Navegando por Palavras-chave "Cromatografia De Imunoafinidade"

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    Padrão de glicosilação da glicoproteína do vírus da raiva produzida em células drosophila melanogaster S2: diferentes sistemas de expressão e condições de cultivo tese
    (Universidade Federal de São Paulo (UNIFESP), 2019-11-13) Silva, Livia Pilatti Mendes Da [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Rabies virus glycoprotein (RVGP) produced in animal cells had become an attractive alternative for development of a new vaccine against rabies with lower biosecurity risk and lower costs. In recombinant proteins production host cell lineage and cultivation parameters and conditions are important factors for glycoform profile of final protein, which impacts directly in its function. Objectives: Determine and compare the glycosylation pattern of rabies virus glycoprotein (RVGP) expressed in insect Drosophila melanogaster Schneider 2 (S2), in different expression systems and culture conditions. Methods: S2 cells expressing RVGP in cell membrane using inducible (S2MtRVGP-imm) or constitutive (S2AcRVGP-2) promoter, and expressing the soluble RVGP ectodomain, using an inducible promoter (S2BipMt_RVGP-Ecto), stablished in previous studies, were used in the present work. S2BipMt_RVGP-Ecto cells were cultivated in schott flasks and S2MtRVGP-imm cells were cultivated in schott and spinner flasks, in both cases with different induction parameters and cultivation time. S2AcRVGP-2 cells grown in bioreactor, in batch or fed-batch with glutamine supplementation. An immunoaffinity chromatography methodology for membrane rRVGP (rRVGP-S2Mt and rRVGP-S2Ac) were stablished using a commercial monoclonal antibody attached to a HiTrap NHSactivated HP (GE). Soluble ectodomain (rRVGP-S2Ecto) were purified by Immobilized Metal Affinity Chromatography (IMAC-Ni+2). Glycosylation pattern were analyzed by hydrophobic interaction chromatography (HILIC) or by HILIC accoupled with a mass spectrometry (UPLC-FLR-MS/MS). Results: For S2BipMt_RVGP-Ecto cells, the variation in inductor concentration presented biological effect that reflected on cell growth and rRVGP production. Inductor concentration and cell concentration at induction resulted in an increase in rRVGP production speed by S2MtRVGP-imm cells. Cultivation on spinner flasks results in a higher production of rRVGP-S2Mt comparing with schott flasks cultivation. The strategy of low initial glutamine concentration with supplementation during the S2AcRVGP-2 cell cultivation decreased the ammonium formation after the end of exponential phase, with an impact on growth and rRVGP production. Immunoaffinity chromatography methodology purification generated samples with high level of purity and efficiency up to 99 % of trimeric rRVGP-S2Mt. For rRVGP-S2Ac the same methodology worked with less efficiency and low level of purity. IMAC-Ni+2 used for rRVGP-S2Ecto presented low level of purity due to the low amount of expressed glycoprotein. In glycan pattern analyzes, samples of rRVGP-S2Ecto presented in higher proportion paucimannosidic structures: F(6)M2, M3 and F(6)M3. In rRVGP-S2Mt samples, the structures F(6)M2 e M3 were detected in higher proportion. rRVGP-S2Ac samples presented higher proportion of structures M2, F(6)M2 e M3. Conclusions: Increase of inductor concentration and of cell concentration at induction time results in a biological effect, by increasing expression speed of rRVGP. Specific production speed of rRVGP has a direct influence on decrease of N-glycans processing. Furthermore, different cultivation modes resulted in variation in glycan pattern, and a drastic reduction in initial glutamine concentration resulted in an increase on glycoforms with five or more mannose residues.