Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L- 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies. Graphical Abstract Piping and instrumentation diagram of the experimental setup and data generated by the different sensors. A VP UV/Vis spectrometer (FlowVPE, yellow) measures the protein concentration. From the data of the light scattering photometer (Zetasizer, green) in the on-line measurement loop, the apparant molecular weight and z-average are calculated. The density sensor (microLDS) measures density and viscosity of the fluid in the on-line loop.

中文翻译：

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用于UF / DF的多属性PAT，可监测蛋白质的浓度，粒径和缓冲液交换。

超滤/渗滤（UF / DF）在生物制药的生产中起着重要作用。在这些步骤中，通过过程分析技术（PAT）监视关键过程参数和质量属性，可以促进过程开发并确保生产过程中质量的一致性。在这项研究中，实验室规模的错流过滤（CFF）设备配备了可变光程（VP）紫外线和可见光（UV / Vis）光谱仪，光散射光度计和液体密度传感器（microLDS）。根据测得的信号，监测蛋白质浓度，缓冲液交换，表观分子量和流体动力学半径。在三个案例研究中对该设置进行了测试。首先，在UF / DF运行中使用了溶菌酶，以显示在线和离线测量的可比性。相应的相关系数超过0.97。接下来，在两个DF步骤中监测尿素诱导的葡萄糖氧化酶（GOx）蛋白质大小的变化。在此，静态光散射（SLS）和动态光散射（DLS）的相关系数≥0.92。蛋白质浓度的相关系数为0.82，这可能是由于时间依赖性蛋白质沉淀所致。最后，使用单克隆抗体（mAb）进行了案例研究，以显示这种设置的全部潜力。再次，离线和在线测量与所有相关系数都超过0.92的情况非常吻合。可以在线监测3至120 g L-1范围内的蛋白质浓度。在DF期间观察到mAb的表观分子量随缓冲液的增加，为过程开发和稳定性评估提供有趣的补充信息。总而言之，开发的设置为评估不同的UF / DF过程提供了强大的测试系统，并且可能是制定过程控制策略的良好起点。实验装置的图形化抽象管道图和仪器图，以及由不同传感器生成的数据。VP UV / Vis光谱仪（FlowVPE，黄色）测量蛋白质浓度。根据在线测量回路中的光散射光度计（Zetasizer，绿色）的数据，计算表面分子量和Z均值。密度传感器（microLDS）测量在线回路中流体的密度和粘度。开发的设置为评估不同的UF / DF过程提供了强大的测试系统，并且可能是制定过程控制策略的良好起点。实验装置的图形化抽象管道图和仪器图，以及由不同传感器生成的数据。VP UV / Vis光谱仪（FlowVPE，黄色）测量蛋白质浓度。根据在线测量回路中的光散射光度计（Zetasizer，绿色）的数据，计算表面分子量和Z均值。密度传感器（microLDS）测量在线回路中流体的密度和粘度。开发的设置为评估不同的UF / DF过程提供了强大的测试系统，并且可能是制定过程控制策略的良好起点。实验装置的图形化抽象管道和仪器示意图，以及由不同传感器生成的数据。VP UV / Vis光谱仪（FlowVPE，黄色）测量蛋白质浓度。根据在线测量回路中的光散射光度计（Zetasizer，绿色）的数据，计算表面分子量和Z均值。密度传感器（microLDS）测量在线回路中流体的密度和粘度。VP UV / Vis光谱仪（FlowVPE，黄色）测量蛋白质浓度。根据在线测量回路中的光散射光度计（Zetasizer，绿色）的数据，计算表面分子量和Z均值。密度传感器（microLDS）测量在线回路中流体的密度和粘度。VP UV / Vis光谱仪（FlowVPE，黄色）测量蛋白质浓度。根据在线测量回路中的光散射光度计（Zetasizer，绿色）的数据，计算表面分子量和z平均。密度传感器（microLDS）测量在线回路中流体的密度和粘度。