Adeno-associated virus separation using cation exchangers.

Abstract

The present disclosure provides a method for purifying adeno-associated virus, comprising purifying a complete AAV capsid from an enriched AAV fraction or preparation comprising empty and complete AAV capsids. The present disclosure also provides a method for purifying adeno-associated virus, comprising purifying an empty AAV capsid from an enriched AAV fraction or preparation comprising empty and complete AAV capsids. The method comprises separating using one or more monovalent cations and one or more divalent cations to obtain purified complete or empty AAV capsids.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 229,303, filed August 4, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Adeno-associated virus (AAV) is a small non-enveloped virus that packages a linear single-stranded DNA genome. AAV belongs to the genus Dependovirus of the family Parvoviridae, since productive infection by AAV occurs only in the presence of a helper virus, such as adenovirus or herpesvirus. Even in the absence of a helper virus, AAV (serotype 2) can achieve latent infection by integrating into chromosome 19q13.4 of the host human genome. AAV is the only mammalian DNA virus known to be capable of site-specific integration (Non-Patent Document 1).

[0003] To make AAV safe for clinical use, it has been genetically modified at several locations in its genome. For example, in many viral vectors, the Rep gene required for viral replication and the elements required for site-specific integration have been removed from the AAV genome. These recombinant AAVs (rAAVs) exist in an extrachromosomal state and have extremely low integration efficiency into genomic DNA. Thus, the possibility of rAAV inducing random mutagenesis in host cells is reduced, if not completely eliminated. Due to these characteristics and lack of pathogenicity, rAAVs show great potential in many preclinical and clinical applications as gene therapy vectors. In the clinic, new serotypes and self-complementary vectors are being tested. In parallel with this ongoing vector development, there is also a continuing effort to develop a scalable manufacturing process that can efficiently produce high titer quantities of rAAV vectors with high purity and titer.

[0004] Although great efforts have been made to design efficient and large-scale methods for purifying AAV products suitable for human administration, there remains a need for better AAV purification methods. For example, current methods for producing AAV in cell culture have been shown to produce "empty" capsids, which induce T cell-mediated immune responses against capsid antigens, leading to low-grade hepatotoxicity and partial loss of expression (Non-Patent Document 2). Therefore, an AAV purification method that includes a step of removing empty AAV capsids from the final AAV product is desirable. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Daya and Berns,Clinical Microbiology Reviews, pages 583-593(2008) [Non-Patent Document 2] Wright, Molec Therapy 22(1):1-2(2014) Summary of the Invention [Means for solving the problem]

[0006] A hallmark of AAV vector production in cell culture is the excessive formation of "empty" capsids that lack the vector genome. Such empty capsids fail to confer the therapeutic benefit associated with transgene production. Although the impact of empty capsids on clinical outcome is unclear, they are a concern in gene therapy because they may increase innate or adaptive immune responses against the vector. Wright,Molec Therapy 22(1):1-2(2014).

[0007] Provided herein is a method for purifying complete AAV capsids from an AAV preparation that contains complete and empty AAV capsids to provide an AAV product that is substantially free of empty AAV capsids, the method comprising: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the full AAV capsid and the empty AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions whereby full AAV capsids are purified from empty AAV capsids.

[0008] Provided herein is a method for separating full and empty AAV capsids in an AAV preparation, the method comprising: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the full AAV capsid and the empty AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that full AAV capsids are separated from empty AAV capsids.

[0009] Provided herein is a method for separating empty and full AAV capsids in an AAV preparation, comprising: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the empty AAV capsid and the full AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions whereby empty AAV capsids are purified from full AAV capsids.

[0010] Provided herein is a method for separating empty and full AAV capsids in an AAV preparation, comprising: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the empty AAV capsid and the full AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that empty AAV capsids are separated from full AAV capsids.

[0011] In some embodiments, the methods of the present disclosure are also useful for purifying AdV particles, lentivirus particles, gammaretrovirus vector particles, herpes simplex virus (HSV) particles, simian virus 40 (SV40) particles, alphavirus particles, togavirus particles, Ross River virus particles, and vaccinia virus particles. In some embodiments, the methods of the present disclosure are also useful for producing vector vaccines.

[0012] In some embodiments, the one or more monovalent cations of the first solution are Na + , K + , N.H. 4 + , Li + , Cs + In some embodiments, the one or more monovalent cations of the first solution are selected from the group consisting of Na + It is.

[0013] In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 5 mM to about 1500 mM, In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 30 mM.

[0014] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ , Co 2+ In some embodiments, the one or more divalent cations of the first solution are selected from the group consisting of Ca 2+ It is.

[0015] In some embodiments, the one or more divalent cations in the first solution are at a total concentration of about 1 mM to about 30 mM, hi some embodiments, the one or more divalent cations in the first solution are at a total concentration of about 2 mM.

[0016] In some embodiments, the first solution has a pH of about 5.0 to about 8.5, In some embodiments, the first solution has a pH of about 6.0.

[0017] In some embodiments, the first solution further comprises one or more surfactants. In some embodiments, the one or more surfactants are selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polyoxyethylene glycol tert-octylphenol ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, polyoxyethylene(20) sorbitan monopalmitate, polyoxyethylene(20) sorbitan monostearate, polyoxyethylene(20) sorbitan tristearate, polyoxyethylene(20) sorbitan trioleate, polyoxyethylene(20)-sorbitan-monooleate (Tween 80 / Polysorbate 80), poloxamer 124, poloxamer 188, poloxamer 407, cremophor, Triton N-101 reduced, Triton X-100, and combinations thereof. In some embodiments, the surfactant is polysorbate 80.

[0018] In some embodiments, the one or more surfactants are in a total amount of about 0.0025% to about 0.0075% w / w. In some embodiments, the one or more surfactants are in a total amount of about 0.005% w / w.

[0019] In some embodiments, the cation exchange column comprises a resin having charged groups, the charged groups being sulfonic acid groups, sulfate groups, sulfopropyl groups, carboxylate groups, phosphate groups, or combinations thereof. In some embodiments, the cation exchange column comprises a resin, the resin being Capto S, Eshmuno S, Mustang S, Poros 50HS, Poros 50 XS, S-Sepharose FF, Source S, Capto MMC, Toyopearl Gigacap S, Gigacap CM, Toyopearl SP, Toyopearl CM, MacroPrep S, UNOsphereS, MacroprepCM, Fractogel EMD SO3, Fractogel EMD COO, Fractogel EMD SE Hicap, Cellufine Sulfate, CM and SP Trisacryl, CM and S HyperD, S and CM Sepharose CL, CM Sepharose FF, S and CM CAPTO™, MonoS, Nuvia S, Cellufine phosphat, Cellufine MAX-S r, Cellufine MAX-S h, Cellufine MAX DexS-HbP, Cellufine MAX In some embodiments, the antibody is selected from the group consisting of DexS-VirS, Toyopearl Sulfate 650, and Heparin Sepharose High Performance. In some embodiments, CaptoS. In some embodiments, Eshmuno S. In some embodiments, Mustang S.

[0020] In some embodiments, the one or more monovalent cations of the second solution are Na + , K + , N.H. 4 + , Li + , Cs + In some embodiments, the monovalent cation of the second solution is selected from the group consisting of Na + It is.

[0021] In some embodiments, the one or more divalent cations of the second solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ In some embodiments, the one or more divalent cations of the second solution are selected from the group consisting of Ca 2+ It is.

[0022] In some embodiments, the second solution has a pH of about 5.0 to about 8.5, In some embodiments, the second solution has a pH of about 6.0.

[0023] In some embodiments, the second solution further comprises one or more surfactants. In some embodiments, the one or more surfactants are selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polyoxyethylene glycol tert-octylphenol ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, polyoxyethylene(20) sorbitan monopalmitate, polyoxyethylene(20) sorbitan monostearate, polyoxyethylene(20) sorbitan tristearate, polyoxyethylene(20) sorbitan trioleate, polyoxyethylene(20)-sorbitan-monooleate (Tween 80 / Polysorbate 80), poloxamer 124, poloxamer 188, poloxamer 407, cremophor, Triton N-101 reduced, Triton X-100, and combinations thereof. In some embodiments, the surfactant is polysorbate 80.

[0024] In some embodiments, the one or more surfactants are in a total amount of about 0.0025% to about 0.0075 w / w%, hi some embodiments, the one or more surfactants are in a total amount of about 0.005 w / w%.

[0025] In some embodiments, the addition of the second solution is performed at a constant concentration of one or more monovalent cations.

[0026] In some embodiments, the one or more monovalent cations of the second solution are at a constant total concentration of about 5 mM to about 1500 mM. In some embodiments, the one or more monovalent cations of the second solution are at a constant total concentration of about 30 mM.

[0027] In some embodiments, the addition of the second solution is performed at a constant concentration of one or more divalent cations.

[0028] In some embodiments, the one or more divalent cations of the second solution are at a constant total concentration of about 1 mM to about 30 mM, in some embodiments, the one or more divalent cations of the second solution are at a constant total concentration of about 2 mM.

[0029] In some embodiments, the addition of the second solution comprises a stepwise increase in concentration of one or more monovalent cations.

[0030] In some embodiments, the initial total concentration of the one or more monovalent cations in the second solution is about 15 mM to about 60 mM. In some embodiments, the initial total concentration of the one or more monovalent cations in the second solution is about 30 mM. In some embodiments, the intermediate total concentration of the one or more monovalent cations in the second solution is about 100 mM to about 300 mM. In some embodiments, the intermediate total concentration of the one or more monovalent cations in the second solution is about 200 mM. In some embodiments, the final total concentration of the one or more monovalent cations in the second solution is about 500 mM to about 1500 mM. In some embodiments, the final total concentration of the one or more monovalent cations in the second solution is about 1000 mM.

[0031] In some embodiments, the addition of the second solution comprises a stepwise increase in the total concentration of one or more divalent cations.

[0032] In certain embodiments, the initial total concentration of the one or more divalent cations in the second solution is about 1 mM to about 10 mM. In certain embodiments, the intermediate total concentration of the one or more divalent cations in the second solution is about 10 mM to about 20 mM. In certain embodiments, the final total concentration of the one or more divalent cations in the second solution is about 20 mM to about 30 mM.

[0033] In some embodiments, the addition of the second solution comprises a continuous linear increase in the total concentration of one or more monovalent cations.

[0034] In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is about 30 mM to about 200 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is about 30 mM to about 80 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is about 30 mM to about 40 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is about 30 mM to about 200 mM in 5 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is about 30 mM to about 80 mM in 5 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over five column volumes.

[0035] In some embodiments, the addition of the second solution comprises a continuous linear increase in the total concentration of one or more divalent cations.

[0036] In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 30 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 15 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 5 mM in 40 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 30 mM in 5 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 15 mM in 5 column volumes. In certain embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is about 1 mM to about 5 mM in 5 column volumes.

[0037] In certain embodiments, the AAV capsid is derived from the group consisting of AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, genetically modified AAV, chemically modified AAV, genetically and chemically modified AAV, and combinations thereof.In certain embodiments, the AAV capsid is derived from AAV8.In certain embodiments, the AAV capsid is derived from AAV9.In certain embodiments, the AAV capsid is derived from AAV6.

[0038] In certain embodiments, the method comprises: (a) concentrating empty AAV capsids by ultrafiltration, anion exchanger, and / or cation exchanger; (b) buffer exchange into an amine-free buffer; (c) preparing an immunoadsorption column comprising immobilizing empty AAV capsids on an activated resin.

[0039] Also provided herein is an AAV preparation comprising the complete AAV capsid purified according to the method described herein.In certain embodiments, the AAV preparation further comprises a pharma- ceutically acceptable carrier.In certain embodiments, the AAV preparation is substantially free of empty AAV capsid.

[0040] Also provided herein is a pharmaceutical composition comprising the AAV product, formulation, or composition produced by the method described herein.In certain embodiments, the AAV pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.In certain embodiments, the AAV pharmaceutical composition is substantially free of empty AAV capsids. [Brief description of the drawings]

[0041] [Figure 1] The complete chromatogram of Example 2 is shown. [Diagram 2] 2 shows a chromatograph of the elution region of Example 2. [Diagram 3] 4 shows the area under the curve (AUC) profile of fraction E2. [Figure 4] The AUC profile of fraction E3 is shown. [Diagram 5] The AUC profile of fraction E4 is shown. [Figure 6] The AUC profile of fraction E5 is shown. [Figure 7] The complete chromatogram of Example 3 is shown. [Figure 8] 2 shows a chromatograph of the elution region of Example 3. [Figure 9] The AUC profile of fraction E2 is shown. [Figure 10] The AUC profile of fraction E3 is shown. [Figure 11] The AUC profile of fraction E5 is shown. [Figure 12] The complete chromatogram of Example 4 is shown. [Figure 13] 1 shows a chromatograph of the elution region of Example 4. [Figure 14] The AUC profile of fraction E2 is shown. [Figure 15] The AUC profile of fraction E3 is shown. [Figure 16] The AUC profile of fraction E4 is shown. [Figure 17] The AUC profile of fraction E5 is shown. [Figure 18] The complete chromatogram of Example 5 is shown. [Figure 19A] 1 shows a chromatograph of the elution region of Example 5. [Figure 19B] 1 shows a chromatograph of the elution region of Example 5. [Figure 20] The AUC profile of fraction E1 is shown. [Figure 21] The AUC profile of fraction E2 is shown. [Figure 22] The AUC profile of fraction E3 is shown. [Diagram 23] The AUC profile of fraction E4 is shown. [Figure 24] The AUC profile of fraction E5 is shown. [Diagram 25] The complete chromatogram of Example 6 is shown. [Figure 26A] 1 shows a chromatograph of the elution region of Example 6. [Figure 26B] 1 shows a chromatograph of the elution region of Example 6. [Figure 27] The AUC profile of fraction E1 is shown. [Figure 28] The AUC profile of fraction E2 is shown. [Figure 29] The AUC profile of fraction E3 is shown. [Diagram 30] The AUC profile of fraction E4 is shown. [Diagram 31] The AUC profile of fraction E5 is shown. [Diagram 32] The AUC profile of fraction E6 is shown. [Figure 33A] 1 shows a chromatograph of the elution region of Example 7. [Figure 33B]1 shows a chromatograph of the elution region of Example 7. [Diagram 34] The AUC profile of fraction E1 is shown. [Diagram 35] The AUC profile of fraction E2 is shown. [Diagram 36] The AUC profile of fraction E3 is shown. [Figure 37] The AUC profile of fraction E4 is shown. [Figure 38] The AUC profile of fraction E5 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] definition Before describing the compositions and methods of the present invention, it is to be understood that the present invention is not limited to the specific processes, compositions, or methodologies described, but may vary. It is also to be understood that the terms used in this description are for the purpose of describing the particular versions or embodiments only, and are not intended to limit the scope of the embodiments herein, which are limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the embodiments of the present invention, the preferred methods, devices, and materials are described below. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein constitutes an admission that the embodiments of the present invention are not entitled to antedate such disclosure by prior invention.

[0043] As used herein, the following terms have the meanings indicated.

[0044] In the context of describing this disclosure (particularly in the context of the claims which follow), the use of the terms "a," "an," and "the," and similar referents, are intended to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0045] In some embodiments, when aspects of the disclosure are described as "comprising" a certain feature or variations thereof (e.g., comprises), it is also contemplated that the embodiment "consists of" or "consists essentially of" that feature.

[0046] The term "about" as used herein is intended to qualify the numerical value it modifies, indicating that such value varies within a margin of error. When no specific error, such as standard deviation for the average value shown in a data figure or table, is described, the term "about" shall be understood to mean a numerical value of plus or minus 10% of the number with which it is used. Thus, about 50% means a range of 45% to 55%.

[0047] The term "alkyl", as used herein, alone or in combination, refers to a straight or branched chain alkyl radical. The alkyl group may be optionally substituted as defined herein. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, nonyl, and the like. The term "alkylene", as used herein, alone or in combination, refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon bonded at two or more positions, such as methylene (-CH 2 Unless otherwise specified, the term "alkyl" can include "alkylene" groups.

[0048] As used herein, the terms "capsid," "capsid particle," and "particle" are used interchangeably and refer to an AAV particle that is composed of at least one intact AAV capsid shell.

[0049] As used herein, the term "empty" with respect to AAV or AAV capsid or AAV particle refers to one that lacks all (i.e., complete) vector genome. Empty AAV or empty AAV capsid or empty AAV particle cannot provide therapeutic benefit. As used herein, the term "complete" or "complete AAV capsid" with respect to AAV or AAV capsid or AAV particle refers to one that contains a majority of the entire vector genome. Complete AAV capsid can provide therapeutic benefit to a recipient patient. In certain embodiments, "complete" also includes "incomplete vector DNA" or "truncated vector DNA". In certain embodiments, complete vector DNA and incomplete and / or truncated vector DNA can be distinguished by additional analytical methods. Such methods include, but are not limited to, capillary electrophoresis, AUC (analytical ultracentrifugation), % agarose DNA (native or alkaline), gels, Southern blots, dot blot hybridization, UV spectrophotometry, weak anion exchange chromatography, and DNA sizing by mass spectrometry (see Resolving Adeno-Associated Viral Particle Diversity with Charge Detection Mass Spectrometry Elizabeth E. Pierson et.al Anal. Chem., 2016, 88(13), pp 6718-6725; incorporated herein in its entirety for all purposes).

[0050] The terms "patient" and "subject" are used interchangeably and in the conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of the AAV products, formulations, or compositions of the present disclosure, and include both human and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes; livestock animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds including chickens, turkeys and other quails, poultry such as ducks, geese, wild birds, and game birds. The terms do not denote a particular age. Thus, adults, young, and newborns are included.

[0051] An AAV product, AAV formulation, or any AAV-containing composition is "substantially free" of empty AAV capsids if it contains less than about 30% empty AAV capsids.

[0052] The recitation of ranges of values ​​herein is intended only to serve as a shorthand way of referring individually to each individual value and each endpoint within the range, unless otherwise indicated herein, and each individual value and each endpoint is incorporated herein as if it were individually recited herein. For example, it is specifically understood that any numerical value described herein includes all values ​​from the lower limit to the upper limit, i.e., all possible combinations of numerical values ​​between the recited lower limit and upper limit are considered to be expressly recited in this application. For example, if a concentration range is described as about 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are expressly recited in this application. The above recited values ​​are merely examples of specifically indicated values.

[0053] All methods described herein can be carried out in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "etc.") are intended only to more clearly illustrate the disclosure, and do not impose limitations on the scope of the disclosure unless otherwise asserted. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the disclosure.

[0054] Preferred embodiments of the present disclosure are described herein, including the best mode for carrying out the disclosure known to the inventors. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will adopt such variations as appropriate, and the inventors intend for the present disclosure to be carried out in ways other than as specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or clearly contradicted by context.

[0055] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0056] Provided herein are methods for producing an adeno-associated virus (AAV) product, formulation, or composition, methods for purifying AAV, methods for purifying full AAV capsids from AAV preparations or fractions containing full and empty AAV capsids, methods for purifying empty AAV capsids from AAV preparations or fractions containing full and empty AAV capsids, and methods for preparing an immunoabsorption column using both purified empty AAV capsids and purified full AAV capsids.

[0057] In certain embodiments, a method for purifying full AAV capsids from an AAV preparation or fraction comprising full and empty AAV capsids to provide an AAV product, formulation, or composition that is substantially free of empty AAV capsids, comprising: (a) providing a first solution comprising full AAV capsids, empty AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the full AAV capsid and the empty AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions whereby full AAV capsids are purified from empty AAV capsids.

[0058] In certain embodiments, a method for separating full and empty AAV capsids in an AAV preparation or fraction comprises: (a) providing a first solution comprising full AAV capsids, empty AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the full AAV capsid and the empty AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that full AAV capsids are separated from empty AAV capsids.

[0059] In some embodiments, the one or more monovalent cations of the first solution are Na + , K + , N.H. 4 + , N(C 1-5 Alkyl) 4 + , Li + , Cs + , Cu + , Ag + , Au + and combinations thereof.

[0060] In some embodiments, the one or more monovalent cations of the first solution are Na + , K + , N.H. 4 + , Li + , Cs + and combinations thereof.

[0061] In some embodiments, the one or more monovalent cations of the first solution are Na + , K + , N.H. 4 + and combinations thereof.

[0062] In some embodiments, the one or more monovalent cations of the first solution are Na + It is.

[0063] In some embodiments, the one or more monovalent cations of the first solution are K + It is.

[0064] In some embodiments, the one or more monovalent cations of the first solution are NH 4+ It is.

[0065] In some embodiments, the one or more monovalent cations in the first solution are from about 5 mM to about 1500 mM, from about 50 mM to about 1500 mM, from about 100 mM to about 1500 mM, from about 200 mM to about 1500 mM, from about 300 mM to about 1500 mM, from about 400 mM to about 1500 mM, from about 500 mM to about 1500 mM, from about 600 mM to about 1500 mM, from about 700 mM to about 1500 mM, from about 800 mM to about 1500 mM, from about 900 mM to about 1500 mM, from about 1000 mM to about 1500 mM, from about 1100 mM to about 1500 mM, from about 1200 mM to about 1500 mM, from about 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about The total concentration is from 5 mM to about 500 mM, from about 5 mM to about 400 mM, from about 5 mM to about 300 mM, from about 5 mM to about 200 mM, from about 5 mM to about 100 mM, from about 5 mM to about 50 mM, 30 mM to about 200 mM, 30 mM to about 80 mM, 30 mM to about 60 mM, or a value within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0066] In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 5 mM to about 1500 mM.

[0067] In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 30 mM to about 200 mM.

[0068] In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 30 mM to about 80 mM.

[0069] In some embodiments, the one or more monovalent cations in the first solution are at a total concentration of about 30 mM to about 60 mM.

[0070] In some embodiments, the one or more monovalent cations of the first solution are at a total concentration of about 200 mM.

[0071] In some embodiments, the one or more monovalent cations of the first solution are at a total concentration of about 80 mM.

[0072] In some embodiments, the one or more monovalent cations of the first solution are at a total concentration of about 60 mM.

[0073] In some embodiments, the one or more monovalent cations of the first solution are at a total concentration of about 30 mM.

[0074] In some embodiments, the one or more monovalent cations of the first solution are Na +and about 5 mM to about 1500 mM, about 50 mM to about 1500 mM, about 100 mM to about 1500 mM, about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 400 mM to about 1500 mM, about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, , about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about 5 mM to about 500 mM, about 5 mM to about 400 mM, about 5 mM to about 300 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, 30 mM to about 200 mM, 30 mM to about 80 mM, 30 mM to about 60 mM, or a total concentration within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0075] In some embodiments, the one or more monovalent cations of the first solution are Na + and a total concentration of about 5 mM to about 1500 mM.

[0076] In some embodiments, the one or more monovalent cations of the first solution are Na + and a total concentration of about 30 mM to about 200 mM.

[0077] In some embodiments, the one or more monovalent cations of the first solution are Na+ and a total concentration of about 30 mM to about 80 mM.

[0078] In some embodiments, the one or more monovalent cations of the first solution are Na + and a total concentration of about 30 mM to about 60 mM.

[0079] In some embodiments, the one or more monovalent cations of the first solution are Na + for a total concentration of approximately 200 mM.

[0080] In some embodiments, the one or more monovalent cations of the first solution are Na + for a total concentration of approximately 80 mM.

[0081] In some embodiments, the one or more monovalent cations of the first solution are Na + for a total concentration of approximately 60 mM.

[0082] In some embodiments, the one or more monovalent cations of the first solution are Na + for a total concentration of approximately 30 mM.

[0083] In some embodiments, the one or more monovalent cations of the first solution are K +and about 5 mM to about 1500 mM, about 50 mM to about 1500 mM, about 100 mM to about 1500 mM, about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 400 mM to about 1500 mM, about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, , about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about 5 mM to about 500 mM, about 5 mM to about 400 mM, about 5 mM to about 300 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, 30 mM to about 200 mM, 30 mM to about 80 mM, 30 mM to about 60 mM, or a total concentration within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0084] In some embodiments, the one or more monovalent cations of the first solution are K + and a total concentration of about 5 mM to about 1500 mM.

[0085] In some embodiments, the one or more monovalent cations of the first solution are K + and a total concentration of about 30 mM to about 200 mM.

[0086] In some embodiments, the one or more monovalent cations of the first solution are K+ and a total concentration of about 30 mM to about 80 mM.

[0087] In some embodiments, the one or more monovalent cations of the first solution are K + and a total concentration of about 30 mM to about 60 mM.

[0088] In some embodiments, the one or more monovalent cations of the first solution are K + for a total concentration of approximately 200 mM.

[0089] In some embodiments, the one or more monovalent cations of the first solution are K + for a total concentration of approximately 80 mM.

[0090] In some embodiments, the one or more monovalent cations of the first solution are K + for a total concentration of approximately 60 mM.

[0091] In some embodiments, the one or more monovalent cations of the first solution are K + for a total concentration of approximately 30 mM.

[0092] In some embodiments, the one or more monovalent cations of the first solution are NH 4 +and about 5 mM to about 1500 mM, about 50 mM to about 1500 mM, about 100 mM to about 1500 mM, about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 400 mM to about 1500 mM, about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, , about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about 5 mM to about 500 mM, about 5 mM to about 400 mM, about 5 mM to about 300 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, 30 mM to about 200 mM, 30 mM to about 80 mM, 30 mM to about 60 mM, or a total concentration within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0093] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + and a total concentration of about 5 mM to about 1500 mM.

[0094] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + and a total concentration of about 30 mM to about 200 mM.

[0095] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + and a total concentration of about 30 mM to about 80 mM.

[0096] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + and a total concentration of about 30 mM to about 60 mM.

[0097] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + for a total concentration of approximately 200 mM.

[0098] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + for a total concentration of approximately 80 mM.

[0099] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + for a total concentration of approximately 60 mM.

[0100] In some embodiments, the one or more monovalent cations of the first solution are NH 4 + for a total concentration of approximately 30 mM.

[0101] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ , Co 2+ , B.E. 2+ , Ga 2+ , Pb 2+ , Sr 2+ , Ti 2+ , Sr 2+and combinations thereof.

[0102] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ , Co 2+ and combinations thereof.

[0103] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ and combinations thereof.

[0104] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ It is.

[0105] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ It is.

[0106] In some embodiments, the one or more divalent cations in the first solution are at a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0107] In some embodiments, the one or more divalent cations in the first solution are at a total concentration of about 1 mM to about 30 mM.

[0108] In some embodiments, the one or more divalent cations in the first solution are at a total concentration of about 1 mM to about 10 mM.

[0109] In some embodiments, the one or more divalent cations of the first solution are at a total concentration of about 2 mM.

[0110] In some embodiments, the one or more divalent cations of the first solution are Ca 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0111] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ and a total concentration of about 1 mM to about 30 mM.

[0112] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ and a total concentration of about 1 mM to about 10 mM.

[0113] In some embodiments, the one or more divalent cations of the first solution are Ca 2+ for a total concentration of approximately 2 mM.

[0114] In some embodiments, the one or more divalent cations of the first solution are Mg 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0115] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ and a total concentration of about 1 mM to about 30 mM.

[0116] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ and a total concentration of about 1 mM to about 10 mM.

[0117] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ for a total concentration of approximately 2 mM.

[0118] In some embodiments, the one or more divalent cations of the first solution are Mg 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0119] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ and a total concentration of about 1 mM to about 30 mM.

[0120] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ and a total concentration of about 1 mM to about 10 mM.

[0121] In some embodiments, the one or more divalent cations of the first solution are Mg 2+ for a total concentration of approximately 2 mM.

[0122] In some embodiments, the one or more divalent cations of the first solution are Zn 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0123] In some embodiments, the one or more divalent cations of the first solution are Zn 2+ and a total concentration of about 1 mM to about 30 mM.

[0124] In some embodiments, the one or more divalent cations of the first solution are Zn 2+ and a total concentration of about 1 mM to about 10 mM.

[0125] In some embodiments, the one or more divalent cations of the first solution are Zn 2+ for a total concentration of approximately 2 mM.

[0126] In some embodiments, the one or more divalent cations of the first solution are Mn 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0127] In some embodiments, the one or more divalent cations of the first solution are Mn 2+ and a total concentration of about 1 mM to about 30 mM.

[0128] In some embodiments, the one or more divalent cations of the first solution are Mn 2+ and a total concentration of about 1 mM to about 10 mM.

[0129] In some embodiments, the one or more divalent cations of the first solution are Mn 2+ for a total concentration of approximately 2 mM.

[0130] In some embodiments, the one or more divalent cations of the first solution are Cu 2+and is a total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0131] In some embodiments, the one or more divalent cations of the first solution are Cu 2+ and a total concentration of about 1 mM to about 30 mM.

[0132] In some embodiments, the one or more divalent cations of the first solution are Cu 2+ and a total concentration of about 1 mM to about 10 mM.

[0133] In some embodiments, the one or more divalent cations of the first solution are Cu 2+ for a total concentration of approximately 2 mM.

[0134] In some embodiments, the first solution has a pH of about 5.0 to about 8.5, about 5.5 to about 8.5, about 6.0 to about 8.5, about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.5 to about 8.5, about 8.0 to about 8.5, about 5.0 to about 8.0, about 5.0 to about 7.5, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.0 to about 5.5, or a value within one of these ranges. Specific examples include about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or a range between any two of these values.

[0135] In some embodiments, the first solution has a pH of about 6.0.

[0136] In some embodiments, the first solution further comprises one or more surfactants.

[0137] In some embodiments, the cation exchange column comprises a resin having charged groups, the charged groups being sulfonic acid groups, sulfate groups, sulfopropyl groups, carboxylate groups, phosphate groups, or combinations thereof.

[0138] In some embodiments, the cation exchange column comprises a resin, the resin being CaptoS, Eshmuno S, Mustang S, Poros 50HS, Poros 50 XS, S-Sepharose FF, Source S, Capto MMC, Toyopearl Gigacap S, Gigacap CM, Toyopearl SP, Toyopearl CM, MacroPrep S, UNOsphereS, MacroprepCM, Fractogel EMD SO3, Fractogel EMD COO, Fractogel EMD SE Hicap, Cellufine Sulfate, CM and SP Trisacryl, CM and S HyperD, S and CM Sepharose CL, CM Sepharose FF, S and CM CAPTO™, MonoS, Nuvia S, Cellufine phosphat, Cellufine MAX-S r, Cellufine MAX-S h, Cellufine MAX DexS-HbP, Cellufine MAX DexS-VirS, Toyopearl Sulfate 650, or Heparin Sepharose High Performance.

[0139] In some embodiments, the resin is CaptoS.

[0140] In some embodiments, the resin is Eshmuno S.

[0141] In some embodiments, the resin is Mustang S.

[0142] In some embodiments, the one or more monovalent cations of the second solution are Na + , K + , N.H. 4 + , N(C 1-5 Alkyl) 4 + , Li + , Cs + , Cu + , Ag 1 , Au 1 and combinations thereof.

[0143] In some embodiments, the one or more monovalent cations of the second solution are Na + , K + , N.H. 4 + , Li + , Cs + and combinations thereof.

[0144] In some embodiments, the monovalent cation of the second solution is Na + It is.

[0145] In some embodiments, the one or more divalent cations of the second solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ , Co 2+ , B.E. 2+ , Ga 2+ , Pb 2+ , Sr 2+ , Ti 2+ , Sr 2+ and combinations thereof.

[0146] In some embodiments, the one or more divalent cations of the second solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ , B.A. 2+ , Sr 2+ and combinations thereof.

[0147] In some embodiments, the one or more divalent cations of the second solution are Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ and combinations thereof.

[0148] In some embodiments, the divalent cation of the second solution is Ca 2+ It is.

[0149] In some embodiments, the divalent cation of the second solution is Mg 2+ It is.

[0150] In some embodiments, the divalent cation of the second solution is Zn 2+ It is.

[0151] In some embodiments, the divalent cation of the second solution is Mn 2+ It is.

[0152] In some embodiments, the second solution has a pH of about 5.0 to about 8.5, about 5.5 to about 8.5, about 6.0 to about 8.5, about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.5 to about 8.5, about 8.0 to about 8.5, about 5.0 to about 8, about 5.0 to about 7.5, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6, about 5.0 to about 5.5, or a value within one of these ranges. Specific examples include about 5, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or a range between any two of these values.

[0153] In some embodiments, the second solution has a pH of about 6.

[0154] In some embodiments, the second solution further comprises one or more surfactants.

[0155] In some embodiments, the addition of the second solution is performed at a constant total concentration of one or more monovalent cations.

[0156] In some embodiments, the one or more monovalent cations in the second solution are from about 5 mM to about 1500 mM, from about 50 mM to about 1500 mM, from about 100 mM to about 1500 mM, from about 200 mM to about 1500 mM, from about 300 mM to about 1500 mM, from about 400 mM to about 1500 mM, from about 500 mM to about 1500 mM, from about 600 mM to about 1500 mM, from about 700 mM to about 1500 mM, from about 800 mM to about 1500 mM, from about 900 mM to about 1500 mM, from about 1000 mM to about 1500 mM, from about 1100 mM to about 1500 mM, from about 1200 mM to about 1500 mM, A constant total concentration of 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about 5 mM to about 500 mM, about 5 mM to about 400 mM, about 5 mM to about 300 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, or a value within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0157] In some embodiments, the one or more monovalent cations of the second solution are at a total concentration of about 5 mM to about 1500 mM.

[0158] In some embodiments, the one or more monovalent cations of the second solution are at a constant total concentration of about 30 mM.

[0159] In some embodiments, the addition of the second solution provides a constant total concentration of Na + It will be carried out.

[0160] In some embodiments, the Na of the second solution + is about 5 mM to about 1500 mM, about 50 mM to about 1500 mM, about 100 mM to about 1500 mM, about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 400 mM to about 1500 mM, about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, A constant total concentration of about 1400 mM to about 1500 mM, about 5 mM to about 1400 mM, about 5 mM to about 1300 mM, about 5 mM to about 1200 mM, about 5 mM to about 1100 mM, about 5 mM to about 1000 mM, about 5 mM to about 900 mM, about 5 mM to about 800 mM, about 5 mM to about 700 mM, about 5 mM to about 600 mM, about 5 mM to about 500 mM, about 5 mM to about 400 mM, about 5 mM to about 300 mM, about 5 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 50 mM, or a value within one of these ranges. Specific examples include about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0161] In some embodiments, the Na of the second solution + is a constant total concentration of about 5 mM to about 1500 mM.

[0162] In some embodiments, the Na of the second solution + is a constant total concentration of approximately 30 mM.

[0163] In some embodiments, the addition of the second solution is performed at a constant total concentration of one or more divalent cations.

[0164] In some embodiments, the one or more divalent cations in the second solution are at a constant total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0165] In some embodiments, the one or more divalent cations of the second solution are at a total concentration of about 1 mM to about 30 mM.

[0166] In some embodiments, the one or more divalent cations of the second solution are at a constant total concentration of about 2 mM.

[0167] In some embodiments, the addition of the second solution provides a constant total concentration of Ca 2+ It will be carried out.

[0168] In some embodiments, the Ca of the second solution 2+ is a constant total concentration of about 1 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0169] In some embodiments, the Ca of the second solution 2+ is a constant total concentration of about 1 mM to about 30 mM.

[0170] In some embodiments, the Ca of the second solution 2+ is a constant total concentration of approximately 2 mM.

[0171] In some embodiments, the addition of the second solution comprises a stepwise increase in the total concentration of one or more monovalent cations.

[0172] In some embodiments, the initial total concentration of one or more monovalent cations in the second solution is about 15 mM to about 60 mM, about 20 mM to about 60 mM, about 25 mM to about 60 mM, about 30 mM to about 60 mM, about 35 mM to about 60 mM, about 40 mM to about 60 mM, about 45 mM to about 60 mM, about 50 mM to about 60 mM, about 55 mM to about 60 mM, about 15 mM to about 55 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, or a value within one of these ranges. Specific examples can include about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, or a range between any two of these values.

[0173] In some embodiments, the initial total concentration of the one or more monovalent cations in the second solution is from about 15 mM to about 60 mM.

[0174] In some embodiments, the initial total concentration of the one or more monovalent cations in the second solution is about 30 mM.

[0175] In some embodiments, the intermediate total concentration of one or more monovalent cations in the second solution is about 100 mM to about 300 mM, about 125 mM to about 300 mM, about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300 mM, about 275 mM to about 300 mM, about 100 mM to about 275 mM, about 100 mM to about 250 mM, about 100 mM to about 225 mM, about 100 mM to about 200 mM, about 100 mM to about 175 mM, about 100 mM to about 150 mM, about 100 mM to about 125 mM, or a value within one of these ranges. Specific examples can include about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, or a range between any two of these values.

[0176] In some embodiments, the intermediate total concentration of the one or more monovalent cations in the second solution is from about 100 mM to about 300 mM.

[0177] In some embodiments, the intermediate total concentration of the one or more monovalent cations in the second solution is about 200 mM.

[0178] In some embodiments, the final total concentration of one or more monovalent cations in the second solution is about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, or about 1600 mM to about 1600 mM. 500 mM, about 1400 mM to about 1500 mM, about 500 mM to about 1400 mM, about 500 mM to about 1300 mM, about 500 mM to about 1200 mM, about 500 mM to about 1100 mM, about 500 mM to about 1000 mM, about 500 mM to about 900 mM, about 500 mM to about 800 mM, about 500 mM to about 700 mM, about 500 mM to about 600 mM, or a value within one of these ranges. Specific examples can include about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0179] In some embodiments, the final total concentration of the one or more monovalent cations in the second solution is from about 500 mM to about 1500 mM.

[0180] In some embodiments, the final total concentration of the one or more monovalent cations in the second solution is about 1000 mM.

[0181] In some embodiments, the addition of the second solution is Na + The method includes gradually increasing the total concentration of

[0182] In some embodiments, the Na of the second solution+ The initial total concentration is about 15 mM to about 60 mM, about 20 mM to about 60 mM, about 25 mM to about 60 mM, about 30 mM to about 60 mM, about 35 mM to about 60 mM, about 40 mM to about 60 mM, about 45 mM to about 60 mM, about 50 mM to about 60 mM, about 55 mM to about 60 mM, about 15 mM to about 55 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, or a value within one of these ranges. Specific examples can include about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, or a range between any two of these values.

[0183] In some embodiments, the Na of the second solution + The initial total concentration is about 15 mM to about 60 mM.

[0184] In some embodiments, the Na of the second solution + The initial total concentration is about 30 mM.

[0185] In some embodiments, the Na of the second solution + The intermediate total concentration is about 100 mM to about 300 mM, about 125 mM to about 300 mM, about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300 mM, about 275 mM to about 300 mM, about 100 mM to about 275 mM, about 100 mM to about 250 mM, about 100 mM to about 225 mM, about 100 mM to about 200 mM, about 100 mM to about 175 mM, about 100 mM to about 150 mM, about 100 mM to about 125 mM, or a value within one of these ranges. Specific examples can include about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, or a range between any two of these values.

[0186] In some embodiments, the Na of the second solution + The intermediate total concentration is about 100 mM to about 300 mM.

[0187] In some embodiments, the Na of the second solution + The mean total concentration is approximately 200 mM.

[0188] In some embodiments, the Na of the second solution + The final total concentration of is about 500 mM to about 1500 mM, about 600 mM to about 1500 mM, about 700 mM to about 1500 mM, about 800 mM to about 1500 mM, about 900 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1100 mM to about 1500 mM, about 1200 mM to about 1500 mM, about 1300 mM to about 1500 mM, about 1400 mM to about 1500 mM, about 1500 mM to about 1500 mM, about 1600 mM to about 1500 mM, about 1700 mM to about 1500 mM, about 1800 mM to about 1500 mM, about 1900 mM to about 1500 mM, about 2000 mM to about 2000 mM, about 2100 mM to about 2000 mM, about 2200 mM to about 2000 mM, about 2300 mM to about 2000 mM, about 2400 mM to about 2500 mM, about 2500 mM to about 2600 mM, about 2600 mM to about 2700 mM, about 2700 mM to about 2800 mM, about 2800 mM to about 2900 mM, about 2900 mM to about 3000 mM, about 3000 mM to about 3000 mM, about 3100 mM to about 3100 mM, about 3200 mM to about 3200 mM, about 3300 mM to about 3300 mM, about 3400 mM to about 3400 mM, about 3500 mM to about 3500 mM, about 3600 mM to about 3600 00 mM, about 500 mM to about 1400 mM, about 500 mM to about 1300 mM, about 500 mM to about 1200 mM, about 500 mM to about 1100 mM, about 500 mM to about 1000 mM, about 500 mM to about 900 mM, about 500 mM to about 800 mM, about 500 mM to about 700 mM, about 500 mM to about 600 mM, or a value within one of these ranges. Specific examples include about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, or a range between any two of these values.

[0189] In some embodiments, the Na of the second solution + The final total concentration is about 500 mM to about 1500 mM.

[0190] In some embodiments, the Na of the second solution + The final total concentration is approximately 1000 mM.

[0191] In some embodiments, the addition of the second solution comprises a stepwise increase in the total concentration of one or more divalent cations.

[0192] In some embodiments, the initial total concentration of the one or more divalent cations in the second solution is about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, or a range between any two of these values.

[0193] In some embodiments, the initial total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 10 mM.

[0194] In some embodiments, the intermediate total concentration of one or more divalent cations in the second solution is between about 10 mM and about 20 mM, between about 11 mM and about 20 mM, between about 12 mM and about 20 mM, between about 13 mM and about 20 mM, between about 14 mM and about 20 mM, between about 15 mM and about 20 mM, between about 16 mM and about 20 mM, between about 17 mM and about 20 mM, between about 18 mM and about 20 mM, or between about 20 mM and about 20 mM. mM, about 19 mM to about 20 mM, about 10 mM to about 19 mM, about 10 mM to about 18 mM, about 10 mM to about 17 mM, about 10 mM to about 16 mM, about 10 mM to about 15 mM, about 10 mM to about 14 mM, about 10 mM to about 13 mM, about 10 mM to about 12 mM, about 10 mM to about 11 mM, or a value within one of these ranges. Specific examples include about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, or a range between any two of these values.

[0195] In some embodiments, the intermediate total concentration of the one or more divalent cations in the second solution is from about 10 mM to about 20 mM.

[0196] In some embodiments, the final total concentration of the one or more divalent cations in the second solution is about 20 mM to about 30 mM, about 21 mM to about 30 mM, about 22 mM to about 30 mM, about 23 mM to about 30 mM, about 24 mM to about 30 mM, about 25 mM to about 30 mM, about 26 mM to about 30 mM, about 27 mM to about 30 mM, about 28 mM to about 30 mM, or about 31 mM to about 31 mM. mM, about 29 mM to about 30 mM, about 20 mM to about 29 mM, about 20 mM to about 28 mM, about 20 mM to about 27 mM, about 20 mM to about 26 mM, about 20 mM to about 25 mM, about 20 mM to about 24 mM, about 20 mM to about 23 mM, about 20 mM to about 22 mM, about 20 mM to about 21 mM, or a value within one of these ranges. Specific examples include about 20 mM, about 21 mM, about 22 mM, about 23, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0197] In some embodiments, the final total concentration of the one or more divalent cations in the second solution is about 20 mM to about 30 mM.

[0198] In some embodiments, the addition of the second solution comprises Ca 2+ The method includes gradually increasing the total concentration of

[0199] In some embodiments, the Ca of the second solution 2+ The initial total concentration is about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, or a value within one of these ranges. Specific examples can include about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, or a range between any two of these values.

[0200] In some embodiments, the Ca of the second solution 2+ The initial total concentration is about 1 mM to about 10 mM.

[0201] In some embodiments, the Ca of the second solution 2+ The intermediate total concentration is about 10 mM to about 20 mM, about 11 mM to about 20 mM, about 12 mM to about 20 mM, about 13 mM to about 20 mM, about 14 mM to about 20 mM, about 15 mM to about 20 mM, about 16 mM to about 20 mM, about 17 mM to about 20 mM, about 18 mM to about 20 mM, about 19 mM to about 20 mM, about 10 mM to about 19 mM, about 10 mM to about 18 mM, about 10 mM to about 17 mM, about 10 mM to about 16 mM, about 10 mM to about 15 mM, about 10 mM to about 14 mM, about 10 mM to about 13 mM, about 10 mM to about 12 mM, about 10 mM to about 11 mM, or a value within one of these ranges. Specific examples can include about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, or a range between any two of these values.

[0202] In some embodiments, the Ca of the second solution 2+ The intermediate total concentration is about 10 mM to about 20 mM.

[0203] In some embodiments, the Ca of the second solution 2+The final total concentration of is about 20 mM to about 30 mM, about 21 mM to about 30 mM, about 22 mM to about 30 mM, about 23 mM to about 30 mM, about 24 mM to about 30 mM, about 25 mM to about 30 mM, about 26 mM to about 30 mM, about 27 mM to about 30 mM, about 28 mM to about 30 mM, about 29 mM to about 30 mM, about 20 mM to about 29 mM, about 20 mM to about 28 mM, about 20 mM to about 27 mM, about 20 mM to about 26 mM, about 20 mM to about 25 mM, about 20 mM to about 24 mM, about 20 mM to about 23 mM, about 20 mM to about 22 mM, about 20 mM to about 21 mM, or a value within one of these ranges. Specific examples can include about 20 mM, about 21 mM, about 22 mM, about 23, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, or a range between any two of these values.

[0204] In some embodiments, the Ca of the second solution 2+ The final total concentration is about 20 mM to about 30 mM.

[0205] In some embodiments, the addition of the second solution comprises a continuous linear increase in the total concentration of one or more monovalent cations.

[0206] In some embodiments, the continuous linear increase in the total concentration of one or more monovalent cations in the second solution is from about 30 mM to about 200 mM in 40 column volumes, from about 40 mM to about 200 mM in 40 column volumes, from about 50 mM to about 200 mM in 40 column volumes, from about 75 mM to about 200 mM in 40 column volumes, from about 100 mM to about 200 mM in 40 column volumes, from about 125 mM to about 200 mM in 40 column volumes, from about 150 mM to about 200 mM in 40 column volumes, and from about 200 mM to about 200 mM in 40 column volumes. In one embodiment, the amount of the glycerol may be about 175 mM to about 200 mM in a column volume, about 30 mM to about 175 mM in a column volume of 40, about 30 mM to about 200 mM in a column volume of 150, about 30 mM to about 125 mM in a column volume of 40, about 30 mM to about 100 mM in a column volume of 40, about 30 mM to about 80 mM in a column volume of 40, about 30 mM to about 75 mM in a column volume of 40, about 30 mM to about 50 mM in a column volume of 40, about 30 mM to about 40 mM in a column volume of 40, or a value within one of these ranges.

[0207] In some embodiments, the continuous linear increase in the total concentration of one or more monovalent cations in the second solution can be from about 30 mM to about 200 mM in 30 column volumes, from about 40 mM to about 200 mM in 30 column volumes, from about 50 mM to about 200 mM in 30 column volumes, from about 75 mM to about 200 mM in 30 column volumes, from about 100 mM to about 200 mM in 30 column volumes, from about 125 mM to about 200 mM in 30 column volumes, from about 150 mM to about 200 mM in 30 column volumes, and from about 200 mM to about 200 mM in 30 column volumes. In one embodiment, the amount of the glycerol may be about 175 mM to about 200 mM in a column volume, about 30 mM to about 175 mM in 30 column volumes, about 30 mM to about 200 mM in 150 column volumes, about 30 mM to about 125 mM in 30 column volumes, about 30 mM to about 100 mM in 30 column volumes, about 30 mM to about 80 mM in 30 column volumes, about 30 mM to about 75 mM in 30 column volumes, about 30 mM to about 50 mM in 30 column volumes, about 30 mM to about 40 mM in 30 column volumes, or a value within one of these ranges.

[0208] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution can be from about 30 mM to about 200 mM in 20 column volumes, from about 40 mM to about 200 mM in 20 column volumes, from about 50 mM to about 200 mM in 20 column volumes, from about 75 mM to about 200 mM in 20 column volumes, from about 100 mM to about 200 mM in 20 column volumes, from about 125 mM to about 200 mM in 20 column volumes, from about 150 mM to about 200 mM in 20 column volumes, and from about 200 mM to about 200 mM in 20 column volumes. In one embodiment, the amount of the glycerol may be about 175 mM to about 200 mM in a column volume, about 30 mM to about 175 mM in 20 column volumes, about 30 mM to about 200 mM in 150 column volumes, about 30 mM to about 125 mM in 20 column volumes, about 30 mM to about 100 mM in 20 column volumes, about 30 mM to about 80 mM in 20 column volumes, about 30 mM to about 75 mM in 20 column volumes, about 30 mM to about 50 mM in 20 column volumes, about 30 mM to about 40 mM in 20 column volumes, or a value within one of these ranges.

[0209] In some embodiments, the continuous linear increase in the total concentration of one or more monovalent cations in the second solution can be from about 30 mM to about 200 mM in 10 column volumes, from about 40 mM to about 200 mM in 10 column volumes, from about 50 mM to about 200 mM in 10 column volumes, from about 75 mM to about 200 mM in 10 column volumes, from about 100 mM to about 200 mM in 10 column volumes, from about 125 mM to about 200 mM in 10 column volumes, from about 150 mM to about 200 mM in 10 column volumes, and from about 100 mM to about 200 mM in 10 column volumes. about 175 mM to about 200 mM in 10 column volumes, about 30 mM to about 175 mM in 10 column volumes, about 30 mM to about 200 mM in 150 column volumes, about 30 mM to about 125 mM in 10 column volumes, about 30 mM to about 100 mM in 10 column volumes, about 30 mM to about 80 mM in 10 column volumes, about 30 mM to about 75 mM in 10 column volumes, about 30 mM to about 50 mM in 10 column volumes, about 30 mM to about 40 mM in 10 column volumes, or a value within one of these ranges.

[0210] In some embodiments, the successive linear increases in the total concentration of the one or more monovalent cations in the second solution are from about 30 mM to about 200 mM in 5 column volumes, from about 40 mM to about 200 mM in 5 column volumes, from about 50 mM to about 200 mM in 5 column volumes, from about 75 mM to about 200 mM in 5 column volumes, from about 100 mM to about 200 mM in 5 column volumes, from about 125 mM to about 200 mM in 5 column volumes, from about 150 mM to about 200 mM in 5 column volumes, and from about 150 mM to about 200 mM in 5 column volumes. about 175 mM to about 200 mM in 5 column volumes, about 30 mM to about 175 mM in 5 column volumes, about 30 mM to about 200 mM in 150 column volumes, about 30 mM to about 125 mM in 5 column volumes, about 30 mM to about 100 mM in 5 column volumes, about 30 mM to about 80 mM in 5 column volumes, about 30 mM to about 75 mM in 5 column volumes, about 30 mM to about 50 mM in 5 column volumes, about 30 mM to about 40 mM in 5 column volumes, or a value within one of these ranges.

[0211] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM over 40 column volumes.

[0212] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over 40 column volumes.

[0213] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 40 mM over 40 column volumes.

[0214] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM over 30 column volumes.

[0215] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over 30 column volumes.

[0216] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 40 mM over 30 column volumes.

[0217] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM over 20 column volumes.

[0218] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over 20 column volumes.

[0219] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 40 mM over 20 column volumes.

[0220] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM over 10 column volumes.

[0221] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over 10 column volumes.

[0222] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 40 mM over 10 column volumes.

[0223] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM over five column volumes.

[0224] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 80 mM over five column volumes.

[0225] In some embodiments, the continuous linear increase in the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 40 mM over five column volumes.

[0226] In some embodiments, the amount of column volumes can be reduced once the exact separation characteristics of a particular construct (e.g., AAV construct) have been evaluated. By way of example and not limitation, if the difference in molar concentration of monovalent cations between the equilibration buffer and the elution buffer is, for example, 5 mM to 10 mM, the column volumes of the gradient can be reduced to (or less than) 5 column volumes (e.g., 30 mM Na in 5 column volumes). + to 40 mM Na + gradient to .

[0227] In some embodiments, the addition of the second solution is Na + The method includes continuously linearly increasing the total concentration of

[0228] In some embodiments, the Na of the second solution +The continuous linear increase in the total concentration of is about 30 mM to about 200 mM at 40 column volumes, about 40 mM to about 200 mM at 40 column volumes, about 50 mM to about 200 mM at 40 column volumes, about 75 mM to about 200 mM at 40 column volumes, about 100 mM to about 200 mM at 40 column volumes, about 125 mM to about 200 mM at 40 column volumes, about 150 mM to about 200 mM at 40 column volumes, and about 175 mM to about 200 mM at 40 column volumes. , about 30 mM to about 175 mM at 40 column volumes, about 30 mM to about 200 mM at 150 column volumes, about 30 mM to about 125 mM at 40 column volumes, about 30 mM to about 100 mM at 40 column volumes, about 30 mM to about 80 mM at 40 column volumes, about 30 mM to about 75 mM at 40 column volumes, about 30 mM to about 50 mM at 40 column volumes, about 30 mM to about 40 mM at 40 column volumes, or a value within one of these ranges.

[0229] In some embodiments, the Na of the second solution + The continuous linear increase in the total concentration of is about 30 mM to about 200 mM in 30 column volumes, about 40 mM to about 200 mM in 30 column volumes, about 50 mM to about 200 mM in 30 column volumes, about 75 mM to about 200 mM in 30 column volumes, about 100 mM to about 200 mM in 30 column volumes, about 125 mM to about 200 mM in 30 column volumes, about 150 mM to about 200 mM in 30 column volumes, and about 175 mM to about 200 mM in 30 column volumes. , about 30 mM to about 175 mM at 30 column volumes, about 30 mM to about 200 mM at 150 column volumes, about 30 mM to about 125 mM at 30 column volumes, about 30 mM to about 100 mM at 30 column volumes, about 30 mM to about 80 mM at 30 column volumes, about 30 mM to about 75 mM at 30 column volumes, about 30 mM to about 50 mM at 30 column volumes, about 30 mM to about 40 mM at 30 column volumes, or a value within one of these ranges.

[0230] In some embodiments, the Na of the second solution +The continuous linear increase in the total concentration of is about 30 mM to about 200 mM in 20 column volumes, about 40 mM to about 200 mM in 20 column volumes, about 50 mM to about 200 mM in 20 column volumes, about 75 mM to about 200 mM in 20 column volumes, about 100 mM to about 200 mM in 20 column volumes, about 125 mM to about 200 mM in 20 column volumes, about 150 mM to about 200 mM in 20 column volumes, and about 175 mM to about 200 mM in 20 column volumes. , about 30 mM to about 175 mM at 20 column volumes, about 30 mM to about 200 mM at 150 column volumes, about 30 mM to about 125 mM at 20 column volumes, about 30 mM to about 100 mM at 20 column volumes, about 30 mM to about 80 mM at 20 column volumes, about 30 mM to about 75 mM at 20 column volumes, about 30 mM to about 50 mM at 20 column volumes, about 30 mM to about 40 mM at 20 column volumes, or a value within one of these ranges.

[0231] In some embodiments, the Na of the second solution + The continuous linear increase in the total concentration of is about 30 mM to about 200 mM in 10 column volumes, about 40 mM to about 200 mM in 10 column volumes, about 50 mM to about 200 mM in 10 column volumes, about 75 mM to about 200 mM in 10 column volumes, about 100 mM to about 200 mM in 10 column volumes, about 125 mM to about 200 mM in 10 column volumes, about 150 mM to about 200 mM in 10 column volumes, and about 175 mM to about 200 mM in 10 column volumes. , about 30 mM to about 175 mM at 10 column volumes, about 30 mM to about 200 mM at 150 column volumes, about 30 mM to about 125 mM at 10 column volumes, about 30 mM to about 100 mM at 10 column volumes, about 30 mM to about 80 mM at 10 column volumes, about 30 mM to about 75 mM at 10 column volumes, about 30 mM to about 50 mM at 10 column volumes, about 30 mM to about 40 mM at 10 column volumes, or a value within one of these ranges.

[0232] In some embodiments, the Na of the second solution +A continuous linear increase in the total concentration of is from about 30 mM to about 200 mM in 5 column volumes, from about 40 mM to about 200 mM in 5 column volumes, from about 50 mM to about 200 mM in 5 column volumes, from about 75 mM to about 200 mM in 5 column volumes, from about 100 mM to about 200 mM in 5 column volumes, from about 125 mM to about 200 mM in 5 column volumes, from about 150 mM to about 200 mM in 5 column volumes, from about 175 mM to about 200 mM in 5 column volumes, about 30 mM to about 175 mM at 5 column volumes, about 30 mM to about 200 mM at 150 column volumes, about 30 mM to about 125 mM at 5 column volumes, about 30 mM to about 100 mM at 5 column volumes, about 30 mM to about 80 mM at 5 column volumes, about 30 mM to about 75 mM at 5 column volumes, about 30 mM to about 50 mM at 5 column volumes, about 30 mM to about 40 mM at 5 column volumes, or a value within one of these ranges.

[0233] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 200 mM over 40 column volumes.

[0234] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 80 mM over 40 column volumes.

[0235] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 40 mM over 40 column volumes.

[0236] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 200 mM over 30 column volumes.

[0237] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 80 mM over 30 column volumes.

[0238] In some embodiments, the Na of the second solution +The continuous linear increase in total concentration is from about 30 mM to about 40 mM over 30 column volumes.

[0239] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 200 mM over 20 column volumes.

[0240] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 80 mM over 20 column volumes.

[0241] In some embodiments, the Na of the second solution + The continuous linear increase in the total concentration of is from about 30 mM to about 40 mM over 20 column volumes.

[0242] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 200 mM over 10 column volumes.

[0243] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 80 mM over 10 column volumes.

[0244] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration of is from about 30 mM to about 400 in 10 column volumes.

[0245] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 200 mM over 5 column volumes.

[0246] In some embodiments, the Na of the second solution + The continuous linear increase in total concentration is from about 30 mM to about 80 mM over five column volumes.

[0247] In some embodiments, the Na of the second solution + The continuous linear increase in the total concentration of is from about 30 mM to about 40 mM over 5 column volumes.

[0248] In some embodiments, the addition of the second solution comprises a continuous linear increase in the total concentration of one or more divalent cations.

[0249] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM in 40 column volumes, from 5 mM to about 30 mM in 40 column volumes, from 10 mM to about 30 mM in 40 column volumes, from 15 mM to about 30 mM in 40 column volumes, from 20 mM to about 30 mM in 40 column volumes, from 25 mM to about 30 mM in 40 column volumes, from 1 mM to about 25 mM in 40 column volumes, from 1 mM to about 20 mM in 40 column volumes, from 1 mM to about 15 mM in 40 column volumes, from 1 mM to about 10 mM in 40 column volumes, from 1 mM to about 5 mM in 40 column volumes, or a value within one of these ranges.

[0250] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM in 30 column volumes, from 5 mM to about 30 mM in 30 column volumes, from 10 mM to about 30 mM in 30 column volumes, from 15 mM to about 30 mM in 30 column volumes, from 20 mM to about 30 mM in 30 column volumes, from 25 mM to about 30 mM in 30 column volumes, from 1 mM to about 25 mM in 30 column volumes, from 1 mM to about 20 mM in 30 column volumes, from 1 mM to about 15 mM in 30 column volumes, from 1 mM to about 10 mM in 30 column volumes, from 1 mM to about 5 mM in 30 column volumes, or a value within one of these ranges.

[0251] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM in 20 column volumes, from 5 mM to about 30 mM in 20 column volumes, from 10 mM to about 30 mM in 20 column volumes, from 15 mM to about 30 mM in 20 column volumes, from 20 mM to about 30 mM in 20 column volumes, from 25 mM to about 30 mM in 20 column volumes, from 1 mM to about 25 mM in 20 column volumes, from 1 mM to about 20 mM in 20 column volumes, from 1 mM to about 15 mM in 20 column volumes, from 1 mM to about 10 mM in 20 column volumes, from 1 mM to about 5 mM in 20 column volumes, or a value within one of these ranges.

[0252] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM in 10 column volumes, from 5 mM to about 30 mM in 10 column volumes, from 10 mM to about 30 mM in 10 column volumes, from 15 mM to about 30 mM in 10 column volumes, from 20 mM to about 30 mM in 10 column volumes, from 25 mM to about 30 mM in 10 column volumes, from 1 mM to about 25 mM in 10 column volumes, from 1 mM to about 20 mM in 10 column volumes, from 1 mM to about 15 mM in 10 column volumes, from 1 mM to about 10 mM in 10 column volumes, from 1 mM to about 5 mM in 10 column volumes, or a value within one of these ranges.

[0253] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM in 5 column volumes, from 5 mM to about 30 mM in 5 column volumes, from 10 mM to about 30 mM in 5 column volumes, from 15 mM to about 30 mM in 5 column volumes, from 20 mM to about 30 mM in 5 column volumes, from 25 mM to about 30 mM in 5 column volumes, from 1 mM to about 25 mM in 5 column volumes, from 1 mM to about 20 mM in 5 column volumes, from 1 mM to about 15 mM in 5 column volumes, from 1 mM to about 10 mM in 5 column volumes, from 1 mM to about 5 mM in 5 column volumes, or a value within one of these ranges.

[0254] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM over 40 column volumes.

[0255] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 15 mM over 40 column volumes.

[0256] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 5 mM over 40 column volumes.

[0257] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM over 30 column volumes.

[0258] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 15 mM over 30 column volumes.

[0259] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 5 mM over 30 column volumes.

[0260] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM over 20 column volumes.

[0261] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 15 mM over 20 column volumes.

[0262] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 5 mM over 20 column volumes.

[0263] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM over 10 column volumes.

[0264] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 15 mM over 10 column volumes.

[0265] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 5 mM over 10 column volumes.

[0266] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 30 mM over five column volumes.

[0267] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 15 mM over five column volumes.

[0268] In some embodiments, the continuous linear increase in the total concentration of the one or more divalent cations in the second solution is from about 1 mM to about 5 mM over five column volumes.

[0269] In some embodiments, the addition of the second solution comprises Ca 2+ The method includes continuously linearly increasing the total concentration of

[0270] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM in 40 column volumes, 5 mM to about 30 mM in 40 column volumes, 10 mM to about 30 mM in 40 column volumes, 15 mM to about 30 mM in 40 column volumes, 20 mM to about 30 mM in 40 column volumes, 25 mM to about 30 mM in 40 column volumes, 1 mM to about 25 mM in 40 column volumes, 1 mM to about 20 mM in 40 column volumes, 1 mM to about 15 mM in 40 column volumes, 1 mM to about 10 mM in 40 column volumes, 1 mM to about 5 mM in 40 column volumes, or a value within one of these ranges.

[0271] In some embodiments, the Ca of the second solution 2+The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM in 30 column volumes, 5 mM to about 30 mM in 30 column volumes, 10 mM to about 30 mM in 30 column volumes, 15 mM to about 30 mM in 30 column volumes, 20 mM to about 30 mM in 30 column volumes, 25 mM to about 30 mM in 30 column volumes, 1 mM to about 25 mM in 30 column volumes, 1 mM to about 20 mM in 30 column volumes, 1 mM to about 15 mM in 30 column volumes, 1 mM to about 10 mM in 30 column volumes, 1 mM to about 5 mM in 30 column volumes, or a value within one of these ranges.

[0272] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM in 20 column volumes, 5 mM to about 30 mM in 20 column volumes, 10 mM to about 30 mM in 20 column volumes, 15 mM to about 30 mM in 20 column volumes, 20 mM to about 30 mM in 20 column volumes, 25 mM to about 30 mM in 20 column volumes, 1 mM to about 25 mM in 20 column volumes, 1 mM to about 20 mM in 20 column volumes, 1 mM to about 15 mM in 20 column volumes, 1 mM to about 10 mM in 20 column volumes, 1 mM to about 5 mM in 20 column volumes, or a value within one of these ranges.

[0273] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM in 10 column volumes, 5 mM to about 30 mM in 10 column volumes, 10 mM to about 30 mM in 10 column volumes, 15 mM to about 30 mM in 10 column volumes, 20 mM to about 30 mM in 10 column volumes, 25 mM to about 30 mM in 10 column volumes, 1 mM to about 25 mM in 10 column volumes, 1 mM to about 20 mM in 10 column volumes, 1 mM to about 15 mM in 10 column volumes, 1 mM to about 10 mM in 10 column volumes, 1 mM to about 5 mM in 10 column volumes, or a value within one of these ranges.

[0274] In some embodiments, the Ca of the second solution 2+The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM in 5 column volumes, 5 mM to about 30 mM in 5 column volumes, 10 mM to about 30 mM in 5 column volumes, 15 mM to about 30 mM in 5 column volumes, 20 mM to about 30 mM in 5 column volumes, 25 mM to about 30 mM in 5 column volumes, 1 mM to about 25 mM in 5 column volumes, 1 mM to about 20 mM in 5 column volumes, 1 mM to about 15 mM in 5 column volumes, 1 mM to about 10 mM in 5 column volumes, 1 mM to about 5 mM in 5 column volumes, or a value within one of these ranges.

[0275] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM over 40 column volumes.

[0276] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 15 mM over 40 column volumes.

[0277] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 5 mM over 40 column volumes.

[0278] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM over 30 column volumes.

[0279] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 15 mM over 30 column volumes.

[0280] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 5 mM over 30 column volumes.

[0281] In some embodiments, the Ca of the second solution 2+The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM over 20 column volumes.

[0282] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 15 mM over 20 column volumes.

[0283] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 5 mM over 20 column volumes.

[0284] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM at 0 column volumes.

[0285] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 15 mM at 0 column volumes.

[0286] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 5 mM at 0 column volumes.

[0287] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in the total concentration of is from about 1 mM to about 30 mM over 5 column volumes.

[0288] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 15 mM over 5 column volumes.

[0289] In some embodiments, the Ca of the second solution 2+ The continuous linear increase in total concentration is from about 1 mM to about 5 mM over 5 column volumes.

[0290] In some embodiments, the AAV capsid is derived from the group consisting of AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, genetically modified AAV, chemically modified AAV, genetically and chemically modified AAV, and combinations thereof.

[0291] In some embodiments, the AAV capsid is derived from AAV8.

[0292] In some embodiments, the AAV capsid is derived from AAV9.

[0293] In some embodiments, the AAV capsid is derived from AAV6.

[0294] In some embodiments, the one or more surfactants (e.g., in the first solution or the second solution) are selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polyoxyethylene glycol tert-octylphenol ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, polyoxyethylene(20) sorbitan monopalmitate, polyoxyethylene(20) sorbitan monostearate, polyoxyethylene(20) sorbitan tristearate, polyoxyethylene(20) sorbitan trioleate, polyoxyethylene(20)-sorbitan-monooleate (Tween 80 / Polysorbate 80), poloxamer 124, poloxamer 188, poloxamer 407, cremophor, Triton N-101 reduced, Triton X-100, and combinations thereof.

[0295] In some embodiments, the one or more surfactants (eg, in the first solution or the second solution) are selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 124, and combinations thereof.

[0296] In some embodiments, the one or more surfactants (eg, of the first solution or the second solution) is polysorbate 80.

[0297] In some embodiments, the one or more surfactants (eg, of the first solution or the second solution) is polysorbate 20.

[0298] In some embodiments, the one or more surfactants (eg, of the first solution or the second solution) is polysorbate 124.

[0299] In some embodiments, the one or more surfactants (e.g., in the first solution or the second solution) are present at about 0.0025 w / w% to about 0.0075 w / w%, about 0.003 w / w% to about 0.0075 w / w%, about 0.0035 w / w% to about 0.0075 w / w%, about 0.004 w / w% to about 0.0075 w / w%, about 0.005 w / w% to about 0.005 w / w%, about 0.006 w / w% to about 0.0075 w / w%, about 0.007 ... .0045w / w% ~ approx. 0.0075w / w%, approx. 0.005w / w% ~ approx. 0.0075w / w%, approx. 0.0055w / w% ~ approx. 0.0075w / w%, approx. 0.006w / w% ~ approx. 0.0075w / w%, approx. 0.0065w / w% ~ approx. 0.0075w / w%, approx. 0.007w / w% ~ approx. 0.0075w / w%, approx. 0 .0025w / w% ~ approx. 0.0070w / w%, approx. 0.0025w / w% ~ approx. 0.0065w / w%, approx. 0.0025w / w% ~ approx. 0.006w / w%, approx. 0.0025w / w%~Approx. 0.0065w / w%, Approx. 0.0025w / w%~Approx. 0.006w / w%, Approx. 0.0025w / w%~Approx. 0.0055w / w%, Approx. The total amount is from 0.0025 w / w% to about 0.005 w / w%, from about 0.0025 w / w% to about 0.0045 w / w%, from about 0.0025 w / w% to about 0.004 w / w%, from about 0.0025 w / w% to about 0.0035 w / w%, from about 0.0025 w / w% to about 0.003 w / w%, or a value within one of these ranges. Specific examples include about 0.0025 w / w%, about 0.003 w / w%, about 0.0035 w / w%, about 0.004 w / w%, about 0.0045 w / w%, about 0.005 w / w%, about 0.0055 w / w%, about 0.006 w / w%, about 0.0065 w / w%, about 0.007 w / w%, about 0.0075 w / w%, or a range between any two of these values.

[0300] In some embodiments, the one or more surfactants (eg, in the first solution or the second solution) are in a total amount of about 0.005 w / w%.

[0301] In certain embodiments, a method for purifying empty AAV capsids from an AAV preparation or fraction comprising empty and complete AAV capsids to provide an AAV product, formulation, or composition that is substantially free of complete AAV capsids, comprising: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the empty AAV capsid and the full AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions whereby empty AAV capsids are purified from full AAV capsids.

[0302] In some embodiments, the purified AAV product, formulation, or composition comprises less than about 50%, about 45%, about 40%, about 35%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% empty AAV capsids. In some embodiments, the purified AAV product, formulation, or composition comprises less than about 30% empty AAV capsids. In some embodiments, the purified AAV product, formulation, or composition comprises less than about 20% empty AAV capsids. In some embodiments, a purified AAV product, preparation, or composition contains less than about 6% empty AAV capsids.

[0303] In certain embodiments, a method for separating empty and full AAV capsids in an AAV preparation or fraction comprises the steps of: (a) providing a first solution comprising empty AAV capsids, full AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions such that the empty AAV capsid and the full AAV capsid bind to the column; (c) applying a second solution containing one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that empty AAV capsids are separated from full AAV capsids.

[0304] In certain embodiments, low conductivity zones may select for full or substantially full capsids, while high conductivity zones may select for empty or substantially empty capsids.

[0305] In some embodiments, the empty AAV capsid is derived from the group consisting of AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, genetically modified AAV, chemically modified AAV, genetically and chemically modified AAV, and combinations thereof.

[0306] In some embodiments, the empty AAV capsid is derived from AAV8.

[0307] In some embodiments, the empty AAV capsid is derived from AAV9.

[0308] In some embodiments, the empty AAV capsid is derived from AAV6.

[0309] Some embodiments provide a method of preparing an immunoabsorption column, comprising: (a) concentrating the empty AAV capsids of any of the embodiments disclosed herein by ultrafiltration; (b) buffer exchange into an amine-free buffer; (c) immobilizing the empty AAV capsid on an activated resin.

[0310] Some embodiments provide a method of preparing an immunoabsorption column, comprising: (a) concentrating the empty AAV capsid of any of the embodiments disclosed herein by anion exchange; (b) buffer exchange into an amine-free buffer; (c) immobilizing the empty AAV capsid on an activated resin.

[0311] Some embodiments provide a method of preparing an immunoabsorption column, comprising: (a) concentrating the empty AAV capsid of any of the embodiments disclosed herein by a cation exchanger; (b) buffer exchange into an amine-free buffer; (c) immobilizing the empty AAV capsid on an activated resin.

[0312] Some embodiments provide a method of preparing an immunoabsorption column, comprising: (a) concentrating the intact AAV capsid of any of the embodiments disclosed herein by ultrafiltration; (b) buffer exchanging with a buffer suitable for treatment of the patient.

[0313] 1. A method for preparing an immunoabsorption column comprising the steps of: (a) concentrating the intact AAV capsid of any of the embodiments disclosed herein by anion exchange; (b) buffer exchanging with a buffer suitable for treatment of the patient.

[0314] Some embodiments provide a method of preparing an immunoabsorption column, comprising: (a) concentrating the complete AAV capsid of any of the embodiments disclosed herein by a cation exchanger; (b) buffer exchanging with a buffer suitable for treatment of the patient.

[0315] In some embodiments, immobilization of the empty AAV capsid onto the activated resin occurs at a temperature of about 2°C to about 37°C, about 5°C to about 37°C, about 10°C to about 37°C, about 15°C to about 37°C, about 20°C to about 37°C, about 25°C to about 37°C, about 30°C to about 37°C, about 35°C to about 37°C, about 2°C to about 35°C, about 2°C to about 30°C, about 2°C to about 25°C, about 2°C to about 20°C, about 2°C to about 15°C, about 2°C to about 10°C, about 2°C to about 5°C, about 5°C to about 35°C, about 10°C to about 30°C, about 15°C to about 25°C, or a value within one of these ranges. Specific examples include about 2°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, or a range between any two of these values.

[0316] In some embodiments, immobilization of empty AAV capsids onto an activated resin occurs from about 2 hours (hrs) to about 20 hours, from about 2 hours to about 19 hours, from about 2 hours to about 18 hours, from about 2 hours to about 17 hours, from about 2 hours to about 16 hours, from about 2 hours to about 15 hours, from about 2 hours to about 14 hours, from about 2 hours to about 13 hours, from about 2 hours to about 12 hours, from about 2 hours to about 11 hours, from about 2 hours to about 10 hours, from about 2 hours to about 9 hours, from about 2 hours to about 8 hours, from about 2 hours to about 7 hours, from about 2 hours to about 6 hours, from about 2 hours to about 5 hours, from about 2 hours to about 4 hours, from about 2 hours to about 3 hours, The reaction is carried out for a reaction time of about 3 hours to about 20 hours, about 4 hours to about 20 hours, about 5 hours to about 20 hours, about 6 hours to about 20 hours, about 7 hours to about 20 hours, about 8 hours to about 20 hours, about 9 hours to about 20 hours, about 10 hours to about 20 hours, about 11 hours to about 20 hours, about 12 hours to about 20 hours, about 13 hours to about 20 hours, about 14 hours to about 20 hours, about 15 hours to about 20 hours, about 16 hours to about 20 hours, about 17 hours to about 20 hours, about 18 hours to about 20 hours, about 19 hours to about 20 hours, or a value within one of these ranges. Specific examples can include about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, or a range between any two of these values.

[0317] In some embodiments, the amine-free buffer is selected from the group consisting of phosphate buffers, citrate buffers, carbonate buffers, acetate buffers, borate buffers, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and combinations thereof.

[0318] In some embodiments, the amine-free buffer is at a concentration of about 50 mM to about 150 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, about 75 mM to about 150 mM, about 100 mM to about 150 mM, about 75 mM to about 125 mM, or a value within one of these ranges. Specific examples include about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or a range between any two of these values.

[0319] In some embodiments, the amine-free buffer is at a concentration of about 100 mM.

[0320] In some embodiments, the amine-free buffer further comprises NaCl.

[0321] In some embodiments, NaCl is at a concentration of about 100 mM to about 200 mM, about 125 mM to about 200 mM, about 150 mM to about 200 mM, about 100 mM to about 175 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, or a value within one of these ranges. Specific examples include about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or a range between any two of these values.

[0322] In some embodiments, NaCl is at a concentration of about 150 mM.

[0323] In some embodiments, the amine-free buffer has a pH of about 7.0 to about 8.5, about 7.25 to about 8.5, about 7.5 to about 8.5, about 7.75 to about 8.5, about 8.0 to about 8.5, about 8.25 to about 8.5, about 7.0 to about 8.25, about 7.0 to about 8, about 7.0 to about 7.75, about 7.0 to about 7.5, about 7.0 to about 7.25, or a value within one of these ranges. Specific examples include about 7.0, about 7.25, about 7.5, about 7.75, about 8.0, about 8.25, about 8.5, or a range between any two of these values.

[0324] In some embodiments, the amine-free buffer has a pH of about 8.0 to about 8.5.

[0325] In some embodiments, the activated resin is selected from the group consisting of CNBr-Sepharose FF, NHS-Sepharose FF, Praesto® CNBr, Poros EP, and Poros AL.

[0326] In some embodiments, the amine-free buffer has a pH of about 6.5, about 7, about 7.5, about 8, or a range between any two of these values.

[0327] In some embodiments, the amine-free buffer has a pH of about 7.5.

[0328] In some embodiments, the anion exchanger is selected from the group consisting of Fractogel TMAE, Poros PI, Q Sepharose HP, Poros HQ, Toyopearl GigaCap Q 650, Cellufine Max Q, and Praesto Q.

[0329] In some embodiments, the anion exchanger is selected from the group consisting of Fractogel TMAE, Poros PI, and Poros HQ.

[0330] In some embodiments, the anion exchanger is Fractogel TMAE.

[0331] In some embodiments, the anion exchanger is Poros PI.

[0332] In some embodiments, the anion exchanger is Poros HQ.

[0333] In some embodiments, the cation exchanger is selected from the group consisting of Capto S, Eshmuno S, Mustang S, Cellufine sulfate, Cellufine phosphate, Toyopearl sulfate 650, Poros XS, Poros HS, and Praesto SP.

[0334] In some embodiments, the cation exchanger is selected from the group consisting of Capto S and Eshmuno S.

[0335] In some embodiments, the cation exchanger is Capto S.

[0336] In some embodiments, the cation exchanger is Eshmuno S.

[0337] By way of example, and not limitation, suitable candidate buffers for patient treatment can be found in WO2018128689A1 and WO2020014479A1, which are hereby incorporated by reference in their entireties for all purposes intended.

[0338] In some embodiments, the preferred buffer for treating patients is L-histidine.

[0339] In some embodiments, a buffer suitable for treating a patient has a concentration of about 5 mM to about 25 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM, or a value within one of these ranges. Specific examples include about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM, or a range between any two of these values.

[0340] In some embodiments, the buffer suitable for treatment of a patient further comprises NaCl.

[0341] In some embodiments, NaCl is at a concentration of about 100 mM to about 200 mM, about 125 mM to about 200 mM, about 150 mM to about 200 mM, about 100 mM to about 175 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, or a value within one of these ranges. Specific examples include about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or a range between any two of these values.

[0342] In some embodiments, NaCl is at a concentration of about 150 mM.

[0343] In some embodiments, a buffer suitable for treating a patient has a pH of about 6.5 to about 9.0, about 6.5 to about 8.0, about 6.9 to about 7.7, or about 7.0 to about 7.5, or a value within one of these ranges. Specific examples include about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, or a range between any two of these values.

[0344] In some embodiments, a buffer suitable for treating a patient has a pH of about 7.0.

[0345] In some embodiments, the amine-free buffer has a pH of about 8.0 to about 7.5.

[0346] Some embodiments are directed to methods for purifying empty AAV capsids from an AAV preparation or fraction, wherein the purified AAV product, formulation, composition, etc. contains less than about 40%, about 35%, about 30%, about 25%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% empty AAV capsids. In some embodiments, a purified AAV product, preparation, or composition contains between about 1% and about 30%, between about 4% and about 30%, between about 1% and about 20%, between about 1% and about 6%, between about 2% and about 20%, between about 3% and about 20%, between about 4% and about 20%, between about 5% and about 20%, between about 6% and about 20%, between about 4% and about 19%, between about 5% and about 19%, between about 4% and about 12%, between about 5% and about 12%, between about 4% and about 11%, or between about 5% and about 11% empty AAV capsids.

[0347] AAV formulations and AAV products Provided herein are AAV preparations comprising complete AAV capsids purified according to the methods described herein.

[0348] Also provided herein are pharmaceutical compositions comprising the AAV products produced by the methods described herein.

[0349] In exemplary embodiments, the AAV formulation or composition or product of the present disclosure comprises an additional pharma- ceutically acceptable ingredient. In exemplary aspects, the AAV formulation or composition or product comprises any one or combination of the following: acidifiers, additives, adsorbents, aerosol propellants, air displacing agents, alkalizing agents, anti-caking agents, anti-coagulating agents, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, pigments, emollients, emulsifiers, emulsion stabilizers. , a filler, a film former, a flavor enhancer, a flavoring agent, a glidant, a gelling agent, a granulating agent, a humectant, a lubricant, a mucoadhesive agent, an ointment base, an ointment, an oleaginous vehicle, an organic base, a lozenge base, a pigment, a plasticizer, an abrasive, a preservative, a sequestering agent, a skin penetration agent, a solubilizing agent, a solvent, a stabilizer, a suppository base, a surface active agent, a surfactant, a suspending agent, a sweetening agent, a therapeutic agent, a thickening agent, an isotonicity agent, a toxicological agent, a thickening agent, a water absorbing agent, a water miscible cosolvent, a water softener, or a humectant. In some embodiments, the AAV formulation or composition or AAV product of the disclosure comprises any one or combination of the following components: gum arabic, acesulfame potassium, acetyl tributyl citrate, acetyl triethyl citrate, agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, diluted alcohol, aleuritic acid, alginic acid, aliphatic polyester, alumina, aluminum hydroxide, aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite, bentonite. Tomagma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, sodium butylparaben, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, calcium phosphate dibasic anhydrous, calcium phosphate dibasic dehydrate, calcium phosphate tribasic, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate, calcium sulfate hemihydrate, canola oil, carbomer,Carbon dioxide, calcium carboxymethylcellulose, sodium carboxymethylcellulose, beta-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride hexidine, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons (CFC), chlorophenoxyethanol, chloroxylenol, corn syrup solid, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agent, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrin, dextrates, dextrin, dextrose, anhydrous dextrose, diazolidinyl urea, dibutyl phthalate, sebacine dibutyl acetate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-β-cyclodextrin, cyclodextrin compounds such as Captisol (registered trademark), dimethyl ether, dimethyl phthalate, dipotassium edetate, disodium edetate, disodium hydrogen phosphate, calcium docusate, potassium docusate, sodium docusate, dodecyl gallate, dodecyltrimethylammonium bromide, calcium disodium edetate, edetic acid, eglumin, ethyl alcohol, ethyl cellulose, ethyl gallate, Ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, potassium ethylparaben, sodium ethylparaben, ethyl vanillin, fructose, liquid fructose, ground fructose, pyrogen-free fructose, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, vegetable saturated fatty acid glyceride mixture, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitate stearate, glycine, glycol,Glycofurol, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, high fructose syrup, human serum albumin, hydrocarbons (HC), dilute hydrochloric acid, hydrogenated vegetable oil type II, hydroxyethyl cellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, imidurea, indigo carmine, ion exchangers, iron oxide, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohol, anhydrous lanolin, lecithin, aluminum magnesium silicate, magnesium carbonate, neutral magnesium carbonate, anhydrous magnesium carbonate, magnesium hydroxide carbonate, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, Malt, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methyl methacrylate, methyl oleate, methylparaben, potassium methylparaben, sodium methylparaben, microcrystalline cellulose and sodium carboxymethylcellulose, mineral oil, light mineral oil, mineral oil and lanolin alcohol, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, oleic acid, palmitic acid, paraffin, oleic acid ... latum, petrolatum and lanolin alcohols, pharmaceutical glazes, phenol, liquefied phenol, phenoxyethanol, phenoxypropanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamers, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates, polyethylene-polyoxypropylene-block polymers, polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,Polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearate, polyvinyl alcohol, polyvinylpyrrolidone, potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, potassium citrate, anhydrous potassium citrate, potassium hydrogen phosphate, potassium metabisulfite, monobasic potassium phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl gallate, propylparaben, potassium propylparaben, sodium propylparaben, protamine sulfate, rapeseed oil, Ringer's solution, saccharin, ammonium saccharin, calcium saccharin, sodium saccharin, safflower oil, saponite, serum proteins, sesame oil, colloidal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate Um, Sodium Bicarbonate, Sodium Bisulfite, Sodium Chloride, Sodium Citrate Anhydrous, Sodium Citrate Dehydrate, Sodium Chloride, Sodium Cyclamate, Sodium Disodium Ethyl Sulfate, Sodium Dodecyl Sulfate, Sodium Lauryl Sulfate, Sodium Metabisulfite, Sodium Phosphate Dibasic, Sodium Phosphate Monobasic, Sodium Tribasic, Sodium Propionate Anhydrous, Sodium Propionate, Sodium Sorbate, Sodium Starch Glycolate, Sodium Stearyl Fumarate, Sodium Sulfite, Sorbic Acid, Sorbitan Esters (Sorbitan Fatty Esters), Sorbitol, Sorbitol Solution 70%, Soybean Oil, Spermacetoacetate, Starch, Corn Starch, Potato Starch, Pregelatinized Starch, Sterilizable Corn Starch, Stearic Acid, Refined Stearic Acid, Stearyl Alcohol, Sucrose, Sugar, Compressed Sugar, Powdered Sugar, Granulated Sugar, Invert Sugar, Sugar Tabs, Sunset Yellow FCF, Synthetic Paraffin, Talc, Tartaric Acid, Tartrazine, Tetrafluoroethane (HFC), Theobroma Oil, Thimerosal, Titanium Dioxide, Alpha Tocopherol, Tocopherol Acetate, Alpha Tocopherol Succinate, Beta Tocopherol, Delta Tocopherol, Gamma Tocopherol, Tragacanth, Triacetin, Tributyl Citrate,Triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin, trimethyltetradecylammonium bromide, Tris buffer, trisodium edetate, vanillin, type I hydrogenated vegetable oil, water, soft water, hard water, carbon dioxide free water, pyrogen free water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for irrigation, wax, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax, non-ionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or any excipient from Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various components used in formulating pharma- ceutically acceptable AAV formulations or AAV products or AAV compositions and known techniques for their preparation, and is incorporated by reference in its entirety for all intended purposes. Any conventional agent is contemplated for use in the pharmaceutical AAV formulations or AAV products or AAV compositions unless it is incompatible with the pharmaceutical AAV formulations or AAV products or AAV compositions. In an exemplary embodiment, the AAV formulations or AAV products or AAV compositions of the present disclosure do not include one or a combination of the above components. In an exemplary embodiment, the AAV formulations or AAV products or AAV compositions of the present disclosure do not include any of these components. In an exemplary embodiment, the pharmaceutical AAV formulations or AAV products or AAV compositions of the present disclosure do not include dextran. In an exemplary embodiment, the pharmaceutical AAV formulations or AAV products or AAV compositions of the present disclosure do not include calcium chloride.

[0350] Quantitative and / or qualitative methods The methods of the disclosure include one or more quality control steps, e.g., measuring the concentration, dose, and / or potency of an AAV fraction, preparation, product, formulation, or composition obtained after one or more steps (e.g., after each step) of the purification process, including the final step to generate an AAV preparation, product, formulation, or composition for administration.

[0351] The methods of the present disclosure may include an ELISA assay specific for AAV (e.g., AAV antigen) to quantify the number of AAV capsids. In certain embodiments, the ELISA assay may include, but is not limited to, enzyme-linked immunosorbent assay, direct ELISA, indirect ELISA, sandwich ELISA, and / or competitive ELISA. In certain embodiments, the ELISA is a sandwich ELISA.

[0352] In certain embodiments, the AAV antigen is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or chimeric AAV antigen.In certain embodiments, the AAV antigen is AAV8 antigen.In certain embodiments, the AAV antigen is derived from recombinant AAV (rAAV).In certain embodiments, the AAV antigen is derived from genetically engineered AAV, or chemically modified AAV, or both.

[0353] In certain embodiments, the ELISA comprises an antibody specific for an AAV epitope. In certain embodiments, the AAV epitope is a conformational epitope present on assembled AAV capsids. In certain embodiments, the AAV epitope is a linear epitope present on assembled AAV capsids. Examples of such epitopes include, but are not limited to, capsid virion proteins VP1, VP2, and / or VP3. In certain embodiments, AAV is genetically engineered to express additional virion proteins on the surface of the capsid, and these engineered proteins can be used / detected in an ELISA assay. In certain embodiments, AAV is chemically modified to express variants of virion proteins, and can be used / detected in an ELISA assay (e.g., VP1', VP2', VP3', etc.). In certain embodiments, the antibody identifies a serotype-specific capsid virion protein.

[0354] ELISA can replace qPCR as a method for determining the dosage and / or potency of AAV fraction, preparation, product, formulation, or composition.The ELISA technique of the present invention has significantly less variability than qPCR methods.In certain embodiments, the method of the present disclosure comprises evaluating AAV fraction, preparation, product, formulation, or composition by AAV-specific ELISA.In certain embodiments, all methods disclosed herein do not include a qPCR step.

[0355] In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after ultracentrifugation by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after depth filtration by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after concentrating the AAV fraction or preparation using an ultrafiltration / diafiltration system by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after a tangential flow filtration (TFF) step by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after negative anion exchange (AEX) chromatography by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.In certain embodiments, the method of the present disclosure comprises testing the AAV fraction or preparation obtained after the polishing step by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.In certain embodiments, the method of the present disclosure comprises testing the purified AAV fraction or preparation by AAV-specific ELISA to determine the number of AAV capsids in the AAV fraction or preparation.

[0356] Additional methods for quantifying the number of AAV capsids include, but are not limited to, surface plasmon resonance (SPR) (e.g., BIACORE, OCTET), differential scanning fluorimetry (e.g., Prometheus NT48, Nanotemper), magnetic immunoassay (MIA), and cloned enzyme donor immunoassay (CEDIA). These methods can be used in addition to or instead of the ELISA quantification assay.

[0357] In certain embodiments, the methods of the disclosure include measuring full versus empty AAV capsids (e.g., the percentage or ratio of full versus empty AAV capsids). Methods for assessing or confirming the percentage or ratio of full:empty AAV capsids in an AAV fraction or preparation include, but are not limited to, cryo-transmission electron microscopy (CryoTEM), negative stain TEM, capillary electrophoresis, analytical ultracentrifugation, or a combination thereof.

[0358] In certain embodiments, the method for measuring complete AAV capsid versus empty AAV capsid is CryoTEM. In certain embodiments, the method involves using both CryoTEM and the above-mentioned AAV-specific ELISA. In certain embodiments, the ELISA is a sandwich ELISA. In certain embodiments, the sandwich ELISA comprises an antibody specific for an AAV epitope. In certain embodiments, the AAV epitope is a conformational epitope present on assembled AAV capsid.

[0359] One advantage of using CryoTEM is that it does not require negative staining.CryoTEM also has the ability to quantify the amount of complete AAV capsid.In certain embodiments, using CryoTEM eliminates the overestimation of the amount of complete AAV capsid caused by false positive signals.

[0360] The method of the present disclosure includes the method of evaluating the number or percentage of complete AAV capsids versus empty AAV capsids.In certain embodiments, the method includes CryoTEM.In certain embodiments, the method includes (i) embedding AAV fraction or preparation in a substrate on an inert support, (ii) flash-freezing the embedded AAV fraction or preparation, (iii) using cryo-transmission electron microscopy to image the embedded AAV fraction or preparation, and (iv) quantifying the percentage of complete AAV capsids versus empty AAV capsids.

[0361] For step (i), examples of substrates suitable for use in the method include, but are not limited to, non-crystalline amorphous ice. Examples of inert supports suitable for use in the method include, but are not limited to, carbon films, thermoplastics, and polyvinyl formals (e.g., polymers formed from polyvinyl alcohol and formaldehyde as copolymers with polyvinyl acetate, such as, but not limited to, Formvar or Vinylec, carbon stabilized polyvinyl formal, silicon monoxide on polyvinyl formal, pure carbon films, carbon type A: carbon support film (e.g., with removable polyvinyl formal on the opposite side of the grid), carbon type B: polyvinyl formal film (e.g., coated with a thicker carbon layer), and silicon monoxide on carbon type A). In certain embodiments, step (i) above involves mounting the sample on a thin carbon support film in a temperature (e.g., below (-196°C)) and humidity controlled environment. In certain embodiments, the humidity level may be relative humidity. For step (ii), the sample may be flash frozen. In certain embodiments, flash freezing may involve using liquid ethane, liquid nitrogen, liquid propane, or helium at near liquid nitrogen temperatures. For example, a container of liquid ethane, liquid nitrogen, liquid propane, or helium is surrounded by liquid nitrogen.

[0362] In certain embodiments, the AAV fraction or preparation is rapidly frozen (e.g., 104-106 K per second) so that ice crystals cannot form. In certain embodiments, amorphous ice is produced by either rapidly cooling liquid water or compressing regular ice at low temperatures. In certain embodiments, after excess AAV fraction or preparation is removed such that a portion of the sample remains attached, the grid is vitrified in liquid ethane and then stored in liquid nitrogen. For a discussion of additional steps for performing CryoTEM, see Cabra and Samso, J. Visualized Experiments, (2015) 95(e52311):1-11, which is incorporated by reference in its entirety for all purposes.

[0363] CryoTEM analysis of AAV particles can be used to assess the overall morphology of the sample, i.e., the presence of various AAV forms (generally including spherical and deformed AAV particles, subunit structures, and larger forms that are less structurally defined). For example, full capsids exhibit internal density with no clear boundary between the shell and the core. AAV capsids that exhibit a clear outer shell and minute internal density are classified as empty capsids.

[0364] CryoTEM can also be used to evaluate indeterminate capsids. For example, CryoTEM can be used to count "full," "empty," and "indeterminate" capsids.

[0365] CryoTEM can also be used to determine the level of particle packaging, either by manually classifying the particles or by using automated image analysis methods.

[0366] In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after ultracentrifugation by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after depth filtration by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after concentrating the AAV fraction or preparation using an ultrafiltration / diafiltration system by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after a tangential flow filtration (TFF) step by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the methods of the disclosure include testing the AAV fraction or preparation obtained after negative anion exchange (AEX) chromatography by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the methods of the disclosure include testing the AAV fraction or preparation obtained after the polishing step by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the methods of the disclosure include testing the purified AAV fraction or preparation by CryoTEM to determine the quantity and / or quality of AAV capsids in the AAV fraction or preparation.

[0367] In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after ultracentrifugation by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after depth filtration by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after concentrating the AAV fraction or preparation using an ultrafiltration / diafiltration system by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method of the present disclosure includes testing the AAV fraction or preparation obtained after a tangential flow filtration (TFF) step by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the disclosed method includes testing the AAV fraction or preparation obtained after negative anion exchange (AEX) chromatography by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the disclosed method includes testing the AAV fraction or preparation obtained after the polishing step by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In certain embodiments, the disclosed method includes testing the purified AAV fraction or preparation by AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation.

[0368] Analytical ultracentrifugation (AUC) allows the separation of proteins according to their sedimentation coefficient. For the same protein, the sedimentation coefficient can be correlated with the aggregation state. Briefly, the samples are diluted with the corresponding sample buffer and then transferred to a cell assembly with a built-in quartz window, loaded into a rotor and spun at a constant speed. Protein molecules of different sizes move towards the bottom of the cell with different sedimentation velocities, which are continuously monitored during centrifugation by a UV detector at 280 nm. The collected data set allows computational analysis to obtain the differential sedimentation coefficient distribution c(s) by deconvoluting the sedimentation and diffusion processes. This identifies the various species of the sample and indicates their s values ​​and populations. The distribution of sedimentation coefficients is integrated to obtain the results of the analysis in terms of the relative area percentage and the S value of the peak maximum.

[0369] AAV preparation Further provided herein is an AAV preparation produced by the method of the present disclosure. AAV preparation and AAV fraction are used interchangeably for the purposes of this disclosure. In certain embodiments, the AAV fraction is a further concentrated AAV preparation or a portion of AAV removed from the AAV preparation. In certain embodiments, a method for producing an AAV preparation includes: (i) transfecting a host cell with at least one plasmid containing a gene of interest; (ii) harvesting a cell culture supernatant or cell suspension containing AAV capsids to produce an AAV fraction or preparation; (iii) quantifying the total number of AAV capsids in the AAV fraction or preparation using an AAV-specific ELISA assay; and (iv) preparing a desired concentration of an AAV product, formulation, or composition based on the total number of AAV capsids determined in step (iii). In certain embodiments, the method includes concentrating the AAV fraction or preparation. In certain embodiments, the method includes removing at least a portion of the empty capsids from the AAV fraction or preparation. In certain embodiments, the amount of empty capsids is removed to create an AAV fraction, preparation, product, AAV formulation or AAV composition having a particular concentration of full capsids and / or a particular ratio of full capsids:empty capsids, hi certain embodiments, the AAV fraction or preparation is diluted to the desired dose with an appropriate buffer.

[0370] In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) in the AAV fraction, preparation, product, formulation, or composition. In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) is CryoTEM. In certain embodiments, the dosage and / or potency is not determined by qPCR. In certain embodiments, the percentage of full AAV capsids is about 40% to about 100%. In certain embodiments, the percentage of full AAV capsids is about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In certain embodiments, the percentage of complete AAV capsids is between about 60% and about 80%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods disclosed herein are used to generate AAV fractions, preparations, products, formulations, or compositions that contain a consistent amount of complete capsids between the AAV fractions, preparations, products, formulations, or compositions.

[0371] In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) in the AAV fraction, preparation, product, formulation, or composition. In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) is CryoTEM. In certain embodiments, the dosage and / or potency is not determined by qPCR. In certain embodiments, the percentage of full AAV capsids is about 40% to about 100%. In certain embodiments, the percentage of full AAV capsids is about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In certain embodiments, the percentage of complete AAV capsids is between about 60% and about 80%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods disclosed herein are used to generate AAV fractions, preparations, products, formulations, or compositions that contain a consistent amount of complete capsids among the AAV fractions or preparations.

[0372] Further provided herein is a method of administering an AAV product, formulation, or composition produced by the method of the present disclosure. In certain aspects, the method of the present invention involves administering a dose of an AAV product, formulation, or composition to a subject in need thereof, and includes (i) obtaining a purified AAV preparation, (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay, and (iii) administering a dose of the AAV product, formulation, or composition to the subject. In certain embodiments, the preparation in steps (i) and (ii) is an AAV fraction or preparation that can be used to make a final AAV product, formulation, or composition, and this AAV fraction or preparation is further purified or diluted to form an AAV product, formulation, or composition for steps (i), (ii), and / or (iii). In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) in the AAV preparation, product, formulation, or composition. In certain embodiments, the method includes assessing or confirming the percentage or ratio of full AAV capsids versus empty AAV capsids (full:empty) by CryoTEM. In certain embodiments, the concentration, dose and / or potency are not determined by qPCR. In certain embodiments, the percentage of full AAV capsids is about 40% to about 100%. In certain embodiments, the percentage of full AAV capsids is about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In certain embodiments, the percentage of full AAV capsids is between about 60% to about 80%. In certain embodiments, at least about 60% of the AAV capsids are full AAV capsids.In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids.

[0373] In certain embodiments, the potency of an AAV preparation, product, formulation, or composition can be further verified by in vitro and / or in vivo biopotency assays. For example, the steps may involve 1) determining the content of AAV in the preparation (e.g., by ELISA), 2) determining the percentage of complete capsids (e.g., by CryoTEM), and 3) confirming biological activity (e.g., by in vivo and / or in vitro biopotency assays).

[0374] In certain embodiments, for the methods, fractions, preparations, products, formulations, or compositions disclosed herein, the concentration of the AAV preparation is about 1×10 10 cp / ml ~ approx. 1 x 10 20 In certain embodiments, for the methods, fractions, preparations, products, formulations, or compositions disclosed herein, the concentration of the AAV preparation, product, formulation, or composition is between about 1×10 11 cp / ml ~ approx. 1 x 10 19 cp / ml, approximately 1 × 10 12 cp / ml ~ approx. 1 x 10 18 cp / ml, approximately 1 × 10 13 cp / ml ~ approx. 1 x 10 17 cp / ml, or approximately 1 × 10 14 cp / ml ~ approx. 1 x 10 16 In certain embodiments, for the methods and preparations, products, formulations, or compositions disclosed herein, the concentration of the AAV preparation, product, formulation, or composition is between about 1×10 12cp / ml ~ approx. 1 x 10 15 cp / ml, approximately 1×10 13 cp / ml ~ approx. 1 x 10 15 cp / ml, or approximately 1 × 10 12 cp / ml ~ approx. 1 x 10 13 In certain embodiments, for the methods and preparations, products, formulations, or compositions disclosed herein, the concentration of the AAV preparation, product, formulation, or composition is between about 1×10 14 cp / ml ~ approx. 5 x 10 14 cp / ml, approximately 2 × 10 14 cp / ml ~ approx. 3 x 10 14 cp / ml, or approximately 3.5 × 10 14 cp / ml ~ approx. 5 x 10 15 In certain embodiments, cp / ml can be total capsids per ml or complete capsids per ml. In certain embodiments, cp / ml is total capsids per ml.

[0375] In certain embodiments, the AAV preparation, product, formulation, or composition comprises at least about 10 L of AAV produced from about 1000 L of starting material (e.g., cell culture). 12 1000 L of starting material (e.g., cell culture), or at least about 10 13 It contains 10 virus particles (vp).

[0376] In certain embodiments, for the methods or preparations, products, formulations, or compositions disclosed herein, the dose of the AAV preparation, product, formulation, or composition is about 1×10 5 cp / kg ~ approx. 1×10 25 In certain embodiments, for the methods or preparations, products, formulations, or compositions disclosed herein, the dose of the AAV preparation, product, formulation, or composition is between about 1×10 6 cp / kg ~ approx. 1×10 24 cp / kg, approximately 1×10 7 cp / kg ~ approx. 1×10 23cp / kg, approximately 1×10 8 cp / kg ~ approx. 1×10 22 cp / kg, approximately 1×10 9 cp / kg ~ approx. 1×10 21 cp / kg, approximately 1×10 10 cp / kg ~ approx. 1×10 20 cp / kg, approximately 1×10 11 cp / kg ~ approx. 1×10 19 cp / kg, approximately 1×10 12 cp / kg ~ approx. 1×10 18 cp / kg, approximately 1×10 13 cp / kg ~ approx. 1×10 17 cp / kg, or approximately 1 × 10 14 cp / kg ~ approx. 1×10 16 In certain embodiments, for the methods and preparations, products, formulations, or compositions disclosed herein, the dose of the AAV preparation, product, formulation, or composition is between about 1×10 12 cp / kg ~ approx. 1×10 20 cp / kg, approximately 1×10 13 cp / kg ~ approx. 1×10 19 cp / kg, approximately 1×10 14 cp / kg ~ approx. 1×10 18 cp / kg, or approximately 1 × 10 15 cp / kg ~ approx. 1×10 17 In certain embodiments, for the methods and preparations, products, formulations, or compositions disclosed herein, the dose of the AAV preparation, product, formulation, or composition is between about 1×10 10 cp / kg ~ approx. 1×10 16 In certain embodiments, for the methods and preparations, products, formulations, or compositions disclosed herein, the dose of the AAV preparation, product, formulation, or composition is at least about 1×10 6 cp / kg, at least about 1 × 10 7 cp / kg, at least about 1 × 10 8 cp / kg, at least about 1 × 10 9 cp / kg, at least about 1 × 10 10 cp / kg, at least about 1 × 10 11cp / kg, at least about 1 × 10 12 cp / kg, at least about 1 × 10 13 cp / kg, at least about 1 × 10 14 cp / kg, at least about 1 × 10 15 cp / kg, at least about 1 × 10 16 cp / kg, at least about 1 × 10 17 cp / kg, at least about 1 × 10 18 cp / kg, at least about 1 × 10 19 cp / kg, at least about 1 × 10 20 cp / kg, at least about 1 × 10 21 cp / kg, at least about 1 × 10 22 cp / kg, at least about 1 × 10 23 cp / kg, at least about 1 × 10 24 cp / kg, or at least about 1 × 10 25 In certain embodiments, cp / kg can be total capsids per kg of subject or complete capsids per kg of subject. In certain embodiments, cp / kg is total capsids per kg of subject.

[0377] In certain embodiments, the AAV preparation, product, formulation, or composition of the present disclosure is highly pure and highly titered, and is suitable for clinical use in subjects. In certain embodiments, the AAV preparation, product, formulation, or composition comprises a homogenous population and highly pure AAV capsid particles. In certain embodiments, the AAV preparation, product, formulation, or composition comprises full-length vector DNA. In exemplary embodiments, the AAV preparation, product, formulation, or composition is substantially free of unwanted contaminants, including, but not limited to, AAV capsid particles containing truncated or incomplete vector DNA, AAV particles containing incomplete protein composition and oligomerization structures, or contaminating viruses, e.g., non-AAV, lipid enveloped viruses. In exemplary embodiments, the AAV preparation, product, formulation, or composition contains a large amount of the coding cDNA of the protein of interest. In certain embodiments, the AAV preparation, product, formulation, or composition of the present disclosure is suitable for administration to a subject. In certain embodiments, the AAV preparation, product, formulation, or composition is sterile and / or Good Manufacturing Practice (GMP) grade. In certain embodiments, the AAV preparation, product, formulation, or composition complies with the requirements set forth in the United States Pharmacopoeia Chapter 1046 or the European Pharmacopoeia for gene therapy medicinal products, or the requirements set forth by the United States Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). In certain embodiments, the AAV preparation, product, formulation, or composition is a ready-to-use preparation, product, formulation, or composition for direct administration to a subject with little or no processing or manipulation.

[0378] Source of AAV In the method of the present disclosure, the AAV can be any AAV serotype. In certain embodiments, the AAV described herein is AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, or a chimeric AAV vector. In certain embodiments, the AAV is wild type. In certain embodiments, the AAV is recombinant AAV (rAAV). In certain embodiments, the AAV is modified by genetic engineering and / or chemically modified. In certain embodiments, the AAV comprises a modified capsid, for example, a genetically engineered or chemically modified AAV capsid. In certain embodiments, the AAV is AAV8 serotype. In certain embodiments, the AAV is AAV9 serotype.

[0379] With respect to the methods of the invention, the AAV fraction or preparation is, in exemplary embodiments, a concentrated AAV fraction or preparation. In certain embodiments, the AAV fraction or preparation is at least about 1 x 10 per mL. 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 Pieces, or about 1 x 10 16 In certain embodiments, the AAV fraction or preparation contains at least about 1 x 10 total AAV capsids per mL. 12 The AAV capsid may include empty AAV capsids and complete AAV capsids.

[0380] In certain embodiments, AAV refers to the AAV fraction or preparation produced by transfected host cells.In certain embodiments, AAV fraction or preparation refers to the supernatant or cell suspension collected from a cell culture containing host cells transfected with a triple plasmid system, one plasmid of the system contains a gene or cDNA of interest, and one plasmid encodes capsid protein VP1, capsid protein VP2, and / or capsid protein VP3.In certain embodiments, VP1, VP2, and / or VP3 are AAV8 VP1, VP2, and / or VP3.Triple plasmid transfection for rAAV production is known in the art. See, e.g., Qu et al., 2015 (supra), and Mizukami et al., "A Protocol for AAV vector production and purification." PhD dissertation, Division of Genetic Therapeutics, Center for Molecular Medicine, 1998; and Kotin et al., Hum Mol Genet 20(R1):R2-R6(2011). In certain embodiments, transfection can be performed using inorganic compounds, such as calcium phosphate, or organic compounds, polyethylenimine (PEI), or non-chemical means, such as electroporation.

[0381] In certain embodiments, the host cell is an adherent cell. In certain embodiments, the host cell is a suspension cell. In certain embodiments, the host cell is a HEK293 cell or an Sf9 cell (e.g., a baculovirus-infected Sf9 cell) or a HeLa or BHK (herpes virus system). In certain embodiments, the cell culture comprises a serum- and protein-free culture medium. In certain embodiments, the medium is a synthetic medium and does not contain animal-derived components, such as hydrolysates.

[0382] In certain embodiments, a fraction or preparation containing rAAV particles refers to a fraction or preparation containing HEK293 cells transfected with a triple plasmid system. In certain embodiments, a fraction or preparation containing AAV particles is obtained about 2 to about 7 days after transfection of HEK293 cells or when the cell culture reaches about 5×10 6 A fraction or preparation of a harvest having a cell density of 1000 cells / mL or greater and having a cell viability of about 50% or greater is shown.

[0383] In certain embodiments, AAV is prepared by triple plasmid transfection followed by harvesting 1-7 days later, hi certain embodiments, AAV is prepared from cell lysate.

[0384] In certain embodiments, AAV is prepared as follows: HEK293 cells are allowed to attach and grow in commercially available culture medium. The medium may be synthetic and may be free of animal-derived components, such as serum and proteins. Cells are cultured at approximately 3×10 6 ~Approx. 12×10 6 cells / ml, e.g., about 6×10 6 ~About 10×10 6 The cells are then cultured until the cell density reaches approximately 3–5×10 6The cells are split at a ratio of approximately 1:2 to obtain 1000 cells / ml. After splitting, the cells can be transfected with three plasmids, including (1) a helper plasmid capable of providing one or more helper virus functions essential for AAV production, (2) a plasmid encoding one or more genes involved in viral encapsidation, replication, and packaging, and (3) a plasmid containing a gene of interest (GOI) to be packaged in the resulting rAAV particles. For example, the GOI can be a vector DNA containing human blood clotting factor IX Padua in a single-stranded self-complementary form, present with the vector DNA. As another example, the GOI can be a vector DNA containing human blood clotting factor IX Padua in a double-stranded self-complementary form, the total length of the vector DNA being 4.8 kB. As another example, the GOI can be a vector DNA containing B-domain deleted human blood clotting factor VIII in a single-stranded self-complementary form, the total length of the vector DNA being 4.8 kB. Other GOIs may be used. Transfection may be performed transiently, such as by using a cationic polymer. Prior to elution, the HEK293 cell line may be cultured for at least about 1 day, e.g., 3-5 days, before harvesting. EXAMPLES

[0385] Example 1: General purification procedure The following examples are provided merely to illustrate the present invention and are not intended to limit its scope in any way.

[0386] AAV8 production was performed in HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the target protein and AAV8-VP1, VP2 and VP3. The clarified cell culture supernatant was concentrated and diafiltered with a Pall Omega T-Series Cassette 100kDa. The viral particles were loaded onto a membrane absorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting flow-through containing AAV8 was used as the load for the following affinity purification step.

[0387] A 32 mm inner diameter column with a bed height of 60 mm and a volume of approximately 200 ml containing POROS™ CaptureSelect™ AAV8 Affinity Matrix (catalog number A30794. Thermo Fisher) was equilibrated with at least 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing POROS™ CaptureSelect™ AAV8 Affinity Matrix (catalog number A30794. Thermo Fisher). The column was then re-equilibrated with 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 5 column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 5 column volumes of Wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 5 column volumes of Wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol (pH 8.5).

[0388] Elution was performed by applying 5 column volumes of the following elution buffer: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl (pH 8.0) to the column.

[0389] The above steps are summarized in Table 1. [Table 1]

[0390] To provide all AAV subtypes with suitable binding characteristics to cation exchangers (CEX), buffer exchange of the eluates into 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) was performed on VIVACELL100 (10K) cartridges (Sartorius).

[0391] Example 2: AAV8, vector genome 2.6 kB, Capto S, in the presence of calcium The following test procedure was performed. Note that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 100 mm bed height, 0.95 cm area column was filled with Capto S cation exchange resin (catalog 17-5441-01; Cytiva). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.5 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Capto S cation exchange resin.

[0392] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0.

[0393] Elution was then performed using a gradient elution from 40 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) to 200 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0). Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0).

[0394] For all steps, the linear flow rate was 60 cm / h.

[0395] The buffer compositions are summarized in Table 2. [Table 2]

[0396] Table 3 shows the chromatographic scheme for the CEX separation. [Table 3]

[0397] Table 4 shows the ddPCR / AAV8:AG ratios. [Table 4]

[0398] Table 5 shows the percentage of full and empty capsids as determined using AUC. [Table 5]

[0399] The results are shown in Figures 1 to 6, and the yields are shown in Table 6.

[0400] Figures 1-6 show runs with data for the majority of fractions. Load, FT = flow-through, W = wash, E = eluate, E1-E9 = pool - fractions of peaks according to chromatogram. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0401] Figures 1 and 2 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0402] [Table 6]

[0403] Separation of the 2.6 kB vector genome containing AAV8 on a cation exchanger Capto S in the presence of calcium demonstrated high resolution for separation of empty and complete AAV8 capsids. The yields of fractions E1, E2, and E3 were 44.4% by ddPCR and 9.1% by AAV8 antigen.

[0404] Example 3: AAV8, vector genome size 2.6 kB, Capto S, in the absence / presence of chelated divalent cations The following test procedure was performed. Note that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 100 mm bed height, 0.95 cm area column was filled with Capto S cation exchange resin (catalog 17-5441-01; Cytiva). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.5 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 0.005 column volumes of 30 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Capto S cation exchange resin.

[0405] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80, pH 6.0.

[0406] Elution was then performed using a gradient elution from 30 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80 (pH 6.0) to 200 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80 (pH 6.0) over 40 column volumes.

[0407] Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM EDTA, 0.005% polysorbate 80 (pH 6.0).

[0408] For all steps, the linear flow rate was 60 cm / h.

[0409] The buffer compositions are summarized in Table 7. [Table 7]

[0410] Table 8 shows the chromatographic scheme for the CEX separation. [Table 8]

[0411] Table 9 shows the ddPCR / AAV8:AG ratios. [Table 9]

[0412] Table 10 shows the percentage of full and empty capsids as determined using AUC. [Table 10]

[0413] The results are shown in Figures 7 to 11, and the yields are shown in Table 11.

[0414] Figures 7-11 show the runs with data for the most interesting fractions: L_nativ = affinity eluate, Load (starting material = affinity eluate dialyzed against equilibration buffer), FT = flow-through, W = wash, E = eluate, E1-E3 = peak fractions (chromatograms). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0415] Figures 7 and 8 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0416] [Table 11]

[0417] Addition of EDTA instead of calcium showed significantly reduced resolution, indicating a reduced ability of the AAV8 construct to bind onto Capto S. Significant amounts of AAV8 were observed in the flow-through.

[0418] Example 4: AAV8, vector genome size >4.8 kB, Capto S, in the presence of calcium Preparation of Load Material AAV8 production was performed in HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the target protein and AAV8-VP1, VP2 and VP3. The clarified cell culture supernatant was concentrated and diafiltered with a Pall Omega T-Series Cassette 100kDa. The viral particles were loaded onto a membrane absorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting flow-through containing AAV8 was used as the load for the following affinity purification step.

[0419] A 32 mm inner diameter column with a bed height of 60 mm and a volume of approximately 200 ml containing POROS™ CaptureSelect™ AAV8 Affinity Matrix (catalog number A30794. Thermo Fisher) was equilibrated with at least 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing POROS™ CaptureSelect™ AAV8 Affinity Matrix (catalog number A30794. Thermo Fisher). The column was then re-equilibrated with 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 5 column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 5 column volumes of Wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 5 column volumes of Wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol (pH 8.5).

[0420] Elution was performed by applying 5 column volumes of the following elution buffer: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl (pH 8.0) to the column, details of which are given in Table A. TIFF2024532700000013.tif169165

[0421] To provide all AAV subtypes with suitable binding characteristics to cation exchangers (CEX), buffer exchange of the eluates into 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) was performed on VIVACELL100 (10K) cartridges (Sartorius).

[0422] The following test procedure was performed. Note that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 101 mm bed height, 0.95 cm area column was filled with Capto S cation exchange resin (catalog 17-5441-01; Cytiva). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.6 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Capto S cation exchange resin.

[0423] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0.

[0424] Elution was then performed using a gradient elution from 40 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) to 200 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0). Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0).

[0425] For all steps, the linear flow rate was 60 cm / h.

[0426] The buffer compositions are summarized in Table 12. [Table 12]

[0427] Table 13 shows the chromatographic scheme for the CEX separation. [Table 13]

[0428] Table 14 shows the ddPCR / AAV8:AG ratios. [Table 14]

[0429] Table 15 shows the percentage of full and empty capsids as determined using AUC. [Table 15]

[0430] The results are shown in Figures 12 to 17, and the yields are shown in Table 16.

[0431] Figures 12-17 show runs with data for the majority of fractions: L native = affinity eluate. Load = (starting material), FT = flow-through, W = wash, E = eluate, E1-E5 = peak fractions (chromatograms). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0432] Figures 12 and 13 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0433] [Table 16]

[0434] Example 5: AAV8 vector genome size 2.6 kB, Eshmuno S, in the presence of calcium The following test procedure was performed. It should be noted that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 100 mm bed height, 0.95 cm area buffer was prepared with Eshmuno S cation exchange resin (catalog 1.20078; Merck-Millipore). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.5 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Eshmuno S cation exchange resin.

[0435] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0.

[0436] Elution was then performed using a gradient elution from 40 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) to 200 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0). Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0).

[0437] For all steps, the linear flow rate was 60 cm / h.

[0438] The buffer compositions are summarized in Table 17. [Table 17]

[0439] Table 18 shows the chromatographic scheme for the CEX separation. [Table 18]

[0440] Table 19 shows the ddPCR / AAV8:AG ratios. [Table 19]

[0441] Table 20 shows the percentage of full and empty capsids as determined using AUC. [Table 20]

[0442] The results are shown in Figures 18 to 24, and the yields are shown in Table 21.

[0443] Figures 18-24 show runs with data for the majority of fractions: L native = AAV8 affinity eluate. Load (starting material, after dialysis into equilibration buffer), FT = flow-through, W = wash, E = eluate, E1-E9 = peak fractions (chromatograms). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0444] Figures 18 and 19 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0445] [Table 21]

[0446] Example 6: AAV8, vector genome size 2.6 kB, Capto S, in the presence of calcium The following test procedure was performed. Note that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 100 mm bed height, 0.95 cm area column was filled with Capto S cation exchange resin (catalog 17-5441-01; Cytiva). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.5 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Capto S cation exchange resin.

[0447] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0.

[0448] Elution was then performed using a gradient elution from 40 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) to 80 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0). Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0).

[0449] For all steps, the linear flow rate was 60 cm / h.

[0450] The buffer compositions are summarized in Table 22. [Table 22]

[0451] Table 23 shows the chromatographic scheme for the CEX separation. [Table 23]

[0452] Table 24 shows the ddPCR / AAV8:AG ratios. [Table 24]

[0453] Table 25 shows the percentage of full and empty capsids as determined using AUC. [Table 25]

[0454] The results are shown in Figures 25 to 32, and the yields are shown in Table 26.

[0455] Figures 25-32 show runs with data for the majority of fractions. Load (starting material, FT = flow-through, W = wash, E = eluate, E1-E6 = peak fractions (chromatograms). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0456] Figures 25 and 26 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0457] [Table 26]

[0458] Example 7: AAV9, vector genome size 2.6 kB, Eshmuno S, in the presence of calcium Preparation of Load Material AAV9 production was performed in HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the target protein and AAV9-VP1, VP2 and VP3. The clarified cell culture supernatant was concentrated and diafiltered with a Pall Omega T-Series Cassette 100kDa. The viral particles were loaded onto a membrane absorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting flow-through containing AAV9 was used as the load for the following affinity purification step.

[0459] The following test procedures were performed. Please note that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. An 11 mm ID column with a bed height of 57 mm and a volume of approximately 5.4 ml containing POROS™ CaptureSelect™ AAVX Affinity Matrix (catalog number A36742, Thermo Fisher) was equilibrated with at least 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing POROS™ CaptureSelect™ AAVX Affinity Matrix (catalog number A36742 Thermo Fisher). The column was then re-equilibrated with 5 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 5 column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 5 column volumes of Wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. An additional wash of 5 column volumes of 100 mM sodium acetate and 0.1% Tween 80, pH 6.0 was applied. All these steps were performed at room temperature.

[0460] The temperature was reduced to +2°C to +8°C, the flow rate was reduced to 5 cm / hr, and elution was performed by applying 10 column volumes of 100 mM sodium acetate and 0.1% Tween 80 (pH 6.0) to the column. The procedure is detailed in Table B. TIFF2024532700000029.tif138165

[0461] To provide all AAV subtypes with suitable binding characteristics to cation exchangers (CEX), buffer exchange of the eluates into 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) was performed on VIVACELL100 (10K) cartridges (Sartorius).

[0462] The following test procedure was performed. It should be noted that all buffers disclosed in this example were made at room temperature and the pH of all buffers was measured at room temperature. First, a 100 mm bed height, 0.95 cm area buffer was prepared with Eshmuno S cation exchange resin (catalog 1.20078; Merck-Millipore). 2 A column with an internal diameter of 11 mm and a volume of approximately 9.5 ml was equilibrated (activated) with 5 column volumes of a buffer containing 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The column was then equilibrated with at least 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0. The load (AAV conditioned with 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0) was applied to the column containing Eshmuno S cation exchange resin.

[0463] The column was then washed with 5 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80, pH 6.0.

[0464] Elution was then performed using a gradient elution from 40 column volumes of 30 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0) to 200 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0). Post-elution was performed with 10 column volumes of 1000 mM sodium acetate, 2 mM calcium acetate, 0.005% polysorbate 80 (pH 6.0).

[0465] For all steps, the linear flow rate was 60 cm / h.

[0466] The buffer compositions are summarized in Table 27. [Table 27]

[0467] Table 28 shows the chromatographic scheme for the CEX separation. [Table 28]

[0468] Table 29 shows the ddPCR / AAV9:AG ratios. [Table 29]

[0469] Table 30 shows the percentage of full and empty capsids as determined using AUC. [Table 30]

[0470] The results are shown in Figures 33 to 38, and the yields are shown in Table 31.

[0471] Figures 33-38 show runs with data for the majority of fractions. Load (starting material, FT = flow-through, W = wash, E = eluate, E1-E6 = peak fractions (chromatograms). The vg / cp ratio indicates the AAV with the highest amount of full capsid (full capsid fraction). The higher the value, the higher the amount of full capsid.

[0472] Figures 33 and 34 are chromatograms of a CEX run. They contain data for a complete run including the regeneration procedure. Curves for UV280nm, UV254nm, conductivity, pH, pressure, fluorescence and fraction are shown. X-axis: UV280nm (left), conductivity (right), Y-axis: volume (ml).

[0473] [Table 31]

Claims

1. 1. A method for purifying complete AAV capsids from an AAV preparation comprising complete AAV capsids and empty AAV capsids to provide an AAV product that is substantially free of empty AAV capsids, comprising: (a) providing a first solution comprising complete AAV capsids, empty AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions in which the full AAV capsid and the empty AAV capsid bind to the column; (c) applying a second solution comprising one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that the intact AAV capsids are purified from the empty AAV capsids; Including, adding the second solution comprises gradually increasing the total concentration of the one or more monovalent cations; or The method, wherein adding the second solution comprises continuously linearly increasing the total concentration of the one or more monovalent cations.

2. The one or more divalent cations of the first solution are Ca ２＋ , Mg ２＋ , Zn ２＋ , Mn ２＋ , Cu ２＋ , Fe ２＋ , Ba ２＋ , Sr ２＋ , Co ２＋ 10. The method of claim 1, wherein the hydroxyl group is selected from the group consisting of:

3. The one or more divalent cations of the first solution are Ca ２＋ The method of claim 2, wherein

4. 10. The method of claim 1, wherein the one or more divalent cations in the first solution are at a total concentration of about 1 mM to about 30 mM.

5. 5. The method of claim 4, wherein the one or more divalent cations in the first solution are at a total concentration of about 2 mM.

6. The one or more divalent cations of the second solution are Ca ２＋ , Mg ２＋ , Zn ２＋ , Mn ２＋ , Cu ２＋ , Fe ２＋ , Ba ２＋ , Sr ２＋ 10. The method of claim 1, wherein the hydroxyl group is selected from the group consisting of:

7. The one or more divalent cations of the second solution are Ca ２＋ The method of claim 6, wherein

8. 8. The method of claim 7, wherein the one or more divalent cations in the second solution are at a total concentration of about 1 mM to about 30 mM.

9. 9. The method of claim 8, wherein the one or more divalent cations in the second solution are at a constant total concentration of about 2 mM.

10. The one or more monovalent cations of the first solution and / or the second solution are Na ＋ , K. ＋ , N.H. ４ ＋ , Li ＋ , Cs ＋ 10. The method of claim 1, wherein the hydroxyl group is selected from the group consisting of:

11. The one or more monovalent cations of the first solution and / or the second solution are Na ＋ The method of claim 10, wherein

12. 10. The method of claim 1, wherein the one or more monovalent cations in the first solution are at a total concentration of about 5 mM to about 1500 mM.

13. 13. The method of claim 12, wherein the one or more monovalent cations in the first solution are at a total concentration of about 30 mM.

14. 10. The method of claim 1, wherein the one or more monovalent cations of the second solution are at a total concentration of about 5 mM to about 1500 mM.

15. The method of claim 14, wherein the total concentration of the one or more monovalent cations in the second solution is from about 30 mM to about 200 mM.

16. The method of claim 1, wherein the first solution and / or the second solution has a pH of about 5.0 to about 8.

5.

17. The method of claim 1 , wherein the first solution and / or the second solution further comprise one or more surfactants.

18. 18. The method of claim 17, wherein the surfactant is polysorbate 80.

19. 18. The method of claim 17, wherein the one or more surfactants are in a total amount of about 0.0025% w / w to about 0.0075% w / w.

20. 2. The method of claim 1, wherein the cation exchange column comprises a resin having charged groups, the charged groups being sulfonic acid groups, sulfate groups, sulfopropyl groups, carboxylic acid groups, phosphate groups, or a combination thereof.

21. 10. The method of claim 1, wherein adding the second solution comprises incrementally increasing the total concentration of the one or more monovalent cations.

22. 10. The method of claim 1, wherein adding the second solution comprises incrementally increasing the total concentration of the one or more divalent cations.

23. 10. The method of claim 1, wherein the addition of the second solution comprises continuously linearly increasing the total concentration of the one or more monovalent cations.

24. 10. The method of claim 1, wherein the addition of the second solution comprises continuously linearly increasing the total concentration of the one or more divalent cations.

25. 2. The method of claim 1, wherein the AAV capsid is derived from the group consisting of AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, genetically modified AAV, chemically modified AAV, genetically and chemically modified AAV, and combinations thereof.

26. 2. The method of claim 1, wherein the AAV product comprises less than about 30% empty AAV capsids.

27. 1. A method for purifying empty AAV capsids from an AAV preparation comprising empty AAV capsids and complete AAV capsids to provide an AAV product that is substantially free of empty AAV capsids, or for separating empty AAV capsids and complete AAV capsids in an AAV preparation, comprising: (a) providing a first solution comprising empty AAV capsids, complete AAV capsids, one or more monovalent cations, and one or more divalent cations; (b) loading the first solution onto a cation exchange column under conditions in which the empty AAV capsids and the complete AAV capsids bind to the column; (c) applying a second solution comprising one or more monovalent cations and one or more divalent cations to the cation exchange column under conditions such that the empty AAV capsids are purified or separated from the complete AAV capsids; The method comprising:

28. 1. A method for preparing an immunoabsorption column, comprising: (a) concentrating the empty AAV capsid of claim 27 by ultrafiltration, an anion exchanger, or a cation exchanger; (b) buffer exchange into an amine-free buffer; (c) immobilizing the empty AAV capsids on an activated resin; The method comprising:

29. 1. A method for preparing an immunoabsorption column, comprising: (a) concentrating the intact AAV capsid of claim 1 by ultrafiltration, anion exchange, or cation exchange; (b) buffer exchange with a buffer suitable for patient treatment; The method comprising:

Images