Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

Abstract

The effect of the sugars sucrose, glucose, and trehalose on the structural and colloidal stability of lysozyme has been investigated using differential scanning calorimetry and quasi-elastic light scattering, respectively. While sugars are known to increase the temperature at which thermal denaturation of protein occurs, it is not clear if, under the same solution conditions, greater colloidal stability is achieved. The measurements were carried out on lysozyme in three different buffer solutions, 0.05 M sodium acetate (pH 4.6), 0.05 M sodium acetate with 5% (w/v) NaCl, and 10 mM sodium phosphate (pH 7.0). The results show that enhancement of structural stability in the presence of sugars is pH, salt concentration, and sugar dependent. Enhancement of colloidal stability in the presence of sugars, while also pH and salt concentration dependent, as expected, only correlates with increases in the structural stability when the solution behavior is not dominated by highly stabilizing electrostatic repulsive interactions. ■ INTRODUCTIONSugars are widely used excipients in the formulation of biotherapeutic products.1−7 Therapeutic proteins are produced and used for the treatment of various human diseases 8 such as insulin for the treatment of diabetes, 9 monoclonal antibodies for rheumatoid arthritis, 10 interferon-α for leukemia, 11 and interferon-β for multiple sclerosis.12 Sugars are generally used as protein structure stabilizers, 2,13 since additional stability is conferred to a protein during lyophilization by the addition of the sugar.14 Two hypotheses have been proposed to describe the stabilizing effect of sugars on proteins during lyophilization; one such hypothesis states that sugar acts as a water substituent and stabilizes proteins by forming hydrogen bonds at specific sites on the surface of the protein.14−16 Another hypothesis, referred to as the "vitrification hypothesis", states that disaccharides form sugar glasses, thereby immobilizing the protein molecules and providing protection against destabilizing reactions. 15,16 Lysozyme is a globular protein with a molecular weight of approximately 14.7 kDa. Lysozyme has been used as a model protein for this study, since its biophysical properties are well understood and the specific contribution of the sugar to its stability over a range of solution conditions can easily be compared with that of the native protein. Lysozyme is a widely studied protein due to its rich phase behavior 17,18 including crystallization, 19 liquid−liquid phase separation, 20−22 and the formation of equilibrium clusters 23−25 and gels. 24 The solution behavior of lysozyme is also well understood in terms of its interaction with salt ions. 26−28Protein stability by itself is a generic term, and it can be thought of in several ways. The stabilization of protein structure against thermal denaturation is referred to as its thermal or structural stability. 37 and presence of excipients. 6 At pH values away from the isoelectric point of the protein, there is an increase in cha...

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

“…In previous work, we have shown that while saccharides consistently increase T m , an increase in colloidal stability is only observed when the interaction potential is not dominated by electrostatic repulsion [20]. The presence of 20% v/v trehalose or maltose resulted in an increase in T m values; however, this increased thermal stability was not always accompanied by an increase in the proportion of refolded lysozyme.…”

“…The buffer type and pH range (pH 5 to pH 9) were chosen for a number of reasons. In previous work we have shown that for lysozyme, T m is pH dependent [20]. In that study, different buffer salts were used to obtain the desired pH (i.e.…”

Proteins in Saccharides Matrices and the Trehalose Peculiarity: Biochemical and Biophysical Properties

“…Data suggest that structural and colloidal stabilization depends on many other factors, such as pH, salt concentration, beside obviously the type of sugar [194].…”

How fluorescent labelling alters the solution behaviour of proteins

“…8,25,33,34 These studies have shown that the effective inter-protein interaction potential results from the complex interplay of both attractive and repulsive contributions. 35 Proteins are inherently anisotropic, since each surface exposed amino acid contributes to varying degrees to the overall net-interaction potential. 9 This anisotropy has been shown to have a direct influence on protein phase behaviour.…”