Fermentació alcohòlica amb cèl·lules immobilitzades

Al present treball presentem l'estudi sobre la posada a punt d'un sistema de fermentació alcohòlica amb cel•lules immobilitzades. En primer lloc estudiem el creixement en discontinu i l'activitat fermentativa de la soca de llevats, aïllada de most de vi d'alta graduació de la comarca del Priorat, que s'empra en el treball. També estudiem la seva modelització matemàtica. Es posa a punt un mètode d'immobilització de cèl•lules senzill i suau, utilitzant gels de carragenat. Es fa una caracterització mecànica i hidrodinàmica de les partícules amb cèl•lules immobilitzades i s'estudia l'estat i l'activitat dels llevats al seu interior. Per últim, es posa a punt una instal•lació experimental i es porta a terme la fermentació alcohòlica en continu d'una solució de glucosa, emprant un reactor tubular reblert amb cèl•lules immobilitzades.In this work the alcoholic fermentation using immobilized yeast cells is studied. The yeast strain used in the fermentation experiments has been isolated from a high grade wine and its growth and fermentation performance are studied in batch culture, as well as their mathematical modelization. The cell immobilization is carried out by a method using carrageenan gel beads which is very simple and mild. The mechanical and hydrodynamic characterization of the immobilized cell particles is done and the distribution and activity of the the cells inside them is studied. Finally, the continuous fermentation of a glucose solution in a tubular reactor filled with immobilized cells is carried out at different flow rates

An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures

Altres ajuts: Fundació la Caixa (LCF/BQ/ES17/11600003)Virus-like particles (VLPs) have gained interest over the last years as recombinant vaccine formats, as they generate a strong immune response and present storage and distribution advantages compared to conventional vaccines. Therefore, VLPs are being regarded as potential vaccine candidates for several diseases. One requirement for their further clinical testing is the development of scalable processes and production platforms for cell-based viral particles. In this work, the extended gene expression (EGE) method, which consists in consecutive media replacements combined with cell retransfections, was successfully optimized and transferred to a bioreactor operating in perfusion. A process optimization using design of experiments (DoE) was carried out to obtain optimal values for the time of retransfection, the cell specific perfusion rate (CSPR) and transfected DNA concentration, improving 86.7% the previously reported EGE protocol in HEK293. Moreover, it was successfully implemented at 1.5L bioreactor using an ATF as cell retention system achieving concentrations of 6.8·10 10 VLP/mL. VLP interaction with the ATF hollow fibers was studied via confocal microscopy, field emission scanning electron microscopy, and nanoparticle tracking analysis to design a bioprocess capable of separating unassembled Gag monomers and concentrate VLPs in one step

Bioprocessos industrials : biocombustibles

La biotecnologia ha tingut ja un important impacte en la millora o substitució de processos químics industrials. En aquest capítol es revisen els aspectes més importants de l'anomenada biotecnologia blanca, i les seves principals aplicacions en diferents sectors: enzims industrials, indústria quimicofarmacèutica, producció de biopolímers i biocombustibles.Biotecnology has already made an important impact in the improvement or replacement of industrial chemical processes. In this chapter, the most important aspects of White Biotechnology are reviewed, as well as the main applications in different sectors: industrial enzymes, chemico-phamaceutical industry, biopolymer production, and biofuels

Quality Assessment of Virus-Like Particles at Single Particle Level : A Comparative Study

Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals

At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification

At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 10 particles/mL. Simple buffers and operation make this process suitable for large scale purposes

PEI-mediated transient transfection of High Five cells at bioreactor scale for HIV-1 VLP production

High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100-200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins

Gene silencing strategies to increase HIV-1 VLP production in HEK 293 cells

The HIV-1 Gag polyprotein, recombinantly expressed in mammalian cell platforms, is self-assembled, generating HIV-1 Gag virus like particles (VLPs). The expression of HIV-1 Gag polyprotein is achieved by DNA/PEI-based transient transfection of HEK 293 cells in suspension. In this study, the use of small interfering RNA (siRNA) has been investigated as an alternative to the use of VPA and caffeine as production enhancers in HIV-1 Gag VLP production. Two shRNA sequences against HDAC5 and PDE8A genes, cloned in the expression vector containing Gag-GFP gene, were tested