Pall Biotech , Southampton Road , Portsmouth , PO6 4BQ , U.K.
Department of Molecular Sciences , Macquarie University , Macquarie Park , Sydney , New South Wales 2109 , Australia.
Anal Chem. 2019 Nov 5;91(21):13794-13802. doi: 10.1021/acs.analchem.9b03259. Epub 2019 Oct 17.
Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.
评估蛋白质的物理稳定性是生物治疗剂开发、制造和配方的最重要挑战之一。在这里，我们描述了一种结合和自动化圆二色性和固有蛋白质荧光光谱法的方法。通过从 96 孔板中自动注入样品到光学兼容的毛细管流动池，可以从与传统技术相比大大减少的样品量中以无人值守的方式收集有关蛋白质二级和三级结构状态的补充信息。我们验证了该方法的准确性和重现性。此外，我们展示了结构筛选如何用于在两个案例研究中监测蛋白质的展开，这两个案例研究使用 (i) 离液剂（脲）和 (ii) 用于 Protein A 色谱法中单克隆抗体 (mAb) 纯化的低 pH 缓冲液。
