Skip to main content
SpringerLink
Log in

Effect of Ethylenediamine on Chemical Degradation of Insulin Aspart in Pharmaceutical Solutions

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

To examine the effect of different amine compounds on the chemical degradation of insulin aspart at pharmaceutical formulation conditions.

Methods

Insulin aspart preparations containing amine compounds or phosphate (reference) were prepared and the chemical degradation was assessed following storage at 37°C using chromatographic techniques. Ethylenediamine was examined at multiple concentrations and the resulting insulin–ethylenediamine derivates were structurally characterized using matrix assisted laser desorption ionization time-of-flight mass spectroscopy. The effects on ethylenediamine when omitting glycerol or phenolic compounds from the formulations were investigated.

Results

Ethylenediamine was superior in terms of reducing formation of high molecular weight protein and insulin aspart related impurities compared to the other amine compounds and phosphate. Monotransamidation of insulin aspart in the presence of ethylenediamine was observed at all of the six possible Asn/Gln residues with AsnA21 having the highest propensity to react with ethylenediamine. Data from formulations studies suggests a dual mechanism of ethylenediamine and a mandatory presence of phenolic compounds to obtain the effect.

Conclusions

The formation of high molecular weight protein and insulin aspart related impurities was reduced by ethylenediamine in a concentration dependant manner.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

AMPD:

2-amino-2-methyl-1,3-propanediol

BIS-TRIS:

2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol

DaMo :

monoisotropic mass in Dalton

DesPhe:

desPheB1-N-oxalyl-ValB2

DTT:

1,4-dithiothreitol

GPC:

gel permeation chromatography

HMWP:

high molecular weight protein

IARI:

insulin aspart related impurities

IEC:

ion exchange chromatography

MALDI-TOF:

matrix assisted laser desorption ionization time of flight

OR:

other related impurities

RP-HPLC:

reverse phase high-performance liquid chromatography

TCEP:

tris(2-carboxyethyl)phosphine

T.E.A.:

tetraethylammonium

References

  1. W. Wang. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int. J. Pharm. 185:129–188 (1999) doi:10.1016/S0378-5173(99)00152-0.

    Article  PubMed  CAS  Google Scholar 

  2. J. Brange, B. Skelbaek-Pedersen, L. Langkjær, U. Damgaard, H. Ege, S. Havelund, L. G. Heding, K. H. Jørgensen, J. Lykkeberg, J. Markussen, and M. Pingel. Galenics of insulin. the physico-chemical and pharmaceutical aspects of insulin and insulin preparations. Springer, Berlin, 1987.

    Google Scholar 

  3. J. Brange, and L. Langkjær. Insulin structure and stability. In Y. J. Wang, and R. Pearlman (eds.), Stability and Characterization of Protein and Peptide Drugs: Case Histories, Plenum, New York, 1993, pp. 315–350.

    Google Scholar 

  4. J. Brange, L. Langkjaer, S. Havelund, and A. Volund. Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations. Pharm. Res. 9:715–726 (1992) doi:10.1023/A:1015835017916.

    Article  PubMed  CAS  Google Scholar 

  5. 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) doi:10.1002/jps.2600840303.

    Article  PubMed  CAS  Google Scholar 

  6. R. T. Darrington, and B. D. Anderson. Effects of insulin concentration and self-association on the partitioning of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimmer. Pharm. Res. 12:1077–1084 (1995) doi:10.1023/A:1016231019677.

    Article  PubMed  CAS  Google Scholar 

  7. 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) doi:10.1023/A:1018909220255.

    Article  PubMed  CAS  Google Scholar 

  8. J. Brange. Chemical-stability of insulin. 4. Mechanisms and kinetics of chemical-transformations in pharmaceutical formulation. Act. Pharm. N. 4:209–222 (1992).

    CAS  Google Scholar 

  9. M. U. Jars, A. Hvass, and D. Waaben. Insulin aspart (Asp(B28) human insulin) derivatives formed in pharmaceutical solutions. Pharm. Res. 19:621–628 (2002) doi:10.1023/A:1015302012070.

    Article  PubMed  CAS  Google Scholar 

  10. J. Bello, and H. R. Bello. Chemical modification and cross-linking of proteins by impurities in glycerol. Arch. Biochem. Biophys. 172:608–610 (1976) doi:10.1016/0003-9861(76)90114-4.

    Article  PubMed  CAS  Google Scholar 

  11. 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) doi:10.1023/A:1015887001987.

    Article  PubMed  CAS  Google Scholar 

  12. J. Brange, and L. Langkjaer. Chemical stability of insulin. 3. Influence of excipients, formulation, and pH. Act. Pharm. N. 4:149–158 (1992).

    CAS  Google Scholar 

  13. J. Brange, O. Hallund, and E. Sorensen. Chemical stability of insulin. 5. Isolation, characterization and identification of insulin transformation products. Act. Pharm. N. 4:223–232 (1992).

    CAS  Google Scholar 

  14. R. C. Beavis, M. D. Kneirman, D. Sharknas, M. A. Heady, B. H. Frank, and M. R. DeFelippis. A novel protein cross-linking reaction in stressed Neutral Protamine Hagedorn formulations of insulin. J. Pharm. Sci. 88:331–336 (1999) doi:10.1021/js9802603.

    Article  PubMed  CAS  Google Scholar 

  15. A. S. Acharya, and J. M. Manning. Reaction of glycolaldehyde with proteins: latent crosslinking potential of alpha-hydroxyaldehydes. Proc. Natl. Acad. Sci. U S A. 80:3590–3594 (1983) doi:10.1073/pnas.80.12.3590.

    Article  PubMed  CAS  Google Scholar 

  16. R. E. Ratner, T. M. Phillips, and M. Steiner. Persistent cutaneous insulin allergy resulting from high- molecular-weight insulin aggregates. Diabetes. 39:728–733 (1990) doi:10.2337/diabetes.39.6.728.

    Article  PubMed  CAS  Google Scholar 

  17. G. Schernthaner. Immunogenicity and allergenic potential of animal and human insulins. Diabet. Care. 16(Suppl 3):155–165 (1993).

    Google Scholar 

  18. W. G. Reeves. The immune response to insulin: characterisation and clinical consequences. Diabet. Annual. 2:81–93 (1986).

    Google Scholar 

  19. D. C. Robbins, S. M. Cooper, S. E. Fineberg, and P. M. Mead. Antibodies to covalent aggregates of insulin in blood of insulin-using diabetic patients. Diabetes. 36:838–841 (1987) doi:10.2337/diabetes.36.7.838.

    Article  PubMed  CAS  Google Scholar 

  20. M. Maislos, P. M. Mead, D. H. Gaynor, and D. C. Robbins. The source of the circulating aggregate of insulin in type I diabetic patients is therapeutic insulin. J. Clin. Invest. 77:717–723 (1986) doi:10.1172/JCI112366.

    Article  PubMed  CAS  Google Scholar 

  21. J. Markussen, I. Diers, P. Hougaard, L. Langkjaer, K. Norris, L. Snel, A. R. Sorensen, E. Sorensen, and H. O. Voigt. Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30. Protein Eng. 2:157–166 (1988) doi:10.1093/protein/2.2.157.

    Article  PubMed  CAS  Google Scholar 

  22. M. G. Sheffer, and H. Kaplan. Unusual chemical properties of the amino groups of insulin implications for structure function relationship. Can. J. Biochem. 57:489–496 (1979).

    Article  PubMed  CAS  Google Scholar 

  23. The Merck Index. An encyclopedia of chemicals, drugs and biologicals. Merck Research Laboratories, Whitehouse Station, 2001.

    Google Scholar 

  24. Y. K. Chan, G. Oda, and H. Kaplan. Chemical properties of the functional groups of insulin. Biochem. J. 193:419–425 (1981).

    PubMed  CAS  Google Scholar 

  25. K. Huus, S. Havelund, H. B. Olsen, M. van de Weert, and S. Frokjaer. Chemical and thermal stability of insulin: effects of zinc and ligand binding to the insulin zinc-hexamer. Pharm. Res. 23:2611–2620 (2006) doi:10.1007/s11095-006-9098-y.

    Article  PubMed  CAS  Google Scholar 

  26. G. D. Smith, and G. G. Dodson. Structure of a rhombohedral R6 insulin/phenol complex. Proteins. 14:401–408 (1992) doi:10.1002/prot.340140309.

    Article  PubMed  CAS  Google Scholar 

  27. G. D. Smith, E. Ciszak, L. A. Magrum, W. A. Pangborn, and R. H. Blessing. R6 hexameric insulin complexed with m-cresol or resorcinol. Acta Crystallogr., Sect D: Biol. Crystallogr. 56:1541–1548 (2000) doi:10.1107/S0907444900012749.

    Article  CAS  Google Scholar 

  28. J. L. Whittingham, D. J. Edwards, A. A. Antson, J. M. Clarkson, and G. G. Dodson. Interactions of phenol and m-cresol in the insulin hexamer, and their effect on the association properties of B28 Pro -> Asp insulin analogues. Biochemistry. 37:11516–11523 (1998) doi:10.1021/bi980807s.

    Article  PubMed  CAS  Google Scholar 

  29. M. F. Dunn. Zinc-ligand interactions modulate assembly and stability of the insulin hexamer—a review. BioMetals. 18:295–303 (2005) doi:10.1007/s10534-005-3685-y.

    Article  PubMed  CAS  Google Scholar 

  30. T. Blundell, G. Dodson, D. Hodgkin, and D. Mercola. Insulin the structure in the crystal and its reflection in chemistry and biology. Adv. Protein Chem. 26:279–402 (1972).

    Article  CAS  Google Scholar 

  31. K. C. Waterman, and R. C. Adami. Accelerated aging: prediction of chemical stability of pharmaceuticals. Int. J. Pharm. 293:101–125 (2005) doi:10.1016/j.ijpharm.2004.12.013.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Dorte Aarup Valore and Trine Tølløse for preparing the insulin formulations and conducting the stability studies, Tina Lykke Larsen for performing the HPLC analyses, and Dorte Christensen for technical assistance during the characterization studies.

Author information

Authors and Affiliations

  1. Pharmaceutics, Diabetes Department, Novo Nordisk A/S, Novo Alle, room 6B1.58.1, 2880, Bagsvaerd, Denmark

    Christian Poulsen

  2. Protein Chemistry, Diabetes Department, Novo Nordisk A/S, 2880, Bagsvaerd, Denmark

    Dorte Jacobsen

  3. Structural Characterization, Novo Nordisk A/S, 2760, Maaloev, Denmark

    Lisbeth Palm

Authors
  1. Christian Poulsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dorte Jacobsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisbeth Palm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian Poulsen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Poulsen, C., Jacobsen, D. & Palm, L. Effect of Ethylenediamine on Chemical Degradation of Insulin Aspart in Pharmaceutical Solutions. Pharm Res 25, 2534–2544 (2008). https://doi.org/10.1007/s11095-008-9670-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-008-9670-8

KEY WORDS

Search

Navigation