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Poster: Rapid and Scalable Transient Transfection Technology for High Titer Protein Production in HEK, CHO and Other Cell Types.
Applications > Protein Production > Viral Vector and VLP Production
Virus-like particles (VLPs) and viral vectors are used for a variety of applications including basic research, gene therapy
and vaccines. Several of the greatest challenges to manufacturing recombinant viruses are the transfection of VLP- or
virus-producing cell lines with multiple DNA/RNA plasmids that encode the viral particle as well as the ability to produce sufficiently large quantities of virus. Additionally, if the VLP or virus is to be used in a clinical setting, the safety
and toxicity of the virus production system must be considered. MaxCyte electroporation is easily scalable, for both adherent and suspension cells, and can rapidly co-transfect cells with multiple plasmids. MaxCyte technology consistently results in high cell transfection efficiencies and viability from starting volumes of less than 100 microliters up to multi-liter volumes using a single platform.
MaxCyte Electroporation for Viral Vector and VLP Production:
- Fully scalable, able to transfect up to 1x1010 cells in < 30 minutes
- Compatible with a wide variety of virus-producing cell lines
- Highly efficient and reproducible transfection of multiple plasmids
- Able to transfect large plasmids
- Uses a software-controlled, closed environment
- FDA-cleared transfection technology
- Scalable Transfection
- Bulk Lentiviral Production
- Protein Expression: STX vs. PEI
- Clinical-Grade Production
Small and Large-scale Lentiviral Production using Suspension Cells
A single MaxCyte transfection instrument can perform both small scale electroporation runs for assay development and initial studies as well as large-scale (flow) electroporation for high titer virus production. Migration to large-scale electroporation is seamless and requires no further assay optimization to maintain the same level of superior transfection performance. MaxCyte scalable transfection is compatible with adherent and suspension cells including HEK cells, insect cell lines and other commonly used virus packaging cell lines.HEK suspension cells, which are better suited to large-scale lentivector manufacturing relative to comparable adherent cells, were co-transfected with a 4 plasmid lentiviral system using small- and large-scale MaxCyte electroporation. Using a single electroporation protocol, small- and large-scale transfection produced nearly identical high titer viral stocks. These data highlight the seamless scalability of MaxCyte instruments.
Figure 1: Small and Large Scale Lentiviral Vector Production in HEK Suspension Cells. Suspension-adapted 293FT
cells were expanded in T175 shake flasks and transfected in 100μl or 10mL electroporation reactions with equivalent
concentrations of a 4 plasmid mixture encoding an HIV-based lentiviral vector system (plasmids courtesy of Dr.
Ken Cornetta, Indiana University School of Medicine). Virus was collected every 24 hours for 4 days and titrated
Flow Electroporation for Large-scale Lentiviral Vector Production
Figure 2 summarizes the cell culture methods used for large-scale flow electroporation of a 4 plasmid mixture encoding the components of an HIV-based lentiviral vector system which contains a GFP transgene. Two independent transfection were performed to demonstrate the high level of transfection reproducibility produced using MaxCyte electroporation.
Figure 2: Large Scale Lentiviral Vector Production in HEK Adherent Cells. A). HEK 293FT adherent cells were seeded in 10 tier
cell factories 2-3 days prior to EP. Cells were trypsinized, harvested and co-transfected with 4 plasmids (HIV-based lentivector
system; plasmids courtesy of Dr. Ken Cornetta, Indiana University School of Medicine) using MaxCyte flow electroporation
(CL-2 processing assembly). The transfected cells were cultured for 3 days post EP. Media was collected daily and transducing
units measured. B). Two independent transfections were performed using the methods depicted in panel A.
Superior Production of Recombinant Viral Protein using MaxCyte Electroporation
MaxCyte scalable electroporation can be used to produce a wide range of proteins including viral proteins. The broad cell type compatibility provide users with flexibility to efficiently produce proteins of interest in a variety of cellular systems including HEK, CHO and Jurkat cell lines, common virus packaging cell lines and insect cells.MaxCyte electroporation results in high levels of transfection efficiency and cell viability. Figure 3 summarizes the results of a comparison of secreted viral coat protein production following transfection with MaxCyte electroporation or polyethylenimine (PEI). Electroporation led to DNA concentration dependent production of the coat protein which exceeded protein titers obtained using an optimized PEI transfection protocol. Additionally, electroporation had a lower impact on growth kinetics relative to PEI transfection.
Figure 3: Recombinant Viral Protein Production via Electroporation vs. PEI. Suspension-adapted HEK 293F cells were transfected with varying
concentrations of a viral coat protein expression plasmid using MaxCyte STX electroporation or via an optimized polyethyleneimine (PEI)
method. Cells were cultured for approximately 5 days. A). Culture media was collected without replacement at various times post transfection
and viral protein titers measured via ELISA. B). Cells were transfected via electroporation or an optimized PEI method with and without a viral
coat expression plasmid. Viable cell number was determined for approximately 5 days post transfection.
Clinical-Grade Production
Manufacturing of viral vectors for use in patients can be challenging due to a variety of production and regulatory requirements. Bench top viral production methods are relatively simple; however, they do not provide the scalability and consistency required for clinical scale-up. Additionally, regulatory concerns about safety (carcinogenicity, unintended activity, and toxicity) have resulted in the standard requirement for additional toxicology studies which lengthen clinical development timelines and increase costs. MaxCyte transfection systems use of a biologically neutral technology that has a USFDA registered drug master file to overcome these challenges, and provide an approach that dramatically accelerates clinical development.MaxCyte electroporation is easily scalable, for both adherent and suspension cells, and transfects cells with multiple plasmids in a software-controlled, sterile, closed environmentMaxCyte technology consistently produces high cell transfection efficiencies and viability. With higher cell loading efficiencies and higher post-loading cell viability, larger lots of viral vector per run can be made, reducing the number of manufacturing runs required to make quantities suitable for clinical trials and commercialization.
