L-ASNase II purification through chromatographic processes with supported and solution ionic liquids

• Grant number: 24/17115-0
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: February 01, 2025
• End date: July 31, 2025
• Field of knowledge: Engineering - Biomedical Engineering - Bioengineering
• Principal Investigator: Valéria de Carvalho Santos Ebinuma
• Grantee: Enzo Corvello
• Supervisor: Mara Guadalupe Freire Martins
• Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Institution abroad: Universidade de Aveiro (UA), Portugal
• Associated to the scholarship: 23/14606-0 - Development of a purification platform of L-asparaginase II from Aliivibrio fischeri produced by Bacillus subtilis, BP.MS

Abstract

L-asparaginase (L-ASNase) is an enzyme capable of catalyzing the hydrolysis of asparagine, making it an important biopharmaceutical for the pharmaceutical industry, especially in the treatment of acute lymphoblastic leukemia. However, new production sources for this enzyme are required due to the development of hypersensitivity to current formulations, making it necessary to develop new biopharmaceuticals that are less immunogenic and with high activity. Since its production occurs through microorganisms, several purification steps are necessary to obtain a highly pure protein, which is essential for its application. To achieve this, there are low-resolution methods that perform the initial purification of the molecule of interest by removing some of the contaminants present in the medium, such as precipitation, where strategies can be combined to concentrate the target protein while removing undesired molecules. High-resolution methods, on the other hand, aim to obtain the target molecule with high purity, with various chromatographic methods being used for this purpose due to their high specificity. However, purification is the primary contributor to the final cost of the products, as it involves expensive columns and often results in loss of the target molecule during the process. Thus, it is evident that producing L-ASNase with high purity, coupled with processes that reduce the final product cost, is of great importance. Aiming to improve this stage by reducing costs and increasing purity, this project will evaluate the use of supported ionic liquids as matrices for chromatographic separation, aiming to increase specificity for the molecule of interest, that can be used with silica or polymeric matrices, maintaining selectivity and reducing the required volume. Another alternative to be evaluated is centrifugal partition chromatography, which takes advantage of the molecule's partitioning differences between two miscible phases under a centrifugal force, avoiding the use of columns that increase operational costs.