E. coli Secretion Technology
Wacker Biosolutions has developed an E. coli-based protein secretion technology, explained Susanne Dilsen, Ph.D., head of production. Referred to as Esetec®, it is a recovery and purification procedure for high-efficiency Fab isolation.
“Our process consists of the Esetec expression system, a fermentation process, a recovery procedure, and a scalable purification process,” Dr. Dilsen said.
Esetec boasts a specially designed E. coli K12 strain, dedicated expression vectors and a toolbox of helper plasmids, supplying chaperones, disulfide isomerases, and foldases. This strain secretes proteins directly into the medium rather than into the periplasm. The accompanying toolbox is an extensive collection of protease deletion strains, signal peptides, chaperones, and secretion components.
An important feature of the process is screening for the most satisfactory clones, working up the ladder in stages. The primary screening stage is a high-throughput, random method, covering 1,000 to 3,000 clones per week, for optimizing titer. The second level of screening focuses on functionality and can handle 30 to 60 clones per week.
Finally, the strain selection for the fermentation component handles only three to nine clones per week and aims at selection for titer, function, and quality.
Nothing is left to happenstance in the configuration process. All inoculation ratios and preculture conditions are standardized and a chemically defined medium free of animal proteins is employed. Temperatures and temperature down-shift before induction have been identified, as have the effects of additives (e.g., reducing agents, stabilizers, antifoaming reagents). These were tested and the cell-harvesting conditions standardized during the initial design phases of the process.
Dr. Dilsen described case studies using the Esetec technology. The first, a collaboration with Bayer HealthCare, focused on the production of a recalcitrant Fab, sporting five disulfide bonds.
“In this case we were aiming for yields in excess of 0.5 g/L in the culture supernatant, favoring large-scale production,” she added. “This required the development of appropriate analytical methods. The first yields were less than satisfactory, so we used the toolbox to improve the heavy chain expression, folding, and secretion.”
The modifications, which increased yields by twofold, included altering the promoter to increase the expression of the heavy chain while at the same time co-expressing helper elements, folding, and secretion of the Fab, which is now correctly assembled and fully functional.
In a second case study, Dr. Dilsen and her colleagues collaborated with Morphosys to construct mono- and bivalent antibody fragment formats with different specificity, framework, and physico-chemical properties. In one series of investigations with the Esetec platform, a 40-fold increase was obtained that generated functional Fab products.