Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

Singh, Somnath; Singh, Jagdish

doi:10.1208/pt040342

Cited by 56 publications

(34 citation statements)

References 31 publications

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“…DSC has been employed to assess protein structural stability by several researchers. 32,33 The thermal denaturation of a protein occurs at the thermal transition temperature (T m ) which is calculated as the midpoint of the thermogram peak, and integration of the area under the peak gives the calorimetric enthalpy (ΔH). Figure 1 shows the melting of lysozyme under several buffer conditions.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…This is consistent with the literature value of 73.7 ± 0.6°C and 59 ± 5 kcal·mol −1 ·°C −1 measured for lysozyme in 4.85% (w/v) NaCl. 32 One hypothesis for the mechanism of structural stabilization of lysozyme in the presence of sugars is the concept of preferential hydration of protein. 13,30 The preferential hydration mechanism proposes that sugars are excluded from the environment around the protein and are replaced by water.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

James

McManus

2012

J. Phys. Chem. B

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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...

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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

James

McManus

2012

J. Phys. Chem. B

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“…In the evaluation of the influence of polyols on lysozyme by Singh and Singh (46), thermodynamic mDSC measurements and biological activity analysis correspond to each other because it was shown that polyols are capable of providing protection against various degradation mechanisms causing protein conformational destabilization associated with a decline in biological activity. Additionally, mDSC predictions of the stability of interleukin-1 receptor type I (IL-1R) in the presence of various preservatives could be verified by means of SE-HPLC data (55).…”

Section: Discussionmentioning

confidence: 98%

A Critical Evaluation of Tm(FTIR) Measurements of High-Concentration IgG1 Antibody Formulations as a Formulation Development Tool

2006

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Purpose. Fourier-transform infrared (FTIR) spectroscopy was applied for the determination of protein melting temperature (T m(FTIR) ) and to assess the stability predictability of a 100-mg/mL liquid IgG 1 antibody formulation. Methods. T m(FTIR) values of various formulations (different pH, buffers, excipients) were compared to the results of a stability study under accelerated conditions (40-C/75% relative humidity), using size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfateYpolyacrylamide gel electrophoresis (SDS-PAGE) for the detection of soluble aggregates and covalent modifications. Results. The highest T m(FTIR) was achieved at pH 5.5, and, similarly, SE-HPLC and SDS-PAGE results suggested a pH optimum between 5.5 and 6.0. Transition temperatures were comparable for all tested buffers. However, the decrease in the monomer fraction upon thermal storage was the lowest for citrate buffers. Whereas sugars and polyols resulted in an increase in T m(FTIR) and enhanced monomer fraction after storage, amino acids showed a destabilization according to SE-HPLC analysis, albeit no change or even an increase in the melting temperature was observed. Conclusions.All examples gave evidence that T m(FTIR) values did not necessarily correspond to the storage stability at 40-C analyzed by means of SE-HPLC and SDS-PAGE. T m values, e.g., determined by FTIR, should only be employed as supportive information to the results from both real-time and accelerated stability studies.

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“…Also changes of the protein hydration properties in the presence of excipients have been considered with regard to stability [34][35][36].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

A thermodynamic analysis of the binding interaction between polysorbate 20 and 80 with human serum albumins and immunoglobulins: A contribution to understand colloidal protein stabilisation

Garidel

Hoffmann

Blume

2009

Biophysical Chemistry

106

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Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

Cited by 56 publications

References 31 publications

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

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

A Critical Evaluation of Tm(FTIR) Measurements of High-Concentration IgG1 Antibody Formulations as a Formulation Development Tool

A thermodynamic analysis of the binding interaction between polysorbate 20 and 80 with human serum albumins and immunoglobulins: A contribution to understand colloidal protein stabilisation

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