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

Author(s)

Magri, Agnes ^[1] ; Pimenta, Marcela V. ^[2] ; Santos, Joao H. P. M. ^{[2, 3]} ; Coutinho, Joao A. P. ^[3] ; Ventura, Sonia P. M. ^[3] ; Monteiro, Gisele ^[2] ; Rangel-Yagui, Carlota O. ^[2] ; Pereira, Jorge F. B. ^[1]

Affiliation

^[1] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, BR-14800903 Araraquara, SP - Brazil

^[2] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Tecnol Bioquim Farmaceut, Sao Paulo, SP - Brazil

^[3] Univ Aveiro, CICECO Aveiro Inst Mat, Dept Chem, Aveiro - Portugal

• Document type: Journal article
• Source: JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY; v. 95, n. 4, p. 1016-1027, APR 2020.
• Web of Science Citations: 3

Abstract

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 (AU)

• FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
• Grantee: Adalberto Pessoa Junior
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/15104-0 - Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases.
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/16424-7 - Optimization and scale-up of liquid-liquid extraction process with ionic liquids (ILs) as a sustainable tool for the separation of the anti-leukemia biopharmaceutical L-asparaginase (ASPase)
• Grantee: Jorge Pereira
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/25994-2 - Development of novel platforms for PEGylation of proteins with therapeutic potential using microfluidics
• Grantee: João Henrique Picado Madalena Santos
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/19793-3 - Optimization and scale-up of novel Ionic-Liquid-based purification processes for recombinant green fluorescent protein produced by Escherichia coli "GFPurIL"
• Grantee: Sandro Roberto Valentini
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/07749-2 - Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants