Planta de producción de carbonato sódico

Aquest projecte presenta el procés de producció del carbonat sòdic a partir del mètode Solvay. Aquest procés es caracteritza perquè a partir de matèries primes molt bàsiques, com són el clorur sòdic i la pedra caliça, s'obté un producte de gran mercat, el carbonat sòdic. En el projecte s'estudien tant el disseny dels equips que seràn part del procès de producció com els sistemes de control utilitzats, impactes mediambientals que es puguin generar o l'avaluació econòmica de l'acceptació del procés en el mercat, entre d'altres

An engineered HIV-1 Gag-based VLP displaying high antigen density induces strong antibody-dependent functional immune responses

Antigen display on the surface of Virus-Like Particles (VLPs) improves immunogenicity compared to soluble proteins. We hypothesised that immune responses can be further improved by increasing the antigen density on the surface of VLPs. In this work, we report an HIV-1 Gag-based VLP platform engineered to maximise the presence of antigen on the VLP surface. An HIV-1 gp41-derived protein (Min), including the C-terminal part of gp41 and the transmembrane domain, was fused to HIV-1 Gag. This resulted in high-density MinGag-VLPs. These VLPs demonstrated to be highly immunogenic in animal models using either a homologous (VLP) or heterologous (DNA/VLP) vaccination regimen, with the latter yielding 10-fold higher anti-Gag and anti-Min antibody titres. Despite these strong humoral responses, immunisation with MinGag-VLPs did not induce neutralising antibodies. Nevertheless, antibodies were predominantly of an IgG2b/IgG2c profile and could efficiently bind CD16-2. Furthermore, we demonstrated that MinGag-VLP vaccination could mediate a functional effect and halt the progression of a Min-expressing tumour cell line in an in vivo mouse model

Differential N- and O-glycosylation signatures of HIV-1 Gag virus-like particles and coproduced extracellular vesicles

Acord transformatiu CRUE-CSICAltres ajuts: "la Caixa" Foundation (ID 100010434)Human immunodeficiency virus 1 (HIV-1) virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP-based vaccine production technology

Strategies for improving production levels of HIV-1 VLPs by transient transfection of HEK 293 suspension cultures

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2017-2018Les partícules similars a virus (VLP) ofereixen un gran potencial com a candidates per a la nova producció de vacunes. En aquest treball es presenta el desenvolupament i l'optimització d'un protocol de producció de VLP de Gag d'VIH-1 mitjançant l'expressió gènica transitòria en cultius de cèl·lules HEK 293 en suspensió. La transfecció transitòria permet una generació ràpida de proteïnes recombinants en quantitat i qualitat suficient per realitzar assajos preclínics, i és particularment interessant en les fases primerenques del desenvolupament. Aquest treball es divideix en quatre capítols principals. Al primer capítol, el medi comercial lliure de sèrum Freestyle 293 s'optimitza mitjançant l'ús de components d'origen no animal. L'ús de medis químicament definits i sense components derivats d'animals és un requisit bàsic per una vacuna destinada als éssers humans. La densitat cel·lular màxima assolida utilitzant el medi de cultiu optimitzat (suplementat amb 0,9X de mescla de lípids, 19,8 mg/L d'insulina recombinant i 1,6 mg/L de transferrina recombinant) és 5,4×106 cèl·lules/ml en discontinu, gairebé el doble del que s'assoleix utilitzant el medi sense suplementar (2.9×106 cèl·lules / ml). A més a més, després de l'optimització del medi, també s'ha millorat el protocol de transfecció. La millor producció s'aconsegueix quan les cèl·lules es transfecten a la meitat de la fase logarítmica (2-3 x 106 cèl·lules/ml) amb recanvi de medi en el moment de la transfecció utilitzant 1 g/(ml de cultiu) d'ADN i 2 g/(mL de cultiu) de polietilenimina. Mitjançant l'ús d'aquest protocol, els títols de VLP es van incrementar 2,4 vegades, obtenint 2,7 × 109 VLP/ml. Tant el medi de cultiu optimitzat com el protocol de transfecció s'utilitzen a la resta del treball. Al capítol dos, la cinètica del procés de transfecció transitòria s'estudia amb l'objectiu de caracteritzar i comprendre el procés complet a nivell intracel·lular que condueix a la producció de VLP, per tal de determinar els punts importants per poder millorar el procés. Els poliplexes comencen a interactuar amb la membrana cel·lular just després de la seva addició al cultiu. Després d'una hora i mitja es detecten al citoplasma de les cèl·lules i arriben al nucli al voltant de 4 hores després de la transfecció. Després de 10 hores es pot detectar la fluorescència GFP dins de les cèl·lules, però no s'observa la sortida de les VLPs de les cèl·lules, per gemmació, fins 48 hores després de la transfecció. El moment òptim de recollida del producte s'ha fixat a les 72 hores post transfecció ja que la producció de VLPs és màxima mentre es manté una viabilitat del cultiu alta. Al capítol 3, s'estudia la millora de la producció de VLPs utilitzant compostos específics. Es proven dos grups de potenciadors de la transfecció, un grup utilitzat per facilitar l'entrada de complexes de ADN/PEI a la cèl·lula o al nucli i un altre grup seleccionat per augmentar els nivells d'expressió gènica. Entre els vuit additius utilitzats (tricostatina A, àcid valproic, butirat de sodi, DMSO, acetat de liti, cafeïna, hidroxiurea i Nocodazol) s'identifica una combinació òptima que permet obtenir el màxim d'expressió. L'addició de 20 mM d'acetat de liti, 3,36 mM d'acid valproic i 5,04 mM de cafeina augmenta els nivells de producció 4 vegades, mentre la viabilitat del cultiu es manté al 94%. Atès que l'expressió transitòria (TGE) es basa en l'expressió episomal d'ADN plàsmidic, està limitada a un període curt de producció, d'en general unes 96 h, fet que limita la productivitat. Al capítol 4, es desenvolupa un protocol nou, anomenat expressió gènica extesa (EGE). L'objectiu de l'EGE és perllongar el període de producció, combinant el recanvi de medi i la transfecció repetida del cultiu per millorar la producció de proteïnes. L'avantatge d'aquesta metodologia és avaluada per a la producció de tres productes recombinants model: la GFP intracel·lular, la GFP secretada, i una partícula similar a virus Gag-GFP (VLP). Utilitzant aquesta nova estratègia EGE, el període de producció es perllonga entre 192 i 240 h amb un augment de producció d'entre 4-12 vegades en els nivells de producció, depenent del tipus de producte considerat.Virus-like particles (VLPs) offer great potential as candidates for new vaccine production. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in HEK 293 suspension cultures is presented. Transient transfection enables a rapid generation of recombinant proteins of sufficient quantity and quality to perform pre-clinical trials, and it is particularly VLP production, and to determine important time points to drive process improvement. Polyplexes start to interact with the cell membrane just after addition to the culture. After 1.5 hpt complexes are detected in the cytoplasm of the cells and reach the nucleus around 4 hours post transfection. After 10 hours post transfection GFP fluorescence is detected inside the cells, but generalized budding of VLPs from the cells is not observed until 48 hours post transfection. The optimal harvest time is determined as 72 hpt as VLP production is highest while high viability of the culture is maintained. In chapter three, the enhancement of VLP production using specific compounds is studied. Two main groups of transfection enhancers are tested, selected on the basis that they can either facilitate the entry of PEI/DNA transfection complexes into the cell or nucleus or they can increase the levels of gene expression. Among the eight transfection-enhancers tested (Trichostatin A, Valproic acid, Sodium Butyrate, DMSO, Lithium Acetate, Caffeine, Hydroxiurea and Nocodazole) an optimal combination of compounds exhibiting the greatest effect on gene expression is subsequently identified. The addition of 20 mM Lithium Acetate, 3.36 mM Valproic Acid and 5.04 mM Caffeine increases production levels by 4 fold, while maintaining cell culture viability at 94 %. As transient gene expression (TGE) is based on episomal plasmid DNA expression, conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. In chapter four, a novel gene expression approach termed extended gene expression (EGE) is proposed. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell culture with plasmid DNA to improve overall protein production. The benefit of this methodology is evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particle (VLP). Using this novel EGE strategy, the production period is prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. interesting in the early development phases. This work is divided in four main chapters. In the first chapter, the serum-free commercial medium Freestyle 293 is optimized using non-animal derived components as supplements. The use of chemically defined animal derived component free media and supplements is a basic requirement for any further use of a vaccine for humans. The maximum cell density attained using the optimized medium (supplemented with 0.9X of Lipid mixture, 19.8 mg/L of r-insulin and 1.6 mg/L of r- transferrin) was 5.4 × 106 cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×106 cells/mL). Moreover, after the medium optimization, the transfection protocol is also improved. Best production performance is attained when cells were transfected at mid-log phase (2-3 × 106 cells/mL) with medium exchange at the time of transfection using 1 μg/(mL of culture) of plasmid DNA and 2 μg/(mL of culture) of polyethylenimine. By using this protocol, VLP titers are increased 2.4-fold, obtaining 2.7 × 109 VLPs/mL. The optimized medium and transfection protocol defined in this chapter are used in the rest of the work. In chapter two, the kinetics of the transient transfection process is studied with the aim to characterize and understand the complete process at intracellular level leading to the this methodology is evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particle (VLP). Using this novel EGE strategy, the production period is prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered

Extended gene expression for HIV-1 VLPs scale-up and production enhancement using shRNA and chemical additives

Gag polyprotein from HIV-1 can generate Virus-Like Particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved using different strategies for increasing product titers. One of them is the so-called Extended Gene Expression (EGE), based on repeated medium exchanges and retransfections of the cell culture to prolong the production phase. Another approach to improve transient transfection results is media supplementation with gene expression enhancers such as valproic acid and caffeine, despite their detrimental effect on cell viability. Valproic acid is a histone deacetylase inhibitor while caffeine has a phosphodiesterase inhibition effect. The work presented has three main objectives. First, the combination of the EGE protocol with valproic acid and caffeine supplementation to maximize VLP production; second, the replacement of these chemical additives by shRNA for obtaining the same inhibition action and third the bioreactor scale-up of the process. The combination of the EGE protocol with caffeine and valproic acid supplementation resulted in a 1.5-fold improvement in HIV-1 VLP production compared with the EGE protocol alone, which is an 18-fold improvement over a conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.5-fold without any detrimental effect on cell viability, which results in obtaining higher quality VLPs. Finally, the combination of shRNA with EGE resulted in more than 14-fold improvement compared with the batch standard protocol traditionally used. This protocol enables the production of high-quality HIV-1 VLPs avoiding toxic effects of the additives but maintaining high product titers. Please click Additional Files below to see the full abstract

A four-step purification process for gag vlps : from culture supernatant to high-purity lyophilized particles

Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 10 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles

Identification of HIV-1-Based Virus-like Particles by Multifrequency Atomic Force Microscopy

Virus-like particles (VLPs) have become a promising platform for vaccine production. VLPs are formed by structural viral proteins that inherently self-assemble when expressed in a host cell. They represent a highly immunogenic and safe vaccine platform, due to the absence of the viral genome and its high protein density. One of the most important parameters in vaccine production is the quality of the product. A related bottleneck in VLP-based products is the presence of cellular vesicles as a major contaminant in the preparations, which will require the set up of techniques allowing for specific discrimination of VLPs from host vesicular bodies. In this work novel, to our knowledge, multifrequency (MF) atomic force microscopy (AFM) has permitted full structural nanophysical characterization by its access to the virus capsid of the HIV-based VLPs. The assessment of these particles by advanced amplitude modulation-frequency modulation (AM-FM) viscoelastic mapping mode has enhanced the imaging resolution of their nanomechanical properties, opening a new window for the study of the biophysical attributes of VLPs. Finally, the identification and differentiation of HIV-based VLPs from cellular vesicles has been performed under ambient conditions, providing, to our knowledge, novel methodology for the monitoring and quality control of VLPs

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