Plasmid DNA and co-delivery of several plasmid DNA
Large-scale virus production
Mainly adenovirus, Adeno-Associated Viruses (AAV), Lentivirus, influenza virus, retrovirus, Virus-like particles (VLP)
Mammalian producer cell lines (HEK-293, HEK-293 derivatives, BHK, VERO cell lines, virus-specific packaging cell lines.
|Number of transfections|
100 mL of PEIpro® transfection reagent is sufficient to transfect on average 50 L of cell culture
5 ± 3 °C, 3 years from manufacturing date
PEIpro® transfection reagent is the leading chemical-based DNA transfection reagent that offers flexibility and scalability to develop a robust and sustainable Process Development for viral vector production. PEIpro® benefits from extensive research development in PEI polymer chemistry and formulation to achieve highest transfection efficiency in both adherent and suspension cell culture systems. Protocols established with PEIpro® during Process Development are directly implementable for large-scale manufacturing of preclinical and clinical batches of viral vectors with the higher quality grades of PEIpro®: PEIpro®-HQ and PEIpro®-GMP.
|Reference Number||Amount of reagent|
Optimized PEI-based transfection reagent for scalable virus production
With its unique property to efficiently condense several plasmids of different sizes, PEIpro® transfection reagent is ideal for optimal co-transfection of several plasmids containing the gene of interest and necessary viral components to produce full recombinant virions. PEIpro® has benefited from extensive developments that made it the unique PEI-based transfection reagent that can offer the flexibility and robustness that is needed during Process Development and Scale-up; it is compatible with commercially available adherent and suspension virus production systems for lentivirus and AAV viral vectors(Table 1). Published work on AAV and lentivirus production with PEIpro®, and on other types of viruses (adenovirus, retrovirus virus-like particle (VLP)) production, are available under the Bibliography tab.
Table 1. PEIpro® is the reagent of choice for virus production runs in most cell culture systems in both adherent and suspension cells. Irrespective of the cell culture-based system and production scale, PEIpro® and PEIpro®-HQ have led to efficient viral vector yields superior to 107 IG/mL and 109 VG/mL, respectively for lentiviruses and AAV (*to reach highest AAV titer yields in suspension cell culture systems, we recommend to use FectoVIR®-AAV transfection reagent).
Suspension cell culture-based systems can offer considerable advantages in simplifying the scale-up process, in terms of cell culture conditions and harvesting. For this several animal-free synthetic media are commercially available to maintain high productivity in cells lines adapted for growth in suspension. Depending on the type of viral vector produced in suspension cell systems, the transfection reagent is critical to reach high viral vector yields. For lentivirus viral vectors produced in suspension cell sytems, high virus production yields can be reached by transfecting suspension-adapted HEK-293T cells with PEIpro® in BalanCD® HEK293 (Irvine Scientific) (Fig. 1).
Fig. 1: Lentivirus production in HEK-293T cells grown in suspension in BalanCD® HEK293 (Irvine Scientific®). HEK-293T cells were thawed directly into each medium and passaged every 3 to 4 days before going into a 2L benchtop bioreactor. Cells were seeded and cultured for 3 days before being transfected by PEIpro®). For transfection, a set of four plasmids was used. Lentiviral titers were measured 48 and 72 hours post-transfection (Data kindly provided by Genethon).
NEW! FectoVIR®-AAV transfection reagent is a novel class of transfection reagent specifically designed to increase recombinant AAV production in suspension cell systems. The increased production yield is reproducible at different scales, for the production of several AAV serotypes (Read more on FectoVIR®-AAV).
Fixed-bed bioreactors are also an attractive scale-up model as they allow for increased volumetric productivity with low shear stress cultivation of adherent cells. Development of scalable viral vector production has been successfully implemented in the fixed-bed reactor iCELLis® (Pall) in combination with the use of PEIpro®. For example, during the scale-up production of a recombinant AAV-8 (rAAV-8) from 0.8 m2 to 4 m2 iCELLis® Nano bioreactor, the established PEIpro® transfection protocol could be readily transferred without further optimization. The virus yields obtained in the 4 m2 iCELLis® Nano bioreactor demonstrated the process scalability and robustness. For more details, see here.
Optimized for transfection compared to other commercial PEIs and Calcium Phosphate
PEIpro® vs. commercial PEIs
The extensive research development invested in PEIpro® and the manufacturing process developed by Polyplus-transfection® has made it the unique PEI-based transfection reagent that benefits from extensive research development in polymer chemistry and formulation, in terms of polymer structure, deacetylation degree, polydispersity index and molecular weight. As a result, PEIpro® is the gold reference among commercially available PEIs and requires lowest DNA amount and volume of reagent to ensure reproducibly high production yields (Fig. 2).
Fig. 2: PEIpro® requires less reagent and similar to lower DNA amount compared to other PEIs. Suspension HEK-293 cells were seeded at 1 x 106 cells/ml in serum free medium and transfected with PEIpro®, PEI “Max” and L-PEI 25 kDa (Polysciences, Warrington, PA) resuspended at 1 mg/ml. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay.
PEIpro® vs. Calcium Phosphate
Calcium phosphate (CaPO4) is known to be inefficient for transfection of cells grown in suspension, costly in the amount of plasmid DNA required, and inconsistent due to several fluctuating parameters during complexation (pH, calcium and phosphate concentrations). Compared to CaPO4, PEIpro® is efficient with high transfection efficiencies ranging from 70 to 90% both in adherent and in suspension cells, which correspondingly leads to higher virus titer yields (Fig. 3).
Fig. 3: PEIpro® gives higher virus yields in comparison to CaPO4-based chemical transfection. Lentiviruses were produced in adherent HEK-293 cells grown in serum-free culture medium, using 15 µg DNA and 30 µl PEIpro® per 75 cm2 flask. Virus yields were determined by flow cytometry of supernatants 48 h after transfection.
Great lot-to-lot consistency
PEIpro® is manufactured and formulated using a highly controlled and strict manufacturing process. By ensuring reproducibility from one run to the next, irrespective of the operator, PEIpro® greatly facilitates process standardization; a key advantage for large-scale production. Moreover, PEIpro® is released using advanced quality controls including a specification for transfection efficiency that guarantees excellent lot-to-lot consistency (Fig. 4).
Fig. 4: Excellent lot-to-lot protein yield reproducibility using PEIpro®. Suspension HEK-293 cells were seeded at 1 x 106 cells/ml in serum-free medium and transfected with PEIpro® following the standard protocol. IgG3-Fc production was assayed 48 h after transfection using protein G affinity quantification (HPLC).
PEIpro® is fully synthetic and free of any animal-origin component. The quality of PEIpro® is continuously assessed and documented during manufacturing with the appropriate control testings. Further systematic lot management and release testing is performed for each lot produced. A qualified transfection assay ensures reliability and reproducibility of the transfection efficiency under conditions suitable for biomanufacturing. PEIpro® undergoes sterility, mycoplasma and bacterial endotoxin testing according to pharmacopoeia standards. A full list of QCs is available upon request.
In addition, Polyplus-transfection® is ISO 9001 Quality Management System accredited since 2002; this level of certification assures global customers that the supplier has established reliable and effective processes for product development, manufacturing, sales and customer support.
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