Production optimization of rotavirus-like particles: a system biology approach

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Systems Biology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaRotavirus-like particles (RLPs), a vaccine candidate against rotavirus disease, were produced by infecting Spodoptera frugiperda Sf-9 cells with genetically engineered recombinant baculoviruses. RLPs are spherically shaped particles composed by three viral proteins (vp) of rotavirus, vp2, vp6 and vp7, arranged in a triple layered structure. A diversity of protein structures, other than the correctly assembled RLP, are observed at the end of a typical production run suggesting that the protein assembly process is rather inefficient. Contaminants such as trimers of vp6 and vp7, vp6 tube-like structures, single-layered vp2 particles, double layered particles of vp2 and vp6 or RLPs lacking one or more subunits represent almost 88% of the total mass of proteins expressed. Thus, optimal control of protein expression concomitant with efficient particle assembly are critical factors for economical RLP production in the baculovirus/insect cells system

Mecanismos de formação, redução e simulação da lesão cervical, resultantes de acidentes rodoviários com impacto lateral

Contexto: os acidentes rodoviários, com veículos ligeiros de passageiros, constituem um domínio que desafia a sociedade na procura continuada de soluções que mitiguem os seus impactos. Dados da Comissão Europeia revelam que, em 2011 e apenas no território dos países pertences à União Europeia, mais de 1,5 milhões de pessoas sofreram algum tipo de lesão em consequência de acidentes rodoviários, das quais, cerca de 250 mil terão sofrido lesões classificadas como graves. Esta dissertação visa a formulação de hipóteses para a redução da lesão cervical, em ocupantes de veículos sujeitos a colisão lateral e a validação de diretrizes para a conceção e o desenvolvimento, no âmbito da engenharia e do design, de uma solução de segurança passiva. Metodologia: recorrendo à literatura publicada, é analisada a anatomia, a fisiologia e a biomecânica da região cervical, bem como o controlo neuromuscular. São também revistos aspetos relevantes dos mecanismos de formação da lesão cervical, da sua classificação, das propriedades mecânicas dos tecidos e da ferramenta principal deste estudo que é a simulação e os modelos numéricos antropomórficos, de elementos finitos (FE). É apresentado o estado da arte do seu desenvolvimento para a simulação da biomecânica cervical e a compreensão dos mecanismos que conduzem à lesão e, posteriormente, à sua redução. Finalmente, são testados um conjunto de hipóteses para a redução do risco de lesão, no modelo ES-2re. Conclusões: a variação do módulo de elasticidade dos elementos viscoelásticos da região cervical do modelo ES-2re, revelou que o aumento global da rigidez cervical provoca um aumento dos valores máximos de deslocação, aceleração e velociade do CG da cabeça. Não é possível, no entanto, comparar o aumento da rigidez cervical avaliado nesta investigação com os testes onde essa rigidez acontece em resultado da ativação muscular. Por falta de correspondência, não é possível verificar a validade das hipóteses apresentadas em 4.4

Mecanismos de formação, redução e simulação da lesão cervical, resultantes de acidentes rodoviários com impacto lateral

Contexto: os acidentes rodoviários, com veículos ligeiros de passageiros, constituem um domínio que desafia a sociedade na procura continuada de soluções que mitiguem os seus impactos. Dados da Comissão Europeia revelam que, em 2011 e apenas no território dos países pertences à União Europeia, mais de 1,5 milhões de pessoas sofreram algum tipo de lesão em consequência de acidentes rodoviários, das quais, cerca de 250 mil terão sofrido lesões classificadas como graves. Esta dissertação visa a formulação de hipóteses para a redução da lesão cervical, em ocupantes de veículos sujeitos a colisão lateral e a validação de diretrizes para a conceção e o desenvolvimento, no âmbito da engenharia e do design, de uma solução de segurança passiva. Metodologia: recorrendo à literatura publicada, é analisada a anatomia, a fisiologia e a biomecânica da região cervical, bem como o controlo neuromuscular. São também revistos aspetos relevantes dos mecanismos de formação da lesão cervical, da sua classificação, das propriedades mecânicas dos tecidos e da ferramenta principal deste estudo que é a simulação e os modelos numéricos antropomórficos, de elementos finitos (FE). É apresentado o estado da arte do seu desenvolvimento para a simulação da biomecânica cervical e a compreensão dos mecanismos que conduzem à lesão e, posteriormente, à sua redução. Finalmente, são testados um conjunto de hipóteses para a redução do risco de lesão, no modelo ES-2re. Conclusões: a variação do módulo de elasticidade dos elementos viscoelásticos da região cervical do modelo ES-2re, revelou que o aumento global da rigidez cervical provoca um aumento dos valores máximos de deslocação, aceleração e velociade do CG da cabeça. Não é possível, no entanto, comparar o aumento da rigidez cervical avaliado nesta investigação com os testes onde essa rigidez acontece em resultado da ativação muscular. Por falta de correspondência, não é possível verificar a validade das hipóteses apresentadas em 4.4

Process intensification combined with Adaptive Laboratory Evolution enhance VLP- based vaccine candidates production in insect cells

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Process intensification for production of a peste des petites ruminants virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, a novel/scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in 2L STB (BIOSTAT DCU-3, Sartorius) using Nigeria 75/1 strain. Engineering correlations (e.g. shear stress and Eddy size) were used to optimize culture conditions. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. PBS washing, TrypLE Select and trypsin inhibitor concentrations, and stirring rates were the variables explored. Perfusion culture was evaluated in 2L STB (equipped with spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP2, Sartorius). Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates of 0.03 h-1 (similar to serum-containing cultures). The new in situ cell detachment method was successfully implemented, with yields above 80%; no impact on cell re-attachment or virus productivity was observed. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining the new in situ cell detachment method with perfusion culture will enable the scale-up to 20L STB directly from a 2L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. The potential of depth filtration for PPRV clarification (upon microcarriers sedimentation) could be confirmed, with yields up to 90%. Process scalability will be validated at the 20L scale in Sartorius BIOSTAT C-Plus (using engineering correlations such as shear stress and Kolmogorov-Eddy size as scale-up criteria) by comparing cells growth, metabolic and PPRV production kinetics to those achieved in 2L STB. In conclusion, the novel/scalable vaccine production process herein proposed has potential to assist the upcoming vaccination program for eradication of PPRV disease

Towards an integrated continuous manufacturing process of adeno- associated virus (AAVs)

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Bioprocess engineering of insect cells for accelerating vaccines development

The majority of novel protein-based biologics require animal cell technology for discovery and production. The costs and complexity associated to these production platforms are commonly high making mandatory further improvements in product yields. Technological breakthroughs and/or new producer hosts allied to classical systems/molecular biology tools and engineering methodologies are therefore necessary for accelerating biologics manufacturing process, including vaccines. Two case studies will be presented where bioprocess engineering assisted/accelerated vaccines development. In the first case study, the aim was to generate an Influenza VLP in which the surface antigens are presented in their native conformation as membrane-bound proteins and are comprised of several hemagglutinin (HA) variants specifically designed to target B cells capable of mounting broad neutralising antibodies. The platform herein adopted for production of enveloped Influenza VLP enclosed certain drawbacks, namely (1) instability of baculovirus vectors encompassing multiple HA genes, and (2) low expression levels and recovery yields. To tackle these issues, a set of bioprocess engineering schemes were designed and subsequently implemented, which included (1) combination of stable and baculovirus-mediated expression of HA in insect High Five cells for production of difficult to express multi-HA VLPs (pentavalent VLP of H3 subtype), (2) DoE for identifying best infection strategy and evolutionary engineering of insect cells phenotype, and (3) development of a scalable, “universal” and “All-Filtration” purification platform of Influenza VLPs. In the second case study, the aim was to develop a fast and flexible insect cell platform for production of enveloped VLPs pseudo-typed with membrane proteins of interest. Stable insect cell lines have been successfully generated using site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins, the Influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes have been designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve Gag-VLP production in the developed stable insect cells. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBBBIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/201

Enhancing production of the malaria asexual blood-stage vaccine candidate PfRipr5 in insect cells by modulating expression vector and culture temperature

Despite the recent approval of the first malaria vaccine RTS,S/AS01, its efficacy in children and infants is still modest. Therefore, continued development of new, improved malaria vaccines, including asexual blood-stage vaccines such as the one herein targeted, is essential to reach desired levels of protection against disease and mortality. In this study, the insect cell-baculovirus expression vector system (IC-BEVS) was used to produce a malaria asexual blood-stage vaccine candidate based on PfRipr5 antigen and compared to traditional mammalian (HEK293) cell system. PfRipr5 could be expressed to higher levels in IC-BEVS, with higher protein purity and reactivity to a conformational anti-PfRipr monoclonal antibody than its mammalian counterpart. The performance of IC-BEVS was further improved by modulating the expression vector sequence and culture temperature. The addition to the expression vector of (i) one alanine (A) amino acid residue adjacent to the signal peptide cleavage site, and (ii) a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag, resulted in up to 2.2-fold increase in the expression of secreted PfRipr5. In addition, lowering temperature from standard 27 °C to 22 °C at the time of infection improved PfRipr5 productivity by up to 1.7-fold. Noteworthy, a synergistic effect was attained by combining both optimization strategies, enabling to increase expression of extracellular PfRipr5 by up to 4-fold and process yield post-purification by 5.2-fold, while maintaining same degree of protein purity and reactivity. This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost expression of secreted proteins

Advances in bioprocessing, analytics and formulation of influenza HA-VLP vaccine candidates produced by insect cells

The emergence of new influenza strains demands the continued development of novel, flexible, and scalable platforms for vaccine production. In this study, we describe advancements in the manufacturing process of influenza hemagglutinin (HA)-displaying virus-like particle (VLP)-based vaccines produced by insect cells, from upstream and downstream processing to analytics and formulation. Aiming to improve influenza HA-VLPs production, evolutionary engineering and process intensification have been applied. Adaptation of stable Sf-9 cells producing HA-VLPs to hypothermic growth resulted in up to 12-fold higher expression. Likewise, adaptation of parental High Five cells to neutral pH induced a 3-fold higher specific HA-VLPs production rate following infection with baculovirus. In both case studies, the adaptation process had no impact on VLPs activity and morphology. Noteworthy, stable adapted Sf-9 cells could be cultured in perfusion (up to 100x106 cell/mL) and continuous (~20x106 cell/mL) operation modes with cell-specific productivity similar to batch mode. Please click Download on the upper right corner to see the full abstract

A modular approach for efficient production of multi-HA Influenza VLP-based vaccines

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains. Promising approaches based on the expression of multiple hemagglutinins (HA) (alone or in combination with neuraminidase and matrix M1 proteins), in a single vector or virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can be an issue due to tandem repetition of promoter sequences leading to its instability. By combining stable with transient expression we can rationally distribute the number of genes to be expressed by each system and thus mitigate this risk. Therefore, we developed a modular system using stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs. First, a stable pool of High Five cells expressing two HA was established by random integration and intracellular HA expression confirmed by immunofluorescence microscopy. This cell pool was then infected at CCI of 2 or 3×106 cells/mL with M1-encoding baculovirus to evaluate the incorporation of stable expressed HA in the M1 core, thus generating Influenza VLPs. Similar levels of Influenza VLPs could be detected in culture medium by hemagglutination assay regardless of the CCI used. Aiming to increase HA production, infections at a higher CCI were attempted by implementing a feeding strategy designed based on the exhaustion of key nutrients, analyzed by 1H-NMR spectroscopy. Noteworthy, the shake flask cultures that were supplemented and infected at a CCI of 4×106 cells/mL showed a 8-fold increase in HA levels when compared to above tested conditions. The robustness of our modular system was then challenged by infecting the stable High Five cell pool with a baculovirus encoding M1 plus three HA proteins. Results obtained at CCI of 4×106 cells/mL with supplementation showed a 4-fold increase in HA levels when compared to standard infection conditions (CCI of 2 and 3×106 cells/mL). Finally, to demonstrate the scalability of the strategy herein designed, cultures in fully controlled 2L stirred tank bioreactors were performed, and a 1.5-fold improvement in HA levels was obtained when compared to shake flask cultures. Overall, this work demonstrates the suitability of combining a stable insect cell line with baculovirus-mediated expression as a faster platform for production of multi-HA Influenza VLPs surpassing standard methods such as coinfections or the use of larger, unstable vectors. Acknowledgements This work was supported by EU-funded project EDUFLUVAC (FP7-HEALTH-2013-INNOVATION)