Scalable purification method for aav8
AssigneeThe Trustees of the University of Pennsylvania
InventorsMartin Lock, Mauricio Alvira
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Abstract
A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.
Claims26
A method for separating AAV8 viral particles having packaged genomic sequences from genome-deficient AAV8 capsid intermediates, said method comprising:
- subjecting a mixture comprising recombinant AAV8 viral particles and AAV8 capsid intermediates to fast performance liquid chromatography, wherein the AAV8 viral particles and AAV8 intermediates are bound to an anion exchange resin equilibrated at a pH of about 10.2 and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280, wherein the AAV8 full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
A method for separating AAV8 viral particles from AAV8 capsid intermediates, said method comprising:
- (a) mixing a suspension comprising recombinant AAV8 viral particles and AAV8 capsid intermediates and a Buffer A having a pH of about 10.2;
- (b) loading the suspension of (a) onto a strong anion exchange resin column;
- (c) washing the loaded anion exchange resin with Buffer 1% B which comprises a salt having and a pH of about 10.2;
- (d) applying an increasing salt concentration gradient to the loaded and washed anion exchange resin, wherein the salt gradient is sufficient to elute the rAAV8 particles;
- and
- (e) collecting rAAV8 particles which are at least about 90% purified from AAV8 intermediates.
A method for separating recombinant AAV8 viral particles containing DNA comprising pharmacologically active genomic sequences from genome-deficient (empty) AAV8 capsid intermediates, said method comprising:
- (a) forming a loading suspension comprising:
- recombinant AAV8 viral particles and empty AAV8 capsid intermediates which have been purified to remove non-AAV materials from an AAV producer cell culture in which the particles and intermediates were generated;
- and a Buffer A comprising 20 mM Bis-Tris propane (BTP) and a pH of about 10.2;
- (b) loading the suspension of (a) onto a strong anion exchange resin, said resin being in a vessel having an inlet for flow of a suspension and/or solution and an outlet permitting flow of eluate from the vessel;
- (c) washing the loaded anion exchange resin with Buffer 1% B which comprises 10 mM NaCl and 20 mM BTP with a pH of about 10.2;
- (d) applying an increasing salt concentration gradient to the loaded and washed anion exchange resin, wherein the salt gradient ranges from 10 mM to about 190 mM NaCl, inclusive of the endpoints, or an equivalent;
- and
- (e) collecting the rAAV particles from eluate, said rAAV particles being purified away from 9 intermediates.
Dependencies are inferred from the claim text.