How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions.

This review aims to provide an overview of current knowledge on stabilization of proteins by sugars in the solid state in relation to stress conditions commonly encountered during drying and storage. First protein degradation mechanisms in the solid state (i.e. physical and chemical degradation routes) and traditional theories regarding protein stabilization (vitrification and water replacement hypotheses) will be briefly discussed. Secondly, refinements to these theories, such as theories focusing on local mobility and protein-sugar packing density, are reviewed in relationship to the traditional theories and their analogies are discussed. The last section relates these mechanistic insights to the stress conditions against which these sugars are used to provide protection (i.e. drying, temperature, and moisture). In summary sugars should be able to adequately form interactions with the protein during drying, thereby maintaining it in its native conformation and reducing both local and global mobility during storage. Generally smaller sugars (disaccharides) are better at forming these interactions and reducing local mobility as they are less inhibited by steric hindrance, whilst larger sugars can reduce global mobility more efficiently. The principles outlined here can aid in choosing a suitable sugar as stabilizer depending on the protein, formulation and storage condition-specific dominant route of degradation.

Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.

For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5 nm) and the size of the protein domains (∼2.5 × 2.5 × 5 nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein.

In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formation between protein and sugar, predictive of protein storage stability.

Sugars are often used as stabilizers of protein formulations during freeze-drying. However, not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy during freeze-drying, it is shown here that hydrogen bond formation during freeze-drying, under secondary drying conditions in particular, can be related to the preservation of the functionality and structure of proteins during storage. The disaccharide trehalose was best capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4kDa. The molecularly rigid oligo- and polysaccharides dextran 5kDa and 70kDa, respectively, formed the least amount of hydrogen bonds and provided least stabilization of the protein. It is concluded that smaller and molecularly more flexible sugars are less affected by steric hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it better.

Inulin, a flexible oligosaccharide. II: Review of its pharmaceutical applications.

Inulin is a flexible oligosaccharide which has been used primarily in food for decades. Recently new applications in the pharmaceutical arena were described. In a previous review (Mensink et al. (2015). Carbohydrate Polymers, 130, 405) we described the physicochemical characteristics of inulin, characteristics which make inulin a highly versatile substance. Here, we review its pharmaceutical applications. Applications of inulin that are addressed are stabilization of proteins, modified drug delivery (dissolution rate enhancement and drug targeting), and lastly physiological and disease-modifying effects of inulin. Further uses of inulin include colon specific drug administration and stabilizing and adjuvating vaccine formulations. Overall, the uses of inulin in the pharmaceutical area are very diverse and research is still continuing, particularly with chemically modified inulins. It is therefore likely that even more applications will be found for this flexible oligosaccharide.

Inulin, a flexible oligosaccharide I: Review of its physicochemical characteristics.

Inulin, a fructan-type polysaccharide, consists of (2→1) linked ß-d-fructosyl residues (n=2-60), usually with an (1↔2) α-d-glucose end group. The applications of inulin and its hydrolyzed form oligofructose (n=2-10) are diverse. It is widely used in food industry to modify texture, replace fat or as low-calorie sweetener. Additionally, it has several applications in other fields like the pharmaceutical arena. Most notably it is used as a diagnostic agent for kidney function and as a protein stabilizer. This work reviews the physicochemical characteristics of inulin that make it such a versatile substance. Topics that are addressed include morphology (crystal morphology, crystal structure, structure in solution); solubility; rheology (viscosity, hydrodynamic shape, gelling); thermal characteristics and physical stability (glass transition temperature, vapor sorption, melting temperature) and chemical stability. When using inulin, the degree of polymerization and processing history should be taken into account, as they have a large impact on physicochemical behavior of inulin.