Scalable manufacturing platform for viral vector purification and viral vectors so purified for use in gene therapy

摘要

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.

主权项

1. A method for producing a highly purified adeno-associated (AAV) vector formulation, said method comprising the steps of:
(a) harvesting cells and cell culture supernatant comprising recombinant AAV vector particles; (b) concentrating said cells and said cell culture supernatant harvested in step (a) via tangential flow filtration to produce a concentrated harvest; (c) lysing said concentrated harvest produced in step (b) by microfluidization 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.