与完善的色谱方法相反的蛋白质结晶具有降低生产成本同时达到相当的高纯度的益处。然而,监测结晶过程仍然是一个挑战,因为产生的晶体可能会干扰分析测量。特别是用于从含有各种杂质的复杂原料中捕获蛋白质,建立可靠的过程分析技术(PAT)来监测蛋白质结晶过程可能很复杂。在非均相混合物中,重要的产品特征可以通过多变量分析和化学计量学发现,从而有助于发展一个透彻的过程理解。在这个项目中,结合离线分析建立了分析设置,在线紫外可见光(UV/Vis)光谱,和在线拉曼光谱,以监测存在多个相和物种的搅拌分批结晶过程。作为一个示例过程,从澄清的大肠杆菌(E.大肠杆菌)在五个不同的实验中以300毫升的规模进行裂解,随着实验条件在初始裂解物溶液制备方法和沉淀剂浓度方面的变化。由于紫外可见光谱对颗粒敏感,基于错流过滤(cross-flowfilteration)的旁路使得能够在线分析液相,从而提供关于核酸与蛋白质比率的裂解物组成的信息。原位拉曼光谱的主成分分析(PCA)支持与产品特定信息相关的光谱和波数范围的识别,并揭示了实验遵循可比较的,当晶体存在时的光谱趋势。基于预处理的拉曼光谱,优化了偏最小二乘(PLS)回归模型以实时监测靶分子浓度.离线样品分析通过自动图像分析以及LkADH和宿主细胞蛋白(HCP)的浓度提供了有关晶体数量和晶体几何形状的信息。尽管存在含有散射晶体和各种杂质的复杂裂解物悬浮液,有可能监测目标分子浓度在一个异质,使用光谱方法的多相过程。有了离线分析设置,粒子敏感在线,和在线分析仪,结晶捕获过程可以在几何形状方面更好地表征,产量,和晶体的纯度。
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300 mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals.