Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131, Karlsruhe, Germany.
Bioprocess Biosyst Eng. 2021 Mar;44(3):525-536. doi: 10.1007/s00449-020-02465-8. Epub 2020 Nov 25.
The protein cloud-point temperature (T) is a known representative of protein-protein interaction strength and provides valuable information during the development and characterization of protein-based products, such as biopharmaceutics. A high-throughput low volume T detection method was introduced in preceding work, where it was concluded that the extracted value is an apparent T (T). As an understanding of the apparent nature is imperative to facilitate inter-study data comparability, the current work was performed to systematically evaluate the influence of 3 image analysis strategies and 2 experimental parameters (sample volume and cooling rate) on T detection of lysozyme. Different image analysis strategies showed that T is detectable by means of total pixel intensity difference and the total number of white pixels, but the latter is also able to extract the ice nucleation temperature. Experimental parameter variation showed a T depression for increasing cooling rates (0.1-0.5 °C/min), and larger sample volumes (5-24 μL). Exploratory thermographic data indicated this resulted from a temperature discrepancy between the measured temperature by the cryogenic device and the actual sample temperature. Literature validation confirmed that the discrepancy does not affect the relative inter-study comparability of the samples, regardless of the image analysis strategy or experimental parameters. Additionally, high measurement precision was demonstrated, as T changes were detectable down to a sample volume of only 5 μL and for 0.1 °C/min cooling rate increments. This work explains the apparent nature of the T detection method, showcases its detection precision, and broadens the applicability of the experimental setup.
蛋白浊点温度 (T) 是蛋白-蛋白相互作用强度的已知代表，在生物制药等蛋白类产品的开发和表征过程中提供有价值的信息。先前的工作中引入了一种高通量小体积 T 检测方法，结论是提取的值是表观 T (T)。由于理解表观性质对于促进研究间数据可比性至关重要，因此进行了当前的工作，以系统地评估 3 种图像分析策略和 2 个实验参数（样品体积和冷却速率）对溶菌酶 T 检测的影响。不同的图像分析策略表明，可以通过总像素强度差和白色像素总数来检测 T，但后者也能够提取冰核温度。实验参数变化表明，随着冷却速率（0.1-0.5°C/min）的增加和样品体积（5-24μL）的增大，T 会降低。探索性的热成像数据表明，这是由于低温装置测量的温度与实际样品温度之间存在差异造成的。文献验证证实，无论使用哪种图像分析策略或实验参数，这种差异都不会影响样品的相对研究间可比性。此外，还证明了该方法具有较高的测量精度，因为即使在样品体积仅为 5μL 和冷却速率增量为 0.1°C/min 的情况下，T 变化也可以被检测到。这项工作解释了 T 检测方法的表观性质，展示了其检测精度，并拓宽了实验装置的适用性。
