Scalable purification method for AAV8
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.
1. A method for separating recombinant adeno-associated virus (AAV) 8 (rAAV8) viral particles having packaged genomic sequences from genome-deficient AAV8 capsid intermediates, said method comprising:
(a) performing filtration through a series of depth filters to afford a clarified rAAV8 production culture harvest;
(b) treating the clarified rAAV8 production culture harvest of (a) with a nuclease or a combination of nucleases to digest contaminating high molecular weight nucleic acid and to afford a mixture comprising rAAV8 viral particles and genome-deficient AAV8 capsid intermediates;
(c) concentrating the nuclease-treated mixture of (b) via tangential flow filtration (TFF) to afford a concentrated rAAV8 feedstream;
(d) contacting the concentrated rAAV8 feedstream of (c) comprising the rAAV8 viral particles and genome-deficient AAV8 capsid intermediates with a high-performance AAV8-capsid binding affinity resin to afford purified mixture;
(e) performing anion exchange chromatography by anion exchange resin on the affinity purified mixture of (d) containing the rAAV8 viral particles at a pH of 10.0 to 10.4;
(f) monitoring an eluate of (e) for ultraviolet absorbance at 260 nm (A260) and at 280 nm (A280); and
(g) collecting rAAV8 viral particles from fractions eluted when the ratio of A260 to A280 (A260/A280) reaches an inflection point.
2. The method according to claim 1 , wherein the inflection point is when the curve ratio of A260/A280 changes from less than 1 to greater than 1.
3. The method according to claim 1 , wherein the anion exchange chromatography of (e) is performed at a salt gradient which has an ionic strength equivalent to at least about 20 mM to about 190 mM NaCl, or a salt gradient having an ionic strength equivalent thereto, and wherein the ionic strength is calculatable using a formula I=½Σ i=1 n c i z i 2 , where c i is the molar concentration of ion i (M, mol/L), z i is the charge number of that ion, and I is taken over all ions in the solution.
4. The method according to claim 1 , wherein the genome-deficient AAV8 capsid intermediates are eluted from the anion exchange resin when a salt gradient reaches an ionic strength equivalent to about 50 mM NaCl or greater, or a salt gradient having an ionic strength equivalent thereto, and wherein the ionic strength is calculatable using a formula I=½Σ i=1 n c i z i 2 , where c i is the molar concentration of ion i (M, mol/L), z i is the charge number of that ion, and I is taken over all ions in the solution.
5. The method according to claim 1 , wherein the method has a sample loading flow rate less than or equal to the elution flow rate.
6. The method according to claim 1 , wherein the anion exchange resin is a quaternary amine strong anion exchange resin.
7. The method according to claim 1 , wherein the anion exchange resin is in a column.
8. The method according to claim 1 , wherein the affinity capture is performed using a high performance affinity resin.
9. A method for separating recombinant adeno-associated virus (AAV) 8 (rAAV8) viral particles from genome-deficient AAV8 capsid intermediates, said method comprising:
(a) performing filtration through a series of depth filters to afford a clarified rAAV8 production culture harvest;
(b) treating the clarified rAAV8 production culture harvest of (a) with a nuclease or a combination of nucleases to digest contaminating high molecular weight nucleic acid and to afford a mixture comprising rAAV8 viral particles and genome-deficient AAV8 capsid intermediates;
(c) contacting the mixture of (b) comprising the rAAV8 viral particles and genome deficient AAV8 capsid intermediates with a high-performance AAV8-capsid binding affinity resin to afford a purified mixture;
(d) mixing an affinity purified mixture of (c) comprising rAAV8 viral particles and genome-deficient AAV8 capsid intermediates and a first buffer at a neutral pH;
(e) loading the suspension of (d) onto a strong anion exchange resin column;
(f) washing the loaded strong anion exchange resin with a second buffer having a pH of about 10.2 and further comprising salt having an amount equivalent to 10 mM to 40 mM NaCl or a salt equivalent and Bis-Tris Propane (BTP);
(g) applying an increasing salt concentration gradient to the loaded and washed strong anion exchange resin, wherein the salt gradient is sufficient to elute the rAAV8 viral particles; and
(h) collecting rAAV8 viral particles which are at least about 90% purified from genome-deficient AAV8 capsid intermediates.
10. The method according to claim 9 , wherein the rAAV8 viral particles and genome-deficient AAV8 capsid intermediates of step (c) have been affinity purified at a high salt concentration.
11. The method according to claim 9 , wherein the strong anion exchange resin is a quaternary amine ion exchange resin.
12. The method according to claim 11 , wherein the strong anion exchange resin column comprises trimethylamine and a support matrix comprising poly(glycidyl methacrylate-co-ethylene dimethacrylate).
13. The method according to claim 9 , wherein the salt gradient of (g) is from about 10 mM to about 190 mM NaCl or a salt equivalent.
14. The method according to claim 9 , wherein the strong anion exchange resin column is a monolith column and wherein column loading, washing, and elution occur in about 60 column volumes.
15. The method according to claim 9 , wherein the elution in (g) has an elution flow rate which is from about 10 mL/min to about 40 mL/min.
16. The method according to claim 15 , wherein the elution flow rate is about 20 mL/min.
17. The method according to claim 1 , wherein the rAAV8 viral particles and the genome-deficient AAV8 capsid intermediates of step (d) have been affinity purified at a high salt concentration.
18. The method according to claim 1 , wherein the series of depth filters of (a) comprise filters of 0.2 μm or greater pore size.
19. The method according to claim 1 , wherein the series of depth filters of (a) comprise filters in the range of about 0.045 μm to about 0.2 μm pore size.
20. The method according to claim 1 , wherein the anion exchange chromatography of (e) is performed at a pH of about 10.2.
21. The method according to claim 1 , wherein genome-deficient AAV8 capsid intermediates are eluted from the anion exchange resin when a salt gradient reaches an ionic strength equivalent to about 50 mM KCl or greater, or a salt gradient having an ionic strength equivalent thereto, and wherein the ionic strength is calculatable using a formula
I
=
1
2
∑
i
=
1
n
c
i
z
i
2
,
where ci is the molar concentration of ion i (M, mol/L), z i is the charge number of that ion, and I is taken over all ions in the solution.
22. The method according to claim 9 , wherein the series of depth filters of (a) comprise filters of 0.2 μm or greater pore size.
23. The method according to claim 9 , wherein the series of depth filters of (a) comprise filters in the range of about 0.045 μm to about 0.2 μm pore size.
24. The method according to claim 9 , wherein the washing buffer of (f) comprising BTP comprises 20 mM to 50 mM BTP.