Sonia Gutiérrez-Granados

Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium

Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8μg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively.

Development and validation of a quantitation assay for fluorescently tagged HIV-1 virus-like particles

Upon expression, the Gag polyprotein of HIV-1 assembles spontaneously in the vicinity of the plasma membrane giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. Robust VLP production and purification processes are required to generate VLPs of sufficient quality and quantity for both pre-clinical and clinical evaluation. The availability of simple, fast and reliable quantitation tools is critical to develop, optimize and monitor such processes. Traditionally, enzyme-linked immunosorbent assays (ELISA) are used to quantify p24 antigen concentrations, which reflect tightly virus particle concentrations. However, this quantitation technique is not only time-consuming, laborious and costly but it is also prone to methodological variability. As an alternative, the development and validation of a fluorescence-based quantitation assay for Gag VLPs is presented here. A Gag polyprotein fused to the enhanced green fluorescent protein was used for generation of fluorescent VLPs. A purified standard reference Gag-GFP VLP material was prepared and characterized in house. The method was validated according to ICH guidelines. The validation characteristics evaluated included accuracy, precision, specificity, linearity, range and limit of detection. The method showed to be specific for Gag-GFP. The fluorescence signal correlated well with p24 concentrations measured using a reference p24 ELISA assay. The method showed little variability compared to ELISA and was linear over a 3-log range. The limit of detection was ~10 ng of p24/mL. Finally, fluorescence-based titers were in good agreement with those obtained using transmission electron microscopy and nanoparticle tracking analysis. This simple, rapid and cost-effective quantitation assay should facilitate development and optimization of bioprocessing strategies for Gag-based VLPs.

Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

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 96 h, 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 240 h with a 4–12‐fold increase in production levels, depending on the product type considered. Biotechnol. Bioeng. 2015;112: 934–946.

Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics

Abstract Transient gene expression (TGE) in animal cell cultures has been used for almost 30 years to produce milligrams and grams of recombinant proteins, virus-like particles and viral vectors, mainly for research purposes. The need to increase the amount of product has led to a scale-up of TGE protocols. Moreover, product quality and process reproducibility are also of major importance, especially when TGE is employed for the preparation of clinical lots. This work gives an overview of the different technologies that are available for TGE and how they can be combined, depending on each application. Then, a critical assessment of the challenges of large-scale transient transfection follows, focusing on suspension cell cultures transfected with polyethylenimine (PEI), which is the most widely used methodology for transfection. Finally, emerging opportunities for transient transfection arising from gene therapy, personalized medicine and vaccine development are reviewed.

Production of HIV virus-like particles by transient transfection of CAP-T cells at bioreactor scale avoiding medium replacement

Human-derived CAP-T cell line has been demonstrated to be a powerful platform for high-titer production of HIV virus-like particles (VLPs) by PEI-mediated transient transfection. Scale-up of transfection processes is key to ensure the necessary quantities for pre-clinical and clinical testing. One of the major operational challenges of large-scale transient transfection is the medium replacement step that is often required before transfection. In this work, CAP-T cells were cultured in 1L bioreactor with addition of sodium bicarbonate and surface aeration, which were observed to improve cell state for transfection. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17+1% of PEM) compatible with cell growth and PEI-mediated transient transfection. In the conditions developed in this work, 0.5×106cells/mL were seeded in 1L bioreactor. Two days later, ∼2×106cells/mL were transfected without medium exchange, using 0.5pg of DNA/cell and 3pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4×1010VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved.

Characterization and quantitation of fluorescent Gag virus-like particles

Background Upon expression, the Gag polyprotein of HIV-1 spontaneously assembles giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. In order to develop robust VLP manufacturing processes, the availability of simple, fast and reliable quantitation tools is crucial. Traditionally, commercial p24 ELISA kits are used to estimate Gag VLP concentrations. However, this quantitation technique is time-consuming, laborious, costly and prone to methodological variability. Reporter proteins are frequently used during process development to allow a straightforward monitoring and quantitation of labeled products. This alternative was evaluated in the present work by using a Gag-GFP fusion construct.