optimization study of the “in-process” and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40°C in a nominal nitrogen headspace (≈0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7–7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

freeze-drying stability of proteins lyoprotectants protein formulation human growth hormone (hGH) 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

REFERENCES

  1. 1.
    J. Geigert. Overview of the stability and handling of recombinant protein drugs. J. Parenter. Sci. Tech. 43:220–224 (1989).Google Scholar
  2. 2.
    K. Hellman, D. S. Miller, and K. A. Cammack. The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora. Biochim. Biophys. Acta 749:133–142 (1983).Google Scholar
  3. 3.
    P. Labrude and C. Vigneron. Stability and functional properties of haemoglobin freeze-dried in the presence of four protective substances after prolonged storage: Dose-effect relationships. J. Pharm. Pharmacol. 35:23–27 (1982).Google Scholar
  4. 4.
    J. F. Carpenter, L. M. Crowe, and J. H. Crowe. Stabilization of phosphofructokinase with sugars during freeze-drying: Characterization of enhanced protection in the presence of divalent cations. Biochim. Biophys. Acta 923:109–115 (1987).Google Scholar
  5. 5.
    M. W. Townsend and P. P. DeLuca. Use of lyoprotectants in the freeze-drying of a model protein, ribonuclease A. J. Parenter. Sci. Tech. 42:190–199 (1988).Google Scholar
  6. 6.
    T. I. Pristoupil, M. Kramlova, H. Fortova, and S. Ulrych. Haemoglobin lyophilized with sucrose: The effect of residual moisture on storage. Haematologia 18:45–52 (1985).Google Scholar
  7. 7.
    M. J. Hageman. The role of moisture in protein stability. Drug Dev. Ind. Pharm. 14:2047–2070 (1988).Google Scholar
  8. 8.
    F. Franks. Freeze drying: from empiricism to predictability. Cryo-Letters 11:93–110 (1990).Google Scholar
  9. 9.
    T. I. Pristoupil, S. Ulrych, and M. Kramlova. Haemoglobin stabilization during lyophilization with saccharides. Perturbation effect of polyethylene glycols. Coll. Czech. Chem. Commun. 46:1856–1859 (1981).Google Scholar
  10. 10.
    J. F. Carpenter and J. H. Crowe. Fundamental differences in the interactions of stabilizing solutes with proteins during freeze-thawing versus during freeze-drying. Cryobiology 25:537–538 (1988).Google Scholar
  11. 11.
    D. S. Johnson and F. Castelli. The influence of sugars on the properties of freeze dried lysozyme and hemoglobin. Thermochim. Acta 144:195–208 (1989).Google Scholar
  12. 12.
    G. W. Becker, P. M. Tackitt, W. W. Bromer, D. S. Lefeber, and R. M. Riggin. Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone. Biotechnol. Appl. Biochem. 10:326–337 (1988).Google Scholar
  13. 13.
    G. W. Becker, R. R. Bowsher, W. C. MacKellar, M. L. Poor, P. M. Tackitt, and R. M. Riggin. Chemical, physical, and biological characterization of a dimeric form of biosynthetic human growth hormone. Biotechnol. Appl. Biochem. 9:478–487 (1987).Google Scholar
  14. 14.
    M. J. Pikal, K. Dellerman, and M. L. Roy. Effects of moisture and oxygen on the stability of freeze-dried formulations of human growth hormone. Biologicals (in press).Google Scholar
  15. 15.
    R. M. Riggin, G. K. Dorulla, and D. J. Miner. A reversed-phase high-performance liquid chromatographic method for characterization of biosynthetic human growth hormone. Anal. Biochem. 167:199–209 (1987).Google Scholar
  16. 16.
    R. M. Riggin, C. J. Shaar, G. K. Dorulla, D. S. Lefeber, and D. J. Miner. High-performance size-exclusion chromatographic determination of the potency of biosynthetic human growth hormone products. J. Chromatogr. 435:307–318 (1988).Google Scholar
  17. 17.
    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
  18. 18.
    C. A. Angell, E. J. Sare, and R. D. Bressel. Concentrated electrolyte solution transport theory: Directly measured glass temperatures and vitreous ice. J. Phys. Chem. 71:2759–2761 (1967).Google Scholar
  19. 19.
    H. Levine and L. Slade. Principles of “crystallization” technology from structure/property relationships of carbohydrate/water systems—a review. Cryo-Letters 9:21–63 (1988).Google Scholar
  20. 20.
    M. J. Pikal. Freeze-drying of proteins. II. Formulation selection. Biopharm. 3:26–30 (1990).Google Scholar
  21. 21.
    S. Charm and B. Wong. Enzyme inactivation with shearing. Biotechnol. Bioeng. 12:1103–1109 (1970).Google Scholar
  22. 22.
    M. Hagenlocher and R. Pearlman. Use of a substituted cyclodextrin for stabilization of solutions of recombinant human growth hormone. Abstract BT219, 4th Annual meeting of the AAPS, Atlanta, Oct. 22–26, 1989.Google Scholar
  23. 23.
    D. N. Brems, S. M. Plaisted, H. A. Havel, and C. C. Tomich. Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 85:3367–3371 (1988).Google Scholar
  24. 24.
    R. Pearlman and T. H. Nguyen. Formulation strategies for recombinant proteins: human growth hormone and tissue plasminogen activator. In D. Marshak and D. Liu (eds.), Therapeutic Peptides and Proteins: Formulations, Delivery, and Targeting, Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, pp. 23–30.Google Scholar
  25. 25.
    T. J. Ahern and A. M. Klibanov. The mechanism of irreversible enzyme inactivation at 100°C. Science 228:1280–1284 (1985).Google Scholar

Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Michael J. Pikal
    • 1
  • Karen M. Dellerman
    • 1
  • Michael L. Roy
    • 1
  • Ralph M. Riggin
    • 1
  1. 1.Lilly Research LaboratoriesEli Lilly and Co.Indianapolis

Personalised recommendations