Abstract

Protein drugs are important therapeutic agents however; they may degrade during formulation processing. The objective of this study was to investigate the correlation between secondary structure alterations and the retentions of biological activity of protein upon the application of thermal stress. Catalase, horseradish peroxidase and α- chymotrypsin were employed as model proteins. Each protein was heated in a solid and solution state at a temperature of 70 °C for 1 h. Attenuated total reflectance Fourier transform infrared spectroscopy, size-exclusion chromatography and biological activity assay were performed. Results showed that heat-exposure of protein solids at 70 °C caused minimum changes in secondary structure and biological activity was almost retained. However, thermal exposure of protein aqueous solution induced significant changes in the secondary structure indicated by area overlap values and caused considerable reduction in the biological activity. The changes in secondary structures were found to be in full alignment with the loss of biological activity for both protein solids as well as aqueous solutions. Catalase lost entire biological activity upon heating in the solution state. In conclusion, the findings of the present study indicate a direct correlation between protein secondary structure alterations and the retention of biological activity which can be taken into account during the development and delivery of protein drugs formulations.

Graphical Abstract

中文翻译：

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研究蛋白质药物的二级结构变化，作为热暴露后其生物活性的指标。

摘要

蛋白药物是重要的治疗剂。它们可能在配方加工过程中降解。这项研究的目的是研究热应力作用下二级结构改变与蛋白质生物活性保留之间的相关性。过氧化氢酶，辣根过氧化物酶和α-胰凝乳蛋白酶被用作模型蛋白。将每种蛋白质以固态和溶液状态在70°C的温度下加热1小时。进行了衰减全反射傅里叶变换红外光谱，尺寸排阻色谱法和生物活性测定。结果表明，蛋白质固体在70°C的热暴露引起二级结构的变化最小，生物活性几乎得以保留。然而，蛋白质水溶液的热暴露引起区域重叠值指示的二级结构发生重大变化，并导致生物活性大大降低。发现二级结构的变化与蛋白质固体以及水溶液的生物活性的丧失完全一致。在溶液状态下加热时，过氧化氢酶丧失了全部生物活性。总之，本研究的发现表明蛋白质二级结构的改变与生物活性的保留之间存在直接的相关性，在蛋白质药物制剂的开发和交付过程中可以考虑这一点。发现二级结构的变化与蛋白质固体以及水溶液的生物活性的丧失完全一致。在溶液状态下加热时，过氧化氢酶丧失了全部生物活性。总之，本研究的发现表明蛋白质二级结构的改变与生物活性的保留之间存在直接的相关性，在蛋白质药物制剂的开发和交付过程中可以考虑这一点。发现二级结构的变化与蛋白质固体以及水溶液的生物活性的丧失完全一致。在溶液状态下加热时，过氧化氢酶丧失了全部生物活性。总之，本研究的发现表明蛋白质二级结构的改变与生物活性的保留之间存在直接的相关性，在蛋白质药物制剂的开发和交付过程中可以考虑这一点。

图形概要