Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process

Abstract

Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to seven-fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins. www.biotechnology-journal.com Biotechnology Journalwww.advancedsciencenews.com Biotechnol. J. 2017, 12, 1700082 for IB resolubilization to unfold the protein followed by a dilution step to reduce the chaotropic conditions which initiates protein folding. However, such refolding buffers commonly require also elevated salt concentrations [5] which therefore results in feed streams being high in conductivity, making ion exchange chromatography an unsuitable option for the next unit operation. Ion exchange chromatography is attractive for industry due to the costefficient resins with high capacities. Hence, additional process steps such as dilution or diafiltration have to be introduced to reduce salt concentration in the solution and allow efficient binding of the protein to the ion exchange resins [6]. Despite the high potential of continuous downstream processing, it is still rarely applied in the manufacturing of biologics. Other industries such as the food and chemical sector, have already successfully implemented integrated processes which create highly flexible and efficient production [6,7]. In continuous operation equipment size can be down scaled, which reduces not only investment costs but also operating costs due to buffer savings [8,9]. Additionally optimized cycle times improve also productivity [10,11]. Not only FDA has addressed this topic [12,13], it also raised the interest of numerous researchers in both academia and industry [14,15]. Even it is not realized on a large production scale yet, various different strategies for continuous biomanufacturing have been developed in recent years [8,[16][17][18][19][20][21].In this work, we describe a novel alternati...

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