Obtenção de anticorpo monoclonal murino anti-cd25 através do cultivo do hibridoma pc-61 em biorreatores spinner em meio livre de soro
Damada, Pedro Henrique
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Cancer is one of the deadliest diseases in the world. Its treatment includes surgical, chemotherapeutic and radiotherapeutic methods, which, although efficient, are expensive, long and can have several side effects to the patient. In order to seek new methodologies, immunotherapy, a treatment based on stimulation of the immune system to control cancer, has been one of the most interesting. One of the most used immunotherapeutic methods for this purpose is based on the use of monoclonal antibodies (mAbs), high specificity glycoproteins that are gaining space and arousing interest due to the positive results have already obtained for different diseases, such as multiple sclerosis and breast cancer. The objective of this work was to optimize the culture of PC-61 hybridomas in spinner flask for the production of the anti-CD25 mAb, with serum free commercial medium (SFM) and with RPMI medium supplemented with fetal bovine serum (FBS), used as control. Two modes of operation were used: batch and fed batch, in order to compare the results and propose a better methodology of cultivation. In addition, the purification of the antibodies was done using the techniques of ion exchange chromatography and immunoprecipitation with protein G in Sepharose. As a result, by batch culture, it was possible to identify the behavior of the cells and to stipulate the best conditions to carry out batch culture. Thus, it was possible to prolong the pre-stationary phase of the culture where a higher production of monoclonal antibody was observed. The SFM medium presented the best results compared to RPMI (with FBS) in batch culture, it was found that the production of mAbs in SFM was 3 times higher than in RPMI (with FBS). Furthermore, through these cultures, cells have been noted to direct their metabolism to produce the antibody when the nutrient level in the culture medium is reduced, and stress conditions (without causing cell death) can lead to increased production of antibody. In relation to purification, the immunoprecipitation technique with G protein proved to be very efficient, mainly for the separation of the antibodies present in the supernatant of the SFM culture, which has low protein content. Therefore, the use of the SFM medium is a great alternative to the culture media supplemented with FBS, since, in certain conditions, it allows the obtaining of larger amounts of antibody, and ensures greater ease in the application of purification techniques, reducing production costs.