Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark; Technical University of Denmark, Department of Chemistry, Kemitorvet 207, 2800 Kongens Lyngby, Denmark.
Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark; Technical University of Denmark, Department of Chemistry, Kemitorvet 207, 2800 Kongens Lyngby, Denmark.
Eur J Pharm Biopharm. 2022 Feb;171:1-10. doi: 10.1016/j.ejpb.2021.08.018. Epub 2021 Nov 23.
High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments.
高通量筛选对于测量工业相关蛋白质及其变体的稳定性是必要的，这是在开发过程中进行质量评估的必要手段。许多技术在自动化、测量时间和样品消耗方面的进步，使得在最小蛋白量的情况下，能够快速进行测量。然而，许多方法包括自动化数据分析，这可能忽略了蛋白质在某些条件下的重要行为方面。在这项研究中，我们将小角 X 射线散射（SAXS）应用于高通量筛选工作流程中，通常不用于评估工业筛选中的蛋白质行为，以解决不同高通量方法之间结果不一致和重现性的问题。作为一个案例研究，我们使用了广泛应用于工业生物催化的Thermomyces lanuginosus 和 Rhizomucor miehei 的脂肪酶。我们表明，即使不对 SAXS 数据进行任何耗时的建模分析，初始分析也可以提供关于颗粒间相互作用的有价值的信息。我们得出结论，自动化和数据处理方面的最新进展，使得 SAXS 能够更广泛地作为一种工具，用于深入了解蛋白质配方开发非常有用的信息。考虑到由于台式仪器的商业可用性，SAXS 的可及性不断提高，这一点尤其重要。
