Method for separating intact and empty AAV particles

Abstract

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

Description

[0001] The present invention belongs to the field of analytical methods. In more detail, herein is reported a method for separating intact recombinant AAV particles from empty recombinant AAV particles using a method comprising an isocratic elution hold. Background Art

[0002] Gene therapy brings unprecedented opportunities for novel treatment methods. Based on the concept of rescuing functional mutations by co-expressing the correct gene, viral vectors are needed to ensure the correct delivery of therapeutic genes in order to restore biological function. In this context, recombinant adeno-associated virus (rAAV) is the most widely used vector. Their biomanufacturing process requires the insertion of therapeutic genes into rAAV (complete AAV particles, i.e., rAAV particles containing encapsidated nucleic acids). However, a certain percentage of rAAV (empty AAV particles, i.e., rAAV particles that do not contain encapsidated nucleic acids) and partially filled rAAV (partially filled AAV particles) that do not contain the desired gene may also be produced, which may affect the efficiency of the therapy. Therefore, it is necessary to monitor the determination of the complete / empty ratio of rAAV particles to ensure consistent product quality and efficacy.

[0003] Various analytical strategies have been proposed to characterize the empty rAAV particle content, including electron microscopy, quantitative polymerase chain reaction (PCR), Elisa assay, UV absorption spectrophotometry, analytical ultracentrifugation, or charge detection mass spectrometry (CDMS) (Gimpel, AL, et al., Mol. Ther.-Methods Clin. Dev. 20 (2021) 740–754; Fu, X., et al., Hum Gene Ther Methods. 30 (2019) 144-152).

[0004] In general, differences in the range of 0.4 pH units could be observed between intact and empty rAAV particles. This feature can be used to attempt to separate and quantify intact and empty rAAV particles by anion exchange chromatography (AEX) (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974–4984; Khatwani, SL, et al., Mol. Ther.-Methods Clin. Dev. 21 (2021) 548–558; Urabe, M., et al., Mol. Ther. 13 (2006) 823–828; Dickerson, R., et al., Biotechnol. J. 16 (2021) 2000015; Kaludov, N., et al., Hum. Gene Ther. 13 (2002) 1235–1243; Lock, M, et al., Hum. Gene Ther. Methods. 23 (2012) 56–64).

[0005] In AEX, rAAV particles follow an “on / off” retention behavior (Snyder, LR, et al., Anal. Chem. 55 (1983) 1412A-1430A; Fekete, S., et al., Anal. Chem. 93 (2021) 1277–1284).

[0006] WO 2021 / 158915 discloses recombinant adeno-associated virus compositions and methods for producing and using the same.

[0007] US2021 / 355503 discloses compositions and methods for making gene therapy vectors.

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

[0009] US2021 / 009964 discloses the separation and quantification of empty viral capsid particles and complete viral capsid particles.

[0010] Dickerson, R., et al. reported the separation of empty recombinant adeno-associated virus particles and complete recombinant adeno-associated virus particles using isocratic elution anion exchange chromatography (Biotechnol J 16(2021)2000015). Summary of the invention

[0011] This article reports an anion exchange chromatography (AEX) method comprising a novel elution scheme. Without being bound by this theory, it is assumed that the method takes advantage of the fact that the viral capsid follows an "on / off" retention behavior. Based on this, an elution method has been found that allows complete rAAV particles to be separated from empty rAAV particles unprecedentedly. The advantage of the method according to the present invention is that, for example, the resolution gain is 3.7 compared to the resolution obtainable using a linear gradient. Using the method according to the present invention, complete rAAV particles and empty rAAV particles can be accurately and accurately baseline separated and quantified, thereby allowing applications, such as, for example, as a quality control (QC) method, among others.

[0012] Accordingly, the present invention encompasses at least the following embodiments:

[0013] 1. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography (AEX) step, wherein the method comprises the following steps:

[0014] a) applying a solution comprising 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),

[0015] b) applying a first buffer solution to the rAAV-loaded AEX chromatography column, whereby the rAAV particles remain bound to the AEX chromatography material, 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, an optional elution salt and an optional salt,

[0016] c) applying a first intermediate buffer solution having an increasing conductivity starting at a first conductivity and ending at a second conductivity to the washed rAAV-loaded AEX chromatography column to obtain an intermediate conditioned rAAV-loaded AEX chromatography column, wherein the increase in conductivity is linear and is increased by adding an elution salt having an increasing concentration,

[0017] d) applying a second buffer solution to the intermediate conditioned rAAV-loaded AEX chromatography column to obtain a half-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,

[0018] e) applying a second intermediate buffer solution having an 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 and is increased by adding an elution salt having an increasing concentration,

[0019] The second buffer solution has a relative strength of about 8 mS / cm to about 10 mS / cm

[0020] Conductivity of 9.5mS / cm

[0021] wherein in step c) or / and d), empty rAAV particles are eluted,

[0022] wherein the applying in step d) lasts for at least 2 minutes,

[0023] wherein in step e), intact rAAV particles are eluted,

[0024] wherein the conductivity is increased in step e) by at least about 20 mS / cm.

[0025] 2. The method of embodiment 1, wherein the rAAV particles are of serotype 8.

[0026] 3. The method according to any one of embodiments 1 to 2, wherein the anion exchange chromatography is strong anion exchange chromatography.

[0027] 4. The method according to any one of embodiments 1 to 3, wherein the anion exchange chromatography material is a stationary phase composed of microporous ethylvinylbenzene and 55% divinylbenzene

[0028] The polymer matrix is ​​cross-linked and has quaternary ammonium groups that provide anion exchange functionality.

[0029] 5. The method according to any one of embodiments 1 to 4, wherein the AEX chromatography column has a size of about 50 mm in length and about 4-5 mm in diameter.

[0030] 6. The method according to any one of embodiments 1 to 5, wherein the AEX chromatography material has a particle size of about 10 μm.

[0031] 7. The method according to any one of embodiments 1 to 6, wherein the method is used for / provides baseline separation of intact rAAV particles and empty rAAV particles.

[0032] 8. The method according to any one of embodiments 1 to 7, wherein the method is used for the quantification of intact rAAV particles and empty rAAV particles.

[0033] 9. The method according to any one of embodiments 1 to 8, wherein the method is a high-throughput method for the quantification of complete rAAV particles and empty rAAV particles rAAV.

[0034] 10. The method of any one of embodiments 1 to 9, wherein the method is a quality control (QC) method for rAAV preparations.

[0035] 11. The method according to any one of embodiments 1 to 10, wherein the solution comprising one or both of empty rAAV particles and intact rAAV particles comprises about 0.001%

[0036] (w / v) buffered solution of non-ionic detergent.

[0037] 12. The method according to any one of embodiments 1 to 11, wherein the solution comprising one or both of empty rAAV particles and intact rAAV particles is a solution comprising about 0.001%

[0038] (w / v) of non-ionic detergent in phosphate buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4).

[0039] 13. The method according to any one of embodiments 1 to 12, wherein the solution comprising one or both of empty rAAV particles and intact rAAV particles is a solution comprising about 0.001%

[0040] (w / v) buffered solution of poloxamer 188 or polysorbate 20.

[0041] 14. The method according to any one of embodiments 1 to 13, wherein the solution comprising one or both of empty rAAV particles and intact rAAV particles is a solution comprising about 0.001%

[0042] (w / v) of poloxamer 188 or polysorbate 20 in phosphate buffered saline (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4).

[0043] 15. The method according to 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.

[0044] 16. The method according to any one of embodiments 1 to 15, wherein the first and second buffer solutions and the first and second intermediate buffer solutions 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.

[0045] 17. The method according to any one of embodiments 1 to 16, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise a pH value of 9.0 to 10.0.

[0046] 18. The method according to any one of embodiments 1 to 17, wherein the first and second buffers

[0047] The solution and the first and second intermediate buffer solutions comprise a pH value of 9.2 to 9.6.

[0048] 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 comprise a pH value of about 9.4.

[0049] 20. The method according to any one of embodiments 1 to 19, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise an organic salt as elution salt.

[0050] 21. The method according to 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 as elution salt.

[0051] 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 contain as elution salts a

[0052] An organic chloride with a molecular weight of 170 g / mol.

[0053] 23. The method according to any one of embodiments 1 to 22, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise tetraethylammonium chloride as elution salt.

[0054] 24. The method according to any one of embodiments 1 to 22, wherein the first and second buffer solutions and the first and second intermediate buffer solutions comprise tetramethylammonium chloride as elution salt.

[0055] 25. The method according to any one of embodiments 1 to 23, wherein the method is carried out at about 0.3

[0056] The flow rate was mL / min.

[0057] 26. The method according to any one of embodiments 1 to 25, wherein the first buffer solution comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4.

[0058] 27. The method according to any one of embodiments 1 to 26, wherein the second buffer solution comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4.

[0059] 28. The method according to any one of embodiments 1 to 27, wherein the buffer substance is N-

[0060] (1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, the eluting salt is tetramethylammonium chloride, and the salt is magnesium chloride.

[0061] 29. The method of any one of embodiments 1 to 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.

[0062] 30. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0063] wherein the method comprises the following series of steps: applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, a first isocratic elution step, a first linear gradient, a second isocratic elution step, and a second linear gradient,

[0064] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0065] in

[0066] i) rAAV particles have serotype 8, or / and

[0067] ii) the anion exchange chromatography material is a strong anion exchange material, or / and

[0068] iii) an anion exchange chromatography material having a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with 55% divinylbenzene polymer matrix, having quaternary ammonium groups providing anion exchange functionality, or / and

[0069] iv) the AEX chromatography column has a size of about 50 mm in length and about 4-5 mm in diameter, or / and

[0070] v) the AEX chromatography material has a particle size of about 10 μm, or / and

[0071] vi) the method is used to / provides baseline separation of intact rAAV particles and empty rAAV particles, or / and

[0072] vii) the method is used for the quantification of intact rAAV particles and empty rAAV particles, or / and viii) the method is a high throughput method for the quantification of intact rAAV particles and empty rAAV particles, or / and

[0073] ix) the method is a quality control (QC) method for rAAV preparations, or / and x) the solution comprising empty and / or intact rAAV particles is a buffer solution comprising about 0.001% (w / v) of a non-ionic detergent, or / and

[0074] xi) the solution containing empty and / or intact rAAV particles is a phosphate buffered saline solution (comprising 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20, or / and

[0075] xii) the solutions used in the isocratic elution step and the linear gradient contain a buffer substance with a pKa value of about 9, or / and

[0076] xiii) the solutions used in the isocratic elution step and the linear gradient 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, or / and

[0077] xiv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.0 to 10.0, or / and

[0078] xv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.2 to 9.6, or / and

[0079] xvi) the solutions used in the isocratic elution step and the linear gradient have a pH of about 9.4, or / and

[0080] xvii) the solutions used in the isocratic elution step and the linear gradient contain an organic salt, or / and xviii) the solutions used in the isocratic elution step and the linear gradient contain an organic chloride as salt, or / and

[0081] xix) the solutions used in the isocratic elution step and the linear gradient contain an organic chloride having a molecular weight below 170 g / mol, or / and

[0082] xx) the solutions used in the isocratic elution step and the linear gradient contain tetraethylammonium chloride, or / and

[0083] xxi) the solutions used in the isocratic elution step and the linear gradient contain tetramethylammonium chloride, or / and

[0084] xxii) the method is carried out at a flow rate of about 0.3 mL / min, or / and

[0085] xxiii) the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0086] xxiv) the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0087] 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

[0088] xxvi) the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0089] 31. A method according to embodiment 30, comprising items i), iii), xi), xiii), xvi), xxi), xxii), xxiv), xxv) or / and xxvi).

[0090] 32. The method according to embodiment 30, comprising items i), iii), xi), xiii), xvi), xxi), xxii), xxiv), xxv) and xxvi).

[0091] 33. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0092] wherein the method comprises the following series of steps: applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, a first isocratic elution step, a first linear gradient, a second isocratic elution step, and a second linear gradient,

[0093] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0094] in

[0095] i) rAAV particles have serotype 8, or / and

[0096] iii) an anion exchange chromatography material having a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with 55% divinylbenzene polymer matrix, having quaternary ammonium groups providing anion exchange functionality, or / and

[0097] iv) the AEX chromatography column has a size of about 50 mm in length and about 4-5 mm in diameter, or / and

[0098] v) the AEX chromatography material has a particle size of about 10 μm, or / and

[0099] x) the solution comprising empty and / or intact rAAV particles is a buffer solution comprising about 0.001% (w / v) of a non-ionic detergent, or / and

[0100] xi) the solution containing empty and / or intact rAAV particles is a phosphate buffered saline solution (comprising 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing about 0.001% (w / v) poloxamer 188 or polysorbate 20, or / and

[0101] xii) the solutions used in the isocratic elution step and the linear gradient contain a buffer substance with a pKa value of about 9, or / and

[0102] xiii) the solutions used in the isocratic elution step and the linear gradient 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, or / and

[0103] xiv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.0 to 10.0, or / and

[0104] xv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.2 to 9.6, or / and

[0105] xvi) the solutions used in the isocratic elution step and the linear gradient have a pH of about 9.4, or / and

[0106] xix) the solutions used in the isocratic elution step and the linear gradient contain an organic chloride having a molecular weight below 170 g / mol, or / and

[0107] xx) the solutions used in the isocratic elution step and the linear gradient contain tetraethylammonium chloride, or / and

[0108] xxi) the solutions used in the isocratic elution step and the linear gradient contain tetramethylammonium chloride, or / and

[0109] xxii) the method is carried out at a flow rate of about 0.3 mL / min, or / and

[0110] xxiii) the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0111] xxiv) the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0112] 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

[0113] xxvi) the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0114] 34. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0115] wherein the method comprises the following series of steps: applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, a first isocratic elution step, a first linear gradient, a second isocratic elution step, and a second linear gradient,

[0116] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0117] in

[0118] i) rAAV particles have serotype 8, or / and

[0119] iii) an anion exchange chromatography material having a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with 55% divinylbenzene polymer matrix, having quaternary ammonium groups providing anion exchange functionality, or / and

[0120] iv) the AEX chromatography column has a size of about 50 mm in length and about 4-5 mm in diameter, or / and

[0121] v) the AEX chromatography material has a particle size of about 10 μm, or / and

[0122] xi) The solution containing empty and / or intact rAAV particles is a phosphate buffered saline solution (containing 137

[0123] mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4), or / and

[0124] xiii) the solutions used in the isocratic elution step and the linear gradient 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, or / and

[0125] xiv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.0 to 10.0, or / and

[0126] xv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.2 to 9.6, or / and

[0127] xvi) the solutions used in the isocratic elution step and the linear gradient have a pH of about 9.4, or / and

[0128] xix) The solutions used in the isocratic elution step and the linear gradient contain a

[0129] g / mol molecular weight of organic chloride, or / and

[0130] xxi) the solutions used in the isocratic elution step and the linear gradient contain tetramethylammonium chloride, or / and

[0131] xxii) the method is carried out at a flow rate of about 0.3 mL / min, or / and

[0132] xxiii) the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0133] xxiv) the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0134] 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

[0135] xxvi) the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0136] 35. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0137] wherein the method comprises the following series of steps: applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, a first isocratic elution step, a first linear gradient, a second isocratic elution step, and a second linear gradient,

[0138] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0139] in

[0140] i) rAAV particles have serotype 8, or / and

[0141] iii) an anion exchange chromatography material having a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with 55% divinylbenzene polymer matrix, having quaternary ammonium groups providing anion exchange functionality, or / and

[0142] iv) the AEX chromatography column has a size of about 50 mm in length and about 4-5 mm in diameter, or / and

[0143] v) the AEX chromatography material has a particle size of about 10 μm, or / and

[0144] xi) the solution containing the empty and / or intact rAAV particles is a phosphate-buffered saline solution (comprising 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing about 0.001% (w / v) of poloxamer 188, or / and xiii) the solution used in the isocratic elution step and the linear gradient contains N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance, or / and xiv) the solution used in the isocratic elution step and the linear gradient has a pH value of 9.0 to 10.0, or / and

[0145] xv) the solutions used in the isocratic elution step and the linear gradient have a pH value of 9.2 to 9.6, or / and

[0146] xvi) the solutions used in the isocratic elution step and the linear gradient have a pH of about 9.4, or / and

[0147] xxi) the solutions used in the isocratic elution step and the linear gradient contain tetramethylammonium chloride, or / and

[0148] xxii) the method is carried out at a flow rate of about 0.3 mL / min, or / and

[0149] xxiii) the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0150] xxiv) the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4, or / and

[0151] 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

[0152] xxvi) the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0153] 36. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0154] wherein the method comprises the following series of steps: applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, a first isocratic elution step, a first linear gradient, a second isocratic elution step, and a second linear gradient, wherein the empty rAAV particles are eluted during the first linear gradient and the intact rAAV particles are eluted during the second linear gradient,

[0155] wherein the rAAV particles are of serotype 8, or

[0156] in

[0157] i) an anion exchange chromatography material having a stationary phase composed of a microporous ethylvinylbenzene cross-linked with 55% divinylbenzene polymer matrix, having quaternary ammonium groups providing anion exchange functionality, and

[0158] ii) the AEX chromatography column has dimensions of about 50 mm in length and about 4-5 mm in diameter, and

[0159] iii) the AEX chromatography material has a particle size of about 10 μm, and

[0160] iv) The solution containing empty and / or intact rAAV particles is a phosphate buffered saline solution (containing 137 mM NaCl, 2.7

[0161] mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), and

[0162] v) The solutions used in the isocratic elution step and the linear gradient contain N-

[0163] (1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, and

[0164] vi) the solutions used in the isocratic elution step and the linear gradient have a pH of about 9.4, and

[0165] vii) the solutions used in the isocratic elution step and the linear gradient comprise tetramethylammonium chloride, and

[0166] viii) the method is carried out at a flow rate of about 0.3 mL / min, and

[0167] ix) the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM salt and has a pH value of about 9.4, and

[0168] and

[0169] x) the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM salt and has a pH value of about 9.4, and

[0170] 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, and

[0171] xii) the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0172] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of 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 disclosed subject matter, so that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 Empty (red line), intact (blue line), and a mixture of intact and empty rAAV8 particles (purple line) were analyzed using the method according to Example 2 using 4 different column hardware (i.e., PP (A), PS (B), AX (C), and QS (D)); E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty rAAV particle ratio.

[0174] Figure 2 Results using different buffer types. Empty (red line), intact (blue line) and a mixture of intact and empty rAAV8 particles (purple line) were analyzed in AEX mode using five different buffer types (i.e., AMPD, BTP, AMPSO, CHES and CAPSO) with an elution method according to Example 2; E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio, na = not applicable.

[0175] Figure 3 Results using AMPSO buffer at different pH values. Empty (red line), intact (blue line) and a mixture of intact and empty rAAV8 particles (purple line) were analyzed in AEX mode at five different pH values ​​(i.e., 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 applicable.

[0176] Figure 4 Results using AMPSO buffer at pH 9.4 and different elution salts. Empty (red line), intact (blue line) and a mixture of intact and empty rAAV8 particles (purple line) were analyzed in AEX mode using the elution method according to Example 2 using three different elution salts; E = empty rAAV8 particles, F = intact rAAV8 particles, Rs = peak resolution, F / E = intact / empty ratio.

[0177] Figure 5 Results at different mobile phase flow rates using AMPSO buffer at pH 9.4 and TMAC as elution salt. A mixture of intact rAAV8 particles and empty rAAV8 particles was analyzed in AEX mode using the elution method according to Example 2 using three different flow rates, namely 0.7 mL / min (grey line), 0.3 mL / min (green line) and 0.1 mL / min (orange line).

[0178] Figure 6 . Overlay of chromatograms using different isocratic elution conditions. A mixture of intact and empty rAAV8 particles was analyzed in AEX mode by using AMPSO buffer pH 9.4 in combination with TMAC as elution salt and the elution pattern according to the invention was maintained with a 4 min isocratic elution step (red line) at different % (v / v) B, i.e. 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.

[0179] Figure 7 . Overlay of chromatograms using different durations of the second isocratic elution step. A mixture of intact rAAV8 particles and empty rAAV8 particles was analyzed in AEX mode by using AMPSO buffer pH 9.4 in combination with TMAC as elution salt, and the elution mode according to the invention included isocratic elution steps maintained at 18% (v / v) B for 4 min (blue line), 6 min (grey line), 8 min (green line) and 10 min (orange line); E = empty rAAV8 particles, F = intact rAAV8 particles.

[0180] Figure 8 Empty (red line), intact (blue line) and a mixture of intact and empty rAAV8 particles (purple line) were analyzed in AEX mode according to Example 2 (comparison) (A), according to Example 3 (comparison) (B) and according to Example 4 (according to the present invention) by using three different elution strategies; E=empty rAAV8 particles, F=intact rAAV8 particles, Rs=peak resolution, F / E=intact / empty ratio.

[0181] Figure 9. Quantification of the percentage of intact particles in different mixtures of empty and intact rAAV8 particles obtained from two different suppliers, namely Virovek (AB) and Sirion (CD). The elution method according to the invention was applied together with a second isocratic elution step at 18.5% (v / v) B for Sirion samples and 18% (v / v) B for Virovek samples; E = empty rAAV8 particles, F = intact rAAV8 particles, F / E = intact / empty ratio. DETAILED DESCRIPTION

[0182] definition

[0183] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" may be used interchangeably.

[0184] The term "about" means a range of + / -20% of the value that follows. In certain embodiments, the term "about" means a range of + / -10% of the value that follows. In certain embodiments, the term "about" means a range of + / -5% of the value that follows.

[0185] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "comprising" also encompasses the term "including..." The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly set forth.

[0186] The terms "empty particles" and "empty recombinant AAV particles" used interchangeably refer to AAV particles (i.e., vectors) that have an AAV protein capsid but lack, in whole or in part, nucleic acids encoding proteins or transcribed into target transcripts flanked by AAV ITRs. Therefore, empty particles cannot function to transfer nucleic acids encoding proteins or transcribed into target transcripts into target cells.

[0187] The term "endogenous" means that something occurs naturally within a cell; is naturally produced by the cell; similarly, an "endogenous locus / cell endogenous locus" is a locus that occurs naturally in a cell.

[0188] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a specific cell, but is introduced into the cell by a DNA delivery method (e.g., by transfection, electroporation, or transduction by a viral vector). Therefore, an exogenous nucleotide sequence is an artificial sequence, wherein the artificiality can be derived from, for example, a combination of subsequences from different sources (e.g., a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence for a green fluorescent protein is an artificial nucleic acid) or from a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or a cDNA) or a partial deletion, or a nuclear base mutation. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence can have an "endogenous" counterpart with the same base composition, but wherein the sequence becomes an "exogenous" sequence, for example, by being introduced into a cell via recombinant DNA technology.

[0189] The terms "intact particle" and "intact recombinant AAV particle" are used interchangeably to refer to an AAV particle (i.e., a vector) having an AAV protein capsid and having packaged therein a nucleic acid encoding a protein or transcribed into a target transcript flanked by AAV ITRs. Thus, the intact particle is capable of transferring the packaged nucleic acid encoding a protein or transcribed into a target transcript into a target cell.

[0190] The terms "intact particle / empty particle ratio" and "ratio of intact recombinant AAV particles to empty recombinant AAV particles", which are used interchangeably, refer to the mathematical ratio of the number of intact recombinant AAV particles to the total number of recombinant AAV particles (intact and empty) in a sample containing recombinant AAV particles or in a recombinant AAV particle preparation. Since the number of intact recombinant AAV particles can be at most the same as the total number of recombinant AAV particles, the ratio can be at most 1. In general, the ratio is less than 1 and is expressed as a percentage. The number of intact recombinant AAV particles is determined by determining the amount of nucleic acid packaged by the recombinant AAV particles in the sample or preparation. This can be done by PCR, in particular digital droplet PCR (ddPCR). The total number of recombinant AAV particles is determined by determining the amount of capsid protein in the sample or preparation. This can be done by ELISA, in particular by a capsid protein-specific ELISA.

[0191] "Recombinant AAV vectors" are derived from the wild-type genome of a virus (such as AAV) by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace the wild-type genome with a non-native nucleic acid (such as a nucleic acid transcribed into a transcript or encoding a protein). For AAV, one or two inverted terminal repeat (ITR) sequences of the wild-type AAV genome are usually retained in the recombinant AAV vector. "Recombinant" AAV vectors are distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-native (i.e., heterologous) sequence relative to the viral genomic nucleic acid. Therefore, the incorporation of non-native sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as a "rAAV vector."

[0192] Recombinant vector (e.g., AAV) sequences can be packaged - referred to herein as "particles" - for subsequent cell infection (transduction) ex vivo, in vitro or in vivo. When recombinant vector sequences are encapsulated or packaged into AAV particles, the particles may also be referred to as "rAAV". Such particles include 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.

[0193] As used herein, the term "serotype" is a distinction based on serologically different AAV capsids. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference AAV serotype or other AAV serotypes.

[0194] Under the traditional definition, serotype refers to the target virus that has been tested for serum, which has specificity for neutralizing activity to all existing and characterized serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may or may not be serological differences with any of the currently existing serotypes. Therefore, in the case where a new virus (such as AAV) does not have serological differences, the new virus (such as AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on mutant viruses with modified capsid sequences to determine whether they have another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" refers broadly to serologically different viruses (such as AAV) and serologically no different viruses (such as AAV), which may be within a subgroup or variant of a given serotype.

[0195] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated directly or in the form of a single strand or RNA to form a virus (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture a recombinant virus particle, the virus particle does not include a "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but it itself is not packaged or encapsulated into a virus (e.g., AAV) particle. Therefore, "vector" refers to a nucleic acid packaged or encapsulated by a virus particle (e.g., AAV).

[0196] Recombinant cells

[0197] Generally speaking, for efficient and large-scale production of recombinant AAV particles (rAAV particles), cells express and, if possible, also secrete the rAAV particles. Such cells are referred to as "recombinant cells" or "recombinant production cells."

[0198] To generate "recombinant producer cells," appropriate mammalian cells are transfected with the nucleic acid sequences required for production of the rAAV particles (including the desired AAV helper functions).

[0199] For expressing coding sequence, i.e. open reading frame, additional regulatory elements are needed, such as promoter and polyadenylation signal (sequence). Therefore, open reading frame is operably connected to the additional regulatory elements for transcription. This can be achieved by being integrated into the so-called expression cassette. The minimum regulatory elements required for the expression cassette to work in mammalian cells are the promoter working in the mammalian cell, the promoter is located at the upstream of the open reading frame, i.e. 5', and the polyadenylation signal (sequence) working in the mammalian cell, the polyadenylation signal is located at the downstream of the open reading frame, i.e. 3'. In addition, the terminator sequence may be present in the 3' of the polyadenylation signal (sequence). For expression, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in an operably connected form.

[0200] Similarly, nucleic acids that are transcribed into RNA encoding non-proteins are referred to as "RNA genes". Also for the expression of RNA genes, additional regulatory elements are required, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and positioning of such elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are also typically integrated into expression cassettes.

[0201] In the case of AAV particles, which consist of different (monomeric) capsid polypeptides and single-stranded DNA molecules and also require other adenoviral helper functions for production and packaging, a number of expression cassettes are required, which contain different open reading frames / coding sequences. In this case, at least an expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, the required helper functions and the VA RNA. Thus, a separate expression cassette is required for each of the helper functions E1A, E1B, E2A, E4orf6, VA RNA, rep and cap genes. HEK293 cells constitutively express the E1A and E1B helper functions.

[0202] Adeno-associated virus (AAV)

[0203] For a general review of AAV and adenovirus or herpes helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Additional 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. The construction of recombinant AAV vectors is described in the following literature: US 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.

[0204] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can only replicate in cells where certain viral functions are provided by a co-infecting helper virus, such as adenovirus, herpes virus, and in some cases poxvirus, such as vaccinia. Nevertheless, AAV can replicate in almost any cell line of human, simian, or rodent origin, provided that the appropriate helper virus functions are present.

[0205] If helper viral genes are not present, AAV establishes latency in its host cells. Its genome integrates into a specific site in chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV-2, other integration sites have been found, such as, for example, on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0206] AAV is divided into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than a hundred sequences corresponding to different branches of AAV have been identified.

[0207] The capsid protein type and symmetry determine the tissue tropism of the corresponding AAV. For example, AAV-2, AAV-4, and AAV-5 are specific for the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific for the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific for cardiac tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific for the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific for the lung.

[0208] Pseudotyping refers to a process that involves cross-packaging of the AAV genome between different serotypes, i.e., the genome is packaged with capsid proteins of different origin.

[0209] The wild-type AAV genome size is about 4.7 kb. The AAV genome further comprises 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 open reading frame encoding the Rep protein provides four proteins of different sizes, which are called Rep78, Rep68, Rep52 and Rep40. These are involved in the replication, rescue and integration of AAV. The open reading frame encoding the Cap protein provides four proteins, called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein capsid of the AAV particle. The combined rep and cap open reading frames are flanked by so-called inverted terminal repeats (ITRs) at their 5' and 3' ends. 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.

[0210] For example, in the case of AAV of serotype 2 (AAV-2), each ITR has a length of 145 nucleotides and is flanked by a coding sequence region of about 4470 nucleotides. Of the 145 nucleotides of 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 non-paired nucleotides are represented as D sequences.

[0211] The AAV genome contains three transcription promoters P5, P19 and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of rep and cap genes.

[0212] ITR sequences must be present in the coding region in cis. ITR provides a functional origin of replication (ori), the signal required for integration into the target cell genome, and effective excision and rescue from host cell chromosomes or recombinant plasmids. ITR further comprises an origin of replication-like element, such as a Rep protein binding site (RBS) and a terminal resolution site (TRS). It has been found that ITR itself can have the function of a transcription promoter in an AAV vector (Flotte et al., J.Biol.Chem.268 (1993) 3781-3790; Flotte et al., Proc.Natl.Acad.Sci.USA 93 (1993) 10163-10167).

[0213] The trans organization of the rep and cap gene products is required for the replication and encapsidation of the viral single-stranded DNA genome, respectively.

[0214] The rep locus comprises two internal promoters, called P5 and P19. It comprises open reading frames for four proteins. Promoter P5 is operably connected to a nucleic acid sequence that provides a non-spliced ​​4.2 kb mRNA encoding Rep protein Rep78 (chromatin nickase that blocks the cell cycle) and a spliced ​​3.9 kb mRNA encoding Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably connected to a nucleic acid sequence that provides a non-spliced ​​mRNA encoding Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding Rep protein Rep40 (DNA helicase for accumulation and packaging).

[0215] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex 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).

[0216] The larger Rep proteins Rep68 and Rep78 can specifically bind to the hairpin conformation of AAV ITR. They exhibit defined enzymatic activity, which is required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for the formation of infectious particles (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0217] All Rep proteins, primarily Rep78 and Rep68, are believed to exhibit regulatory activities such as induction and repression of AAV genes and inhibition of cell growth (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).

[0218] Recombinant overexpression of Rep78 produces a phenotype of reduced cell growth caused by inducing DNA damage, thereby arresting host cells in the S phase, thereby promoting latent infection of the virus (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0219] Tratschin et al. 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, it has been reported that after stable integration of AAV, expression for some cell lines is only very low (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0220] The cap locus contains a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence that provides 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, non-spliced ​​mRNA transcripts), VP2 (72 kDa, from spliced ​​mRNA transcripts) and VP3 (61 kDa, from alternative start codons) by alternative splicing and using alternative start codons. VP1 to VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for about 90% of the total protein of the viral particle. However, all three proteins are essential for effective capsid production.

[0221] Inactivation of all three capsid proteins VP1 to VP3 reportedly prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 (“Lip negative” or “Inf negative”) still allow single-stranded DNA to assemble into viral particles, thereby greatly reducing infectious titers.

[0222] The AAP open reading frame encodes the assembly activation protein (AAP). It is about 22 kDa in size and transports native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0223] A single AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" strand or the "minus" strand. AAV virus particles containing DNA molecules are infectious. Within infected cells, the parental infectious single strand is converted to a double strand and subsequently amplified. Amplification produces a large number of double-stranded DNA molecules, which displace the single strand and are packaged into capsids.

[0224] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long term. One disadvantage of using AAV vectors is that the size of the transgene that can be introduced into cells is limited.

[0225] Viral vectors such as parvovirus particles, including AAV serotypes and variants thereof, provide a way to deliver nucleic acids to protein-encoding cells in vitro, in vitro, and in vivo, so that the cells express the encoded protein. AAV is a virus that can be used as a gene therapy vector because they can penetrate cells and introduce nucleic acids / genetic material so that nucleic acids / genetic material can be stably maintained in cells. In addition, for example, these viruses can introduce nucleic acids / genetic material into specific sites. Since AAV is not related to human pathogenic diseases, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0226] AAV particles used as vehicles for efficient gene delivery have many characteristics required for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also showed no persistent toxicity and little or no detectable immune response. AAV is known to infect a variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0227] Recombinant AAV particles generally do not include viral genes associated with pathogenesis. Such vectors generally have one or more of the wild-type AAV genes that are completely or partially deleted, such as rep and / or cap genes, but retain at least one functional flanking ITR sequence, which is necessary for rescuing, replicating, and packaging the recombinant vector into AAV particles. For example, only the essential parts of the vector are included, such as ITR and LTR elements, respectively. Therefore, the AAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0228] Recombinant AAV vectors and methods and uses thereof include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as, for example, 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 different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be the same as one or more of the capsid proteins that package the vector. In addition, the recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, and at least one of the three capsid proteins can be 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0229] 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, such as amino acid insertions, additions, substitutions and deletions), for example, as shown in WO 2013 / 158879, WO 2015 / 013313 and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0230] In certain embodiments of all aspects and embodiments of the invention, the rAAV particle comprises a capsid sequence having 70% or more sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74 or 7m8 capsid sequence.

[0231] In certain embodiments of all aspects and embodiments of the invention, the rAAV particles comprise an ITR sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0232] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any medicament that does not itself induce an immune response that is harmful to the individual receiving the composition and can be administered without excessive toxicity.

[0233] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, the entire contents of which are incorporated herein by reference.

[0234] Recombinant AAV particles (rAAV particles)

[0235] Different methods for generating rAAV particles are known in the art. For example, transfection using AAV plasmids and AAV auxiliary sequences is combined with co-infection with an AAV helper virus (e.g., adenovirus, herpes virus or vaccinia virus), or transfection using recombinant AAV plasmids, AAV helper plasmids and auxiliary function plasmids. Non-limiting methods for generating rAAV particles are described in, for example, US 6,001,650, US 6,004,797, WO 2017 / 096039 and WO 2018 / 226887. After recombinant rAAV particle production (i.e., generating particles in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatants and purified.

[0236] For the production of recombinant AAV particles, it is necessary to express the Rep and Cap proteins, the accessory proteins E1A, E1B, E2A and E4orf6, and the adenovirus VA RNA in a single mammalian cell. The accessory proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Therefore, any promoter can be used.

[0237] In general, in order to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of these plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deleted AAV elements (i.e., open reading frames for Rep and Cap proteins) required for offspring recombinant genome replication and subsequent packaging are contained in the second plasmid in trans. Overexpression of Rep protein causes an inhibitory effect on cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing genes of helper viruses (i.e., E1, E4orf6, E2A and VA from adenovirus).

[0238] To reduce the number of plasmids required, Rep, Cap, and adenoviral helper genes can be combined on a single plasmid.

[0239] Alternatively, the host cell may have stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, denoted 293, was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (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. This covers E1A and E1B genes and adenovirus packaging signals (Louis, N. et al., Virology 233 (1997) 423-429).

[0240] When HEK293 cells are used, the deleted E2A, E4orf6 and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6 and VA (see, 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. 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).

[0241] To restrict transgene activity to specific tissues, ie, to limit integration sites, the transgene can be operably linked to an inducible or tissue-specific promoter (see, eg, Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0242] E1A, E1B, E2 and E4

[0243] The coding sequences (open reading frames) of E1A and E1B may be derived from human adenoviruses, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and the sequence of an exemplary human Ad2 is found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 as reported in EP 1 230 354 and plasmids pGS119 and pGS122 as reported in WO2007 / 056994 may also be used as sources of E1A and E1B open reading frames.

[0244] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. The expression of 12S and 13S proteins causes the activation of other viral functions E1B, E2, E3 and E4. In addition, the expression of 12S and 13S proteins forces the cell to enter the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell will die (apoptosis).

[0245] E1B is the second viral helper gene expressed. It is activated by E1A-derived proteins 12S and 13S. The mRNA derived from the E1B gene can be spliced ​​in two different ways, resulting in a first 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in regulating the cell cycle, blocking mRNA trafficking in late infected cells, and preventing E1A-induced apoptosis. The E1B 19kDa protein is involved in preventing E1A-induced apoptosis.

[0246] The E2 genes encode different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication.

[0247] The E4 gene also encodes several proteins. The 34 kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55 kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0248] Adenovirus VA RNA

[0249] Virus-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed from a type 2 polymerase III promoter by RNA polymerase III (see, 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.

[0250] The structure, function and evolution of adenovirus-associated RNA were studied using a phylogenetic approach by the following literature: Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099) used a phylogenetic approach to study the structure, function and evolution of adenovirus-associated RNA. They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence. The disclosure is hereby incorporated by reference into this application in its entirety.

[0251] VA RNA, VAI and VAII consist of 157 to 160 nucleotides (nt).

[0252] Depending on the serotype, adenovirus contains one or two VA RNA genes. VA RNAI is thought to play a major 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).

[0253] VA RNA is not essential, but plays an important role in efficient viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, adenoviruses lacking VA RNA fail to grow during the initial steps of vector generation, where only a few copies of the viral genome are present per cell, probably because viral genes other than VARNA that block cellular antiviral mechanisms may not be adequately expressed (see Maekawa, A. et al. Nature Sci. Rep. 3 (2013) 1136).

[0254] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral 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-deficient adenoviruses through FLP recombinase-mediated excision of the VA RNA locus.

[0255] Human adenovirus 2VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0256] Methods for producing rAAV particles

[0257] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with transgenes (Carter, BJ, "Handbook of Parvoviruses", P. Tijssen ed., CRC Press, pp. 155-168 (1990)). In addition, it is reported that ITRs must be maintained to retain the functions of transgene replication, rescue, packaging and integration into the target cell genome.

[0258] When cells containing the corresponding viral helper genes are transduced with AAV vectors, or vice versa, when cells containing integrated AAV provirus are transduced with appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0259] The production cells contain rep and cap gene sequences, and a transgenic cassette flanked by ITR sequences on one or more plasmids retained by drug selection. The production of rAAV particles in these cell lines generally occurs after they have been infected with the required helper functions. Therefore, the cells are infected with a replication-competent AdV (usually wild-type Ad5) or a plasmid containing the corresponding helper genes to provide helper viral proteins and initiate rAAV particle production. The packaging cell line is different from the production cell line because it only contains the rep and cap genes.

[0260] More generally, cells transfected or transduced with DNA for recombinant production of AAV particles can be referred to as "recombinant cells". Such cells may be any mammalian cell that has been used as a receptor for nucleic acids (plasmids) encoding packaging proteins (such as AAV packaging proteins), nucleic acids (plasmids) encoding auxiliary proteins, and nucleic acids (plasmids) encoding proteins or transcribed into target transcripts (i.e., transgenes placed between two AAVITRs). The term includes the descendants of the original cells that have been transduced or transfected. It should be understood that the descendants of a single parent cell may not necessarily be exactly the same as the original parent in morphology or genome or total nucleic acid complement due to natural, accidental or deliberate mutations.

[0261] Many cell growth mediums suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. The example of such culture medium includes serum-free eukaryotic growth medium, such as for maintaining mammalian (e.g., human) cell viability or providing mammalian (e.g., human) cell growth medium. Non-limiting examples include Ham's F12 or F12K culture medium (Sigma-Aldrich), FreeStyle (FS) F17 culture medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. Such culture medium 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.

[0262] To produce rAAV particles, three plasmids are co-transfected into mammalian cells. The transgenic plasmid encodes an expression cassette cloned between the AAV ITRs, while the rep and cap genes are provided in trans by co-transfection of the second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also called the helper plasmid, contains minimal helper viral factors (usually adenovirus E2A, EV, and VA genes) but lacks the AAV ITRs.

[0263] The present invention relates to a method for transferring DNA to mammalian cells. In some embodiments of all aspects and embodiments, electroporation, nuclear transfection or microinjection for nucleic acid transfer / transfection are used. In some embodiments of all aspects and embodiments, inorganic substances (such as calcium phosphate / DNA coprecipitation), cationic polymers (such as polyethyleneimine, DEAE-dextran) or cationic lipids (lipofection) for nucleic acid transfer / transfection are used. Calcium phosphate and polyethyleneimine are the most commonly used reagents (see, for example, Baldi et al., Biotechnol.Lett.29 (2007) 677-684) for the transfection of larger-scale nucleic acid transfer, wherein polyethyleneimine is preferred.

[0264] Growing in serum-free suspension culture and using PEI as the transfection reagent improves the efficiency and reproducibility of transfection conditions, allowing real-time scale-up of AAV production using shake flasks, wave-type, or stirred tank bioreactors.

[0265] The composition may further comprise plasmids or / and cells. Such plasmids and cells may be contacted with free PEI.

[0266] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid and inhibits histone deacetylase activity. For this reason, it is often added to mammalian cell cultures as an enhancer for recombinant protein production.

[0267] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0268] In certain embodiments of all aspects and embodiments, the encoded accessory proteins include adenoviral E1A and E1B, adenoviral E2 and / or E4, VA RNA and / or non-AAV accessory proteins.

[0269] The usual conditions for culturing eukaryotic cells at about 37° C., 95% humidity and 8 vol.-% CO2 can be used for culturing. Cultivation can be carried out in serum-containing or serum-free medium, adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel, such as, for example, in a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaking container or a rotating container or a so-called roller bottle. Transfection can be carried out in a high-throughput format and screening, for example, in a 96 or 384-well format, respectively.

[0270] The methods according to the invention may include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any one of the following: AAV serotypes 1 to 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 an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0271] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0272] The ITR may be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. The AAV particles may include any VP1, VP2 and / or VP3 capsid proteins having 75% or more sequence identity with any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 and AAV 7m8 AAV serotypes.

[0273] After producing recombinant virus (e.g., AAV) particles, if desired, various conventional methods can be used to purify and / or isolate virus (e.g., rAAV) particles from host cells. Such methods include column chromatography, CsCl gradients, iodixanol gradients, etc.

[0274] For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column. (See, for example, WO 02 / 12455 and US 2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US2012 / 0135515; and US2013 / 0072548). In addition, if infectious viruses are used to express packaging proteins and / or auxiliary proteins, various methods can be used to inactivate residual viruses. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer. This treatment effectively inactivates the helper virus because AAV is heat stable, while the helper adenovirus is heat unstable.

[0275] One goal of rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated by AAV.

[0276] Considering that rAAV particles account for only a small part of the biomass, rAAV particles need to be purified to a purity level that can be used as a clinical human gene therapy product (see, for example, Smith PH et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0277] As an initial step, cultured cells producing rAAV particles are typically harvested and optionally combined with harvested cell culture supernatant (culture medium) in which cells (suspension cells or adherent cells) producing rAAV particles have been cultured. The harvested cells and optional cell culture supernatant can be used as is, lysed or concentrated as appropriate. In addition, if infection is used to express auxiliary functions, residual helper viruses can be inactivated. For example, adenoviruses can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer, which only inactivates the helper virus because AAV is heat stable, while helper adenoviruses are heat unstable.

[0278] By lysing the cells in the culture broth harvested according to the method of the present invention, to release rAAV particles. During cell lysis or after cell lysis, a nuclease (such as, for example, benzonase) is added to degrade the contaminated DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation to obtain a clarified cell lysate. In a specific example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1-10.0 μm pore size filter, for example, a 0.45 μm and / or pore size 0.2 μm filter) to produce a clarified lysate.

[0279] The lysate (optionally clarified) contains AAV particles (including rAAV vectors as well as empty capsids) and production / process-related impurities, such as soluble cellular components from host cells, which may include cell proteins, lipids and / or nucleic acids, and cell culture medium components, among others. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including rAAV vectors) from the impurities using chromatography. Prior to the first chromatography step, the clarified lysate may be diluted or concentrated with an appropriate buffer.

[0280] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent and sequential chromatography steps may be used to purify the rAAV particles.

[0281] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0282] If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography in any order or sequence or combination.

[0283] For example, during downstream processing, empty capsids are removed from intact capsids based on their different isoelectric points (pi) in anion exchange chromatography. The average pI calculated for all serotypes is 5.9 for intact capsids and 6.3 for empty capsids (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984).

[0284] The role of cation exchange chromatography is to separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of a sulfonate functional group, 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 ThermoFisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto SImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial preparations of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). and Family. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphate resins (based on phosphate functional groups), methanesulfonate (S) resins, and sulfopropyl (SP) resins.

[0285] The effect of anion exchange chromatography is to separate AAV particles from proteins, cells and other components present in the lysate and / or column eluate of the clarification from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thus control the amount of empty capsids in the eluate. For example, the anion exchange column with the rAAV particles combined therewith can be washed with a solution of NaCl comprising a moderate concentration (for example, about 100mM to 125mM, such as 110mM to 115mM), and a portion of empty capsids can be eluted when flowing through without eluting rAAV particles in large quantities. Subsequently, the rAAV particles bonded to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (for example, about 130mM to 300mM NaCl), thereby producing the column eluate of the rAAV particles comprising rAAV vectors with empty capsids reduced or depleted and proportionally increased amounts.

[0286] 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 (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchangers available from GE Healthcare). Healthcare, Marlborough, MA, 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). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0287] Manufacturing processes for purifying recombinant AAV particles intended as products for treating human disease should achieve the following goals: 1) consistent particle purity, potency, and safety; 2) manufacturing process scalability; and 3) acceptable manufacturing costs.

[0288] WO 2019 / 006390 reports an exemplary process for purification of recombinant AAV particles.

[0289] 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 determining 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).

[0290] In order to determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis, which consists of any gel (e.g., gradient gel) capable of separating the three capsid proteins, and then running the gel until the sample is separated and blotted onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds the primary antibody contains a tool for detecting the primary antibody. Semi-quantitative detection of the binding between the primary and secondary antibodies determines the amount of capsids. Another method is to use an analytical HPLC or analytical ultracentrifuge using a SEC column.

[0291] Method according to the invention

[0292] Twelve different AAV serotypes are known and are characterized by specific target tissue tropisms.

[0293] AAV is composed of a protein capsid containing a viral genome. The capsid is composed of about 60 copies of three viral proteins VP1, VP2, and VP3 (respectively in a ratio of 1: 1: 10), assembled into an icosahedron with a diameter of 26 nm. The genome consists of a single-stranded DNA of about 4.7 kb. The structure of the genome is relatively simple because it consists of three genes flanked by two reverse terminal repeats (ITRs) at the ends (Straus, SE, et al., Proc. Natl. Acad. Sci. 73 (1976) 742–746). The first gene is the Rep gene, which encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are produced by the same sequence but by different promoters and by alternative splicing (Dismuke, D., et al., Curr. Gene Ther. 13 (2014) 434–452). They can be used to target viral integration, viral replication, transcription, and packaging of viral DNA into viral capsids (Dismuke, D., et al., Curr. Gene Ther. 13 (2014) 434–452). The second gene is the Cap gene, which encodes three viral capsid proteins (VP1, VP2, and VP3), which are different due to translation and alternative splicing of different start codons, despite the same gene sequence. The third gene encodes assembly activation 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).

[0294] Given its simplicity, the wild-type AAV genome can be easily engineered and replaced by an artificial AAV genome, thereby generating an artificial recombinant AAV (rAAV) lacking viral DNA encoding viral proteins (Rep and Cap). In fact, the ITR sequence of the viral genome is retained to maintain transcriptional activity, while the rest of the viral sequence is replaced by an expression cassette containing a therapeutic gene, which is called a transgene (Naso, MF, et al., BioDrugs.31 (2017) 317-334). Transgenes allow the expression of desired therapeutic entities, such as, for example, polypeptides or proteins, for example, proteins that may be missing or non-functional proteins associated with specific pathological states (Maguire, AM, et al., Mol.Ther.29 (2021) 442–463).

[0295] During the production of rAAV particles, the artificial AAV genome may not be properly integrated into the AAV capsid. A mixture of empty rAAV particles and complete rAAV particles is thus produced. The proportion of empty rAAV particles may correspond to 10% to 90% of the total rAAV particles (Flotte, TR, Hum. Gene Ther. 28 (2017) 147–148). Therefore, empty rAAV particles and partially filled rAAV particles are product-related impurities that may reduce the effective concentration of the final drug, compete for binding sites, and reduce the efficacy of the drug (Gao, K., et al., Mol. Ther.-Methods Clin. Dev. 1 (2014) 9). According to the FDA, these impurities need to be monitored and reported in a complete / empty (F / E) ratio (FDA Industry Guide: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), (2020)).

[0296] Due to their similarities, empty rAAV particles are difficult to avoid or eliminate (Schnoedt, M. and Buening, H., Hum. Gene Ther. Methods. 28 (2017) 101-108). Complete rAAV particles and empty rAAV particles have the same size. However, since complete rAAV particles are loaded with negatively charged DNA, they have a slightly lower pI than empty rAAV particles. In general, differences in the range of 0.4 pH units can be observed. This feature is the basis for separating and quantifying complete rAAV particles and empty rAAV particles in the method according to the present invention.

[0297] The present invention is based at least in part on the discovery that the combination of a linear gradient with an isocratic elution step allows for an unprecedented separation of intact / empty rAAV particles, in a preferred embodiment rAAV particles of serotype 8 (rAAV8). The method according to the invention allows for a precise and accurate baseline separation and quantification of intact and empty rAAV particles. One application of the method according to the invention is a high throughput method or / quality control (QC) method for rAAV.

[0298] The following is presented using recombinant AAV particles with capsids of serotype 8. This is presented only to illustrate the invention and should not be construed as limiting the general applicability of the method according to the invention. The true scope is set out in the appended claims.

[0299] Four strong anion exchange adsorbents, all functionalized with positively charged quaternary groups and differing in matrix composition (monolithic or nonporous beads), were tested for their efficacy in resolving empty and intact rAAV8 particles.

[0300] The column materials are stainless steel, PEEK or PEEK lined stainless steel (SS), which should not have an effect on the separation characteristics. PEEK stands for polyetheretherketone. PEEK tubing has become a standard in the operation of many HPLC systems and exhibits high column strength, as well as high tensile strength and high bending modulus, making it very suitable for catheter and other tubing applications that require good torque response and pushability. Extruded PEEK tubing is used in medical applications where extremely high rigidity is required.

[0301] Table 1. List of chromatographic columns and their characteristics (chemical composition: all columns have quaternary positively charged N-based functional groups). PEEK and SS represent polyetheretherketone and stainless steel, respectively.

[0302]

[0303] To work within the column pressure limitations, the flow rates used for the PP and AX columns were adjusted to 0.7 mL / min, respectively, and the flow rates used for the QS and PS columns were adjusted to 0.5 mL / min and 0.35 mL / min, respectively. The method of Example 2 was used.

[0304] like Figure 1 As shown, adequate peak resolution (Rs) can only be obtained using PP (Rs = 1.07) and PS (Rs = 1.00) columns.

[0305] QS columns contain a non-porous polymer based chromatographic material. In more detail, the stationary phase / chromatographic material is a monodisperse, non-porous methacrylate based resin particle, whose surface consists of a multilayer open access network of anion exchange groups. The surface has been modified with quaternary ammonium. QS is a strong anion exchanger.

[0306] AX columns contain a chromatographic material based on a synthetic stationary phase consisting of a highly cross-linked particle core based on a copolymer of poly(styrene divinyl benzene) (PS-DVB) coated with a hydrophilic polymer layer and densely packed quaternary ammonium functional groups chemically bound thereto. AX is a strong anion exchanger.

[0307] It has been reported that the AX column can be used to analyze the intact / 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).

[0308] 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 an uninterrupted, interconnected network of through pores, also known as channels. The material can be a methacrylate polymer. The monolith consists of aggregates of spheres. Most spheres are engineered to be less than 500 nm. The spheres are essentially non-porous. The pore volume is about 60% of the column volume. The modal pore size ranges from about 1.6 μm to 2.7 μm, with the most abundant pore size being about 2 μM. The surface has been modified with quaternary amines. PS is a strong anion exchanger.

[0309] In a preferred embodiment according to all aspects and embodiments of 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.

[0310] The PP column contains a chromatographic material based on a synthetic stationary phase consisting of a highly crosslinked particle core based on a copolymer of ethylvinylbenzene and divinylbenzene (EVB-DVB) coated with a crosslinked hydrophilic boundary layer and a linear anion exchange phase with quaternary ammonium functional groups grafted thereon. In more detail, the PP stationary phase consists of a microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix covered with a highly hydrophilic neutral polymer 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.

[0311] In a preferred embodiment according to all aspects and embodiments of the present invention, the stationary phase / chromatographic material is a synthetic stationary phase consisting of a highly crosslinked particle core based on a copolymer of styrene and divinylbenzene (EVB-DVB) coated with a crosslinked hydrophilic boundary layer and a linear anion exchange phase with quaternary ammonium functional groups grafted thereon. In certain embodiments, the stationary phase consists of a microporous ethylvinylbenzene crosslinked with a 55% divinylbenzene polymer matrix, which is covered with a highly hydrophilic neutral polymer to form a hydrophilic layer, onto which controlled polymer chains with quaternary ammonium groups are grafted. In certain embodiments, the solid phase is a particle. In certain embodiments, the particle size of the solid phase, i.e., the particle diameter, is about 10 μm.

[0312] In the following, the method according to the present invention is illustrated using a PP column.

[0313] It has been found that dilution of rAAV8 particles in a buffer comprising PBS (phosphate buffered saline; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) supplemented with 0.001% (w / v) poloxamer 188, produces more stable samples over time. Under such dilution conditions, only limited changes in the rAAV8 intact (RSD = 8.6%) and empty (RSD = 8.3%) peak areas were observed, and the intact / empty ratio was much more accurately measured (RSD = 3.4%) compared to water as a diluent.

[0314] Table 2. Summary of results of sample stability studies.

[0315]

[0316] Therefore, a poloxamer-supplemented buffer was chosen as the preferred dilution solvent, and the rest of the rAAV8 analysis was always performed within 3 days from sample preparation to limit variability associated with sample stability.

[0317] Different buffers, pH values ​​and salts were tested.

[0318] Table 3. List of buffer salts and their chemical structures.

[0319]

[0320] The reference BTP buffer was compared with four alternative biological buffers (i.e., AMPD, AMPSO, CHES, and CAPSO) having different pKa values ​​and chemical structures (see Table 2). The chromatograms obtained with these different buffered mobile phases using the elution method according to Example 2 are shown in Figure 2 The intact / empty ratio (F / E) and chromatographic resolution (Rs) were determined. It can be seen that the intact / empty ratio was not significantly affected by the use of different buffers (ranging from 1.08 to 1.28). However, resolution and Rs values ​​were only obtained for BTP buffer and AMPSO buffer (Rs = 0.80 and 0.84, respectively).

[0321] In certain embodiments, the buffer in the methods according to the invention is 1,3-bis[tris(hydroxymethyl)methylamino]propane.

[0322] In a preferred embodiment of all aspects and embodiments of the invention the buffer in the method according to the invention is N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0323] In the following, the method according to the current invention is exemplified using AMPSO buffer.

[0324] A mobile phase consisting of AMPSO buffer was evaluated at different pH values ​​(ranging from pH 8.6 to pH 9.4) at 0.2 pH unit intervals. The elution method according to Example 2 was used. Figure 3 As shown, a slight decrease in retention time was observed when increasing the mobile phase pH. This behavior is consistent with an AMPSO pKa of 9. When using a mobile phase buffered at a pH below its pKa (here 8.6 and 8.8), a higher amount of the neutral form of AMPSO is present in solution, while using a mobile phase buffered at a pH above its pKa (here 9.2 and 9.4), a higher amount of the negatively charged form of AMPSO is expected to be in solution. Since only the negatively charged form of AMPSO in the buffer competes with the analyte for binding to the positively charged stationary phase, a slight decrease in retention time was observed when increasing the mobile phase pH. Although the intact / empty ratio was not significantly affected by the pH change (ranging from 1.00 to 1.09), higher resolution was obtained at pH 9.4 (Rs = 0.93) compared to pH 9.0 (Rs = 0.70).

[0325] 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 higher. 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 a preferred embodiment, the mobile phase has a pH value of about 9.4.

[0326] In the following, a pH value of 9.4 is used to illustrate the method according to the invention.

[0327] Three different salt types (NaCl, KCl, and tetramethylammonium chloride (TMAC)) were tested as eluents for separation of intact and empty rAAV8 particles. Figure 4 In FIG. 5 , the corresponding chromatograms obtained by using different elution salts are shown.

[0328] It is noteworthy that, although the intact / empty ratio was not significantly affected (ranging from 0.96 to 1.09), 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).

[0329] In certain embodiments of all aspects and embodiments of the 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 with a molecular weight less than 200 g / mol. In certain embodiments, the organic salt is an organic chloride with a molecular weight less than 170 g / mol. In certain embodiments, the organic salt is tetraethylammonium chloride. In a preferred embodiment, the organic salt is tetramethylammonium chloride.

[0330] In the following, the method according to the invention is illustrated using tetramethylammonium chloride as elution salt.

[0331] Three different mobile phase flow rates were tested, namely 0.7 mL / min, 0.3 mL / min, and 0.1 mL / min. Figure 5 Shown in.

[0332] A flow rate of 0.3 mL / min was selected as the best compromise between chromatographic resolution, peak intensity and mobile phase consumption.

[0333] Large proteins exhibit a specific elution behavior under reversed phase liquid chromatography (RPLC) conditions, where retention is extremely sensitive to the mobile phase composition (see, e.g., Snyder, LR, et al., Anal. Chem. 55 (1983) 1412A-1430A; Fekete, S., et al., Anal. Chem. 93 (2021) 1277–1284). This special retention behavior is described as an "on / off" or "bind and elute" mechanism and can be explained as follows: the retention of large solutes in weak eluents is almost infinite (this corresponds to the "on" or "bind" state, in which the protein species is completely adsorbed at the column inlet), while only a limited increase in eluent strength results in a significant decrease in retention (this corresponds to the "off" or "elution" state, because the protein migrates to the column outlet without any further interaction).

[0334] It is reported that a strategy combining an isocratic elution step with a very short steep gradient segment in the protein elution composition can adjust the selectivity as desired (Fekete, S., et al., Anal. Chem. 91 (2019) 12954–12961; Nguyen, JM, et al., J. Chromatogr. B. 1173 (2021) 122694). This type of strategy has only been applied once in AEX to separate empty and complete capsids of recombinant adeno-associated viruses (Khatwani, SL, et al., Mol. Ther.-Methods Clin. Dev. 21 (2021) 548-558).

[0335] Based on two initial AEX gradients from 0% to 60% B in 15 minutes and 45 minutes, respectively, and measurements of the retention times of the two substances, the following gradient was derived: 98% (v / v) mobile phase A (A) and 2% (v / v) mobile phase B (B) for 1 min at the beginning of the method. Thereafter, a linear change to about 82% (v / v) A and 18% (v / v) B in 5 minutes, an isocratic elution was maintained for about 4 minutes, and a further linear change to about 40% (v / v) A and 60% (v / v) B in 5 minutes.

[0336] These conditions have been verified experimentally.

[0337] Therefore, the present invention provides a method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0338] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0339] Wherein the empty recombinant AAV particles are eluted during the first linear gradient and the intact recombinant AAV particles are eluted during the second linear gradient.

[0340] It is important to note that the second isocratic elution step has the effect of allowing the entire amount of empty rAAV particles to be eluted from the column before changing the conditions to elute the intact rAAV particles from the column.

[0341] Different isocratic elution holding condition compositions around the derived values ​​were tested, namely 17.0% (v / v), 17.5% (v / v), 18% (v / v) and 18.5% (v / v) B. The corresponding chromatograms are shown in Figure 6 The overlap in is shown.

[0342] It can be seen that complete separation of intact and empty rAAV8 particle peaks was achieved independent of the isocratic elution hold conditions applied.

[0343] In order to assess the most suitable conditions, the complete / empty ratio and chromatographic resolution were used. When the isocratic elution step was performed at 17% (v / v) B or 17.5% (v / v) B, the peak corresponding to the empty particles (the first elution peak) was broadened compared to the other tested conditions. As a result, the complete / empty ratio was too high. When the isocratic elution step was performed at 18.5% (v / v) B, the peak shape obtained was good and the resolution was maximum (Rs was 5.02), but the complete / empty ratio was inconsistent with theoretical expectations (only 0.68). When the isocratic elution step was performed at 18% (v / v) B, the resolution was good (Rs was 3.72), no obvious peak tailing / broadening was observed, and the complete / empty ratio was close to the expected value of 1 (1.14).

[0344] In addition to the ionic strength of the second isocratic elution step, the duration of the second isocratic elution step can also be used to change selectivity and resolution. In more detail, the duration of the second isocratic elution step affects the retention of the next peak to be eluted (i.e., the complete particle peak). It has no effect on the previously eluted peak (i.e., the empty particle peak).

[0345] To demonstrate this, the duration of the second isocratic elution step was evaluated using durations of 4, 6, 8 and 10 minutes. Figure 7 The corresponding chromatograms are shown in .

[0346] When the duration of the second isocratic elution step was increased, a decrease in the intensity and area of ​​the peak corresponding to intact rAAV particles was observed, whereas the peak corresponding to empty rAAV particles was not affected (see Figure 7 Without being bound by this theory, it is assumed that a too long isocratic elution step leads to an incorrect full / empty ratio (too low a value).

[0347] In order to show the improvement obtained with the method according to the invention, in a linear gradient elution method (Example 2, Figure 8 A), adjusted linear gradient elution method (Example 3, Figure 8 B) and the method according to the present invention (Example 4, Figure 8 C) to compare.

[0348] It can be seen that in Figure 8 The gain in chromatographic resolution between the chromatograms reported in A and 8B is modest, while the ratio of intact / empty remains comparable. However, when the method according to the invention is applied, the resolution improvement is significant (Rs of 3.72), while the ratio of intact / empty remains comparable (1.14).

[0349] In order to examine the applicability of the method according to the present invention for rAAV particle characterization, two different types of samples, including both complete rAAV8 particles and empty rAAV8 particles, were obtained from two different suppliers, namely Sirion Biotech and Virovek.

[0350] For the particles obtained from Sirion Biotech, the retention time of the empty rAAV particles was slightly longer than that of the particles from the Virovek supplier. This could be explained by the vector production process, which results in a different amount of negative charge on the capsid, which could cause a somewhat greater retention of the samples. Therefore, the separation conditions for the intact and empty particles of rAAV from Sirion Biotech were slightly different, and the isocratic elution step was set to 18.5% (v / v) of mobile phase B to allow sufficient elution of the Sirion empty particles.

[0351] Eleven mixtures of both intact particles and empty particles in different percentages (intact capsids ranged from 0% to 100%) in PBS buffer were analyzed. Figure 9 shows the corresponding chromatograms for all the mixtures, where the 0:100 and 100:0 empty / intact samples correspond to pure intact rAAV particles and empty rAAV particles, respectively. As shown in the chromatograms, the intact capsid samples from Virovek already contained about 10% of empty particles, while this value dropped to only 1% to 2% for the Sirion Biotech samples. On the other hand, the empty particle samples from both providers already contained more than 14% of intact particles. In Figure 9, a graphical representation of the experimental and theoretical percentages of intact rAAV particles in the mixture is also provided. The experimental values ​​were obtained only from the peak areas. It can be seen that the peak areas of the two peaks vary linearly (R for the two different samples). 2 The peak area percentages of empty rAAV8 particles and intact rAAV8 particles also changed accordingly, which was obtained by the method according to the present invention. These observations confirm that the area of ​​each peak is additive, linear and specific for empty rAAV8 particles and intact rAAV8 particles. This confirms that the method according to the present invention can be used in a QC environment because it is accurate, linear and sufficiently robust.

[0352] In summary, the present invention at least provides the following contents:

[0353] 1. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0354] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0355] Wherein the empty recombinant AAV particles are eluted during the first linear gradient and the intact recombinant AAV particles are eluted during the second linear gradient.

[0356] 2. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0357] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0358] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0359] The solution containing empty and / or intact rAAV particles contains about 0.001% (w / v)

[0360] A solution of non-ionic detergent in phosphate-buffered saline.

[0361] 3. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0362] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0363] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0364] Among them, the rAAV particles were of serotype 8.

[0365] 4. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0366] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0367] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0368] The anion exchange chromatography material has a stationary phase which is composed of a microporous ethylvinylbenzene cross-linked with a 55% divinylbenzene polymer matrix and has quaternary ammonium groups providing anion exchange functionality.

[0369] 5. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0370] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0371] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0372] The solutions used in the isocratic elution step and the linear gradient comprise a buffer substance having a pKa value of about 9.

[0373] 6. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0374] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0375] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0376] The solution used in the isocratic elution step and the linear gradient contained N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance.

[0377] 7. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0378] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0379] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0380] The solutions used in the isocratic elution step and the linear gradient had a pH value of 9.0 to 9.6.

[0381] 8. A method for separating intact recombinant adeno-associated virus particles and empty recombinant adeno-associated virus particles using an anion exchange chromatography step,

[0382] wherein the method comprises the following series of steps: a) applying a solution comprising empty and / or intact rAAV particles to an anion exchange chromatography material in a chromatography column, b) performing a first isocratic elution step, c) applying a first linear gradient, d) performing a second isocratic elution step, and e) applying a second linear gradient,

[0383] wherein empty recombinant adeno-associated virus particles are eluted during the first linear gradient and intact recombinant adeno-associated virus particles are eluted during the second linear gradient,

[0384] The solutions used in the isocratic elution steps and the linear gradients contained tetramethylammonium chloride as elution salt.

[0385] 9. The method of any one of embodiments 1 to 8, wherein the solution comprising empty and / or intact rAAV particles is a phosphate-buffered saline solution comprising approximately 0.001% (w / v) non-ionic detergent.

[0386] 10. The method according to any one of embodiments 1 to 9, wherein the non-ionic detergent is selected from poloxamer 188 and polysorbate 20.

[0387] 11. The method of any one of embodiments 1 to 10, wherein the rAAV particles are of serotype 8.

[0388] 12. A method according to any one of embodiments 1 to 11, wherein the anion exchange chromatography material has a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with a 55% divinylbenzene polymer matrix with quaternary ammonium groups providing anion exchange functionality.

[0389] 13. The method according to 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.

[0390] 14. The method according to any one of embodiments 1 to 13, wherein the method

[0391] i) provide baseline separation of intact rAAV particles and empty rAAV particles, or

[0392] ii) for the quantification of intact and empty rAAV particles, or

[0393] iii) is a high throughput method for the quantification of complete rAAV particles and empty rAAV particles, or

[0394] iv) is the quality control (QC) method for rAAV preparations.

[0395] 15. The method according to any one of embodiments 1 to 14, wherein the solution used in the isocratic elution step and the linear gradient comprises a buffer substance having a pKa value of about 9.

[0396] 16. The method according to any one of embodiments 1 to 15, wherein the solution used in the isocratic elution step and the linear gradient comprises N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as buffer substance.

[0397] 17. The method according to any one of embodiments 1 to 16, wherein the solution used in the isocratic elution step and the linear gradient has a pH value of 9.0 to 9.6.

[0398] 18. The method according to any one of embodiments 1 to 17, wherein the solution used in the isocratic elution step and the linear gradient comprises an organic chloride with a molecular weight lower than 170 g / mol as elution salt.

[0399] 19. The method according to any one of embodiments 1 to 18, wherein the solution used in the isocratic elution step and the linear gradient comprises tetramethylammonium chloride as elution salt.

[0400] 20. The method according to any one of claims 1 to 19, wherein the method comprises

[0401] The flow rate was mL / min.

[0402] 21. The method according to any one of claims 1 to 20, wherein the solution applied in the first isocratic elution step comprises about 65 mM buffer substance, about 10 mM elution salt, about 2 mM magnesium chloride and has a pH value of about 9.4,

[0403] 22. The method according to any one of claims 1 to 21, wherein the solution applied in the second isocratic elution step comprises about 65 mM buffer substance, about 90 mM elution salt, about 2 mM magnesium chloride and has a pH value of about 9.4.

[0404] 23. The method of any one of claims 1 to 22, wherein the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes and the second linear gradient has a length of about 5 minutes.

[0405] ***

[0406] The examples and drawings are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It is to be understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0407] ***

[0408] Material

[0409] Bis-Tris propane (BTP, ≥99.0%), 2-amino-2-methyl-1,3-propanediol (AMPD, ≥99.0%), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropane sulfonic acid (AMPSO, ≥99.0%), 2-(cyclohexylamino)ethanesulfonic acid (CHES, BioUltra, ≥99.5%), 3-(cyclohexylamino)-2-hydroxy-1-propane sulfonic acid (CAPSO, ≥99% anhydrous basis), magnesium chloride hexahydrate (BioXtra, ≥99.0%), hydrochloric acid solution (1N), sodium chloride (BioUltra, for molecular biology, ≥99.5%), potassium chloride (BioUltra, for molecular biology, ≥99.5%), and tetramethylammonium chloride (TMAC, LiChropur, ≥99.0%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium hydroxide solution (1 N) was obtained from VWR Chemicals. Phosphate buffered saline (PBS) and poloxamer 188 were obtained from Roche Diagnostics GmbH (Penzberg, Germany). Water was provided by a Milli-Q purification system from Millipore (Bedford, MA, USA).

[0410] Samples containing intact 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.

[0411] Anion exchange chromatography was performed on an ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) equipped with an autosampler including a 50 μL injection loop, a quaternary solvent delivery pump, and a fluorescence detector (FD). Data were acquired using FLR excitation at 280 nm and emission at 350 nm.

[0412] Table 1. List of chromatographic columns and their characteristics (chemical composition: all columns are made of quaternary ammonium). PEEK and SS represent polyetheretherketone and stainless steel, respectively.

[0413]

[0414]

[0415] The column was kept at room temperature during analysis. 20 μL of 1.00E+12 vp / mL sample was injected in all cases, corresponding to a column load of 2.00E+10 vp. Acquisition was performed in salt gradient mode and applied to intact, empty and mixed rAAV particle samples. Data acquisition and instrument control were performed by Empower Pro 3 software (Waters).

[0416] Example 1

[0417] Sample preparation

[0418] The concentration of rAAV particles in the samples was expressed as the number of viral particles per mL (vp / mL).

[0419] Samples were stored at -80°C. Prior to analysis, samples were diluted to 1.00E+12 vp / mL in an appropriate solvent (phosphate buffered saline containing 0.001% poloxamer 188 or corresponding mobile phase).

[0420] For "mixed samples" containing both intact rAAV particles and empty rAAV particles, appropriate volumes of intact rAAV particles containing the diluted sample and empty rAAV particles containing the diluted sample were taken and mixed to obtain the desired ratio of intact rAAV particles to empty rAAV particles in the aliquot.

[0421] All samples were stored at 4 °C after sample preparation and analyzed within 72 h of preparation.

[0422] Example 2 - Comparative Example

[0423] Standard Methods

[0424] Mobile phase A (A) consisted of 65 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP), 2 mM magnesium chloride hexahydrate aqueous solution, 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 aqueous solution, adjusted to pH 9.0 with 1 M sodium hydroxide solution.

[0425] The standard method first holds 2% (v / v) B for 3 minutes. Then, a linear gradient is applied by increasing the fraction of mobile phase B from 2% (v / v) to 56% (v / v) in 30 min. This is followed by a wash step at 100% (v / v) B for 5 min and a column re-equilibration step at 2% (v / v) B for 6 min. The column is kept at room temperature and the flow rate is set to a constant value, such as 0.7 mL / min.

[0426] Example 3 - Comparative Example

[0427] Optimizing standard methods

[0428] Mobile phase A (A) consisted of 65 mM N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2 mM magnesium chloride hexahydrate aqueous solution, and 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 aqueous solution, and adjusted to pH 9.4 with 1 M sodium hydroxide solution.

[0429] The column is kept at room temperature and the flow rate is set to a constant value, such as 0.3 mL / min. The same linear gradient as described in Example 2 is applied.

[0430] Example 4

[0431] Method according to the invention

[0432] Mobile phase A (A) consisted of 65 mM N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2 mM magnesium chloride hexahydrate aqueous solution, and 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 aqueous solution, and adjusted to pH 9.4 with 1 M sodium hydroxide solution.

[0433] The method according to the invention is started by applying a mixture of 98% (v / v) A and 2% (v / v) B for 1 minute. Thereafter, the concentration of B is increased from 2% (v / v) to 18% (v / v) in 5 min. Thereafter, the concentration of 18% (v / v) B is maintained for 4 min. Thereafter, the concentration of B is increased from 18% (v / v) to 60% (v / v) in 5 min, followed by a washing step at 100% (v / v) B for 5 min and a re-equilibrium step at 2% (v / v) B for 5 min.

[0434] The column is maintained at room temperature and a constant flow rate is applied, such as 0.3 mL / min.

[0435]

Claims

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

2. The method according to claim 1, wherein the nonionic detergent is selected from poloxamer 188 and polysorbate 20.

3. The method according to any one of claims 1 to 2, wherein the rAAV particles are of serotype 8.

4. A method according to any one of claims 1 to 3, wherein the anion exchange chromatography material has a stationary phase consisting of a microporous ethylvinylbenzene cross-linked with a 55% divinylbenzene polymer matrix, with quaternary ammonium groups providing anion exchange functionality.

5. The method according to any one of claims 1 to 4, wherein the anion exchange chromatography column has dimensions of about 50 mm in length and about 4 to 5 mm in diameter, and the anion exchange chromatography material has a particle size of about 10 μm.

6. The method according to any one of claims 1 to 5, wherein the method i) provide baseline separation of intact rAAV particles and empty rAAV particles, or ii) for the quantification of intact and empty rAAV particles, or iii) is a high throughput method for the quantification of complete rAAV particles and empty rAAV particles, or iv) is the quality control (QC) method for rAAV preparations.

7. The method according to any one of claims 1 to 6, wherein the solution used in the isocratic elution step and the linear gradient comprises a buffer substance having a pKa value of about 9.

8. The method according to any one of claims 1 to 7, wherein the solution used in the isocratic elution step and the linear gradient contains N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid as a buffer substance.

9. The method according to any one of claims 1 to 8, wherein the solution used in the isocratic elution step and the linear gradient has a pH value of 9.0 to 9.

6.

10. The method according to any one of claims 1 to 9, wherein the solution used in the isocratic elution step and the linear gradient comprises as elution salt an organic chloride having a molecular weight lower than 170 g / mol.

11. The method according to any one of claims 1 to 10, wherein the solution used in the isocratic elution step and the linear gradient comprises tetramethylammonium chloride as elution salt.

12. The method according to any one of claims 1 to 11, wherein the method is performed at a flow rate of about 0.3 mL / min.

13. The method according to any one of claims 1 to 12, wherein the solution applied in the first isocratic elution step comprises about 65 mM of the buffer substance, about 10 mM of the elution salt, about 2 mM of magnesium chloride and has a pH value of about 9.

4.

14. The method according to any one of claims 1 to 13, wherein the solution applied in the second isocratic elution step comprises about 65 mM of the buffer substance, about 90 mM of the elution salt, about 2 mM of magnesium chloride and has a pH value of about 9.

4.

15. The method according to any one of claims 1 to 14, wherein the first isocratic elution step has a length of about 1 minute, the first linear gradient has a length of about 5 minutes, the second isocratic elution step has a length of about 2 to 5 minutes, and the second linear gradient has a length of about 5 minutes.

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