Produção de fragmento recombinante de anticorpo em Pichia pastoris

Abstract

FEITOSA, V.A. Production of recombinant antibody fragment in Pichia pastoris. 2014. 84 p. Thesis (Master) Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, 2014. Composition and pH culture medium for the production of antiLDL(-) antibody fragment (scFv) were studied in recombinant yeast Pichia pastoris. The experiments that defined medium composition, pH and the initial cell concentration in the induction phase were carried out in baffled shaker flasks at 250 rpm, with temperature at 30°C for growth phase and 20°C for induction phase, during 72 hours with daily addition of 1% (v/v) methanol. A design of experiments employing for medium modification with independent variables: soy extract, casamino acids and urea, which replaced the YNB and biotin present in the standard medium (BMMY) was conducted for medium formulation. Even though, there was an increase in medium production with soy extract, the medium containing 10 g.L casamino acids was selected since it favors the purification step. Through a pH range study (3.0 to 8.0), it was determined that pH 8.0 during induction phase offered higher levels. Then, the best results from shaker flask cultivation (BMMY-CA with casamino acids and pH 8.0) were carried out in 1L bioreactor, showing a biomass and a scFv production increase compared to the standard, BMMY (pH 6.0). Finally, the medium BMMY-CA (pH 8,0) was submitted to a volume scale-up into a 10L bioreactor, which was also used to evaluate kinetic parameters (rates of growth, substrate consumption and production as well as productivity). In conclusion, by optimizing culture medium and operating conditions it was possible to increase yield and scale-up the production of scFv anti-LDL(-) in bioreactor.