Chemical Stability of Peptides in Polymers. 1. Effect of Water on Peptide Deamidation in Poly(vinyl alcohol) and Poly(vinyl pyrrolidone) Matrixes MEI C. LAI, † MICHAEL J. HAGEMAN, †,‡ RICHARD L. SCHOWEN, † RONALD T. BORCHARDT, † AND ELIZABETH M. TOPP* ,† Contribution from Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, and Pharmaceutical Development I, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49001. Received May 27, 1998. Final revised manuscript received July 21, 1999. Accepted for publication July 21, 1999. Abstract 0 This paper examines the effect of water content, water activity, and glass transition temperature (T g ) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 °C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing T g . The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or T g ) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of T g from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 °C, suggesting that the physical state of formulations could be used to predict chemical stability. Introduction Many proteins are formulated with polymers to protect against degradation during storage and/or processing or to provide a matrix for controlled release. Moisture content, polymer composition, and temperature can affect the stability of solid protein formulations by influencing the rates of chemical degradation reactions, such as asparagine deamidation. 1-4 Hydration of these solid formulations can easily occur during processing, storage, or after in vivo implantation. However, although the importance of poly- mer selection and moisture content is recognized, a com- plete mechanistic understanding of their effects on the chemical stability of proteins has not been developed. This manuscript addresses this mechanistic issue by examining the deamidation of a model hexapeptide (Val-Tyr-Pro-Asn- Gly-Ala, Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinylpyrrolidone) (PVP) matrixes at various hydration levels at a constant temperature of 50 °C. Water can affect the chemical stability of solid protein formulations in at least three ways: (1) as a solvent, (2) as a reactant in a reaction such as hydrolysis, and/or (3) as a plasticizer. 1,5 Water as a plasticizer of amorphous solids induces a physical transition from a brittle, dynami- cally constrained glassy state to a more mobile, less viscous, rubbery state at some temperature T g , the glass transition temperature. 1 Because many reactions require sufficient mobility of the reactants to proceed, an increase in mobility with water content could promote chemical reactivity. 1,6,7 The decrease in T g with increasing water content is often cited as an important factor in protein degradation in solids. 1,2,4,8 The effect of water as a plasticizer may be qualitatively described by changes in T g produced by a given amount of water. The effect of water as a solvent or reactant should be related to its concentration (water content) or chemical potential (water activity). We report here the effects of water on the stability of the Asn- hexapeptide in solid formulations as a function of water content, water activity, and T g . Deamidation at asparagine residues is one of the most prevalent chemical instabilities in proteins and peptides. The Asn-hexapeptide was selected as a model compound because its deamidation kinetics and mechanisms have been well characterized in solution. 9,10 This degree of mechanistic understanding makes the Asn-hexapeptide ideally suited for a study of the effect of water on reaction kinetics and mechanisms in solid polymer matrixes. PVP and PVA were selected as model polymers because they are commonly used pharmaceutical excipients with a simple chemical structure. Both polymers are linear, amorphous, polar, and hydrophilic (Scheme 1). Each con- sists of a vinyl backbone with pendant functional group (N-pyrrolidone for PVP and hydroxyl for PVA) at a 1,3- separation. This paper examines the effect of water on Asn-hexapep- tide deamidation in PVA and PVP at 50 °C. The relation- ships between water content, water activity, and T g in these two polymer formulations will first be determined to characterize the effect of water on these polymer matrixes. Then the relationship between deamidation rates and residual moisture will be explored by correlating deami- dation rates with formulation water content, water activity, * To whom correspondence should be addressed. Telephone: (785) 864-3644. Fax: (785) 864-5736. E-mail: topp@ukans.edu. † The University of Kansas. ‡ Pharmacia & Upjohn, Inc. Scheme 1sStructures of poly(vinyl alcohol) (PVA) and poly(vinyl pyrroli- done) (PVP). © 1999, American Chemical Society and 10.1021/js980227g CCC: $18.00 Journal of Pharmaceutical Sciences / 1073 American Pharmaceutical Association Vol. 88, No. 10, October 1999 Published on Web 09/01/1999