Method for determining purity of AAV virus

Abstract

The invention provides a method for determining the purity of an AAV virus. The invention provides optimized size exclusion chromatography mobile phase composition and chromatographic conditions, by optimizing the salt ion concentration of the mobile phase and adding the organic phase, the conditions of poor chromatographic column adsorption and separation degree and serious trailing in the AAV virus detection process are improved, the accuracy and reproducibility of the method are improved, and the method has application prospects in the field of virus detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for determining the purity of AAV virus. Background Art

[0002] In therapeutic viral drugs, viral aggregates are generally considered to affect drug efficacy or even cause immunogenicity and are treated as product-related impurities that need to be removed in downstream purification processes.

[0003] SEC detection (size exclusion chromatography) is a common method for detecting aggregate content. During the product development, production and storage process, the detection results of the SEC method are directly related to the development and locking of downstream processes, product release and product stability, etc. Therefore, a stable and accurate SEC detection method is particularly important.

[0004] SEC detection uses a fluorescence detector or UV detector. The detection method is simple, the detection cost is low, and the throughput is high. It is mostly used for product process monitoring and release detection. SEC-MALS detection can also be used for virus aggregate detection, but it requires a UV detector and the like to be used in conjunction with the MALS detector. The equipment cost is high, which is about 20 times the cost of the ordinary SEC method. The SEC-MALS method is generally used for product characterization research. The experimental operation is relatively complicated and is not often used for release detection. In addition, the MALS detector equipment cost is high, which is about 20 times the cost of the fluorescence detector. The ultraspeed analytical centrifuge (AUC) can also be used to determine the purity of the virus, but its detection time is long, about 7 hours, which is much longer than the detection time of about 30 minutes for one sample of the ordinary SEC method; and the AUC detection throughput is low, and only 7 samples can be detected in one experiment, while the ordinary SEC detection can detect dozens of samples in one experiment.

[0005] In daily use, SEC is a commonly used purity determination method for protein drugs, and the method is relatively mature in protein detection. However, for viral drugs, since viral particles contain protein molecules and double-stranded DNA molecules, the viral components are relatively complex (including hollow, solid, partial and other complex components), and their relative molecular weight is much larger than the molecular weight of a single protein, which brings challenges to liquid chromatography analysis. In actual SEC analysis, there are usually problems such as chromatographic column adsorption, poor method separation, and severe tailing.

[0006] Prior art CN116056775A discloses a method for separating adenovirus by SEC, which uses a stationary phase bonded with hydroxyl-terminated PEO, and adds amino acids (such as lysine) to the mobile phase to improve the separation effect. However, this method requires the use of a special stationary phase, which increases the cost to a certain extent. At the same time, the addition of amino acids may require sample purification of the product, and the operation process is more complicated.

[0007] Therefore, there is a need in the art to establish a virus SEC detection method that improves chromatographic column adsorption and tailing phenomena and enhances separation. Summary of the invention

[0008] The purpose of the present invention is to provide a method for determining the purity of AAV virus.

[0009] In a first aspect of the present invention, a size exclusion chromatography system for virus purity determination is provided, the chromatography system comprising:

[0010] 1) Size exclusion chromatography column;

[0011] 2) a mobile phase having an ionic strength of 150 mM to 500 mM and comprising an organic solvent;

[0012] 3) Sample solution.

[0013] In another preferred embodiment, the sample contains adeno-associated virus (AAV) particles.

[0014] In another preferred embodiment, the ionic strength of the mobile phase is 150 mM-500 mM, preferably 150 mM.

[0015] In another preferred embodiment, the mobile phase comprises:

[0016] i) Phosphate buffer (PB);

[0017] ii) a salt selected from the group consisting of sodium chloride, potassium chloride, or a combination thereof.

[0018] In another preferred embodiment, in the mobile phase, the concentration of phosphate buffer is 30-70 mM, preferably 40-60 mM, and more preferably 50 mM.

[0019] In another preferred embodiment, the mobile phase comprises:

[0020] Sodium dihydrogen phosphate dihydrate 1.0 g / L-1.5 g / L, preferably 1.25 g / L;

[0021] Disodium hydrogen phosphate dodecahydrate 10-20 g / L, preferably 15.04 g / L.

[0022] In another preferred embodiment, the concentration of sodium chloride or potassium chloride in the mobile phase is 100-200 mM, preferably 125-175 mM, and more preferably 150 mM.

[0023] In another preferred embodiment, the organic solvent is an alcohol solvent.

[0024] In another preferred embodiment, the organic solvent is selected from the following group: methanol, ethanol, or a combination thereof.

[0025] In another preferred embodiment, the volume concentration of the organic solvent in the mobile phase is 3-10%, preferably 4-7%, and more preferably 5%.

[0026] In another preferred embodiment, the size exclusion chromatography column comprises a filler based on Waters ethylene bridged hybrid (BEH) particle technology and a diol-based bonded phase covered on its surface.

[0027] In another preferred embodiment, the size exclusion chromatography column is XBridge BEH SEC 3.5μm column, Part No.: 186007643.

[0028] In another preferred embodiment, the size exclusion chromatography system further comprises a fluorescence detector or a UV detector, preferably a fluorescence detector.

[0029] In another preferred embodiment, in the size exclusion chromatography system, the fluorescence excitation wavelength of the fluorescence detector is set at 260-290 nm, preferably 280 nm.

[0030] In another preferred embodiment, the size exclusion chromatography system has one or more of the following effects when detecting the purity of a virus, preferably an AAV virus:

[0031] 1) Separation ≥ 1.5, preferably ≥ 1.7;

[0032] 2) The tailing factor is 0.8-1.5, preferably 1-1.4;

[0033] 3) The number of theoretical plates is ≥2000, more preferably ≥2300.

[0034] In the second aspect of the present invention, a method for determining virus purity is provided, the method comprising the step of performing detection using the size exclusion chromatography system as described in the first aspect of the present invention.

[0035] In another preferred embodiment, the method uses a fluorescence detector or a UV detector for detection, preferably a fluorescence detector.

[0036] In another preferred embodiment, in the chromatographic conditions of the method, the flow rate of the mobile phase is 0.3-0.7 ml / min, preferably 0.4-0.6 ml / min, and more preferably 0.5 ml / min.

[0037] In another preferred embodiment, in the chromatographic conditions of the method, the fluorescence excitation wavelength is 260-290 nm, preferably 280 nm.

[0038] In another preferred embodiment, the virus is adeno-associated virus (AAV).

[0039] In another preferred embodiment, the method has one or more of the following effects when detecting the purity of a virus, preferably an AAV virus:

[0040] 1) Separation ≥ 1.5, preferably ≥ 1.7;

[0041] 2) The tailing factor is 0.8-1.5, preferably 1-1.4;

[0042] 3) The number of theoretical plates is ≥2000, more preferably ≥2300.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0045] Figure 1 Shown is a chromatogram using a 50 mM PB, 150 mM NaCl mobile phase.

[0046] Figure 2 Shown is the reproducibility using 50 mM PB, 150 mM NaCl mobile phase.

[0047] Figure 3 Shown is a chromatogram using 50 mM PB, 300 mM NaCl mobile phase.

[0048] Figure 4 Shown is a chromatogram using 50 mM PB, 500 mM NaCl mobile phase.

[0049] Figure 5 Shown is a chromatogram using 50 mM PB, 500 mM KCl mobile phase.

[0050] Figure 6 Shown is a chromatogram using a 50 mM PB, 150 mM NaCl, 10% acetonitrile mobile phase.

[0051] Figure 7 Shown is a chromatogram using a 50 mM PB, 150 mM NaCl, 5% ethanol mobile phase.

[0052] Figure 8Reproducibility is shown using a 50 mM PB, 150 mM NaCl, 5% ethanol mobile phase.

[0053] Fig. 9 Shown is a chromatogram using a 50 mM PB, 150 mM NaCl, 5% methanol mobile phase.

[0054] Fig.10 Reproducibility is shown using a 50 mM PB, 150 mM NaCl, 5% methanol mobile phase.

[0055] Fig.11 Shown is a chromatogram using a 50 mM PB, 150 mM KCl, 5% ethanol mobile phase.

[0056] Fig.12 Reproducibility is shown using a 50 mM PB, 150 mM KCl, 5% ethanol mobile phase.

[0057] Fig.13 Spectra obtained using a UV detector at a detection wavelength of 260 nm are shown.

[0058] Fig.14 Spectra obtained using a UV detector at a detection wavelength of 280 nm are shown.

[0059] Fig.15 Spectra obtained using a UV detector at a detection wavelength of 215 nm are shown.

[0060] Fig.16 Shown is a chromatogram obtained using a Tosoh TSKgel G4000SWXL column.

[0061] Fig.17 Shown is a chromatogram obtained using a Wyatt WTC050N5 column.

[0062] Fig.18 Shown are spectra obtained using a flow rate of 0.4 ml / min.

[0063] Fig.19 Shown are spectra obtained using a flow rate of 0.6 ml / min.

[0064] Fig. 20 A comparison of the effects of using 215nm and 280nm excitation wavelengths is shown.

[0065] Fig.21 The fitting results of the linear detection of the present invention are shown. DETAILED DESCRIPTION

[0066] After extensive and in-depth research, the inventors have developed a method for determining the purity of AAV virus for the first time. In the size exclusion chromatography detection method of the present invention, the optimized mobile phase composition and chromatographic conditions are utilized, especially by optimizing the salt ion concentration of the mobile phase and adding an organic phase, which improves the chromatographic column adsorption, poor separation, and severe tailing during the AAV virus detection process, and increases the accuracy and reproducibility of the method. On this basis, the present invention is completed.

[0067] the term

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0069] As used herein, the terms “contain,” “have,” or “include” include “comprises,” “mainly consist of,” “substantially consist of,” and “consist of;” “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0070] Size Exclusion Chromatography

[0071] As used herein, the terms "size exclusion chromatography (SEC)" and "size exclusion chromatography" are used interchangeably and refer to a chromatographic technique that achieves separation based on the difference in retention of analytes of different molecular sizes in the pores of a chemically inert porous stationary phase. The size exclusion chromatography column contains many holes of different sizes or three-dimensional mesh gels. Size exclusion chromatography separates samples based on the pore volume and pore size distribution of the stationary phase gel, the molecular weight of the sample and its distribution, and the matching of each other. Within the molecular weight range determined by the stationary phase, the elution order is distributed according to the molecular size. When the sample molecular weight is so large that it is completely excluded from the micropores of the stationary phase (total exclusion), its retention volume is equal to the void volume between the particles in the chromatographic column; while molecules with a molecular weight small enough to completely enter the micropores, their retention volume is the sum of the void volume and the volume of the micropores in the stationary phase. The molecular weight measurement range of the stationary phase is the molecular weight range between full penetration and total exclusion.

[0072] As used herein, the term "resolution" refers to an indicator used to evaluate the degree of separation between the substance to be tested and the substance to be separated, and is a key indicator for measuring the separation efficiency of a chromatographic system. Unless otherwise specified, the resolution between the chromatographic peak of the substance to be tested and the adjacent chromatographic peak should not be less than 1.5.

[0073] As used herein, the term "tailing factor" refers to an indicator used to evaluate the symmetry of a chromatographic peak. Generally, when peak height is used as a quantitative parameter, the value should be between 0.95 and 1.05 unless otherwise specified. When peak area is used as a quantitative parameter, general peak tailing or fronting will not affect the peak area integration, but severe tailing will affect the judgment of the baseline and the start and end of the chromatographic peak and the accuracy of the peak area integration. In this case, the tailing factor should be specified under the product text.

[0074] The detection object of the method of the present invention is a virus particle, which is much larger than conventional small molecule drugs, and the tailing phenomenon is more serious than that of small molecule drugs in SEC detection. The method of the present invention can greatly reduce the tailing phenomenon for virus detection, reduce the tailing factor to an acceptable range, and improve the quantitative accuracy. There is no unified numerical range standard for the tailing factor range in the pharmacopoeias of various countries. The effect of the method of the present invention can basically meet the requirements for chromatographic detection. For example, the United States Pharmacopoeia (USP) requires that the tailing factor of certain substances is not greater than 2.0; the European Pharmacopoeia (EP) and the British Pharmacopoeia (BP) stipulate that when performing relevant substance or content detection, the symmetry factor of the chromatographic peak of the quantitative reference solution in the chromatogram should be 0.8-1.5 unless otherwise specified. In drug analysis chromatographic experiments, it is generally believed that chromatographic peaks with a tailing factor less than 2.0 can be accurately quantified according to peak area. In actual chromatographic experiments, for different substances, specific tailing factor values ​​with limiting significance need to be specified in combination with factors such as the characteristics of the compound itself, the number of theoretical plates, and the degree of separation.

[0075] Mobile phase

[0076] The present invention provides an optimized chromatography system, especially an optimized mobile phase, to achieve SEC detection of AAV virus.

[0077] The mobile phase provided by the invention has an optimized ionic strength range and contains an organic solvent.

[0078] The mobile phase ionic strength of the present invention is in the range of 150-500 mM. The ionic strength range can be adjusted by adjusting the type and concentration of salt in the mobile phase. Sodium chloride and potassium chloride can be used to adjust the ionic strength, but are not limited thereto. Any buffer conventionally used in the art, such as phosphate buffer, can be used in the mobile phase of the present invention.

[0079] In one embodiment, the mobile phase comprises 1.0 g / L-1.5 g / L sodium dihydrogen phosphate dihydrate, 10-20 g / L disodium hydrogen phosphate dodecahydrate, and 100-200 mM sodium chloride or potassium chloride.

[0080] The organic solvent used in the mobile phase of the present invention can be ethanol, acetonitrile, or a combination thereof, but is not limited thereto. In a preferred embodiment, the mobile phase of the present invention comprises ethanol at a volume concentration of 3%-10%, preferably 5%.

[0081] Detection Methods

[0082] The present invention provides a method for determining the purity of AAV virus, wherein the method utilizes the chromatography system described in the first aspect of the present invention for detection.

[0083] The method of the present invention can be detected using a fluorescence detector or a UV detector. In a preferred embodiment, a fluorescence detector is used for detection.

[0084] In one embodiment, the mobile phase flow rate of the method of the present invention can be 0.3-0.7 ml / min, such as 0.4, 0.5 or 0.6 ml / min. In one embodiment, the fluorescence excitation wavelength used in the method of the present invention is 260-290 nm, such as 280 nm.

[0085] The chromatographic column of the present invention can be selected from waters XBridge SEC 3.5μm, its filler is: based on Waters ethylene bridged hybrid (BEH) particle technology and a diol-based bonded phase covered on its surface.

[0086] The sample tray temperature, injection volume, column temperature, elution mode, etc. can be adjusted according to actual needs, and such adjustments are routinely mastered by those skilled in the art.

[0087] The method of the present invention can overcome the problems of Blank interference, poor separation, obvious tailing, etc., and obtain purity determination results with high accuracy and good reproducibility. The method of the present invention can achieve a separation of ≥1.5 and a tailing factor of 0.8-1.5.

[0088] The main advantages of the present invention include:

[0089] 1) The present invention provides an optimized size exclusion chromatography AAV purity detection method, which effectively improves the problems of chromatographic column adsorption, poor tailing factor, poor separation and blank solution impurity peaks by optimizing the mobile phase ion strength, organic phase and other detection parameters.

[0090] 2) The present invention can detect the purity of AAV virus using only size exclusion chromatography. Compared with the use of SEC-MALS, the cost is effectively reduced to about one-twentieth, and the convenience of operation is improved; compared with AUC technology, the equipment cost is reduced to one-forty-tenth, and the detection results of a large number of samples can be obtained in a short time at high speed and high throughput.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0092] General experimental methods

[0093] Pipette 150 μl AAV virus sample (sample concentration greater than 1×10 12 Capsids / mL) into the liner tube (do not generate bubbles), place it in a sample bottle, and inject 10 μl.

[0094] In the following examples, unless otherwise specified, the experiments were carried out according to the procedures shown in Table 1 below.

[0095] Table 1 SEC detection program parameters

[0096]

[0097] Example 1 Optimization of salt ion concentration parameters in mobile phase

[0098] 1.1. Mobile phase (50 mM PB, 150 mM NaCl)

[0099] Preparation of mobile phase: weigh 1.25g sodium dihydrogen phosphate dihydrate, 15.04g disodium hydrogen phosphate dodecahydrate, 8.76g sodium chloride, add ultrapure water to 1000ml and mix well. Filter with a 0.2μm filter membrane.

[0100] Results: The mobile phase results showed that Blank had impurity peaks (such as Figure 1 ), and the method has serious column adsorption, resulting in poor reproducibility (such as Figure 2 ).

[0101] 1.2. Mobile phase (50 mM PB, 300 mM NaCl)

[0102] Preparation of mobile phase: weigh 1.25 g of sodium dihydrogen phosphate dihydrate, 15.04 g of disodium hydrogen phosphate dodecahydrate, and 17.52 g of sodium chloride, add ultrapure water to 1000 ml and mix well. Filter with a 0.2 μm filter membrane.

[0103] Results: The detection spectrum is as follows Figure 3 shown.

[0104] 1.3. Mobile phase (50 mM PB, 500 mM NaCl)

[0105] Preparation of mobile phase: weigh 1.25 g sodium dihydrogen phosphate dihydrate, 15.04 g disodium hydrogen phosphate dodecahydrate, 29.20 g sodium chloride, add ultrapure water to 1000 ml and mix well. Filter with a 0.2 μm filter membrane.

[0106] Results: The detection spectrum is as follows Figure 4 shown.

[0107] 1.4. Mobile phase (50 mM PB, 500 mM KCl)

[0108] Preparation of mobile phase: weigh 1.25 g sodium dihydrogen phosphate dihydrate, 15.04 g disodium hydrogen phosphate dodecahydrate, and 37 g potassium chloride, add ultrapure water to 1000 ml and mix well. Filter with a 0.2 μm filter membrane.

[0109] Results: The detection spectrum is as follows Figure 5 shown.

[0110] The results of this example show that under too high ionic strength, serious tailing phenomenon occurs, and the separation effect is poor. The mobile phase system of 50mM PB and 150mM NaCl has relatively less serious tailing, but there are blank impurities and chromatographic column adsorption, which still needs further optimization.

[0111] In this embodiment, the ionic strength of the mobile phase is changed, and the separation degree and tailing factor change as shown in Table 2:

[0112] Table 2

[0113] Different ionic strength mobile phases Main peak separation USP tailing factor USP Theoretical Plate Number 50mM PB, 150mM NaCl 1.710938e+00 1.497954e+00 2.541661e+03 50mM PB, 300mM NaCl 1.372345e+00 2.073519e+00 1.111950e+03 50mM PB, 500mM NaCl 1.565562e+00 1.805977e+00 1.531213e+03 50mM PB, 500mM KCl 1.317878e+00 2.074541e+00 1.051099e+03

[0114] The results in Table 2 show that the main peak separation, USP tailing factor, and USP theoretical plate number of 50 mm / L PB+150 mm / L NaCl are better than those of other mobile phases. However, the mobile phase reproducibility of this component is poor, and there are mixed peaks in the blank. In Example 2, further optimization is performed by adding an organic solvent.

[0115] Example 2 Optimization of organic solvent parameters in mobile phase

[0116] 2.1. Mobile phase (50 mM PB, 150 mM NaCl, 10% acetonitrile)

[0117] Preparation of mobile phase: weigh 1.25g sodium dihydrogen phosphate dihydrate, 15.04g disodium hydrogen phosphate dodecahydrate, 8.76g sodium chloride, add ultrapure water to 900ml and mix well. Filter with 0.2μm filter membrane and add 100ml acetonitrile.

[0118] Results: The detection spectrum is as follows Figure 6 As shown, an unknown miscellaneous peak appears after the main peak.

[0119] 2.2 Mobile phase (50 mM PB, 150 mM NaCl, 5% ethanol)

[0120] Weigh 1.25 g of sodium dihydrogen phosphate dihydrate, 15.04 g of disodium hydrogen phosphate dodecahydrate, and 8.76 g of sodium chloride, add ultrapure water to 950 ml and mix well. Filter with a 0.2 μm filter membrane and add 50 ml of anhydrous ethanol.

[0121] Results: The detection spectrum is as follows Figure 7 As shown, the reproducibility results are Figure 8 shown.

[0122] Further experiments showed that the ranges of 50mmol / L PB, 100-200mmol / L NaCl, and 3-10% ethanol can achieve separation effects similar to that of this system.

[0123] 2.3 Mobile phase (50 mM PB, 150 mM NaCl, 5% methanol)

[0124] Weigh 1.25 g of sodium dihydrogen phosphate dihydrate, 15.04 g of disodium hydrogen phosphate dodecahydrate, and 8.76 g of sodium chloride, add ultrapure water to 950 ml and mix well. Filter with a 0.2 μm filter membrane and add 50 ml of anhydrous methanol.

[0125] Results: The detection spectrum is as follows Fig. 9 As shown, the reproducibility results are Fig.10 shown.

[0126] Further experiments showed that the range of 50mmol / L PB, 100-200mmol / L NaCl, and 3-10% methanol can achieve a separation effect similar to that of this system.

[0127] 2.3 Mobile phase (50 mM PB, 150 mM KCl, 5% ethanol)

[0128] Weigh 1.25 g of sodium dihydrogen phosphate dihydrate, 15.04 g of disodium hydrogen phosphate dodecahydrate, and 11.11 g of potassium chloride, add ultrapure water to 950 ml and mix well. Filter with a 0.2 μm filter membrane and add 50 ml of anhydrous ethanol.

[0129] Results: The detection spectrum is as follows Fig.11 As shown, the reproducibility results are Fig.12 As shown, the use of KCl showed equally good experimental results compared to the use of NaCl in Example 2.2.

[0130] The results of this example show that adding different organic phases to the mobile phase has a significant impact on the detection results. The organic solvent parameters in the mobile phase are optimized, and the changes in separation degree and tailing factor are shown in Table 3:

[0131] Table 3

[0132]

[0133] The results in Table 3 show that the separation degrees of the mobile phases 50mM Pb, 150mM NaCl, 5% ethanol, 50mM Pb, 150mM NaCl, 5% methanol and 50mM Pb, 150mM KCl, 5% ethanol are all greater than 1.5, all having good separation effects and good reproducibility.

[0134] Example 3 Optimization of detection process

[0135] 3.1 Detector

[0136] The detection method is the same as in Example 1.1, except that the fluorescence detector is replaced by a UV detector, and the detection wavelengths are 260nm, 280nm and 215nm respectively. Figure 13-15 shown.

[0137] The resolution and tailing factor changes using different wavelengths of the UV detector are shown in Table 4:

[0138] Table 4

[0139]

[0140] The results in Table 4 show that when using a UV detector, the tailing of the detection wavelengths of 260 nm, 280 nm, and 215 nm is severe, which may affect the correctness of the integration.

[0141] 3.2 Chromatographic columns

[0142] The detection method is the same as in Example 2.2, using a fluorescence detector for detection, and replacing the chromatographic columns with the following chromatographic columns for detection:

[0143] 1) TSKgel G4000SWXL (8 μm, 45 nm, 7.8 × 300 mm);

[0144] 2) Chromatographic column: Wyatt WTC050N5 (5 μm, 50 nm, 4.6×300 mm).

[0145] Test results such as Figure 16-17 Using different chromatographic columns, the separation and tailing factor changes are shown in Table 5:

[0146] Table 5

[0147]

[0148] The results in Table 5 show that it is difficult to obtain good separation using the Tosoh and Wyatt chromatographic columns, and both are not suitable for the AAV detection method of the present invention.

[0149] 3.3 Flow rate

[0150] The detection method is the same as in Example 2.2, wherein the flow rate is changed to 0.4 ml / min and 0.6 ml / min. The detection results are as follows Fig.18 , Fig.19 shown.

[0151] Using different flow rates, the separation and tailing factor changes are shown in Table 6:

[0152] Table 6

[0153] Different flow rates Main peak separation USP tailing factor USP Theoretical Plate Number Flow rate 0.4ml / min 1.590954e+00 1.412053e+01 1.995985e+03 Flow rate 0.6ml / min 1.547628e+00 1.387931e+01 2.308847e+03

[0154] The results in Table 6 show that the separation is good within the flow rate range of 0.4 ml / min to 0.6 ml / min, and the tailing factor is less than 1.5, which meets the method requirements.

[0155] 3.4 Fluorescence excitation wave

[0156] The detection method is the same as in Example 2.2, and the detection effects of 280nm and 215nm excitation wavelengths are compared. Fig. 20 As shown, the peak effect of 280nm excitation wavelength is better.

[0157] Example 4 Linearity and Repeatability Verification

[0158] The detection method is as in Example 2.2, and good linearity and repeatability data are shown by detecting the AAV control substance.

[0159] 1. Linearity: as shown in Table 7 and Fig.21 As shown, R 2 >0.995, indicating that the method of the present invention has good linearity.

[0160] Table 7 Linearity test results

[0161]

[0162] 2. Repeatability: As shown in Table 8, the purity RSD% (n=6) ≤ 2.0%, indicating that the method of the present invention has good repeatability.

[0163] Table 8 Repeatability test results

[0164]

[0165] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A size exclusion chromatography system for virus purity determination, characterized in that: The chromatographic system comprises: 1) Size exclusion chromatography column; 2) a mobile phase having an ionic strength of 150 mM to 500 mM and comprising an organic solvent; 3) Sample solution.

2. The size exclusion chromatography system according to claim 1, characterized in that The sample contains adeno-associated virus (AAV) particles.

3. The size exclusion chromatography system according to claim 1, characterized in that The mobile phase comprises: i) Phosphate buffer (PB); ii) a salt selected from the group consisting of sodium chloride, potassium chloride, or a combination thereof.

4. The size exclusion chromatography system according to claim 3, characterized in that In the mobile phase, the concentration of sodium chloride or potassium chloride is 100-200 mM, preferably 125-175 mM, and more preferably 150 mM.

5. The size exclusion chromatography system according to claim 1, characterized in that The organic solvent is an alcohol solvent.

6. The size exclusion chromatography system according to claim 1, characterized in that The organic solvent is selected from the following group: methanol, ethanol, or a combination thereof.

7. The size exclusion chromatography system according to claim 1, characterized in that In the mobile phase, the volume concentration of the organic solvent is 3-10%, preferably 4-7%, and more preferably 5%.

8. The size exclusion chromatography system according to claim 1, characterized in that The size exclusion chromatography system has one or more of the following effects when detecting the purity of a virus (preferably an AAV virus): 1) Separation ≥ 1.5, preferably ≥ 1.7; 2) The tailing factor is 0.8-1.5, preferably 1-1.4; 3) The number of theoretical plates is ≥2000, more preferably ≥2300.

9. A method for determining virus purity, characterized in that: The method comprises the step of performing detection using the size exclusion chromatography system as claimed in claim 1.

10. The method according to claim 9, characterized in that In the chromatographic conditions of the method, the flow rate of the mobile phase is 0.3-0.7 ml / min, preferably 0.4-0.6 ml / min, and more preferably 0.5 ml / min.

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