Back to results
US 11,028,372 B2Granted
Scalable purification method for AAVRH10
AssigneeThe Trustees of the University of Pennsylvania
InventorsMartin Lock, Mauricio Alvira
Life of this patent
Estimating the term…
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 AAVrh10 (rAAVrh10) viral particles containing DNA comprising pharmacologically active genomic sequences from genome-deficient AAVrh10 capsid intermediates, said method comprising:
- (a) separating rAAVrh10 viral particles and AAVrh10 capsid intermediates from non-AAV contaminants obtained from an AAV producer cell culture which comprises an expression cassette encoding an AAVrh10 capsid protein operably linked to expression control sequences which direct expression thereof in the producer cell, an expression cassette comprising sequences for packaging in the AAVrh10 capsid, and an expression cassette comprising helper viruses necessary for production and packaging of the rAAVrh10 into a viral particle, wherein the separation is performed using an AAVrh10-capsid binding affinity resin comprising an AAV specific antibody to afford purified materials of (a);
- (b) forming a loading mixture comprising:
- a suspension which comprises (i) materials of (a) purified by the AAVrh10-capsid binding affinity resin and (ii) a buffer comprising 20 mM Bis-Tris propane (BTP) at a pH of about 10.0, whereby the mixture has a pH of about 10.0;
- (c) loading the mixture of (b) onto a quaternary amine anion exchange column, said column being in a vessel having an inlet for a loading flow of a suspension and/or solution and an outlet permitting for an elution flow of eluate from the vessel;
- (d) washing the loaded anion exchange column with a buffer comprising 10 mM NaCl and 20 mM BTP, wherein the buffer has a pH of about 10.0;
- (e) applying an increasing salt concentration gradient to the loaded and washed anion exchange column, wherein the salt gradient is an elution gradient and ranges from about 10 mM to about 190 mM NaCl, inclusive of the endpoints, or a salt gradient having an ionic strength equivalent thereto, and wherein the ionic strength is calculatable using the following formula:
- I
- =
- 1
- 2
-
- ∑
- i
- =
- 1
- n
-
- c
- i
-
- z
- i
- 2
- ,
- where ci is the molar concentration of ion i (M, mol/L), zi is the charge number of that ion, and I is taken over all ions in the solution;
- and
- (f) monitoring eluate for ultraviolet absorbance at about A260 and at about A280 and collecting the rAAVrh10 particles from a fraction which is eluted when the peak for A260 crosses over and exceeds the peak for A280, said rAAVrh10 particles being purified away from AAVrh10 intermediates.
24 more elements
A method for separating recombinant AAVrh10 (rAAVrh10) viral particles containing DNA comprising pharmacologically active genomic sequences from genome-deficient AAVrh10 capsid intermediates, said method comprising:
- (a) separating a mixture comprising rAAVrh10 viral particles and AAVrh10 capsid intermediates from non-AAV contaminants generated in a production cell system which comprises an expression cassette, wherein the separation is performed using an AAvrh10-capsid binding affinity capture resin comprising a ligand for AAVrh10, said affinity capture comprising:
- (i) equilibrating the AAVrh10-capsid binding affinity resin with a buffer which comprises about 200 mM to about 600 mM NaCl at a pH of 7.5 prior to applying the material to the affinity resin and applying the material to the affinity resin;
- (ii) washing the loaded resin with a buffer which comprises about 800 mM NaCl to about 1200 mM NaCl at a pH of 7.5;
- (iii) washing the affinity resin of (a)(ii) with a buffer which comprises about 200 mM to about 600 mM NaCl, 20 mM Sodium Citrate, at a pH of 2.5;
- and
- (iv) collecting the resulting eluate of (a)(iii) which comprises the rAAVrh10 viral particles and the AAVrh10 capsid intermediates, wherein the collected rAAVrh10 viral particles and the AAVrh10 capsid intermediates contain less than about 10% contamination from non-AAV viral and cellular proteinaceous and nucleic acid materials;
- and
- (b) purifying the material of (a) via an anion exchange column which comprises the following:
- (i) forming a loading mixture comprising:
- a suspension which comprises the collected materials of (a) and a buffer comprising 20 mM Bis-Tris propane (BTP) at a pH of 10.0, whereby the mixture has a pH of 10.0;
- (ii) loading the mixture of (b)(i) onto a quaternary amine strong anion exchange column;
- (iii) washing the loaded anion exchange column with a buffer comprising 10 mM NaCl and 20 mM BTP, wherein the buffer has a pH of 10.0;
- (iv) applying an increasing salt concentration gradient to the loaded and washed anion exchange column, wherein the salt gradient generated via 1% to 19% of a buffer comprising 1M NaCl, 20 mM Bis-Tris-Propane (BTP) and having a pH of 10.0, and wherein the loading flow rate equals the elution flow rate;
- and
- (v) collecting the rAAVrh10 viral particles from a fraction which is eluted when the peak for A260 crosses over and exceeds the peak for A280, said rAAVrh10 viral particles being purified away from AAVrh10 intermediates.
12 more elements
Dependencies are inferred from the claim text.