Skip to main content
SpringerLink
Log in

Stabilization of Lactate Dehydrogenase Following Freeze-Thawing and Vacuum-Drying in the Presence of Trehalose and Borate

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose. The purpose of this work was to investigate the effects of trehalose and trehalose/sodium tetraborate mixtures on the recovery of lactate dehydrogenase (LDH) activity following freeze-thawing and centrifugal vacuum-drying/rehydration. The storage stability of LDH under conditions of either high relative humidity or high temperature was also studied.

Methods. LDH was prepared in buffered aqueous formulations containing trehalose alone and trehalose/'borate' mixtures. Enzymatic activity was measured immediately following freeze-thawing and vacuum-drying/rehydration processes, and also after vacuum-dried formulations were stored in either high humidity or high temperature environments. Also, glass transition temperatures (Tg) were measured for both freeze-dried and vacuum-dried formulations.

Results. The Tgvalues of freeze-dried trehalose/borate mixtures are considerably higher than that of trehalose alone. Freezing and vacuum-drying LDH in the presence of 300 mM trehalose resulted in the recovery of 80% and 65% of the original activity, respectively. For vacuum-dried mixtures, boron concentrations below 1.2 mole boron/ mole trehalose had no effect on recovered LDH. After several weeks storage in either humid (100% relative humidity) or warm (45°C) environments, vacuum-dried formulations that included trehalose and borate showed greater enzymatic activities than those prepared with trehalose alone. We attribute this stability to the formation of a chemical complex between trehalose and borate.

Conclusions. The high Tgvalues of trehalose/borate mixtures offer several advantages over the use of trehalose alone. Most notable is the storage stability under conditions of high temperature and high relative humidity. In these cases, formulations that contain trehalose and borate are superior to those containing trehalose alone. These results have practical implications for long-term storage of biological materials.

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. T. Tamiya, N. Okahashi, R. Sakuma, T. Aoyama, T. Akahane, and J. Matsumoto. Freeze denaturation of enzymes and its prevention with additives. Cryobio. 22:446-456 (1985).

    Google Scholar 

  2. K. Izutsu, S. Yoshioka, and T. Terao. Stabilizing effect of amphiphilic excipients on the freeze-thawing and freeze-drying of lactate dehydrogenase. Biotech. Bioeng. 43:1102-1107 (1994).

    Google Scholar 

  3. R. H. M. Hatley and F. Franks. The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants. FEBS Letters 257:171-173 (1989).

    PubMed  Google Scholar 

  4. D. Greiff and R. T. Kelley. Cryotolerance of enzymes. I. Freezing of lactic dehydrogenase. Cryobio. 2:335-341 (1966).

    Google Scholar 

  5. J. H. Crowe, J. F. Carpenter, L. M. Crowe, and T. J. Anchordoguy. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobio. 27:219-231 (1990).

    Google Scholar 

  6. T. Arakawa and S. N. Timasheff. Stabilization of protein structure by sugars. Biochem. 21:6536-6544 (1982).

    Google Scholar 

  7. J. H. Crowe, L. M. Crowe, J. F. Carpenter, and C. A. Wistrom. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem J. 242:1-10 (1987).

    PubMed  Google Scholar 

  8. G. Xie and S. N. Timasheff. The thermodynamic mechanism of protein stabilization by trehalose. Biophys. Chem. 64:25-43 (1997).

    PubMed  Google Scholar 

  9. S. Y. Gerlsma. Reversible denaturation of ribonuclease in aqueous solutions as influenced by polyhydric alcohols and some other additives. J. Biol. Chem. 243:957-961 (1968).

    PubMed  Google Scholar 

  10. J. F. Back, D. Oakenfull, and M. B. Smith. Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18:5191-5196 (1979).

    PubMed  Google Scholar 

  11. T. Arakawa, Y. Kita, and J. F. Carpenter. Protein-solvent interactions in pharmaceutical formulations. Pharm. Res. 8:285-291 (1991).

    PubMed  Google Scholar 

  12. J. F. Carpenter and J. H. Crowe. The mechanism of cryoprotection of proteins by solutes. Cryobio. 25:244-255 (1988).

    Google Scholar 

  13. D. P. Miller, J. J. de Pablo, and H. R. Corti. Thermophysical properties of trehalose and its concentrated aqueous solutions. Pharm Res. 14:578-590 (1997).

    PubMed  Google Scholar 

  14. S. B. Leslie, E. Israeli, B. Lighthart, J. H. Crowe, and L. M. Crowe. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl. and Environ. Microbiol. 61:3592-3597 (1995).

    Google Scholar 

  15. L. M. Crowe, D. S. Reid, and J. H. Crowe. Is trehalose special for preserving dry biomaterials? Biophys. J. 71:2087-2093 (1996).

    PubMed  Google Scholar 

  16. J. S. Clegg. The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Comp. Biochem. Physiol. 14:135-143 (1965).

    PubMed  Google Scholar 

  17. G. Bianchi, A. Gamba, C. Murellie, F. Salamini, and D. Bartels. Novel carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. Plant J. 1:355-359 (1991).

    Google Scholar 

  18. J. H. Crowe. Anhydrobiosis: An unsolved problem. Am. Naturalist 105:563-573 (1971).

    Google Scholar 

  19. J. F. Carpenter and J. H. Crowe. Modes of stabilization of a protein by organic solutes during desiceation. Cryobio. 25:459-470 (1988).

    Google Scholar 

  20. H. Levine and L. Slade. Another view of trehalose for drying and stabilizing biological molecules. BioPharm 5:36-40 (1992).

    Google Scholar 

  21. B. J. Aldous, A. D. Auffret, and F. Franks. The crystallization of hydrates from amorphous carbohydrates. Cryo-Letters 16:181-186 (1995).

    Google Scholar 

  22. J. L. Green and C. A. Angell. Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly. J. Phys. Chem. 93:2880-2882 (1989).

    Google Scholar 

  23. K. Tanaka, T. Takeda, and K. Miyajima. Cryoprotective effect of saccharides on denaturation of catalase during freeze-drying. Chem. Pharm. Bull. 39:1091-1094 (1991).

    Google Scholar 

  24. J. H. Crowe, S. B. Leslie, and L. M. Crowe. Is vitrification sufficient to preserve liposomes during freeze drying? Cryobio. 31:355-366 (1994).

    Google Scholar 

  25. J. F. Carpenter, S. J. Prestrelski, and T. Arakawa. Separation of freezing-and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. Arch. Biochem. Biophys. 303:456-464 (1993).

    PubMed  Google Scholar 

  26. J. F. Carpenter, B. Martin, L. M. Crowe, and J. H. Crowe. Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures. Cryobio. 24:455-464 (1987).

    Google Scholar 

  27. J. Boeseken. The use of boric acid for the determination of the configuration of carbohydrates. Adv. Carbohydrate Chem. 4:189-210 (1949).

    Google Scholar 

  28. O. Noble and F. Taravel. Complex formation between guar D-galacto-D-mannan and borate ion. Thermodynamic data. Carbohydrate Res. 184:236-243 (1988).

    Google Scholar 

  29. E. Pezron, A. Ricard, F. Lafuma, and R. Audebert. Reversible gel formation induced by ion complexation 1. Borax-Galactomannan interactions. Macromol. 21:1121-1125 (1988).

    Google Scholar 

  30. J. G. Dawber, S. I. E. Green, J. C. Dawber, and S. Gabrail. A polarimetric and 11B and 13C nuclear magnetic resonance study of the reaction of the tetrahydroxyborate ion with polyols and carbohydrates. J. Chem. Soc., Faraday Trans. I, 84:41-56 (1988).

    Google Scholar 

  31. S. T. Ohnishi and J. K. Barr. A simplified method for quantitating protein using the biuret and phenol reagents. Analytical Biochem. 86:193-200 (1978).

    Google Scholar 

  32. L. van den Berg and D. Rose. Effect of freezing on the pH and composition of sodium and potassium phosphate solutions: The reciprocal system KH2PO4-Na2HPO4-H2O. Arch. Biochem. Biophys. 81:319-329 (1959).

    PubMed  Google Scholar 

  33. N. Murase and F. Franks. Salt precipitation during the freeze-concentration of phosphate buffer solutions. Biophysical Chem. 34:293-300 (1989).

    Google Scholar 

  34. K. Lippert and E. A. Galinski. Enzyme stabilization by ectoinetype compatible solutes: protection against heating, freezing and drying. Appl. Microbiol. Biotechnol. 37:61-65 (1992).

    Google Scholar 

  35. F. S. Soliman and L. van den Berg. Factors affecting freezing damage of lactic dehydrogenase. Cryobio. 8:73-78 (1970).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin, 53706

    Danforth P. Miller & Juan J. de Pablo

  2. Department of Chemical Engineering, University of California-Berkeley, Berkeley, California, 94720-1462

    Rebeccah E. Anderson

Authors
  1. Danforth P. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebeccah E. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juan J. de Pablo
    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

Miller, D.P., Anderson, R.E. & de Pablo, J.J. Stabilization of Lactate Dehydrogenase Following Freeze-Thawing and Vacuum-Drying in the Presence of Trehalose and Borate. Pharm Res 15, 1215–1221 (1998). https://doi.org/10.1023/A:1011987707515

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1011987707515

Search

Navigation