The use of E. coli for manufacturing therapeutic proteins is an economically attractive alternative to mammalian cell-based production. E. coli grows fast, has no demanding culture requirements, and produces high protein yields. Additionally, the purification process does not involve virus-clearance steps, which usually results in significantly lower costs per gram of product. These advantages make E. coli the system of choice for the production of proteins that do not need post-translational modifications.
Traditional E. coli expression involves the recovery of the product as intracellular inclusion bodies that require refolding. Frequently, the efficiency of the refolding step is very low and only small amounts of correctly folded protein are recovered.
Periplasmic expression in E. coli eliminates the need for refolding, as the proteins are correctly folded and/or assembled in the periplasm; however, fermentation yields are typically lower compared to inclusion-body processes due to the physical restriction of the periplasmic space.
To overcome these production limitations, Wacker Biotech has developed an E. coli expression technology termed Esetec® that secretes correctly folded proteins in high titers directly into the culture supernatant. This feature simplifies the bioprocess while rendering high yields of biologically active proteins.
The high secretion efficiency of Wacker's engineered strains, together with the lack of spatial constraint, results in superior yields of high-quality products at an affordable cost. Since soluble proteins are recovered in their native form from the culture supernatant, there is no need for cell disruption or refolding (Figure 1). This translates into simpler primary recovery and purification schemes, thus improving the overall process efficiency.
The primary recovery of recombinant proteins secreted into the culture supernatant usually encompasses centrifugation steps for cell separation and a series of filtration techniques for clarification, such as tangential flow filtration and depth filtration. The main goal of this filtration cascade is to remove cells or cell debris before starting the chromatographic sequence, to prevent clogging the first column, and to avoid unpredictable elution profiles.
Wacker has further developed and optimized the classical tangential flow filtration (TFF) to a simple and upscalable operation for primary recovery of proteins produced with Esetec strains. The supernatant containing the product is clarified by just a single tangential flow microfiltration step. This sole method efficiently removes all remaining host cells and cell debris, and also depletes large amounts of process-related impurities such as host-cell proteins, host-cell DNA, and endotoxins.
While these process-related impurities are efficiently removed, the product is recovered at high titers with negligible losses and the clarified supernatant is used without further conditioning for capture chromatography. By sparing additional conditioning steps such as dilution, depth filtration or diafiltration, the overall process performance is improved in terms of higher yields, minimized processing time, and reduced cost of goods.