In biopharmaceutical production, most research in the field of process analytical technologies (PAT) has been focused on monitoring and control of chromatographic steps. UF/DF (ultrafiltration/diafiltration) steps have not received as much attention yet, even though they are generally applied at least once during the production process.<br/>However, monitoring UF/DF is desirable for development and in manufacturing. During development, purified product, especially for the final UF/DF step, is expensive and only available in little quantities. Therefore, in-line measurements of product concentration, product degradation and the buffer exchange state save product, time and reduces causes of error because no sampling and off-line analytics are necessary. For manufacturing, the monitoring of UF/DF is important because no further purification is performed after the final UF/DF step. Therefore, contaminants introduced by the step, like degraded product, cannot be depleted and remain in the final product.<br/>In this study, we developed a UF/DF setup consisting of a commercial lab-scale cross-flow filtration device, an in-line variable pathlength (VP) spectrometer, and an on-line measurement loop. In the on-line measurement loop, a light-scattering spectrometer and viscosity/density meter were implemented. VP UV/Vis spectroscopy provided information on the protein concentration in a large dynamic range and on changes in the tertiary structure of the protein. Static light scattering (SLS) and Dynamic Light Scattering (DLS) gave information about aggregation or fragmentation of the product. By leveraging the Zimm-equation, the protein concentration was used to correct the scattered-light intensity for concentration-induced changes in intensity providing a concentration corrected scattered-light intensity. Density measurements gave insights on the progress of the buffer exchange.<br/>To confirm the versatility of the setup, three case studies are discussed. First, a proof-of-concept study was conducted using Lysozyme as aggregation-stable protein to show the influence of concentration and buffer exchange on the sensors. Then the unfolding of glucose-oxidase by diafiltration with urea was monitored with this set-up. Finally a UF/DF step with a monoclonal antibody (mAb) was controlled by concentration and buffer exchange progress while measuring light scattering for aggregation and fragmentation prediction.

UF/DF monitoring and control by variable pathlength UV/Vis spectroscopy, light scattering and density measurements

2019 Spring National Meeting

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