Method for analyzing DNA using enhanced high performance chromatography system

Abstract

The present invention relates to improved and enhanced chromatography systems for the analysis of DNA, such as DNA released from AAV virions. The present invention avoids past problems by using a large aperture medium, different column flow rates, and a buffer comprising a surfactant and an increased concentration of an inorganic salt, and typically has a column pressure of about 1450 to about 1550 psi.

Description

[0001] This application claims priority to U.S. Application Serial No. 63 / 540,259, filed September 25, 2023, which is incorporated herein by reference. Technical Field

[0002] This invention relates to improved and enhanced chromatographic systems for analyzing DNA, such as DNA released from AAV virions. Background Technology

[0003] Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus used as a gene delivery vector for research and therapeutic agents. (Weitzman and Linden, Adeno-Associated Virus Biology (chapter 1)) Meth. Molec. Biol. 807: 1-23 (2011). Recombinant AAV has shown promise for human gene therapy based on its safety profile and potential for long-term efficacy in animal models and patients. Wang et al., Nature , 18:358-78 (2019). Recombinant AAV includes at least one gene of interest (GOI) and an AAV inverted terminal repeat (ITR) flanked by the gene of interest.

[0004] SEC-UPLC has been used to analyze cell-free DNA in AAV virus preparations. See Xu et al., Int'l J. Pharmaceutics 615:121464 (2022). Other methods, such as the determination of cell-free DNA using fluorescent dyes, have also been employed. See Bee. et al., Int'l J. Pharmaceutics 110 :3183-87 (2021).

[0005] Previous methods used 5 µm, 4.6 x 300 nm SEC columns with 500 Å pore sizes. Run buffer based on 2X Dulbecco's Phosphate Buffered Saline (DPBS) was used, with injection volumes containing 3.7 × 10⁻⁶ Å at a flow rate of 0.5 mL / min. 12 The prepared vector genome (vg) was loaded onto the column. The salt concentration of the buffer was 140 mM (millimoles).

[0006] Previous SEC-UPLC methods have exhibited inconsistent performance and are limited by problems such as column clogging, pressure buildup up to approximately 1700 psi, and limited interpeak resolution. Therefore, these older methods cannot provide reliable characterization and quantification of free DNA in AAV preparations. This invention addresses and overcomes these limitations. Summary of the Invention

[0007] This invention provides a method for analyzing free DNA in a preparation containing AAV virus particles, wherein the method comprises the following steps: (a) loading the preparation containing AAV virus particles into a size exclusion column with a pore size greater than 500 angstroms, wherein the preparation is mixed with a running buffer before loading; (b) running the preparation on the size exclusion column at a flow rate of less than 0.5 mL / min, wherein pressure buildup in the size exclusion column does not cause column clogging; and (c) detecting the elution of free AAV DNA and AAV virus particles from the size exclusion column.

[0008] This invention provides a method in which the preparation of rAAV virus particles comprises a buffer, a cryoprotectant, a salt, a nonionic surfactant, and a pH of about 6.0 to about 8.0. The cryoprotectant may be sucrose at a concentration of about 0.5% to about 5.0% (w / v). The concentration of sucrose may be about 1.0% to about 3.5% (w / v), about 1.0% to about 3.0% (w / v), about 1.0% to about 2.5% (w / v), about 1.0% to about 2.0% (w / v), about 1.0% to about 1.5% (w / v), about 1.0% (w / v), about 1.5% (w / v), about 2.0% (w / v), about 2.5% (w / v), about 3.0% (w / v), and additional ranges formed based on the above values.

[0009] Size exclusion chromatography column media can have nominal pore sizes greater than 500 Å, such as about 550 Å, about 600 Å, about 650 Å, about 700 Å, about 800 Å, about 850 Å, about 900 Å, 950 Å, about 1000 Å, about 1050 Å, about 1100 Å, about 1150 Å, about 1200 Å, about 1250 Å, about 1300 Å, about 1350 Å, about 1400 Å, about 1450 Å, or about 1500 Å. The aperture range includes, but is not limited to, 550 Å to 1500 Å, 700 Å to 1500 Å, 550 Å to 1100 Å, 700 Å to 1100 Å, or 900 Å to 1100 Å, as well as additional sub-ranges formed based on the above values.

[0010] The flow rate should be less than 0.5 ml / min to reduce pressure buildup. Preferably, the average flow rate is about 0.49 ml / min, 0.48 ml / min, 0.47 ml / min, 0.46 ml / min, 0.45 ml / min, 0.44 ml / min, 0.43 ml / min, 0.42 ml / min, 0.41 ml / min, 0.40 ml / min, 0.39 ml / min, 0.38 ml / min, 0.37 ml / min, 0.36 ml / min, 0.35 ml / min, 0.34 ml / min, 0.33 ml / min, 0.32 ml / min, 0.31 ml / min, 0.30 ml / min, 0.29 ml / min, 0.28 ml / min, 0.27 ml / min, 0.26 ml / min, 0.25 ml / min, 0.24 ml / min, 0.23ml / min, 0.22 ml / min, 0.21 ml / min, 0.20 The flow rates are 0.19 ml / min, 0.18 ml / min, 0.17 ml / min, 0.16 ml / min, 0.15 ml / min, 0.14 ml / min, 0.13 ml / min, 0.12 ml / min, 0.11 ml / min, or 0.10 ml / min. Any of the above flow rate ranges can be used. For example, the flow rate can be 0.30 ml / min to 0.40 ml / min, 0.31 ml / min to 0.39 ml / min, 0.32 ml / min to 0.38 ml / min, 0.33 ml / min to 0.37 ml / min, 0.34 ml / min to 0.36 ml / min, 0.33 ml / min to 0.35 ml / min, 0.34 ml / min to 0.35 ml / min, 0.35 ml / min to 0.38 ml / min, 0.35 ml / min to 0.37 ml / min, or 0.35 ml / min to 0.36 ml / min, as well as additional sub-ranges formed based on the above values.

[0011] The running buffer may contain one or more nonionic surfactants, such as poloxamer, preferably poloxamer 188. The running buffer may also have salt concentrations exceeding 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, or 400 mM. The salt content range may include, but is not limited to, 140 mM to 400 mM, 150 mM to 400 mM, 180 mM to 400 mM, 200 mM to 380 mM, 220 mM to 360 mM, 240 mM to 360 mM, 260 mM to 360 mM or 260 mM to 340 mM, and additional sub-ranges formed based on the above values.

[0012] The run buffer may contain inorganic salts. Optionally, the run buffer may contain two or more types of inorganic salts, such as sodium chloride and sodium phosphate. Attached Figure Description

[0013] Figure 1 This is a graph depicting the elution of recombinant AAV8 virions and the elution of free DNA detected using a photodiode array (PDA) measuring absorbance at 260 nm. The graph depicts stressed recombinant AAV virions (10 freeze-thaw cycles of AAV8) and control recombinant AAV (thawed from -80°C).

[0014] Figure 2 It is a graph depicting the elution of recombinant AAV8 virions and free AAV DNA detected by a PDA measuring absorbance at 260 nm and 280 nm.

[0015] Figure 3 It is a graph depicting the elution of recombinant AAV8 virions and the elution of free DNA detected by a PDA measuring absorbance at 280 nm and an Acquity fluorescence detector with excitation wavelength of 280 nm and emission wavelength of 350 nm.

[0016] Figure 4This is a graph depicting the elution of DNA from two DNA samples with a dominant recombinant AAV8 peak under stress, detected using a PDA measuring absorbance at 260 nm. The graph shows stress samples subjected to two temperature treatments: one at 50°C for 30 minutes and the other at 50°C for 30 minutes, followed by DNase treatment at 37°C for 30 minutes. Figure 4 The chart also depicts the control recombinant AAV (thawed from -80°C).

[0017] Figures 5A-5C Characterization data of genomic DNA leakage from AAV are shown. Figure 5A This is a bar graph depicting the quantification of genomic DNA leakage from a recombinant AAV8 sample with a genomic titer of 7.5E+12 vg / mL subjected to freezing / thawing, stirring, and heat stress conditions. The dashed lines facilitate comparison between the test sample and the control. Figure 5B This is a bar graph depicting the quantification of genomic DNA leakage from recombinant AAV8 with a genomic titer of 3.0E+13 vg / mL after various freeze-thaw cycles. The dashed lines facilitate comparison between test samples and controls. Figure 5C This is a bar graph depicting data from samples subjected to freeze / thaw stress conditions in an AAV formulation containing 1.5% w / v sucrose. X: Number of cycles; D: Days; W: Weeks; F / T: Freeze / thaw. For each data point, n=6 (three samples were prepared for each condition, and the run was repeated for each sample).

[0018] Figure 6 This is a graph showing the genomic DNA ejection and thermal desorption profile of AAV8 as measured by differential scanning fluorometry (DSF) using SYBR gold dye. The dashed lines indicate the T-phase ejection of genomic DNA. 起始 Solid line indicates T m .

[0019] Figures 7A-7B Data on the performance evaluation of the SEC cell-free DNA method are presented. Figure 7A This is a graph showing the linear relationship between the AAV sample injection volume and the calculated amount of free DNA. Figure 7B This is a graph illustrating the reproducibility of a method that tests AAV samples subjected to the same 10 freeze / thaw cycles of stress over a two-month period.

[0020] Figure 8 This is a graph showing the linear relationship between the mass of 2 kb double-stranded (ds) DNA (in nanograms (ng)) and the peak area in liquid chromatography (LC).

[0021] Figure 9It is a bar graph depicting the calculated free DNA concentration (µg / ml) for samples with different injection volumes (µl).

[0022] Figure 10 This is a bar graph describing the quantification of genomic DNA leakage in AAV5 samples using the SEC cell-free DNA method before and after 4 freeze / thaw cycles, and in formulations containing and without 1.5% w / v sucrose. X: Number of cycles; F / T: Freeze / thaw.

[0023] Figure 11 This is a graph showing the linear relationship between the reported percentage of intact capsid (intact%) and the absorbance ratio at 260 nm / 230 nm. An asterisk indicates two test samples where the theoretical intact % value was reported using an orthogonal method.

[0024] Figures 12A-12B : Figure 12A This is a graph depicting the SEC chromatogram of an AAV5 sample containing dimers (arrows) detected at an absorbance of 280 nm. Figure 12B This is a graph depicting the characterization of AAV5 dimers using the SEC free DNA method, which employs fluorescence detection at excitation at 280 nm and emission at 350 nm, for both control AAV5 samples and AAV5 samples treated with DNase.

[0025] Figure 13 It is a graph depicting the linear relationship between the AAV sample injection volume and the absorption area of ​​the dimer peak at 230 nm. Detailed Implementation

[0026] 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 to which this invention pertains.

[0027] definition

[0028] In the context of numerical values ​​and ranges, the term "about" refers to a value or range that is approximately or close to the listed value or range, such that the invention can proceed as contemplated, such as having the desired rate, quantity, density, degree, increase, decrease, percentage, value, or the presence of a form, variant, temperature, or time quantity, as is apparent from the teachings contained herein. For example, "about" can mean a value that is higher or lower than the stated value, which ranges from approximately + / - 10% or more or less, depending on the capability of execution. Thus, the term covers values ​​other than those caused solely by systematic errors.

[0029] "Recombinant AAV vector" refers to an AAV virus that includes at least one gene of interest (GOI) and an AAV inverted terminal repeat (ITR) sequence flanked by the GOI.

[0030] "Free DNA" refers to DNA species that are neither bound to nor inside the viral capsid and can be separated from the viral capsid using SEC-UPLC.

[0031] A “gene of interest” (GOI) encodes a “protein of interest.” A “protein of interest” or “peptide of interest” (POI) can have any amino acid sequence and includes any protein, polypeptide, or peptide that is desired to be expressed. This includes, but is not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins, and animal (including human) proteins. Protein types may include, but are not limited to, antibodies (including derivatives and fragments), receptors, Fc-containing proteins, trap proteins (including small trap proteins), fusion proteins, antagonists, inhibitors, enzymes (such as enzymes used in enzyme replacement therapy), factors, repressor proteins, activators, ligands, reporter proteins, select proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters, and contractile proteins. Derivatives, components, domains, chains, and fragments of the above substances are also included. The sequence can be natural, semi-synthetic, or synthetic.

[0032] "Inorganic salts" are salts lacking carbon-hydrogen bonds and can be halide salts of alkali metals or alkaline earth metals. Inorganic salts include beryllium, lithium, sodium, and potassium salts of acetic acid; sodium bicarbonate and potassium bicarbonate; lithium carbonate, sodium carbonate, potassium carbonate, and cesium carbonate; lithium chloride, sodium chloride, potassium chloride, cesium chloride, and magnesium chloride; sodium fluoride and potassium fluoride; sodium nitrate, potassium nitrate, and calcium nitrate; sodium phosphate and potassium phosphate; and calcium sulfate and magnesium sulfate. Inorganic salts can combine in solution to form part of a buffer solution, such as a buffer solution containing sodium chloride and sodium phosphate. Inorganic salts can also combine with organic salts having at least one carbon-hydrogen bond.

[0033] A surfactant is a substance that reduces the surface tension of the fluid to which it is dissolved and / or reduces the interfacial tension between two liquids (liquid and gas or liquid and solid). Surfactants can be ionic or nonionic. Exemplary nonionic surfactants that may be included in the formulations of this invention include, for example, alkyl poly(ethylene oxide), alkyl polyglycosides (e.g., octyl glucoside and decyl maltodextrin), fatty alcohols (such as cetyl alcohol and oleyl alcohol), cocoamide MEA, cocoamide DEA, and cocoamide TEA. Specific nonionic surfactants that may be included in the formulations of this invention include, for example, polysorbates, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamer, such as poloxamer 188 and poloxamer 407; polyethylene glycol-polypropylene glycol; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitol monolaurate, and polyoxyethylene sorbitol monolaurate.

[0034] All numerical limits and ranges shown herein include all numerical values ​​within or around that range or limit. The ranges and limits described herein explicitly name and define all integer, decimal, and fractional values ​​defined and covered by that range or limit. Therefore, unless otherwise specified herein, the listing of numerical ranges is intended to refer to each individual value falling within that range, and each individual value is incorporated into the description as if it were listed separately herein. All listed numerical values ​​explicitly define and name all ranges arising from the listed numerical values.

[0035] describe

[0036] This invention provides a novel SEC cell-free DNA assay that achieves reliable quantification of genomic DNA leakage from AAV capsids by optimizing column properties, mobile phase composition, and column regeneration procedures to improve assay sensitivity and separation efficiency. The optimized method of this invention uses DNA standards to generate calibration curves and achieves satisfactory assay performance in terms of sensitivity, precision, and linearity for quantifying cell-free DNA released from AAVs, and has proven applicable to a variety of AAV serotypes. This invention is suitable for all AAV serotypes, including, for example, AAV1, AAV2, AAV2quad(YF), AAV27m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, avian AAV, sea lion AAV, bearded dragon AAV, and their variants. Furthermore, this invention demonstrates that the optimized SEC assay can also be applied to characterize various quality properties of AAV, including quantifying the percentage of intact capsids and AAV dimers, and can serve as an invaluable tool for supporting the development of AAV gene therapy products.

[0037] Compared to past methods hampered by column clogging, pressure buildup to approximately 1700 psi, and limited interpeak resolution, this invention offers improved performance. Column clogging and high pressure are considered to be the result of AAV virus particles adsorbing onto the surface of the chromatographic medium, particularly for media with pore sizes of 500 Å or smaller. This invention avoids these problems by using large-pore columns, varying column flow rates, and buffer solutions containing surfactants and increased concentrations of inorganic salts, and typically achieves column pressures of approximately 1450 to approximately 1550 psi. For example, column pressures can be approximately 1400 psi, approximately 1450 psi, approximately 1500 psi, approximately 1550 psi, approximately 1600 psi, approximately 1650 psi, or approximately 1700 psi.

[0038] According to the present invention, the running buffer may contain one or more inorganic salts, with a total salt concentration of about 140 mM to about 400 mM, preferably about 180 mM to about 350 mM. For example, the running buffer may also have a salt content of more than 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM or 400 mM. Preferred subranges include 200 mM to 350 mM, 225 to 350 mM, 250 mM to 350 mM, 300 mM to 350 mM, 305 mM to 345 mM, 310 mM to 340 mM, 315 mM to 335 mM, and 320 mM to 330 mM. The ranges described herein explicitly name and articulate all integer, decimal, and fractional values ​​defined and covered by those ranges. For example, 300 mM to 350 mM includes 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349 and 350 mM, as well as fractional values ​​between them.

[0039] One or more surfactants may be used, and the total surfactant concentration may be from about 0.001% to about 0.1%; or from about 0.005% to about 0.05%. For example, the preparations of the present invention may contain about 0.005%; about 0.006%; about 0.007%; about 0.008%; about 0.009%; about 0.010%; about 0.011%; about 0.012%; about 0.013%; about 0.014%; about 0.015%; about 0.016%; about 0.017%; about 0.018%; about 0.019%; about 0.020%; about 0.021%; about 0.22%; about 0.023%; about 0.024%; about 0.025%; about 0.026%; about 0.0 27%; approximately 0.028%; approximately 0.029%; approximately 0.030%; approximately 0.031%; approximately 0.32%; approximately 0.033%; approximately 0.034%; approximately 0.035%; approximately 0.036%; approximately 0.037%; approximately 0.038%; approximately 0.039%; 0.040%; approximately 0.041%; approximately 0.042%; approximately 0.043%; approximately 0.044%; approximately 0.045%; approximately 0.046%; approximately 0.047%; approximately 0.048%; approximately 0.049% or approximately 0.050% of total surfactant.

[0040] 10 can be used 12 Up to 10 14 A column loading concentration of 1 viral genome per milliliter. Preferably, approximately 3.5 x 10⁻⁶. 12 vg / ml up to 9 x 10 13 vg / ml. For example, preferred subranges include 4 x 10 12 vg / ml up to 8 x 10 13 vg / ml; 5 x 10 12 vg / ml up to 7.5 x 10 13 vg / ml; 6 x 10 12 vg / ml up to 7 x 10 13 vg / ml; 7 x 10 12 vg / ml up to 7 x 10 13 vg / ml; 7 x 10 12 vg / ml up to 6 x 10 13 vg / ml; 7.5 x 10 12 vg / ml up to 5 x 10 13 vg / ml; 7.5 x 10 12 vg / ml up to 4 x 10 13 vg / ml; 7.5 x 10 12 vg / ml up to 3.5 x 1013 vg / ml; 7.5 x 10 12 vg / ml up to 3 x 10 13 vg / ml; 8 x 10 12 vg / ml up to 3 x 10 13 vg / ml; 9 x 10 12 vg / ml up to 3 x 10 13 vg / ml; or 1 x 10 13 vg / ml up to 3 x 10 13 vg / m.

[0041] The pore size (usually indicated by the manufacturer as the nominal pore size) of the size exclusion column media should be greater than 500 Å. Preferably, the pore size of the media is about 550 Å, about 600 Å, about 650 Å, about 700 Å, about 800 Å, about 850 Å, about 900 Å, 950 Å, about 1000 Å, about 1050 Å, about 1100 Å, about 1150 Å, about 1200 Å, about 1250 Å, about 1300 Å, about 1350 Å, about 1400 Å, about 1450 Å, or about 1500 Å. The aperture should be greater than 500 Å, such as approximately 550 Å, approximately 600 Å, approximately 650 Å, approximately 700 Å, approximately 800 Å, approximately 850 Å, approximately 900 Å, 950 Å, approximately 1000 Å, approximately 1050 Å, approximately 1100 Å, approximately 1150 Å, approximately 1200 Å, approximately 1250 Å, approximately 1300 Å, approximately 1350 Å, approximately 1400 Å, approximately 1450 Å, or approximately 1500 Å. Aperture ranges include, but are not limited to, 550 Å to 1500 Å, 700 Å to 1500 Å, 550 Å to 1100 Å, 700 Å to 1100 Å, or 900 Å to 1100 Å, as well as additional sub-ranges based on the above values.

[0042] The flow rate should be less than 0.5 ml / min to reduce pressure buildup. Preferably, the average flow rate is about 0.49 ml / min, 0.48 ml / min, 0.47 ml / min, 0.46 ml / min, 0.45 ml / min, 0.44 ml / min, 0.43 ml / min, 0.42 ml / min, 0.41 ml / min, 0.40 ml / min, 0.39 ml / min, 0.38 ml / min, 0.37 ml / min, 0.36 ml / min, 0.35 ml / min, 0.34 ml / min, 0.33 ml / min, 0.32 ml / min, 0.31 ml / min, 0.30 ml / min, 0.29 ml / min, 0.28 ml / min, 0.27 ml / min, 0.26 ml / min, 0.25 ml / min, 0.24 ml / min, 0.23ml / min, 0.22 ml / min, 0.21 ml / min, 0.20 The flow rates are 0.19 ml / min, 0.18 ml / min, 0.17 ml / min, 0.16 ml / min, 0.15 ml / min, 0.14 ml / min, 0.13 ml / min, 0.12 ml / min, 0.11 ml / min, or 0.10 ml / min. Any of the above flow rate ranges can be used. For example, flow rates can be 0.30 ml / min to 0.40 ml / min, 0.31 ml / min to 0.39 ml / min, 0.32 ml / min to 0.38 ml / min, 0.33 ml / min to 0.37 ml / min, 0.34 ml / min to 0.36 ml / min, 0.33 ml / min to 0.35 ml / min, 0.34 ml / min to 0.35 ml / min, 0.35 ml / min to 0.38 ml / min, 0.35 ml / min to 0.37 ml / min, or 0.35 ml / min to 0.36 ml / min. Pressure buildup refers to the accumulation of back pressure inside the column. Column clogging means that the column is blocked by particles from the sample or mobile phase, and cannot function properly or results in high column pressure.

[0043] buffer solution

[0044] The preparation of rAAV viral particles may contain about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM ± 1 mM Tris buffer. The preparation may contain about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM ± 1 mM sodium phosphate buffer. The preparation may contain 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, or about 15 mM Tris buffer. The preparation may contain 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, or about 15 mM sodium phosphate buffer. The preparation may also contain approximately 10 mM ± 2 mM Tris buffer. The preparation may contain approximately 10 mM ± 2 mM sodium phosphate buffer.

[0045] pH

[0046] The preparations of rAAV viral particles disclosed herein have physiologically compatible pH values. Preparations containing a buffer are provided, the buffer being suitable for maintaining the pH of the preparation between about 6.0 and about 8.0. The pH of the preparations disclosed herein can be about 6.0 to about 7.0, about 6.5 to about 7.5, about 6.6 to about 7.0, about 6.7 to about 7.0, about 6.8 to about 7.0, about 6.9 to about 7.0, about 7.0 to about 7.5, about 7.0 to about 7.1, or about 7.0 to about 7.3. The pH of the preparations can be about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, 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, or about 8.0. The pH of the preparations disclosed herein can be about 7.0 ± 0.1. The pH of the protein preparations disclosed herein can be about 7.0 ± 0.05. The pH of the protein preparations disclosed herein can be about 6.9 ± 0.1. The pH of the protein preparations disclosed herein can be about 6.9 ± 0.05.

[0047] cryoprotectant

[0048] The preparation of rAAV virus particles disclosed herein may include one or more cryoprotectants, such as one or more sugars.

[0049] The addition of one of several sugars (e.g., between about 1% and about 10%) improves the stability of the preparations of this disclosure. The preparations of this disclosure may contain about 1% to about 10% of one or more sugars. Any sugar may be used in the preparation, such as monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, mannitol, sorbitol, xylose, maltose, lactose, sucrose, dextran, trehalose, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethyl cellulose. The sugar may be sucrose, trehalose, or a combination thereof.

[0050] Sugars may be used alone or in combination. Sugars or combinations thereof may be present in the preparation at concentrations of about 0.10% to about 1.0% (w / v), about 0.20% to about 1.0% (w / v), about 0.30% to about 1.0% (w / v), about 0.40% to about 1.0% (w / v), about 0.50% to about 1.0% (w / v), about 0.60% to about 1.0% (w / v), about 0.70% to about 1.0% (w / v), about 0.80% to about 1.0% (w / v), about 0.90% to about 1.0% (w / v), about 1.0% to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.5% to about 7.5% (w / v), about 3% (w / v) to about 7% (w / v), or about 4% to about 6% (w / v). The preparations disclosed herein may contain approximately 1.0% (w / v), approximately 1.1% (w / v), approximately 1.2% (w / v), approximately 1.3% (w / v), approximately 1.4% (w / v), approximately 1.5% (w / v), approximately 1.6% (w / v), approximately 1.7% (w / v), approximately 1.8% (w / v), approximately 1.9% (w / v), approximately 2.0% (w / v), approximately 2.1% (w / v), approximately 2.2% (w / v), approximately 2.3% (w / v), approximately 2.4% (w / v), approximately 2.5% (w / v), approximately 2.6% (w / v), approximately 2.7% (w / v), approximately 2.8% (w / v), approximately 2.9% (w / v), approximately 3.0% (w / v), approximately 3.1% (w / v), approximately 3.2% (w / v), and approximately 3.3%. (w / v), approximately 3.4% (w / v), approximately 3.5% (w / v), approximately 3.6% (w / v), approximately 3.7% (w / v), approximately 3.8% (w / v), approximately 3.9% (w / v), approximately 4.0% (w / v), approximately 4.1% (w / v), approximately 4.2% (w / v), approximately 4.3% (w / v), approximately 4.4% (w / v), approximately 4.5% (w / v), approximately 4.6% (w / v), approximately 4.7% (w / v), approximately 4.8% (w / v), approximately 4.9% (w / v), approximately 5.0% (w / v), approximately 5.1% (w / v), approximately 5.2% (w / v), approximately 5.3% (w / v), approximately 5.4% (w / v), approximately 5.5% (w / v), approximately 5.6% (w / v), approximately 5.7% (w / v), approximately 5.8% (w / v), approximately 5.9% (w / v), approximately 6.0% (w / v), approximately 6.1% (w / v), approximately 6.2% (w / v), approximately 6.3% (w / v), approximately 6.4% (w / v), approximately 6.5% (w / v), approximately 6.6% (w / v), approximately 6.7% (w / v), approximately 6.8% (w / v), approximately 6.9% (w / v), approximately 7.0% (w / v), approximately 7.1% (w / v), approximately 7.2% (w / v), approximately 7.3% (w / v), approximately 7.4% (w / v), approximately 7.5% (w / v), approximately 7.6% (w / v), approximately 7.7% (w / v), approximately 7.8% (w / v), approximately 7.9% (w / v), approximately 8.0% (w / v), approximately 8.1% (w / v), approximately 8.2% (w / v), approximately 8.3% (w / v), approximately 8.4% (w / v), approximately 8.5% (w / v), approximately 8.6% (w / v), approximately 8.7% (w / v), approximately 8.8% (w / v), approximately 8.9% (w / v), approximately 9.0% (w / v), approximately 9.1% Sugars in the following proportions (w / v): approximately 9.2% (w / v), approximately 9.3% (w / v), approximately 9.4% (w / v), approximately 9.5% (w / v), approximately 9.6% (w / v), approximately 9.7% (w / v), approximately 9.8% (w / v), approximately 19.9% ​​(w / v), or approximately 10% (w / v).

[0051] The preparations disclosed herein may include about 0.10% to about 1.0% (w / v), about 0.20% to about 1.0% (w / v), about 0.30% to about 1.0% (w / v), about 0.40% to about 1.0% (w / v), about 0.50% to about 1.0% (w / v), about 0.60% to about 1.0% (w / v), about 0.70% to about 1.0% (w / v), about 0.80% to about 1.0% (w / v), about 0.90% to about 1.0% (w / v), or about 1.0% to about 10% (w / v) of sucrose. The preparations may contain about 0.5% w / v ± 0.1%, about 1.0% w / v ± 0.1%, about 1.5% w / v ± 0.1%, or about 2.0% w / v ± 0.1% w / v of sucrose.

[0052] Salt

[0053] Preparations disclosed herein may include one or more pharmaceutically acceptable salts.

[0054] Pharmaceutically acceptable salts may include, but are not limited to, metal salts such as sodium, potassium, and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine, and N-substituted guanidine, acetamidine, and N-substituted acetamidine, pyridine, methylpyridine, ethanolamine, triethanolamine, dicyclohexylamine, and N,N′-dibenzylethylenediamine. Pharmaceutically acceptable salts (with a basic nitrogen center) may include, but are not limited to, inorganic acid salts such as hydrochlorides, hydrobromic acid salts, sulfates, and phosphates; organic acid salts such as trifluoroacetates and maleates; sulfonates such as methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, and naphthalenesulfonates; amino acid salts such as arginine, alanine, aspartate, and glutamate; and carbohydrate salts such as gluconates and galacturons. Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sodium, ammonium, potassium, calcium, and magnesium salts (e.g., sodium chloride, ammonium chloride, potassium chloride, calcium chloride, and magnesium chloride; sodium acetate, ammonium acetate, potassium acetate, calcium acetate, and magnesium acetate; sodium citrate, ammonium citrate, potassium citrate, calcium citrate, and magnesium citrate; sodium phosphate, ammonium phosphate, potassium phosphate, calcium phosphate, and magnesium phosphate; sodium fluoride, ammonium fluoride, potassium fluoride, calcium fluoride, and magnesium fluoride; sodium bromide, ammonium bromide, potassium bromide, calcium bromide, and magnesium bromide; and sodium iodide, ammonium iodide, potassium iodide, calcium iodide, and magnesium iodide). Pharmaceutically acceptable salts may be sodium chloride or arginine hydrochloride (L-arginine hydrochloride).

[0055] The preparations disclosed herein may also include one or more pharmaceutical viscosity reducers, such as sodium chloride, lysine, proline, etc.

[0056] The preparations disclosed herein may contain approximately 10 mM to approximately 300 mM, approximately 50 mM to approximately 150 mM, approximately 50 mM to approximately 100 mM, approximately 50 mM to approximately 200 mM, approximately 50 mM to approximately 250 mM, approximately 50 mM to approximately 300 mM, approximately 100 mM to approximately 200 mM, approximately 100 mM to approximately 250 mM, approximately 100 mM to approximately 300 mM, approximately 150 mM to approximately 200 mM, approximately 150 mM to approximately 250 mM, approximately 150 mM to approximately 300 mM, approximately 250 mM to approximately 300 mM, approximately 75 mM to approximately 100 mM, approximately 175 mM to approximately 200 mM, approximately 175 mM to approximately 225 mM, approximately 200 mM to approximately 225 mM, approximately 225 mM to approximately 275 mM, approximately 275 mM to approximately 300 mM. The preparations disclosed herein may comprise pharmaceutically acceptable salts of about 0 mM or about 175 mM to about 275 mM.The preparations disclosed herein may contain approximately 50 mM, approximately 51 mM, approximately 52 mM, approximately 53 mM, approximately 54 mM, approximately 55 mM, approximately 56 mM, approximately 57 mM, approximately 58 mM, approximately 59 mM, approximately 60 mM, approximately 61 mM, approximately 62 mM, approximately 63 mM, approximately 64 mM, approximately 65 mM, approximately 66 mM, approximately 67 mM, approximately 68 mM, approximately 69 mM, approximately 70 mM, approximately 71 mM, approximately 72 mM, approximately 73 mM, approximately 74 mM, approximately 75 mM, approximately 76 mM, approximately 77 mM, approximately 78 mM, approximately 79 mM, approximately 80 mM, approximately 81 mM, approximately 82 mM, approximately 83 mM, approximately 84 mM, approximately 85 mM, approximately 86 mM, approximately 87 mM, approximately 88 mM, approximately 89 ... mM, approximately 90 mM, approximately 91 mM, approximately 92 mM, approximately 93 mM, approximately 94 mM, approximately 95 mM, approximately 96 mM, approximately 97 mM, approximately 98 mM, approximately 99 mM, approximately 100 mM, approximately 101 mM, approximately 102 mM, approximately 103 mM, approximately 104 mM, approximately 105 mM, 106 mM, approximately 107 mM, approximately 108 mM, approximately 109 mM, approximately 110 mM, approximately 115 mM, approximately 120 mM, approximately 125 mM, approximately 130 mM, approximately 135 mM, approximately 140 mM, approximately 145 mM, approximately 150 mM, approximately 155 mM, approximately 160 mM, approximately 165 mM, approximately 170 mM, approximately 175 mM, approximately 180 mM, approximately 185 Pharmaceutically acceptable salts of approximately 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, or 300 mM.

[0057] The preparations disclosed herein may contain about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM of sodium chloride. The preparations disclosed herein may contain about 100 mM ± 5 mM, about 110 mM ± 5 mM, about 120 mM ± 5 mM, about 130 mM ± 5 mM, about 140 mM ± 5 mM, about 150 mM ± 5 mM, about 160 mM ± 5 mM, about 170 mM ± 5 mM, about 180 mM ± 5 mM, about 190 mM ± 5 mM, about 200 mM ± 5 mM, or about 210 mM ± 5 mM of sodium chloride.

[0058] surfactants

[0059] The preparations disclosed herein may include one or more surfactants.

[0060] The preparations disclosed herein may contain a stable concentration of a pharmaceutically acceptable nonionic surfactant. Pharmaceutically acceptable nonionic surfactants that may be used in the preparations discussed herein may include, but are not limited to, polysorbate 80 (Tween 80; PS80), polysorbate 81 (Tween 81; PS81), polysorbate 82 (Tween 82; PS82), polysorbate 20 (Tween 20; PS20), and various poloxamers (e.g., poloxamer 188), or mixtures thereof.

[0061] The preparations disclosed herein may contain about 0.01% (w / v) to about 0.30% (w / v) of a nonionic surfactant. The preparations may contain about 0.01% to 0.30% (w / v), about 0.01% to 0.05% (w / v), about 0.05% to 0.10% (w / v), about 0.10% to 0.15% (w / v), about 0.15% to 0.20% (w / v), about 0.20% to 0.25% (w / v), or about 0.25% to 0.30% (w / v) of a nonionic surfactant. The preparations disclosed herein may contain approximately 0.05% (w / v), approximately 0.06% (w / v), approximately 0.07% (w / v), approximately 0.08% (w / v), approximately 0.09% (w / v), approximately 0.10% (w / v), approximately 0.11% (w / v), approximately 0.12% (w / v), approximately 0.13% (w / v), approximately 0.14% (w / v), approximately 0.15% (w / v), approximately 0.16% (w / v), approximately 0.17% (w / v), approximately 0.18% (w / v), approximately 0.19% (w / v), approximately 0.20% (w / v), approximately 0.21% (w / v), approximately 0.22% (w / v), approximately 0.23% (w / v), approximately 0.24% (w / v), approximately 0.25% (w / v), and approximately 0.26%. The preparations disclosed herein may contain about 0.20% w / v ± 0.01% w / v of a nonionic surfactant.

[0062] The preparations disclosed herein may contain about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), or about 0.01%. (w / v) poloxamer 188. Preparations disclosed herein may contain about 0.005% w / v ± 0.001% w / v of polysorbate 80 or about 0.005% w / v ± 0.001% w / v of poloxamer 188.

[0063] The present invention is further described through the following embodiments, which are intended to support and illustrate the invention, but do not limit the invention in any way.

[0064] Example

[0065] Materials and reagents

[0066] Recombinant AAV8 and AAV5 viral vectors encapsulating approximately 4.0 kb of single-stranded DNA (ssDNA) transgenes were produced by triple transfection of HEK293 cells in suspension cell cultures, followed by purification using affinity chromatography and anion exchange chromatography. Kolliphor® poloxamer 188 (P188) BIO was obtained from BASF (Geismar, LA). Tris base and Tris hydrochloride were from Avantor JT Baker® (Radnor Township, PA). All other excipients and reagents were pharmacopoeia grade. 2.0 kb double-stranded DNA (dsDNA) standards and Invitrogen™ DNase I were from ThermoFisher Scientific (Waltham, MA). Crystal Zenith® (CZ) cycloolefin polymer (COP) vials, aluminum Flip-Off® seals, and serum NovaPure® stoppers were from West Pharmaceutical Services (Exton, PA).

[0067] Sample preparation

[0068] In formulation 1 (F1) containing 10 mM Tris, 180 mM sodium chloride, 0.005% w / v P188, and pH 7.3, or in formulation 2 (F2) containing 1.5% w / v sucrose in addition to the same excipients as in F1, the AAV8 and AAV5 vectors were prepared to target titers of 7.5E+12 or 3E+13 vector genomes (vg) / mL, respectively. After preparation, 0.4 mL of AAV8 or AAV5 vector was filled into 2 mL CZ vials and sealed with a 13 mm serum NovaPure stopper and a 13 mm aluminum Flip-Off sealing cap.

[0069] Generation of stress-induced AAV samples

[0070] To generate freeze / thaw stress AAV samples, vials containing AAV8 and AAV5 were placed in a freezer at -80°C for at least 1.5 hours, then transferred to a benchtop at room temperature for at least 1 hour to complete each freeze / thaw cycle. To generate stir stress samples, AAV8 and AAV5 vials were placed on a track shaker set to 300 rpm for 1 and 2 days. To generate heat stress samples, AAV8 and AAV5 sample vials were incubated at 37°C for 1 and 2 weeks, or at 50°C for 30 minutes. Control AAV samples were kept at -80°C.

[0071] DNA enzyme treatment

[0072] To digest any unencapsulated DNA that may be present in the sample, the AAV8 and AAV5 vectors were treated with DNase I according to the manufacturer's instructions. In short, 30 μL of the DNase I reaction mixture was added to the AAV sample in a 20 μL polymerase chain reaction (PCR) tube. After gently vortexing for 15 seconds, the reaction mixture was incubated in a thermal cycler set to 37°C for 30 minutes, and then cooled to 4°C until analysis.

[0073] Size exclusion chromatography (SEC)

[0074] SEC was performed on a Waters™ ACQUITY UPLC system equipped with photodiode array detectors (PDA, absorption wavelengths of 230 nm, 260 nm, and 280 nm) and fluorescence detectors (FLD, excitation wavelength of 280 nm and emission wavelength of 350 nm). Cell-free DNA was separated from AAV using an SRT SEC column (1000 Å, 5 µm, 4.6 x 300 mm, Sepax Technologies, Newark, DE). All sample vials were stored in an autosampler set to 4°C. AAV samples were analyzed using a mobile phase containing 20 mM sodium phosphate, 300 mM sodium chloride, 0.02% w / v P188, pH 7.3, at a flow rate of 0.35 mL / min, with a total run time of 40 min per sample.

[0075] Optimization of the SEC method

[0076] Optimizing the SEC method for quantifying free DNA released from AAV serves two purposes. The first is to prevent pressure buildup or blockage in the column, which has been identified as a major problem with previously reported SEC methods, likely due to the inherent high surface adsorption tendency of AAV samples. The second is to improve the separation efficiency between the free DNA peak and the AAV peak.

[0077] To achieve these objectives, the present invention optimizes the mobile phase by including 300 mM sodium chloride and 0.02% w / v P188 to prevent column pressure buildup. High salt content is typically included in SEC mobile phases, which effectively prevents adsorption onto the column by reducing charge-mediated nonspecific interactions. Furthermore, mobile phases containing high salt are more compatible with AAV sample analysis because the high salt content helps prevent AAV aggregation. Surfactants (such as P188) work by reducing the surface tension at the stationary phase interface of the SEC column, and thus inhibit the adsorption of AAV and free DNA due to competition for interfacial binding with the surfactant. Notably, 0.02% w / v P188 plays a role in reducing adsorption onto the column, as significant pressure buildup is observed when it is omitted from the mobile phase or at lower concentrations (such as 0.005% w / v). Additionally, the column is optimized for operating, regeneration, and storage conditions that significantly affect column performance, such as leading to a significant increase in back pressure or loss of separation resolution. For example, although the free DNA and major AAV particle peaks eluted within a 13-minute run, the run time was extended to 40 minutes to ensure that any residual levels of precipitated protein or other contaminants that might accumulate on the column and require extended elution times were completely removed before subsequent injections began. After the SEC run, the column was washed with 3 column volumes of water, followed by 3 column volumes of 20% ethanol at a flow rate of 0.2 mL / min to prevent microbial growth. By applying this optimized SEC protocol, excellent separation resolution of both peaks was obtained, with the free DNA peak eluting at approximately 7 minutes and the AAV particle peak eluting at approximately 9.5 minutes. (As Xu) et al. (Xu et al. 2022. Genome DNA leakage of Adeno-Associated virus under freeze-thaw stress. Int J Pharm As described in 615:121464), the identity of the free DNA peaks has been verified using NGS.

[0078] As a result of method optimization, a novel and improved SEC method for cell-free DNA was developed, which exhibits excellent separation efficiency between cell-free DNA and AAV particle peaks. Enhanced accuracy and reproducibility of the assay were achieved without pressure accumulation. Furthermore, the optimized mobile phase is relatively mild at neutral pH 7.3 and is free of organic solvents, which matches the pH of the AAV formulation tested in the experiments disclosed herein. Additionally, 0.02% w / v P188 showed good compatibility with several AAV serotypes. Therefore, the optimized SEC mobile phase carries minimal or no risk of conformational changes or denaturation of AAV particles.

[0079] The invention is further described through the following embodiments, which are not intended to limit the invention in any way and are applicable to all sections of this specification and aspects of the invention. The order of execution of the following embodiments may be changed or combined, as determined by those skilled in the art based on the teachings and data contained herein.

[0080] Example 1

[0081] This embodiment provides evidence that the present invention can detect and distinguish cell-free DNA from AAV virions. The concentration was 7.5 E+10 in a storage buffer of 10 mM Tris, 180 mM NaCl, and 0.005% poloxamer 188 at pH 7.3. 12 Recombinant AAV8 at vg / ml was subjected to 10 freeze-thaw cycles (10X freeze / thaw). Excessive freeze / thaw stresses the viral capsid and leads to capsid instability, thereby releasing DNA. The control recombinant AAV8 preparation was thawed without undergoing repeated freeze-thaw cycles.

[0082] Figure 1 Representative chromatograms were collected at 260 nm absorbance from an exemplary control AAV8 sample stored at -80°C and an AAV sample after 10 freeze / thaw cycles. Baseline separation was achieved between the free DNA peak eluted at approximately 7 min and the main AAV particle peak eluted at approximately 9.5 min. The free DNA from the stress sample and the control sample had sufficient retention time difference to be easily distinguishable from the main AAV peak. A significant increase in both the area and height of the free DNA peak was observed in the freeze / thaw-stressed AAV sample compared to the control AAV sample. Furthermore, the signal-to-noise ratio of the free DNA peak detected in the stressed AAV sample was approximately 69.3, significantly higher than 10, indicating the feasibility of applying this improved SEC method to quantify free DNA released from AAV samples.

[0083] Example 2

[0084] This embodiment provides validation of free DNA peaks. Three orthogonal methods were used to validate the free DNA peaks. In the evaluation study, a method involving 10 freeze / thaw cycles was used. Figure 2 and Figure 3 ) or 50℃ treatment ( Figure 4 The AAV sample was analyzed. First, the purity of the free DNA was assessed using the ratio of absorbance at 260 nm and 280 nm. For DNA, a 260 / 280 ratio of approximately 1.8 is generally considered "pure." A significantly lower ratio indicates the presence of proteins or other contaminants that strongly absorb at or near 280 nm. Figure 2 As shown, the 260 / 280 ratio of the main peak is 1.3, which is typical for AAV particles containing capsid proteins and genomic DNA. In contrast, the 260 / 280 ratio of the free DNA peak is 1.8, indicating that the peak is pure DNA. Secondly, nucleic acids are generally considered non-fluorescent because their intrinsic fluorescence is extremely weak compared to that of proteins. Therefore, fluorescence signals excited at 280 nm and emitted at 350 nm are collected. Figure 3 In fact, fluorescence was detected only at the main AAV particle peak, and not at the free DNA peak, confirming that the free DNA peak contained pure DNA without protein. Finally, the AAV8 sample was stress-treated at 50°C for 30 minutes, followed by treatment with DNase to remove any free DNA released from the AAV capsid. Figure 4 As shown, after heat stress, the level of free DNA increased significantly; after DNase treatment, the level of free DNA decreased to near baseline, further confirming that the free DNA peak contained pure DNA without protein.

[0085] Example 3

[0086] This embodiment relates to genomic DNA leakage of AAV under various stress conditions. Once an optimized SEC method was established, this assay was applied to evaluate genomic DNA leakage of AAV8 under a variety of stress conditions, including up to 10 freeze / thaw cycles, up to 2 days of agitation, and up to 2 weeks of heat treatment at 37°C. Figure 5A Additionally, AAV8 samples treated at 50°C for 30 minutes were included as a positive control, as the initiation temperature for AAV8 capsid genome expulsion was previously determined to be approximately 50°C. Figure 5AResults from this experiment showed that the AAV8 capsid is particularly sensitive to freeze / thaw stress. After 5 and 10 freeze / thaw cycles, an increase in free DNA was observed of approximately 1.9-fold (from 0.28 µg / mL to 0.53 µg / mL) and 3.9-fold (from 0.28 µg / mL to 1.10 µg / mL), respectively. In contrast, neither orbital oscillation nor heat treatment at 37°C induced substantial release of genomic DNA compared to AAV8 samples stored at -80°C. Freeze / thaw induced DNA leakage was confirmed by high genomic titer samples (genomic titer 3.0E+13 vg / mL) subjected to various freeze-thaw cycles. Figure 5B Therefore, the stabilization of AAV during freezing / thawing becomes crucial for reducing the risk associated with genomic DNA leakage. Data are shown in Table 1 below and plotted as a bar graph. Figure 5A middle.

[0087] Table 1

[0088]

[0089] Example 4

[0090] Compared to the control, this embodiment is at 3 x 10 13 Recombinant AAV8 virions were tested at vg / ml for 2, 5, or 10 freeze / thaw cycles. Freeze / thaw stress caused capsid instability, resulting in the release of more DNA with increasing freeze / thaw cycles.

[0091] The data is shown in Table 2 below and plotted as a bar chart. Figure 5B middle.

[0092] Table 2

[0093]

[0094] This example demonstrates that free AAV DNA increases with the number of freeze / thaw cycles.

[0095] Example 5

[0096] This example demonstrates the use of cryoprotectants to mitigate genomic DNA leakage. An important decision in designing robust and stable AAV formulations is the selection of excipients based on their properties and established protein stabilization mechanisms. Cryoprotectants, particularly sucrose, are chosen as excipients to stabilize proteins, especially those sensitive to freezing / thawing stress. Cryoprotectants function through various mechanisms, including preferential exclusion from protein surfaces, formation of a glass matrix around protein molecules, or hydrogen bonding with proteins. Surfactants such as P188 are also commonly chosen for AAV formulations. As described herein, surfactants reduce surface tension at interfaces caused by mechanical disturbance or freezing / thawing processes, and thus inhibit protein adsorption at interfaces or surface-induced protein denaturation due to competition for binding at the interface with surfactants. Because the AAV formulation evaluated in this study contained similar levels of P188 as commonly found in clinically and approved AAV products (0.005% w / v P188 in F1), AAV8 samples were prepared with a buffer containing 1.5% w / v sucrose, while maintaining P188 at the same level (F2). The sucrose concentration was chosen to provide adequate stabilization while maintaining the isotonicity of the formulation to accommodate desired routes of administration, such as intravenous delivery. AAV8 samples in the 1.5% w / v sucrose formulation underwent up to four freeze / thaw cycles, followed by analysis of cell-free DNA using the SEC method. Figure 5C The AAV8 sample in the sucrose-free formulation showed a 1.9-fold increase after 5 freeze / thaw cycles. Figure 5A In contrast, adding 1.5% w / v sucrose to the AAV8 formulation effectively mitigated the release of genomic DNA due to freeze / thaw stress, and therefore no significant increase in free DNA was observed after four freeze / thaw cycles. Similar results were observed in AAV5. Figure 10 This study confirmed that 1.5% sucrose is sufficient to reduce the potential risk associated with genomic DNA leakage under freezing / thawing stress and can be included in AAV formulations designed for long-term cryopreservation.

[0097] Example 6

[0098] This example demonstrates the evaluation of the SEC method performance. To assess whether the optimized SEC method was sufficient for quantifying cell-free DNA, the method performance was characterized using AAV8 samples at 3E+13 vg / mL after 10 freeze / thaw cycles, focusing on linearity, sensitivity, and precision. A series of six injections were performed, with volumes increasing from 5 µl to 40 µL. The amount of genomic DNA released was then calculated according to the formula described above and plotted against the injection volume. Figure 7AAs shown in the figure, a strong linear correlation was observed after linear regression analysis, with an R² value of 0.99997. The data are also shown in Table 3. Furthermore, in samples with a minimum injection volume of 5 µL, the signal-to-noise ratio of the free DNA peak was greater than 10, indicating that the optimized SEC method has satisfactory sensitivity for the quantification of released genomic DNA.

[0099] Table 3

[0100]

[0101] To evaluate the precision of the optimized SEC method, six replicates were performed on each AAV8 sample under various stress conditions at 7.5E+12 vg / mL. Figure 5A The coefficient of variation (CV) for each stressed AAV8 sample was determined, and finding it within 7% demonstrated excellent reproducibility of the method. Next, the intermediate precision of the method was evaluated by measuring the same frozen / thawed stressed AAV8 samples over a two-month period. Figure 7B As shown, the results indicate a highly consistent profile of the free DNA peaks, with negligible 1% variation. Overall, the results demonstrate that the optimized SEC method can provide reliable results for the quantification of free DNA, or in other words, genomic DNA leakage from the AAV capsid. These stress studies were also conducted with AAV5 at approximately the same target titer. Figure 10 Genomic DNA leakage at levels similar to those observed in AAV8 was detected. This optimized SEC method was also used to characterize genomic DNA leakage in several other AAV serotypes, such as AAV9, AAV2, and AAV1. These data support reliable cell-free DNA quantification for other AAV serotypes using the optimized SEC method.

[0102] Example 7

[0103] This example demonstrates the development of a method for quantifying cell-free DNA. The applicability of the optimized SEC method to quantifying cell-free DNA was evaluated. First, the correlation between the amount of injected DNA standard and the peak area measured at absorbance at 260 nm was assessed. Figure 8 To quantify free DNA, DNA standards are needed to establish a calibration curve. A 2 kb dsDNA standard was selected by comparing commercially available dsDNA and ssDNA standards due to its high purity (100%). Results from this experiment indicate a strong linear relationship between the amount of DNA and the measured peak area, yielding a coefficient of determination (R²) of 0.9991 after linear regression analysis, as shown in the equation disclosed in Example 8.

[0104] Using calibration curves generated from dsDNA standards, a formula based on Beer's Law was developed to calculate the amount of released cell-free DNA. As shown in the equations disclosed in Example 8, a conversion factor was introduced into the formula, representing the ratio between the molar absorptivity of dsDNA and ssDNA, to account for the use of dsDNA in generating the calibration curve, while ssDNA was released from the AAV capsid during the study. Applying this formula, the amount of cell-free DNA released from an AAV8 sample of 3E+13 vg / mL after 10 freeze / thaw cycles was calculated to be approximately 4 µg / mL, which is related to approximately 6% of the total genomic DNA in the AAV sample. The presence of small amounts of contaminants in the dsDNA standards can contribute to the total absorbance at 260 nm, resulting in a slightly lower cell-free DNA value calculated based on the calibration curves generated using dsDNA standards. However, the values ​​reported herein using this cell-free DNA quantification method provide valuable information for developing risk assessment and mitigation strategies to achieve robust and stable AAV products.

[0105] Example 8

[0106] This embodiment involves Beer's Law, namely... A = Calculation of the estimated concentration of εbC double-stranded DNA. A It is absorbance. ε It is the molar absorptivity (L / (mol)) cm), b It is the optical path length in centimeters, and C This is a concentration expressed in mol / L. See Gallagher, Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy, Current Protocols in Cell Biology (2000).

[0107] The equations used are as follows:

[0108]

[0109] Notice:

[0110]

[0111] Figure 8 The mass of the injected 2 kb dsDNA was depicted in relation to the liquid chromatography (LC) area, showing the peak area (µV). A good linear relationship exists between the time (seconds) and the mass of DNA injected (ng). This decisive linear formula allows for the calculation of estimated ssDNA concentrations.

[0112] Example 9

[0113] This example involves estimating the concentration of single-stranded DNA based on Beer's Law. See Example 8. The concentration is 3 x 10⁻⁶. 13 Recombinant AAV8 virions at a concentration of vg / ml were subjected to 10 freeze / thaw cycles, which stressed the viral capsid and led to DNA release. Injection volumes of 5 µl, 10 µl, 15 µl, 20 µl, 25 µl, 30 µl, 35 µl, and 40 µl were tested. See Table 4.

[0114] Table 4

[0115]

[0116] Figure 9 This is a bar graph depicting the concentration (µg / ml) of free AAV8 DNA calculated at different injection volumes (µl). The concentrations are based on the following:

[0117]

[0118] Example 10

[0119] This embodiment tested the method's ability to work with AAV5. Repeated vials were used under each condition. Repeated runs on LC (full sequence repeats) were also performed. A similar trend to that observed with AAV8 was observed. The results are depicted in bar graph form. Figure 10 middle.

[0120] Example 11

[0121] This embodiment provides an application of the SEC-UPLC method to test the percentage of intact capsids and the formation of AAV dimers using the SEC method.

[0122] Since the system used for optimizing the SEC method is equipped with a fluorescence detector and a PDA detector capable of three-wavelength detection, experiments were conducted to explore whether the signals collected during SEC analysis could be used to characterize additional quality properties of AAV beyond free DNA. Three UV absorption wavelengths at 230, 260, and 280 nm were selected due to the strong absorption of peptide bonds at 230 nm, the high absorption of nucleic acids at 260 nm, and the major absorption of capsid proteins at 280 nm.

[0123] Based on the understanding that there is a linear relationship between the absorbance ratio at 260 nm / 230 nm and the percentage of intact AAV capsid (intact%), UV-Vis is used to assess the integrity of AAV. However, a drawback of batch analysis methods (such as UV-Vis) is that any impurities present in the sample can interfere with the results. In this case, the optimized SEC method effectively separates AAV from free DNA and other impurities, thus significantly minimizing interference from impurities. Therefore, experiments were conducted using the SEC free DNA method to evaluate whether a linear correlation can be established between the absorbance ratio of the main AAV particle peak at 260 nm / 230 nm and the integrity percentage of AAV.

[0124] Two AAV5 samples (Sample 1 with a low integrity percentage of 6.3% and Sample 2 with a higher integrity percentage of 77.8%) were mixed at different ratios (Table 5) to generate a series of AAV5 samples with different theoretical integrity percentages. The integrity percentage values ​​in the two AAV5 reference standards were determined using mass spectrometry (MP), an orthogonal method to AUC, for quantifying the integrity percentage and empty-to-full ratio of AAV. The series of AAV5 samples with different integrity percentages prepared above were analyzed using the SEC method to create calibration curves. The ratio of the integral area under the curve of the main AAV peak at 260 nm / 230 nm was determined and plotted against the theoretical integrity percentage calculated for each AAV5 sample (Table 5), showing a strong linear correlation with R² of 0.9996 ( ). Figure 11 To validate the SEC method used to determine the integrity percentage of AAV samples, two AAV5 samples with unknown integrity percentages were analyzed using the SEC method. Based on calibration curves generated using two AAV5 reference standards as described herein, the integrity percentages of these two AAV5 samples were determined to be 32.6% and 73.1%, respectively. Figure 11 The same two AAV5 samples were then analyzed using mass spectrometry, yielding similar integrity % values. These results provide strong evidence that the optimized SEC method can potentially provide quantitative analysis of the integrity % of AAV samples. The integrity % values ​​reported using the SEC-free DNA method proved to be highly comparable to those reported using existing methods, such as mass spectrometry commonly used for integrity % characterization of AAV.

[0125] Table 5 provides the quantification of integrity percentage using the SEC cell-free DNA method. The theoretical integrity percentage range for AAV samples was used to create a calibration curve for quantifying integrity percentage.

[0126] Table 5

[0127]

[0128] Example 12

[0129] This example evaluates the performance of a SE-UPLC method for AAV dimer quantification. An AAV5 sample containing the identified dimer was analyzed using this SEC cell-free DNA method. Experiments were performed in duplicate using serial injections from 5 µL to 40 µL. Results indicated that the method achieved good sensitivity, precision (%CV < 6%), and linearity (R² > 0.999). Figure 13 The linear relationship of the dimer peak area was depicted.

[0130] For biopharmaceutical analysis, SEC can be used to characterize small soluble aggregates or high molecular weight (HMW) substances such as dimers and oligomers. Therefore, experiments were conducted to evaluate whether the SEC cell-free DNA method could be applied to the characterization of HMW species in AAV samples. An AAV5 sample containing identified dimers was analyzed using the SEC cell-free DNA method. An additional peak eluted at approximately 9 minutes was observed, which was well separated from the cell-free DNA peak eluted at approximately 7 minutes and the main AAV particle peak eluted at approximately 9.5 minutes. Figure 12A The additional peak exhibited a positive fluorescence signal and remained unchanged after DNase treatment. Figure 12B This further confirms that the additional peak is due to the formation of AAV5 dimers rather than DNA impurities. The performance of the method used to quantify the dimers in AAV5 samples was then evaluated. A series of injections of AAV5 samples were performed, with the injection volume increasing from 5 µL to 40 µL, to generate a calibration curve plotting the integrated area under the AAV dimer peak at absorbance of 230 nm against the injection volume. Figure 13 For the quantification of AAV dimers, a wavelength of 230 nm was chosen instead of 280 nm because the absorption intensity of AAV dimers at 230 nm is significantly higher than that at 280 nm, resulting in a greater signal-to-noise ratio. The results show that the SEC cell-free DNA method exhibits good sensitivity, precision, and linearity for the quantification of dimers in AAV5 samples. The method disclosed herein can also be applied to other AAV serotypes for the quantification of dimers or oligomers (if present).

Claims

1. A method for analyzing cell-free DNA in a preparation containing recombinant AAV (rAAV) viral particles, wherein the method comprises the following steps: (a) The preparation containing rAAV virus particles is loaded into a size exclusion column with a pore size greater than 500 angstroms, wherein the preparation is mixed with a running buffer before loading; (b) The preparation is run on the size exclusion column at a flow rate of less than 0.5 mL / min, wherein pressure buildup in the size exclusion column does not occur and column clogging does not occur; and (c) Detection of the elution of free rAAV DNA and rAAV virus particles from the size exclusion column.

2. The method of claim 1, wherein the size exclusion column medium has a nominal pore size of at least about 700 angstroms.

3. The method of claim 1, wherein the size exclusion column medium has a pore size of at least about 900 angstroms.

4. The method of claim 1, wherein the size exclusion column has a pore size of about 900-1100 angstroms.

5. The method of claim 1, wherein the size exclusion column has a pore size of at least about 1000 angstroms.

6. The method according to claims 1 to 5, wherein the flow rate in step (b) is 0.45 ml / min.

7. The method according to claims 1 to 5, wherein the flow rate in step (b) is 0.40 mL / min.

8. The method according to claims 1 to 5, wherein the flow rate in step (b) is 0.35 ml / min.

9. The method according to claims 1 to 5, wherein the flow rate in step (b) is 0.30 ml / min.

10. The method of claims 1 to 9, wherein the running buffer comprises a surfactant.

11. The method according to claims 1 to 10, wherein the running buffer has a total salt content of more than 140 mM.

12. The method according to claims 1 to 10, wherein the running buffer has a total salt content of more than 150 mM.

13. The method according to claims 1 to 10, wherein the running buffer has a total salt content of more than 200 mM.

14. The method according to claims 1 to 10, wherein the running buffer has a total salt content of more than 250 mM.

15. The method according to claims 1 to 10, wherein the running buffer has a total salt content of more than 300 mM.

16. The method of claims 11 to 15, wherein the running buffer comprises two types of salts.

17. The method of claim 16, wherein the salt is sodium chloride and sodium phosphate.

18. The method according to claims 1 to 10, wherein the running buffer comprises poloxamer.

19. The method of claim 18, wherein the poloxamer is poloxamer 188.

20. A method for determining the stability of rAAV, comprising measuring uncoated DNA using SE-UPLC at 260 nm UV and analyzing the free DNA as claimed in claim 1.

21. The method of claim 20, wherein the preparation of rAAV virus particles comprises a buffer, a cryoprotectant, a salt, a nonionic surfactant, and a pH of about 6.0 to about 8.

0.

22. The method of claim 21, wherein the cryoprotectant is sucrose at a concentration of about 0.5% to about 5.0% (w / v).

23. The method of claim 21, wherein the concentration of sucrose is about 1.0 to about 3.5% (w / v), about 1.0 to about 3.0% (w / v), about 1.0 to about 2.5% (w / v), about 1.0 to about 2.0% (w / v), about 1.0 to about 1.5% (w / v), about 1.0% (w / v), about 1.5% (w / v), about 2.0% (w / v), about 2.5% (w / v), or about 3.0% (w / v).

Images