April 1, 2017 (Vol. 37, No. 7)

E. Coli-Based Refolding Technology Relies on Antibiotic- and Phage-Free Plasmid System

Wacker Biotech and Fresenius Medical Care Adsorber Tec last month reached a key milestone in their collaboration by producing an scFv-class antibody treatment, which is used in apheresis products for selective dialysis. Employing its E. coli-based Foldtec® technology, Wacker overhauled a process employed by Fresenius Medical Care to facilitate production on an industrial scale.

To learn more about Foldtec, GEN interviewed Ilona Koebsch, Ph.D., manager, business development, and Sebastian Schuck, Ph.D., head of business development, both from Wacker Biotech.

Carrying out industrial-scale protein refolding with Foldtec® technology allows microbially produced pharmaceutical proteins to be manufactured cost-efficiently and reliably in high yields, without the need for antibiotics or phage components, according to Wacker Biotech Officials. [Wacker Chemie]

GEN: Will you describe the Foldtec technology in more detail as it applies to biomanufacturing?

Drs. Koebsch and Schuck: Poorly soluble substances form aggregated inclusion bodies (IBs) in microbial cells containing incorrectly and/or incompletely folded target proteins. Refolding is the art of transferring these denatured and misfolded recombinant proteins back into their biologically active form. This is of particular importance for complex biopharmaceuticals that cannot be produced in their soluble state, such as small, hydrophobic, toxic, or highly complex proteins.

 Wherever in vitro refolding is needed for recombinant manufacturing of such challenging pharmaceutical proteins, Wacker offers its Foldtec technology. It comprises highly efficient E. coli producer strains, an antibiotic- and phage-free plasmid maintenance system, and comprehensive refolding know-how.

Various physical parameters, additives, and buffer components influence the outcome and efficacy of refolding processes. Selection and proper adjustment of these conditions is of utmost importance for the cost-efficient industrial manufacturing of biopharmaceuticals.

Many refolding protocols show poor efficacy or cannot be transferred to commercial scale, either due to extremely high working volumes or expensive buffer additives. Screening to identify the most suitable combinations can be tedious and time consuming. It requires an in-depth understanding of protein folding and the technical requirements and limitations of large-scale manufacturing.

Foldtec technology meets these refolding challenges: Based on optimized E. coli strains in combination with a plasmid maintenance system and years of experience in designing refolding processes, Wacker can now cost-efficiently and reliably produce aggregating pharmaceutical proteins in high yields and purities. In combination with a set of plasmids, the system shows high expression levels without the need for antibiotics or phage components. Supported by Wacker’s high-cell-density processes, expression results in high-quality inclusion bodies containing up to 12 grams of target protein per liter culture broth.

GEN: What is it specifically about Foldtec that makes it so suitable for meeting refolding’s challenges?

Drs. Koebsch and Schuck: Foldtec provides solutions for the sophisticated manufacturing of biopharmaceuticals by refolding, in synergy with Wacker’s high-cell-density fermentation systems. As mentioned above, the Foldtec technology is based on E. coli strains optimized for high-quality inclusion bodies and superior yields (up to 12 g/L) as a starting material for the refolding step.

During in vitro refolding, high protein concentrations promote aggregation while low concentrations promote proper folding. Contrary to that, low protein concentrations during refolding are highly undesirable in industrial scale.

Highly diluted refolding reactions require large vessels as well as high amounts of buffers and additives. Foldtec avoids high dilutions without compromising yields by combining innovative refolding techniques (e.g., pulse refolding) with a tailored screening approach for rapid identification of ideal refolding conditions

GEN: How is Foldtec able to avoid the use of antibiotics during the process?

Drs. Koebsch and Schuck: While conventional systems depend on the use of antibiotics, Foldtec can do without them entirely. Genetic integrity and plasmid retention are achieved by a proprietary plasmid maintenance system developed by Wacker. It allows primary inclusion body titers similar to a BL21(DE3) conventional system, identical growth and genetic stability, but without any antibiotics. This feature contributes to Foldtec’s high safety, environmental compatibility and meets the strictest of regulatory requirements.

Furthermore, the new system is phage-free, which is a significant advantage for all microbial manufacturing operations. Phage reactivation cannot occur with Foldtec as there is no residual phage DNA present in our strains.                   

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