Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Université catholique de Louvain, Brussels, Belgium.
Biotechnol Bioeng. 2012 Feb;109(2):502-16. doi: 10.1002/bit.23336. Epub 2011 Oct 16.
In high-throughput screening (HTS) assays, the use of ultraviolet absorption spectroscopy (UA) is commonly limited to concentration and turbidity measurements. Our aim was to evaluate microplate-based UA and its second-derivative [(2d)UA] for measuring the conformational stability of two recombinant antigenic proteins in the presence of 44 excipients. Protein conformational stability was assessed by (2d)UA upon titration with guanidine hydrochloride. (2d)UA was compared with tryptophan fluorescence spectroscopy (TF) and differential scanning fluorimetry (DSF), both commonly used techniques for measuring protein conformational stability. The HTS data were corrected for plate, row and column effects by applying a median polish procedure. Irrespective of the unfolding method applied, similar stabilizing excipients were identified by all analytical methods for a given antigen. The native forms of both antigens were destabilized by arginine, hydroxypropyl-β-cyclodextrin, and sodium docusate, and were protected by polyols. The median polish correction improved the quality of the prediction models and the screening resolution. The higher sensitivities of TF and DSF compared with (2d)UA allowed the identification of a larger number of stabilizing excipients. However, similar screening resolutions (z'-factor > 0.8) were observed for 2dUA, TF, and DSF in a HTS of excipients applied to one of the antigens. Therefore, (2d)UA deserves more attention in HTS studies focused on protein conformational stability.
在高通量筛选 (HTS) 测定中，通常将紫外吸收光谱 (UA) 用于浓度和浊度测量。我们的目的是评估基于微孔板的 UA 及其二阶导数 [(2d)UA] 在 44 种赋形剂存在下测量两种重组抗原蛋白构象稳定性的能力。通过盐酸胍滴定测量蛋白构象稳定性的 (2d)UA。(2d)UA 与色氨酸荧光光谱法 (TF) 和差示扫描荧光法 (DSF) 进行比较，这两种方法通常用于测量蛋白构象稳定性。通过应用中值抛光程序校正了板、行和列效应对 HTS 数据的影响。无论应用何种展开方法，所有分析方法都为给定的抗原识别出相似的稳定赋形剂。两种抗原的天然形式都被精氨酸、羟丙基-β-环糊精和十二烷基硫酸钠破坏，并被多元醇保护。中值抛光校正提高了预测模型和筛选分辨率的质量。与 (2d)UA 相比，TF 和 DSF 的更高灵敏度允许鉴定更多的稳定赋形剂。然而，在对一种抗原应用赋形剂的 HTS 中，(2d)UA、TF 和 DSF 的相似筛选分辨率 (z'因子>0.8) 被观察到。因此，(2d)UA 在专注于蛋白构象稳定性的 HTS 研究中值得更多关注。
