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

Grant number: 16/22065-5
Support type:Regular Research Grants
Duration: April 01, 2017 - December 31, 2019
Field of knowledge:Engineering - Chemical Engineering
Principal Investigator:Carlota de Oliveira Rangel Yagui
Grantee:Carlota de Oliveira Rangel Yagui
Home Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

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)

Articles published in Agência FAPESP about the research grant
Alternative process purifies proteins used as biosensors 

Scientific publications (4)
(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)
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: 0.
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: 0.
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: 0.
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: 2.

Please report errors in scientific publications list by writing to: cdi@fapesp.br.