Enhanced performance of polymer-polymer aqueous two-phase systems using ionic liquids as adjuvants towards the purification of recombinant proteins


Protein biopharmaceuticals, among which interferon alpha-2b (IFN alpha-2b) that can be used in the treatment of chronic hepatitis C and hairy cell leukemia, have become an indispensable product of current medicine. However, their current high costs derived from the lack of cost-effective downstream strategies still limits their widespread use. Polymer-based aqueous two-phase systems (ATPS) or aqueous biphasic systems (ABS) can be used in biopharmaceuticals purification. This work investigates the application of ionic liquids (ILs) as adjuvants (at 5 wt%) in ATPS constituted by polyethylene glycol with a molecular weight of 600 g mol(-1) (PEG 600) and polypropylene glycol with a molecular weight of 400 g mol(-1) (PPG 400) at constant pH (8) to purify the recombinant protein IFN alpha-2b from Escherichia coli lysates. IFN alpha-2b was produced from isopropyl beta-d-1-thiogalactopyranoside (1 mM)-induced Escherichia coli BL21 (DE3) cultures and recovered from inclusion bodies after mechanical lysis, involving glass beads and a solubilization step with urea (8 M) in alkaline pH (12.5). PEGPPG-based ATPS involving ILs as adjuvants were subsequently applied for IFN alpha-2b purification, in which the target protein tends to migrate to the PEG-rich phase (being the phase also enriched in IL) and the remaining proteins tend to precipitate at the interface (fitting within the three-phase partitioning approach). In comparison with the ATPS without adjuvant, most systems comprising ILs as adjuvants lead to enhancements in the purification factors of IFN alpha-2b, namely from 2.28 +/- 0.06 up to 6.77 +/- 0.49. The purity of IFN alpha-2b is maximized using ILs composed of aromatic cations and anions with high hydrogen-bond basicity. The secondary structure of IFN alpha-2b is preserved during the purification step, as appraised by circular dichroism and western-blot studies. Overall, the obtained results demonstrate the ILs ability to tune the characteristics of the ATPS coexisting phases towards improved purification processes, paving the way for their investigation in the purification of other highvalue biological products.



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Castro, LS; Pereira, P; Passarinha, LA; Freire, MG; Pedro, AQ

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This work was developed within the scope of the project CICECOAveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, the CICS-UBI project UIDB/00709/2020, and the CEMMPRE project UID/EMS/00285/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The authors also acknowledge the financial support from the project IL2BioPro -PTDC/BII-BBF/030840/2017funded by FEDER, through COMPETE2020 -Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds (OE), through FCT/MCTES.

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