Recombinant adeno-associated virus compositions and methods for producing same
Disclosed herein are compositions comprising recombinant adeno-associated virus (rAAV), as well as recombinant baculovirus systems and methods of using the same for producing and purifying such compositions. Also disclosed herein are assays for testing the titer and potency of such compositions.
1 . A therapeutic composition comprising recombinant adeno-associated virus (rAAV) particles, wherein the rAAV particles comprise an adeno-associated virus 9 (AAV9) capsid protein and an expression cassette encoding a gene product of interest, wherein the therapeutic composition comprises more than 1E+13 viral genomes per milliliter (vg/mL) rAAV particles,
wherein the therapeutic composition comprises less than 15% empty rAAV particles and at least 80% full rAAV particles,
wherein the gene product of interest is human glucocerebrosidase (GCase), and the expression cassette comprises the nucleotide sequence of SEQ ID NO: 2,
wherein the therapeutic composition is produced by infecting Sf9 insect cells with a population of Baculovirus vectors and lysing the insect cells to produce a cellular lysate comprising the rAAV particles, wherein the cellular lysate is subjected to a method comprising the steps of:
(i) contacting an affinity chromatography column with the cellular lysate, wherein the affinity column comprises a binding agent specific for a capsid protein of the rAAV particles under conditions under which the rAAV particles bind to the affinity chromatography column;
(ii) eluting the bound rAAV particles from the column thereby producing a first eluate;
(iii) performing anion-exchange chromatography on the first eluate to produce a second eluate, wherein the second eluate comprises fewer empty rAAV particles than the first eluate; and
(iv) concentrating the second eluate by performing tangential flow filtration using a flow buffer comprising Tris, MgCl 2 , NaCl, and Poloxamer 188, thereby producing the therapeutic composition comprising rAAV particles, and
wherein the therapeutic composition comprises less than or equal to 1.3 ng residual host cell DNA from the insect cells per 1E+14 vg/ml of the rAAV particles of the therapeutic composition.
2 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises from about 1E+13 vg/mL to about 1E+14 vg/mL.
3 . The therapeutic composition of claim 1 , wherein the therapeutic composition is in a container.
4 . The therapeutic composition of claim 3 , wherein the therapeutic composition comprises less than 6000 particles that are larger than 10 μm per container, and less than 600 particles that are larger than 25 μm per container.
5 . The therapeutic composition of claim 3 , wherein the therapeutic composition in the container has an extractable volume equal to or greater than 1.0 mL.
6 . The therapeutic composition of claim 1 , wherein the therapeutic composition is sterile.
7 . The therapeutic composition of claim 1 , wherein the therapeutic composition does not promote microbial growth.
8 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises an endotoxin level less than 0.5 endotoxin units per milliliter (EU/mL).
9 . The therapeutic composition of claim 1 , wherein more than 1.0E+13 vg/mL of the therapeutic composition comprises the gene product.
10 . The therapeutic composition of claim 1 , wherein the therapeutic composition has a 50% cell culture infectious dose (TCID50) titer from about 1,000 viral genomes per infectious unit (vg/IU) to about 6,000 vg/IU or from about 4,500 vg/IU to about 10,000 vg/IU.
11 . The therapeutic composition of claim 1 , wherein activity of the human GCase is at least 110% relative to a reference standard, wherein the reference standard is a purified rAAV encoding GCase.
12 . The therapeutic composition of claim 1 , wherein the therapeutic composition has an infectious titer from about 8.0E+9 infectious unit per milliliter (IU/mL) to about 1.2E+10 IU/mL.
13 . The therapeutic composition of claim 1 , wherein the therapeutic composition has an osmolality between about 300 mOsm/kg and about 500 mOsm/kg.
14 . The therapeutic composition of claim 1 , wherein the therapeutic composition has a pH between about 7 and about 9.
15 . The therapeutic composition of claim 1 , wherein the therapeutic composition is free from visible particulate matter.
16 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises less than or equal to 3% aggregates.
17 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises a total protein level from about 300 μg/mL to about 1000 μg/mL.
18 . The therapeutic composition of claim 1 , wherein the therapeutic composition has a purity of more than 90% v/v.
19 . The therapeutic composition of claim 18 , wherein the therapeutic composition does not comprise any single impurity greater than about 5% v/v.
20 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises from about 0.0007% to about 0.0012% of Poloxamer 188.
21 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises less than 5.5×104 copies of Rhabdovirus RNA per mL of the therapeutic composition.
22 . The therapeutic composition of claim 1 , wherein the therapeutic composition comprises less than or equal to 45 ng residual host cell protein from the insect cells per 1E+13 viral genomes (vg) of the rAAV particles.