Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Fritz-Haber-Weg 2, 76131, Karlsruhe, Germany.
Sanofi-Aventis Deutschland GmbH, Industriepark Höchst, 65926, Frankfurt am Main, Germany.
Anal Bioanal Chem. 2020 Apr;412(9):2123-2136. doi: 10.1007/s00216-019-02318-8. Epub 2020 Feb 18.
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. 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.
超滤/渗滤 (UF/DF) 在生物制药生产中起着重要作用。通过过程分析技术 (PAT) 在这些步骤中监测关键工艺参数和质量属性，可以促进工艺开发并确保生产过程中的质量一致。在这项研究中，实验室规模的错流过滤 (CFF) 装置配备了可变光程 (VP) 紫外可见 (UV/Vis) 光谱仪、光散射光度计和液体密度传感器 (microLDS)。基于测量信号，监测了蛋白质浓度、缓冲液交换、表观分子量和流体动力半径。该装置在三个案例研究中进行了测试。首先，溶菌酶在 UF/DF 运行中被用于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 步骤中监测了尿素诱导的葡萄糖氧化酶 (GOx) 蛋白尺寸的变化。在这里，静态光散射 (SLS) 和动态光散射 (DLS) 的相关系数均≥0.92。蛋白质浓度的相关系数为 0.82，可能是由于蛋白质的时间依赖性沉淀。最后，用单克隆抗体 (mAb) 进行了案例研究，展示了该装置的全部潜力。同样，离线和在线测量结果非常吻合，所有相关系数均超过 0.92。蛋白质浓度可以在 3 至 120 g/L 的大范围内进行在线监测。在 DF 过程中观察到 mAb 的表观分子量随缓冲液的增加而增加，为工艺开发和稳定性评估提供了有趣的补充信息。总之，所开发的装置为评估不同 UF/DF 工艺提供了强大的测试系统，并且可能是开发过程控制策略的良好起点。
