Detection method of virus full shell rate

Abstract

The invention provides a method for detecting the full shell rate of a virus. The virus has a capsid and a nucleic acid molecule contained in the capsid, and the detection method comprises the following steps: marking the capsid of the virus in a sample containing the virus by using a first dye, and detecting a fluorescent particle group signal of a fluorescent positive particle group marked by the first dye; destroying the capsid of the virus, then labeling the nucleic acid molecule with a second dye, and detecting a fluorescent particle swarm signal of a fluorescent positive particle swarm labeled with the second dye; and respectively carrying out data fitting on the fluorescent particle swarm signals of the particle swarms which are marked by the first dye and the second dye and show fluorescence positive, and calculating the virus full shell rate according to the areas under the curves of the two fitting curves or the number of events between two points where the two fitting curves are intersected with the abscissa of the corresponding fluorescence intensity distribution map.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for detecting virus capsid rate. Background Art

[0002] Viruses having a capsid are widely used in the field of gene therapy. Examples of viruses having a capsid include adeno-associated viruses, lentiviruses, adenoviruses, and poxviruses.

[0003] Adeno-associated virus (AAV) is a member of the helper-dependent parvovirus B19 family of viruses with a particle size of approximately 20 nm, a non-enveloped icosahedral structure, and a dense, single-stranded DNA interior. Recombinant adeno-associated virus (rAAV) vectors offer advantages such as high expression efficiency, long-term sustained and stable expression, low immunogenicity, no reported cases of pathogenicity or tumorigenicity, and the potential for large-scale production, making them one of the most promising vectors in gene therapy. According to statistics, over 100 clinical studies involving rAAV have been documented worldwide, demonstrating remarkable efficacy in the treatment of Leber's congenital amaurosis and lipoprotein lipase deficiency.

[0004] The quality control technology of adeno-associated virus products is the most important factor restricting their clinical transformation and application. Internationally, in terms of the quality control of adeno-associated virus products, the Adenovirus Reference Material Working Group (ARMWG) first developed a standard sample guide. After that, the U.S. Food and Drug Administration (FDA), the National Institute of Health Recombinant DNA Advisory Committee (NIH-RAC), and the National Gene Vector Laboratory (FNGCL) jointly established the AAV Reference Standard Working Group (AAVRSWG). Based on this guideline, the standardization of AAV was further promoted, especially for the unified and standardized AAV titer determination and clinical dosage usage standards.

[0005] The study found that the requirement for virus production in clinical trials is 10 3 and 10 5 DRP / cell (DNase-Resistant Particles / cell). The dosage in clinical trials is generally 2×10 11 DRP / kg, the required single batch output is about 10 15DRP. Different tissues have different requirements for the titer of rAAV. In ophthalmic clinical trials, only about 2×10 8 DRP, while the treatment of muscle or liver diseases requires 1×10 14 Therefore, the titer control of adeno-associated virus products is considered to be an important factor affecting their clinical therapeutic effects.

[0006] Numerous studies have shown that during the preparation and purification of rAAV vectors, in addition to the amplification and enrichment of solid AAV particles, additional empty AAV particles are inevitably produced. Therefore, the full AAV titer directly affects the effectiveness and safety of rAAV vectors. Only viral particles containing intact genomes can successfully deliver the target gene into the host cell nucleus for gene expression.

[0007] At present, the following methods are mainly used to detect the full shell rate:

[0008] Optical density (OD) is a quantitative method for measuring the titer of capsid protein and the content ratio of adeno-associated virus by measuring the absorption of nucleic acid and protein at 260nm and 280nm. This method requires a sample concentration higher than 5×10 11 VG / mL, the detection consumes a lot of sample, and the OD linear range is small and the coefficient of variation is high.

[0009] Transmission electron microscopy is a direct tool for measuring the relative number of empty capsids of adeno-associated virus, but this method is time-consuming and the equipment is expensive, which is not conducive to large-scale promotion and application, and the statistical representativeness is not high.

[0010] Analytical ultracentrifugation (AUC) combined with ultraviolet absorption or Raleigh interference assays can accurately characterize the various components of adeno-associated virus (AAV) including empty virus particles, solid virus particles, virus particle aggregates, viral fragments, and genomic fragments. However, this method suffers from high sample consumption, low detection throughput, and high cost.

[0011] In summary, the field of gene therapy biotechnology still needs to develop improved methods for detecting parameters such as viral capsid rate, which can detect viral capsid rate in an efficient, accurate, easy to operate, small sample volume and good repeatability manner. Summary of the Invention

[0012] The inventors overcame the technical difficulty of the existing technology that cannot detect the number of virus particles, especially the number of adeno-associated virus particles, using scattered light by labeling the virus capsid and the nucleic acid inside the virus capsid and detecting the virus capsid fullness rate after removing the free nucleic acid and free dye.

[0013] Secondly, the present application removes the interference of free nucleic acid on the detection results of intracapsid nucleic acid by removing free nucleic acid before labeling with the second dye, and the obtained results are highly accurate and reproducible.

[0014] Finally, the present application can eliminate the interference of unfilled virus particles in virus-containing samples after labeling with the second dye, and the interference of impurities, cell debris, free nucleic acids, free dyes, etc. in the detection environment, as well as the interference of impurities, cell debris, free nucleic acids, free dyes, etc. in the detection environment in virus-containing samples after labeling with the first dye by performing data fitting on the signal data of the fluorescent particle group, which is beneficial to improving the accuracy and reproducibility of the virus full shell rate, especially the full shell rate detection results of adeno-associated virus.

[0015] The present invention provides a method for detecting a virus capsid rate, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, and the method comprises:

[0016] (1) labeling the capsid of the virus in the sample containing the virus with a first dye;

[0017] (2) after destroying the viral capsid, labeling the nucleic acid molecule contained in the viral capsid with a second dye;

[0018] (3) detecting the fluorescent signal of the first dye-positive marker in the sample to obtain a fluorescence intensity distribution graph A, and detecting the fluorescent signal of the second dye-positive marker in the sample to obtain a fluorescence intensity distribution graph B;

[0019] (4) Perform data fitting on the above fluorescence intensity distribution graphs A and B to obtain fitting curve A f 、B f , according to the fitting curve A f 、B f The area under the curve, or the fitted curve A f 、B f The virus capsid rate was calculated based on the number of events between two points where the horizontal axis of the corresponding fluorescence intensity distribution graph intersected.

[0020] Preferably, the sample to be detected can be purified to remove impurities and / or free nucleic acids and free dyes before or after the labeling with the first dye, and before or after the labeling with the second dye.

[0021] The method of the present invention calculates the full-capsid rate by data fitting, eliminating the influence of the following substances in the sample to be detected after being labeled with the first dye or the second dye: unfull-capsid virus particles; and the influence of the following substances in the detection environment: impurities, free nucleic acids, free dyes, etc., thereby improving the accuracy and reproducibility of the detection results.

[0022] Specifically, the present invention includes the following aspects.

[0023] In a first aspect of the present invention, a method for detecting a viral property of a virus is provided, wherein the viral property is a capsid rate, and the virus has a capsid and a nucleic acid molecule contained within the capsid.

[0024] The method comprises:

[0025] (1) labeling the capsid of the virus in a sample containing the virus with a first dye, wherein the first dye specifically binds to a surface recognition molecule of the capsid of the virus and emits fluorescence, and optionally removing free dye and free nucleic acid in the sample after labeling with the first dye;

[0026] (2) destroying the viral capsid, and then labeling the nucleic acid molecule contained in the viral capsid with a second dye, wherein the second dye can specifically bind to the nucleic acid molecule and emit fluorescence;

[0027] (3) detecting the signal of a fluorescent particle group that exhibits fluorescence after being labeled with a first dye in the sample to be tested containing the virus within the flow volume within the unit detection time, and obtaining a fluorescence intensity distribution graph A; detecting the signal of a fluorescent particle group that exhibits fluorescence after being labeled with a second dye in the sample to be tested containing the virus within the flow volume within the unit detection time, and obtaining a fluorescence intensity distribution graph B;

[0028] Optionally, the sample to be tested containing the virus is purified to remove impurities before labeling;

[0029] Preferably, the horizontal axis of the fluorescence intensity distribution graph A and the fluorescence intensity distribution graph B is the fluorescence signal intensity; the vertical axis is the number of fluorescence-positive events;

[0030] (4) Perform data fitting on the fluorescence intensity distribution graph A and the fluorescence intensity distribution graph B to obtain fitting curves A and B respectively. f and B f , obtain the area under the curve S1 and S2; or obtain the flow volume of the sample under test in the corresponding unit detection time on the fitting curve A f and B f The number of events N1 and N2 between the two points where the horizontal axis of the corresponding fluorescence intensity distribution graph intersects;

[0031] Calculate the virus full shell rate in the sample to be tested based on the test data.

[0032] In the method of the present invention, the first dye is directly conjugated with the surface recognition molecule to emit fluorescence, or the first dye has a recognition group and is indirectly conjugated with the surface recognition molecule to emit fluorescence.

[0033] Preferably, the recognition group specifically binds to the surface recognition molecule.

[0034] Preferably, the recognition group is selected from one or more of an antibody or antibody fragment, antigen, ligand, ligand receptor or polysaccharide that specifically binds to the surface recognition molecule; more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule, and further preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of AVV virus.

[0035] The second dye can specifically bind to the nucleic acid molecule, and is preferably a nucleic acid fluorescent dye.

[0036] use

[0037] Measuring viral capsid fill rate has a significant impact on product quality and clinical use. Controlling and monitoring capsid fill rate is crucial in the development, production, and clinical application of viruses such as AAV, ensuring product quality and clinical treatment success.

[0038] Measuring the full shell rate by the method of the present invention is conducive to optimizing conditions to improve the accuracy of virus dosage, improve product safety, optimize clinical treatment effects, reduce the risk of immune response, and provide a basis for drug administration regimen and dosage determination.

[0039] Advantages of the present invention

[0040] Compared with the prior art, the technical solution provided by the present invention has at least one of the following beneficial technical effects:

[0041] (1) The method provided by the present invention has the advantages of high efficiency, accuracy, easy operation, short time consumption, small sample amount and good repeatability for detecting virus full shell rate.

[0042] (2) The present invention can eliminate the interference of free nucleic acids on the results and improve the accuracy and reproducibility of detection by removing free nucleic acids in the step of labeling with the first dye and / or the step of labeling with the second dye.

[0043] (3) The present invention can reduce background interference and improve detection accuracy and reproducibility by removing impurities such as cell debris, floating cells, extracellular vesicles, miscellaneous proteins, lipids, salts, and free dyes and free nucleic acids in the step of labeling with the first dye and / or the step of labeling with the second dye.

[0044] (4) The present invention detects the full shell rate by labeling the nucleic acid in the virus shell and the virus capsid and removing the free nucleic acid and free dye, thereby overcoming the technical difficulty that the existing technology cannot detect the number of adeno-associated virus particles using scattered light. The detection method provided by the present invention has high accuracy and good repeatability in the detection results.

[0045] (5) The present invention uses a fitting method suitable for normally distributed data or a Gaussian fitting method to count the number of events (S2 or N2) of fully encapsidated viruses, especially fully encapsidated adeno-associated viruses, to eliminate the effects of unencapsulated virus particles and impurities in the detection environment, cell debris, free nucleic acids, free dyes, incompletely packaged viruses, and virus aggregates on the virus encapsulation rate. Compared with calculating the encapsulation rate by counting all fluorescent particles (including not only fully encapsulated virus particles but also unencapsulated virus particles and impurities in the detection environment, cell debris, free nucleic acids, free dyes, etc.), this avoids overestimation of the encapsulation rate and improves the accuracy and precision of the detection.

[0046] In addition, the present invention uses a fitting method suitable for normally distributed data or a Gaussian fitting method to count the total number of virus particles (S1 or N1), especially adeno-associated virus particles, thereby eliminating the influence of impurities, cell debris, free nucleic acids, free dyes, incompletely packaged viruses and virus aggregates in the detection environment on the full shell rate results, thereby improving the accuracy and precision of the detection.

[0047] (6) The detection method provided by the present invention is used to detect viruses in cells, such as the full shell rate of rAAV virus in cells. The results obtained are not significantly different from the theoretical values, with high accuracy, and are suitable for a variety of virus detection scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the fluorescence intensity distribution diagram of the fluorescent particle group of the rAAV mixture 1c in Example 1 after Gaussian fitting.

[0049] Figure 2 This is the fluorescence intensity distribution diagram of the fluorescent particle group of the rAAV mixture 2b in Example 1 after Gaussian fitting.

[0050] Figure 3 This is the fluorescence intensity distribution diagram of the fluorescent particle group of the rAAV mixed solution 1e in Example 2 after Gaussian fitting.

[0051] Figure 4 This is the fluorescence intensity distribution diagram of the fluorescent particle group of the rAAV mixture 2b-2 in Example 2 after Gaussian fitting.

[0052] Figure 5 This is a graph showing the AUC analysis results for Comparative Example 1.

[0053] Figure 6 This is the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1f in Example 4 after Gaussian fitting.

[0054] Figure 7 This is the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 4b in Example 4 after Gaussian fitting.

[0055] Among them, Figures 1-4 and Figure 6-Figure 7 In the fluorescence intensity distribution graph, the horizontal axis is the fluorescence signal intensity; the vertical axis is the number of events labeled by the dye. DETAILED DESCRIPTION

[0056] The present application will be further described in detail below through the accompanying drawings and examples, through which the features and advantages of the present application will become more clear and distinct.

[0057] The word "exemplary" is used exclusively herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

[0058] In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0059] In this application, "room temperature" refers to ambient temperature, which may be 20°C-30°C; in some embodiments, 22°C-28°C; in some embodiments, 24°C-26°C; and in some embodiments, 25°C.

[0060] Throughout this application, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. Based on the disclosed figures, the numerical value of each number may vary by less than ±10%, or by amounts deemed reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0061] In this application, the term "optional", "optional" or "optionally" means that the event or situation described later can but does not have to occur. For example, "optional surfactant" means that the surfactant may or may not be present.

[0062] The term "wt%" means percentage by mass. The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0063] definition

[0064] Viruses having a capsid and a nucleic acid molecule contained within the capsid may include lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, and the like.

[0065] The term "rAAV" means recombinant adeno-associated virus.

[0066] The term "AAV" means adeno-associated virus.

[0067] The term "full-shell virus" refers to a virus that has a capsid and an intact nucleic acid molecule inside the virus.

[0068] The term "event count" refers to the total number of particles (or cells) passing through the laser detection area recorded by an instrument, such as a flow cytometer, during an assay. Each particle passing through the laser generates a signal, which is recorded by the instrument and is called an "event." Event counts are typically expressed in units of "numbers" (or "counts").

[0069] The term "capsid fill rate" refers to the percentage of virus particles with a capsid and intact nucleic acid molecules inside the virus (full capsid adeno-associated virus) to the total number of virus particles.

[0070] The term "solid virus" refers to a virus having a capsid and a nucleic acid molecule inside the virus, including a virus having an incomplete nucleic acid inside and a virus having a complete nucleic acid inside.

[0071] The term "solid rate" refers to the percentage of viruses with capsids and nucleic acid molecules inside the virus (including viruses with incomplete nucleic acids inside (incompletely packaged viruses) and viruses with complete nucleic acids inside (completely packaged viruses)) to the total number of virus particles.

[0072] The single-molecule detection system includes an optical system, a photodetector, and a signal acquisition and analysis system. The optical system includes at least one fluorescence channel. The photodetector is used to detect fluorescence signals in the sample and convert them into electrical signals. The signal acquisition and analysis system is capable of collecting and analyzing the electrical signals.

[0073] The term "dilution factor" refers to the ratio of the concentration before dilution to the concentration after dilution.

[0074] Viral genome titer refers to the number of viral genome copies per unit volume (such as per milliliter) of sample. For example, 10 6 viral genome copies, then the viral genome titer of this sample is 10 6 copies / mL or 10 6 vg / ml.

[0075] Viral capsid titer refers to the total number of viral capsid particles in a unit volume (such as per milliliter) of sample, and its unit is, for example, particles / ml, including complete viral particles containing the viral genome and empty viral particles without the genome.

[0076] VG / mL (Vector Genomes per mL): The number of viral vector genomes per unit volume, commonly used for titer determination of adeno-associated viral vectors, is a physical unit.

[0077] GC / mL (Genomic Copies per mL): The number of genomic copies of a viral vector per unit volume. It is commonly used for titer determination of adeno-associated viral vectors and is a physical unit.

[0078] VP / mL (Virus Particles per mL): The number of virus particles per unit volume. It can be used to determine the titer of adenovirus vectors and is a physical unit.

[0079] The term "Gaussian fitting" refers to a mathematical method used to fit data to a Gaussian curve (normal distribution curve) to describe the distribution characteristics of the data. The basic principle of Gaussian fitting is to fit a large number of data points that conform to a Gaussian distribution to obtain a specific Gaussian function.

[0080] The first embodiment of the present invention is a method for detecting the virus capsid rate, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, and the method comprises:

[0081] (1) First dye labeling (capsid labeling): a first test sample containing the virus is mixed with a first dye, and the first dye specifically binds to and labels the surface recognition molecule of the capsid of the virus in the test sample, thereby obtaining a test sample labeled with the first dye for detection in step (3);

[0082] Optionally, before or after mixing, the sample is diluted to a detection working concentration at a dilution factor α relative to the sample to obtain a labeled diluted sample I for detection in step (3), wherein α is greater than or equal to 1.

[0083] Preferably, after the first dye labeling, free dye and free nucleic acid in the sample to be tested are removed. The removal method can be performed using the method described in the "Purification used in this method" section.

[0084] Preferably, the removal of free dye and free nucleic acid is performed using a separation column separation method. As a separation column separation method, for example, the test sample labeled with the first dye can be passed through a dye and nucleic acid removal spin column, centrifuged, and the outflowing liquid collected.

[0085] In one embodiment, the virus type having a capsid may include: lentivirus, adenovirus, adeno-associated virus, poxvirus, etc., preferably adeno-associated virus.

[0086] The adeno-associated virus can be a natural virus, a recombinant virus, an artificially modified virus such as a viral vector with an artificially edited segment inserted, a viral vaccine, etc., or a virus strain with desired properties selected in cells or microorganisms.

[0087] In one embodiment, the first dye specifically binds to the surface recognition molecule. The surface recognition molecule of the viral capsid is one or a combination of two or more selected from proteins and carbohydrates.

[0088] Preferably, the surface recognition molecule of the viral capsid is a protein.

[0089] The first dye is directly conjugated with the surface recognition molecule to emit fluorescence, or the first dye has a recognition group and is indirectly conjugated with the surface recognition molecule to emit fluorescence.

[0090] In some embodiments, the recognition group is selected from one or more of an antibody or antibody fragment, an antigen, a ligand, a ligand receptor, or a polysaccharide that specifically binds to the surface recognition molecule; more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule; and even more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of AVV. Further preferably, the first dye is composed of a recognition group and a fluorescent dye that emits fluorescence.

[0091] Fluorescent dyes commonly used in the art can be used without particular limitation, and include, for example, organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, polymer dots, and the like. Organic fluorescent molecules include, but are not limited to, YF488 and FITC. Fluorescent proteins include, but are not limited to, phycoerythrin, phycocyanin, red fluorescent protein, green fluorescent protein, and mCherry.

[0092] As the first dye, those described in the section "Dye for labeling - first dye" can be used.

[0093] In one embodiment, the method may include purifying the sample containing the virus to remove impurities before or after labeling with the first dye.

[0094] In one embodiment, the method may include removing free dye and free nucleic acid after labeling with the first dye.

[0095] As the methods for purification and removal of free dye and free nucleic acid, those described in the section "Purification used in the present method" can be used;

[0096] (2) Second dye labeling (labeling of nucleic acid in the shell):

[0097] taking a second sample to be tested containing the virus, and destroying the viral capsid;

[0098] The second test sample is mixed with a second dye, and the second dye is combined with the nucleic acid molecule contained in the capsid of the virus and labeled to obtain a test sample labeled with the second dye, which is used for the detection in step (3).

[0099] Preferably, free nucleic acid is removed from the sample before the viral capsid is destroyed.

[0100] Preferably, the sample is purified before labeling with the second dye, more preferably the purification is performed by magnetic separation, which can be performed using those commonly used in the art, such as those described in the "Purification for this method" section.

[0101] The second sample to be tested can be the first sample to be tested, the sample to be tested labeled with the first dye, the diluted sample I or the solution obtained by diluting the first sample to be tested, or the supernatant obtained by purifying the first sample to be tested to remove impurities.

[0102] Optionally, before or after the second dye labeling, the corresponding test sample is diluted to a detection working concentration at a dilution factor β relative to the second test sample to obtain a labeled diluted sample II for detection in step (3), wherein β is greater than or equal to 1.

[0103] In one embodiment, the method for destroying the viral capsid is selected from a nucleic acid extraction reagent method or a thermal lysis method. The nucleic acid extraction reagent method includes a method based on a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid phase matrix, or a combination of two or more. The heating temperature of the thermal lysis method can be 50°C-130°C. Preferably, the heating temperature is selected from 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C or 130°C.

[0104] Optionally, the method comprises removing free dye and free nucleic acid after labeling the capsid of the virus in the sample comprising the virus with the second dye.

[0105] In one embodiment, the method for removing free nucleic acids is selected from the group consisting of enzyme digestion and separation column separation. Methods for removing impurities include those described in the section "2) Purification for Removal of Free Dyes and Free Nucleic Acids".

[0106] The second dye can specifically bind to the nucleic acid molecule contained in the viral capsid. Preferably, the second dye is a nucleic acid fluorescent dye.

[0107] In some embodiments, the nucleic acid fluorescent dye comprises one or a combination of two or more of a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, or a DNA double helix minor groove binding dye. As the second dye, those described in the "Dyes for Labeling - Second Dye" section can be used;

[0108] (3) Detection:

[0109] The single-molecule detection system is used to detect the fluorescent particle group in the flow volume V1 of the sample to be tested or the diluted sample I labeled with the first dye within the unit detection time, and the fluorescent particle group signals are counted to obtain a fluorescence intensity distribution graph A. The fluorescent particle group in the fluorescent particle group in the flow volume V2 of the sample to be tested or the diluted sample II labeled with the second dye within the unit detection time is also detected, and the fluorescent particle group signals are counted to obtain a fluorescence intensity distribution graph B.

[0110] The horizontal axis of the fluorescence intensity distribution graphs A and B is the fluorescence signal intensity; the vertical axis is the number of fluorescence-positive dye-labeled events.

[0111] (4) Data processing

[0112] The fluorescence intensity distribution graph A and the fluorescence intensity distribution graph B in step (3) are respectively fitted to obtain the fitting curve A. f and fitting curve B f , and respectively calculate the areas S1 and S2 under the fitting curves, or respectively count the number of events N1 and N2 of the first dye-labeled test sample or mixed solution 1 and the second dye-labeled test sample or mixed solution 2 between the two points where the fitting curves intersect the abscissas of the corresponding fluorescence intensity distribution graphs,

[0113] In one embodiment, the data fitting can be performed using a fitting method suitable for normally distributed data, preferably the data fitting is performed using a Gaussian fitting method;

[0114] As the Gaussian fitting method, the method described in the "Data Fitting" section can be used;

[0115] Calculation: Calculate the full shell rate according to the following formula 1 or formula 2:

[0116] The virus full shell rate is calculated according to the following formula 1:

[0117] Virus full shell rate (%) = ((S2 / V2)*β) / ((S1 / V1)*α)*100% ... ... Formula 1

[0118] Among them, S1 is the fitting curve A f The area under the curve is , V1 is the flow volume of the diluted sample I per unit time;

[0119] S2 is the fitting curve B f The area under the curve is , V2 is the flow volume of the diluted sample II per unit time;

[0120] The virus full shell rate can also be calculated according to the following formula 2:

[0121] Virus full shell rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... ... Formula 2

[0122] Among them, N1 is the fitting curve A f The number of events between the two points intersecting the horizontal axis, N2 is the ... f The number of events between the two points intersecting the horizontal axis, V1 is the flow volume of the diluted sample I per unit time, S1 is the flow volume of the fitting curve A f The area under the curve of

[0123] S2 is the fitting curve B f V2 is the flow volume of the diluted sample II per unit time.

[0124] In one embodiment, the above method further comprises a purification step, such as one or more of the following steps:

[0125] a) purifying the virus-containing sample to remove impurities before or after labeling the capsid of the virus in the virus-containing sample with a first dye;

[0126] b) after labeling the capsid of the virus in the sample containing the virus with the first dye, removing free dye and free nucleic acid;

[0127] c) purifying the virus-containing sample to remove impurities before labeling the virus capsid in the virus-containing sample with the first dye, and removing free dye and free nucleic acid after labeling with the first dye; or

[0128] d) purifying the virus-containing sample to remove impurities before or after disrupting the capsid and labeling the nucleic acid molecules contained within the viral capsid with a second dye;

[0129] As the method for the above-mentioned purification to remove impurities and to remove free dyes and free nucleic acids, methods commonly used in the art, such as those described in the section "Purification used in the present method", can be used.

[0130] Virus

[0131] The method of the present invention can be applied to viruses having a capsid and a nucleic acid molecule contained within the capsid. The types of viruses that can be detected by the method of the present invention may include: lentivirus, adenovirus, adeno-associated virus, poxvirus, etc., preferably adeno-associated virus.

[0132] In some embodiments, the virus in the sample to be tested is an adeno-associated virus. The serotype of the adeno-associated virus can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ18, AAV-PHPeB, AAV-PHP.S, AAV2-retro, AAV2-QuadYF, or AAV2.7m8.

[0133] In some embodiments, the dilution of the test sample, such as the diluted sample, the test sample, the quantitative solution, etc., can use phosphate buffered saline (PBS) as a diluent.

[0134] In some embodiments, the detection working concentration of the virus in the step of labeling with the first dye and / or the step of labeling with the second dye is independently selected from 1×10 5 -5×10 9 Genome titer / mL.

[0135] In some embodiments, the detection working concentrations in the step of labeling with the first dye and / or the step of labeling with the second dye are independently selected from 1×10 5 Genome titer / mL, 5×10 5 Genome titer / mL, 1×10 6 Genome titer / mL, 5×10 6 Genome titer / mL, 1×10 7 Genome titer / mL, 5×10 7 Genome titer / mL, 1×10 8 Genome titer / mL, 5×10 8 Genome titer / mL, 1×10 9 Genome titer / mL or 5×10 9 Genome titer / mL.

[0136] In some embodiments, the test sample can be derived from: process intermediates, preparations, cell cultures, body fluids, tissues, or cell lysates.

[0137] Dyes used for labeling

[0138] -First Dye

[0139] The first dye in the method of the present invention can specifically bind to the surface recognition molecule of the virus in the sample to be tested, and preferably the first dye is an optical dye.

[0140] In some embodiments, the optical dye comprises a fluorescent molecule, a fluorescent material, or a combination thereof. In some embodiments, the optical dye comprises one or a combination of two or more of a fluorescent molecule, a fluorescent protein, a quantum dot, or a polymer dot.

[0141] Preferably, the first dye carries a recognition group, which may include an antibody or antibody fragment, antigen, ligand, ligand receptor or polysaccharide that specifically binds to the surface recognition molecule.

[0142] The surface recognition molecule of the viral capsid can be one or a combination of two or more of proteins and carbohydrates.

[0143] The first dye can be specifically bound to the surface recognition molecule in the following manner: the first dye is directly conjugated to the surface recognition molecule; or the first dye carries a recognition group and is indirectly conjugated to the surface recognition molecule.

[0144] In some embodiments, the surface recognition molecule of the viral capsid comprises at least one of functional groups such as hydroxyl, carboxyl, amino, and thiol.

[0145] The recognition group specifically binds to the surface recognition molecule, and preferably the recognition group is selected from one or more of an antibody or antibody fragment, an antigen, a ligand, a ligand receptor or a polysaccharide; more preferably, the recognition group is an antibody or an antibody fragment; further preferably, the recognition group is an antibody or an antibody fragment that specifically binds to the surface recognition molecule of the AAV virus.

[0146] In some embodiments, the first dye comprises a fluorescent dye portion and a recognition group portion, preferably the recognition group portion comprises an AAV antibody, and the AAV antibody can be CaptureSelect TM Biotin-anti-AAVX conjugate.

[0147] In one embodiment, the first dye is a fluorescent antibody dye comprising an AAVX antibody and a fluorescent dye portion that emits fluorescence.

[0148] CaptureSelect TMBiotin anti-AAVX conjugate (ThermoFisher) binds specifically to different AAV serotypes including AAV1, AAV2, AAV2_HSPG, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, and AAVrh10 with high affinity and selectivity.

[0149] As a preparation of fluorescent antibody dyes, for example, AAVX antibodies can be combined with It is prepared by mixing optical dyes such as 647A SE.

[0150] The first dye may not comprise a recognition group moiety. 647A SE is a type of fluorescent dye with amino-reactive activity. The SE group of this type of dye can react with the amino group to produce a stable amide bond. In this embodiment, only 647A SE marks the protein of the virus capsid, or 647ASE is used in conjunction with antibodies to prepare fluorescent antibody dyes.

[0151] In some preferred embodiments, the recognition group portion is an AAV antibody. In some embodiments, the AAV antibody clone number includes at least one selected from ADK1a, A20, A20R, ADK4, ADK5a, ADK5b, ADK6, ADK8 and ADK8 / 9.

[0152] In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV1, and the AAV antibody clone number is ADK1a. In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV2, and the AAV antibody clone numbers are A20, A20R, and combinations thereof. In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV3, and the AAV antibody clone numbers are A20, A20R, and combinations thereof. In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV4, and the AAV antibody clone number is ADK4. In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV5, and the AAV antibody is selected from clone numbers ADK5a, ADK5b, and combinations thereof. In some embodiments, the serotype of the adeno-associated virus in the sample to be tested is AAV6, and the AAV antibody is ADK6. In some embodiments, the serotype of the adeno-associated virus in the test sample is AAV8, and the AAV antibody is selected from clone numbers ADK8, ADK8 / 9, and combinations thereof. In some embodiments, the serotype of the adeno-associated virus in the test sample is AAV9, and the AAV antibody is selected from clone numbers ADK9, ADK8 / 9, and combinations thereof.

[0153] The working concentration, incubation temperature, incubation time, etc. of the first dye can be within the range commonly used in the art without special limitation. The working concentration can be, for example, 0.01 to 10 μg / μL, the incubation temperature can be, for example, in the range of 4°C to 37°C, and the incubation time can be, for example, 5 to 60 minutes.

[0154] In one embodiment, the method of the present invention comprises, after disrupting the viral capsid, detecting the number of particles in the sample that fluoresce positively to a first dye. The number of particles in the sample that fluoresce positively to the first dye can be used to determine the amount of intact viral capsid present in the sample.

[0155] When viral particles bound to the first dye undergo capsid disruption and disintegrate, the first dye will exhibit diffuse, non-granular fluorescence in the sample solution. Therefore, after capsid disruption, the fluorescence channel detecting the first dye can be used to monitor the luminescence of the sample containing the first dye after capsid disruption. Particles that still exhibit fluorescence corresponding to the first dye indicate viral particles whose capsids have not been successfully disrupted.

[0156] -Second dye

[0157] The second dye in the method of the present invention is capable of specifically binding to nucleic acid molecules in the sample to be tested, such as nucleic acid molecules contained within the capsid of the virus. Preferably, the second dye is a nucleic acid fluorescent dye. The nucleic acid fluorescent dye can be any commonly used one in the art and is not particularly limited.

[0158] In some embodiments, the nucleic acid fluorescent dye comprises one or a combination of two or more of a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, and a DNA double helix minor groove binding dye.

[0159] In some embodiments, the nucleic acid fluorescent dye includes a member selected from the group consisting of Acridine Orange, Actinomycin D, 7-AAD (7-aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide, BOBO-3 Iodide, DAPI (4', 6-Diamidino-2-Phenylindole, Dihydrochloride), Dihydroethidine (Hydroethidine), Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride,Trihydrate, Trihydrate-FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace Blue Fluorescent NisslStain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615RedFluorescent Nissl Stain, NeuroTrace Deep-Red Fluorescent Nissl Stain, POPO-1Iodide, POPO-3Iodide, PO-PRO-1Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR GreenII, SYBR Safe DNA Gel Stain, SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13.SYTO 14.SYTO 16.SYTO 21.SYTO 24.SYTO BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, TO-PRO-1Iodide, TO-PRO-3Iodide, TOTO-1Iodide, TOTO-3Iodide, YO-PRO-1Iodide, YO-PRO-3Iodide, YOYO-1Iodide, YOYO-3Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask RedStain, ethidium bromide, or a combination of two or more.

[0160] Preferably, the nucleic acid fluorescent dye includes but is not limited to: SYTO9, SYTO16, SYTO21, SYTO24, SYTOXGreen, SYTO BC, SYTO RNASelect, and PicoGreen.

[0161] The working concentration, incubation temperature, incubation time, etc. of the nucleic acid fluorescent dye can be within the commonly used ranges in the art and are not particularly limited. The working concentration can be 0.1 to 10 μM, the working temperature can be in the range of 4° C. to 37° C., and the incubation time can be 1 to 60 min.

[0162] Purification used in this method

[0163] The method preferably includes a purification step. The purification step used in the method may include: 1) purification to separate the target virus from impurities; and 2) purification to remove free dyes and free nucleic acids, which are described below.

[0164] The impurities described in the present invention are selected from cell debris, floating cells, extracellular vesicles, miscellaneous proteins, lipids, salts and combinations thereof.

[0165] By separating impurities such as cell debris, floating cells, extracellular vesicles, miscellaneous proteins, lipids, salts, etc. from the target virus, and removing free optical dyes and free nucleic acids, it is helpful to reduce background interference and improve detection accuracy and reproducibility.

[0166] 1) Separation of target virus and purification of impurities

[0167] In the present invention, "isolation" mainly refers to the purification of the virus from a mixture, mixed solution, cell fluid, cell lysate, etc. containing the virus.

[0168] In some embodiments, step (1) of performing the first dye labeling or step (2) of performing the second dye labeling comprises purification to separate the target virus from impurities.

[0169] The purification methods used in step (1) or step (2) to separate the target virus from impurities can be independently selected from: magnetic separation, centrifugation, delamination, precipitation, solid-liquid separation, or a combination of two or more thereof.

[0170] The solid-liquid separation method may include chromatography, ultrafiltration or membrane filtration. The chromatography method may include liquid chromatography. The liquid chromatography method may include one or a combination of two or more of affinity liquid chromatography, ion exchange liquid chromatography, size exclusion liquid chromatography, hydrophobic liquid chromatography and reversed phase liquid chromatography.

[0171] In the present invention, it is preferred to specifically separate the target virus from impurities by magnetic separation.

[0172] The magnetic separation method may include magnetic bead separation and solid-phase chip separation. The magnetic bead separation method may include surface-loaded functional group magnetic bead separation. Surface-loaded functional group magnetic bead separation methods may include antigen-antibody magnetic bead separation, ligand-receptor magnetic bead separation, enzyme-substrate magnetic bead separation, protein-inhibitor magnetic bead separation, electron donor-electron acceptor magnetic bead separation, or structural complementation magnetic bead separation.

[0173] In some embodiments, the antigen-antibody magnetic bead separation method includes the following steps: after mixing the magnetic beads carrying the adeno-associated virus antibodies with the sample to be tested, the magnetic beads carrying the adeno-associated virus antibodies capture the adeno-associated virus in the sample to be tested, and adsorbing and separating the magnetic beads carrying the adeno-associated virus antibodies through an external magnetic field; or

[0174] The method comprises the following steps: mixing adeno-associated virus antibodies with a sample to be tested, allowing the adeno-associated virus antibodies to capture the adeno-associated virus in the sample to be tested, adding modified magnetic beads that can bind to the adeno-associated virus antibodies, and adsorbing and separating the modified magnetic beads through an external magnetic field.

[0175] The antigen-antibody magnetic bead separation method is a separation technology based on antigen-antibody interaction, which achieves efficient separation and purification of target proteins through the specific binding between magnetic beads and target proteins.

[0176] In some embodiments, preferably, the antigen-antibody magnetic bead separation method uses micro-nano-sized magnetic beads for separation. Micro-nano-sized magnetic beads can be those commonly used in the art, without particular limitation, and can be selected from, for example, carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads, thiol magnetic beads, tosyl magnetic beads, protein A / G magnetic beads, Oligo(dT) magnetic beads, antibody-coupled magnetic beads, SA magnetic beads, etc.

[0177] The term "SA magnetic beads" refers to streptavidin nanoparticle magnetic beads. The term "SA magnetic bead method" refers to labeling a target substance (such as adeno-associated virus, protein, nucleic acid, etc.) with biotin (biotin and the target substance can be linked using antibodies and disulfide bonds). This mixed solution containing the biotin-labeled target substance is then exposed to SA magnetic beads, where biotin specifically binds to streptavidin, capturing the target substance on the bead surface. An external magnetic field is then used to adsorb the SA magnetic beads, separating the target substance-bound beads from unbound impurities, thereby purifying the target substance.

[0178] The method of purification using SA magnetic beads may, for example, include the following steps: mixing the sample to be tested with biotin carrying antibodies and disulfide bonds, so that the virus is labeled with biotin carrying antibodies and disulfide bonds, and obtaining the biotin-labeled sample to be tested; contacting the obtained sample to be tested with SA magnetic beads, so that biotin and streptavidin specifically bind to each other, and the virus is captured on the surface of the SA magnetic beads; using an external magnetic field to adsorb the SA magnetic beads, and separating the SA magnetic beads bound to the target substance.

[0179] In some embodiments, step (1) comprises purifying the sample to be tested using SA magnetic beads before labeling with the first dye. Thus, step (1) comprises:

[0180] The test sample is purified using the SA magnetic bead method to obtain a purified sample containing virus-SA magnetic beads ③, i.e., the first test sample; the first test sample is mixed with a first dye, so that the first dye specifically binds to and labels the surface recognition molecule of the virus; a disulfide bond cleavage reagent is added to destroy the disulfide bond between the SA magnetic beads ③ and the virus bound thereto; and the supernatant A is separated by adsorption using a magnetic stand for detection in step (3).

[0181] Optionally, the supernatant A is diluted to a detection working concentration by a dilution factor of α (α is greater than or equal to 1) relative to the sample to be tested, and a diluted sample I is obtained for detection in step (3).

[0182] Optionally, in the above step (1), after the operation of “mixing the first sample to be tested with the first dye, allowing the optical dye to specifically bind to and label the surface recognition molecule of the virus”, the operation of “incubating, adsorbing on a magnetic stand, discarding the supernatant, and resuspending” is repeated 0-5 times (0 times: not performing the operation of “incubating, adsorbing on a magnetic stand, discarding the supernatant, and resuspending”).

[0183] In some embodiments, step (2) comprises purifying the sample to be tested using the SA magnetic bead method before or after labeling with the second dye. The purification operation is the same as that used in step (1) of purifying the sample to be tested using the SA magnetic bead method, except that after adsorption on a magnetic rack, supernatant B is separated instead of supernatant A, and supernatant B is diluted to obtain diluted sample II.

[0184] 2) Removal of free dyes and purification of free nucleic acids

[0185] In the present invention, "purification by removing free dyes and free nucleic acids" mainly refers to the purification by removing free dyes and free nucleic acid components other than the virus and the nucleic acid molecules contained inside the virus from a mixture, mixed solution, cell fluid, cell lysate, etc. containing the virus.

[0186] In some embodiments, the step (1) further comprises: after labeling the virus in the sample to be tested with the first dye, removing free dye and free nucleic acid.

[0187] In some embodiments, the step (2) further comprises: removing free dye and free nucleic acid before or after labeling the nucleic acid in the virus in the sample to be tested with a nucleic acid fluorescent dye.

[0188] In some embodiments, the method for removing free dye and free nucleic acid in step (1) or step (2) is selected from the group consisting of: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, and dilution methods, or a combination of two or more thereof.

[0189] In some embodiments, the method for removing free nucleic acids in step (1) or step (2) is selected from enzymatic digestion or separation column separation; for example, in the separation column separation method, a dye and nucleic acid removal centrifugal column is used for separation.

[0190] Examples of the dye and nucleic acid removal spin column include, but are not limited to, BeyoDesalt G-25 columns.

[0191] Methods for destroying the capsid

[0192] In some embodiments, the method of destroying the viral capsid in the method of the present invention comprises: a nucleic acid extraction reagent method or a thermal lysis method.

[0193] -Nucleic acid extraction reagent method

[0194] In some embodiments, the nucleic acid extraction reagent method includes a method based on one or a combination of two or more of a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid matrix.

[0195] In some embodiments, the lysis reagent includes one or a combination of two or more of cetyltriethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), guanidine isothiocyanate, phenol, chloroform, isoamyl alcohol, cesium chloride-ethidium bromide (CsCl-EB), alkali, and water.

[0196] In some embodiments, the divalent ion reagent comprises a transition metal divalent salt or an alkaline earth metal divalent salt. In some embodiments, the transition metal divalent salt is selected from a manganese salt or a zinc salt. In some embodiments, the alkaline earth metal divalent salt is selected from a magnesium salt or a calcium salt.

[0197] In some embodiments, the enzyme reagent comprises one or a combination of two or more of subtilisin, subtilisin, or alkaline serine protease. In some embodiments, the enzyme reagent further comprises one or a combination of two or more of proteinase K, proteinase R, proteinase T, subtilisin A, Nagarse, subtilisin B, or thermolysin.

[0198] In some embodiments, the detergent comprises one or a combination of two or more of cetyltrimethylammonium bromide, Tween-type surfactant, Triton X-100, sodium lauryl sulfate, nonylphenol polyoxyethylene ether, or polyoxyethylene nonionic detergent.

[0199] In some embodiments, the solid phase matrix comprises beads composed of one or a combination of two or more of magnetic microbeads, silica, diatomaceous earth, chromatography columns, glass particles, anion exchange resins, membranes, chips, carbon nanotubes, well plates, slides, chromatography matrices, or any combination thereof.

[0200] - Thermal cracking method

[0201] In some embodiments, the heating temperature of the thermal cracking method is 50° C.-130° C. In some embodiments, the heating temperature of the thermal cracking method is 0° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., or 130° C.

[0202] In some embodiments, the heat time of the thermal cracking method is 1 minute to 60 minutes. In some embodiments, the heat time of the thermal cracking method is 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

[0203] Detection

[0204] In some embodiments, the sample to be tested is labeled with the first dye and the second dye and then detected using a single molecule detection system.

[0205] In some embodiments, a quantitative virus solution with a known full-shell rate, such as an rAAV quantitative solution, can be labeled with the first dye and the second dye and then detected using a single-molecule detection system to locate the position of the peak formed by the full-shell virus particles in the sample to be tested in the fluorescence intensity distribution diagram.

[0206] In some embodiments, the single-molecule detection system comprises an optical system, a photodetector, and a signal acquisition and analysis system. The optical system comprises at least one fluorescence channel. The photodetector is configured to detect a fluorescence signal in a sample labeled with a first dye or a sample labeled with a second dye, and convert the fluorescence signal into an electrical signal. The signal acquisition and analysis system is configured to acquire and analyze the electrical signal and convert the electrical signal into a fluorescence intensity distribution graph.

[0207] In some embodiments, the optical system comprises a light source emitter and at least one fluorescence channel.

[0208] In some embodiments, the signal acquisition and analysis system is capable of acquiring and analyzing electrical signals, and performing calculations based on the conversion of the electrical signals to obtain the number of fluorescence events in the fluorescent particle population.

[0209] FITC and PC5 are fluorescent channels that can be used in the methods of the present invention. These two channels can be used on commonly used detection instruments in the art, such as the fluorescence channels on the NanoFCM Flow NanoAnalyzer. The FITC fluorescent channel can detect at least the fluorescence of AF488 and SYTO BC fluorescent nucleic acid dyes; the PC5 fluorescent channel can detect at least the fluorescence of AF647.

[0210] In one embodiment, the detection channel parameters may be: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488.

[0211] -Concentration standard solution

[0212] There is no special limitation on the fluorescent microspheres in the concentration standard solution, and fluorescent microsphere standards commonly used in virus quality control can be used, such as NBD & Cy5 quality control fluorescent microsphere standards.

[0213] In some embodiments, the concentration of the standard fluorescent microspheres in the concentration standard solution is 1×10 5 -5×10 9 In some embodiments, the concentration of the standard fluorescent microspheres in the concentration standard solution is 1×10 5 Particles / mL, 5×10 5 Particles / mL, 1×10 6 Particles / mL, 5×10 6 Particles / mL, 1×10 7 Particles / mL, 5×10 7 Particles / mL, 1×10 8 Particles / mL, 5×10 8 Particles / mL, 1×109 particles / mL or 5×10 9 particles / mL.

[0214] The concentration standard solution can be obtained by diluting with a common diluent, such as phosphate buffered saline (PBS).

[0215] Data fitting

[0216] In the present application, the data fitting is performed using a fitting method suitable for normally distributed data. Preferably, the data fitting includes Gaussian fitting.

[0217] The present invention uses a fitting method suitable for normally distributed data or a Gaussian fitting method to count the number of fully encapsidated adeno-associated virus particles (S2 or N2), thereby eliminating the influence of the number of incompletely encapsidated adeno-associated virus particles (in a sample containing incompletely encapsidated adeno-associated virus particles, the fluorescence intensity of the peak formed by the incompletely encapsidated adeno-associated virus particles in the fluorescence intensity distribution diagram is lower than the fluorescence intensity of the peak formed by the fully encapsidated adeno-associated virus particles in the fluorescence intensity distribution diagram), incompletely packaged viruses and virus aggregates on the adeno-associated virus full-capsid rate, thereby improving the accuracy and precision of the detection.

[0218] In addition, the present invention uses a fitting method suitable for normally distributed data or a Gaussian fitting method to count the total number of adeno-associated virus particles (S1 or N1), thereby eliminating the influence of impurities, cell debris, free nucleic acids, free dyes, incompletely packaged viruses and virus aggregates in the detection environment on the results, thereby improving the accuracy and precision of the detection.

[0219] The fitting method of the normally distributed data or Gaussian fitting of the present invention can be implemented using professional software such as Python and MATLAB. The above fitting effect can also be achieved by polynomial regression, gradient descent, Newton method, Gauss-Newton method, Loewenberg-Marquardt method (LM algorithm), genetic algorithm, etc.

[0220] use

[0221] Taking the adeno-associated virus (AAV) full capsid rate as an example, the important uses of the virus full capsid rate in several aspects of product quality and clinical use are explained:

[0222] (1) Viral activity, potency, and therapeutic effects

[0223] A high capsid repletion rate generally means more viral particles with complete structure and function. A fully capsidated AAV is more likely to carry the correct therapeutic gene and effectively deliver and express the gene after entering the target cell. In contrast, a low capsid repletion rate may result in reduced viral activity, affecting therapeutic efficacy.

[0224] Full-shell AAV may also have more advantages in terms of stability, and can better resist the influence of external environmental factors such as temperature and pH changes, thereby maintaining higher activity during storage and transportation.

[0225] (2) Purity and safety

[0226] A higher fill rate helps improve product purity. Impure AAV preparations may contain unassembled viral proteins, empty capsids, or other impurities that may trigger an immune response or cause adverse effects in patients.

[0227] Increasing the full capsid fraction can reduce the presence of non-functional particles and lower the potential risk of immunogenicity. The immune system may respond to incomplete or abnormal viral particles, thus affecting the safety and efficacy of treatment.

[0228] (3) Dosage regimen and dosage determination

[0229] Accurate determination of capsid fill can help optimize dosing regimens and determine appropriate dosages. Understanding AAV capsid fill can more accurately assess viral activity and potency, allowing for personalized treatment plans tailored to the patient's specific circumstances.

[0230] Therefore, the virus full shell rate detected by the method of the present invention has an important impact on product quality and clinical use.

[0231] Improving the capsid fill rate can enhance viral activity and potency, improve product purity and safety, optimize clinical treatment outcomes, reduce the risk of immune responses, and provide a basis for dosing regimens and dosage determination. During the research and development, production, and clinical application of viruses such as AAV, the control and monitoring of the capsid fill rate should be given high priority to ensure product quality and clinical treatment success.

[0232] Example

[0233] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0234] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention. Gaussian fitting in the following examples or comparative examples of the present invention can be implemented using Python or MATLAB software.

[0235] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0236] The term "M" stands for mol / L, i.e., moles per liter. "vg / ml" stands for genome copies per milliliter. PBS or PBS buffer stands for phosphate buffered saline, which is an aqueous solution containing 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4.

[0237] The term "OD" means absorbance, such as "OD230nm" means absorbance at 230nm.

[0238] Reagents and instruments used in the examples or comparative examples

[0239] 1) Reagents:

[0240] 1.1) Fluorescent labeling reagents

[0241] 647ASE( 647A succinimidyl ester) was purchased from BIORIGIN;

[0242] SYTO BC Green Fluorescent Nucleic Acid Stains (SYTO BC fluorescent nucleic acid stains) were purchased from Invitrogen.

[0243] 1.2) Fluorescent antibody dyes

[0244] CaptureSelect TM Biotin anti-AAVX conjugate antibody: (This conjugate shows binding reactivity to AAV serotypes including AAV1, AAV2, AAV2_HSPG, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, and AAVrh10) purchased from ThermoFisher. AAV8 antibody: purchased from Genscript, MonoRab rm AAV8 (intact particle) Antibody (72E8)

[0245] Preparation of YF647-AAV8 antibody (according to the procedure provided on the BIORIGIN official website "YF647A Antibody Labeling Kits (YF647A Antibody Labeling Kit) Instructions"):

[0246] a. Prepare the antibody in reaction buffer: dilute the antibody in 0.1 M NaHCO3 solution (pH ~ 8.3).

[0247] b. Transfer AAV8 antibody (Genscript, MonoRab rm AAV8 (intact particle) Antibody (72E8)) (2.5 mg / mL) to a Add the 647ASE dye to the vial. Mix well and incubate with agitation at room temperature for 1 hour.

[0248] c. Separate the labeled antibody by running the solution through a dialysis column.

[0249] d. Store the labeled antibody at 2°C-6°C in the dark.

[0250] Preparation of YF647-AAVX antibody (according to the procedure provided on the BIORIGIN official website "YF647A Antibody Labeling Kits (YF647A Antibody Labeling Kit) Instructions"):

[0251] a. Prepare the antibody in reaction buffer: dilute the antibody in 0.1 M NaHCO3 solution (pH ~ 8.3).

[0252] b. AAVX antibody (CaptureSelect TM Biotin anti-AAVX conjugate) (2.5 mg / mL) was transferred to a Add the 647ASE dye to the vial. Mix well and incubate with agitation at room temperature for 1 hour.

[0253] c. Separate the labeled antibody by running the solution through a dialysis column.

[0254] d. Store the labeled antibody at 2°C-6°C in the dark.

[0255] 1.3) Other

[0256] EZ-Link TM NHS-SS-biotin: biotin disulfide-active ester was purchased from ThermoFisher Scientific.

[0257] Biotin-SS-AAV8 Antibody: Preparation of Biotin-Disulfide Bond-AAV8 Antibody (according to the procedure provided on the ThermoFisherScientific official website, "User Guide: EZ-Link NHS-SS-Biotin"):

[0258] a. Before use, prepare 10 mM NHS SS biotin solution.

[0259] b. Add 100 μL of 10 mM NHS SS biotin to the AF647-AAV8 antibody per mL of reaction volume.

[0260] c. Incubate the reaction at room temperature for 30 minutes to obtain biotin-disulfide bond-AAV8 antibody.

[0261] 2) Instruments or equipment:

[0262] Dye and nucleic acid removal spin column: BeyoDesalt G-25 column (Superfine, 5 ml); Shanghai Beyotime Biotechnology Co., Ltd.

[0263] SA magnetic beads: Streptavidin Magnetic Beads: MCE company, product number: HY-K0208;

[0264] Single molecule detection system: Flow NanoAnalyzer (manufacturer: NanoFCM Inc);

[0265] Centrifuge 5810R refrigerated centrifuge was purchased from Eppendorf;

[0266] A Tecnai G2 Spirit transmission electron microscope was purchased from FEI Czech Ltd.

[0267] Comparative Example 1: AUC method for detecting full shell rate

[0268] rAAV concentrate: the same rAAV concentrate to be labeled as described in Example 1 (obtained by diluting the stock solution, stock solution: manufacturer: Paigen Bio, AAV8 [scAAV.CAG.EGFP.WPRE.SV40pA], product number: PGS08001)

[0269] Sample preparation: The OD values ​​of the solution were measured by optical density measurement at 260 nm ranging from 0.1 to 1.0. The test sample (rAAV concentrate) was adjusted to a target concentration greater than 5 × 10 by direct dilution or further concentration using an Amicon Ultra-0.5 / 30K MWCO centrifugal filter device. 11 vg / mL.

[0270] Sedimentation velocity AUC data acquisition:

[0271] Sedimentation velocity analytical ultracentrifugation (AUC) analysis was performed on a Beckman Coulter XL-I. 400 μL of sample was loaded into the sample sector of the two sector velocity chambers, and 400 μL of PBS buffer was loaded into the corresponding reference sector. The sample was placed in a four-well rotor and allowed to equilibrate in the instrument at 20°C and full vacuum for 1 hour.

[0272] Sedimentation velocity centrifugation was performed at 20,000 RPM, 20°C, with a 0.003 cm radius step setting, without delay and without repetition. Absorbance (230 nm) and Raleigh interferometer optics were used to simultaneously record the radius concentration as a function of time until the smallest sedimenting component passed through the optical window (1.2 hours). The assay throughput was limited to a single sample per run, based on the absorbance scan collection time of greater than 1 minute and the size and rapid sedimentation of AAV.

[0273] AUC Data Analysis: The shell fill rate was determined by analyzing approximately 75 scans from each detection method using the SEDFIT continuous size C(S) distribution model. Second-order (2nd) derivative regularization was applied to the fit, with a confidence level of 0.68 for the F-statistic. The following C(S) parameters were kept constant: resolution = 200S, Smin = 1, Smax = 200, and friction ratio = 1.0. RI and TI noise reduction were applied, and the meniscus position was allowed to float, with the optimal position automatically selected by the software.

[0274] This model fits the data to the Lamm equation, and the resulting size distribution is a "distribution of sedimentation coefficients," where the area under each peak is proportional to the concentration expressed in fringes or OD230nm units. The sedimentation coefficient (expressed in Svedberg units) and relative concentration (expressed in OD units) of each component in the distribution are determined.

[0275] Each AUC run was determined independently, and the following attributes of each assay were monitored to ensure the quality of the results: goodness of fit (rmsd), the ratio of OD230nm / interference signal (represented as fringes) for each peak (A230 / IF ratio), consistency of sedimentation coefficient for each species between runs, and the overall quality of the scan.

[0276] Absorbance optics (230 nm): The molar concentration and actual percentage of the intact vector peak were calculated from the absorbance data using the extinction coefficient. The molar absorptivity extinction coefficients for empty capsids and intact viral particles (intact vector) were calculated based on published formulas. Extinction coefficients were applied to both empty capsids and intact AAV peaks. The SEDFIT algorithm was used to determine C(S) values. The molar concentrations of both intact vector and empty capsids were calculated using Beer's law, and from these values, the full capsid fraction in the rAAV concentrate was calculated to be 75.02% ( Figure 5 ).

[0277] The method takes more than 6 hours from preparing the sample to obtaining the test result.

[0278] Example 1: rAAV full shell rate ( 647ASE-labeled capsid)

[0279] rAAV concentrate to be labeled: (obtained after dilution of the stock solution, stock solution: Manufacturer: Paigen Bio, AAV8 [scAAV.CAG.EGFP.WPRE.SV40pA], Product Number: PGS08001)

[0280] 647ASE is a type of fluorescent dye with amino-reactive activity. The SE group of this type of dye can react with the amino group to produce a stable amide bond. In this example, 647ASE is used to label the capsid protein in the viral capsid.

[0281] Step (1):

[0282] Take 100 μL of the rAAV concentrate to be labeled, add 10 μL of 1M NaHCO3 aqueous solution, and mix thoroughly. Then add 2 μL of 10 μg / μL Mix 647ASE thoroughly and react in the dark at room temperature for 15 min to obtain rAAV mixture 1a.

[0283] Take the dye and nucleic acid removal centrifugal column, add rAAV mixture 1a, centrifuge at 1000g for 2 minutes to remove free 647ASE and free nucleic acid, and collect the liquid flowing out as rAAV mixed solution 1b.

[0284] The rAAV mixture 1b was diluted 62.5-fold with PBS (ie, 70-fold dilution relative to the rAAV concentrate to be labeled) to obtain the rAAV mixture 1c (ie, diluted sample I).

[0285] Step (2):

[0286] A portion of the aforementioned rAAV mixture 1b was diluted to 4.5 times with PBS buffer, and 100 μl of the diluted rAAV product was taken to obtain rAAV mixture 2a.

[0287] The rAAV mixed solution 2a was heated at 95°C for 10 minutes to obtain the rAAV DNA sample.

[0288] Take 10 μl of rAAV DNA sample, dilute it 10-fold with PBS buffer, add 1 μl of 100 μM SYTO BC Green Fluorescent Nucleic Acid Stains aqueous solution for labeling (i.e., it is diluted 50.904-fold relative to the rAAV concentrate to be labeled), and incubate at 37°C in the dark for 5 min to obtain rAAV mixture 2b (i.e., diluted sample II).

[0289] Preparation of concentration standard solution:

[0290] The concentration was 2.14×10 10 The NBD&Cy5 quality control fluorescent microsphere standard with a concentration of 100 particles / mL was diluted 100 times to obtain a concentration standard solution.

[0291] Step (3): Detection

[0292] Flow NanoAnalyzer was used to detect rAAV mixture 1c, rAAV mixture 2b and concentration standard solution respectively.

[0293] The detection parameters are: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using PC5 fluorescence channel. PC5 fluorescence channel is 647 excitation signals, used to characterize the number of events of rAAV, and analyze the The particle population with 647 positive signals was detected in the PC5 fluorescence channel and the detection results were recorded. The fluorescence of SYTO fluorescent nucleic acid fluorescent dye was detected in the FITC fluorescence channel to characterize the number of rAAV nucleic acid events. The particle population with SYTO positive signals was analyzed in the FITC fluorescence channel and the detection results were recorded.

[0294] The detection result includes the distribution of the fluorescence intensity of a single event relative to the positive fluorescence signal, and can be output as a fluorescence intensity distribution graph, wherein the horizontal axis of the fluorescence intensity distribution graph is the fluorescence signal intensity and the vertical axis is the number of fluorescence-positive (dye-labeled) events.

[0295] Step (4): Data processing

[0296] Gaussian fitting was performed on the fluorescence intensity distribution of the fluorescent particle group of rAAV mixture 1c, and the results are shown in Figure 1 , and the area under the fitting curve S1 and the number of events N1 between the two points where the fitting curve intersects the horizontal axis are obtained respectively. The results are shown in Tables 1 and 2.

[0297] The fluorescence intensity distribution of the fluorescent particle group of rAAV mixture 2b was fitted with Gaussian fitting, and the results are shown in Figure 2 , and the area under the fitting curve S2 and the number of events N2 between the two points where the fitting curve intersects the horizontal axis are obtained respectively. The results are shown in Tables 1 and 2.

[0298] (i) The full shell rate was calculated according to Formula 1. The results are shown in Table 1.

[0299] Virus full shell rate (%) = ((S2 / V2)*β) / ((S1 / V1)*α)*100% ... Formula 1

[0300] in,

[0301] S1 is the area under the curve obtained by fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1c detected by the single molecule detection system within a unit detection time.

[0302] V1 is the flow volume of the mixed solution 1c passing through the single molecule detection system per unit time,

[0303] ɑ is the dilution ratio of the mixed solution 1c relative to the sample to be tested,

[0304] S2 is the area under the curve obtained by fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 2b detected by the single molecule detection system within a unit detection time.

[0305] V2 is the flow volume of the mixed solution 2b passing through the single molecule detection system per unit time,

[0306] β is the dilution factor of the mixed solution 2b relative to the sample to be tested.

[0307] Table 1: Full shell rate calculated according to formula 1

[0308]

[0309]

[0310] (ii) The full shell rate was calculated according to Formula 2. The results are shown in Table 2.

[0311] Virus full shell rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 2

[0312] in,

[0313] N1 is the number of events between the two points where the fitting curve obtained by data fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1c detected by the single molecule detection system within a unit detection time intersects with the abscissa of the fluorescence intensity distribution diagram,

[0314] V1 is the flow volume of the mixed solution 1c passing through the single molecule detection system per unit time,

[0315] ɑ is the dilution ratio of the mixed solution 1c relative to the sample to be tested,

[0316] N2 is the number of events between the two points where the fitting curve obtained by data fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 2b detected by the single molecule detection system within a unit detection time intersects with the abscissa of the fluorescence intensity distribution diagram,

[0317] V2 is the flow volume of the mixed solution 2b passing through the single molecule detection system per unit time,

[0318] β is the dilution factor of the mixed solution 2b relative to the sample to be tested.

[0319] Table 2: Full shell rate calculated according to formula 2

[0320]

[0321] Detection time: The detection time of step (3) is 2 minutes, and the total time of steps (1) to (4) is about 34 minutes.

[0322] Result analysis:

[0323] The AUC method is currently considered in this field to be the standard for evaluating rAAV capsid rate and product-specific impurity analysis, but it has problems such as long detection time and cumbersome operation.

[0324] Compared with the full shell rate detected by the AUC method, there is no significant difference in the full shell rate detected by the detection method of Example 1, indicating that the detection method of Example 1 has high accuracy. In addition, compared with the AUC method, the detection method of Example 1 has the advantages of simple operation, short time consumption, high efficiency, and small sample consumption.

[0325] Example 2: rAAV capsid fullness rate (using fluorescent antibody for capsid labeling)

[0326] Step (1):

[0327] 100 μL of the labeled rAAV test solution (same as in Example 1) was added to a centrifuge for removing dyes and nucleic acids. Free nucleic acids were removed by centrifugation, and the effluent liquid was collected to obtain a rAAV mixed solution 1 d-1.

[0328] 100 μL of rAAV mixture 1d-1 was taken, 20 μL of YF647-AAVX antibody was added, and incubated for 10 min as rAAV mixture 1d-2.

[0329] The labeled rAAV mixture 1d-2 was diluted 43.5-fold with PBS (ie, 52.2-fold diluted relative to the rAAV concentrate to be labeled) to obtain the rAAV mixture 1e (ie, diluted sample I).

[0330] Step (2):

[0331] A portion of the aforementioned rAAV mixture 1d-2 was diluted to 4.5 times with PBS buffer, and 100 μl of the diluted rAAV product was taken to obtain 2 g of rAAV mixture.

[0332] Heat 2 g of the rAAV mixture at 95°C for 10 minutes to obtain the rAAV DNA sample.

[0333] Take 10 μl of rAAV DNA sample, dilute it 10-fold with PBS buffer, add 1 μl of 100 μSYTO BC Green Fluorescent Nucleic Acid Stains aqueous solution for labeling (i.e., it is diluted 50.904-fold relative to the rAAV concentrate to be labeled), and incubate at 37°C in the dark for 5 min to obtain the intracapsid nucleic acid labeling solution, which is recorded as rAAV mixture 2b-2 (i.e., diluted sample II).

[0334] Preparation of concentration standard solution:

[0335] The concentration standard solution is the same as that used in Example 1.

[0336] Step (3): Detection

[0337] The rAAV mixture 1e, rAAV mixture 2b-2 and concentration standard solution were detected respectively using Flow NanoAnalyzer (the detection data of the concentration standard solution in Example 2 were the same as those in Example 1).

[0338] The detection parameters are: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using PC5 fluorescence channel. PC5 fluorescence channel is 647 excitation signals, used to characterize the number of events of rAAV, and analyze the The particle population with 647 positive signals was detected in the PC5 fluorescence channel and the detection results were recorded. The fluorescence of SYTO fluorescent nucleic acid fluorescent dye was detected in the FITC fluorescence channel to characterize the number of rAAV nucleic acid events. The particle population with SYTO positive signals was analyzed in the FITC fluorescence channel and the detection results were recorded.

[0339] Step (4): Data processing

[0340] Gaussian fitting was performed on the fluorescence intensity distribution of the fluorescent particle group of rAAV mixture 1e, and the results are shown in Figure 3 , and the area under the fitting curve S1 and the number of events N1 between the two points where the fitting curve intersects the horizontal axis are obtained respectively. The results are shown in Tables 3 and 4.

[0341] Gaussian fitting was performed on the fluorescence intensity distribution of the fluorescent particle population of rAAV mixture 2b-2, and the results are shown in Figure 4, and the area under the fitting curve S2 and the number of events N2 between the two points where the fitting curve intersects the horizontal axis are obtained respectively. The results are shown in Tables 3 and 4.

[0342] (i) Calculate the full shell rate according to formula 1

[0343] The full shell rate was calculated according to formula 1 and the results are shown in Table 3.

[0344] Virus full shell rate (%) = ((S2 / V2)*β) / ((S1 / V1)*α)*100% ... Formula 1

[0345] in,

[0346] S1 is the area under the curve obtained by fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1e detected by the single molecule detection system within a unit detection time.

[0347] V1 is the flow volume of the mixed solution 1e passing through the single molecule detection system per unit time,

[0348] ɑ is the dilution multiple of the mixed solution 1e relative to the sample to be tested,

[0349] S2 is the area under the curve of the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 2b-2 detected by the single molecule detection system within the unit detection time after data fitting,

[0350] V2 is the flow volume of the mixed solution 2b-2 passing through the single molecule detection system per unit time; β is the dilution factor of the mixed solution 2b-2 relative to the sample to be tested.

[0351] Table 3: Full shell rate calculated according to formula 1

[0352]

[0353] (ii) The full shell rate was calculated according to Formula 2. The results are shown in Table 3.

[0354] Virus full shell rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 2

[0355] in,

[0356] N1 is the number of events between the two points where the fitting curve obtained by data fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1e detected by the single molecule detection system within a unit detection time intersects with the abscissa of the fluorescence intensity distribution diagram.

[0357] V1 is the flow volume of the mixed solution 1e passing through the single molecule detection system per unit time,

[0358] ɑ is the dilution factor of the mixed solution 1e relative to the sample to be tested,

[0359] N2 is the number of events between the two points where the fitting curve obtained by data fitting the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 2b-2 detected by the single molecule detection system within a unit detection time intersects with the abscissa of the fluorescence intensity distribution diagram,

[0360] V2 is the flow volume of the mixed solution 2b-2 passing through the single molecule detection system per unit time,

[0361] β is the dilution factor of the mixed solution 2b-2 relative to the sample to be tested.

[0362] Table 4: Full shell rate calculated according to formula 2

[0363]

[0364] Result analysis:

[0365] (1) The results show that the number of rAAV product capsid events measured in Example 2 is not significantly different from that measured in Example 1. The method of Example 2 of the present application can mark viruses such as rAAV product capsids and accurately measure their event number.

[0366] The inventors noted that the method of Example 2 using a fluorescent antibody dye containing an antibody fragment that specifically binds to the viral capsid for labeling is simpler and faster (see Figure 2 ).

[0367] (2) Compared with the full shell rate detected by the AUC method, there is no significant difference in the full shell rate detected by the detection method of Example 2, indicating that the detection method of Example 2 has high accuracy.

[0368] In addition, compared with the AUC method, the detection method of Example 2 has the advantages of simple operation, shorter time consumption, high efficiency, and less sample consumption.

[0369] Example 3: Accuracy and repeatability test of the detection method of Example 1

[0370] Sample preparation: rAAV concentrate with known full shell rate (the same as the rAAV concentrate to be labeled as described in Example 1) and empty shell rAAV product (manufacturer: Paizhen Bio, AAV8 empty shell standard, product number: PGS08002) were mixed to form rAAV quantitative solutions with full shell rates (theoretical full shell rates) of 57.5%, 45.2%, 28.8%, 12.3% and 4.1%, respectively. The solvent of the above quantitative solutions was PBS.

[0371] The rAAV quantitative solutions with different shell filling rates were used as test samples, and were tested according to the detection method provided in Example 1 (calculated using Formula 1) and the method provided in Comparative Example 1. The samples at each concentration point were measured four times. The results are shown in Table 5.

[0372] Table 5 Accuracy and repeatability test in rAAV quantitative solution (n=4)

[0373]

[0374]

[0375] SD (Standard Deviation): A statistical indicator used to measure the degree of dispersion of data. It indicates the degree of deviation of each value in a data set from the mean of the data set.

[0376] Result analysis: The detection method provided by the present invention has high accuracy and good repeatability.

[0377] Example 4: Detection of the full shell rate of rAAV products in cell lysates

[0378] Step (1)

[0379] 1) Preparation of rAAV simulation samples:

[0380] The rAAV product with a known full capsid rate (54%) (the same rAAV concentrate to be labeled as described in Example 1, manufactured by Paizhen Bio, serotype: AAV8, product number: PGS08001, different batch number) containing 1×10 11 The rAAV8 (rAAV8) with a genome titer of 500 genomic titers / ml) was mixed with the supernatant of HEK-293T cell lysate to obtain a rAAV simulation sample with a known capsid rate (54%). The above rAAV quantitative solution (i.e., the rAAV simulation sample) was used as the test sample and tested as follows.

[0381] 2) Sample specific purification: Take 30 μl 2×10 10 SA magnetic beads with a concentration of 100 μl / ml were added to 500 μl of PBS containing 0.1 wt% BSA (bovine serum albumin), and the mixture was adsorbed on a magnetic stand for 1 min. The supernatant was discarded.

[0382] Repeat the above steps (add 500 μl of PBS containing 0.1 wt% BSA, adsorb on the magnetic stand for 1 min, and discard the supernatant) 3 times to obtain SA magnetic beads ①.

[0383] Resuspend the SA magnetic beads ① with 200 μl of PBS containing 0.2 wt% BSA, add 10 μl of Biotin-SS-AAV8 antibody, incubate with rotation mixing at 4°C for 5 min, adsorb on the magnetic stand for 1 min, discard the supernatant, and resuspend with 500 μl of PBS containing 0.2 wt% BSA.

[0384] Repeat the above steps (incubate with rotation at 4°C for 5 min, adsorb on a magnetic rack for 1 min, discard the supernatant, and resuspend in PBS containing 0.2% BSA) three times. In the first two repetitions, the volume of PBS containing 0.2 wt% BSA used for resuspension was 500 μl. In the third repetition, after discarding the supernatant, the volume of PBS containing 0.2 wt% BSA used for resuspension was 200 μl, to obtain SA magnetic bead mixture ②;

[0385] Divide SA magnetic bead mixture ② into two tubes, 100 μl each, and perform the following steps on each tube:

[0386] Take 100ul of the SA magnetic bead mixture ② and add 1ml of the above rAAV quantitative solution with known shell filling rate, rotate and mix at 4℃ for 8min, adsorb on the magnetic stand for 1min, discard the supernatant, and resuspend with 400μl PBS.

[0387] Repeat the above steps (rotating and mixing at 4°C, incubating for 8 minutes, adsorbing on a magnetic stand for 1 minute, discarding the supernatant, and resuspending with PBS) 4 times. In the first 3 repetitions, the volume of PBS used for resuspension was 400 μl. In the last repetition, after discarding the supernatant, the two tubes of SA magnetic beads were combined into one tube, and then the SA magnetic beads were resuspended with 200 μl of PBS to obtain SA magnetic beads ③.

[0388] 3) Sample capsid labeling: Take 50 μl of the SA magnetic beads ③, add 20 μl of YF647-AAV8 antibody to the SA magnetic beads ③, and incubate at room temperature for 8 minutes. Adsorb on a magnetic stand for 1 minute, discard the supernatant, and resuspend with 200 μl PBS. Repeat the above steps (incubate at room temperature for 8 minutes. Adsorb on a magnetic stand for 1 minute, discard the supernatant, and resuspend with PBS) 3 times. In the first two repetitions, the volume of PBS used for resuspension is 200 μl. In the last repetition, after discarding the supernatant, resuspend with 50 μl PBS and add 4 μl of 2.5M DTT (disulfide bond cleavage reagent). React at room temperature for 1.5 hours to obtain reaction solution A. Adsorb on a magnetic stand to obtain the supernatant of reaction solution A. Take part of the supernatant of reaction solution A and dilute it 43.5 times with PBS to obtain mixed solution 1f (i.e., diluted sample I).

[0389] Step (2)

[0390] Sample intracapsid nucleic acid labeling: A portion of the supernatant of reaction solution A was taken to replace the rAAV mixed solution 1d-2 in step (2) of Example 2. The remaining operations were the same as step (2) of Example 2 to obtain an intracapsid nucleic acid labeling solution, recorded as rAAV mixed solution 4b (i.e., diluted sample II).

[0391] Step (3)

[0392] Sample detection: The mixed solution 1f and rAAV mixed solution 4b were detected according to the detection parameters in step (4) of Example 2. The results are shown in Figure 6 and Figure 7 .

[0393] Step (4)

[0394] Calculations were performed according to Equation 1 and Equation 2, and the results are shown in Tables 6 and 7, respectively.

[0395] (i) Calculate the full shell rate according to formula 1

[0396] The full shell rate was calculated according to Formula 1 and the results are shown in Table 8.

[0397] Virus full shell rate (%) = ((S2 / V2)*β) / ((S1 / V1)*α)*100% ... Formula 1

[0398] in,

[0399] S1 is the area under the curve after data fitting of the fluorescence intensity distribution diagram of the fluorescent particle group in the mixed solution 1f detected by the single molecule detection system within a unit detection time; V1 is the flow volume of the mixed solution 1f passing through the single molecule detection system within a unit time; ɑ is the dilution factor of the mixed solution 1f relative to the sample to be tested (i.e., rAAV quantitative solution),

[0400] S2 is the area under the curve after data fitting of the fluorescence intensity distribution diagram of the fluorescent particle group in the rAAV mixture 4b detected by the single molecule detection system within a unit detection time; V2 is the flow volume of the rAAV mixture 4b passing through the single molecule detection system within a unit time; β is the dilution factor of the rAAV mixture 4b relative to the sample to be tested (i.e., the rAAV quantitative solution).

[0401] Table 6: Full shell rate calculated according to formula 1

[0402]

[0403] (ii) Calculate the full shell rate according to formula 2

[0404] The full shell rate was calculated according to Formula 2 and the results are shown in Table 9.

[0405] Virus full shell rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% Formula 2;

[0406] in,

[0407] N1 is the number of events between the two points where the fitting curve intersects the horizontal axis after data fitting of the fluorescence intensity distribution diagram of the fluorescent particle group in the supernatant of reaction solution A detected by the single molecule detection system within a unit detection time; V1 is the flow volume of the supernatant of reaction solution A passing through the single molecule detection system per unit time; ɑ is the dilution factor of the supernatant of reaction solution A relative to the sample to be tested (i.e., rAAV quantitative solution);

[0408] N2 is the number of events between the two points where the fitting curve intersects the horizontal axis after data fitting of the fluorescence intensity distribution diagram of the fluorescent particle group in the rAAV mixture 4b detected by the single-molecule detection system within a unit detection time; V2 is the flow volume of the rAAV mixture 4b passing through the single-molecule detection system within a unit time; β is the dilution factor of the rAAV mixture 4b relative to the sample to be tested (i.e., the rAAV quantitative solution).

[0409] Table 7: Full shell rate calculated according to formula 2

[0410]

[0411] Results: The detection method provided by the present invention was used to detect the rAAV capsid filling rate in cells. The results obtained were not significantly different from the theoretical filling rate, indicating high accuracy. This shows that the method of the present invention is applicable to viruses in a mixed state with cell lysate and can obtain accurate results.

[0412] Comparative Example 2: No removal of free nucleic acids and free dyes

[0413] The sample preparation and detection were carried out according to the method of Example 1, except that After 647ASE was reacted at room temperature in the dark, no spin column was used to remove free 647A SE and free nucleic acid, and the remaining procedures were the same as in Example 1. The test was repeated four times. The results are shown in Table 8.

[0414] Table 8: Test results (n=4)

[0415]

[0416] Result analysis: Without removing free dyes In the case of 647ASE and free nucleic acid, the capping rate could not be detected.

[0417] In summary, according to the results of the above embodiments and comparative examples, the detection method provided by the present invention has good accuracy and repeatability, is easy to operate, saves time, requires a small amount of sample, has high detection efficiency, avoids overestimation of the full shell rate, and has excellent and unexpected technical effects.

[0418] The present application has been described above in conjunction with preferred embodiments, but these embodiments are merely exemplary and serve only as an illustrative example. On this basis, various replacements and improvements can be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A method for detecting the full capsid rate of a virus, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, the method comprising: (1) labeling the capsid of the virus in the sample containing the virus with a first dye, wherein the first dye specifically binds to the surface recognition molecule of the capsid and emits fluorescence; (2) destroying the viral capsid, and then labeling the nucleic acid molecule contained in the viral capsid with a second dye, wherein the second dye can specifically bind to the nucleic acid molecule and emit fluorescence; (3) detecting the positive fluorescence signal after labeling with the first dye to obtain a fluorescence intensity distribution graph A, and detecting the positive fluorescence signal after labeling with the second dye to obtain a fluorescence intensity distribution graph B; (4) Perform data fitting on the fluorescence intensity distribution graph A and the fluorescence intensity distribution graph B to obtain fitting curve A f and fitting curve B f , according to the fitting curve A f and fitting curve B f The area under the curve, or the fitted curve A f and fitting curve B f The virus filling rate was calculated based on the number of events between two points that intersected the abscissa of the corresponding fluorescence intensity distribution graph.

2. The method according to claim 1, wherein the method comprises: In step (1), after or before labeling the viral capsid in the sample with the first dye, the sample is diluted to a detection working concentration at a dilution factor α relative to the sample to obtain a labeled diluted sample I, wherein α is greater than or equal to 1; and In step (2), after or before labeling the nucleic acid molecule with a second dye, the sample is diluted to a detection working concentration at a dilution factor β relative to the sample to obtain a labeled diluted sample II, wherein β is greater than or equal to 1.

3. The method according to claim 1 or 2, wherein in step (3), detecting the fluorescent signal that is positive after dye labeling includes detecting the fluorescent particle group in the flow volume V of the diluted sample passing through the single molecule detection system within a unit detection time through a single molecule detection system.

4. The method according to claim 3, wherein the single molecule detection system comprises an optical system, a photodetector and a signal acquisition and analysis system, The optical system comprises at least one fluorescence channel; the photodetector is used to detect the fluorescence signal in the sample and convert the fluorescence signal into an electrical signal; the signal acquisition and analysis system is capable of acquiring and analyzing the electrical signal.

5. The method according to claim 3, further comprising: The virus full shell rate is calculated by formula 1, Virus full shell rate (%) = ((S2 / V2)*β) / ((S1 / V1)*α)*100%…………Formula 1 Among them, S1 is the fitting curve A f The area under the curve is , V1 is the flow volume of the diluted sample I passing through the single molecule detection system per unit time; S2 is the fitting curve B f V2 is the flow volume of the diluted sample II passing through the single molecule detection system per unit time.

6. The method according to claim 3, comprising: The virus full shell rate is calculated by formula 2, Virus full shell rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%………… Formula 2 Where N1 is the fitting curve A f The number of events between the two points that intersect the abscissa of the corresponding fluorescence intensity distribution graph, N2 is the number of events between the two points that intersect the abscissa of the corresponding fluorescence intensity distribution graph, and ... f the number of events between two points that intersect the abscissa of the corresponding fluorescence intensity distribution graph; V1 is the flow volume of the diluted sample I passing through the single molecule detection system per unit time; V2 is the flow volume of the diluted sample II passing through the single molecule detection system per unit time.

7. The method according to any one of claims 1 to 6, comprising one or more of the following steps: a) purifying the sample containing the virus to remove impurities or free nucleic acid before or after labeling with the first dye; b) removing free dye and free nucleic acid from the sample containing the virus after labeling with the first dye; c) purifying the virus-containing sample to remove impurities before labeling with the first dye, and removing free dye and free nucleic acid from the virus-containing sample after labeling with the first dye; and d) purifying the sample containing the virus to remove impurities before or after labeling with the second dye; wherein the impurities are selected from cell debris, floating cells, extracellular vesicles, miscellaneous proteins, lipids, salts and combinations thereof, Preferably, the method comprises step a) and step b).

8. The method according to any one of claims 1 to 7, wherein The virus is selected from adeno-associated virus, lentivirus, adenovirus, and poxvirus, and preferably the virus is adeno-associated virus.

9. The method according to any one of claims 1 to 8, wherein The first dye is directly conjugated with the surface recognition molecule to emit fluorescence; or the first dye has a recognition group and is indirectly conjugated with the surface recognition molecule to emit fluorescence, The recognition group specifically binds to the surface recognition molecule, Preferably, the recognition group is selected from one or more of an antibody or antibody fragment, an antigen, a ligand, a ligand receptor or a polysaccharide; more preferably, the recognition group is an antibody or an antibody fragment; further preferably, the recognition group is an antibody or an antibody fragment that specifically binds to the surface recognition molecule of the adeno-associated virus, Further preferably, the first dye consists of a recognition group and a fluorescent dye.

10. The method according to any one of claims 1 to 9, wherein The second dye is a nucleic acid fluorescent dye, Preferably, the second dye is one or a combination of two or more selected from cyanine dyes, non-permeable dyes, permeable dyes, intercalating dyes, and DNA double helix minor groove binding dyes.

11. The method according to any one of claims 1 to 10, wherein The method for destroying the capsid is selected from the nucleic acid extraction reagent method or the thermal lysis method.

12. The method according to claim 11, wherein The heating temperature of the thermal cracking method is 50°C-130°C. Preferably, the heating temperature is selected from 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C or 130°C. The heating time of the thermal cracking method is 1 minute to 60 minutes, preferably, the heating time of the thermal cracking method is selected from 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

13. The method according to claim 11, wherein the nucleic acid extraction reagent method comprises a method based on one or a combination of two or more of a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid phase matrix.

14. The method according to any one of claims 7 to 13, wherein: In different steps, the method for removing free dyes and free nucleic acids is independently selected from: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, dilution method, or a combination of two or more thereof; The method for removing free nucleic acids is preferably selected from the group consisting of: enzymatic digestion and separation column separation, more preferably separation column separation.

15. The method according to any one of claims 7 to 14, wherein: In different steps, the purification is independently selected from: magnetic separation, centrifugation, delamination, precipitation, solid-liquid separation, or a combination of two or more thereof; preferably, magnetic separation.

16. The method according to claim 15, wherein The magnetic separation method is selected from: magnetic bead separation method and solid phase chip separation method, or a combination of two or more thereof. The magnetic bead separation method includes a surface-loaded functional group magnetic bead separation method; The surface-loaded functional group magnetic bead separation method is selected from: antigen-antibody magnetic bead separation method, ligand-receptor magnetic bead separation method, enzyme-substrate magnetic bead separation method, protein-inhibitor magnetic bead separation method, electron donor-electron acceptor magnetic bead separation method or structural complementary magnetic bead separation method, or a combination of two or more thereof.

17. The method according to claim 16, wherein The antigen-antibody magnetic bead separation method comprises: After mixing the magnetic beads carrying virus antibodies with a sample containing the virus, the magnetic beads carrying virus antibodies are adsorbed and separated by an external magnetic field; or After the viral antibodies are mixed with a sample containing the virus, modified magnetic beads that can bind to the viral antibodies are added, and the modified magnetic beads are adsorbed and separated by an external magnetic field.

18. The method according to any one of claims 1 to 17, wherein the data fitting adopts a fitting method suitable for normally distributed data or a Gaussian fitting method.

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