Defensa de la Tesi de Jesús Lavado García
04.10.2021  - 

El día 06/10/2021, presentará su Tesis Doctoral Jesús Lavado García, desarrollada en el grupo Grupo de Ingeniería Celular y Tisular


La defensa de la Tesis de Jesús Lavado García, con el título "Bioprocess Engineering for HIV-1 Gag VLP production in HEK293 cells" tendrá lugar en Sala de Grados de la Escuela de Ingeniería el día 6 de octubre a las 10: 00h y se pordrá seguir también a través del'enllaç siguiente: https://mcgill.zoom.us/j/87811029420?from=addon

La dirección de la Tesis han sido llevada a cabo por el Dr. Frances Gòdia y la Dra. Laura Cervera y el Tribunal que evaluará la tesis estará formado por:

  • Prof. Amine Kamen (University McGill, Montreal, Canada)
  • Prof Massimo Morbidelli (ETH Zurich, Switzerland and Politecnico de Milano, Italy)
  • Prof. Paula Alves (IBET, Oeiras, Portugal)

Resumen:

The importance of vaccine technology has recently become a matter of high interest due to the outbreak of the COVID-19 pandemic. However, the development of vaccine technology against infectious diseases throughout history has never stopped. It has influenced the way the population interacts and the establishment of social rules. The research in the field of vaccine technology is deeply tied to the research in bioprocess engineering, as in a globalized world, an effective vaccine is the one that can be produced at large scale, reaching as many people as possible through efficient vaccination policies. This PhD thesis is focused on the study of the bioprocess for the production of human immunodeficiency virus (HIV-1) virus-like particles (VLPs) based on the HIV-1 polyprotein Gag using HEK293 cells and transient transfection. These VLPs are non-infectious membrane-bound nanostructures whose conformation resembles the native HIV-1 virus albeit lacking the viral genetic material. In order to use these particles as potential vaccines, they can be later modified presenting exogenous antigens in their membrane to elicit an immune response against different diseases. In order to optimize VLP production, multiple aspects of the bioprocess must be studied, from molecular factors like cellular pathways to the modulation of operational parameters at bioreactor scale. In this work, this undertaken challenge can be divided in three main parts.

The first part, comprising chapters one and two, focused on the optimization of the VLP production process at cellular level through proteomics and metabolic engineering. Here, the effects of transient transfection and production of VLPs was studied at molecular level using multiplexed quantitative proteomics to identify metabolic bottlenecks influencing VLP production. Once these pathways were identified, metabolic engineering and design of experiments (DoE) approaches were applied to optimize transfection, cell budding efficiency and VLP production.

The second part, comprising chapter three, focused on the intensification and optimization of VLP production at bioreactor level, implementing a perfusion-based bioprocess. The culture media used, the DNA complexation agent and culture conditions in the bioreactor were studied. Also, operational parameters like the cell specific perfusion rate (CSPR), the amount of DNA used for transfection and the time of retransfection were also optimized using a DoE approach. Moreover, different cell retention devices were tested to work towards the implementation of a continuous harvest mode of operation.

The third and last part, comprising chapters four, five and six, focused on the characterization of the product aiming to design an efficient downstream process. Parallel reaction monitoring (PRM) was used to establish a method of determining Gag stoichiometry in the VLPs for different production platforms, paving the way for improvements in quality control and process analytics technologies (PAT) in a future large scale bioprocess. Moreover, the coproduced extracellular vesicles (EVs) were studied through multiplexed quantitative proteomics to better understand the nature of the main source of impurities in a VLP preparation. Additionally, EV and VLP glycosylations were analyzed, to characterize the two nanostructures glycosylation signature and help gain some insight towards the design of a specific separation method for VLP purification.

The results hereby presented contribute to the development of a promising vaccine platform based on  Gag VLPs and pave the way towards the advance and evolution of vaccine manufacturing.

 
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