Superior performance of ionic liquids over deep eutectic solvents in stabilizing L-Asparaginase for sustainable biopharmaceutical applications

Abstract

The stability of protein-based biopharmaceuticals is a critical factor limiting their formulation, storage, and clinical performance. This work shows the performance of cholinium-based ionic liquids (ILs) versus deep eutectic solvents (DESs) as sustainable stabilizing agents for L-asparaginase (L-ASNase), a therapeutic enzyme used in the treatment of leukemia. Three cholinium-based ILs, namely cholinium chloride ([Ch]Cl), cholinium bicarbonate ([Ch][HCO3]), and cholinium glycolate ([Ch][Gly]), and two analogous DESs, namely [Ch]Cl-Glycerol and [Ch]Cl-Glycolic acid (GA), were investigated. These were applied in a range of concentrations (0-80 wt %) to determine their effectiveness in enhancing the enzyme stability and activity. Alterations in both the structure and activity of L-ASNase were observed in response to the hydration content of ILs and DESs, using several spectroscopic and computational techniques, with optimal performance observed at 40 wt %. Notably, [Ch]Cl, [Ch]Cl-Glycerol and [Ch][HCO3] revealed as promising to be incorporated into ASNase formulations, with [Ch]Cl-Glycerol exhibiting enhanced molecular polarity and establishing extended hydrogen bonding with the protein. On the other hand, both GA and [Ch]Cl-GA lead to the protein loss of stability. The most promising formulations, with ILs and DESs at 40 wt %, were evaluated under different temperature conditions (25 °C and 4 °C), for 5 h and 10 days. The best results were achieved with [Ch]Cl, which significantly enhanced the conformational and colloidal stability of L-ASNase, and with the thermal stability being remarkably increased by 9 °C. Reconstituted L-ASNase can be stored for up to 5 days at 4 °C in aqueous solutions of [Ch]Cl (40 wt %), offering potential cost savings and improved treatment administration.Furthermore, the structure and size of L-ASNase were maintained for at least 3 h at room temperature. Although it depends on the protein under study and conditions investigated, the set of results shown in this work demonstrate that ILs perform as better stabilizers of the ASNase biopharmaceutical than equivalent DESs. Our findings show the potential of choliniumbased formulations in extending the stability and improving the activity of L-ASNase, paving the way for sustainable solutions in the biopharmaceutical sector.