An integrated process combining the reaction and purification of PEGylated proteins

Author(s)

Santos, Joao H. P. M. ^{[1, 2]} ; Mendonca, Carlos M. N. ^{[1, 2]} ; Silva, Amanda R. P. ^[1] ; Oliveira, Ricardo P. S. ^[1] ; Pessoa Jr, Adalberto ; Coutinho, Joao A. P. ^[2] ; Ventura, Sonia P. M. ^[2] ; Rangel-Yagui, Carlota O. ^[3]

Affiliation

^[1] Univ Sao Paulo, Dept Biochem & Pharmaceut Technol, Av Prof Lineu Prestes 580 Bloco 16, BR-05508000 Sao Paulo, SP - Brazil

^[2] Univ Aveiro, CICECO Aveiro Inst Mat, Dept Chem, P-3810193 Aveiro - Portugal

^[3] Pessoa Jr, Jr., Adalberto, Univ Sao Paulo, Dept Biochem & Pharmaceut Technol, Av Prof Lineu Prestes 580 Bloco 16, BR-05508000 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: GREEN CHEMISTRY; v. 21, n. 23, p. 6407-6418, DEC 7 2019.
• Web of Science Citations: 0

Abstract

A downstream process combining PEGylation reaction and the use of enzyme conjugates acting as phase-forming components of aqueous biphasic systems (ABS) is proposed here. In this approach, citrate buffer (pH = 7.0) was used simultaneously to stop the reaction (avoiding the use of hydroxylamine) and as a phase forming agent inducing the phase separation of the PEGylated proteins. The partition of the bioconjugates was assessed using two model enzymes of small size {[}cytochrome c (Cyt-c) and lysozyme (LYS)], and two of large size {[}l-asparaginase (ASNase) and catalase (CAT)] as well as reactive PEG of 5, 10, 20 and 40 kDa. The effect of the reaction time on the PEGylation and recovery steps was also evaluated. All reactive PEGs allowed high selectivity in the separation of PEGylated proteins from native proteins (S > 100). A positive effect in terms of selectivity was found for longer reaction times. It allowed greater amounts of PEGylated proteins, with an increase of the PEG-protein rich-phase volume (top phase), allowing 100% of recovery of PEGylated proteins. More selective systems were obtained for Cyt-c and LYS (S > 100) compared to those for ASNase and CAT (40 < S < 60); nevertheless for all, the native and PEGylated proteins had their biological activity preserved. Envisioning the industrial potential evaluation, an integrated process diagram was defined combining the PEGylation reaction with the purification of the protein conjugates. Two different scenarios were investigated considering the PEGylation reaction performance. For both approaches (complete and incomplete PEGylation reaction), high recovery yields and purities were achieved for the PEGylated conjugates (92.1 +/- 0.4% < %RecT(Cyt-c-PEG) < 98.1 +/- 0.1%; 84.6% < purity < 100%) and for the unreacted enzyme (%RecB(Cyt-c) = 81 +/- 1%; purity = 97.7%), while maintaining their structural integrity. (AU)

• FAPESP's process: 16/22065-5 - N-terminal pegylation of proteins and purification by aqueous two-phase systems
• Grantee: Carlota de Oliveira Rangel Yagui
• Support Opportunities: Regular Research Grants
• 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: 18/25994-2 - 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