Human thioredoxin (hTrx) is an acidic redox protein with therapeutic value, successfully expressed in recombinant <i>E. coli</i>. Downstream process development is complex due to the susceptibility of hTrx to oxidation and aggregation. Additionally, the low hydrophathicity index (- 0.096) and minimal net charge (-6.8 at pH 8) are major challenges to purification of hTrx by single-mode chromatography resins.<br/>Mixed-mode chromatography resins expand the profile of interaction mechanisms with enhanced hydrophobic interactions and salt tolerant binding. Extensive screening and optimization is crucial in selecting the binding and elution conditions for these resins.<br/>High throughput screening (HTS) on 96-well filter plates allows a scaled down model of interactions and comprehensive analysis of the optimal operating conditions. Commercial mixed-mode resins: CaptoAdhere, HEA HyperCel, PPA HyerCel, CFT and Toyopearl NH2-750F were screened by HTS for selectivity. HEA HyperCel was chosen as the capture chromatography, which under optimized binding conditions, provided a 73% purity in a single chromatography step. The HTS of elution buffer conditions revealed that 95% recovery could be achieved by salt and arginine. The role of buffers in hTrx aggregation and interaction mechanisms with the resin was also investigated. Optimal process parameters derived from HTS experiments, including the resin binding capacity, were tested and confirmed by packed-bed column chromatography. This work demonstrates the application of mixed-mode resins for direct capture of non-mAb proteins from crude lysates.

Process development for isolation of recombinant thioredoxin using mixed-mode resins: High throughput screening and scale up

2019 Spring National Meeting

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