Development of Direct Liquid–Liquid Extraction (LLE) of an Enzymatic Cascade En Route to Enlicitide Using an Automated, High-Throughput Screening Platform

Abstract A liquid–liquid extraction process was developed to isolate a macrocyclic peptide product generated via biocatalysis in the synthesis of enlicitide decanoate. Applying traditional purification methods for enzymatic cascades is challenging due to the presence of proteins, salts, and organic impurities that complicate downstream processing. To address this complexity, we employed a vision-guided, automated, high-throughput screening platform that enabled the systematic evaluation of hundreds of process conditions. Through comprehensive screening, we successfully identified optimal conditions for the selective isolation of the macrocyclic peptide product, achieving 93% recovery with no detectable protein residue. This approach served to streamline purification and enhanced overall process efficiency, demonstrating the utility of automated high-throughput screening for addressing complex purification challenges in biocatalytic process development.