The manufacture of recombinant proteins, especially the ones lacking affinity handles, often requires multiple chromatography steps and complicated feedstream conditioning, resulting in high production costs and relatively low yields. In this presentation we will describe the development of a two-step workflow for the purification of an acidic recombinant protein from <i>E. coli</i> fermentate, in which the target protein accounts for just 2% of total soluble proteins. Capture chromatography was performed with a hydrophobic anion exchanger, Nuvia aPrime 4A. At modest salt concentrations and neutral pH, the majority of impurities such as host cell proteins, lipids, and nucleic acids, were eliminated in the column flow-through fractions. Interestingly, the target protein was not eluted from this column by high concentration of sodium chloride, a salting-in salt commonly used for dissociating proteins from an ion exchange column. Instead, a divalent metal ion containing buffer was found effective for recovering the target protein molecules. The eluate from the capture step was loaded directly onto the second mixed-mode media, for polish purification, resulting in over 95% purity and close to quantitative recovery of the target protein. The possible mechanisms for the selective binding and elution of target protein, and hence the strategies for chromatography method optimization, will be discussed in this presentation. Both the mixed-mode chromatography media are macroporous particles, which offer efficient mass transfer in a wide range of operation flow rates. Moreover, the gentle chromatography conditions and the easy step transition allow maximum protection of the target protein integrity. This purification workflow is robust and scalable.

Efficient purification of a recombinant protein from <i>E. coli</i> fermentate with mixed-mode chromatography resins

2019 Spring National Meeting

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Efficient purification of a recombinant protein from E. coli fermentate with mixed-mode chromatography resins