Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human ß-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

Greening the synthesis of peptide therapeutics: an industrial perspective.

Solid-phase peptide synthesis (SPPS) is generally the method of choice for the chemical synthesis of peptides, allowing routine synthesis of virtually any type of peptide sequence, including complex or cyclic peptide products. Importantly, SPPS can be automated and is scalable, which has led to its widespread adoption in the pharmaceutical industry, and a variety of marketed peptide-based drugs are now manufactured using this approach. However, SPPS-based synthetic strategies suffer from a negative environmental footprint mainly due to extensive solvent use. Moreover, most of the solvents used in peptide chemistry are classified as problematic by environmental agencies around the world and will soon need to be replaced, which in recent years has spurred a movement in academia and industry to make peptide synthesis greener. These efforts have been centred around solvent substitution, recycling and reduction, as well as exploring alternative synthetic methods. In this review, we focus on methods pertaining to solvent substitution and reduction with large-scale industrial production in mind, and further outline emerging technologies for peptide synthesis. Specifically, the technical requirements for large-scale manufacturing of peptide therapeutics are addressed.