Methods of size exclusion chromatography for the characterization of recombinant adeno-associated virus compositions

Patent metadata

Field	Value
Publication number	US-12565645-B2
Application number	US-202017602199-A
Country	US
Kind code	B2
Filing date	Apr 10, 2020
Priority date	Apr 11, 2019
Publication date	Mar 3, 2026
Grant date	Mar 3, 2026

Abstract

The present disclosure relates to using size exclusion chromatography to isolate AAV genome, to determine the vector genome size purity of a composition comprising isolated rAAV particles, to assess the folding or secondary structure of vector genomes inside the capsids, and to determine vector genome titer (Vg) of a composition comprising isolated rAAV particles.

First claim

What is claimed is:

1 . A method of isolating recombinant adeno associated virus (rAAV) genome comprising
a) subjecting a composition comprising rAAV particles to a condition under which the rAAV particles are denatured; b) subjecting the composition comprising the denatured rAAV particles to size exclusion chromatography (SEC), wherein the SEC produces an eluate; and c) detecting DNA in the eluate by spectrophotometry, wherein the mobile phase for the SEC comprises a salt, organic solvent, or detergent.

2 . The method of claim 1 , further comprising recovering the AAV genome.

3 . The method of claim 1 , wherein the detecting DNA in the eluate by spectrophotometry comprises measuring the eluate's UV absorbance at one or both of about 260 nm and at about 280 nm.

4 . A method of determining the vector genome size uniformity of a composition comprising isolated rAAV particles, wherein the method comprises
a) subjecting the composition to a condition under which the rAAV particles are denatured; b) subjecting the composition comprising the denatured rAAV particles to size exclusion chromatography (SEC), wherein the SEC produces an eluate; c) measuring the eluate's UV absorbance at one or both of about 260 nm and at about 280 nm; and d) determining the vector genome size uniformity of the composition comprising rAAV particles, wherein the mobile phase for the SEC comprises a salt, organic solvent, or detergent.

5 . A method of assessing the folding or secondary structure of AAV vector genomes inside the capsids, wherein the method comprises
a) subjecting the composition to a condition under which the rAAV particles are denatured; b) subjecting the composition comprising the denatured rAAV particles to size exclusion chromatography (SEC), wherein the SEC produces an eluate; and c) measuring the eluate's UV absorbance at one or both of about 260 nm and at about 280 nm, wherein the mobile phase for the SEC comprises a salt, organic solvent, or detergent.

6 . A method of determining vector genome titer (Vg) of a composition comprising isolated rAAV particles, wherein the method comprises
a) subjecting the composition to a condition under which the rAAV particles are denatured; b) subjecting the composition comprising the denatured rAAV particles to size exclusion chromatography (SEC), wherein the SEC produces an eluate; c) measuring the eluate's UV absorbance at one or both of about 260 nm and at about 280 nm; and d) determining the Vg of the composition comprising rAAV particles, wherein the mobile phase for the SEC comprises a salt, organic solvent, or detergent.

7 . The method of claim 1 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises
a) exposing the composition to a condition that substantially maximizes the dsDNA signal detectable during SEC; b) exposing the composition to a temperature that is no more than 10° C. above the minimum temperature needed to denature substantially all viral particles in the composition; c) subjecting the composition to a condition that results in a % Fragment DNA determination that correlates with the % Partial-filled capsid of the same composition determined by analytical ultracentrifugation (AUC); d) exposing the composition to thermal denaturation that substantially maximizes the dsDNA signal detectable during SEC; or e) subjecting the composition to thermal denaturation that results in a % Fragment DNA determination that correlates with the % Partial-filled capsid of the same composition determined by AUC.

8 . The method of claim 1 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises
a) incubating the composition at a temperature between about 65° C. and about 95° C.; b) incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes; or c) incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes in the presence of a detergent.

9 . The method of claim 4 , wherein the mobile phase for the SEC comprises a salt, organic solvent, and detergent.

10 . The method of claim 4 , wherein the mobile phase comprises between about 5 mM and about 50 mM sodium phosphate, between about 100 mM and about 500 mM sodium chloride, between about 0.01% and about 0.5% polysorbate 80, and between about 5% and about 50% methanol, and has a pH between about 6.0 and 8.5.

11 . The method of claim 4 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises
a) exposing the composition to a condition that substantially maximizes the dsDNA signal detectable during SEC; b) exposing the composition to a temperature that is no more than 10° C. above the minimum temperature needed to denature substantially all viral particles in the composition; c) subjecting the composition to a condition that results in a % Fragment DNA determination that correlates with the % Partial-filled capsid of the same composition determined by analytical ultracentrifugation (AUC); d) exposing the composition to thermal denaturation that substantially maximizes the dsDNA signal detectable during SEC; or e) subjecting the composition to thermal denaturation that results in a % Fragment DNA determination that correlates with the % Partial-filled capsid of the same composition determined by AUC.

12 . The method of claim 4 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises incubating the composition at a temperature between about 65° C. and about 95° C.

13 . The method of claim 12 , wherein at least about 95% of the viral particles in the sample are denatured by the denaturation process.

14 . The method of claim 12 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes.

15 . The method of claim 12 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes in the presence of a detergent.

16 . The method of claim 15 , wherein the detergent comprises sodium dodecyl sulfate (SDS), trimethyl ammonium bromide (ETMAB), polysorbate 80, polysorbate 20, poloxamer 188, or a combination thereof.

17 . The method of claim 4 , wherein the mobile phase comprises between about 5 mM and about 50 mM sodium phosphate, between about 100 mM and about 500 mM sodium chloride, between about 0.01% and about 0.5% polysorbate 80, and between about 5% and about 50% methanol, and has a pH between about 6.0 and 8.5.

18 . The method of claim 5 , wherein subjecting the composition to a condition under which the rAAV particles are denatured comprises
a) incubating the composition at a temperature between about 65° C. and about 95° C.; b) incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes; or c) incubating the composition at a temperature between about 65° C. and about 95° C. for between about 5 minutes and 60 minutes in the presence of a detergent.

19 . The method of claim 6 , wherein subjecting the composition to a condition under which the rAAV particles are denature

Assignees

• Regenxbio Inc

Inventors

• Zhi Li
• Wu Zhuchun

Classifications

• G01N2030/027

  Liquid chromatography · CPC title

• G01N30/74

  Optical detectors {(measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J)} · CPC title

• G01N1/44

  Sample treatment involving radiation, e.g. heat · CPC title

• C12Q1/701

  Specific hybridization probes · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title