Intensifying aqueous two-phase extraction by adding decisive excipients for enhancement of stability and solubility of biomolecules.

• Optimized ATPS extraction performance for ATPE of biomolecules through excipients. • Rapid identification of suitable excipients by methodical selection. • Improved conformational and colloidal biomolecule stability. • Decreased biomolecule precipitation and increased yield through improved stability. • Simplified process handling by biomolecule stability and solubility improvements Despite the benefits of aqueous two-phase extraction (ATPE) for purification of high-value biomolecules, phase-formers that are used to generate the aqueous two-phase system (ATPS) often reduce the stability and solubility of the biomolecule. Within this work, we thus evaluated the use of suitable excipients to increase the biomolecule stability and solubility during ATPE, whilst not negatively affecting yield and selectivity. Applying this new physically sound approach enabled a rapid selection of L-arginine as suitable excipient for the ATPE of Immunoglobulin G. It was demonstrated that both the (conformational and colloidal) stability and solubility of Immunoglobulin G in the ATPS were increased, thus maximizing yield, minimizing precipitation, and improving the extraction performance of the ATPS. The applicability and transferability of the approach was confirmed by optimizing the ATPE of an industrial relevant amine dehydrogenase. The optimized ATPS enabled an extraction yield of 79.17 wt% (impurities < 7 wt%) directly from crude extract. Furthermore, using the excipients broadened the ATPE process window, thus simplifying process handling during ATPE and further downstream unit operations. Prospectively, the use of excipients can support the establishment of ATPE as downstream technology. [Display omitted] [ABSTRACT FROM AUTHOR]