CN115337408A

CN115337408A - Adeno-associated virus formulations

Info

Publication number: CN115337408A
Application number: CN202211133080.9A
Authority: CN
Inventors: 岳占龙; 肖啸; 郑静; 郑浩; 刘素丽; 陈晨; 袁龙辉; 李小灵; 王新涛; 杜增民; 蒋威; 吴侠
Original assignee: Shanghai Letter Pharmaceutical Technology Co ltd
Current assignee: Shanghai Letter Pharmaceutical Technology Co ltd
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2022-11-15

Abstract

The present disclosure relates to an aqueous composition for stabilizing adeno-associated virus, and an adeno-associated virus preparation comprising the same. The aqueous compositions of the present disclosure can effectively reduce the loss of potency during storage of AAV formulations. The adeno-associated virus formulations of the present disclosure can maintain good stability after storage for a period of time at room temperature.

Description

Adeno-associated virus formulations

Technical Field

The present disclosure relates to an aqueous composition for stabilizing adeno-associated virus, and an adeno-associated virus preparation comprising the same.

Background

Adeno-associated viruses (AAV) are a class of tiny, non-enveloped and icosahedral-structured viruses. The viral particles have a diameter of between 20 and 26nm and contain a linear single-stranded DNA genome of about 4.7kb in size. Recombinant adeno-associated virus (rAAV) is a gene vector which is further modified on the basis of non-pathogenic wild AAV, is regarded as one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range, low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy research in the world.

AAV formulations for delivery of genes of interest to treat disease are expected to have good stability, minimizing loss of AAV potency during the manufacturing, packaging, and storage processes. The selection of a suitable solvent is critical to ensure stable storage of the AAV formulation. In addition, the storage conditions of the AAV preparation preferably meet the marketing requirements of pharmaceutical products, and preferably do not employ harsh cryopreservation conditions, and are capable of storing at room temperature while maintaining sufficient biological activity of the AAV preparation.

Disclosure of Invention

In view of the above-mentioned needs in the art, it is an object of the present disclosure to provide an aqueous composition for stabilizing AAV, which can effectively reduce the loss of potency during storage of the AAV formulation. It is also an object of the present disclosure to provide an AAV formulation that can maintain good stability after storage for a period of time at room temperature.

In a first aspect, the present disclosure provides an aqueous composition comprising: selected from Tris-HAc, KH ₂ PO ₄ -Na ₃ C ₆ H ₅ O ₇ Or a combination thereof; a surfactant selected from F68, PS20, PS80, or a combination thereof; sodium or potassium salts; selected from Fe ²⁺ 、Ca ²⁺ And Mg ²⁺ One or more of (a); and optionally a macromolecular polymer.

In one embodiment, the concentration of the buffer in the aqueous composition is 2-100mM, preferably 10-30mM, more preferably 20mM.

In one embodiment, the concentration of surfactant in the aqueous composition is from 0.0001% to 0.5% (w/v), preferably from 0.005% to 0.05% (w/v), more preferably 0.01% (w/v).

In one embodiment, the concentration of the sodium or potassium salt in the aqueous composition is 100-800mM, preferably 250-400mM, more preferably 350mM.

In one embodiment, the aqueous composition comprises Fe ²⁺ 、Ca ²⁺ Or Mg ²⁺ Is 0.01-5mM, preferably 0.1-2mM, more preferably 1mM.

In one embodiment, the concentration of the macromolecular polymer in the aqueous composition is between 0.1% and 5% (w/v), preferably between 0.5% and 3% (w/v), more preferably 2% (w/v).

In one embodiment, the aqueous composition further comprises a saccharide. In one embodiment, the saccharide includes sucrose, trehalose, sorbitol, mannitol, glucose, lactose, galactose, maltose and fructose.

In a preferred embodiment, the concentration of saccharide in the aqueous composition is between 0.1% and 10% (w/v), preferably between 1% and 5% (w/v), more preferably 2% (w/v).

In a preferred embodiment, the saccharide is sucrose.

In one embodiment, the aqueous composition has a pH of 4.5 to 8.5, preferably 7.0 to 8.5, more preferably 7.0 to 7.5.

In one embodiment, the sodium salt is selected from the group consisting of sodium chloride, sodium sulfate, sodium nitrate, sodium phosphate and sodium carbonate, preferably sodium chloride. In one embodiment, the potassium salt is potassium chloride.

In one embodiment, the macromolecular polymer is selected from the group consisting of polyethylene glycol, albumin, dextran, methylcellulose, hydroxypropylcellulose, povidone, and sodium alginate, preferably albumin.

In a second aspect, the present disclosure provides an adeno-associated virus preparation comprising: adeno-associated virus and an aqueous composition according to the first aspect.

In one embodiment, the titer of the adeno-associated virus is 1X 10 ⁸ To 5X 10 ¹⁴ vg/mL。

In one embodiment, the adeno-associated virus is a recombinant adeno-associated virus (rAAV).

In one embodiment, the adeno-associated virus formulation is a liquid formulation, a frozen formulation or a lyophilized formulation.

In a third aspect, the present disclosure provides the use of calcium ions or magnesium ions for stabilizing an adeno-associated virus preparation.

In one embodiment, the use of calcium or magnesium ions for stabilizing an adeno-associated virus preparation comprises: for maintaining the capsid titer of the adeno-associated virus in the adeno-associated virus preparation; for maintaining genome titer of adeno-associated virus in adeno-associated virus preparation; for maintaining the activity of adeno-associated virus in adeno-associated virus preparations; monomer percentage for maintaining adeno-associated virus in adeno-associated virus preparation; and/or for increasing the protein denaturation temperature of adeno-associated virus in adeno-associated virus preparations.

In a fourth aspect, the present disclosure provides a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of an adeno-associated virus formulation according to the second aspect.

In one embodiment, the adeno-associated virus formulation is administered by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration.

In a preferred embodiment, the adeno-associated virus preparation is administered by a systemic route, e.g. intravenously.

Drawings

FIG. 1 shows the results of capsid titer (Fortebio) and genome titer (qPCR) assays for samples A through N of example 1 at 37 ℃ for 10d (37 ℃ for 10 days).

FIG. 2 shows the results of the measurement of protein denaturation temperatures (Tonset initial denaturation temperature and Tm denaturation midpoint temperature) of the samples A to N at 37 ℃ for 10d in example 1.

FIG. 3 shows the results of SEC (relative percentage of viral monomers) at 37 ℃ for 10d for samples A to N in example 1.

FIG. 4 shows the results of the measurement of the relative in vitro activities of samples A to N in example 1 at 37 ℃ for 10 days.

FIG. 5 shows the results of capsid titer (Fortebio) and genome titer (qPCR) assays at 37 ℃ for 10d for samples J, O to U in example 2.

FIG. 6 shows the results of the determination of the protein denaturation temperature (Tonset initial denaturation temperature and Tm denaturation midpoint temperature) for the samples J, O to T at 37 ℃ for 10d in example 2.

FIG. 7 shows the results of SEC (relative percentage of viral monomers) at 37 ℃ for samples J, O to T in example 2 at 10 d.

FIG. 8 shows the results of the determination of the relative in vitro activity of the samples J, O to U in example 2 at 37 ℃ for 10 d.

FIG. 9 shows the results of measurement of capsid titer (Fortebio) and genome titer (qPCR) at 37 ℃ for 10d for samples 1 to 7 in example 3.

FIG. 10 shows the results of the measurement of the relative in vitro activity of samples 1 to 7 at 37 ℃ for 10d in example 3.

FIG. 11 shows the results of the SEC (relative percentage of viral monomers) measurements at 37 ℃ for 10d for samples 1 to 5 and 7 in example 3.

FIG. 12 shows the results of the measurement of protein denaturation temperatures (Tonset initial denaturation temperature and Tm denaturation midpoint temperature) of the samples 1 to 5 and 7 in example 3 at 37 ℃ for 10 d.

FIG. 13 shows the results of the measurement of protein denaturation temperatures (Tonset initial denaturation temperature and Tm denaturation midpoint temperature) of the samples 8 to 13 under the condition of 0h in example 4.

FIG. 14 shows the results of the determination of genome titers (qPCR) of samples 8 to 14 at 37 ℃ for 10d in example 4.

FIG. 15 shows the results of the measurement of the relative in vitro activities of samples 8 to 14 in example 4 at 37 ℃ under 10d and 250rpm at 10 d.

Detailed Description

Unless defined otherwise, 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 disclosure belongs.

As used herein, unless otherwise specified, any concentration range, percentage range, proportion range, or integer range is to be understood as including the value of any integer within the range and, where appropriate, the value of a fraction thereof (e.g., one tenth and one hundredth of an integer).

As used herein, the term "AAV" includes naturally occurring adeno-associated viruses and recombinant forms of adeno-associated viruses (rAAV), and includes mutant forms of AAV.

As used herein, "therapeutically effective amount" refers to a dose that produces an effect upon administration to a subject. The exact dosage depends on the purpose of the treatment and can be determined by one skilled in the art using known techniques.

As used herein, "storage" or "preservation" refers to the formulation not being administered to a subject immediately after preparation, but rather being left for a period of time under specified conditions (e.g., a specified temperature, etc.) prior to use.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

In one embodiment, the buffer salt comprises: inorganic acid salts such as phosphate (sodium or potassium), bicarbonate, and the like; organic acid salts such as citrate, acetate, succinate, and the like; amino acids such as histidine, arginine, glycine and the like; acidic buffers such as acetic acid, phosphoric acid, hydrochloric acid, carbonic acid, succinic acid, citric acid, histidine hydrochloride, malic acid, and the like; basic buffers such as sodium hydroxide, tris, HEPES and the like.

In one embodiment, the surfactant is selected from: sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene beeswax derivatives, polyoxyethylene lanolin derivatives, and polyoxyethylene fatty acid amides.

Preferably, the surfactant is selected from polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers. Particularly preferably, the surfactant is selected from polysorbate 20 (PS 20), 40 (PS 40), 60 (PS 60), 80 (PS 80), 85 (PS 85) and poloxamer 188. Most preferably, the surfactant is selected from

polysorbate

20, 80 and poloxamer 188 (Pluronic F68).

In one embodiment, the concentration of the surfactant in the aqueous composition is from 0.0001% to 0.5% (w/v), such as 0.001% (w/v), 0.005% (w/v), 0.01% (w/v), 0.02% (w/v), 0.05% (w/v), 0.10% (w/v), 0.20% (w/v), 0.30% (w/v), 0.40% (w/v).

In one embodiment, the concentration of HSA in the aqueous composition is between 0.1% and 5% (w/v), such as 0.5% (w/v), 1% (w/v), 1.5% (w/v), 2% (w/v), 2.5% (w/v), 3% (w/v), 3.5% (w/v), 4% (w/v), 4.5% (w/v).

In one embodiment, the aqueous composition comprises Fe ²⁺ 、Ca ²⁺ Or Mg ²⁺ Is in a concentration of 0.01-5mM, e.g., 0.05mM, 0.1mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM.

In one embodiment, fe ²⁺ From FeCl ₂ Provided is a method.

In one embodiment, ca ²⁺ From CaCl ₂ Provided is a method.

In one embodiment, mg ²⁺ From MgCl ₂ Provided is a method.

It is well known in the art that AAV capsids function as targeted delivery, encapsulating the genome of interest within the viral capsid to form a composition, and yet have overall properties similar to protein capsids.

It is understood that the adeno-associated virus formulations of the present disclosure comprise AAV, and the capsid protein of the AAV can be that of any AAV serotype.

In some embodiments, the AAV comprises a capsid protein selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV-DJ8, AAV-DJ9, AAVrh8R, AAVrh10, AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv, AAVXL32, AAV. PHP.B and AAV2.1.

In some embodiments, the adeno-associated virus formulation is a reconstituted lyophilized formulation.

In some embodiments, the adeno-associated virus formulation retains significant AAV activity upon prolonged storage.

In some embodiments, the adeno-associated virus formulation is sterile. By "sterile" is meant that the formulation is substantially free of immunogenic components, e.g., substantially free of microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.).

In some embodiments, the aqueous composition or adeno-associated virus formulation of the present disclosure comprises pharmaceutically acceptable additional ingredients.

The present disclosure is described in further detail below with reference to the drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1 stability Effect of different buffers and pH values

It is well known in the art that pH is an important factor affecting the stability of proteins, and that both the physical and chemical stability of proteins are affected by pH. In this example, the effect of different buffer solutions and pH on AAV stability was investigated.

Comparing AAV stability at different pH: buffer 10mM PB-CA (KH) ₂ PO ₄ /Na ₃ C ₆ H ₅ O ₇ (sodium citrate)) buffer range covering pH 5.0-7.5, 10mM AA (HAc/NaAc) buffer range covering pH 4.5/5.0, 10mM Tris-AA (Tris-HAc) buffer range covering pH 8.5. The pH range of the above buffers covered 4.5-8.5 for comparison of AAV stability at different pH.

Comparing AAV stability in different buffers: 10mM AA, 10mM His-AA (His/HAc), 10mM CA (C) ₆ H ₈ O ₇ (citric acid)/Na ₃ C ₆ H ₅ O ₇ ) 10mM SA (succinic acid/NaOH) buffer range covers pH 5.0;10mM PB (KH) ₂ PO ₄ /Na ₂ HPO ₄ ) 10mM Tris-AA, 10mM PB-CA buffer range covered pH 7.5. The above buffers were used to compare the effect of different buffers on AAV stability.

As shown in table 1 below, each sample contained the same titer of rAAV (BBM-H803, capsid AAVXL32.1, described in WO 2019241324 A1, molecular weight about 4000 Kda) with a concentration of salt (NaCl) and surfactant (F68).

TABLE 1 formulation design for samples A-N

The samples were examined for Fortebio (capsid titer), qPCR (genome titer), relative in vitro activity, SEC (relative percentage of viral monomer), protein denaturation temperature (initial denaturation temperature T), immediately after formulation (0 h) and under conditions of storage at 37 ℃ for 10 days (37 ℃ 10 d) _onset And the midpoint denaturation temperature T _m ). The resulting data are shown in table 2 below.

TABLE 2 stability test results for samples A-N

As can be seen from Table 1, under the condition of 0h, the capsid titer (Fortebio), the genome titer (qPCR) and the SEC of different samples have no obvious difference, the protein denaturation temperature shows a certain difference, and the trend is consistent with that under the condition of 10d at 37 ℃.

As shown in FIG. 1, the A-N samples showed a substantially consistent trend in capsid and genome titers at 37 ℃ for 10 days.

Among them, the assay results of the a and B samples showed that the AAV stability at pH 5.0 was superior to pH 4.5. The assay results for the G and H samples showed that AAV stability at pH 7.5 was superior to pH 8.5. The assay results for the I to N samples showed that AAV titer first increased with decreasing pH, reached a maximum titer at pH 5.5 (M samples), and then decreased slightly with decreasing pH, in the pH 7.5-5.0 range.

F. The pH of the G and I samples is 7.5, the difference is that the buffers are different (PB, tris-AA and PB-CA), and the result shows that the AAV stability difference in the three different buffers is not large, but the Tris-AA buffer system with the pH of 7.5 has a little advantage. The pH of the samples B to E were 5.0, except for the different buffers, and the assay showed that the AAV titers in the different buffers were relatively close at the same pH.

The protein denaturation temperature assay results (as shown in FIG. 2) of the A to N samples at 37 ℃ for 10 days were consistent with the trend of the titer assay results. At pH 5.0, the protein denaturation temperature was slightly lower for the C-only sample (His-AA buffer).

As shown in FIG. 3, SEC measurements of samples A to E and of samples M to N (pH 4.5 to 5.5) at 37 ℃ for 10d showed a high relative percentage of virus monomer in the different buffer systems. SEC measurements of the A and B samples showed a higher relative percentage of viral monomers at pH 5.0 compared to pH 4.5. F. The pH of the G and I samples is 7.5, the difference is that the buffers are different (PB, tris-AA and PB-CA respectively), the results show that the relative percentages of the virus monomers in the three different buffers are obviously different, and the I sample (PB-CA buffer) is obviously higher than F and G. The same buffer was used for the I to N samples, but the pH was decreased from 7.5 to 5.0 in order, showing that the relative percentage of viral monomers gradually increased as the pH decreased.

As shown in FIG. 4, the in vitro activity results of the A to N samples at 37 ℃ for 10d are significantly different from the trend of other detection results. The in vitro activity measurements of samples A to E showed that the samples were less active at pH 4.5 and pH 5.0, wherein the same buffer was used for sample A and sample B, but the pH values were different, and the in vitro activity of sample B at pH 5.0 was better than that of sample A at pH 4.5. The in vitro activity assay results for samples F to I show that the G sample (Tris-AA, pH 7.5) shows very significant activity (105%), much higher than the F sample with other buffers of the same pH (15%), I (53%) or the H sample with the same buffer of different pH (30%). The in vitro activity assay results of the samples I to N showed that AAV had good activity at pH 6.0 to 7.0 in the case where all the buffers were PB-CA.

Example 2 stability Effect of different excipients

Sugar (sucrose), surfactant species (F68, PS20, PS 80), divalent salt species (MgCl) were investigated using a 10mM PB-CA buffer system ₂ 、CaCl ₂ ) And the effect of macromolecular proteins (human serum albumin HSA) on AAV stability. In addition, caCl was compared ₂ Stability at different pH.

As shown in table 3 below, each sample contained the same titer of rAAV and the same concentration of NaCl.

TABLE 3 formulation design for sample J, O-U

The samples were examined for Fortebio, qPCR, relative in vitro activity, SEC and protein denaturation temperature immediately after formulation (0 h) and stored at 37 ℃ for 10 days (37 ℃ 10 d). The resulting data are shown in table 4 below.

TABLE 4 stability test results for sample J, O-U

As shown in Table 4, the capsid titer (Fortebio), the genome titer (qPCR) and the SEC of the J and O-T samples were not obviously different under the condition of 0h, the protein denaturation temperature showed a certain difference, and the trend was consistent with that under the condition of 37 ℃ and 10 d.

The measurement results of the J and O samples at 37 ℃ for 10d show that the genome titer can be improved to some extent by adding sucrose. J. The measurement results of the P to R samples show that CaCl is added under the condition of pH 7.0 ₂ Or MgCl ₂ Can remarkably improve capsid titer and genome titer, and is CaCl ₂ Is obviously superior to MgCl ₂ (as shown in fig. 5). However, comparison of the measurement results of the Q sample and the M sample in example 1 shows that CaCl is added under the condition of pH 5.5 ₂ Failure to improve capsid or genome titer. J. The results of the S to U samples showed that the genome titers were slightly increased by either F68 for PS20 or PS80 or by addition of HSA.

The above results show that CaCl was added at pH 7.0 ₂ Or MgCl ₂ Can significantly increase AAV stability, and CaCl ₂ Is obviously superior to MgCl ₂ . However, at pH 5.5, caCl ₂ Hardly play a stabilizing role. Furthermore, addition of sucrose or HSA, or replacement of F68 with PS20 or PS80, may result in a slight increase in AAV stability.

In addition, the protein denaturation temperature measurement results (as shown in FIG. 6) of the J and O-T samples were consistent with the titer measurement results in the trend, but the protein denaturation temperature difference between the samples was small (the protein denaturation temperature of the Q sample was the highest).

SEC measurements of J and O samples at 37 ℃ for 10d showed that the addition of sucrose did not affect the relative percentage of virus monomer. J. The SEC measurements of the P and Q samples showed (as shown in FIG. 7) that CaCl was added at pH 5.5 or pH 7.0 ₂ The relative percentage of viral monomers can be significantly improved (up to 95% or more). J. SEC measurements of P and R samples show that MgCl was added ₂ Can also obviously improve the relative percentage of virus monomers, but the effect is inferior to that of CaCl ₂ . In addition, the SEC measurements of the J, S and T samples showed no significant change in the relative percentage of viral monomer after the F68 was changed to PS20, PS 80.

The in vitro activity measurement results of the J and O-T samples at 37 ℃ for 10d show that the sucrose and MgCl are added ₂ Neither HSA nor F68 to PS20 nor PS80 gave any significant change. As shown in FIG. 8, surprisingly, caCl was added at pH 7.0 ₂ Very outstanding in vitro activity (133%) could be achieved. However, at pH 5.5, the in vitro activity was still low.

Example 3 Effect of component concentrations on AAV stability

To obtain the optimum concentrations of the ingredients, the following sample formulations (table 5) were designed and tested. AAV titers in each sample were 2X 10 ¹³ vg/mL。

TABLE 5 formulation design for samples 1-7

Sample (I)

Buffer solution

pH

Concentration of NaCl

Surface active agent

Supplemental ingredient

1

10mM AA

5.5

300mM

0.01％PS80

-

2

10mM AA

5.5

300mM

0.01％PS80

1mM CaCl

₂

3

10mM AA

5.5

300mM

0.01％PS80

5mM CaCl

₂

4

10mM AA

5.5

300mM

0.05％PS80

-

5

10mM AA

5.5

400mM

0.01％PS80

-

6

10mM AA

5.5

300mM

0.01％PS80

2％HSA

7

20mM Tris-AA

7.4

350mM

0.01％F68

1mM CaCl ₂

The samples were examined for Fortebio, qPCR, relative in vitro activity, SEC and protein denaturation temperature immediately after formulation (0 h) and stored at 37 ℃ for 10 days (37 ℃ 10 d). The resulting data are shown in table 6 below.

TABLE 6 stability test results for samples 1-7

As shown in Table 6, under the condition of 0h, the capsid titer (Fortebio), the genome titer (qPCR), the SEC and the protein denaturation temperature of different samples have no significant difference, and the in vitro activity is higher.

As can be seen by comparing samples 1-3 at 37 ℃ for 10d, 1mM CaCl was added to the AA buffer at pH 5.5 ₂ Does not cause titer change; will be provided withCaCl ₂ Increasing the concentration to 5mM resulted in a slight increase in genome titer, in vitro activity, relative percentage of viral monomers, and protein denaturation temperature (as shown in FIG. 12). As can be seen by comparing

samples

1 and 4, increasing PS80 concentration from 0.01% to 0.05% resulted in a slight increase in both capsid and genome titers with essentially no effect on in vitro activity. As can be seen by comparing

samples

1 and 5, increasing the sodium chloride concentration also resulted in a slight increase in capsid titer, relative percentage of viral monomers, and protein denaturation temperature, and significantly improved in vitro activity. As can be seen from a comparison of

samples

1 and 6, the addition of HSA can significantly improve the in vitro activity.

As shown in fig. 9 to 11, surprisingly, the genome titer, in vitro activity and percentage of viral monomers were significantly higher for sample 7 than for samples 1-6.

₂ Example 4 stability Effect of CaCl in different buffer systems

In this example, caCl was investigated ₂ The stability effect on AAV in different buffer systems (pH, buffer) is observed, and HSA and Tris are used as stabilizing agents to examine the stability. The formulation of each sample is shown in table 7.

TABLE 7 formulation design for samples 8-14

The samples were examined for SEC, fortebio, qPCR, protein denaturation temperature and relative in vitro activity immediately after formulation (0 h), 10 days at 37 ℃ (10 d at 37 ℃), 10 days at room temperature (20-25 ℃) with shaking at 250rpm (10 d at 250rpm), and 5 cycles of repeated freeze-thaw at-25 ℃/4 ℃ (5C at-25 ℃/4 ℃). The resulting data are shown in table 8 below.

TABLE 8 stability test results for samples 8-14

As shown in Table 8, the capsid titer (Fortebio), genome titer (qPCR) and SEC of the samples 8-14 were not significantly different under the condition of 0h, and the protein denaturation temperature was highest at pH 5.5/6.5 (as shown in FIG. 13).

As shown in FIG. 14, in samples 8-14, the genome titer first increased with increasing pH between pH 4.5 and 8.5, reached a maximum at pH 7.5, and then decreased with increasing pH at 37 ℃ for 10 d; the effect of adding 20mM Tris on genome titer is not obvious; the additional addition of HSA may slightly increase the genome titer.

As can be seen from the results of the comparative samples tested at 37 ℃ for 10d, 250rpm for 10d and-25 ℃/4 ℃ for 5C, the freeze-thaw has a very small effect on the quality of the samples, and the shaking has a large effect on the in vitro activity of the samples. As shown in FIG. 15, the in vitro activity trend of the samples was consistent at 37 ℃ 10d and 250rpm10d, increasing with increasing pH between pH 4.5 and 8.5; the effect of adding 20mM Tris on the in vitro activity of the sample is not obvious; the additional addition of HSA may slightly increase the in vitro activity of the sample.

Surprisingly, caCl ₂ The difference in stability under different pH conditions is surprisingly significant.

From the above experimental results, it can be seen that CaCl was added ₂ The AAV stability can be significantly improved, especially at pH 7.5-8.5. In addition, increasing NaCl concentration or addition of HSA increased AAV stability.

All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated by reference herein in their entirety.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Claims

1. An aqueous composition comprising:

selected from Tris-HAc, KH ₂ PO ₄ -Na ₃ C ₆ H ₅ O ₇ Or a combination thereof;

a surfactant selected from F68, PS20, PS80, or a combination thereof;

sodium or potassium salts;

selected from Fe ²⁺ 、Ca ²⁺ And Mg ²⁺ One or more of (1); and

optionally a macromolecular polymer.

2. The aqueous composition of claim 1,

the concentration of the buffer is 2-100mM, preferably 10-30mM;

the concentration of the surfactant is 0.0001-0.5% (w/v), preferably 0.005-0.05% (w/v);

the concentration of the sodium or potassium salt is 100-800mM, preferably 250-400mM;

Fe ²⁺ 、Ca ²⁺ or Mg ²⁺ In a concentration of 0.01-5mM, preferably 0.1-2mM; and/or

The concentration of the optional macromolecular polymer is between 0.1% and 5% (w/v), preferably between 0.5% and 3% (w/v).

3. Aqueous composition according to claim 1 or 2, wherein the aqueous composition further comprises a saccharide, preferably at a concentration of 0.1-10% (w/v), preferably 1-5% (w/v).

4. The aqueous composition of claim 1 or 2, wherein the saccharide comprises sucrose, trehalose, sorbitol, mannitol, glucose, lactose, galactose, maltose and fructose.

5. Aqueous composition according to claim 1 or 2, wherein the pH of the aqueous composition is between 4.5 and 8.5, preferably between 7.0 and 8.5, more preferably between 7.0 and 7.5.

6. Aqueous composition according to claim 1 or 2, wherein the sodium salt is selected from sodium chloride, sodium sulphate, sodium nitrate, sodium phosphate and sodium carbonate, preferably sodium chloride.

7. Aqueous composition according to claim 1 or 2, wherein the macromolecular polymer is selected from the group consisting of polyethylene glycol, albumin, dextran, methylcellulose, hydroxypropylcellulose, povidone and sodium alginate, preferably albumin.

8. An adeno-associated virus preparation comprising: an adeno-associated virus and the aqueous composition of any one of claims 1 to 7.

9. The adeno-associated virus preparation according to claim 8 wherein the titer of the adeno-associated virus is 1 x 10 ⁸ To 5X 10 ¹⁴ vg/mL。

10. The adeno-associated virus formulation according to claim 8 wherein the adeno-associated virus is a recombinant adeno-associated virus.

11. The adeno-associated virus formulation according to any one of claims 8 to 10 wherein the adeno-associated virus formulation is a liquid formulation, a frozen formulation or a lyophilized formulation.

12. Use of calcium or magnesium ions for stabilizing an adeno-associated virus preparation.

13. Use according to claim 12, comprising:

for maintaining the capsid titer of the adeno-associated virus in the adeno-associated virus preparation;

for maintaining genome titer of adeno-associated virus in an adeno-associated virus preparation;

for maintaining the activity of adeno-associated virus in adeno-associated virus preparations;

monomer percentage for maintaining adeno-associated virus in adeno-associated virus preparation; and/or

Used for increasing the protein denaturation temperature of adeno-associated virus in adeno-associated virus preparation.