Methods for separating complete and empty AAV particles

Abstract

A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step is reported herein, comprising a series of steps: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step; a first linear gradient; a second isocratic step; and a second linear gradient; wherein the empty recombinant adeno-associated virus particles are eluted in the first linear gradient and the intact recombinant adeno-associated virus particles are eluted in the second linear gradient. In certain embodiments, the solution applied in the first isocratic step comprises about 65 mM buffer, about 10 mM elution salt, about 2 mM salt, and has a pH of about 9.4. In certain embodiments, the solution applied in the second isocratic step comprises about (65) mM of buffer substance, about (90) mM of elution salt, about (2) mM of salt, and has a pH value of about 9.4. In certain embodiments, the buffer substance is N-(1,1-dimethyl-(2)-hydroxyethyl)-(3)-amino-2-hydroxypropanesulfonic acid, the elution salt is tetramethylammonium chloride, and the salt is magnesium chloride.

Description

[Technical Field]

[0001] The present invention is in the field of analytical methods. More specifically, a method for separating intact and empty recombinant AAV particles using an isocratic hold is reported herein. [Background technology]

[0002] Gene therapy has opened unprecedented opportunities for novel therapeutic approaches. Based on the concept of repairing functional mutations by coexpressing the correct gene, restoring biological function requires the use of viral vectors to ensure proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAVs) are the most widely used vectors. Their biomanufacturing process requires the insertion of therapeutic genes into rAAVs (full AAV particles, i.e., rAAV particles containing encapsidated nucleic acid). However, a proportion of rAAVs that do not contain the desired gene (empty AAV particles, i.e., rAAV particles without encapsidated nucleic acid) as well as partially filled rAAVs (partially filled AAV particles) can also be produced, potentially affecting the efficiency of treatment. Therefore, the full / empty ratio of rAAV particles needs to be monitored to ensure consistent product quality and efficacy.

[0003] To identify the content of empty rAAV particles, various analytical strategies have been proposed, including electron microscopy, quantitative polymerase chain reaction (PCR), ELISA assay, UV absorbance spectrophotometry, analytical ultracentrifugation, or charge detection mass spectrometry (CDMS) (Gimpel, AL et al., Mol. Ther.-Methods Clin. Dev. 20 (2021) 740-754 (Non-Patent Document 1); Fu, X. et al., Hum Gene Ther Methods. 30 (2019) 144-152 (Non-Patent Document 2)).

[0004] Generally, a difference in the range of 0.4 pH units can be observed between full and empty rAAV particles. This feature can be exploited in attempts to separate and quantify intact and empty rAAV particles by anion exchange chromatography (AEX) (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984 (Non-Patent Document 3); Khatwani, S.L. et al., Mol. Ther. - Methods Clin. Dev. 21 (2021) 548-558 (Non-Patent Document 4); Urabe, M. et al., Mol. Ther. 13 (2006) 823-828 (Non-Patent Document 5); Dickerson, R. et al., Biotechnol. J. 16 (2021) 2000015 (Non-Patent Document 6); Kaludov, N. et al., Hum. Gene Ther. 13 (2002) 1235-1243 (Non-Patent Document 7); Lock, M. et al., Hum. Gene Ther.Methods.23(2012)56-64 (Non-Patent Document 8)).

[0005] In AEX, rAAV particles follow an "on / off" retention behavior (Snyder, L.R. et al., Anal. Chem. 55 (1983) 1412A-1430A (Non-Patent Document 9); Fekete, S. et al., Anal. Chem. 93 (2021) 1277-1284 (Non-Patent Document 10)).

[0006] International Publication No. 2021 / 158915 (Patent Document 1) discloses recombinant adeno-associated virus compositions and methods for producing and using the same.

[0007] US Patent Application Publication No. 2021 / 355503 (Patent Document 2) discloses compositions and methods for producing vectors for gene therapy.

[0008] Khatwani Santoshkumar et al. describe an anion-exchange HPLC assay for the separation and quantification of empty and intact capsids in multiple adeno-associated virus serotypes ( Mol. Ther. Meth. Clin. Dev. 21 (2021) 548-558 ).

[0009] US Patent Application Publication No. 2021 / 009964 (Patent Document 3) discloses the separation and quantification of empty and complete viral capsid particles.

[0010] Dickerson, R. et al. report the separation of empty and intact recombinant adeno-associated virus particles using isocratic anion-exchange chromatography (Biotechnol J 16(2021)2000015). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2021 / 158915 [Patent Document 2] U.S. Patent Application Publication No. 2021 / 355503 [Patent Document 3] U.S. Patent Application Publication No. 2021 / 009964 [Non-patent literature]

[0012] [Non-Patent Document 1] Gimpel, AL et al., Mol.Ther.-Methods Clin.Dev.20(2021)740-754 [Non-patent document 2] Fu, X. et al., Hum Gene Ther Methods.30(2019)144-152 [Non-patent document 3] Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984 [Non-patent document 4] Khatwani, SL et al., Mol.Ther.-Methods Clin.Dev.21(2021)548-558 [Non-Patent Document 5] Urabe, M. et al., Mol. Ther. 13 (2006) 823-828 [Non-patent document 6] Dickerson, R. et al., Biotechnol. J. 16 (2021) 2000015 [Non-Patent Document 7] Kaludov, N. et al., Hum. Gene Ther. 13 (2002) 1235-1243 [Non-patent document 8] Lock, M et al., Hum. Gene Ther. Methods. 23 (2012) 56-64 [Non-Patent Document 9] Snyder, L.R. et al., Anal. Chem. 55 (1983) 1412A-1430A [Non-Patent Document 10] Fekete, S. et al., Anal.Chem.93(2021)1277-1284 Summary of the Invention

[0013] An anion exchange chromatography (AEX) method including a novel elution scheme is reported herein. Without being bound by this theory, it is hypothesized that this method exploits the fact that viral capsids follow an "on / off" retention behavior. Based on this, an elution method was discovered that allows for unprecedented separation of intact rAAV particles from empty rAAV particles. An advantage of the method according to the present invention is that it provides a 3.7-fold improvement in resolution compared to, for example, that obtained with a linear gradient. The method according to the present invention allows for accurate and precise baseline separation and quantification of intact and empty rAAV particles, making it particularly applicable as a quality control (QC) method, for example.

[0014] Therefore, the present invention encompasses at least the following embodiments.

[0015] 1. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography (AEX) step, comprising: a) applying a solution containing one or both of recombinant AAV particles without encapsidated nucleic acid (empty rAAV particles) and recombinant AAV particles with encapsidated nucleic acid (intact rAAV particles) to a chromatography column comprising an anion exchange (AEX) chromatography material to produce a recombinant AAV particle-loaded AEX chromatography column (rAAV-loaded AEX chromatography column); b) applying a first buffer solution to the rAAV-loaded AEX chromatography column such that the rAAV particles remain bound to the AEX chromatography column to obtain a washed rAAV-loaded AEX chromatography column, wherein the first buffer solution has a first pH value, a first conductivity, and comprises a buffer substance, optionally an elution salt, and optionally a salt; c) applying a first intermediate buffer solution of increasing conductivity, starting at a first conductivity and ending at a second conductivity, to the washed rAAV-loaded AEX chromatography column to obtain an intermediately conditioned rAAV-loaded AEX chromatography column, wherein the increase in conductivity is linear with the addition of increasing concentrations of elution salt; d) applying a second buffer solution to the intermediately conditioned rAAV-loaded AEX chromatography column to obtain a semi-eluted rAAV-loaded AEX chromatography column, wherein the second buffer solution has a second pH value, a second conductivity, and comprises a buffer substance, an elution salt, and optionally a salt; e) applying a second intermediate buffer solution of increasing conductivity, starting at a second conductivity and ending at a third conductivity, to the semi-eluted rAAV-loaded AEX chromatography column to obtain an eluted AEX chromatography column, wherein the increase in conductivity is linear with the addition of increasing concentrations of elution salt. Including, The second buffer solution has a conductivity increased by approximately 8 mS / cm to 9.5 mS / cm compared to the first buffer solution, In step c) or / and d), empty rAAV particles are eluted, the application in step d) lasts for at least 2 minutes, In step e), intact rAAV particles are eluted, The conductivity increases in step e) by at least about 20 mS / cm. method.

[0016] 2. The method of embodiment 1, wherein the rAAV particles are of serotype 8.

[0017] 3. The method of embodiment 1 or 2, wherein the anion exchange chromatography is strong anion exchange chromatography.

[0018] 4. The method of any one of embodiments 1-3, wherein the anion exchange chromatography material is a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix bearing quaternary ammonium groups that provide anion exchange functionality.

[0019] 5. The method of any one of embodiments 1 to 4, wherein the AEX chromatography column has dimensions of about 50 mm in length and about 4 to 5 mm in diameter.

[0020] 6. The method of any one of embodiments 1 to 5, wherein the AEX chromatography material has a particle size of about 10 μm.

[0021] 7. The method of any one of embodiments 1 to 6, for / providing a baseline separation of complete and empty rAAV particles.

[0022] 8. The method of any one of embodiments 1 to 7, for quantification of complete and empty rAAV particles.

[0023] 9. The method of any one of embodiments 1 to 8, which is a high-throughput method for quantification of complete rAAV particles rAAV and empty rAAV particles rAAV.

[0024] 10. The method of any one of embodiments 1 to 9, which is a quality control (QC) method for rAAV preparations.

[0025] 11. The method of any one of embodiments 1 to 10, wherein the solution containing one or both of the empty rAAV particles and the complete rAAV particles is a buffered solution containing about 0.001% (w / v) non-ionic surfactant.

[0026] 12. The method of any one of embodiments 1-11, wherein the solution containing one or both of the empty and complete rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) non-ionic surfactant.

[0027] 13. The method of any one of embodiments 1-12, wherein the solution containing one or both of empty and complete rAAV particles is a buffered solution containing about 0.001% (w / v) poloxamer 188 or polysorbate 20.

[0028] 14. The method of any one of embodiments 1-13, wherein the solution containing one or both of the empty and full rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20.

[0029] 15. The method of any one of embodiments 1 to 14, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise a buffer substance having a pKa value of about 9.

[0030] 16. The method of any one of embodiments 1 to 15, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise a buffer substance selected from 1,3-bis[tris(hydroxymethyl)methylamino]propane, and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0031] 17. The method of any one of embodiments 1 to 16, wherein the first and second buffer solutions and the first and second intermediate buffer solutions have a pH value of 9.0 to 10.0.

[0032] 18. The method of any one of embodiments 1 to 17, wherein the first and second buffer solutions and the first and second intermediate buffer solutions have a pH value of 9.2 to 9.6.

[0033] 19. The method of any one of embodiments 1 to 18, wherein the first and second buffer solutions and the first and second intermediate buffer solutions have a pH value of about 9.4.

[0034] 20. The method of any one of embodiments 1-19, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise an organic salt as an elution salt.

[0035] 21. The method of any one of embodiments 1 to 20, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise an organic chloride salt as an elution salt.

[0036] 22. The method according to any one of embodiments 1 to 21, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise an organic chloride having a molecular weight of less than 170 g / mol as an eluting salt.

[0037] 23. The method of any one of embodiments 1-22, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise tetraethylammonium chloride as an elution salt.

[0038] 24. The method of any one of embodiments 1-22, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise tetramethylammonium chloride as an elution salt.

[0039] 25. The method of any one of embodiments 1 to 23, carried out at a flow rate of about 0.3 mL / min.

[0040] 26. The method of any one of embodiments 1 to 25, wherein the first buffer solution comprises about 65 mM of buffer substance, about 10 mM of elution salt, about 2 mM of salt, and has a pH value of about 9.4.

[0041] 27. The method of any one of embodiments 1 to 26, wherein the second buffer solution comprises about 65 mM of buffer substance, about 90 mM of elution salt, about 2 mM of salt, and has a pH value of about 9.4.

[0042] 28. The method of any one of embodiments 1-27, wherein the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride.

[0043] 29. The method of any one of embodiments 1-28, wherein step b) has a length of about 1 minute, step c) has a length of about 5 minutes, step d) has a length of at least 2 minutes and at most 5 minutes, and step e) has a length of about 5 minutes.

[0044] 30. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step, a first linear gradient, a second isocratic step, and a second linear gradient series; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; i) the rAAV particles are of serotype 8, or / and ii) the anion exchange chromatography material is a strong anion exchange material, or / and iii) the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide the anion exchange functionality, or / and iv) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter; or / and v) the AEX chromatography material has a particle size of about 10 μm, or / and vi) the method is for / provides a baseline separation of full and empty rAAV particles, or / and vii) the method is for quantification of complete and empty rAAV particles, or / and viii) the method is a high-throughput method for quantification of complete rAAV particles rAAV and empty rAAV particles rAAV, or / and ix) the method is a quality control (QC) method for rAAV preparations, or / and x) the solution containing empty rAAV particles and / or intact rAAV particles is a buffered solution containing about 0.001% (w / v) non-ionic surfactant, or / and xi) the solution containing empty and / or intact rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20, or / and xii) the solutions used in the isocratic steps and in the linear gradient contain a buffer substance with a pKa value of about 9, or / and xiii) the solutions used in the isocratic steps and in the linear gradient contain a buffer substance selected from 1,3-bis[tris(hydroxymethyl)methylamino]propane and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, or / and xiv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.0 to 10.0, or / and xv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.2 to 9.6, or / and xvi) the solutions used in the isocratic steps and the linear gradient have a pH value of about 9.4; or / and xvii) the solutions used in the isocratic steps and linear gradients contain organic salts, and / or xviii) the solutions used in the isocratic steps and in the linear gradient contain organic chloride salts, or / and xix) the solutions used in the isocratic steps and the linear gradient contain organic chlorides with a molecular weight of less than 170 g / mol, or / and xx) the solutions used in the isocratic steps and in the linear gradient contain tetraethylammonium chloride, and / or xxi) the solutions used in the isocratic steps and linear gradients contain tetramethylammonium chloride, or / and xxii) the method is carried out at a flow rate of about 0.3 mL / min; or / and xxiii) the solution applied in the first isocratic step contains about 65 mM of a buffer substance, about 10 mM of an elution salt, about 2 mM of a salt, and has a pH value of about 9.4, or / and xxiv) the solution applied in the second isocratic step contains about 65 mM of a buffer substance, about 90 mM of an elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxv) the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride; or / and xxvi) A method wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

[0045] 31. The method of embodiment 30, comprising items i), iii), xi), xiii), xvi), xxi), xxii), xxiv), xxv), or / and xxvi).

[0046] 32. The method of embodiment 30, comprising items i), iii), xi), xiii), xvi), xxi), xxii), xxiv), xxv) and xxvi).

[0047] 33. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step, a first linear gradient, a second isocratic step, and a second linear gradient series; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; i) the rAAV particles are of serotype 8, or / and iii) the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide the anion exchange functionality, or / and iv) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter; or / and v) the AEX chromatography material has a particle size of about 10 μm, or / and x) the solution containing empty rAAV particles and / or intact rAAV particles is a buffered solution containing about 0.001% (w / v) non-ionic detergent, or / and xi) the solution containing empty and / or intact rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20, or / and xii) the solutions used in the isocratic steps and in the linear gradient contain a buffer substance with a pKa value of about 9, or / and xiii) the solutions used in the isocratic steps and in the linear gradient contain a buffer substance selected from 1,3-bis[tris(hydroxymethyl)methylamino]propane and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, or / and xiv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.0 to 10.0, or / and xv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.2 to 9.6, or / and xvi) the solutions used in the isocratic steps and the linear gradient have a pH value of about 9.4; or / and xix) the solutions used in the isocratic steps and the linear gradient contain organic chlorides with a molecular weight of less than 170 g / mol, or / and xx) the solutions used in the isocratic steps and in the linear gradient contain tetraethylammonium chloride, and / or xxi) the solutions used in the isocratic steps and linear gradients contain tetramethylammonium chloride, or / and xxii) the method is carried out at a flow rate of about 0.3 mL / min; or / and xxiii) the solution applied in the first isocratic step contains about 65 mM of a buffer substance, about 10 mM of an elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxiv) the solution applied in the second isocratic step contains about 65 mM of buffer substance, about 90 mM of elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxv) the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride; or / and xxvi) A method wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

[0048] 34. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step, a first linear gradient, a second isocratic step, and a second linear gradient series; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; i) the rAAV particles are of serotype 8, or / and iii) the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide the anion exchange functionality, or / and iv) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter; or / and v) the AEX chromatography material has a particle size of about 10 μm, or / and xi) the solution containing empty and / or intact rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20, or / and xiii) the solutions used in the isocratic steps and in the linear gradient contain a buffer substance selected from 1,3-bis[tris(hydroxymethyl)methylamino]propane and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, or / and xiv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.0 to 10.0, or / and xv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.2 to 9.6, or / and xvi) the solutions used in the isocratic steps and the linear gradient have a pH value of about 9.4; or / and xix) the solutions used in the isocratic steps and the linear gradient contain organic chlorides with a molecular weight of less than 170 g / mol, or / and xxi) the solutions used in the isocratic steps and linear gradients contain tetramethylammonium chloride, or / and xxii) the method is carried out at a flow rate of about 0.3 mL / min; or / and xxiii) the solution applied in the first isocratic step contains about 65 mM of a buffer substance, about 10 mM of an elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxiv) the solution applied in the second isocratic step contains about 65 mM of buffer substance, about 90 mM of elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxv) the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride; or / and xxvi) A method wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

[0049] 35. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step, a first linear gradient, a second isocratic step, and a second linear gradient series; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; i) the rAAV particles are of serotype 8, or / and iii) the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide the anion exchange functionality, or / and iv) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter; or / and v) the AEX chromatography material has a particle size of about 10 μm, or / and xi) the solution containing empty and / or intact rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188, or / and xiii) the solutions used in the isocratic steps and in the linear gradient contain N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance, or / and xiv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.0 to 10.0, or / and xv) the solutions used in the isocratic steps and linear gradients have a pH value of 9.2 to 9.6, or / and xvi) the solutions used in the isocratic steps and the linear gradient have a pH value of about 9.4; or / and xxi) the solutions used in the isocratic steps and linear gradients contain tetramethylammonium chloride, or / and xxii) the method is carried out at a flow rate of about 0.3 mL / min; or / and xxiii) the solution applied in the first isocratic step contains about 65 mM of a buffer substance, about 10 mM of an elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxiv) the solution applied in the second isocratic step contains about 65 mM of buffer substance, about 90 mM of elution salt, about 2 mM of salt and has a pH value of about 9.4, or / and xxv) the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride; or / and xxvi) A method wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

[0050] 36. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; a first isocratic step, a first linear gradient, a second isocratic step, and a second linear gradient series; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; the rAAV particles are of serotype 8, or i) the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide anion exchange functionality; ii) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter; iii) the AEX chromatography material has a particle size of about 10 μm; iv) the solution containing empty and / or intact rAAV particles is phosphate-buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) containing about 0.001% (w / v) poloxamer 188; v) the solutions used in the isocratic steps and in the linear gradient contain N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance; vi) the solutions used in the isocratic step and the linear gradient have a pH value of about 9.4; vii) the solution used in the isocratic step and the linear gradient contains tetramethylammonium chloride; viii) the method is carried out at a flow rate of about 0.3 mL / min; ix) the solution applied in the first isocratic step comprises about 65 mM of buffer substance, about 10 mM of elution salt, about 2 mM of salt and has a pH value of about 9.4; x) the solution applied in the second isocratic step comprises about 65 mM of buffer substance, about 90 mM of elution salt, about 2 mM of salt and has a pH value of about 9.4; xi) the buffer substance is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride; xii) A method wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

[0051] In addition to the various embodiments described and claimed, the presently disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the presently disclosed subject matter, such that the presently disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing descriptions of specific embodiments of the presently disclosed subject matter have been presented for purposes of illustration and description. They are not intended to be comprehensive or to limit the presently disclosed subject matter to the disclosed embodiments. [Brief explanation of the drawings]

[0052] [Figure 1] Empty rAAV8 particles (red line), intact rAAV8 particles (blue line), and a mixture of intact and empty rAAV8 particles (purple line) were analyzed using four different column hardware: PP (A), PS (B), AX (C), and QS (D) using the method according to Example 2. E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty rAAV particle ratio. [Figure 2]Results using various types of buffers. Empty rAAV8 particles (red line), intact rAAV8 particles (blue line), and a mixture of intact and empty rAAV8 particles (purple line) analyzed in AEX mode with the elution method according to Example 2 using five types of buffers: AMPD, BTP, AMPSO, CHES, and CAPSO. E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio, na = not available. [Figure 3] Results using AMPSO buffer at various pH values. Empty rAAV8 particles (red line), intact rAAV8 particles (blue line), and a mixture of intact and empty rAAV8 particles (purple line) were analyzed in AEX mode at five pHs: 8.6, 8.8, 9.0, 9.2, and 9.4, using the elution method according to Example 2. E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio, na = not available. [Figure 4] Results using AMPSO buffer at pH 9.4 and various elution salts. Empty rAAV8 particles (red line), intact rAAV8 particles (blue line), and a mixture of intact and empty rAAV8 particles (purple line) analyzed in AEX mode by using three different elution salts with the elution method according to Example 2; E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio. [Figure 5] Results using AMPSO buffer at pH 9.4 and TMAC as the elution salt at various mobile phase flow rates. A mixture of intact and empty rAAV8 particles analyzed in AEX mode using the elution method according to Example 2 at three different flow rates: 0.7 mL / min (gray line), 0.3 mL / min (green line), and 0.1 mL / min (orange line). [Figure 6]Overlay of chromatograms under various isocratic conditions. Mixtures of intact and empty rAAV8 particles analyzed in AEX mode by using AMPSO buffer pH 9.4 in combination with TMAC as the eluent salt and the elution mode according to the present invention were subjected to a 4-minute isocratic step (red line) at various % (v / v) B: 17% (v / v) B (black line), 17.5% (v / v) B (blue line), 18% (v / v) B (pink line), and 18.5% (v / v) B (green line). E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio. [Figure 7] Overlay of chromatograms using a second isocratic step of various durations. Mixtures of intact and empty rAAV8 particles analyzed in AEX mode by using AMPSO buffer pH 9.4 in combination with TMAC as the eluent salt and in the elution mode according to the invention, including an isocratic step hold at 18% (v / v) B for 4 minutes (blue line), 6 minutes (gray line), 8 minutes (green line), and 10 minutes (orange line). E = empty rAAV8 particles, F = intact rAAV8 particles. [Figure 8] Empty rAAV8 particles (red line), intact rAAV8 particles (blue line), and a mixture of intact and empty rAAV8 particles (purple line) analyzed in AEX mode using three different elution strategies according to Example 2 (comparative) (A), Example 3 (comparative) (B), and Example 4 (according to the invention). E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio. [Figure 9A] Quantification of the % of intact particles in different empty and intact rAAV8 particle mixtures obtained from two different suppliers, Virovek (AB) and Sirion (CD). The elution method according to the invention was applied with a second isocratic step of 18.5% (v / v) B for the Sirion sample and 18% (v / v) B for the Virovek sample. E = empty rAAV8 particles, F = intact rAAV8 particles, F / E = intact / empty ratio. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. DETAILED DESCRIPTION OF THE INVENTION

[0053] definition It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0054] The term "about" refers to a range of ±20% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±10% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±5% of the preceding numerical value.

[0055] As used herein, the terms "comprise(s)," "having / has," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The term "comprising" also encompasses the term "consisting of." The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0056] The terms "empty particle" and "empty recombinant AAV particle" can be used interchangeably, but refer to AAV particles that have an AAV protein shell but lack the nucleic acid encoding a protein or transcribed into a target transcript adjacent to the AAV ITR, i.e., the whole or part of the vector. Thus, empty particles do not function to transfer the nucleic acid encoding a protein or transcribed into a target transcript into target cells.

[0057] The term "endogenous" refers to something that occurs naturally within a cell; something that is naturally produced by a cell; similarly, an endogenous locus / cell-endogenous locus is a locus that occurs naturally within a cell.

[0058] As used herein, the term "exogenous" indicates that a nucleotide sequence is not native to a particular cell but is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation with a viral vector. Thus, an exogenous nucleotide sequence is an artificial sequence, which may result, for example, from a combination of subsequences of different origins (e.g., the combination of a recombinase recognition sequence with an SV40 promoter and a green fluorescent protein coding sequence is an artificial nucleic acid), or from partial deletion or nucleic acid base mutation of a sequence (e.g., a sequence or cDNA encoding only the extracellular domain of a membrane-bound receptor). The term "endogenous" refers to a nucleotide sequence that originates from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but the sequence has become an "exogenous" sequence by introduction into the cell, for example, via recombinant DNA technology.

[0059] The terms "complete particle" and "complete recombinant AAV particle" can be used interchangeably and refer to an AAV particle having an AAV protein shell and encapsidating a nucleic acid, i.e., a vector, that encodes a protein or is transcribed into a transcript of interest adjacent to the AAV ITRs. Thus, the complete particle can transfer the encapsidated nucleic acid, which encodes a protein or is transcribed into a transcript of interest, into a target cell.

[0060] The terms "full-to-empty ratio" and "ratio of full recombinant AAV particles to empty recombinant AAV particles" can be used interchangeably and refer to the mathematical ratio of the number of full recombinant AAV particles to the total number of recombinant AAV particles (full and empty) in a recombinant AAV particle-containing sample or recombinant AAV particle preparation. Because the number of full recombinant AAV particles can be at most the same as the total number of recombinant AAV particles, the ratio can be at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The number of full recombinant AAV particles is determined by determining the number of recombinant AAV particle-encapsidated nucleic acids in a sample or preparation. This can be done by PCR, particularly digital droplet PCR (ddPCR). The total number of recombinant AAV particles is determined by determining the number of capsid proteins in a sample or preparation. This can be done by ELISA, particularly capsid protein-specific ELISA.

[0061] A "recombinant AAV vector" is obtained from the wild-type genome of a virus such as AAV by using molecular biology methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-natural nucleic acid, such as a nucleic acid transcribed into a transcript or a nucleic acid encoding a protein. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from the wild-type virus AAV genome because all or part of the viral genome is replaced with a non-natural (i.e., heterologous) sequence relative to the viral genome nucleic acid. Therefore, the incorporation of a non-natural sequence defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be called a "rAAV vector."

[0062] Recombinant vectors (e.g., AAV) are packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and may be referred to herein as "particles." When recombinant vector sequences are enclosed or packaged in AAV particles, the particles may also be referred to as "rAAV." Such particles contain proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins, such as AAV VP1, VP2, and VP3.

[0063] As used herein, the term "serotype" refers to a distinction based on serologically distinct AAV capsids. Serological specificity is determined based on the lack of cross-reactivity between antibodies against one AAV compared to other AAVs. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from a reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to a reference or other AAV serotype.

[0064] Under the traditional definition, a serotype means that the virus of interest has been tested against all existing and characterized serotype-specific sera for neutralizing activity, and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, new viruses may or may not be serologically distinct from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, it is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically distinct viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0065] A "vector" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or enclosed, either directly or in single-stranded or RNA form, to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or produce a recombinant viral particle, the viral particle does not contain any portion of the "plasmid" that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone," which is important for the cloning and amplification of the plasmid, a process necessary for propagation and recombinant viral production, but is not itself packaged or enclosed in the viral (e.g., AAV) particle. Thus, a "vector" refers to a nucleic acid packaged or enclosed by a viral particle (e.g., AAV).

[0066] Recombinant cells Generally, for efficient and large-scale production of recombinant AAV particles (rAAV particles), cells that express and possibly secrete the rAAV particles are referred to as "recombinant cells" or "recombinant production cells."

[0067] To generate a "recombinant producer cell," a suitable mammalian cell is transfected with the nucleic acid sequences necessary to produce the rAAV particles, including the necessary AAV helper functions.

[0068] Expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Therefore, the open reading frame is operably linked to the additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream, i.e., 5', of the open reading frame, and a polyadenylation signal (sequence) functional in mammalian cells located downstream, i.e., 3', of the open reading frame. In addition, a terminator sequence may be present 3' from the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0069] Similarly, nucleic acids that are transcribed into non-protein-coding RNA are called "RNA genes." Expression of RNA genes also requires additional regulatory elements, such as a promoter and a transcription termination or polyadenylation signal (sequence). The nature and location of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Therefore, RNA genes are usually also incorporated into expression cassettes.

[0070] For AAV particles, which are composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules and require other adenoviral helper functions for production and encapsidation, multiple expression cassettes containing different open reading frames / coding sequences are required. In this case, at least one expression cassette is required for each of the required helper functions: the transgene, the different polypeptides that form the encapsid of the AAV vector, and the VA RNA. Thus, individual expression cassettes are required for each of the helper functions E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes. HEK293 cells constitutively express E1A and E1B helper functions.

[0071] Adeno-associated virus (AAV) For a general review of AAV and adenovirus or herpesvirus helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The AAV genome is described in Srivastava et al., J. Virol., 45 (1983) 555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also WO 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. Construction of recombinant AAV vectors as described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al., J. Virol. 63 (1989) 3822-3828.

[0072] Adeno-associated virus (AAV) is a replication-deficient parvovirus.It can only replicate in cells where certain viral functions are provided by co-infecting helper viruses such as adenovirus, herpesvirus, and sometimes poxviruses such as vaccinia.Nevertheless, AAV can replicate in virtually any cell line of human, monkey, or rodent origin if appropriate helper virus functions exist.

[0073] In the absence of helper virus genes, AAV establishes latency in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0074] AAVs are classified into different serotypes, which are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and over 100 sequences corresponding to different clades of AAV have been identified.

[0075] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific to the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific to the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific to heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific to the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific to the lung.

[0076] Pseudotyping refers to a process involving cross-packaging of AAV genomes between various serotypes, i.e., the genomes are packaged with capsid proteins of different origins.

[0077] The wild-type AAV genome has a size of approximately 4.7 kb. The AAV genome further contains two overlapping genes, called rep and cap, which contain multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral., 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides four proteins of different sizes, called Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The Cap protein encoding open reading frame provides four proteins, called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A, and VA or the corresponding factors of another helper virus.

[0078] For example, in the case of AAV serotype 2 (AAV-2), each ITR is 145 nucleotides long and flanks a coding sequence region of approximately 4,470 nucleotides. Of the 145 nucleotides in the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are designated as the D sequence.

[0079] The AAV genome has three transcription promoters, P5, P19, and P40, for expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0080] The ITR sequence must be present in cis relative to the coding region. The ITR provides a functional origin of replication (ori), the signal required for integration into the genome of target cells, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. The ITR also contains replication origin-like elements such as Rep protein binding sites (RBS) and terminal release sites (TRS). It has been found that the ITR itself can function as a transcription promoter in AAV vectors (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0081] Replication and encapsidation of the viral single-stranded DNA genome require trans-assembly of the rep and cap gene products, respectively.

[0082] The rep locus contains two internal promoters, designated P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides an unspliced ​​4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase that arrests the cell cycle) and a spliced ​​3.9 kb mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides an unspliced ​​mRNA encoding the Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0083] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0084] The larger Rep proteins, Rep68 and Rep78, can specifically bind to hairpin conformations in the AAV ITRs. They exhibit specific enzymatic activities required for resolving replication at AAV termini. Expression of Rep78 or Rep68 can be sufficient for infectious particle formation (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0085] All Rep proteins, primarily Rep78 and Rep68, are thought to exhibit regulatory activities such as AAV gene induction and repression and inhibitory effects on cell proliferation (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0086] Recombinant overexpression of Rep78 results in a phenotype involving reduced cell proliferation due to the induction of DNA damage, which arrests host cells in S phase and thereby promotes latent infection by the virus (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0087] reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, only very low expression has been reported for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0088] The cap locus contains a single promoter, designated P40. Through alternative splicing and the use of alternative start codons, promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6-kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced ​​mRNA transcript), VP2 (72 kDa from a spliced ​​mRNA transcript), and VP3 (61 kDa from an alternative start codon). VP1 through VP3 constitute the viral capsid, which functions to bind to cell surface receptors and enable intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle protein. Nevertheless, all three proteins are essential for efficient capsid production.

[0089] It has been reported that inactivation of all three capsid proteins, VP1-VP3, prevents the accumulation of single-stranded progeny AAV DNA. Mutations in the VP1 amino terminus ("lipid-negative" or "Inf-negative") still allow assembly of single-stranded DNA into viral particles, thereby significantly reducing infectious titers.

[0090] The AAP open reading frame encodes the assembly activating protein (AAP), which is approximately 22 kDa in size and transports native VP proteins to the nucleolus for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0091] Each AAV particle contains only a single-stranded DNA molecule. This can be either a "plus" or "minus" strand. AAV viral particles containing DNA molecules are infectious. Inside infected cells, the parental infectious single strand is converted to a double-stranded form and then amplified. Amplification results in a large pool of double-stranded DNA molecules, from which the single strand is displaced and packaged into capsids.

[0092] Adeno-associated virus (AAV) vector can transduce dividing cells as well as quiescent cells.The transgene introduced into target cells using AAV vector is expected to be expressed for a long period of time.One drawback of using AAV vector is the size restriction of the transgene that can be introduced into cells.

[0093] Viral vectors, such as parvovirus particles containing AAV serotypes and their variants, provide a means for delivering nucleic acids to cells ex vivo, in vitro, and in vivo, encoding proteins so that cells express the encoded proteins. AAV is a useful virus as a gene therapy vector because it can penetrate cells and introduce nucleic acids / genetic materials so that the nucleic acids / genetic materials can be stably maintained within the cells. Furthermore, these viruses can, for example, introduce nucleic acids / genetic materials into specific sites. Because AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0094] AAV particles, used as vehicles for effective gene delivery, possess several desirable characteristics for such applications, including targeting both dividing and non-dividing cells. Early clinical experience with these vectors has demonstrated no persistent toxicity, and immune responses have been minimal or undetectable. AAV is known to infect a wide variety of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0095] Recombinant AAV particles typically do not contain viral genes related to pathogenesis.Such vectors typically have one or more wild-type AAV genes, such as the rep and / or cap genes, deleted in whole or in part, but retain at least one functional adjacent ITR sequence as required for the rescue, replication and packaging of the recombinant vector into AAV particles.For example, only the essential parts of the vector, such as the ITR element and the LTR element, respectively, are included.Therefore, the AAV vector genome will contain the sequences (e.g., functional ITR sequence) required in cis for replication and packaging.

[0096] Recombinant AAV vectors and their methods and uses include any virus strain or serotype.As a non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8.Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other.As a non-limiting example, recombinant AAV vectors based on one serotype genome can be identical in one or more of the capsid proteins that package the vector.Furthermore, recombinant AAV vector genomes can be based on AAV (for example, AAV2) serotype genomes that are different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome may be based on AAV2, but at least one of the three capsid proteins may be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8, or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8 capsids.

[0097] In certain embodiments of all aspects and embodiments of the invention, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8, and variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions), as described, for example, in WO 2013 / 158879, WO 2015 / 013313, and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0098] In certain embodiments of all aspects and embodiments of the invention, the rAAV particles comprise a capsid sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, or 7m8 capsid sequence.

[0099] In certain embodiments of all aspects and embodiments of the invention, the rAAV particles comprise ITR sequences having 70% or greater sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequences.

[0100] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful, inter alia, for administration and delivery to a subject in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce a harmful immune response in the individual receiving the composition and can be administered without undue toxicity.

[0101] Protocols for the production of adenoviral vectors are described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated herein by reference in their entireties.

[0102] Recombinant AAV particles (rAAV particles) Various methods are known in the art for producing rAAV particles.For example, transfection using AAV plasmid and AAV helper sequence in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection with recombinant AAV plasmid, AAV helper plasmid, and helper function plasmid.Non-limiting methods for producing rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887.After recombinant rAAV particle production (i.e., particle production in cell culture system), rAAV particles can be obtained and purified from host cells and cell culture supernatant.

[0103] The production of recombinant AAV particles requires the expression of Rep and Cap proteins, helper proteins E1A, E1B, E2A and E4orf6, and adenovirus VA RNA in a single mammalian cell. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, particularly the CMV IE promoter, as described by Matsushita et al. (Gene Ther. 5 (1998) 938-945). Therefore, any promoter can be used.

[0104] Typically, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of the progeny recombinant genome, namely the open reading frames for the Rep and Cap proteins, are contained in trans on a second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). Additionally, a third plasmid containing helper virus genes, namely E1, E4orf6, E2A, and VA from adenovirus, is required for AAV replication.

[0105] To reduce the number of plasmids required, the Rep, Cap and adenoviral helper genes may be combined on a single plasmid.

[0106] Alternatively, the host cell may already stably express the E1 gene product. Such a cell is an HEK293 cell.

[0107] The human embryonic kidney clone designated 293 was created in 1977 by integrating adenovirus DNA into human embryonic kidney (HEK) cells (Graham, FL et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome, which includes the E1A and E1B genes and the adenovirus packaging signal (Louis, N. et al., Virology 233 (1997) 423-429).

[0108] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (e.g., Samulski, RJ et al., J. Virol. 63 (1989) 3822-3828; Allen, JM et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K. et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR et al., Gene Ther. 2 (1995) 29-37; Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA et al., Hum. Gene Ther. Ther. 6 (1995) 1531-1541; Ferrari, FK et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A. et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X. et al., J. Virol. 72 (1998) 2224-2232; Grimm, D. et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X. et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors may be used (see, e.g., Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ et al., Gene Ther. 2 (1995) 481-485; Fisher, JK et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370).

[0109] To restrict transgene activity to specific tissues, i.e., to limit the integration site, the transgene can be operably linked to an inducible or tissue-specific promoter (see, e.g., Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0110] E1A, E1B, E2, and E4 The coding sequences for E1A and E1B (open reading frames) can be derived from, for example, a human adenovirus, such as human adenovirus serotype 2 or serotype 5, among others. An exemplary sequence for human Ad5 (adenovirus serotype 5) can be found in GenBank entry X02996, AC_000008, and an exemplary sequence for human Ad2 can be found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146, reported in EP 1230354, and plasmids pGS119 and pGS122, reported in WO 2007 / 056994, can also be used as sources of E1A and E1B open reading frames.

[0111] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are derived from the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins activates other viral functions, E1B, E2, E3, and E4. Furthermore, expression of the 12S and 13S proteins forces cells into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cells will die (apoptosis).

[0112] E1B is the second viral helper gene expressed. It is activated by the E1A-derived proteins 12S and 13S. The E1B gene-derived mRNA can be spliced ​​in two different ways, resulting in a 55-kDa transcript and a 19-kDa transcript. The E1B 55-kDa protein is involved in regulating the cell cycle, preventing the transport of cellular mRNA during the late stages of infection, and preventing E1A-induced apoptosis. The E1B 19-kDa protein is involved in preventing E1A-induced apoptosis of cells.

[0113] The E2 gene encodes various proteins: The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0114] The E4 gene also encodes several proteins: the 34 kDa protein derived from the E4 gene (E4orf6), which, together with the 55 kDa E1B protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the cell nucleus to the cytoplasm.

[0115] Adenovirus VA RNA Virus-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VARNAI) and VAII (VARNAII). Both are transcribed by RNA polymerase III from a type 2 polymerase III promoter (e.g., Machitani, M. et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0116] The structure, function, and evolution of adenovirus-associated RNAs using a phylogenetic approach were investigated by Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099), who provided an alignment and consensus VA RNA sequence based on 47 known human adenovirus serotypes, the disclosure of which is incorporated herein by reference in its entirety.

[0117] VA RNAs, VAI and VAII, consist of 157 to 160 nucleotides (nt).

[0118] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a dominant proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0119] Although VA RNA is not essential, it plays an important role in efficient viral propagation by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral propagation, VA RNA-deficient adenoviruses cannot propagate during the early stages of vector production, when only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNA may not be sufficiently expressed, which blocks cellular antiviral mechanisms (see Maekawa, A. et al. Nature Sci. Rep. 3 (2013) 1136).

[0120] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery, in which HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0121] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 to 10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 to 10820 of the sequence of GenBank entry AC_000008.

[0122] Methods for producing rAAV particles Carter et al. showed that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, BJ, "Handbook of Parvoviruses," ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that the ITRs must be maintained to maintain the functions of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0123] When cells containing the respective viral helper genes are transduced by an AAV vector, or vice versa, when cells containing an integrated AAV provirus are transduced by the appropriate helper virus, the AAV provirus is activated and again enters the lytic infection cycle (Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0124] Producer cells contain the rep and cap gene sequences, as well as a transgene cassette flanked by ITR sequences on one or more plasmids that are maintained through drug selection. Production of rAAV particles in these cell lines generally occurs after their infection with the necessary helper functions. Therefore, cells are infected with either a replication-competent AdV (usually wild-type Ad5) or a plasmid containing the respective helper genes to provide helper virus proteins and initiate rAAV particle production. Packaging cell lines differ from producer cell lines because they contain only the rep and cap genes.

[0125] More generally, cells transfected or transduced with DNA for recombinant production of AAV particles can be referred to as "recombinant cells." Such cells can be any mammalian cell used as a recipient of nucleic acids (plasmids) encoding packaging proteins, such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, and nucleic acids (plasmids) encoding proteins or transcribed into a desired transcription product, i.e., a transgene, located between two AAV ITRs. This term includes the progeny of the original transduced or transfected cell. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genome or total nucleic acid complement to the original parent due to natural, accidental, or deliberate mutations.

[0126] Numerous cell growth media suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining viability or media for providing mammalian (e.g., human) cell growth. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0127] To produce rAAV particles, three plasmids are co-transfected into mammalian cells. The transgene plasmid encodes an expression cassette cloned between the AAV ITRs, while the rep and cap genes are provided in trans by co-transfecting a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also called the helper plasmid, contains minimal helper virus elements, generally the adenovirus E2A, EV, and VA genes, but lacks the AAV ITRs.

[0128] Various methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the methods of the present invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection, or microinjection are used for nucleic acid transfer / transfection. In certain embodiments of all aspects and embodiments, inorganic substances (e.g., calcium phosphate / DNA co-precipitation), cationic polymers (e.g., polyethyleneimine, DEAE-dextran, etc.), or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for larger-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethyleneimine being preferred.

[0129] Growth in serum-free suspension culture and improved efficiency and reproducibility of transfection conditions using PEI as a transfection reagent allow for easy scale-up of AAV production using shake flasks, wave or stirred tank bioreactors.

[0130] The composition may include additional plasmids and / or cells, and such plasmids and cells may be in contact with free PEI.

[0131] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched, short-chain fatty acid that inhibits histone deacetylase activity. For this reason, it is commonly added to mammalian cell cultures as an enhancer of recombinant protein production.

[0132] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap, hi certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0133] The encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0134] Culturing can be carried out using conditions commonly used for culturing eukaryotic cells, such as about 37°C, 95% humidity, and 8% CO 2 by volume.

[0135] Cultivation can be performed in serum-containing or serum-free medium, in adherent or suspension culture. Suspension culture can be performed in any fermentation vessel, such as a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaker or spinner vessel, or so-called roller bottles. Transfection can be performed in a high-throughput format and screening, respectively, for example, in a 96- or 384-well format.

[0136] Methods according to the present invention can include AAV particles of any serotype or variant thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any of AAV serotypes 1-12, AAV VP1, VP2, and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2, and / or VP3 capsid proteins, or wild-type AAV VP1, VP2, and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or AAV pseudotype, and the AAV pseudotype comprises an AAV capsid serotype that is different from the ITR serotype.

[0137] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0138] The ITRs can be any of the AAV2, AAV6, AAV8, or AAV9 serotypes, or a combination thereof. The AAV particles can comprise any VP1, VP2, and / or VP3 capsid proteins having 75% or greater sequence identity to any of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, or AAV 7m8, or comprise modified or variant VP1, VP2, and / or VP3 capsid proteins selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, and AAV 7m8 AAV serotypes.

[0139] After production of recombinant viral (e.g., AAV) particles, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from the host cells using a variety of conventional methods, including column chromatography, CsCl gradients, iodixanol gradients, and the like.

[0140] For example, multiple column purification steps, such as purification using anion exchange columns, affinity columns, and / or cation exchange columns, may be used (see, e.g., International Publication No. WO 02 / 12455 and U.S. Patent Application Publication No. 2003 / 0207439). Alternatively or additionally, an iodixanol or CsCl gradient step may be used (see, e.g., U.S. Patent Application Publication No. 2012 / 0135515 and U.S. Patent Application Publication No. 2013 / 0072548). Furthermore, when using infectious viruses to express packaging and / or helper proteins, various methods may be used to inactivate residual virus. For example, adenovirus may be inactivated by heating to a temperature of approximately 60°C for, e.g., 20 minutes or more. Because AAV is thermostable, but the helper adenovirus is thermolabile, this treatment effectively inactivates the helper virus.

[0141] The goal of the rAAV vector production and purification system is to implement strategies to minimize / control the generation of production-related impurities such as protein, nucleic acid, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities.

[0142] Given that rAAV particles represent a very small fraction of the biomass, they must be purified to a level of purity that allows them to be used as clinical human gene therapy products (e.g., Smith PH et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; reports from the CHMP Gene Therapy Expert Group Meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0143] As a first step, typically, cultured cells producing rAAV particles are harvested, optionally in combination with the harvested cell culture supernatant (medium) in which the rAAV particle-producing cells (suspension or adherent) were cultured. The harvested cells and, optionally, the cell culture supernatant can be used as is, dissolved, or concentrated as needed. Furthermore, if infection is used to express helper function, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to approximately 60°C for, for example, 20 minutes or more; this inactivates only the helper virus, since AAV is thermostable while the helper adenovirus is thermolabile.

[0144] Cells in the harvested culture broth are lysed by the method according to the present invention to release rAAV particles. During or after cell lysis, a nuclease, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris, e.g., by filtering or centrifugation, to produce a clarified cell lysate. In a specific example, the lysate is filtered through a micron-diameter pore size filter (e.g., a filter with a pore size of 0.1 to 10.0 μm, e.g., a filter with a pore size of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0145] The lysate (optionally clarified) contains AAV particles (including rAAV vectors and empty capsids) and production / process-related impurities, such as soluble cellular components from host cells, which may include, inter alia, cellular proteins, lipids, and / or nucleic acids, as well as cell culture medium components. The clarified lysate is then optionally subjected to a purification step to purify the AAV particles (including rAAV vectors) from impurities using chromatography. The clarified lysate can be diluted or concentrated with an appropriate buffer before the first chromatography step.

[0146] After cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent sequential chromatographic steps can be used to purify the rAAV particles.

[0147] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0148] If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography, in any order, sequence, or combination.

[0149] For example, the removal of empty capsids from full capsids during downstream processing is based on their different isoelectric points (pI) in anion exchange chromatography. The average pI calculated across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984).

[0150] Cation exchange chromatography functions to separate AAV particles from cellular and other components present in the clarified lysate and / or column eluate from affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include any sulfonic acid-based resin indicated by the presence of sulfonate functional groups, including aryl- and alkyl-substituted sulfonates such as sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin.

[0151] Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on phosphate functional groups), methyl sulfonate (S) and sulfopropyl (SP) resins.

[0152] Anion exchange chromatography functions to separate AAV particles from proteins, cellular components, and other components present in the clarified lysate and / or column eluate from affinity chromatography, cation exchange chromatography, or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty capsids in the eluate. For example, an anion exchange column to which rAAV particles are bound can be washed with a solution containing a moderate concentration of NaCl (e.g., about 100-125 mM, e.g., 110-115 mM), resulting in the elution of a portion of the empty capsids in the flow-through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted with a solution containing a higher concentration of NaCl (e.g., about 130-300 mM NaCl), producing a column eluate with a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV vector-containing rAAV particles.

[0153] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those generally based on quaternized nitrogen atoms, including, but not limited to, quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (GE DEAE Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0154] A manufacturing process for purifying recombinant AAV particles intended as a product for treating human disease should achieve the following objectives: 1) consistent particle purity, potency, and safety; 2) scalability of the manufacturing process; and 3) acceptable production costs.

[0155] An exemplary process for recombinant AAV particle purification is reported in WO 2019 / 006390.

[0156] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying empty capsids and rAAV particles with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0157] To determine the presence or amount of disassembled / denatured capsid, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel. The gel is then run until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody to bind to the denatured capsid protein (see, for example, Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that binds to the primary antibody provides a means for detecting the primary antibody. The binding between the primary and secondary antibodies is semi-quantitatively detected to determine the amount of capsid. Another method is analytical HPLC using an SEC column or analytical ultracentrifuge.

[0158] Method according to the invention Twelve different AAV serotypes are known and are characterized by specific target tissue tropisms.

[0159] AAVs consist of a protein capsid containing the viral genome. The capsid is composed of approximately 60 copies of three viral proteins, VP1, VP2, and VP3 (at a 1:1:10 ratio, respectively), assembled into an icosahedron with a diameter of 26 nm. The genome consists of a single-stranded DNA of approximately 4.7 kb. The genome structure is relatively simple, consisting of three genes flanked by two inverted terminal repeats (ITRs) at the termini (Straus, SE et al., Proc. Natl. Acad. Sci. 73 (1976) 742-746). The first gene, the Rep gene, encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that are produced from the same sequence but via alternative splicing from different promoters (Dismuke, D. et al., Curr. Gene Ther. 13 (2014) 434-452). They are useful for targeting viral DNA integration into the viral capsid, viral replication, transcription, and viral DNA packaging (Dismuke, D. et al., Curr. Gene Ther. 13 (2014) 434-452). The second gene is the Cap gene, which encodes three distinct viral capsid proteins (VP1, VP2, and VP3) despite the same gene sequence due to translation from various start codons and alternative splicing. The third gene encodes the assembly activating protein (AAP) and is located within the Cap coding sequence (Dismuke, D. et al., Curr. Gene Ther. 13 (2014) 434-452; Weitzman, MD et al., Proc. Natl. Acad. Sci. 91 (1994) 5808-5812).

[0160] Given its simplicity, the wild-type AAV genome can be easily manipulated and replaced with an artificial AAV genome, resulting in an artificial recombinant AAV (rAAV) lacking viral DNA encoding viral proteins (Rep and Cap). The ITR sequences of the viral genome are preserved to maintain transcriptional activity, and the remainder of the viral sequence is replaced by an expression cassette containing a therapeutic gene, called a transgene (Naso, MF et al., BioDrugs. 31 (2017) 317-334). The transgene allows the expression of a desired therapeutic entity, such as a polypeptide or protein, which may be, for example, a defective or non-functional protein associated with a specific pathological condition (Maguire, AM et al., Mol. Ther. 29 (2021) 442-463).

[0161] During rAAV particle production, the artificial AAV genome may not be properly incorporated into the AAV capsid, resulting in a mixture of empty and complete rAAV particles. The proportion of empty rAAV particles can represent 10% to up to 90% of the total rAAV particles (Flotte, TR, Hum. Gene Ther. 28 (2017) 147-148). Therefore, empty and partially complete rAAV particles are product-associated impurities that can reduce the effective concentration of the final drug, compete for binding sites, and reduce drug efficacy (Gao, K. et al., Mol. Ther. - Methods Clin. Dev. 1 (2014) 9). According to the FDA, these impurities must be monitored and reported as the full / empty (F / E) ratio (FDA Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), (2020)).

[0162] Because they are similar to each other, it is difficult to avoid or eliminate empty rAAV particles (Schnoedt, M. and Buening, H., Hum. Gene Ther. Methods. 28 (2017) 101-108). Full and empty rAAV particles have the same size. However, because full rAAV particles are loaded with negatively charged DNA, they have a slightly lower pI than empty rAAV particles. In general, a difference in the range of 0.4 pH units can be observed. This characteristic is the basis for the separation and quantification of full and empty rAAV particles in the method according to the present invention.

[0163] The present invention is based, at least in part, on the discovery that a combination of a linear gradient and an isocratic step allows for unprecedented separation of full and empty rAAV particles in a preferred embodiment of serotype 8 (rAAV8). The method according to the invention allows for accurate and precise baseline separation and quantification of full and empty rAAV particles. One application of the method according to the invention is as a high-throughput or / quality control (QC) method for rAAV.

[0164] The following is presented using recombinant AAV particles with serotype 8 capsids. This is presented solely to illustrate the invention and should not be construed as a limitation of the general applicability of the method according to the invention. The true scope is set forth in the appended claims.

[0165] Four strong anion exchange adsorbents, all functionalized with positively charged quaternary groups and differing in matrix composition (monolith or nonporous beads), were tested for their effectiveness in separating empty and intact rAAV8 particles.

[0166] The column materials were stainless steel, PEEK, or PEEK-lined stainless steel (SS), which should not affect the separation characteristics. PEEK stands for polyetheretherketone. PEEK tubing has become a standard item in the operation of many HPLC systems and exhibits high column strength as well as high tensile strength and flexural modulus, making it ideal for catheters and other tubing applications requiring good torque response and pushability. Extruded PEEK tubing is used in medical applications where very high stiffness is required.

[0167] Table 1: List of chromatography columns and their properties (chemistry: quaternary positively charged N-based functional groups for all columns). PEEK and SS represent polyetheretherketone and stainless steel, respectively. TIFF2025530294000001.tif50170

[0168] To operate within the column pressure limits, the flow rates were adjusted to 0.7 mL / min used for the PP and AX columns and 0.5 mL / min and 0.35 mL / min for the QS and PS columns, respectively. The method of Example 2 was used.

[0169] As shown in Figure 1, adequate peak resolution (Rs) was obtained only with the PP (Rs = 1.07) and PS (Rs = 1.00) columns.

[0170] QS columns contain a non-porous polymer-based chromatographic material. More specifically, the stationary phase / chromatographic material is a monodisperse, non-porous methacrylate-based resin particle whose surface consists of an open-access network of multilayered anion-exchange groups. The surface is modified with a quaternary ammonium salt. QS is a strong anion exchanger.

[0171] AX columns contain chromatographic materials based on a synthetic stationary phase consisting of a highly crosslinked particle core of a poly(styrenedivinylbenzene) (PS-DVB)-based copolymer coated with a hydrophilic polymer layer and densely packed quaternary ammonium functional groups chemically bonded thereto. AX is a strong anion exchanger.

[0172] The AX column has been reported to be useful for analyzing the full / empty capsid ratio in adeno-associated virus serotypes 1 and 6 using biocompatible liquid chromatography (Liau, B, Agilent Application Note DE10415602, 5994-4589EN, February 2022).

[0173] PS columns contain chromatographic materials based on polymer monoliths prepared by an in situ polymerization process. The monolith is a single cylindrical polymer rod containing a continuous, interconnected network of through-holes, also called channels. The material can be a methacrylate polymer. The monolith consists of an aggregate of small spheres, the majority of which are designed to be less than 500 nm in size. The small spheres are essentially nonporous. The pore volume is approximately 60% of the column volume. The modal pore size ranges from approximately 1.6 μm to 2.7 μm, with the most common pore size being approximately 2 μm. The surface is modified with a quaternary amine. PS is a strong anion exchanger.

[0174] In a preferred embodiment of all aspects and embodiments according to the present invention, the stationary phase / chromatographic material is a synthetic stationary phase consisting of a polymer monolith. In certain embodiments, the monolith is a methacrylate polymer. In certain embodiments, the monolith is functionalized with quaternary amine functional groups.

[0175] The PP column contains a chromatographic material based on a synthetic stationary phase consisting of a highly crosslinked particle core of a copolymer based on ethylvinylbenzene and divinylbenzene (EVB-DVB) coated with a crosslinked hydrophilic boundary layer and a linear anion exchange phase grafted thereon with quaternary ammonium functional groups. More specifically, the PP stationary phase consists of microporous ethylvinylbenzene crosslinked with 55% divinylbenzene polymer groups coated with a highly hydrophilic neutral polymer substrate, forming a hydrophilic layer onto which controlled polymer chains with quaternary ammonium groups are grafted to introduce anion exchange functionality. PP is a strong anion exchanger.

[0176] In a preferred embodiment of all aspects and embodiments according to the present invention, the stationary phase / chromatographic material is a synthetic stationary phase consisting of a highly crosslinked particle core of a copolymer based on styrene and divinylbenzene (EVB-DVB) coated with a crosslinked hydrophilic boundary layer and a linear anion exchange phase grafted thereon with quaternary ammonium functional groups. In a specific embodiment, the stationary phase consists of a microporous ethylvinylbenzene crosslinked 55% divinylbenzene polymer substrate covered with a highly hydrophilic neutral polymer forming a hydrophilic layer onto which controlled polymer chains bearing quaternary ammonium groups are grafted. In a specific embodiment, the solid phase is a particle. In a specific embodiment, the particle size of the solid phase, i.e., the diameter of the particles, is about 10 μm.

[0177] In the following, a PP column is used to illustrate the method according to the invention.

[0178] We found that dilution of rAAV8 particles in a buffer containing PBS (phosphate-buffered saline; 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO) supplemented with 0.001% (w / v) poloxamer 188 resulted in a more stable sample over time. Under these dilution conditions, the variability in the rAAV8 full (RSD = 8.6%) and empty (RSD = 8.3%) peak areas was limited, and measurement of the full / empty ratio was much more accurate (RSD = 3.4%) compared to using water as the diluent.

[0179] Table 2: Summary of sample stability test results. TIFF2025530294000002.tif100149

[0180] Therefore, poloxamer-supplemented buffer was selected as the preferred dilution solvent, and remaining rAAV8 analyses were always performed within 3 days of sample preparation to limit variability with respect to sample stability.

[0181] A variety of buffer salts, pH values, and salts were tested.

[0182] Table 3: List of buffer salts and their chemical structures. TIFF2025530294000003.tif80170

[0183] The reference BTP buffer was compared with four alternative biological buffers (see Table 2) with various pKa values ​​and chemical structures: AMPD, AMPSO, CHES, and CAPSO. Chromatograms obtained with these various buffered mobile phases using the elution method described in Example 2 are shown in Figure 2. The full / empty ratio (F / E) and chromatographic resolution (Rs) were determined. The full / empty ratio was found to be significantly unaffected by the use of the various buffers (ranging from 1.08 to 1.28). However, resolution and calculated Rs values ​​were obtained only for the BTP and AMPSO buffers (Rs = 0.80 and 0.84, respectively).

[0184] In a particular embodiment, the buffering agent in the method according to the invention is 1,3-bis[tris(hydroxymethyl)methylamino]propane.

[0185] In one preferred embodiment of all aspects and embodiments of the present invention, the buffering agent in the method according to the present invention is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0186] In the following, the method according to the invention is exemplified using AMPSO buffer.

[0187] A mobile phase consisting of AMPSO buffer was evaluated at various pH values ​​ranging from pH 8.6 to pH 9.4 in 0.2 pH unit intervals. The elution method described in Example 2 was used. As shown in Figure 3, a slight decrease in retention time was observed with increasing mobile phase pH. This behavior is consistent with the pKa of AMPSO being equal to 9. When using mobile phases buffered at pH values ​​below its pKa (here, 8.6 and 8.8), a larger amount of the neutral form of AMPSO is present in solution, whereas with mobile phases buffered at pH values ​​above its pKa (here, 9.2 and 9.4), a larger amount of the negatively charged form of AMPSO is expected in solution. Since only the negatively charged form of AMPSO buffer is competing with the analyte for binding to the positively charged stationary phase, a slight decrease in retention time was observed with increasing mobile phase pH. The full / empty ratio was not significantly affected by pH variation (ranging from 1.00 to 1.09), but higher resolution was obtained at pH 9.4 (Rs = 0.93) compared to pH 9.0 (Rs = 0.70).

[0188] In certain embodiments of all aspects and embodiments of the method according to the invention, the mobile phase has a pH value of 9.0 or greater. In certain embodiments, the mobile phase has a pH value of 9.0 to 10.0. In certain embodiments, the mobile phase has a pH value of 9.2 to 9.6. In one preferred embodiment, the mobile phase has a pH value of about 9.4.

[0189] In the following, a pH value of 9.4 is used to illustrate the method according to the invention.

[0190] Three different salt types were tested as eluents for separating rAAV8 intact and empty particles: NaCl, KCl, and tetramethylammonium chloride (TMAC). Figure 4 shows the corresponding chromatograms obtained by using the various elution salts.

[0191] Notably, the full / empty ratio was not significantly affected (ranging from 0.96 to 1.09), but the use of the organic chloride TMAC resulted in better resolution (Rs = 1.14) compared to the inorganic salts NaCl (Rs = 0.93) and KCl (Rs = 0.90).

[0192] In certain embodiments of all aspects and embodiments of methods according to the invention, the salt that causes elution of the rAAV particles is an organic salt. In certain embodiments, the organic salt is an organic chloride. In certain embodiments, the organic salt is an organic chloride having a molecular weight of less than 200 g / mol. In certain embodiments, the organic salt is an organic chloride having a molecular weight of less than 170 g / mol. In certain embodiments, the organic salt is tetraethylammonium chloride. In a preferred embodiment, the organic salt is tetramethylammonium chloride.

[0193] In the following, tetramethylammonium chloride is used as the eluting salt to illustrate the method according to the invention.

[0194] Three different mobile phase flow rates were tested: 0.7 mL / min, 0.3 mL / min, and 0.1 mL / min. The results are shown in Figure 5.

[0195] A flow rate of 0.3 mL / min was selected as the best compromise between chromatographic resolution, peak intensity, and mobile phase consumption.

[0196] Large proteins exhibit specific elution behavior under reversed-phase liquid chromatography (RPLC) conditions, with retention being highly sensitive to mobile phase composition (see, e.g., Snyder, L.R. et al., Anal. Chem. 55 (1983) 1412A-1430A; Fekete, S. et al., Anal. Chem. 93 (2021) 1277-1284). This specific retention behavior has been described as an "on / off" or "bind and elute" mechanism and can be explained as follows: retention of large solutes is nearly infinite in weak eluents (this corresponds to the "on" or "bind" state, where the protein species is fully adsorbed at the column inlet), while only small increases in eluent strength result in large retention decreases (this corresponds to the "off" or "elute" state, as the protein migrates toward the column outlet without further interactions).

[0197] Strategies combining isocratic steps with very short steep gradient segments to tailor the desired selectivity in protein elution compositions have been reported (Fekete, S. et al., Anal. Chem. 91 (2019) 12954-12961; Nguyen, J. M. et al., J. Chromatogr. B. 1173 (2021) 122694). Such a strategy has been applied once in AEX to separate empty and full capsids of recombinant adeno-associated viruses (Khatwani, S. L. et al., Mol. Ther. - Methods Clin. Dev. 21 (2021) 548-558).

[0198] Based on two initial AEX gradients from 0% to 60% B at 15 and 45 minutes, respectively, and measurements of the retention times of the two species, the following gradient was derived: 98% (v / v) mobile phase A (A) and 2% (v / v) mobile phase B (B) for 1 minute at the beginning of the method, followed by a linear change to approximately 82% (v / v) A and 18% (v / v) B within 5 minutes, an isocratic hold for approximately 4 minutes, and a further linear change to approximately 40% (v / v) A and 60% (v / v) B within 5 minutes.

[0199] These conditions were experimentally verified.

[0200] Accordingly, the present invention provides a method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; A method is provided in which empty recombinant adeno-associated virus particles are eluted in a first linear gradient and complete recombinant adeno-associated virus particles are eluted in a second linear gradient.

[0201] It should be noted that the second isocratic step has the effect of eluting the entire amount of empty recombinant adeno-associated virus particles from the column before changing conditions to elute intact recombinant adeno-associated virus particles from the column.

[0202] Various isocratic hold condition compositions around the derived values, i.e., 17.0% (v / v), 17.5% (v / v), 18% (v / v), and 18.5% (v / v) B, were tested, and the corresponding chromatograms are shown as overlays in Figure 6.

[0203] It can be seen that complete separation of the intact and empty rAAV8 particle peaks was achieved regardless of the isocratic hold conditions applied.

[0204] The full / empty ratio and chromatographic resolution were used to evaluate the most appropriate conditions. When the isocratic step was performed at 17% (v / v) B or 17.5% (v / v) B, the peak corresponding to empty particles (the first eluting peak) was broadened compared to the other test conditions. As a result, the full / empty ratio was very high. When the isocratic step was performed at 18.5% (v / v) B, the resulting peak shape was good and the resolution was maximized (Rs = 5.02), but the full / empty ratio did not match theoretical expectations (only 0.68). When the isocratic step was performed at 18% (v / v) B, the resolution was good (Rs = 3.72), no substantial peak tailing / broadening was observed, and the full / empty ratio was close to the expected value of 1 (1.14).

[0205] In addition to the ionic strength of the second isocratic step, the duration of the second isocratic step can also be used to modify selectivity and resolution. More specifically, the duration of the second isocratic step affects the retention of the next peak to elute, i.e., the full particle peak. This does not affect the previously eluted peak, i.e., the empty particle peak.

[0206] To demonstrate this, the duration of the second isocratic step was evaluated using durations of 4, 6, 8, and 10 minutes, and the corresponding chromatograms are shown in Figure 7.

[0207] When the duration of the second isocratic step was increased, a decrease in the peak intensity and peak area corresponding to full rAAV particles was observed, while the peak corresponding to empty rAAV particles was unaffected (see Figure 7). Without being bound by this theory, it is hypothesized that if the isocratic step is too long, the full / empty ratio will be inaccurate (too low).

[0208] To demonstrate the improvement obtained with the method according to the invention, a comparison was made between the linear gradient elution method (Example 2, Figure 8A), the adjusted linear gradient elution method (Example 3, Figure 8B) and the method according to the invention (Example 4, Figure 8C).

[0209] As can be seen, the gain in chromatographic resolution between the chromatograms reported in Figures 8A and 8B is moderate, while the full / empty ratio remains comparable. However, the improvement in resolution is more pronounced when applying the method according to the invention (Rs of 3.72), while the full / empty ratio remains comparable (1.14).

[0210] To confirm the applicability of the method according to the invention for characterizing rAAV particles, two different types of samples containing both full and empty rAAV8 particles were obtained from two different suppliers (i.e., Sirion Biotech and Virovek).

[0211] The retention time of empty rAAV particles from Sirion Biotech was slightly greater than that from Virovek. This may be explained by the vector production process, which results in a variable negative charge on the capsid, which may induce somewhat greater retention of the sample. Therefore, the separation conditions for full and empty rAAV particles from Sirion Biotech were slightly different. The isocratic step was set to 18.5% (v / v) of mobile phase B, which allowed sufficient elution of Sirion empty particles.

[0212] Eleven mixtures containing both complete and empty particles co-mixed in various ratios (ranging from 0% to 100% complete capsid) in PBS buffer were analyzed. Figure 9 shows the corresponding chromatograms for all co-mixtures with 0:100 and 100:0 empty / complete samples, corresponding to pure complete and empty rAAV particles, respectively. As shown in the chromatograms, the complete capsid sample from Virovek already contains approximately 10% empty particles, while this value drops to only 1–2% for the Sirion Biotech sample. On the other hand, the empty particle samples from the two suppliers already contain over 14% complete particles. Figure 9 also provides a graphical representation of the experimental versus theoretical percentage of complete rAAV particles in the mixtures. The experimental values ​​were obtained exclusively from the peak areas. As can be seen, a linear change in the peak area of ​​both peaks (R2 values ​​higher than 0.995 for the two different samples) with a corresponding change in the peak area of ​​the percentage of empty and complete rAAV particles is obtained with the method according to the present invention. These observations confirm that the areas of each peak are additive, linear, and specific for empty and intact rAAV8 particles, confirming that the method according to the invention is accurate, linear, and sufficiently robust to be used in a QC environment.

[0213] To summarize the above, the present invention provides at least the following:

[0214] 1. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; A method in which empty recombinant adeno-associated virus particles are eluted in a first linear gradient and complete recombinant adeno-associated virus particles are eluted in a second linear gradient.

[0215] 2. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method wherein the solution containing empty rAAV particles and / or complete rAAV particles is phosphate buffered saline containing about 0.001% (w / v) non-ionic surfactant.

[0216] 3. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method wherein the rAAV particles are of serotype 8.

[0217] 4. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; The method, wherein the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide anion exchange functionality.

[0218] 5. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method wherein the solutions used in the isocratic steps and the linear gradient contain a buffer substance having a pKa value of about 9.

[0219] 6. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method wherein the solutions used in the isocratic steps and in the linear gradient contain N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance.

[0220] 7. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method wherein the solutions used in the isocratic steps and the linear gradient have pH values ​​of 9.0 to 9.6.

[0221] 8. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The method comprises the following steps: a) applying a solution containing empty rAAV particles and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient; Empty recombinant adeno-associated virus particles are eluted in a first linear gradient, and complete recombinant adeno-associated virus particles are eluted in a second linear gradient; A method in which the solutions used in the isocratic steps and the linear gradient contain tetramethylammonium chloride as the eluting salt.

[0222] 9. The method of any one of embodiments 1-8, wherein the solution containing empty and / or complete rAAV particles is phosphate-buffered saline containing about 0.001% (w / v) non-ionic surfactant.

[0223] 10. The method of any one of embodiments 1-9, wherein the non-ionic surfactant is selected from poloxamer 188 and polysorbate 20.

[0224] 11. The method of any one of embodiments 1 to 10, wherein the rAAV particles are of serotype 8.

[0225] 12. The method of any one of embodiments 1-11, wherein the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide anion exchange functionality.

[0226] 13. The method of any one of embodiments 1 to 12, wherein the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter, and the AEX chromatography material has a particle size of about 10 μm.

[0227] 14.Antibodies i) provide a baseline separation of full and empty rAAV particles; or ii) for quantitation of intact and empty rAAV particles; or iii) a high-throughput method for quantification of intact and empty rAAV particles; or iv) The method according to any one of embodiments 1 to 13, which is a quality control (QC) method for the preparation of rAAV.

[0228] 15. The method according to any one of embodiments 1 to 14, wherein the solutions used in the isocratic steps and in the linear gradient contain a buffer substance with a pKa value of about 9.

[0229] 16. The method according to any one of embodiments 1 to 15, wherein the solutions used in the isocratic steps and in the linear gradient contain N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance.

[0230] 17. The method according to any one of embodiments 1 to 16, wherein the solutions used in the isocratic steps and in the linear gradient have a pH value of 9.0 to 9.6.

[0231] 18. The method according to any one of embodiments 1 to 17, wherein the solutions used in the isocratic steps and in the linear gradient contain an organic chloride having a molecular weight of less than 170 g / mol as an eluting salt.

[0232] 19. The method of any one of embodiments 1 to 18, wherein the solutions used in the isocratic steps and in the linear gradient contain tetramethylammonium chloride as an eluting salt.

[0233] 20. The method of any one of items 1 to 19, carried out at a flow rate of about 0.3 mL / min.

[0234] 21. The method according to any one of items 1 to 20, wherein the solution applied in the first isocratic step comprises about 65 mM of a buffer substance, about 10 mM of an elution salt, about 2 mM of magnesium chloride and has a pH value of about 9.4.

[0235] 22. The method according to any one of items 1 to 21, wherein the solution applied in the second isocratic step comprises about 65 mM of a buffer substance, about 90 mM of an elution salt, about 2 mM of magnesium chloride and has a pH value of about 9.4.

[0236] 23. The method according to any one of items 1 to 22, wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2 to 5 minutes, and the second linear gradient has a length of about 5 minutes.

[0237] The examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0238] material Bis-trispropane (BTP, 99.0% or higher), 2-amino-2-methyl-1,3-propanediol (AMPD, 99.0% or higher), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO, 99.0% or higher), 2-(cyclohexylamino)ethanesulfonic acid (CHES, BioUltra, 99.5% or higher), 3-(cyclohexylamino)-2-hydroxy-1-propane Caprylic / capsular sulfonic acid (CAPSO, anhydrous basis, ≥99%), magnesium chloride hexahydrate (BioXtra, ≥99.0%), hydrochloric acid solution (1N), sodium chloride (BioUltra, molecular biology grade, ≥99.5%), potassium chloride (BioUltra, molecular biology grade, ≥99.5%), and tetramethylammonium chloride (TMAC, LiChropur, ≥99.0%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium hydroxide solution (1N) was obtained from VWR Chemicals. Phosphate-buffered saline (PBS) and poloxamer 188 were obtained from Roche Diagnostics GmbH (Penzberg, Germany). Water was supplied by a Milli-Q purification system from Millipore (Bedford, MA, USA).

[0239] Samples containing either full or empty rAAV8 particles were obtained from Virovek (Hayward, CA, USA) at a concentration of 2.00E+13 vp / mL and from Sirion Biotech (Graefelfing, Germany) at 5.00E+13 vp / mL.

[0240] Anion exchange chromatography was performed on an ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) equipped with a 50 μL injection loop, a quaternary solvent delivery pump, and an autosampler with a fluorescence detector (FD). Data were acquired using FLR excitation at 280 nm and emission at 350 nm.

[0241] Table 1: List of chromatography columns and their properties (chemistry: quaternary ammonium for all columns). PEEK and SS stand for polyetheretherketone and stainless steel, respectively. TIFF2025530294000004.tif50170

[0242] The chromatography column was maintained at room temperature throughout the analysis. In all cases, 20 μL of sample at 1.00E+12 vp / mL was injected, corresponding to a column load of 2.00E+10 vp. Acquisition was performed in salt gradient mode and applied to full, empty, and mixed rAAV particle samples. Data acquisition and instrument control were performed by Empower Pro3 software (Waters). [Example]

[0243] Example 1 Sample preparation The concentration of rAAV particles in the sample is expressed as the number of viral particles per mL (vp / mL).

[0244] Samples were stored at −80° C. Prior to analysis, samples were diluted to 1.00E+12 vp / mL in the appropriate solvent (phosphate buffer solution containing 0.001% poloxamer 188 or the respective mobile phase).

[0245] For "mixed samples" containing both intact and empty rAAV particles, appropriate volumes of the intact and empty rAAV particle-containing diluted samples were taken and mixed to obtain the desired ratio of intact and empty rAAV particles in the aliquots.

[0246] After sample preparation, all samples were stored at 4°C and analyzed within 72 hours of preparation.

[0247] Example 2 - Comparative Example standard method Mobile phase A (A) consisted of 65 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP), 2 mM magnesium chloride hexahydrate in water, adjusted to pH 9.0 with 1 M sodium hydroxide solution. Mobile phase B (B) consisted of 65 mM BTP, 2 mM magnesium chloride hexahydrate, 500 mM sodium chloride in water, adjusted to pH 9.0 with 1 M sodium hydroxide solution.

[0248] The standard method started with a 3-minute hold at 2% (v / v) B. A linear gradient was then applied by increasing the percentage of mobile phase B from 2% (v / v) to 56% (v / v) over 30 minutes. This was followed by a 5-minute wash step with 100% (v / v) B and a 6-minute column re-equilibration step at 2% (v / v) B. The column was maintained at room temperature, and the flow rate was set to a constant value, such as 0.7 mL / min.

[0249] Example 3 - Comparative Example Optimization Standard Method Mobile phase A (A) consisted of 65 mM Mn-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2 mM magnesium chloride hexahydrate in water, adjusted to pH 9.4 with 1 M sodium hydroxide solution. Mobile phase B (B) consisted of 65 mM AMPSO, 2 mM magnesium chloride hexahydrate, 500 mM tetramethylammonium chloride in water, adjusted to pH 9.4 with 1 M sodium hydroxide solution.

[0250] The column was kept at room temperature and the flow rate was set at a constant value such as 0.3 mL / min. The same linear gradient as described in Example 2 was applied.

[0251] Example 4 Method according to the invention Mobile phase A (A) consisted of 65 mM Mn-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2 mM magnesium chloride hexahydrate in water, adjusted to pH 9.4 with 1 M sodium hydroxide solution. Mobile phase B (B) consisted of 65 mM AMPSO, 2 mM magnesium chloride hexahydrate, 500 mM tetramethylammonium chloride in water, adjusted to pH 9.4 with 1 M sodium hydroxide solution.

[0252] The method according to the present invention began with the application of a mixture of 98% (v / v) A and 2% (v / v) B for 1 minute. The concentration of B was then increased from 2% (v / v) to 18% (v / v) over 5 minutes. The concentration of 18% (v / v) B was then maintained for 4 minutes. The concentration of B was then increased from 18% (v / v) to 60% (v / v) over 5 minutes, followed by a 5-minute wash step with 100% (v / v) B and a 5-minute re-equilibration step with 2% (v / v) B.

[0253] The column was maintained at room temperature and a constant flow rate, eg, 0.3 mL / min, was applied. TIFF2025530294000005.tif46153

Claims

1. 1. A method for separating intact and empty recombinant adeno-associated virus particles using an anion exchange chromatography step, comprising: The following series of steps: a) applying a solution containing empty and whole recombinant adeno-associated virus (rAAV) particles to an anion exchange chromatography material in a chromatography column; b) performing a first isocratic step; c) applying a first linear gradient; d) performing a second isocratic step; and e) applying a second linear gradient Including, the empty recombinant adeno-associated virus particles are eluted in the first linear gradient and the complete recombinant adeno-associated virus particles are eluted in the second linear gradient. The method.

2. The method described in claim 1, wherein the solution containing the empty rAAV particles and complete rAAV particles is phosphate-buffered saline containing approximately 0.001% (w / v) of a non-ionic surfactant selected from poloxamer 188 and polysorbate 20.

3. The method of claim 1 or 2, wherein the rAAV particles are of serotype 8.

4. 3. The method of claim 1 or 2, wherein the anion exchange chromatography material has a stationary phase consisting of microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix having quaternary ammonium groups that provide anion exchange functionality.

5. 3. The method of claim 1, wherein the anion exchange chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter, and the anion exchange chromatography material has a particle size of about 10 μm.

6. i) provide a baseline separation of intact and empty rAAV particles; or ii) for quantitation of intact and empty rAAV particles; or iii) a high-throughput method for quantitation of intact and empty rAAV particles; or iv) quality control (QC) methods for the preparation of rAAV; 3. The method according to claim 1 or 2.

7. 3. The method of claim 1, wherein the solutions used in the isocratic steps and the linear gradient contain a buffer substance having a pKa value of about 9.

8. The method according to claim 1 or 2, wherein the solutions used in the isocratic step and the linear gradient contain N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance.

9. 3. The method according to claim 1, wherein the solutions used in the isocratic steps and the linear gradient have a pH value of 9.0 to 9.

6.

10. 3. The method according to claim 1, wherein the solutions used in the isocratic steps and the linear gradient contain an organic chloride having a molecular weight of less than 170 g / mol as an eluting salt.

11. 3. The method of claim 1, wherein the solutions used in the isocratic steps and linear gradients contain tetramethylammonium chloride as an eluting salt.

12. 3. The method of claim 1 or 2, wherein the method is carried out at a flow rate of about 0.3 mL / min.

13. 3. The method of claim 1, wherein the solution applied in the first isocratic step comprises about 65 mM of the buffer substance, about 10 mM of the elution salt, about 2 mM magnesium chloride, and has a pH value of about 9.

4.

14. 3. The method of claim 1, wherein the solution applied in the second isocratic step comprises about 65 mM of the buffer substance, about 90 mM of the elution salt, about 2 mM magnesium chloride, and has a pH value of about 9.

4.

15. 3. The method of claim 1, wherein the first isocratic step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic step has a length of about 2-5 minutes, and the second linear gradient has a length of about 5 minutes.

Images