期刊
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
卷 109, 期 -, 页码 311-322出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2017.12.089
关键词
Protein stability; Carbohydrates; Preferential exclusion
资金
- National Research Foundation of Korea (NRF)
- Korean government (MSIT) [2015R1A1A1A05000942]
- National Research Foundation of Korea [2015R1A1A1A05000942] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
Carbohydrates are widely used as additives for biopharmaceutical formulations, but the mechanisms by which they confer stability to and their applicability on protein stability remain undiscovered. Herein, we aimed to elucidate these mechanisms, by studying the thermodynamic changes using isothermal titration calorimetry and micro-differential scanning calorimetry. Furthermore, conventional biophysical analyses, namely circular dichroism, dynamic light scattering, and size-exclusion chromatography, were used to investigate the beneficial effects of carbohydrates on protein stability. Four representative carbohydrates (sucrose, fructose, mannitol, and trehalose) were evaluated at three different concentrations on etanercept, a fusion protein. Consequently, sucrose and trehalose increased the exothermic enthalpy while mixing together with protein along with different concentrations. The results were consistent with those of size-exclusion chromatography after accelerated storage. Nevertheless, non-covalent specific interactions between proteins and carbohydrates could not be detected. However, significant increases in exothermic enthalpy were observed due to the carbohydrates, indicating preferential exclusion of water molecules around etanercept moieties. This energy was maximal at the highest concentration of sucrose and trehalose (i.e., 250 mM). Thus, these carbohydrates consistently exhibited a beneficial effect on the aggregation and conformational stability of etanercept. Based on such findings, the stabilizing mechanism of carbohydrates is proposed herein. (C) 2017 Elsevier B.V. All rights reserved.
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