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N-terminal pegylation of proteins and purification by aqueous two-phase systems

Abstract

Biological medicines represent an important breakthrough of the pharmaceutical industry that generated considerable income to patent holders of the recent marketed biomolecules. Nonetheless, the need to keep competitive and to solve intrinsic problems of protein-based biodrugs, such as immunogenicity and biological instability, has led to the development of biobetters as follow-on biodrugs. Within this context, one of the most common strategies in biodrugs production, including biobetters, refers to the covalent linkage of poly(ethylene glycol) (PEG) chains. This strategy known as pegylation allows pharmacokinetic and pharmacodynamic improvement of biodrugs, especially protein-based ones. In this project we will study site-specific N-terminal pegylation of several proteins (BSA, catalase, L-asparaginase and lysozyme), as well as the use of aqueous two-phase systems to purify pegylated proteins from unreacted protein molecules. Initially, pegylation reaction conditions will be investigated with the protein BSA to define best monopegylation conditions. We will investigate the influence of ionic strength (PBS buffer 0.01, 0.1 or 0.2 M), PEG:Protein ratio (25:1 or 50:1) and pH (6.0, 6.5, 7.0, 7.5 or 8.0) on reaction yield and protein stability. At the defined conditions, we will identify the best reaction time to minimize polydispersion (PEG conjugation at unespecific sites). Following, pegylated proteins will be purified by ion exchange and size-exclusion chromatography. The partitioning of PEG-protein species will be investigated in aqueous two-phase PEG/potassium phosphate systems employing different molecular mass PEGs and different phosphate buffer compositions. The possibility of using pegylated proteins as phase-forming components in PEG-protein/potassium phosphate systems will also be investigated. The best conditions obtained will be tested for the pegylation reaction media with no previous purification step. For the enzymes (catalase, L-asparaginase and lysozyme) we will also study the thermodynamics of thermal denaturation as well as the thermodynamics of the enzymatic reaction catalyzed by each enzyme, in pegylated and unpegylated forms. (AU)

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Scientific publications (12)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
SANTOS, JOAO H. P. M.; OLIVEIRA, CAMILA A.; ROCHA, BEATRIZ M.; CARRETERO, GUSTAVO; RANGEL-YAGUI, CARLOTA O. Pegylated catalase as a potential alternative to treat vitiligo and UV induced skin damage. Bioorganic & Medicinal Chemistry, v. 30, JAN 15 2021. Web of Science Citations: 0.
SANTOS, JOAO H. P. M.; MARTINS, MARGARIDA; SILVA, AMANDA R. P.; CUNHA, JHENIFFER R.; RANGEL-YAGUI, CARLOTA O.; VENTURA, SONIA P. M. Imidazolium-based Ionic Liquids as Adjuvants to Form Polyethylene Glycol with Salt Buffer Aqueous Biphasic Systems. JOURNAL OF CHEMICAL AND ENGINEERING DATA, v. 65, n. 8, p. 3794-3801, AUG 13 2020. Web of Science Citations: 0.
SANTOS, JOAO H. P. M.; BRUMANO, LARISSA P.; PESSOA, JR., ADALBERTO; RANGEL-YAGUI, CARLOTA O. Tailoring Protein PEGylation Reaction: An Undergraduate Laboratory Experiment. Journal of Chemical Education, v. 97, n. 5, p. 1443-1447, MAY 12 2020. Web of Science Citations: 0.
HERRERA BELEN, LISANDRA; DE OLIVEIRA RANGEL-YAGUI, CARLOTA; BELTRAN LISSABET, JORGE FELIX; EFFER, BRIAN; LEE-ESTEVEZ, MANUEL; PESSOA, ADALBERTO; CASTILLO, RODRIGO L.; FARIAS, JORGE G. From Synthesis to Characterization of Site-Selective PEGylated Proteins. FRONTIERS IN PHARMACOLOGY, v. 10, DEC 18 2019. Web of Science Citations: 0.
SANTOS, JOAO H. P. M.; MENDONCA, CARLOS M. N.; SILVA, AMANDA R. P.; OLIVEIRA, RICARDO P. S.; PESSOA JR, ADALBERTO; COUTINHO, JOAO A. P.; VENTURA, SONIA P. M.; RANGEL-YAGUI, CARLOTA O. An integrated process combining the reaction and purification of PEGylated proteins. GREEN CHEMISTRY, v. 21, n. 23, p. 6407-6418, DEC 7 2019. Web of Science Citations: 0.
SANTOS, JOAO H. P. M.; FERREIRA, ANA M.; ALMEIDA, MAFALDA R.; QUINTEIRO, PAULA S. G. N.; DIAS, V, ANA C. R.; COUTINHO, JOAO A. P.; FREIRE, MARA G.; RANGEL-YAGUI, CARLOTA O.; VENTURA, SONIA P. M. Continuous separation of cytochrome-c PEGylated conjugates by fast centrifugal partition chromatography. GREEN CHEMISTRY, v. 21, n. 20, p. 5501-5506, OCT 21 2019. Web of Science Citations: 1.
SANTOS, JOAO H. P. M.; CARRETERO, GUSTAVO; VENTURA, SONIA P. M.; CONVERTI, ATTILIO; RANGEL-YAGUI, CARLOTA O. PEGylation as an efficient tool to enhance cytochrome c thermostability: a kinetic and thermodynamic study. JOURNAL OF MATERIALS CHEMISTRY B, v. 7, n. 28, p. 4432-4439, JUL 28 2019. Web of Science Citations: 1.
HERRERA BELEN, LISANDRA; BELTRAN LISSABET, JORGE; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; EFFER, BRIAN; MONTEIRO, GISELE; PESSOA, ADALBERTO; FARIAS AVENDANO, JORGE G. A structural in silico analysis of the immunogenicity of L-asparaginase from Escherichia coli and Erwinia carotovora. BIOLOGICALS, v. 59, p. 47-55, MAY 2019. Web of Science Citations: 1.
TORRES-OBREQUE, KARIN; MENEGUETTI, GIOVANNA PASTORE; CUSTODIO, DEBORA; MONTEIRO, GISELE; PESSOA-JUNIOR, ADALBERTO; DE OLIVEIRA RANGEL-YAGUI, CARLOTA. Production of a novel N-terminal PEGylated crisantaspase. Biotechnology and Applied Biochemistry, v. 66, n. 3, p. 281-289, MAY-JUN 2019. Web of Science Citations: 1.
MENEGUETTI, GIOVANNA PASTORE; PICADO MADALENA SANTOS, JOAO HENRIQUE; TORRES OBREQUE, KARIN MARIANA; VAZ BARBOSA, CHRISTIANO MARCELLO; MONTEIRO, GISELE; POLISELLI FARSKY, SANDRA HELENA; DE OLIVEIRA, ADRIANO MARIM; ANGELI, CLAUDIA BLANES; PALMISANO, GIUSEPPE; MARQUES VENTURA, SONIA PATRICIA; PESSOA-JUNIOR, ADALBERTO; RANGEL-YAGUI, CARLOTA DE OLIVEIRA. Novel site-specific PEGylated L-asparaginase. PLoS One, v. 14, n. 2 FEB 12 2019. Web of Science Citations: 4.
PICADO MADALENA SANTOS, JOAO HENRIQUE; TORRES-OBREQUE, KARIN MARIANA; MENEGUETTI, GIOVANNA PASTORE; AMARO, BEATRIZ PANICHI; RANGEL-YAGUI, CARLOTA OLIVEIRA. Protein PEGylation for the design of biobetters: from reaction to purification processes. Brazilian Journal of Pharmaceutical Sciences, v. 54, n. SI 2018. Web of Science Citations: 7.
SANTOS, JOAO H. P. M.; CARRETERO, GUSTAVO; COUTINHO, JOAO A. P.; RANGEL-YAGUI, CARLOTA O.; VENTURA, SONIA P. M. Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems. GREEN CHEMISTRY, v. 19, n. 24, p. 5800-5808, DEC 21 2017. Web of Science Citations: 6.

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