Evaluation of glycosylation, solubility, stability, and immune response of a recombinant serine protease from Crotalus durissus collilineatus modified by PEGylation

• Grant number: 25/05917-7
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: December 01, 2025
• End date: November 30, 2028
• Field of knowledge: Biological Sciences - Pharmacology - Toxicology
• Principal Investigator: Eliane Candiani Arantes Braga
• Grantee: Isadora Lino Mendes
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development, AP.CCD

Abstract

rCollinein-1 is a recombinant serine protease from Crotalus durissus collilineatus, capable of interfering with the mechanisms that control hemostasis, and is considered a promising alternative for the treatment of vascular and thrombotic disorders. However, its heterogeneous glycosylation pattern may impact immunogenicity, structural, and functional properties. In this context, PEGylation emerges as a promising alternative to enable its clinical use. Based on this, this study aims to evaluate the effects of glycosylation and PEGylation on the activity, solubility, stability, and immunogenicity of rCollinein-1. Expression will be performed in Komagataella phaffii and purification by metal ion affinity chromatography. The 5 kDa mPEG-maleimide polymer will be used in PEGylation, whose efficiency will be evaluated by reverse-phase liquid chromatography (C4). The rCollinein-1-PEG interaction will be simulated by molecular modeling with AutoDock Vina. Proteolytic activity will be measured on fibrinogen and kinetics on the chromogenic substrate S-2302. Solubility and stability will be evaluated using the shake flask method and under acidic, basic, and oxidative stress. Glycans will be characterized by glycopeptide enrichment followed by mass spectrometry. In vivo functionality will be investigated by antithrombotic assays and coagulogram, and immunogenicity by indirect ELISA, quantifying specific antibodies in mouse serum. The results may identify key factors in the characterization of rCollinein-1 and generate unprecedented data on the applicability of PEGylation in glycosylated recombinant SVSPs. (AU)