Part 1, X-ray structural characterisation of some complexes of second- and third-row transition metals ; Part 2, Crystallographic studies into the nature of two industrial bleaching agents

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Starter culture production in fluidized bed reactor with a flocculent strain ofL. plantarum

A lactic starter culture of a flocculentLactobacillus plantarum was produced in a fluidized bed reactor with higher cell volumetric productivities than in a continuous stirred tank reactor. The fluidized bed reactor was operated at optimised parameters obtained in batch reactor performed with and without pH control

Structure determination of Split-soret Cytochrome from a Desulfovibrio species isolated from a human abdominal abcess

The determined structure of the split-soret cytochrome (SSC) isolated from Desulfovibrio desulfuricans ATCC 27774 (D.d.) revealed a new Heme arrangement, which suggests that this protein constitutes a new cytochrome class.. SSC is a 52.6kDa homodimer containing four hemes at one end of the molecule. In each monomer the two hemes have their edges overlapped within van der Waals contacts. The polypeptide chain of each monomer supplies the sixth ligand to the heme-iron of the other monomer. A similar protein was recently purified from a homologous Desulfovibrio clinical strain isolated from an abdominal wall abscess in human patient2. Crystals of this SSC were grown using vapour diffusion method in the presence of agarose gel. Diffraction data were collected using X-ray synchrotron radiation at the ESRF, beamline, ID 14-1. The structure will be solved by molecular replacement using the structure of the D.d. as a starting model

Advancing downstream purification of cell and gene therapy medicinal products

The advent of advanced therapies in the biopharmaceutical industry has moved the spotlight into complex products such as viral vectors or stem cells, holding great promise in a myriad of clinical targets. Currently, the challenge for a widespread application of these new biopharmaceuticals is the development of cost-effective bioprocesses while maintaining product\u27s bioactivity and quality attributes. This presentation will focus on the latest advancements on downstream purification of cell and gene therapy medicinal products, supported by process innovation and the flexibility of old, but robust technologies such as tangential flow filtration (TFF) and chromatography. Improvement of purification yields of virus based biopharmaceuticals can be achieved through the rational development of alternative strategies, combining different modes of operation, such as flow through purification or multi-column chromatography, together with the recent developments of chromatographic media and fundamental understanding of the adsorption phenomena as reported for the case of gene therapy medicinal products. Critical quality attributes of cell based medicinal products cannot be compromised by the processing route chosen. The use of the already established TFF technology has the potential to improve the purity of cell based products, with the evaluation of critical process parameters of cell concentration and washing being of paramount importance. The purity of such products can also be incremented with the use of negative mode expanded bed adsorption chromatography with a new multimodal prototype matrix based on core–shell bead technology as demonstrated for the case of human mesenchymal stem cells. In summary, the advancement of the purification of complex biopharmaceutical entities, such as the ones here reported, can be described as an incremental process, but still with space for inn

High cell density reactor for the production of Lactobacillus plantarum

The production of a flocculent strain of Lactobacillus plantarum was performed in a high cell density reactor: a fluidized bed reactor (FBR) with a settler and an external cell recirculation. Two variables were assessed, the recirculation rate (R) and the dilution rate (D). The effect of the latter is much more important than the effect of the former in ensuring a quick start up in the flocculation process. The cell volumetric productivities obtained with this system increase directly with dilution rate and recirculation rate. The values of cell volumetric productivities obtained are considerably higher than those obtained in continuous stirred tank reactors (CSTR) and much higher than in batch reactors

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)

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

<p>Abstract</p> <p>Background</p> <p>Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for <it>in silico </it>metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.</p> <p>Results</p> <p>A hybrid framework is presented combining classical metabolic flux analysis with projection to latent structures to further link estimated metabolic fluxes with measured productivities. We first explore the functional metabolic decomposition of a baculovirus-producing insect cell line from experimental data, highlighting the TCA cycle and mitochondrial respiration as pathways strongly associated with viral replication. To reduce uncertainty in metabolic target identification, a Monte Carlo sampling method was used to select meaningful associations with the target, from which 66% of the estimated fluxome had to be screened out due to weak correlations and/or high estimation errors. The proposed hybrid model was then validated using a subset of preliminary experiments to pinpoint the same determinant pathways, while predicting the productivity of independent cultures.</p> <p>Conclusions</p> <p>Overall, the results indicate our hybrid metabolic flux analysis framework is an advantageous tool for metabolic identification and quantification in incomplete or ill-defined metabolic networks. As experimental and computational solutions for constructing comprehensive global cellular models are in development, the contribution of hybrid metabolic flux analysis should constitute a valuable complement to current computational platforms in bridging the metabolic state with improved cell culture performance.</p

Leveraging vectored vaccine candidates manufacturing to GMP compatible bioprocesse

Background Vectored vaccines are very efficient in the in vivo delivery of antigens either in the form of antigen protein and peptides or genetic material. The bioprocess of vectored vaccines poses however several challenge since the viral particles to be effective must maintain their infectivity. Lentiviral and adenoviral vectors are among the particles more used in the treatment of cancer diseases modulating the immune system. Both viral vectors are currently produced in transient upstream process. While the adenoviral vectors are produced at high titers the lentiviral vector upstream process still requires further improvement. The non-lytic nature of lentivirus enables the design of stable cell lines which may improve its yields through perfusion and longer term productions, reducing costs. The application of novel methods for the downstream processing such as continuous purification will contribute to increase the yield and lower the overall cost of the manufacturing processes. Experimental approach At the upstream process, many of the challenges lentiviral bioproducts present in its manufacturing are related to the apoptosis-leading cytotoxicity of some of the vector components. Supported on our long track experience and enabling tools developed for gammaretrovirus manufacturing, we undertook the challenge of establishing a constitutive stable lentiviral producer cell line. To address this challenge we proposed to eliminate or reduce the cytotoxicity of the lentiviral vector expression components. At the downstream process lentiviral vectors face the challenges common to retroviridae family of vectors namely short half-lives at room temperature, sensitivity to pH variations and salt concentrations, and shear stress. The purification strategy developed was designed to be based on disposable and easily scalable technologies. A final concentration achieving 108 TU mL-1 was targeted since the concentration step itself allows to reduce the burden on process and improve the transduction efficiency. To address the high doses requirements we will report an improved oncolytic adenovirus purification process for phase I and II clinical trials and present a case on the use of Polysorb 20 as a replacement for Triton X-100 during cell lysis. Product recovery, potency, purity and the effect of manufacturing holding points will be discussed. Results and discussion A lentiviral producer cell line constitutively producing titers above 106 TU.mL-1.day-1 was established. The cell line showed to be stable, consistently maintaining vector productivity over one month in the absence of antibiotics. At the bioreaction process it was possible to maintain the cells continuously producing over 10 days. At downstream we implemented scalable protocols for lentiviral and adenoviral vectors that is easy to transfer to GMP environment, combining microfiltration, anion-exchange, and ultrafiltration membranes technologies toward maximization of infectious virus recovery, allowing generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner. Herein we will present and discuss the challenges on the biomanufacturing of lentiviral as well as adenoviral virus, the strategies and novel technologies to be adopted in order to enable a faster development of novel vectored vaccine candidates focusing on several case studies, supported by process technology innovation