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Kinetics and Mechanisms of Deamidation and Covalent Amide-Linked Adduct Formation in Amorphous Lyophiles of a Model Asparagine-Containing Peptide

Pharmaceutical Research volume 29, pages 2722–2737 (2012)Cite this article

ABSTRACT

Purpose

Asparagine containing peptides and proteins undergo deamidation via a succinimide intermediate. This study examines the role of the succinimide in the formation of covalent, amide-linked adducts in amorphous peptide formulations.

Methods

Stability studies of a model peptide, Gly-Phe-L-Asn-Gly, were performed in lyophiles containing an excess of Gly-Val at ‘pH’ 9.5 and 40°C/40% RH. Reactant disappearance and the formation of ten different degradants were monitored by HPLC. Mechanism-based kinetic models were used to generate rate constants from the concentration vs. time profiles.

Results

Deamidation of Gly-Phe-L-Asn-Gly in lyophiles resulted in L- and D-aspartyl and isoaspartyl-containing peptides and four amide-linked adducts between the succinimide and Gly-Val. The kinetic analysis demonstrated competition between water and terminal amino groups in Gly-Val for the succinimide. The extent of covalent adduct formation was dependent on dilution effects due to its second order rate law.

Conclusion

The cyclic imide formed during deamidation of asparagine containing peptides in lyophiles can also lead to covalent adducts due to reaction with other neighboring peptides. A reaction model assuming a central role for the succinimide in the formation both hydrolysis products and covalent adducts was quantitatively consistent with the kinetic data. This mechanism may contribute to the presence of covalent, non-reducible aggregates in lyophilized peptide formulations.

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ACKNOWLEDGMENTS & DISCLOSURES

The authors thank the faculty at the University of Kentucky Mass Spectrometry Facility for generating the mass spectra and for their helpful discussions. Partial support for this project was provided by the H.B. Kostenbauder Endowed Professorship.

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  1. Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, A323A ASTeCC Bldg., Lexington, Kentucky, 40536-0082, USA

    Michael P. DeHart & Bradley D. Anderson

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  1. Michael P. DeHart
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Correspondence to Bradley D. Anderson.

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DeHart, M.P., Anderson, B.D. Kinetics and Mechanisms of Deamidation and Covalent Amide-Linked Adduct Formation in Amorphous Lyophiles of a Model Asparagine-Containing Peptide. Pharm Res 29, 2722–2737 (2012). https://doi.org/10.1007/s11095-011-0591-6

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  • DOI: https://doi.org/10.1007/s11095-011-0591-6

KEY WORDS

  • aggregation
  • amorphous reaction kinetics
  • covalent adducts
  • deamidation
  • lyophilization