resumo
L-asparaginase is used for the treatment of acute lymphoblastic leukaemia (ALL); however, its formulation presents drawbacks such as a lack of stability and formation of aggregates. Osmolytes are small molecules accumulated by cells in response to environmental stresses and present a protective behaviour, favouring the equilibrium of macromolecules towards the native conformation. Therefore, osmolytes are employed as excipients in pharmaceutical protein formulations. Herein, recombinant L-ASNase II from Erwinia chrysanthemi (ErA II) was analysed with respect to the effect of osmolytes on kinetic and stability of this biopharmaceutical, The aggregation profiles were analysed trough nanotracking particle analysis and dynamic light scattering. The majority of the tested osmolytes increased ErA II specific activity and stability, being more pronounced for sucrose and sorbitol, which increased almost 70% of ErA II activity. The polyol preserved total enzyme activity for 30 days while sucrose preserved 81.1 +/- 5.3% total enzyme activity over this period. Each osmolyte resulted in a specific aggregation profile and the presence of sucrose or sorbitol resulted in a lower quantity of aggregates in the range of 100-300 nm. The present findings may contribute to the improvement of adjuvants in L-ASNase formulations and the optimization of other biopharmaceutical formulations.
palavras-chave
PROTEIN STABILITY; STABILIZATION; EXPRESSION; MECHANISM; PROTECTION; ARGININE; SUCROSE
categoria
Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology; Engineering
autores
Wlodarczyk, SR; Costa-Silva, TA; Pessoa, A; Madeira, P; Monteiro, G
nossos autores
Pedro António Palma Madeira
Investigador Júnioragradecimentos
This work was supported by the State of Sao Paulo Research Foundation (FAPESP/Brazil, process numbers: 2013/24024-6, 2015/07749-2) and is also part of the hematic project FAPESP (2013/086177). This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-Brazil (CAPES), Finance Code 001. The authors would like to thank the National Postdoctoral Program (PNPD/Capes -FCF-USP) [Award Number: 1781837]. G.M. received a Productivity Fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq < GN4 > 309595/2016-9 < GN4 >).