PH conditioning is a crucial step in primary recovery - A case study for a recombinant Fab from E. coli

Abstract

Primary recovery of recombinant proteins from E. coli often describes a major challenge in downstream processing. After product release, the target protein usually accounts for only a small amount of total protein and has to be separated from a complex mixture of host cell proteins (HCPs) and non-proteinogenic impurities, such as DNA and lipids. Non-optimized procedures as well as unfavorable conditions at the extraction step and conditioning cause significant product loss already prior capture. In this study, we investigated pH conditioning during primary recovery for a subsequent cation exchange chromatography (CEX)-based capture of a recombinant Fab produced in E. coli. We showed that pH ≤ 5.0, which is necessary for CEX, led to high product loss due to protein precipitation during cell disruption and pH conditioning. Thus, we developed a procedure that resulted in a 25% increased Fab recovery prior capture based on simple re-arrangement of process steps and the use of a low-cost stabilizing agent. Summarizing, we show the huge potential for simple and cheap improvement of overall downstream process recovery by optimization of pH conditioning during primary product recovery.