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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 (19)
(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)
LIMA, GUILHERME M.; ATRAZHEV, ALEXEY; SARKAR, SUSMITA; SOJITRA, MIRAT; REDDY, REVATHI; TORRES-OBREQUE, KARIN; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; MACAULEY, MATTHEW S.; MONTEIRO, GISELE; DERDA, RATMIR. DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications. ACS Chemical Biology, . (13/08617-7, 16/22065-5, 18/15041-8, 19/09354-6, 18/15104-0)
TORRES-OBREQUE, KARIN M.; MENEGUETTI, GIOVANNA P.; MUSO-CACHUMBA, JORGE J.; FEITOSA, VALKER A.; SANTOS, JOAO H. P. M.; VENTURA, SONIA P. M.; RANGEL-YAGUI, CARLOTA O.. Building better biobetters: From fundamentals to industrial application. DRUG DISCOVERY TODAY, v. 27, n. 1, p. 65-81, . (16/22065-5, 18/25994-2)
SANTOS, JOAO H. P. M.; FEITOSA, VALKER A.; MENEGUETTI, GIOVANNA P.; CARRETERO, GUSTAVO; COUTINHO, JOAO A. P.; VENTURA, SONIA P. M.; RANGEL-YAGUI, CARLOTA O.. Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability. BIOSENSORS-BASEL, v. 12, n. 2, p. 13-pg., . (16/22065-5, 18/25994-2)
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, . (16/22065-5)
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, . (16/22065-5, 18/25994-2)
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, . (16/22065-5, 13/08617-7)
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, . (16/22065-5, 13/08617-7, 18/25994-2)
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, . (16/22065-5)
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, . (16/22065-5)
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, . (16/22065-5)
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, . (16/22065-5)
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, p. 17-pg., . (16/22065-5)
TORRES-OBREQUE, KARIN; KLEINGESINDS, EDUARDO KREBS; SANTOS, JOAO H. P. M.; CARRETERO, GUSTAVO; RABELO, JHENIFFER; CONVERTI, ATTILIO; MONTEIRO, GISELE; PESSOA JR, ADALBERTO; RANGEL-YAGUI, CARLOTA O.. PEGylation versus glycosylation: effect on the thermodynamics and thermostability of crisantaspase. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. N/A, p. 11-pg., . (16/22065-5, 17/20384-9, 13/08617-7, 18/25994-2)
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, . (16/22065-5)
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, . (17/21819-9, 16/22065-5, 13/08617-7, 18/25994-2)
OLIVEIRA, CAMILA AREIAS; FORSTER, CAMILA; LEO, PATRICIA; RANGEL-YAGUI, CARLOTA. Development of triblock polymersomes for catalase delivery based onquality by designenvironment. JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY, v. 43, n. 1, p. 126-135, . (16/03887-4, 16/22065-5)
LIMA, GUILHERME M.; ATRAZHEV, ALEXEY; SARKAR, SUSMITA; SOJITRA, MIRAT; REDDY, REVATHI; TORRES-OBREQUE, KARIN; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; MACAULEY, MATTHEW S.; MONTEIRO, GISELE; DERDA, RATMIR. DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications. ACS Chemical Biology, v. 17, n. 11, p. 12-pg., . (19/09354-6, 16/22065-5, 18/15041-8, 13/08617-7, 18/15104-0)
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, . (16/22065-5)
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; et al. Novel site-specific PEGylated L-asparaginase. PLoS One, v. 14, n. 2, . (16/22065-5, 13/08617-7)

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