Wacker has established a simple and efficient platform approach for the primary recovery of proteins that are expressed and secreted by Esetec strains. The baseline fermentation is usually a fed-batch process in chemically defined mineral salt media, which is driven to a defined harvesting point by accurate µ-control and induction of expression with low IPTG concentration along with a shift to lower temperature.
On conclusion of fermentation, a balance is obtained between product quality, yield, and critical physicochemical and physiological parameters (e.g., nutrient concentration, viscosity, oxygen transfer, cell density, cell viability). The biomass is then removed by centrifugation at room temperature.
The load of impurities derived from Esetec cells in the supernatant is considerably lower than the amount of process-related impurities in whole-cell lysates from conventional E. coli processes. Therefore, it is conceivable that a single clarification step for Esetec processes might not only deplete the solids in the supernatant but also efficiently remove host cell-derived material. Removal of contaminants at this early stage would greatly simplify the subsequent chromatographic sequence, enabling higher product purity to be targeted while production costs are also conveniently reduced.
Tangential flow filtration has been implemented at Wacker as a sole clarification step. The process is performed with hollow-fiber modules. The modular nature of the TFF devices supports upscalability of the technology to accommodate larger volumes of supernatant. The extent of product recovery and impurity removal during TFF is based on the size and shape of the molecules. It, therefore, largely depends on the nominal pore size of the filter media and on the applied process parameters (e.g., shear rate and transmembrane pressure).
The pore size of the membrane (usually below 0.1 µm) and the most suitable operating parameters are identified for each product with a basic scouting approach early on during downstream process development (Figure 2). This is crucial if the TFF is to serve not only for clarification and volume reduction but also as a partial purification step.
Once cells have been removed, the culture supernatant can be used for filtration without further conditioning. The hollow-fiber units used for TFF are pre-assembled, ready-to-use, and disposable. Hence, material and equipment preparations are minimal, and there is no need to develop and validate cleaning procedures. These benefits help streamline the process by eliminating time-consuming and costly steps.
The product yields in the TFF permeates are usually 85% (ranging from 70–90%), while host cell proteins are typically reduced by 35–45%. More than 90% of residual DNA and endotoxins are also depleted in this single step; clearance rates of 99% (2 logs) and 99.99% (4 logs) are commonly observed for residual DNA and endotoxin respectively (Figure 3, top panel). Altogether, the TFF permeates derived from Esetec cells consistently show improved impurity profiles compared to their counterparts from conventional E. coli (Figure 3, bottom panel).
Permeates from TFF can be used for chromatography (capture step) without further conditioning and are compatible with a broad variety of capture matrices. For example, clarified supernatants for Esetec-derived Fabs have shown superior performance in capture experiments using affinity chromatography, with product yields >90%. The permeates can also be loaded onto nonaffinity chromatographic media such as multimodal resins.