Precipitation can be a highly effective capture step in the production of high titer recombinant protein products like monoclonal antibodies. In this work, precipitates with high precipitation yield and consistent morphologies were produced continuously in tubular precipitators using a combination of ZnCl2 and PEG as cross-linking and volume exclusion agents, respectively. Microfiltration was then integrated as part of a continuous operation to concentrate and wash the precipitate slurry to remove non-precipitated impurities, such as host cell proteins (HCP) and DNA, as shown schematically in Figure 1. High degrees of HCP / DNA removal were achieved using multiple tangential flow filtration (TFF) hollow fiber modules in a counter-current configuration, with long term stable operation achieved by operating the membrane modules below the critical filtrate flux for fouling. In the precipitation stage, cross-linking (e.g., ZnCl2) and volume excluding (e.g., polyethylene glycol, PEG) precipitants were added in-line to the harvested cell culture fluid (HCCF) and the resulting suspension was filtered to remove soluble impurities and excess precipitants while retaining the precipitated monoclonal antibody (mAb). In the subsequent washing stage, buffer solution was used in a two-stage counter-current configuration to wash the precipitates, further reducing the concentration of any soluble impurities. In the last stage, the precipitated mAb was re-dissolved by lowering the pH to disrupt the cross-linking. This was followed by a depth filter to remove any precipitated materials that could not be re-solubilized. The critical flux values in the precipitation and washing stages were evaluated using a flux-stepping procedure. It was found that the critical flux was greater for the larger diameter hollow fibers and for precipitates with larger effective particle size. These results provide important insights into the development of effective tangential flow filtration systems for processing large quantities of precipitated protein as would be required for large scale continuous protein purification by precipitation.

Process development of a precipitation-filtration unit for recombinant protein capture step II: Continuous counter-current washing and filtration

2019 Spring National Meeting

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