Skip to main content
SpringerLink
Log in

Surface Denaturation at Solid-Void Interface—A Possible Pathway by Which Opalescent Participates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 °C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22 - 1.78 m2/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103 - 105 °C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. Activase-Alteplase Recombinant. In Physicians' Desk Reference (46th ed.), Medical Economics Data, New Jersey, 1992, pp 1047.

  2. F. Franks. Improved freeze-drying: An analysis of basic scientific principles. Process Biochem. 24:iii–vii (1989).

    Google Scholar 

  3. G. Taborski. Protein alterations at low temperatures: an overview. In O. Fennema (ed.), Proteins at low temperatures, American Chemical Society, Washington, D.C., 1979, pp 1–26.

    Google Scholar 

  4. F. S. Soliman and L. van den Berg. Factors affecting freezing damage of lactic dehydrogenase. Cryobiology 8:73–78 (1971).

    Google Scholar 

  5. D. F. Kimball and R. G. Wolfe. Malate dehydrogenase: A higher molecular weight form produced by freeze-thaw treatment of pig heart supernatant enzyme. Arch. Biochem. Biophys. 181:33–38 (1977).

    Google Scholar 

  6. C. Domenech, X. Bozal, A. Mazo, A. Cortes, and J. Bozal. Factors affecting malate dehydrogenase activity in freezingthawing processes. Comp. Biochem. Physiol. 88B:461–466 (1987).

    Google Scholar 

  7. R. I. N. Greaves. Theoretical aspects of drying by vacuum sublimation. In R. J. C. Harris (ed.), Biological applications of freezing and drying, Academic Press, New York, 1954, pp 87–126.

    Google Scholar 

  8. P. J. Campbell. Biological standards: problems in large-scale production. Develop. biol. Standard. 36:355–363 (1977).

    Google Scholar 

  9. B. M. Eckhardt, J. Q. Qeswein, and T. A. Bewley. Effect of freezing on aggregation of human growth hormone. Pharm. Res. 8:1360–1364 (1991).

    Google Scholar 

  10. P. Douzou. Cryobiochemistry: An introduction, Academic Press, New York, 1977.

    Google Scholar 

  11. F. Franks. Protein stability and function at low temperatures. Cryo-letters 8:108–115 (1987).

    Google Scholar 

  12. T. Koseki, N. Kitabatake, and E. Doi. Freezing denaturation of ovalbumin at acid pH. J. Biochem. 107:389–394 (1990).

    Google Scholar 

  13. M. W. Townsend and P. P. Deluca. Stability of ribonuclease A in solution and the freeze-dried state. J. Pharm. Sci. 79:1083–1086 (1990).

    Google Scholar 

  14. W. R. Liu, R. Langer, and A. M. Klibanov. Moisture-induced aggregation of lyophilized proteins in the solid state. Biotech. Bioeng. 37:177–184 (1991).

    Google Scholar 

  15. M. J. Hageman. The role of moisture in protein stability. Drug Dev. Ind. Pharm. 14:2047–2070 (1988).

    Google Scholar 

  16. D. Greiff. Protein structure and freeze drying: the effects of residual moisture and gases. Cryobiology 8:145–152 (1971).

    Google Scholar 

  17. C. C. Hsu, C. A. Ward, R. Pearlman, H. M. Nguyen, D. A. Yeung, and J. G. Curley. Determining the optimum residual moisture in lyophilized protein pharmaceuticals. Develop. biol. Standard. 74:255–272 (1991).

    Google Scholar 

  18. F. Franks. Freeze-drying: from empiricism to predictability-The significance of glass transitions. Develop. biol. Standard. 74:9–19 (1991).

    Google Scholar 

  19. F. Franks, R. H. M. Hatley, and S. F. Mathia. Materials science and the production of shelf-stable biologicals. Pharm. Technol. 3:32–50 (1992).

    Google Scholar 

  20. M. L. Roy, M. J. Pikal, E. C. Rickard, and A. M. Maloney. The effects of formulation and moisture on the stability of a freezedried monoclonal antibody-vinca conjugate: a test of the WLF glass transition theory. Develop. biol. Standard. 74:323–340 (1991).

    Google Scholar 

  21. J. F. Carpenter and J. H. Crowe. An infrared spectroscopy study of the interactions of carbohydrates with dried proteins. Biochem. 28:3916–3922 (1989).

    Google Scholar 

  22. J. F. Carpenter, T. Arakawa, and J. H. Crowe. Interactions of stabilizing additives with proteins during freeze-thawing and freeze-drying. Develop. biol. Standard. 74:225–239 (1991).

    Google Scholar 

  23. T. Arakawa, S. Prestrelski, W. C. Kenney, and J. F. Carpenter. Factors affecting short-term and long-term stabilities of proteins. Adv. Drug Delivery Reviews 10:1–28 (1993).

    Google Scholar 

  24. G. A. Vehar, M. W. Spellman, B. A. Keyt, C. K. Ferguson, R. G. Keck, R. C. Chloupek, R. Harris, W. F. Bennet, S. E. Builder, and W. S. Hancock. Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology. Cold Spring Harbor Symposia on Quantitative Biology 51:551–562 (1986).

    Google Scholar 

  25. M. W. Spellman, L. J. Basa, C. K. Leonard, J. A. Chakel, J. V. O'Connor, S. Wilson, and H. van Halbeek. Carbohydrate structures of human tissue plasminogen activator expressed in Chinese Hamster Ovary cells. J. Biol. Chem. 264:14100–14111 (1989).

    Google Scholar 

  26. U. Hartmann, B. Nunner, CH. Körber, and G. Rau. Where should the cooling rate be determined in an extended freezing sample. Cryobiology 28:115–130 (1991).

    Google Scholar 

  27. S. H. Maron and C. F. Prutton. Types II–V adsorption isotherms and determination of surface area of adsorbents. In Principles of Physical Chemistry (4 th ed.), Macmillan Company, New York, 1965, pp 817–819.

    Google Scholar 

  28. B. M. Eckhardt, J. Q. Oeswein, D. A. Yeung, T. D. Milby, and T. A. Bewley. A turbidimetric method to determine visual appearance of protein solutions. J. Parent. Sci. Technol. 48:64–70 (1994).

    Google Scholar 

  29. R. H. Carlson, R. L. Garnick, A. J. Jones, and A. M. Meunier. The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer. Anal. Biochem. 168:428–435 (1987).

    Google Scholar 

  30. L. Her and S. L. Nail. Measurement of glass transition temperatures of freeze-concentrated solutes by differential scanning calorimetry. Pharm. Research. 11:54–59 (1994).

    Google Scholar 

  31. J. Q. Oeswein and S. J. Shire. Physical biochemistry of protein drugs. In V. H. Lee (ed.), Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp 167–202.

    Google Scholar 

Download references

Author information

Author notes

  1. Gary S. Koe

    Present address: Chemical engineering Department, University of California, Los Angels, California, 90024

Authors and Affiliations

  1. Department of Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California, 94080

    Chung C. Hsu, Hoc M. Nguyen, Douglas A. Yeung, Dennis A. Brooks, Thomas A. Bewley & Rodney Pearlman

Authors
  1. Chung C. Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hoc M. Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas A. Yeung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dennis A. Brooks
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gary S. Koe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas A. Bewley
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rodney Pearlman
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hsu, C.C., Nguyen, H.M., Yeung, D.A. et al. Surface Denaturation at Solid-Void Interface—A Possible Pathway by Which Opalescent Participates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures. Pharm Res 12, 69–77 (1995). https://doi.org/10.1023/A:1016270103863

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1016270103863

Search

Navigation