Scalable manufacturing platform for viral vector purification and viral vectors so purified for use in gene therapy
First Claim
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1. A method for producing a highly purified adeno-associated (AAV) vector formulation said method comprising the steps of:
- (a) harvesting cells comprising recombinant AAV vector particles;
(b) concentrating said cells harvested in step (a) via tangential flow filtration to produce a concentrated harvest;
(c) lysing said concentrated harvest produced in step (b) to produce a lysate;
(d) filtering said lysate produced in step (c) to produce a clarified lysate;
(e) subjecting said clarified lysate produced in step (d) to ion exchange column chromatography to produce a column eluate comprised of purified AAV vector particles, and optionally concentrating said column eluate by tangential flow filtration to produce a concentrated column eluate;
(f) mixing said column eluate or said concentrated column eluate produced in step (e) with cesium chloride to produce a mixture, and subjecting said mixture to gradient ultracentrifugation to substantially separate said bona fide AAV vector particles from empty capsid AAV vector particles and other AAV vector related impurities;
(g) collecting said bona fide AAV vector particles separated in step (f) and subjecting said collected bona fide AAV vector particles to a buffer exchange by tangential flow filtration;
(h) formulating said bona fide AAV vector particles resulting from step (g) with surfactant to produce an AAV vector formulation; and
(i) filtering said AAV vector formulation produced in step (h) to produce a highly purified AAV vector formulation in which at least 90% of the AAV vector particles in said highly purified AVV vector formulation are bona fide AAV particles.
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Abstract
Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.
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Citations
24 Claims
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1. A method for producing a highly purified adeno-associated (AAV) vector formulation said method comprising the steps of:
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(a) harvesting cells comprising recombinant AAV vector particles; (b) concentrating said cells harvested in step (a) via tangential flow filtration to produce a concentrated harvest; (c) lysing said concentrated harvest produced in step (b) to produce a lysate; (d) filtering said lysate produced in step (c) to produce a clarified lysate; (e) subjecting said clarified lysate produced in step (d) to ion exchange column chromatography to produce a column eluate comprised of purified AAV vector particles, and optionally concentrating said column eluate by tangential flow filtration to produce a concentrated column eluate; (f) mixing said column eluate or said concentrated column eluate produced in step (e) with cesium chloride to produce a mixture, and subjecting said mixture to gradient ultracentrifugation to substantially separate said bona fide AAV vector particles from empty capsid AAV vector particles and other AAV vector related impurities; (g) collecting said bona fide AAV vector particles separated in step (f) and subjecting said collected bona fide AAV vector particles to a buffer exchange by tangential flow filtration; (h) formulating said bona fide AAV vector particles resulting from step (g) with surfactant to produce an AAV vector formulation; and (i) filtering said AAV vector formulation produced in step (h) to produce a highly purified AAV vector formulation in which at least 90% of the AAV vector particles in said highly purified AVV vector formulation are bona fide AAV particles. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24)
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21. A method for producing a highly purified adeno-associated (AAV) vector formulation, said method comprising the steps of:
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(a) harvesting cells comprising recombinant AAV vector particles; (b) concentrating said cells harvested in step (a) via tangential flow filtration to produce a concentrated harvest; (c) lysing said concentrated harvest produced in step (b) to produce a lysate; (d) filtering said lysate produced in step (c) to produce a clarified lysate; (e) subjecting said clarified lysate produced in step (d) to ion exchange column chromatography to produce a column eluate comprised of purified AAV vector particles, and optionally concentrating said column eluate by tangential flow filtration to produce a concentrated column eluate; (f) mixing said column eluate or said concentrated column eluate produced in step (e) with cesium chloride to produce a mixture; (g) subjecting said mixture in step (f) to gradient ultracentrifugation conducted in a single step to substantially separate said bona fide AAV vector particles from empty capsid AAV vector particles and other AAV vector related impurities; (h) collecting said bona fide AAV vector particles separated in step (g) and subjecting said collected bona fide AAV vector particles to a buffer exchange by tangential flow filtration; (i) formulating said bona fide AAV vector particles resulting from step (h) with surfactant to produce an AAV vector formulation; and (j) filtering said AAV vector formulation produced in step (i) to produce a highly purified AAV vector formulation in which at least 90% of the AAV vector particles in said highly purified AVV vector formulation are bona fide AAV particles, and wherein said bona fide AAV vector particles are present in said highly purified AAV vector formulation at a concentration of 1015 particles per mL. - View Dependent Claims (23)
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Specification