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Cambridge, MA, United States

Dressen M.H.C.L.,TU Eindhoven | Van De Kruijs B.H.P.,TU Eindhoven | Meuldijk J.,Process Development group | Vekemans J.A.J.M.,TU Eindhoven | Hulshof L.A.,TU Eindhoven
Organic Process Research and Development

Microwave-irradiated and conventionally heated nucleophilic additions of various C-6 substituted isocytosines (methyl, ethyl, isopropyl, and phenyl) to (di)isocyanates have been compared. As compared to conventional heating, the heterogeneous reaction mixtures showed higher reaction rates on using microwave heating. Variation of C-6 substituent, temperature, and amount of cosolvent influenced significantly the magnitude of these microwave effects. The magnitude of the microwave effect was governed by the solubility and the intrinsic reactivity of the variation in C-6 substitution on isocytosine. Presumably, the liquid layer near the solid surface is the area where selective heating by microwaves is occurring. As a consequence of locally higher temperatures, the solubility of the reactant as well as the rate coefficient of the reaction increase. Thus, higher reaction rates are observed than those corresponding with the measured bulk temperature. The observed microwave effects have a thermal rationale based on direct, fast, and selective heating, and the local heating effect is not found during conventional heating. This finding is crucial for a justified process scale-up scenario. © 2010 American Chemical Society. Source

Gutierrez-Granados S.,Autonomous University of Barcelona | Cervera L.,Autonomous University of Barcelona | Cervera L.,McGill University | Segura M.D.L.M.,Process Development group | And 2 more authors.
Applied Microbiology and Biotechnology

HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 1010 VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics. © 2015 Springer-Verlag Berlin Heidelberg Source

Cervera L.,Autonomous University of Barcelona | Fuenmayor J.,Autonomous University of Barcelona | Gonzalez-Dominguez I.,Autonomous University of Barcelona | Gutierrez-Granados S.,Autonomous University of Barcelona | And 2 more authors.
Applied Microbiology and Biotechnology

The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett–Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94 %. © 2015, Springer-Verlag Berlin Heidelberg. Source

Cervera L.,Autonomous University of Barcelona | Gutierrez-Granados S.,Autonomous University of Barcelona | Berrow N.S.,Barcelona Institute for Research in Biomedicine | Segura M.M.,Process Development group | Godia F.,Autonomous University of Barcelona
Biotechnology and Bioengineering

Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc. Source

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