Foamy viral vector compositions and methods for the manufacture of same
AssigneeChildren's Hospital Medical Center
InventorsJohannes C.M. van der Loo, David William Russell, Punam Malik, Md Nasimuzzaman
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Abstract
Disclosed are methods of preparing FV vector particles. In some aspects, the disclosed methods may include the steps of transfecting a population of eukaryotic cells by contacting said population of eukaryotic cells with one or more transfection reagents to form a transfection mixture, and incubating the transfection mixture to form a transfected cell population; harvesting the FV vector particles from said transfected cell population; purifying the FV vector particles; and concentrating the FV vector particles.
Claims21
A method of preparing FV vector particles, comprising the steps of:
- a. transfecting a population of eukaryotic cells by contacting said population of eukaryotic cells with one or more transfection reagents to form a transfection mixture, and incubating said transfection mixture to form a transfected cell population;
- b. harvesting said FV vector particles from said transfected cell population, wherein said harvesting step is carried out about 70 hours to about 100 hours, or about 70 hours to about 90 hours, or about 70 hours to about 80 hours, or about 72 hours to about 75 hours, post-transfection;
- c. purifying said FV vector particles;
- d. concentrating said FV vector particles.
A method of obtaining an increased titer of FV vector particles, comprising the steps of:
- a. pre-seeding a population of eukaryotic cells for about 20 to about 30 hours, or about 24 hours, wherein said pre-seeding is carried out until said population of eukaryotic cells achieves a cell density of from about 1×105 cells/cm2 to about 2×105 cells/cm2 or about 1.8×105 cells/cm2;
- b. transfecting a population of eukaryotic cells by contacting said population of eukaryotic cells with one or more transfection reagents, wherein said one or more transfection reagents comprise vector and a plasmid comprising codon optimized pCiGAGopt, wherein said plasmid is used at a concentration of about 0.16 to about 10.4 microgram per 75 cm2 culture surface equivalent, preferably 0.65 microgram per 75 cm2 culture surface equivalent to form a transfection mixture, and incubating said transfection mixture to form a transfected cell population;
- c. harvesting said FV vector particles from said transfected cell population, wherein said harvesting step is carried out about 70 hours to about 100 hours, or about 70 hours to about 90 hours, or about 70 hours to about 80 hours, or about 72 hours to about 75 hours, post-transfection;
- d. purifying said FV vector particles, wherein said purification step comprises use of a media comprising heparin;
- e. concentrating said FV vector particles;
- f. diluting said FV vector particles to about 150 mM NaCl.
Dependencies are inferred from the claim text.