Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

Background: PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N-terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy p...

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Autores principales:	Víctor R. Campos-García, Jorge Benavides, José González-Valdez
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Aqueous two-phase systems Biphasic systems Integrated production/purification PEGylated proteins PEGylated therapeutic proteins PEGylation Biotechnology TP248.13-248.65 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/bd7998d3b2444c5fbfeb010764a512ed
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spelling	oai:doaj.org-article:bd7998d3b2444c5fbfeb010764a512ed2021-11-06T04:20:40ZReactive aqueous two-phase systems for the production and purification of PEGylated proteins0717-345810.1016/j.ejbt.2021.09.003https://doaj.org/article/bd7998d3b2444c5fbfeb010764a512ed2021-11-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S0717345821000464https://doaj.org/toc/0717-3458Background: PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N-terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. Results: Selected linear 5 kDa and 20 kDa mPEG – potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the di-PEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods. Conclusions: This novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another example where reactive ATPS can be successfully implemented.How to cite: Campos-García VR, Benavides J, González-Valdez J. Reactive aqueous two-phase systems for the production and purification of PEGylated proteins. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.09.003Víctor R. Campos-GarcíaJorge BenavidesJosé González-ValdezElsevierarticleAqueous two-phase systemsBiphasic systemsIntegrated production/purificationPEGylated proteinsPEGylated therapeutic proteinsPEGylationBiotechnologyTP248.13-248.65Biology (General)QH301-705.5ENElectronic Journal of Biotechnology, Vol 54, Iss , Pp 60-68 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Aqueous two-phase systems Biphasic systems Integrated production/purification PEGylated proteins PEGylated therapeutic proteins PEGylation Biotechnology TP248.13-248.65 Biology (General) QH301-705.5
spellingShingle	Aqueous two-phase systems Biphasic systems Integrated production/purification PEGylated proteins PEGylated therapeutic proteins PEGylation Biotechnology TP248.13-248.65 Biology (General) QH301-705.5 Víctor R. Campos-García Jorge Benavides José González-Valdez Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
description	Background: PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N-terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. Results: Selected linear 5 kDa and 20 kDa mPEG – potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the di-PEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods. Conclusions: This novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another example where reactive ATPS can be successfully implemented.How to cite: Campos-García VR, Benavides J, González-Valdez J. Reactive aqueous two-phase systems for the production and purification of PEGylated proteins. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.09.003
format	article
author	Víctor R. Campos-García Jorge Benavides José González-Valdez
author_facet	Víctor R. Campos-García Jorge Benavides José González-Valdez
author_sort	Víctor R. Campos-García
title	Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
title_short	Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
title_full	Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
title_fullStr	Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
title_full_unstemmed	Reactive aqueous two-phase systems for the production and purification of PEGylated proteins
title_sort	reactive aqueous two-phase systems for the production and purification of pegylated proteins
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/bd7998d3b2444c5fbfeb010764a512ed
work_keys_str_mv	AT victorrcamposgarcia reactiveaqueoustwophasesystemsfortheproductionandpurificationofpegylatedproteins AT jorgebenavides reactiveaqueoustwophasesystemsfortheproductionandpurificationofpegylatedproteins AT josegonzalezvaldez reactiveaqueoustwophasesystemsfortheproductionandpurificationofpegylatedproteins
_version_	1718443940324048896

Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

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