PH-Switchable Phase-Transfer Agents for Host Cell Protein Rejection in the Cascaded Biocatalytic Synthesis of an Active Pharmaceutical Ingredient

Abstract

Given the increased reliance on multienzymatic cascades to synthesize biologically inspired small molecule drug candidates, the pharmaceutical industry requires efficient strategies to control immunogenic protein impurities in active pharmaceutical ingredients. Despite advances in directed enzyme evolution and biocatalysis, it is still economical to use unpurified enzymes overexpressed in host cell lysates in manufacturing processes. Due to similar solubility profiles between protein impurities and the pharmaceutical, separation strategies relying on solubility differences (direct crystallization from water or extraction) often fail. Leveraging the rapid generation of modular acid–base pairs, we describe a pH-responsive extraction strategy using tertiary ammonium phase-transfer agents for efficient purification and isolation of hydrophilic immuno-oncology drug candidate MK-1454 from an aqueous biocatalytic cascade containing crude E. coli cell lysates. We demonstrate this technique to isolate several hundred grams of MK-1454 with undetectable protein impurities for human administration in clinical trials. We further show that this extraction technique is driven by reverse micellization, and may be generally applied to the purification of other hydrophilic small molecules.