Antileukemic biopharmaceuticals production and purification using nanomaterials

• Autor(a) principal: Paixão, Catarina de Brito
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/37153
• topic: Biopharmaceuticals; L-asparaginase; Purification; Supported Ionic Liquids; Merrifield resin

Biopharmaceuticals are a natural alternative to chemically synthesized pharmaceuticals. They can be based on enzymes, hormones, monoclonal antibodies, cytokines, among others; and are already applied in the treatment of many diseases that do not reply to common therapies. The enzyme L-asparaginase (L-ASNase) is a biopharmaceutical already used in treatments of many leukemias, namely Acute Lymphoblastic Leukemia (ALL), or Hodgkin’s disease. Particularly, in ALL, type of leukemia that attacks mostly children and young people, L-ASNase acts as an inhibitor of the carcinogen cells growth. However, the L-ASNase commercially available, and from Escherichia coli, contains a high activity of L-glutaminase, causing side effects, for example edema, fever, diabetes and haemorrhages, in the patients. Therefore, the search for new microorganisms that produce L-ASNase with decreased secondary effects is of extreme importance. In this work, a recombinant L-ASNase was produced from Bacillus Subtilis. After that, as the conventional methods for the purification of this enzyme are very expensive, it was developed a new purification technique for L-ASNase, more economical, through the usage of supported ionic liquids (SIL). Three SILs were synthesized and characterized, and different experimental conditions were tested, in order to optimize the purification process of L-ASNase, namely the cell extract total protein concentration and the material/cell extract ratio. At last, a semi-continuous assay was performed with the optimized conditions. The results obtained demonstrate that optimum experimental conditions for L-ASNase purification occur at a cell extract concentration of 16.7 mg/mL (original total protein concentration after cell lysis) and 50 mg of material per each mL of cell extract, corresponding to a specific activity of L-ASNase of 0.0382 U/mg and a purification fold of 3.46.