Plasmid DNA and co-delivery of several plasmid DNA
Recombinant AAV virus production in suspension cells
Adeno-Associated Viruses (AAV)
Mammalian producer cell lines (HEK-293, HEK-293 derivatives) grown in suspension cultures
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100 mL of FectoVIR®-AAV transfection reagent is sufficient to transfect on average 50 L of cell culture
5 ± 3 °C, stable for at least 9 months when stored appropriately
AAVs are becoming the leading viral vector in the development of gene-based advanced therapy medicinal products (ATMPs). Their success is due to their intrinsic properties: they are primarily non-integrating as they persist within cells as episomes, and have a broad tropism with eleven serotypes that have the ability to infect specific cell types. A major challenge today is manufacturing sufficient quantities of AAV in order to treat larger groups of patients. FectoVIR®-AAV is a novel class of animal free transfection reagent specifically developed for industrial scale production of recombinant AAV (rAAV) viral vectors in suspension HEK-293 cell types. FectoVIR®-AAV transfection reagent guarantees higher rAAV viral titers, improved flexibility and scalability for industrial scale manufacturing.
A Frequently Asked Question section about FectoVIR®-AAV can be found here.
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High performance: Superior yields for rAAV production in suspension culture system
FectoVIR®-AAV transfection reagent is a novel chemical-based and animal-free transfection reagent that has been engineered to address the current limits in rAAV viral vector yields and industrial scalability. FectoVIR®-AAV transfection reagent is the outcome of an extensive development process in which candidates from our proprietary internal chemical library were tested in comparison to the gold standard for virus production, our PEIpro® transfection reagent. In comparison to PEIpro® and other competitors, FectoVIR®-AAV stands out with its unmatched performance for the production of rAAV viral vectors in suspension cell culture systems. FectoVIR®-AAV reproducibly improves substantially AAV production yield with up to a remarkable 10-fold increase compared to other competitors (PEImax) (Fig.1).
Fig 1. Improvement in both viral genome production and packaging efficiency with FectoVIR®-AAV results in a up 10-fold increase in functional rAAV-2-GFP production in comparison with competitors. Suspension HEK-293T cells were transfected with PEIMax, PEIpro® and FectoVIR®-AAV using the recommended conditions. rAAV-2-GFP were harvested 72h post-transfection of suspension HEK-293T cells with FectoVIR®-AAV/DNA complexes prepared in several synthetic media. Functional viral titers (TU/ml) of rAAV-2-GFP were measured in an infectivity assay 72h post-transduction of adherent HEK-293T cells.
Scalability: Developed for small to large scale production
Scalability is essential to guarantee the development of high-yielding and reliable rAAV viral vector manufacturing process for commercialization. Large scale rAAV viral vector manufacturing is dependent on a robust large scale transient transfection process. Large scale transient transfection implies that large volumes of transfection complexes need to be prepared and added to suspension cells within a given time frame for the transfection to be efficient. We’ve addressed both limiting aspects of volume and time constraints in AAV manufacturing by developing FectoVIR®-AAV specifically for large scale transfection.
Transfection complexation volume: Typically, a transfection complexation volume usually represents 10% of the final cell culture volume, which can lead to technical constraints when scaling up production. FectoVIR®-AAV has been optimized to reduce the complexation volume down to 1%, facilitating the preparation of transfection complexes for large scale manufacturing (Fig.2). For example, for a 200 L cell suspension volume, the transfection complexation volume can be optimized down from 20 L (10%) to 2 L (1%).
Fig.2 Optimized FectoVIR®-AAV transfection complex preparation for large scale transient transfection. Total viral genome (VG/cell) of rAAV-2-GFP were quantified 72h post-transfection of suspension HEK-293T cells in commercial synthetic medium. Transfection complexes were prepared in different volumes of complexation: 10%, 5% and 1% of final culture volumes.
Transfection complex stability: Large scale transient transfection requires addition of a larger volume of transfection complexes to suspension cell culture, which means longer transfer time. With FectoVIR®-AAV, the transfer time is no longer a limiting factor that can lead to viral titer yield variability. As shown in Fig.3, FectoVIR®-AAV transfection complexes show tremendous stability: upon mixing FectoVIR®-AAV transfection reagent and plasmid DNA, transfection complexes can be reliably added to cells within 6h with no significant variation in rAAV titer yields.
Fig.3 Long-term stability of FectoVIR®-AAV /DNA complexes for reliable titer yields at industrial scale. rAAV-2-GFP were harvested 72h post-transfection of suspension HEK-293T cells with FectoVIR®-AAV/DNA complexes prepared following the recommended conditions. With varying pre-incubation time of complexes (15 min to 6h). Functional viral titers (TU/ml) of rAAV-2-GFP were measured in an infectivity assay 72h post-transduction of adherent HEK-293T cells.
Flexibility: Compatible with various commercial culture media and cell systems
The choice of a synthetic cell culture medium is specific to each rAAV manufacturing platform. The efficiency of FectoVIR®-AAV/DNA complexes has been thoroughly tested in different commercially available synthetic complexation media, and has demonstrated little variability in titer yields across the range of tested media (Fig 3).
Fig. 4 FectoVIR®-AAV is compatible with several commercially available synthetic media for suspension HEK-293 cells. Recombinant AAV-2 were harvested 72h post-transfection of suspension HEK-293T cells with FectoVIR®-AAV/DNA complexes prepared in several synthetic media. Functional viral titers (TU/ml) of rAAV-2-GFP were measured in an infectivity assay 72h post-transduction of adherent HEK-293 cells.
Polyplus-transfection® has a strong expertise in developing GMP grade transfection reagent, recently marked by the launch of the first GMP compliant transfection reagent for therapeutic virus production (PEIpro®-GMP in 2018). Not only is Polyplus-transfection the provider of the first GMP transfection reagent for the Gene and Cell therapy market, it is also the only transfection reagent supplier that accompanies viral manufacturers from initial process development till commercialization with extensive Technical and Regulatory support.
Our strategy for developing GMP-grade transfection reagent is a fully validated process in compliance with GMP guidelines to ensure traceability from starting material to the final product. We anticipate on the evolution of current GMP guidelines by providing an extensive list of supplementary quality controls in addition to those required.
With our strong regulatory and GMP development team, we are happy to announce that GMP compliant FectoVIR®-AAV is coming soon!
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FectoVIR®-AAV, a giant step for AAV large scale manufacturing
AAVs are the most promising viral vector for gene therapy thanks to their broad tropism and non-integrating properties. The current challenge is manufacturing sufficient amount of recombinant AAVs (rAAVs) viral particles to treat patients. In order to improve productivity, viral manufacturers are working on optimising their upstream process to produce higher amounts of rAAVs by switching to more performant cell suspension systems and transient transfection systems that can be scaled-up for industrial manufacturing. During this webinar, we will demonstrate how our novel transfection reagent FectoVIR-AAV is developped for large scale transient transfection of suspension cell systems to produce higher reproducible amounts of rAAVs.
Advances in gene therapy delivery: next-generation transfection reagent for large-scale AAV manufacturing
The number of ATMP therapeutic-based medicines for inherited genetic disorders is in constant growth, with a global 32% increase in new clinical trials in the last 4 years. ATMPs have demonstrated their success with already more than ten approved for commercialization.
Attendees will learn:
- How to improve AAV viral vector yields by focusing on transfection step.
- How to perform transfection at large scale.
- How to ensure reproducibility at different production scales.
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