received by the International Bureau on 08.01 .2018

We claim:

1. A method for manufacturing a recombinant lentiviral vector, the method comprising:

(a) mixing PEI and plasmid coding for a recombinant lentiviral vector to form a transfection solution; and then

(b) adding line cells to the transfection solution whereby the plasmid transfects the line cells to make producer cells which produce the recombinant lentiviral vector; and

(c) culturing the producer cells in adherent mode in an adherent bioreactor having a fixed bed volume of at least about 5 liters whereby the producer cells produce the recombinant lentiviral vector; and then

(d) harvesting recombinant lentiviral vector.

2. The method of claim 1, wherein the transfection solution is incubated for materially longer than 20 minutes, whereby the transfection solution does not form a DNA-PEI complex precipitate.

3. 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.

4. The method of claim 1, wherein the transfection solution is at least about 20 liters in volume.

5. The method of claim 1, wherein the transfection is substantially complete before adding the transfection solution to the bioreactor.

6. The method of claim 1, wherein the line cells are added to the bioreactor before transfection is complete, and wherein the step of (b) adding line cells to the transfection solution further comprises circulating the transfection solution in the bioreactor until transfection is substantially complete.

7. The method of claim 1, wherein the plasmid DNA concentration in the transfection solution is at least about 300 nanograms of DNA per cm².

8. The method of claim 6, wherein the plasmid DNA concentration in the transfection solution is not more than about 400 nanograms of DNA per cm².

9. The method of using PEI to transfect cells with plasmid DNA, characterized by measuring the formation of DNA-PEI complexes using light scattering.

10. The method of using PEI to transfect cells with plasmid DNA and culturing the cells in the presence of added CO2, characterized by substantially ceasing the addition of CO2, whereby the PEI does not substantially react with the C0₂.

11. The method of using PEI to transfect cells with plasmid DNA in a bioreactor having an automatic pH control mechanism, characterized by allowing the pH of the culture medium

2/2 to fall naturally during or just after transfection, producing a slightly-acidic culture medium which prevents PEI-DNA complex precipitate formation.

12. The method of claim 2, wherein the transfection solution is stirred.

13. The method of claim 1, wherein the transfection solution is incubated until the DNA/PEI particle size decreases to be not more than about 80% of the maximum particle size.