Composition of shelf-stable plasmid dna/polyethyleneimine (pei) particles with defined sizes for viral vector production and method for preparation of the same
AssigneeThe Johns Hopkins University
InventorsHai-Quan Mao, Yining Zhu, Yizong Hu
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Abstract
A scalable method for producing DNA/polycation particles having an optimal, defined particle size with multiple virus assembly plasmids for efficient transfection of viral production cells in suspension cultures. The presently disclosed DNA/polycation particles yield superior and reproducible transfection activity and shelf stability in the suspension form and can be used as an off-the-shelf product. The presently disclosed DNA/polycation particle formulation can potentially simplify and streamline the viral manufacturing process and improve production quality and consistency.
Claims16
A method for preparing a plurality of polycationic/nucleic acid nanoparticles, the method comprising:
- (a) flowing a first stream comprising one or more water-soluble polycationic polymers at a first variable flow rate and a second stream comprising one or more nucleic acids at a second variable flow rate into a first flash nanocomplexation (FNC) mixer to form a plurality of nanoparticles having a first particle size;
- (b) flowing a third stream comprising the plurality of nanoparticles having a first particle size at a third variable flow rate and a fourth stream comprising an assembly buffer at a fourth variable flow rate into a second FNC mixer to form a plurality of assembled nanoparticles;
- (c) incubating the plurality of assembled nanoparticles formed in step (b) for a period of time to form a plurality of assembled nanoparticles having a second particle size;
- and
- (d) flowing a fifth stream comprising the plurality of assembled nanoparticles having a second particle size at a fifth variable flow rate and a sixth stream comprising a stabilization buffer at a sixth variable flow rate into a third FNC mixer to form a plurality of polycationic/nucleic acid nanoparticles.
The method of 4, wherein the one or more nucleic acids comprise a mixture one or more plasmid DNAs, wherein the one or more plasmid DNAs comprise a transfer plasmid and plasmid DNAs encoding a gag protein, a pol protein, a rev protein, and an env protein.
Dependencies are inferred from the claim text.