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The amorphous state is widespread both in natural materials (such as resins, wax, and amber) and artificial materials (paraffin, tar, bitumen, plasticine, adhesives, glass, plastics). Amorphous solids (ASs) are also called 'glassy'. The word 'amorphous' comes from the Greek a- 'without' and morphē – 'shape, form', which reflects the essence of this state: the strict long-range order in the arrangement of substance particles characteristic of crystalline structures is absent. In ASs there is only a relative (short-range) order, reminiscent of local structures in a crystalline lattice (1-3). Thus, ASs are of a heterogeneous nature (4), on the one hand having the elastic properties of crystalline solids, and on the other, properties of a liquid such as fluidity. When heated, ASs gradually soften and pass into a viscous state: in other words, they do not have a strict melting point. In their glass-like state, ASs exhibit the rheological properties of solids while possessing a high molecular mobility (2). An important characteristic of ASs is that they are thermodynamically unstable and tend to crystallise over time, which is usually associated with an increase in density (2).


Crystalline solid (left) and amorphous solid (right); the absence of long-range order and preservation of local (short-range) order are visible in the latter.

Many pharmaceuticals are in an amorphous form or have amorphous constituents (for example, lyophilised pharmaceuticals and biopharmaceuticals) (1,5). Drugs with a low molecular weight typically contain amorphous polymers to improve solubility and bioavailability, and protein molecules are usually enclosed in a lyophilised matrix of amorphous sugars to enhance stability and shelf life (3). In almost all cases, such amorphous pharmaceuticals (APs) contain a certain amount of water, which can be more than 30% (lyophilised solutions of sugars and proteins) (1). The presence of water has a significant effect on the properties of APs, therefore the study of amorphous systems' structure and their interaction with water has attracted increasing attention from the pharmaceutical community in recent years (1).


A schematic representation of the model of liquid water showing hydrogen-bonded clusters and unclustered water molecules. The molecules within the clusters have a tetrahedral structure (although are not drawn as such). The representation is based on the Frank-Wen model of liquid water (6).
Source (1): Nemethy G., Scheraga H.A. (1962). Structure of water and hydrophobic bonding in proteins. 1. A model for the thermodynamic properties of liquid water. J. Chem. Phys., 36: 3382-400.

It is known that water molecules can exist in their free form or in the form of aggregates (clusters) of different sizes and geometries (1, 2). The assumption that water consists of constantly changing clusters of different sizes and shapes with a half-life of about 10–11–10–12 s was made as far back as 1957 (6). The structuring of water molecules occurs due to their ability to form hydrogen bonds. Through these bonds, each water molecule can bind to four others, forming tetrahedral structures similar to the structure of ice (2,7). More recently, Japanese scientists have provided direct evidence that liquid water is indeed a mixture of two types of local structures: a highly ordered tetrahedral structure and a 'disordered' structure devoid of tetrahedral symmetry (7). In a number of amorphous systems, including carbohydrates and their aqueous solutions, synthetic polymers and proteins, water can also have a clustered structure (1,3). In this case, the clustering depends on the amount of water in the AS. As a rule, unclustered molecules are found at a lower water content, while clusters are formed as water content increases (1). In amorphous sorbitol, for example, the transition from unclustered to clustered organisation of water occurs when its content rises to 10% (1, 3). In the polyvinylpyrrolidone (PVP) polymer, cluster organisation occurs at a water content of 0.5 - 10% (8), in glycerol - at 4.7 - 16.4% (9). The transition from unclustered to clustered water in ASs can also occur at a higher water content; for ASs of collagen, water clustering occurs at approximately 35% (1,3).

It is believed that an increase in the water content of an amorphous product increases molecular mobility, which in turn causes a decrease in the stability of the product. However, an analysis of experimental data shows that the relationship between water content and AP stability is rather complex and can be non-linear (1,3). For example, clustering may occur when water content is increased, reducing mobility as a result of increased cluster size. Therefore, the degree of structural organisation of water in the composition of an AP can have a significant effect on properties such as stability, pharmacokinetics, and pharmacodynamics. However, there have been very few studies of water clusters within APs to date (1).

The interaction between water and AP active ingredient molecules can be heterogeneous, depending on the structural organisation of the water (clustered or otherwise), as well as the size of the clusters. Such heterogeneity can lead to the formation of populations of AP active component molecules with different reactivity, especially during hydrolysis or water-catalysed reactions. In this case, the average kinetic curve of the reactions represents the distribution of the individual rate constants, therefore giving a more complex picture (1). Such a complex difference in the shape of the degradation curves between dry samples and samples with increased water content was noted in a study of amide hydrolysis of the model drug zoniporide in its amorphous state (10).

Studying APs with varying water content is standard procedure in pharmaceutical research and development (3). A wide range of sensitivity between different systems was revealed in an analysis of the effect of water on various chemical processes in APs (11). Some drugs exhibit low sensitivity to water, reflecting the weak dependence of reaction rates on changes in water content, while others show a much stronger dependence on water. It has been theorised that small changes in reaction rates with a change in water content correspond to clustering: an increase in water content leads to water clusters forming and increasing in size without a significant increase in water-drug contact, which is expressed in the weak sensitivity of the reaction rate to an increase in water content (11). The deamidation of a model tetrapeptide in amorphous polymeric matrices is such an example of the reaction rate 'levelling off' with an increase in water content. It was found that the degradation rate increased with an increase in water content from 6% to 8%, and remained practically constant after that (12). Another study showed that the rate of chemical degradation of IgG was the same over a relatively wide water content range – from 0.2% to 5.2% (13).

The properties of amorphous solids, due to their non-equilibrium nature, depend on the history of their processing and thermal effects. It was shown that annealing of glassy ASs reduces the rate of chemical degradation, probably due to a decrease in molecular mobility (14). However, the details of the structural changes that lead to improved stability are not fully understood. It has been posited that annealing changes the distribution of water, increasing the extent of its clustering, for example. This can weaken the contacts between water and the amorphous material and, as a consequence, the rate of hydrolytic processes. In addition, water in clusters can have a different structural arrangement (for example, due to different extents of unsatisfied hydrogen bonds) compared to unclustered water molecules, which can lead to differences in the ability of water molecules to participate in hydrolytic chemical processes (1). It can thus be suggested that 'water history' can also play an important role in the stability of APs.

Two methods are commonly used to achieve variable water content when studying the effect of water on stability and other characteristics of lyophilised drugs: interrupting secondary drying during lyophilisation and rehydrating the dried material in the gaseous phase. It is likely that APs which have the same water content but have been prepared in two different ways may differ in stability. However, it should be noted that direct studies of the influence of hydration history on the dynamics, stability and structure (including clustering and water distribution) of ASs are not carried out in practice (1).

Another hypothesis has been put forward about the role of water in the chemical instability of APs (3). For many chemical reactions, water plays a critical role in lowering the activation barrier by facilitating proton transfer. Water can thus serve as a catalyst in such common degradation reactions as hydrolysis and deamidation. According to one hypothesis, it is water clusters, and not individual molecules, that are directly involved in proton transfer reactions (3). It is known that when the water content in ASs reaches a certain threshold value, clusters are consequently formed. Water molecules are predominantly unclustered at lower values and are therefore less catalytically active (3). For example, the critical role of water clusters as a medium for proton transfer can explain the kinetic deamidation curves of some lyophilised proteins which have been observed in experiments (15). It is therefore hypothesised that the chemical instability of APs is directly related to the catalysis of proton transfer by water clusters.

The clustering of water and its local structure may also be relevant to protein cryoprotection. Proteins are usually stored frozen in a sugar or polyhydric alcohol solution. These are two-phase systems consisting of ice and a freeze-dried solution, with a water content of between 20 and 50%, depending on the temperature and solutes present (1). In a study of a representative glass-state sorbitol-water system containing 30% water, water clusters with a structure resembling low-density amorphous ice (glassy water) were discovered. Glassy water is known to have properties that are different from ordinary water. For example, salts are poorly soluble in it, which can significantly affect protein stability. Thus, glassy water will promote ion pairing (minimising dissociation), thereby disrupting electrostatic interactions which are known to significantly contribute to protein stability (1).

In general, systematic studies of water clustering in APs (such as lyophilised mixtures of proteins and sugars) and the effect of this process on the various physicochemical properties of APs are practically absent. In addition, it is unclear which methods should be used to detect water cluster formation in APs. These problems do not allow one to reach unambiguous conclusions about the extent of water clustering in APs, the water content threshold at which cluster formation occurs, or the effect of these processes on the properties of amorphous pharmaceuticals. The assumption that the transition from unclustered water molecules in APs to cluster organisation leads to a change in various properties of amorphous pharmaceuticals is yet to be verified. However, it is already clear that without studying the relationship between water clustering and the properties of APs, our understanding how water affects the stability of amorphous substances will be incomplete.


1.    Authelin, J. R., MacKenzie, A. P., Rasmussen, D. H., Shalaev, E. Y. (2014). Water clusters in amorphous pharmaceuticals. Journal of pharmaceutical sciences, 103(9): 2663–2672.

2.    Healy, A.M., Worku, Z.A., Kumar, D., Madi, A.M. (2017). Pharmaceutical solvates, hydrates and amorphous forms: A special emphasis on cocrystals. Advanced drug delivery reviews, 117: 25–46.

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6.    Frank HS, Wen W-Y III. (1957). Ion-solvent interactions. Structural aspects of ion-slovent interactions: A suggested picture of water structure. Discuss Faraday Soc, 24: 133–140.

7.    Shi, R., Tanaka, H. (2020). Direct Evidence in the Scattering Function for the Coexistence of Two Types of Local Structures in Liquid Water. Journal of the American Chemical Society, 142(6): 2868–2875.

8.    Xiang TX, Anderson BD. (2005). Distribution and effect of water content on molecular mobility in poly(vinylpyrrolidone) glasses: A molecular dynamics simulation. Pharm Res 22(8): 1205–1214.

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10.    Luthra SA, Shalaev EY, Medek A, Hong J, Pikal MJ. (2012). Chemical stability of amorphous materials: Specific and general media effects in the role of water in the degradation of freeze-dried zoniporide. J Pharm Sci, 101(9): 3110–3123.

11.    Ohtake S, Shalaev EY. (2013). Effect of water on the chemical stability of amorphous pharmaceuticals: I. Small molecules. J Pharm Sci, 102: 1139–1154.

12.    DeHart MP, Anderson BD. (2012). Effects of water and polymer content on covalent amide-linked adduct formation in peptide-containing amorphous lyophiles. J Pharm Sci, 101: 3142.

13.    Chang L, Shepherd D, Sun J, Tang Z, Pikal MJ. (2005). Effect of sorbitol and residualmoisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state. J Pharm Sci, 94(7): 1445–1455.

14.    Luthra SA, Hodge IM, Utz M, Pikal MJ. (2008). Correlation of annealing with chemical stability in lyophilized pharmaceutical glasses. J Pharm Sci, 97(12): 5240–5251.

15.    Ohtake S, Feng S, Shalaev E. (2017). Effect of water on the chemical stability of amorphous pharmaceuticals: 2. Deamidation of peptides and proteins. J Pharm Sci., 107(1): 42-56.