Controlling the l-asparaginase extraction and purification by the appropriate selection of polymer/salt-based aqueous biphasic systems


BACKGROUND l-Asparaginase (ASNase) is an important biopharmaceutical for the treatment of acute lymphoblastic leukemia (ALL); however, with some restrictions due to its high manufacturing costs. Aqueous biphasic systems (ABS) have been suggested as more economical platforms for the separation/purification of proteins, but a full understanding of the mechanisms behind the ASNase partition is still a major challenge. Polymer/salt-based ABS with different driving-forces (salting-out and hydrophilicity/hydrophobicity effects) were herein applied to control the partition of commercial ASNase. RESULTS The main results showed the ASNase partition to the salt- or polymer-rich phase depending on the ABS studied, with extraction efficiencies higher than 95%. For systems composed of inorganic salts, the ASNase partition was controlled by the polyethylene glycol (PEG) molecular weight used. Cholinium-salts-based ABS were able to promote a preferential ASNase partition to the polymer-rich phase using PEG-600 and to the salt-rich phase using a more hydrophobic polypropylene glycol (PPG)-400 polymer. It was possible to select the ABS composed of PEG-2000 + potassium phosphate buffer as the most efficient to separate the ASNase from the main contaminant proteins (purification factor = 2.4 +/- 0.2), while it was able to maintain the enzyme activity for posterior application as part of a therapeutic. CONCLUSION Polymer/salt ABS can be used to control the partition of ASNase and adjust its purification yields, demonstrating the ABS potential as more economic platform for the selective recovery of therapeutic enzymes from complex broths. (c) 2019 Society of Chemical Industry




Biotechnology & Applied Microbiology; Chemistry; Engineering


Magri, A; Pimenta, MV; Santos, JHPM; Coutinho, JAP; Ventura, SPM; Monteiro, G; Rangel-Yagui, CO; Pereira, JFB

nossos autores


This work was developed in the scope of the project FAPESP 2014/16424-7 and within the scope of the project CICECO-Aveiro Institute of Materials, Portuguese Foundation for Science and Technology (FCT) Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. A. Magri acknowledges the financial support from Brazilian National Counsil of Technological and Scientific Development (CNPq) 163292/2015-9. The authors are grateful for the financial support of FCT for the doctoral grant of SFRH/BD/102915/2014 of JoAo H. P. M. Santos and the contract under Investigator FCT 2015 contract number IF/00402/2015 of S.P.M. Ventura. This work was also supported by FAPESP/Brazil through the grants 2013/08617-7, 2014/19793-3, 2015/07749-2, 2018/25994-2 and 2018/15104-0. G. Monteiro and C. Rangel-Yagui received a Productivity Fellowship from the CNPq (309595/2016-9 and 301832/201-0)). A. Magri, M. V. Pimenta, G. Monteiro and J. F. B. Pereira also thank the support from CoordenacAo de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) finance code 001.

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