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Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations

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Abstract

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (α-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8–A9 was observed. Formation of this split product is species dependent: bovine > porcine > human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

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REFERENCES

  1. W. O. Storvick and H. J. Henry. Effect of storage temperature on stability of commercial insulin preparations. Diabetes 17:499–502 (1968).

    Google Scholar 

  2. M. Pingel and A. Vølund. Stability of insulin preparations. Diabetes 21:805–813 (1972).

    Google Scholar 

  3. F. Sundby. Separation and characterization of acid-induced insulin transformation products by paper electrophoresis in 7 M urea. J. Biol. Chem. 237:3406–3411 (1962).

    Google Scholar 

  4. L. I. Slobin and F. H. Carpenter. The labile amide in insulin: Preparation of desalanine-desamido-insulin. Biochemistry 2:22–28 (1963).

    Google Scholar 

  5. B. V. Fisher and P. B. Porter. Stability of bovine insulin. J. Pharm. Pharmacol. 33:203–206 (1981).

    Google Scholar 

  6. R. L. Jackson, W. O. Storvick, C. S. Hollinden, L. E. Stroeh, and J. G. Stilz. Neutral regular insulin. Diabetes 21:235–245 (1972).

    Google Scholar 

  7. J. Schlichtkrull, M. Pingel, L. G. Heding, J. Brange, and K. H. Jørgensen. Insulin preparations with prolonged effect. In A. Hasselblatt and F. von Bruchhausen (eds.), Handbook of Experimental Pharmacology, New Series, Vol. XXXII/2, Springer-Verlag, Berlin, Heidelberg, New York, 1975, pp. 729–777.

    Google Scholar 

  8. J. Brange, B. Skelbaek-Pedersen, L. Langkjaer, U. Damgaard, H. Ege, S. Havelund, L. G. Heding, K. H. Jørgensen, J. Lykkeberg, J. Markussen, M. Pingel, and E. Rasmussen. Galenics of Insulin: The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations, Springer-Verlag, Berlin, Heidelberg, New York, London, Paris, Tokyo, 1987.

    Google Scholar 

  9. J. Schlichtkrull, J. Brange, A. H. Christiansen, O. Hallund, L. G. Heding, K. H. Jørgensen, S. M. Rasmussen, E. Sørensen, and A. Vølund. Monocomponent insulin and its clinical implications. Horm. Metab. Res. (Suppl. Ser.) 5:134–143 (1974).

    Google Scholar 

  10. J. Brange, S. Havelund, and P. Hougaard. Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations. Pharm. Res. 9:727–734 (1992).

    Google Scholar 

  11. M. C. Manning, K. Patel, and R. T. Borchardt. Stability of protein Pharmaceuticals. Pharm. Res. 6:903–918 (1989).

    Google Scholar 

  12. A. B. Robinson and C. J. Rudd. Deamidation of glutaminyl and asparaginyl residues in peptides and proteins. Current Topics in Cellular Regulation, Vol. 8, Academic Press, New York, 1974, pp. 247–295.

    Google Scholar 

  13. T. Geiger and S. Clarke. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J. Biol. Chem. 262:785–794 (1987).

    Google Scholar 

  14. J. Brange, L. Langkjær, S. Havelund, and E. Sørensen. Chemical stability of insulin: Neutral insulin solutions. Diabetologia 25:193 (1983) (abstr).

    Google Scholar 

  15. J. Brange, L. Langkjær, S. Havelund, and E. Sørensen. Chemical stability of insulin: Formation of desamidido insulins and other hydrolytic products in intermediate-and long-acting insulin preparations. Diabetes Res. Clin. Pract. Suppl. 1:67 (1985) (abstr).

    Google Scholar 

  16. J. Brange, S. Havelund, E. Hommel, E. Sørensen, and C. Kühl. Neutral insulin solutions physically stabilized by addition of Zn2+. Diabet. Med. 3:532–536 (1986).

    Google Scholar 

  17. R. Lura and V. Schirch. Role of peptide conformation in the rate and mechanism of deamidation of asparaginyl residues. Biochemistry 21:7671–7677 (1988).

    Google Scholar 

  18. S. J. Leach and H. Lindley. The kinetics of hydrolysis of the amide group in proteins and peptides. Part 2. Acid hydrolysis of glycyl-and l-leucyl-l-asparagine. Trans. Faraday Soc. 49:921–925 (1953).

    Google Scholar 

  19. H. T. Wright. Sequence and structure determinants of the nonenzymatic deamidation of asparagin and glutamine residues in proteins. Protein Eng. 4:283–294 (1991).

    Google Scholar 

  20. P. Bornstein and G. Balian. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 47:132–145 (1977).

    Google Scholar 

  21. Y. C. Meinwald, E. R. Stimson, and H. A. Scheraga. Deamidation of the asparaginyl-glycyl sequence. Int. J. Peptide Protein Res. 28:79–84 (1986).

    Google Scholar 

  22. R. C. Stephenson and S. Clarke. Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J. Biol. Chem. 264:6164–6170 (1989).

    Google Scholar 

  23. S. Clarke. Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int. J. Peptide Protein Res. 30:808–821 (1987).

    Google Scholar 

  24. C. E. M. Voorter, W. A. de Haard-Hoekman, P. J. M. van den Oetelaar, H. Bloemendal, and W. W. de Jong. Spontaneous peptide bond cleavage in aging α-crystallin through a succinimide intermediate. J. Biol. Chem. 263:19020–19023 (1988).

    Google Scholar 

  25. N. P. Bhatt, K. Patel, and R. T. Borchardt. Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone. Pharm. Res. 7:593–599 (1990).

    Google Scholar 

  26. A. A. Kossiakoff. Tertiary structure is a principal determinant to protein deamidation. Science 240:191–194 (1988).

    Google Scholar 

  27. A. Wollmer, B. Rannefeld, B. R. Johansen, K. R. Hejnaes, P. Balschmidt, and F. B. Hansen. Phenol-promoted structural transformation of insulin in solution. Biol. Chem. Hoppe-Seyler 368:903–911 (1987).

    Google Scholar 

  28. U. Derewenda, Z. Derewenda, E. J. Dodson, G. G. Dodson, C. D. Reynolds, G. D. Smith, C. Sparks, and D. Swenson. Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer. Nature 338:594–596 (1989).

    Google Scholar 

  29. R. Gregory, S. Edwards, and N. A. Yateman. Demonstration of insulin transformation products in insulin vials by high-performance liquid chromatography. Diabetes Care 14:42–48 (1991).

    Google Scholar 

  30. E. N. Baker, T. L. Blundell, J. F. Cutfield, S. M. Cutfield, E. J. Dodson, G. G. Dodson, D. M. C. Hodgkin, R. E. Hubbard, N. W. Isaacs, C. D. Reynolds, K. Sakabe, N. Sakabe, and N. M. Vijayan. The structure of 2Zn pig insulin crystals at 1.5 Å resolution. Phil. Trans. R. Soc. 319:369–456 (1988).

    Google Scholar 

  31. S. Toma, S. Campagnoli, E. De Gregoriis, R. Gianna, I. Margarit, M. Zamai, and G. Grandi. Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of bacillus subtilis neutral protease. Protein Eng. 2:359–364 (1989).

    Google Scholar 

  32. W. G. Reeves. Immunogenicity of insulin of various origins. Neth. J. Med. 28 (Suppl 1):43–46 (1985).

    Google Scholar 

  33. S. Fankhauser. Neuere Aspekte der Insulintherapie. Schweiz Med. Wochenschr. 99:414–420 (1969).

    Google Scholar 

  34. N. S. Fineberg, S. E. Fineberg, R. J. Mahler, and L. G. Linarelli. Is regular human insulin less immunogenic than repository? Diabetes 35 (Suppl 1):91A (1986) (abstr).

    Google Scholar 

  35. D. C Robbins, S. M. Cooper, S. E. Fineberg, and P. M. Mead. Antibodies to covalent aggregates of insulin in blood of insulinusing diabetic patients. Diabetes 36:838–841 (1987).

    Google Scholar 

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Authors and Affiliations

  1. Novo Research Institute, Novo Alle, DK-2880, Bagsvaerd, Denmark

    Jens Brange, Liselotte Langkj\sgmaelig;r, Svend Havelund & Aage Vølund

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  1. Jens Brange
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  2. Liselotte Langkj\sgmaelig;r
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  3. Svend Havelund
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  4. Aage Vølund
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To whom correspondence should be addressed at Nove Research Institute, Novo Alle, DK-2880Bagsvaerd, Denmark

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Brange, J., Langkj\sgmaelig;r, L., Havelund, S. et al. Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations. Pharm Res 9, 715–726 (1992). https://doi.org/10.1023/A:1015835017916

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