Pharmaceutical Research

, Volume 13, Issue 8, pp 1142–1153 | Cite as

Solid-State Stability of Human Insulin I. Mechanism and the Effect of Water on the Kinetics of Degradation in Lyophiles from pH 2–5 Solutions

  • Robert G. Strickley
  • Bradley D. Anderson


Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range.

Methods. Solutions of 1.0 mg/mL insulin were adjusted to pH 2–5 using HC1, freeze-dried, then exposed to various relative humidities at 35°C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance.

Results. Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH (‘pH’) yielding, at any given water content, solid-state ‘pH’-rate profiles parallel to the solution pH-rate profile. This ‘pH’ dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of ≈4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3–5 and at 35°C involves rate-limiting intramolecular nucleophilic attack of the AsnA21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., PheB1, and GlyAl) of another insulin molecule to generate either deamidated insulin (AspA21) or an amide-linked covalent dimer (e.g., [AspA21-PheB1] or [AspA21-GlyA1]), respectively. The rate of insulin degradation in lyophilized powders at 35°C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20–50%) required to induce a glass transition.

Conclusions. The decomposition of human insulin in lyophilized powders between pH 3–5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.

protein stability solid-state degradation deamidation covalent dimerization acyl transfer intramolecular catalysis 


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  1. 1.
    M. C. Manning, K. Patel and R. T. Borchardt. Stability of protein pharmaceuticals. Pharm. Res. 6:903–918 (1989).Google Scholar
  2. 2.
    R. Pearlman and T. H. Nguyen. Pharmaceutics of protein drugs. J. Pharm. Pharmacol. 44 (Suppl.):178–185 (1992).Google Scholar
  3. 3.
    M. J. Pikal. Freeze-drying of proteins: Process, formulation and stability. In J. L. Cleland, R. Langer, (ed.) Formulation and Delivery of Proteins and Peptides, American Chemical Society, Washington, D.C., 1994, 120–133.Google Scholar
  4. 4.
    Y. C. J. Wang and M. A. Hanson. Parenteral formulations of proteins and peptides: stability and stabilizers. J. Parenteral Sci. Techn. 42:S1–S24 (1988).Google Scholar
  5. 5.
    M. J. Hageman. The role of moisture in protein stability. Drug Dev. Ind. Pharm. 14:2047–2070 (1988).Google Scholar
  6. 6.
    M. J. Hageman. Water Sorption and Solid State Stability of Proteins. In T. J. Ahern, M. C. Manning, (ed.) Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation, Plenum, New York, 1992Google Scholar
  7. 7.
    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
  8. 8.
    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
  9. 9.
    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. Dev. Biol. Standard 74:323–340 (1992).Google Scholar
  10. 10.
    H. Levine and L. Slade. Interpreting the behavior of low moisture foods. In T. M. Hardman, (ed.) Water and Food Quality, Elsevier Applied Science, London, 1989, 71–134.Google Scholar
  11. 11.
    L. Slade, H. Levine and J. W. Finley. Protein-water interactions: Water as a plasticizer of gluten and other protein polymers. In R. D. Phillips, J. W. Finley, (ed.) Protein Quality and the Effects of Processing, Dekker, New York, 1989, 9–124.Google Scholar
  12. 12.
    Y. Roos and M. Karel. Differential scanning calorimetry study of phase transitions affecting quality of dehydrated materials. Biotechnol. Prog. 6:159–163 (1990).Google Scholar
  13. 13.
    K. J. Palmer, W. B. Dye and D. Black. X-ray diffractometer and microscopic investigation of crystallization of amorphous sucrose. J. Agric. Food Chem. 4:77–81 (1956).Google Scholar
  14. 14.
    M. Otsuka and N. Kaneniwa. Hygroscopicity and solubility of noncrystalline cephalexin. Chem. Pharm. Bull. 31:230–236 (1983).Google Scholar
  15. 15.
    B. Makower and W. B. Dye. Equilibrium moisture content and crystallization of amorphous sucrose and glucose. J. Agric. Food Chem. 4:72–77 (1956).Google Scholar
  16. 16.
    E. Fukuoka, M. Makita and S. Yamamura. Glassy state of pharma-ceuticals. III. Thermal properties and stability of glassy pharmaceuticals and their binary glass systems. Chem. Pharm. Bull. 37:1047–1050 (1989).Google Scholar
  17. 17.
    J. A. Rupley, E. Gratton and G. Careri. Water and globular proteins. Trends Biochem. Sci. 8:18–22 (1983).Google Scholar
  18. 18.
    M. J. Pikal, A. L. Lukes and J. E. Lang. Thermal decomposition of amorphous β-lactam antibiotics. J. Pharm. Sci. 66:1312–1316 (1977).Google Scholar
  19. 19.
    B. C. Hancock, S. L. Shamblin and G. Zografi. Molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures. Pharm. Res. 12:799–806 (1995).Google Scholar
  20. 20.
    M. J. Hageman, J. M. Bauer, P. L. Possert and R. T. Darrington. Preformulation studies oriented toward sustained delivery of recombinant somatotropins. J. Agric. Food Chem. 40:348–355 (1992).Google Scholar
  21. 21.
    C. Oliyai and R. T. Borchardt. Solution and solid state chemical instabilities of asparaginyl and aspartyl residues in model peptides. In J. L. Cleland, R. Langer, (ed.) Formulation and Delivery of Proteins and Peptides, American Chemical Society, Washington, D.C., 1994, 46–58.Google Scholar
  22. 22.
    R. T. Darrington and B. D. Anderson. The role of intramolecular nucleophilic catalysis and the effects of self-association on the deamidation of human insulin at low pH. Pharm. Res. 11:784–793 (1994).Google Scholar
  23. 23.
    R. T. Darrington and B. D. Anderson. Effects of insulin concentration and self-association of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimer. Pharm. Res. 12:1077–1084 (1995).Google Scholar
  24. 24.
    R. T. Darrington and B. D. Anderson. Evidence for a common intermediate in insulin deamidation and covalent dimer formation: Effects of pH and aniline trapping in dilute acidic solutions. J. Pharm. Sci. 84:275–282 (1995).Google Scholar
  25. 25.
    S. Brunauer, P. H. Emmett and E. Teller. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60:309–319 (1938).Google Scholar
  26. 26.
    C. van den Berg and S. Bruin. Water activity and its estimation in food systems: Theoretical aspects. In L. B. Rockland, G. F. Stewart, (ed.) Water Activity: Influences of Food Quality, Academic Press, New York, 1981, 1–61.Google Scholar
  27. 27.
    U. Grau. Fingerprint analysis of insulin and proinsulins. Diabetes 34:1174–1180 (1985).Google Scholar
  28. 28.
    G. Careri, E. Gratton, P.-H. Yang and J. A. Rupley. Correlation of IR spectroscopic, heat capacity, diamagnetic susceptibility and enzymatic measurements on lysozyme powder. Nature 284:572–573 (1980).Google Scholar
  29. 29.
    P. L. Poole and J. L. Finney. Sequential hydration of dry proteins: A direct difference IR investigation of sequence homologs lysozyme and α-lactalbumin. Biopolymers 23:1647–1666 (1984).Google Scholar
  30. 30.
    C. A. Oksanen and G. Zografi. The relationship between the glass transition temperature and water vapor absorption by poly(vinylpyrrolidone). Pharm. Res. 7:654–657 (1990).Google Scholar
  31. 31.
    C. A. Oksanen and G. Zografi. Molecular mobility in mixtures of absorbed water and solid poly(vinylpyrrolidone). Pharm. Res. 10:791–799 (1993).Google Scholar
  32. 32.
    H. Levine and L. Slade. Water as a plasticizer: Physico-chemical aspects of low-moisture polymeric systems. In F. Franks, (ed.) Water Science Reviews, Cambridge University Press, Cambridge, 1987, 79–185.Google Scholar
  33. 33.
    C. Ahlneck and G. Zografi. The molecular basis of moisture effects on the physical and chemical stability of drugs in the solid state. Int. J. Pharm. 62:87–95 (1990).Google Scholar
  34. 34.
    M. D. Sorenson and J. J. Led. Structural details of Asp(B9) human insulin at low pH from two-dimensional NMR titration studies. Biochemistry 33:13727–13733 (1994).Google Scholar
  35. 35.
    M. Baudys, T. Uchio, D. Mix, D. Wilson and S. W. Kim. Physical stabilization of insulin by glycosylation. J. Pharm. Sci. 84:28–33 (1995).Google Scholar
  36. 36.
    Y.-K. Chan, G. Oda and H. Kaplan. Chemical properties of the functional groups of insulin. Biochem. J. 193:419–425 (1981).Google Scholar
  37. 37.
    M. J. Pikal, K. M. Dellerman, M. L. Roy and R. M. Riggin. The effects of formulation variables on the stability of freeze-dried human growth hormone. Pharm. Res. 8:427–436 (1991).Google Scholar
  38. 38.
    J. Zhang, T. C. Lee and C.-T. Ho. Thermal deamidation of proteins in a restricted water environment. J. Agric. Food Chem. 41:1840–1843 (1993).Google Scholar
  39. 39.
    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
  40. 40.
    R. G. Strickley, M. Brandl, K. W. Chan, K. Straug and L. Gu. High-performance liquid chromatography (HPLC) and HPLC-mass spectroscopic (MS) analysis of the degradation of the luteinizing hormone-releasing hormone (LH-RH) antagonist RS-26306 in aqueous solution. Pharm. Res. 7:530–536 (1990).Google Scholar
  41. 41.
    C. Oliyai, J. P. Patel, L. Carr and R. T. Borchardt. Chemical pathways of peptide degradation. VII. Solid state chemical instability of an aspartyl residue in a model hexapeptide. Pharm. Res. 11:901–908 (1994).Google Scholar
  42. 42.
    Z. Shahrokh, G. Eberlein, D. Buckley, M. V. Paranandi, D. W. Aswad, P. Stratton, R. Mischak and Y. J. Wang. Major degradation products of basic fibroblast growth factor: Detection of succinimide and iso-aspartate in place of aspartate. Pharm. Res. 11:936–944 (1994).Google Scholar
  43. 43.
    J. A. Straub, A. Akiyama, P. Parmar and G. F. Musso. Chemical pathways of degradation of the bradykinin analog, RMP-7. Pharm. Res. 12:305–308 (1995).Google Scholar
  44. 44.
    C. A. Angell. Formation of glasses from liquids and biopolymers. Science 267:1924–1935 (1995).Google Scholar
  45. 45.
    F. Franks, R. H. M. Hatley and S. F. Mathias. Material science and the production of shelf-stable biologicals. Biopharm. 4:38–55 (1991).Google Scholar
  46. 46.
    L. Slade and H. Levine. Beyond water activity: Recent advances based on an alternative approach to the assessment of food quality and safety. Crit. Rev. Food Sci. and Nutrit. 30:115–360 (1991).Google Scholar
  47. 47.
    K. Kohlhammer, G. Kothe, B. Reck and H. Ringsdorf. Deuteron NMR relaxation studies of combined main-chain/side-chain polymers in the liquid crystalline and glassy state. Ber. Bunsen-Ges. Phys. Chem. 93:1323–1325 (1989).Google Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Robert G. Strickley
    • 1
  • Bradley D. Anderson
    • 3
  1. 1.Department of Pharmaceutics and Pharmaceutical Chemistry College of PharmacyUniversity of UtahSalt Lake City
  2. 2.Genentech, Inc.South San Francisco
  3. 3.Department of Pharmaceutics and Pharmaceutical Chemistry, College of PharmacyUniversity of UtahSalt Lake City

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