Laura Cervera

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.

Optimization of HEK 293 cell growth by addition of non-animal derived components using design of experiments

Background Mammalian cells are a widely used expression platform for the production of recombinant therapeutic proteins or viral particle-based vaccines since they typically perform appropriate protein post-translational modifications and authentic viral particle assembly. Of the available mammalian cells, HEK 293 is one of the most industrially relevant cell lines because it is cGMP compliant and is able to grow in suspension in a variety of commercial serum-free media. Of note, production of human therapeutics in mammalian cell culture has become more and more stringent in past recent years and not only demands serum-free but also animal-component free production conditions to ensure safety. This work is part of a project aimed to optimize HEK 293 cell growth by addition of non-animal derived components to serum-free and protein-free media through design of experiments (DoE) in order to maximize productivity of a recombinant VLP vaccine by PEI-mediated transient transfection.

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.

Intracellular Characterization of Gag VLP Production by Transient Transfection of HEK 293 Cells

Transient transfection is a fast, flexible, and cost‐effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus‐like particles (VLPs). The kinetics of PEI‐mediated transfection following an established in‐house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes’ internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag‐GFP fusion construct that forms fluorescent Gag‐VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post‐transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (total Gag‐GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. Biotechnol. Bioeng. 2017;114: 2507–2517.

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.

Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells

Polyethyleneimine (PEI) has been used extensively for transient gene expression (TGE) in mammalian cell cultures. However, the relationship between DNA/PEI complex preparation and their biological activity has not been fully established. Here, a systematic study of DNA/PEI complexes, their physicochemical properties during formation and their transfection efficiency was performed on a virus-like particle (VLP) production platform. The same chemically defined cell culture medium for DNA/PEI complex formation was used as an alternative to simple ionic solutions to minimize changes in complex properties during transfection. Upon formation, an initial concentration of 1E + 10 DNA/PEI complexes/mL underwent partial aggregation with an average size of 300 nm. The participation of NaCl ions in the evolution of complexes was analyzed by X-ray spectroscopy, stressing the relevance of complexing media composition in TGE strategies. After 15 min incubation, 250 complexes plus aggregates per cell were estimated at the time of transfection. Such heterogeneous preparations cannot be easily characterized; subsequently, nanoparticle tracking analysis (NTA) and cryo-electron microscopy were combined to achieve a complete picture of the preparation. Finally, the contribution of each DNA/PEI complex subpopulation was tested by drug inhibition endocytosis. Interestingly, all complexes delivered DNA efficiently and high size aggregates, which enter through macropinocytosis, when inhibited presented a major contribution to transfection efficiency. There is a need to understand the physicochemical factors that participate in DNA delivery protocols. Hence, this study provides new insights into the characterization of DNA/PEI complexes that will assist in more productive and reproducible TGE strategies.

Enhancement of HIV-1 VLP production using gene inhibition strategies

Gag polyprotein from HIV-1 is able to generate virus-like particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved by the use of 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 is the 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. Here, the combination of the EGE protocol with additive supplementation to maximize VLP production is first tested. As an alternative to the direct additive supplementation, the replacement of these chemical additives by iRNA for obtaining the same inhibition action is also tested. 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, representing an overall 18-fold improvement over conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.3 fold without any detrimental effect on cell viability (91.7%) compared with the batch cultivation (92.0%). Finally, the combination of shRNA with EGE resulted in more than 15.6-fold improvement compared with the batch standard protocol traditionally used. The methodology developed enables the production of high titers of HIV-1 VLPs avoiding the toxic effects of additives.