METHODS FOR PURIFICATION OF RECOMBINANT AAV VECTORS
Provided herein are methods for the purification of recombinant adeno-associated virus (rAAV) vectors that can be used for gene transfer and specifically for gene therapy or vaccination. Recombinant AAV vectors of the invention are substantially free of in-process impurities, including production components such as cellular nucleic acids, cellular proteins, helper virus, and media components.
1 . A method for isolating a population of recombinant adeno-associated virus (rAAV) particles from in-process impurities in a feedstream, comprising the steps of:
(a) contacting a feedstream containing the rAAV particles with an apatite chromatography medium in the presence of polyethylene glycol (PEG), wherein the rAAV particles bind to the apatite chromatography medium; and
(b) eluting the rAAV particles bound to the apatite chromatography medium with an elution buffer containing less than 3% (w/v) PEG.
2 . The method of claim 1 , wherein the apatite chromatography medium is ceramic hydroxyapatite (CHT).
3 . The method of claim 1 , wherein the apatite chromatography medium is ceramic fluoroapatite (CFT).
4 . The method of claim 1 , wherein the specific binding of the apatite chromatography medium to the rAAV particles is between 10 14 and 10 16 DNase-resistant particles per milliliter (DRP/mL).
5 . The method of claim 1 , further comprising a step of binding the rAAV particles in the feedstream eluted from the apatite chromatography medium to an anionic chromatography medium.
6 . The method of claim 1 , wherein the feedstream containing the rAAV particles in step (a) is contacted with an apatite chromatography medium in the presence of polyethylene glycol (PEG) and a basic buffer.
7 . The method of claim 6 , wherein the basic buffer is between pH 7.6 and 10.
8 - 9 . (canceled)
10 . The method of claim 6 , wherein the basic buffer comprises borate.
11 . The method of claim 1 , wherein the PEG has an average molecular weight between about 5,000 (PEG5000) grams per mole and about 15,000 (PEG15000) grams per mole.
12 . (canceled)
13 . The method of claim 1 , wherein the feedstream containing the rAAV particles in step (a) is contacted with the apatite chromatography medium in the presence of between about 3% (w/v) and about 10% (w/v) PEG.
14 - 15 . (canceled)
16 . The method of claim 1 , further comprising a step of washing the apatite chromatography medium with a wash buffer after the feedstream is contacted with the apatite chromatography medium but before eluting the rAAV particles from the apatite chromatography medium.
17 . The method of claim 16 , wherein the apatite chromatography medium is washed one or more times with a wash buffer containing about 7.5% (w/v) PEG and/or a wash buffer containing about 5% (w/v) PEG.
18 . (canceled)
19 . The method of claim 16 , wherein the wash buffer comprises a buffer selected from the group consisting of borate, N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and Tris-HCl.
20 - 24 . (canceled)
25 . The method of claim 19 , wherein the wash buffer further comprises between 100 and 500 mM of a phosphate.
26 . The method of claim 19 , wherein the wash buffer further comprises between 50 and 250 mM NaCl.
27 . The method of claim 1 , wherein the rAAV particles bound to the apatite chromatography medium are eluted with an elution buffer containing low concentrations of PEG or in the absence of PEG.
28 - 29 . (canceled)
30 . The method of claim 27 , wherein the elution buffer further comprises less than 100 mM phosphate.
31 - 32 . (canceled)
33 . A method for isolating a population of recombinant adeno-associated virus (rAAV) particles from in-process impurities in a feedstream, comprising the steps of:
(a) contacting a feedstream containing the rAAV particles with a hydrophobic interaction chromatography (HIC) medium in a high salt buffer, wherein the rAAV particles and the in-process impurities bind to the HIC medium; and
(b) eluting the rAAV particles bound to the HIC medium with a medium salt buffer.
34 . The method of claim 33 , wherein the HIC medium is selected from the group of Tosoh Butyl 650M, Tosoh SuperButyl 650C, Tosoh Phenyl 650C, EMD Fractogel Phenyl, and Tosoh Has(butyl) resin.
35 . The method of claim 33 , wherein the high salt buffer comprises between about 0.5 M and about 2.0 M citrate.
36 - 42 . (canceled)