Fida Biosystems ApS, Generatorvej 6, 2860 Soeborg, Denmark.
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
Molecules. 2022 Apr 13;27(8):2506. doi: 10.3390/molecules27082506.
In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.
在治疗性蛋白的开发过程中，对结构稳定性和完整性的分析评估是一项重要的活动，因为蛋白的稳定性和完整性会影响药物的疗效，最终影响患者的安全。现有的分析方法仅依赖于热或化学扰动后光学性质（如荧光或散射）的相对变化。在这里，我们提出了一种利用泰勒分散分析（TDA）和 LED-UV 荧光检测来评估蛋白稳定性、结构和变性的绝对分析方法。所开发的 TDA 方法可测量在盐酸胍（GuHCl）在线变性过程中蛋白的大小（流体力学半径）和固有荧光的变化。以 pH 为函数，对治疗性抗体阿达木单抗和人血清白蛋白的构象稳定性进行了表征。该方法具有简单的工作流程和低的样品消耗（每个数据点 40ng 蛋白），非常适合在药物早期开发阶段或在样品稀缺的情况下评估与稳定性相关的蛋白特性。
