Effect of Water on the Chemical Stability of Amorphous Pharmaceuticals: 2. Deamidation of Peptides and Proteins

Abstract

Role of water in chemical (in)stability is revisited, with focus on deamidation in freeze-dried amorphous proteins and peptides. Two distinct patterns for deamidation versus water have been reported, that is, a consistent increase in rate constant with water, and a "hockey stick"-type behavior. For the latter, deamidation is essentially independent of water at lower water contents and accelerates when water content increases above a threshold value. Two simple kinetic models are developed to analyze literature-reported relationships between water content and deamidation rate constants. One model is based on catalytic role of water clusters in enabling proton transfer, which is a critical reaction step. Water clusters are formed when water content increases above a threshold value, while unclustered (and less catalytically-active) water molecules are predominant at lower water levels. The second model considers the dual role of water, as both a destabilizer via catalysis and a stabilizer of protein native structure. Considering that both models emphasize the importance of local structure and that local structure is intrinsically related to fast (and non-cooperative) relaxation modes, it is plausible to expect correlations between local mobility, such as beta-relaxation, and amorphous chemical instability.

Freezing of Aqueous Solutions and Chemical Stability of Amorphous Pharmaceuticals: Water Clusters Hypothesis

“…In recent reviews, the role of water in the chemical instability of amorphous materials was reconsidered, with the focus on the amide hydrolysis and deamidation reactions. 80,81 The analysis of several case studies enabled the authors to highlight 2 probable mechanisms, that is, water as plasticizer/antiplasticizer of local mobility and fast relaxation modes (as previously proposed by Cicerone et al 7,8 ) and water as a reaction medium, in particular, in water-supported proton transfer. It was also noted that for proteins, which possess higher-order structure, water could play a dual role as both a destabilizer, via water catalysis, and a stabilizer of protein native structure (role of water in the maintenance of protein higher level structure was reviewed elsewhere 82 ).…”

“…It was also noted that for proteins, which possess higher-order structure, water could play a dual role as both a destabilizer, via water catalysis, and a stabilizer of protein native structure (role of water in the maintenance of protein higher level structure was reviewed elsewhere 82 ). It was concluded 80,81 that the hydrolysis and other relevant reactions in the amorphous solid state are under chemical (rather than diffusion) control (e.g., for amide hydrolysis in zoniporide 43 ) and that global alpha relaxation is probably not a chemical-stabilityedefining factor in these systems. This conclusion is consistent with the emerging view that reactions that do not require whole molecule diffusion (i.e., motion over long length scales) are essentially uncoupled from structural relaxation dynamics.…”

“…The critical role of water clusters as the media for proton transfer has been invoked to explain experimentally observed kinetic curves (original data reported in Pikal et al, 121 Chang et al, 84 and Breen et al 122 ) for chemical degradation via deamidation of several freeze-dried proteins. 81 In these cases, the deamidation rate constants were essentially independent of water in the lower water content range, 84,121 whereas the reaction accelerates upon increasing water content above a threshold. 122 An example of such "hockey stick-type" relationship between degradation rate and water content is presented in Figure 10, which also shows the treatment of the experimental data using a "water content threshold" model.…”

“…Acceleration of deamidation reaction at higher water contents was attributed to the formation of water clusters which facilitate the proton transfer. 81 It should be noted that there are other possible explanations of the hockey-stick type stability curves. One alternative model to describe the acceleration above certain water content is based on the dual role of water (i.e., both stabilizer of the higher-order structure and destabilizer via water catalysis) in the stability of freeze-dried proteins.…”

“…One alternative model to describe the acceleration above certain water content is based on the dual role of water (i.e., both stabilizer of the higher-order structure and destabilizer via water catalysis) in the stability of freeze-dried proteins. 81 Finally, the sharp increase in the deamidation rate ( Fig. 10) coincided with the increase in water content above the Wg value, that is, water content associated with glass-to-liquid transition, thus leading to quite logical "glass transition" interpretation in the original publication by Breen et al 122 Note, however, that more recent studies point to weak correlations between alpha-relaxation and chemical degradation, 78,79 thus weakening the mechanistic foundation of the "glass transition" explanation of chemical instabilities.…”