Large-scale PEI-mediated plasmid transfection
We have found a way to make possible large-scale plasmid transfection using PEI to produce high titer viral vectors in fixed bed or adherent cell culture bioreactors by using PEI as a transfection agent, while avoiding formation of the PEI-plasmid precipitate which in prior art approaches clogged adherent bioreactor substrates. We have also found a way to improve PEI-based transfection by modifying how pH and CO 2 are managed during transfection.
1. A method for manufacturing a recombinant lentiviral vector, the method comprising:
(a) mixing polyethylene imine (PEI) and plasmid DNA coding for a recombinant lentiviral vector to form a transfection solution, wherein the transfection solution is incubated for longer than 20 minutes and the transfection solution is stirred or mixed throughout the incubation time such that the transfection solution does not form a DNA-PEI complex precipitate;
(b) adding cells and the transfection solution to a bioreactor and recirculating the transfection solution until transfection is substantially complete, whereby the plasmid DNA transfects the cells to make producer cells which produce the recombinant lentiviral vector; and then
(c) culturing the producer cells in adherent mode in the bioreactor, wherein the bioreactor has a fixed bed volume of at least 5 liters and whereby the producer cells produce the recombinant lentiviral vector; and then
(d) harvesting recombinant lentiviral vector.
2. The method of claim 1 , wherein the plasmid is present in an amount adequate to produce a PEI:plasmid DNA ratio of about 1:1.5.
3. The method of claim 1 , wherein the transfection solution is at least 20 liters in volume.
4. The method of claim 1 , wherein the plasmid DNA concentration in the transfection solution is at least 300 nanograms of DNA per cm 2 .
5. The method of claim 4 , wherein the plasmid DNA concentration in the transfection solution is not more than 400 nanograms of DNA per cm 2 .
6. The method of claim 1 , further comprising:
measuring the formation of DNA-PEI complexes in the transfection solution using light scattering.
7. The method of claim 1 , wherein step (b) comprises ceasing addition of CO 2 to the transfection solution, whereby the PEI does not react with added CO 2 .
8. The method of claim 1 , wherein the bioreactor comprises an automatic pH control mechanism, and wherein the method further comprises allowing the pH of the culture medium to fall naturally during or after transfection, producing an acidic culture medium which prevents PEI-DNA complex precipitate formation.