By David Frisch, Hyunsic Choi, James M. Sulzberger, William Rushton, Daniel M. Yoshikawa, and Sharon Bola
The purification and manufacture of recombinant proteins, especially those lacking affinity handles, often requires multiple chromatography steps and complicated feedstream conditioning, resulting in high production costs and relatively low recovery. These issuses may be exacerbated by low protein expression, pH sensitivity, and limited stability. Mixed-mode chromatography resinds have become an important tool for the purifications of such proteins. The objective of this study was to purify one such recombinant protein, EPA, from E. coli fermentate to >95% purity and recovery.
A purification protocol to purify a recombinant protein from E. coli was developed and optimized. Mixed-mode, ion exchange, and hydrophobic interaction chromatography (HIC) resins from various commercial vendors were screened for the capture step. Goals were to minimize fermentation dilution, determine acceptable binding conditions, and increase purity and recovery to >75%. A hydrophobic anion exchange mixed-mode resin was selected for the capture step. Optimal wash and elution conditions were determined using a design of experiment (DOE) approach. An elution buffer containing NaCI and MgCI2 was found to efficiently purify the target molecule with high recovery and purity.