| Grant number: | 16/22065-5 |
| Support Opportunities: | Regular Research Grants |
| Start date: | April 01, 2017 |
| End date: | December 31, 2019 |
| Field of knowledge: | Engineering - Chemical Engineering - Chemical Process Industries |
| Principal Investigator: | Carlota de Oliveira Rangel Yagui |
| Grantee: | Carlota de Oliveira Rangel Yagui |
| Host Institution: | Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
| City of the host institution: | São Paulo |
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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