Methods for Purification of AAV Vectors by Anion Exchange Chromatography

A method of preparing a solution comprising a rAAV vector for purification by AEX, the method comprising a step of:

• i) diluting a first solution 2 to 25-fold (e.g., 15-fold) with a dilution solution comprising histidine, Tris and P188;
• and optionally
• ii) filtering the first solution from step i) through a filter to produce a diluted, and optionally filtered solution;
• wherein the pH of the diluted, and optionally filtered solution is increased as compared to the pH of the first solution;
• and wherein the conductivity of the diluted, and optionally filtered solution is decreased as compared to the conductivity of the first solution.

A purified rAAV vector prepared by a method comprising a step of:

• i) loading a solution comprising the rAAV vector to be purified onto an AEX stationary phase in a column;
• ii) performing gradient elution of a material from the stationary phase in the column wherein a first gradient elution buffer, a second gradient elution buffer or a mixture of both are applied to the stationary phase and a concentration of a salt is varied from 0 mM to 500 mM such that the rate of increase in concentration of the salt over the course of the gradient elution is about 10 mM/CV to 50 mM/CV (e.g., about 25 mM/CV);
• iii) collecting at least one fraction of eluate from the column during gradient elution beginning when absorbance of a column flow-through reaches an absorbance threshold;
• and/or
• vi) measuring an absorbance of the at least one fraction of eluate collected from the column and determining an A260/A280 ratio.

An solution comprising a rAAV vector for purification by AEX prepared by a method comprising a step of:

• i) diluting an affinity eluate 2 to 25-fold (e.g., 15-fold) with a dilution solution comprising histidine, Tris, and P188;
• and optionally
• ii) filtering the affinity eluate from step i) through a 0.2 μm filter to produce a diluted, and optionally filtered affinity eluate;
• wherein a pH of the diluted, and optionally filtered affinity eluate is increased as compared to a pH of the affinity eluate;
• and wherein a conductivity of the diluted, and optionally filtered affinity eluate is decreased as compared to a conductivity of the affinity eluate.

A method of regenerating an AEX stationary phase, the method comprising a step of:

• i) post-use sanitizing the stationary phase comprising application of 14.4 to 17.6 CV (e.g., about 16 CV) of a solution comprising about 0.1 M to 1.0 M NaOH to the stationary phase, optionally by upward flow;
• ii) regenerating the stationary phase comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 1 M to 3 M NaCl, 50 mM to 150 mM Tris, pH 8.5 to 9.5 to the stationary phase;
• iii) equilibrating the stationary phase comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 50 mM to 150 mM Tris, pH 8.5 to 9.5 to the stationary phase;
• iv) post-use flushing the stationary phase comprising application of ≥4.5 (e.g., about 5 CV) of water for injection to the stationary phase;
• and/or
• v) applying a storage solution to the stationary phase comprising application 2.7 to 3.3 CV (e.g., about 3 CV) of a solution comprising about 17% to 17.5% ethanol to the stationary phase.

A regenerated AEX stationary phase prepared by a method comprising a step of:

• i) post-use sanitizing of the stationary phase comprising application of 14.4 to 17.6 CV (e.g., about 16 CV) of a solution comprising about 0.1 M to 1.0 M NaOH to the stationary phase, optionally by upward flow;
• ii) regenerating the stationary phase comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 1 M to 3 M NaCl, 50 mM to 150 mM Tris, pH 8.5 to 9.5 to the stationary phase;
• iii) equilibrating the stationary phase comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 50 mM to 150 mM, Tris, pH 8.5 to 9.5 to the stationary phase;
• iv) post-use flushing of the stationary phase comprising application of ≥4.5 (e.g., about 5 CV) of water for injection to the stationary phase;
• and/or
• v) applying a storage solution to the stationary phase comprising application 2.7 to 3.3 CV (e.g., about 3 CV) of a solution comprising about 17% to 17.5% ethanol to the stationary phase.

A method of purifying an rAAV vector by AEX, the method comprising a step of:

• i) loading a solution comprising the rAAV vector to be purified onto an AEX stationary phase in a column;
• ii) performing gradient elution of a material from the stationary phase in the column wherein a percentage of a first gradient elution buffer is varied in a manner inversely proportional to variation in a percentage of a second gradient elution buffer;
• wherein at the start of the gradient elution the percentage of the first gradient elution buffer is about 75% to about 100% and at the end of the gradient elution the percentage of the second gradient elution buffer is about 60% to about 100%;
• and wherein the percentage of the second elution buffer increases at a rate of about 2% to 5% per CV over the gradient elution;
• iii) collecting at least one fraction of eluate from the column when performing the gradient elution beginning when the percentage of the second gradient elution buffer is about 30% to about 35%,
• and wherein the at least one fraction of eluate comprises the rAAV vector to be purified.