Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field. This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process. / <p>QC 20170523</p>

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-02-21T12:43:27Z No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-24T11:24:18Z (GMT) No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-24T11:24:33Z (GMT) No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Made available in DSpace on 2017-04-24T11:31:33Z (GMT). No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) Previous issue date: 2016-08-08 / Não recebi financiamento / Although effective, current vaccinations against rabies, one of the most lethal infectious diseases in the world, present security issues of administration and production costs. In this scenario, modern biotechnology has become a source of new alternatives of great interest for vaccine production. The main antigen capable of conferring neutralizing immune response against infection by rabies virus is the glycoprotein of rabies virus (RVGP), which the production by recombinant DNA technology has been developed by researchers at the Viral Immunology Laboratory (LIV) of the Butantan Institute of São Paulo using various expression systems in Drosophila melanogaster S2 cells. One of the latest developments is S2MtRVGP-H-His cell line, obtained by stable transfection with plasmids containing cDNA from other components of RVGP and histidine tag to facilitate purification, both under control of the inducicle metallothionein promoter. This work aims to study the kinetic characteristics of cell growth and production of recombinant glycoprotein rRVGP rabies virus strain of Drosophila melanogaster S2MtRVGP-H-His, in order to evaluate the potential of a bioreactor with agitation induced by waves (Wave) for the scale-up production of rRVGP. The first stage of the study, involving batch cultures in 20 mL Schott bottle with commercial culture medium Sf900-III, allowed us to determine the optimal temperature of cultivation (28ºC), time of induction of expression (72 h), the specific growth rate ranging from 0.022 to 0,034 h-1; maximum cell density 1.82×107 cel.mL-¹ and rRVGP produced from 0.07 to 0.99 μg.mL-1. Based on these results, was started the second part of the study performed in the Single-use Wave bioreactor, involving batch cultures with 650 mL of Sf900-III, with 60% of dissolved oxygen and pH ranging without control from 6.2 to 7.0. The culture in the bioreactor showed maximum specific growth rate of 0,035 h-1, maximum cell density was 1.1×107cel.mL-¹ and RVGP produced 0.85 μg.mL-1. The production of large scale rRVGP with S2MtRVGP-H-His cells using the Wave bioreactor has shown to be viable, reproducible and with high potential to scale-up. / Embora eficazes, as vacinas atuais contra a raiva, uma das doenças infecciosas mais letais do mundo, apresentam problemas relacionados com a segurança de administração e o custo de produção. Nesse contexto, a biotecnologia moderna se torna uma fonte de alternativas inovadoras de grande interesse para produção de vacinas. O principal antígeno capaz de conferir uma resposta imunológica neutralizante contra o vírus rábico é a glicoproteína do vírus da raiva (RVGP), cuja produção por tecnologia de DNA recombinante vem sendo desenvolvida por pesquisadores do Laboratório de Imunologia Viral (LIV) do Instituto Butantan de São Paulo, utilizando vários sistemas de expressão em células de Drosophila melanogaster S2. Um dos mais recentes desenvolvimentos é a linhagem S2MtRVGP-H-His, obtida mediante transfecção estável com plasmídeos contendo entre outros componentes o cDNA da RVGP e a cauda de histidina para facilitar a purificação, ambos sob controle do promotor indutível da metalotioneína. O presente trabalho tem como objetivo o estudo de características cinéticas de crescimento celular e de produção de glicoproteína recombinante do vírus da raiva rRVGP da linhagem de Drosophila melanogaster S2MtRVGP-H-His com vistas a avaliação do potencial de um biorreator com agitação induzida por ondas (waves) para escalonamento da produção de rRVGP. A primeira etapa dos trabalhos, envolvendo cultivos em batelada em frasco Schott com 20 mL de meio de cultura comercial Sf900-III, permitiu a determinação da temperatura ideal (28ºC), o tempo apropriado de indução da expressão (72 h) e das velocidades específicas de crescimento de 0,022-0,034 h-1, densidade celular máxima de 1,82×107 cel.mL-¹ e rRVGP produzida de 0,07-0,99 μg.mL-1. Com base nesses resultados, iniciou-se a segunda parte dos trabalhos com cultivos em biorreator Wave utilizando 650 mL de meio Sf900-III, com concentração média de oxigênio dissolvido de 60% da saturação com ar e pH variando sem controle de 6,2-7,0. O cultivo no biorreator apresentou velocidade específica máxima de crescimento de 0,035 h-1, densidade celular máxima de 1,1×107 cel.mL-¹ e rRVGP produzida de 0,85 μg.mL-1. A produção da rRVGP em larga escala com células S2MtRVGP-H-His utilizando o biorreator Wave mostrou ser uma alternativa viável, reprodutível e com grande potencial de escalonamento.