Purification of antibodies using aqueous biphasic systems
authors Emanuel V. Capela
supervisors Dr. Mara G. Freire, Master Mafalda R. Almeida
thesis type MSc Thesis in Biochemistry
nationality International
author keywords Antibodies, biopharmaceuticals, immunoglobulin Y, liquid-liquid extraction, purification, aqueous biphasic systems, ionic liquids
abstract Nowadays, the emergence of antibiotic-resistant microorganisms, diseases that do not respond to conventional therapies and individuals whose immune system is unable to generate an efficient response to traditional vaccination are common concerns. Thus, itís essential to develop specific antibodies for use in passive immunotherapy. Although mammalian antibodies are more specific, a potential alternative consists on immunoglobulin Y (IgY), an antibody produced in large quantities and present in the egg yolk of hens, once the obtaining process does not make use of invasive techniques, and the IgY is abundantly produced, which contributes to a reduction on the production costs by the pharmaceutical industries. However, this cost of production of IgY with high purity is still significant due to the absence of an effective purification technique able to separate IgY from other contaminant proteins. The major goal of this work consists in the development of an alternative technology in order to extract and purify antibodies from egg yolk aiming at producing cheaper and more effective biopharmaceuticals. Therefore, in this work, three types of aqueous biphasic systems (ABS) were studied, namely constituted by a polymer and a salt, a polymer, a salt and an ionic liquid (IL) as adjuvant and by an IL and a salt, as alternative liquid-liquid systems for the selective extraction, and thus purification, of IgY from egg yolk. The phase diagrams of the systems were determined, as well as the corresponding tie-lines. The relative hydrophobicity of their coexisting phases was also ascertained by means of the determination of the partition coefficients of a series of dinitrophenylated amino acids. According to the obtained results, it was verified that the purification of IgY wasnít achieved in a single step procedure, and where the major contaminant protein, -livetin, still appears in all systems. However, amongst the several ABS evaluated, the system constituted by [C4mim][N(CN)2] + Na2SO4 allowed the selective migration of -livetin for one of the phases, and then IgY may be purified applying successive extractions. This system represents the improved choice foreseeing the selective extraction and subsequent purification of IgY.
year published 2014