Scalable purification method for AAV9
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.
Claims37
A method for separating recombinant adeno-associated virus serotype 9 (rAAV9) viral particles having packaged genomic sequences from genome-deficient adeno-associated virus serotype 9 (AAV9) capsid intermediates, said method comprising:
- (a) filtering rAAV9 viral particles and genome-deficient AAV9 capsid intermediates harvested from an rAAV9 production culture, wherein the rAAV9 viral particles and the AAV9 capsid intermediates comprise an adeno-associated virus capsid serotype 9 (AAV9 capsid) comprising the amino acid sequence of SEQ ID NO:
- 1 or a sequence at least 99% identical to SEQ ID NO:
- 1, to afford a concentrated liquid suspension media comprising rAAV9 viral particles and genome-deficient AAV9 capsid intermediates,
- (b) contacting the liquid suspension comprising the rAAV9 viral particles and genome-deficient AAV9 capsid intermediates from step (a) with a high-performance binding affinity resin which binds the AAV9 capsid comprising the amino acid sequence of SEQ ID NO:
- 1 or a sequence at least 99% identical to SEQ ID NO:
- 1, to afford a purified mixture containing the rAAV9 viral particles and genome-deficient AAV9 capsid intermediates;
- (c) loading a suspension comprising the rAAV9 viral particles and genome-deficient AAV9 capsid intermediates from the purified mixture obtained in step (b) onto an anion exchange resin for anion exchange chromatography;
- (d) equilibrating the anion exchange resin at a pH of 10.0 to 10.4;
- and
- (e) monitoring an eluate for ultraviolet absorbance at about 260 nm (A260) and about 280 nm (A280) and collecting rAAV9 viral particles from fractions eluted after the ratio of A260 to A280 (A260/A280) reaches an inflection point, thereby separating the rAAV9 viral particles from the genome-deficient AAV9 capsid intermediates.
A method for separating recombinant adeno-associated virus 9 (rAAV9) viral particles having packaged genomic sequences from genome-deficient rAAV9 capsid intermediates, said method comprising:
- (a) purifying a mixture comprising recombinant AAV9 viral particles and rAAV9 capsid intermediates from production system contaminants using an AAV-specific antibody-based affinity capture resin;
- (b) subjecting the mixture comprising recombinant AAV9 viral particles and rAAV9 capsid intermediates to fast performance liquid chromatography, wherein the recombinant AAV9 viral particles and rAAV9 capsid intermediates are bound to a strong anion exchange resin equilibrated at a pH of 10.0 to 10.4;
- (c) applying an increasing salt concentration gradient to the loaded anion exchange resin while monitoring eluate for ultraviolet absorbance at 260 nm (A260) and 280 nm (A280);
- and
- (d) collecting the recombinant AAV9 viral particles from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
Dependencies are inferred from the claim text.