Amrhein Sven, Schwab Marie-Luise, Hoffmann Marc, Hubbuch Jürgen
Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
J Chromatogr A. 2014 Nov 7;1367:68-77. doi: 10.1016/j.chroma.2014.09.042. Epub 2014 Sep 30.
Over the last decade, the use of design of experiment approaches in combination with fully automated high throughput (HTP) compatible screenings supported by robotic liquid handling stations (LHS), adequate fast analytics and data processing has been developed in the biopharmaceutical industry into a strategy of high throughput process development (HTPD) resulting in lower experimental effort, sample reduction and an overall higher degree of process optimization. Apart from HTP technologies, lab-on-a-chip technology has experienced an enormous growth in the last years and allows further reduction of sample consumption. A combination of LHS and lab-on-a-chip technology is highly desirable and realized in the present work to characterize aqueous two phase systems with respect to tie lines. In particular, a new high throughput compatible approach for the characterization of aqueous two phase systems regarding tie lines by exploiting differences in phase densities is presented. Densities were measured by a standalone micro fluidic liquid density sensor, which was integrated into a liquid handling station by means of a developed generic Tip2World interface. This combination of liquid handling stations and lab-on-a-chip technology enables fast, fully automated, and highly accurate density measurements. The presented approach was used to determine the phase diagram of ATPSs composed of potassium phosphate (pH 7) and polyethylene glycol (PEG) with a molecular weight of 300, 400, 600 and 1000 Da respectively in the presence and in the absence of 3% (w/w) sodium chloride. Considering the whole ATPS characterization process, two complete ATPSs could be characterized within 24h, including four runs per ATPS for binodal curve determination (less than 45 min/run), and tie line determination (less than 45 min/run for ATPS preparation and 8h for density determination), which can be performed fully automated over night without requiring man power. The presented methodology provides a cost, time and material effective approach for characterization of ATPS phase diagram on base on highly accurate and comprehensive data. By this means the derived data opens the door for a more detailed description of ATPS towards generating mechanistic based models, since molecular approaches such as MD simulations or molecular descriptions along the line of QSAR heavily rely on accurate and comprehensive data.
在过去十年中，生物制药行业已将实验设计方法与由机器人液体处理工作站（LHS）支持的全自动高通量（HTP）兼容筛选、适当的快速分析和数据处理相结合，发展成为一种高通量工艺开发（HTPD）策略，从而减少了实验工作量、样本量，并提高了整体工艺优化程度。除了HTP技术外，芯片实验室技术在过去几年中也有了巨大的发展，并进一步减少了样本消耗。LHS和芯片实验室技术的结合非常理想，并且在本工作中得以实现，用于表征双水相系统的系线。特别是，提出了一种新的高通量兼容方法，通过利用相密度差异来表征双水相系统的系线。密度由一个独立的微流体液体密度传感器测量，该传感器通过开发的通用Tip2World接口集成到液体处理工作站中。液体处理工作站和芯片实验室技术的这种结合实现了快速、全自动且高度准确的密度测量。所提出的方法用于确定分别由磷酸钾（pH 7）和分子量为300、400、600和1000 Da的聚乙二醇（PEG）组成的双水相系统在有和没有3%（w/w）氯化钠存在下的相图。考虑到整个双水相系统表征过程，两个完整的双水相系统可以在24小时内表征完成，包括每个双水相系统进行四次用于双节线曲线测定的运行（每次运行少于45分钟）以及系线测定（双水相系统制备每次运行少于45分钟，密度测定需要8小时），这些都可以在夜间全自动进行，无需人力。所提出的方法基于高度准确和全面的数据，为双水相系统相图的表征提供了一种经济、省时且节省材料的方法。通过这种方式，所获得的数据为更详细地描述双水相系统以生成基于机理的模型打开了大门，因为诸如MD模拟或QSAR分子描述等分子方法严重依赖于准确和全面的数据。
