Standard production processes for antimicrobial peptides (AMPs) comprise of batch cultivations in upstream and chromatographic purification in downstream. In spite of limitations in scale-up and high costs, chromatography still remains industrial gold standard. To overcome this bottleneck, alternative downstream strategies using aggregating tags and membrane filtration are a promising approach. However, for making use of the full potential of such processes, they have to be engineered from scratch. Therefore, a unique combination of a thioredoxin-tag (trxA), elastin-like-polypeptides (ELPs) and a self splicing intein (ΔI-CM) was assembled using golden gate cloning, to facilitate a soluble AMP expression process in combination with a non-chromatographic downstream process. A tailor made chemically defined media with optimized trace elements increased growth and recombinant production of the ELP-tagged AMP up to 167%. The trxA acts as a solubility enhancer and facilitates the release into the extracellular space by simple osmotic shock procedure. This method proofed to be superior to chemical lysis or mechanical cell disruption by a 2x-fold increase in product per host cell protein (HCP) yield. Reversible temperature dependent phase transition of the ELPs enables membrane filtration steps for purification of the desired product from impurities and, after cleavage by self-splicing inteins, to separate the amp from the tags itself. The interaction of thioredoxin-, ELP- and intein-tag facilitates scalability and economic feasibility, rendering cell disruption apparatuses, chromatographic columns and enzymatic digestion obsolete. Integration of process analytical technology (PAT) and a design of experiments (DoE) based upstream optimization of inductor strength, induction temperature and duration generates a production process with high potential for application in pharmaceutical industry in regard of GMP-compliance.

Upstream optimization and non-chromatographic downstream strategy for production and purification of antimicrobial peptide in E. coli

255th National Meeting (2018)

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