Method for determining intact capsid content in adeno-associated viruses

Abstract

The present invention relates to a method of determining the percentage of intact capsid in an adeno-associated virus (AAV) sample comprising the AAV. The invention is characterized by two basic steps. The first step involves dividing each sample into two equally divided samples. One aliquot is maintained at room temperature (RT) while the other aliquot is heated below the AAV melting point to disrupt the binding affinity of the AAV to the anti-AAV coated probe. Heating the AAV aliquot results in different binding affinity of the empty capsid and the intact capsid on the anti-AAV coated probe. The heated empty capsids retain their binding affinity for the probe, while the heated complete capsids significantly reduce their binding affinity for the probe. The second step involves measuring the wavelength shifts of the heated and RT aliquot samples due to light interference and calculating their ratios. The amount of hollow capsid and intact capsid of an unknown sample can be determined by quantifying the ratio with reference to a standard curve.

Description

Technical Field

[0001] The present invention discloses an immunoassay capable of distinguishing empty viral capsids from complete viral capsids in adeno-associated viruses based on interferometry. Background Art

[0002] Adeno-associated viruses (AAV) have become the vector of choice for gene therapy clinical trials because they are expressed for a long time in humans and are not pathogenic. To date, 12 AAV serotypes have been identified, of which AAV2 is the best characterized and most commonly used serotype for gene therapy applications. Wild-type AAV consists of a single-stranded DNA genome (up to 4.8 kb in size) that is encapsidated within a protein coat consisting of 60 viral proteins (VPs). A mixture of VP1, VP2, and VP3 in an approximate molar ratio of 1:1:10 is organized with icosahedral symmetry to form the viral capsid shell. The VP3 sequence is common to all VPs and is 57 amino acids longer than VP2. Compared to VP2, VP1 is further extended at the N-terminus, with the VP1 unique (VP1u) region containing the phospholipase A2 (PLA2) domain required for infectivity.

[0003] Differences in amino acid sequence and capsid structure mediate the interaction of AAV serotypes with different host cell receptors, resulting in alternative cell or tissue tropism. For example, AAV2 has tropism for skeletal muscle, neurons, and vascular smooth muscle cells, while AAV8 has tropism for the liver. Another important consequence of the differences in capsid structure between the two AAV serotypes is the stability of the capsid structure to thermal and pH changes. For example, AAV5 has the highest melting temperature (Tm) determined by differential fluorescence scanning, while AAV2 has the lowest Tm. This unique property has been used to distinguish AAV serotypes from each other.

[0004] AAV capsids that contain a full-length therapeutic transgene are called intact capsids. The first FDA-approved AAV-related gene therapy is Luxturna, an AAV2 vector. An inherent feature of the AAV manufacturing process is the production of capsids that are not packaged with a therapeutic transgene; such capsids are called empty capsids. Typically, up to 95% of the capsids produced upstream of cell culture may be empty capsids, and this percentage has been shown to vary significantly between independent vector preparations.

[0005] Table 1 shows the main types of capsids generated during recombinant AAV production. (Gimpel et al., Molecular Therapy: Methods & Clinical Development, 20: 740-754, 2021)

[0006] Table 1 .

[0007]

[0008] *Ratios for all capsids are given at the time the rAAV production culture is harvested ("harvest"), i.e. in the cell lysate before any purification, and in the purified product ("purified"), i.e. after purification of intact capsids from the cell lysate but before any polishing steps for almost complete separation of intact capsids from empty capsids.

[0009] The clinical efficacy of these empty capsids is not yet fully understood, but it has been suggested that there may be an enhanced immune response to high concentrations of viral particles. Empty capsids do not provide the expected therapeutic benefit and are therefore considered product-related impurities. Different analytical techniques exist to quantify the amount of empty AAV capsids versus intact AAV capsids throughout the manufacturing process and in the final product.

[0010] Existing methods are usually low-throughput and require concentrated (10 12 Virus particles / mL-10 14 Virus particles / mL) purified samples or long assay times. In the development and manufacture of therapeutic AAV vectors, quantification of AAV titers and determination of AAV empty and intact capsids remain challenging. A high-throughput analytical method is needed that can quantify the amount of empty AAV capsids versus intact AAV capsids in a short assay time and allows the use of crude samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A A biosensor interferometer system is shown. Figure 1B A biosensor interferometer probe is depicted. Figure 1C Another biosensor interferometer probe is depicted.

[0012] Figure 2A It is shown that the first reflected light signal and the second reflected light signal form a spectral interference pattern. Figure 2B The spectral interference pattern is shown shifting from T0 to T1 when the analyte molecule binds to the analyte binding molecule on the interfering distal surface.

[0013] Figure 3 Shown are the melting points of intact and empty capsids of AAV1-AAV10 in different buffers.

[0014] Figure 4 The V value (ratio of wavelength shift of heated sample / wavelength shift of room temperature sample) is plotted versus the % of intact AAV2 capsid. DETAILED DESCRIPTION

[0015] definition

[0016] The terms used in the claims and specification should be interpreted according to their ordinary meanings as understood by those skilled in the art, except for the definitions set forth below.

[0017] As used herein, "about" means within ±10% of the stated value.

[0018] As used herein, an "analyte binding" molecule refers to any molecule that is capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to: (i) an antigen molecule, used to detect the presence of an antibody specific for the antigen; (ii) an antibody molecule, used to detect the presence of an antigen; (iii) a protein molecule, used to detect the presence of a binding partner for the protein; (iv) a ligand, used to detect the presence of a binding partner; or (v) a single-stranded nucleic acid molecule, used to detect the presence of a nucleic acid binding molecule.

[0019] The "aspect ratio" of a shape refers to the ratio of its longer dimension to its shorter dimension.

[0020] "Binding molecule" refers to a molecule that is capable of binding to another molecule of interest.

[0021] As used herein, "binding pair" refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but are not limited to, antigens and antibodies to antigens, ligands and their receptors, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectins and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, digoxin / anti-digoxin.

[0022] As used herein, "empty AAV" and "complete AAV" refer to the DNA content within the AAV protein capsid. Empty AAV means that the viral capsid does not contain DNA. Complete AAV means that the viral capsid contains a full-length single-stranded DNA genome. An X% complete AAV sample means that X% of the capsids have a full-length single-stranded DNA genome. For example, a 60% complete AAV sample means that 60% of the capsids have a full-length single-stranded DNA genome and 40% of the capsids do not have a full-length single-stranded DNA genome.

[0023] As used herein, "immobilization" refers to an agent being fixed to a solid surface. When an agent is immobilized to a solid surface, it is either non-covalently bound or covalently bound to the surface.

[0024] As used herein, "monolithic substrate" refers to a single piece of solid material having one refractive index, such as glass, quartz, or plastic.

[0025] As used herein, "probe" refers to a monolithic substrate having an aspect ratio (length to width) of at least 2 to 1 and coated with a thin film layer on the sensing side. The probe has a distal end and a proximal end. The proximal end (also referred to as the probe tip in this application) has a sensing surface coated with a thin layer of analyte binding molecules.

[0026] "Waveguide" refers to a device (eg, a conduit, coaxial cable, or optical fiber) designed to confine and direct the propagation of electromagnetic waves (eg, light).

[0027] The present invention provides a method for determining the percentage of complete capsids of AAV in a sample containing adeno-associated virus (AAV). The present invention is characterized by two basic steps. The first step involves dividing each sample into two aliquots. One aliquot is kept at room temperature (RT) while the other aliquot is heated to destroy the binding affinity of AAV to an anti-AAV coated probe. Without heating, there is almost no difference in the binding affinity of empty AAV capsids or complete AAV capsids on an anti-AAV coated glass biosensor probe. However, heating the AAV aliquots results in different binding affinities of empty capsids and complete capsids on the anti-AAV coated probe. The heated empty capsids retain their binding affinity to the probe, while the heated complete capsids significantly reduce their binding affinity to the probe. The second step involves measuring the wavelength shift caused by light interference of the heated and RT aliquots, and calculating their ratio. By quantifying the ratio against a standard curve, the amount of empty capsids and complete capsids in an unknown sample can be determined.

[0028] Biosensor Interferometer System

[0029] The present invention is applicable to biosensor interferometer systems. Figure 1A to Figure 1B An example of such a system is illustrated. Figure 1A Depicted is a biosensor interferometer 100 (or simply "interferometer") that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a "probe"). The probe 108 may be connected to the waveguide 106 via a coupling medium.

[0030] The light source 102 can emit white light that is guided by the waveguide 106 toward the probe 108. For example, the light source 102 can be a light emitting diode (LED) configured to generate light within a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a specified spectrum (e.g., 400 nm or less to 700 nm or greater). Alternatively, the interferometer 100 can employ multiple light sources with different characteristic wavelengths, such as LEDs designed to emit light of different wavelengths in the visible range. The same functionality can be achieved with a single light source having appropriate filters for directing light of different wavelengths onto the probe 108.

[0031] The detector 104 is preferably a spectrometer, such as the Ocean Optics USB4000, which is capable of recording the spectrum of the interfering light received from the probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, the detector 104 can be a simple photodetector capable of recording the intensity at each wavelength. In another embodiment, the detector 104 can include multiple filters that allow detection of the intensity at each of the multiple wavelengths.

[0032] The waveguide 106 can be configured to transmit light emitted by the light source 102 to the probe 108, and then transmit light reflected by surfaces within the probe 108 to the detector 104. In some embodiments, the waveguide 106 is a bundle of optical fibers (e.g., a single-mode fiber optic cable), while in other embodiments, the waveguide 106 is a multi-mode fiber optic cable.

[0033] like Figure 1B As shown, the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as an "interference layer"), and a biomolecule layer (also referred to as a "biolayer") composed of analyte molecules 122, which are bound to analyte binding molecules 120. The monolithic substrate 114 is composed of a transparent material through which light can pass. The interference layer is also composed of a transparent material. When light is irradiated on the probe 108, the proximal surface of the interference layer can act as a first reflective surface, and the biolayer can act as a second reflective surface. As further described below, the light reflected by the first reflective surface and the second reflective surface can form an interference pattern that can be monitored by the interferometer 100.

[0034] The interference layer typically includes multiple layers combined in a manner that improves the detectability of the interference pattern. For example, the interference layer is composed of a tantalum pentoxide (Ta2O5) layer 116 and a silicon dioxide (SiO2) layer 118. The tantalum pentoxide layer 116 can be thin (e.g., about 10nm to 40nm) because its main purpose is to improve the reflectivity at the near-side surface of the interference layer. At the same time, the silicon dioxide layer 118 can be relatively thick (e.g., about 650nm to 900nm) because its main purpose is to increase the distance between the first reflective surface and the second reflective surface.

[0035] To illustrate a simple interferometry test, a probe 108 can be suspended in a well 110 that includes a sample 112. During a diagnostic test, analyte molecules 122 will bind to analyte binding molecules 120 along the distal end of the probe 108, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biological layer formed along the distal end of the probe 108 by detecting a shift in the phase characteristics of the interference pattern.

[0036] Figure 1CAnother biosensor interferometer probe is shown. The probe includes a monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and an analyte binding molecule layer coated on the interference layer, wherein the monolithic substrate has a first surface and a second surface arranged substantially parallel to each other at opposite ends of the monolithic substrate. The interference layer will usually be composed of magnesium fluoride (MgF2). When light is irradiated on the interference measurement sensor, the first interface between the monolithic substrate and the interference layer acts as a first reflection surface, and when light is irradiated on the probe, the second interface between the biological layer formed by the combination of the analyte molecules in the sample with the analyte binding molecules and the solution containing the sample acts as a second reflection surface. As described above, the thickness of the biological layer can be estimated based on the interference pattern of the light reflected by the first reflection surface and the second reflection surface.

[0037] The probe 200 includes an interference layer 204 fixed along the distal end of the monolithic substrate 202. Analyte binding molecules 206 can be deposited along the distal surface of the interference layer 204. During the biochemical test process, as analyte molecules 208 in the sample bind to the analyte binding molecules 206, a biolayer will be formed.

[0038] like Figure 1C As shown, the monolithic substrate 202 has a proximal surface (also referred to as the "coupling side") that can be coupled to a waveguide of, for example, an interferometer, and a distal surface (also referred to as the "sensing side") on which additional layers are deposited. Typically, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length to width) of the monolithic substrate 202 is at least 5 to 1. In such embodiments, the monolithic substrate 202 can be said to have a cylindrical form. The cross-section of the monolithic substrate 202 can be circular, elliptical, square, rectangular, triangular, pentagonal, etc. The monolithic substrate 202 preferably has a refractive index that is substantially higher than the refractive index of the interference layer 204, so that the proximal surface of the interference layer 204 effectively reflects light directed onto the probe 200. The preferred refractive index of the monolithic substrate can be higher than 1.5, 1.8, or 2.0. Thus, the monolithic substrate 202 may be composed of a high refractive index material such as glass (refractive index 2.0) rather than a low refractive index material such as quartz (refractive index 1.46) or plastic (refractive index 1.32 to 1.49).

[0039] The interference layer 204 is composed of at least one transparent material, which is coated on the distal surface of the monolithic substrate 202. These transparent materials are deposited on the distal surface of the monolithic substrate 202 in the form of a thin film with a thickness ranging from a fraction of a nanometer (e.g., a monolayer) to several microns. The interference layer 204 can have a thickness of at least 500nm, 700nm, or 900nm. Exemplary thicknesses are between 500nm and 5,000nm (and preferably between 800nm ​​and 1,200nm). In this article, for example, the interference layer 204 has a thickness of approximately 900nm to 1,000nm or 940nm.

[0040] Compared to conventional probes, the interference layer 204 has a refractive index substantially similar to that of the biological layer. This ensures that the reflection from the distal end of the probe 200 is primarily due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte binding molecules 206. In some embodiments, the interference layer 204 is composed of magnesium fluoride (MgF2), while in other embodiments, the interference layer 204 is composed of potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlF6), sodium aluminum fluoride (Na3A1F6), strontium fluoride (SrF2), aluminum fluoride (AlF3), sulfur hexafluoride (SF6), etc. Magnesium fluoride has a refractive index of 1.38, which is substantially the same as the refractive index of the biological layer formed along the distal end of the probe 200. For comparison, the interference layer of a conventional probe is typically composed of silicon dioxide, and the refractive index of silicon dioxide is about 1.4 to 1.5 in the visible light range. Because the interference layer 204 and the biological layer have similar refractive indices, the light will experience minimal scattering as it travels from the interference layer 204 into the biological layer and then from the biological layer back into the interference layer 204 .

[0041] In one embodiment, the probe 200 includes an adhesion layer that is deposited along the distal surface of the interference layer 204 fixed to the monolithic substrate 202. The adhesion layer can be composed of a material that promotes the adhesion of the analyte binding molecules 206. An example of such a material is silicon dioxide. Compared with the interference layer 204, the adhesion layer is usually very thin, so its effect on the light traveling toward or returning from the biological layer will be minimal. For example, the adhesion layer 310 can have a thickness of about 3nm to 10nm, while the interference layer 304 can have a thickness of about 800nm ​​to 1,000nm. The biological layer formed by the analyte binding molecules 306 and the analyte molecules 308 will usually have a thickness of several nm.

[0042] When light is irradiated on probe 200, the near side surface of interference layer 204 can act as a first reflective surface, and the far side surface of biological layer can act as a second reflective surface. The presence, concentration or binding rate of analyte molecule 208 and probe 200 can be estimated based on the interference of the light beam reflected by these two reflective surfaces. When analyte molecule 208 is attached to (or detached from) analyte binding molecule 206, the distance between the first reflective surface and the second reflective surface will change. Since the size of all other components in probe 200 remains the same, the interference pattern formed by the light reflected by the first reflective surface and the second reflective surface is phase-shifted according to the change of the biological layer thickness caused by the binding event.

[0043] In operation, an incident light signal 210 emitted by a light source is transmitted toward the biological layer through the monolithic substrate 202. Within the probe 200, the light will be reflected at the first reflective surface, thereby generating a first reflected light signal 212. The light will also be reflected at the second reflective surface, thereby generating a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte binding molecules 206 and the sample in which the probe 200 is immersed. When binding occurs during a biochemical test, the second reflective surface becomes the interface between the analyte molecules 208 and the sample.

[0044] The first reflected light signal and the second reflected light signal form a spectral interference pattern, such as Figure 2A When the analyte molecules bind to the analyte binding molecules on the far side surface of the interference layer, the optical path of the second reflected light signal will become longer. Figure 2B As shown, the spectral interference pattern shifts from T0 to T1. By measuring the phase shift after a period of time, the association rate of the analyte molecule with the analyte binding molecule fixed on the far side surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. By measuring the phase shift continuously in real time, a kinetic binding curve can be drawn with the offset versus time. The association rate of the analyte molecule with the analyte binding molecule fixed on the far side surface of the interference layer can be used to calculate the analyte concentration in the sample. Therefore, the measurement of phase shift is the detection principle of thin film interferometer.

[0045] Immunoassay

[0046] The present invention relates to a method for determining the percentage of intact capsids of adeno-associated virus (AAV) in a sample containing AAV. The method comprises the following steps: (a) obtaining a first probe and a second probe, the first probe and the second probe having the same fixed amount of the same anti-AAV antibody fixed on the tip of the probe; (b) heating an aliquot of the sample at 2°C-18°C below the melting temperature of AAV; (c) immersing the first probe tip in the heated aliquot of (b) for a first time period under defined binding conditions to capture AAV on the probe, and measuring a first wavelength shift due to light interference; (d) immersing the second probe tip in an unheated aliquot from the sample for a second time period under the same defined binding conditions as in step (c) to capture AAV on the second probe, and measuring a second wavelength shift for the second time period due to light interference; (e) determining the ratio of the first wavelength shift relative to the second wavelength, and quantifying the ratio against a calibration curve to determine the percentage of intact capsids of AAV in the sample.

[0047] In step (a) of the present method, two probes having a small tip for binding an analyte are obtained. The two probes have the same anti-AAV antibody immobilized in the same amount on the tip of the probe, so that they have the same ability and capacity to capture AAV.

[0048] In one embodiment, the tip has a small surface area, with a diameter of ≤5 mm, preferably <2 mm or ≤1 mm. A probe with a small surface area is advantageous because it has less non-specific binding and therefore produces a lower background signal.

[0049] Methods for immobilizing proteins (such as antibodies) to a solid phase (sensing surface of the probe tip) are known in immunochemistry. The protein can be directly bound to the solid phase by adsorption, or it can be indirectly bound to the solid phase via a binding pair. For example, the probe surface can be coated with a first member of the binding pair (e.g., an anti-hapten), and an anti-AAV antibody labeled with a second member of the binding pair (e.g., a hapten) is immobilized on the probe via biotin-streptavidin binding.

[0050] In step (b) of the method, an aliquot of the sample is removed from the sample and heated at about 2°C to 18°C ​​below the melting temperature of AAV for about 2 minutes to up to 3 hours.

[0051] The inventors have found that heating the complete AAV capsid results in a significant decrease in the binding affinity of the complete AAV capsid to the anti-AAV coated probe. In contrast, heating the empty AAV capsid does not result in a significant decrease in the binding affinity of the empty AAV capsid to the anti-AAV coated probe. Typically, heating the complete AAV2 capsid at 50°C-65°C in PBS buffer for 30 minutes results in a decrease in the binding affinity of the complete AAV capsid to the anti-AAV coated probe to 15%-35% compared to the unheated complete AAV2 capsid. Typically, heating the empty AAV2 capsid at 50°C-65°C in PBS buffer for 30 minutes results in a change in the binding affinity of the empty AAV capsid to the anti-AAV coated probe to 75%-90% relative to the unheated sample.

[0052] The heating is performed at a temperature about 2°C-18°C or about 5°C-12°C below the melting temperature of AAV. Figure 3 The melting points of AAV1-AAV9 and AAVrh.10 (complete capsid and empty capsid) in different buffers are shown in (Bennett et al., Molecular Therapy: Methods & Clinical Development, 6: 171-182 (2017))

[0053] For example, when the AAV sample is in PBS buffer, the heating temperature range is about 65°C-80°C for AAV1, about 50°C-65°C for AAV2, about 55°C-70°C for AAV3, about 55°C-72°C for AAV4, about 70°C-85°C for AAV5, about 60°C-75°C for AAV6, about 60°C-75°C for AAV7, about 55°C-70°C for AAV8, about 60°C-75°C for AAV9, and about 60°C-75°C for AAV10.

[0054] The AAV aliquot is heated to change the binding affinity of the intact AAV capsid to the anti-AAV immobilized on the probe. The heating time is 2 minutes to 5 minutes to 1 hour to 3 hours, such as 2 minutes to 1 hour, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 1 hour, or 10 minutes to 30 minutes, or 20 minutes to 40 minutes, depending on the AAV type and AAV concentration.

[0055] In step (c), the first probe tip is immersed in the heated aliquot of (b) for a first period of time under defined binding conditions to capture the AAV on the probe, and a first wavelength shift due to optical interference is measured.

[0056] In step (d), the second probe tip is immersed in an unheated aliquot from the same sample for a second period of time under the same defined binding conditions as in step (c) to capture AAV on the second probe, and a second wavelength shift due to optical interference is measured.

[0057] Unheated aliquots are kept at room temperature (eg, 18°C-40°C) until ready for testing.

[0058] The binding conditions in steps (c) and (d) are the same, and the first time period and the second time period of binding in steps (c) and (d) are the same; and therefore, the difference (if any) in the amount of AAV capsids bound to the anti-AAV coated probe in the heated aliquot and the unheated aliquot is only due to the heating versus non-heating procedures.

[0059] The wavelength shift in steps (c) and (d) can be measured by a kinetic approach or an endpoint approach. Kinetic measurement refers to measuring the change in wavelength shift relative to time, usually in nanometers of wavelength shift per second. Endpoint measurement refers to measuring the wavelength change after a certain period of time, such as nanometers of wavelength shift after 2 minutes to 60 minutes or 5 minutes to 40 minutes.

[0060] In step (e), the ratio of the first wavelength shift relative to the second wavelength shift is determined. The ratio is then quantified against a pre-established calibration curve having the wavelength shift plotted relative to the percentage of intact capsids of AAV in the sample to determine the percentage of intact capsids of AAV in the sample. The calibration curve was pre-established using a probe coated with the same amount of the same anti-AAV antibody under the same heating and binding conditions.

[0061] The present invention is a facile and accurate method for determining the percentage of intact capsids of adeno-associated virus (AAV) in a sample containing AAV.

[0062] The present invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention to the specific procedures described therein.

[0063] Example

[0064] instrument

[0065] GatorPrime and GatorPro systems (Gator Bio, Inc.) were used for BLI measurements. Samples were heated up to 60°C for 30 minutes using the ProFlex PCR system (Invitrogen).

[0066] Example 1. Preparation of reagents and anti-AAV2 probes for immunoassay

[0067] A quartz probe with a diameter of 1 mm and a length of 2 cm with a BLI optical layer at the distal tip was coated with aminopropylsilane using a chemical vapor deposition method (Yield Engineering Systems, 1224P) according to the manufacturer's protocol. Figure 1B and Figure 1C The BLI probe is shown. The probe tip was then functionalized with 1 mg / mL bis(sulfosuccinimidyl) suberate (ThermoFisher Scientific 21580) in 10 mM sodium phosphate buffer at pH 3.8 for 2 minutes. 50 μg / mL of native streptavidin (IBA) was then fixed in 10 mM sodium phosphate buffer at pH 3.8 for 10 minutes. Finally, a biotinylated anti-AAV2 antibody (Genscript A02160) diluted to 10 μg / mL in PBS buffer at pH 7.4 (Sigma-Aldrich P3813) was fixed on the probe for 10 minutes. The probe was washed with PBS and then immersed in a 15% sucrose solution and then dried in a 40°C oven for 20 minutes.

[0068] Example 2. Wavelength shift ratio of room temperature sample and heated sample

[0069] 0% intact (empty) or 100% intact (intact) AAV2 capsids (Sirion Biotech) were diluted to 9.0 × 10 in Q buffer (PBS with 0.2% BSA, 0.02% Tween-20, and 0.02% Proclin-30). 10 vp / mL, 3.0×10 10 vp / mL and 1.0×10 10 vp / mL. Half of the diluted capsids were kept at room temperature (RT) and the other half was heated at 60°C for 30 min.

[0070] Then, 100 μL of each RT sample and each heated sample was pipetted into a 384-well black plate (Greiner) in which anti-AAV2 coated probes were inserted and run on a GatorPlus instrument to determine their wavelength shift according to the protocol in Table 2 below.

[0071] Table 2 .

[0072] Step Number reaction sample Time (seconds) Speed ​​(RPM) 1 Baseline Run Q Buffer 60 1000 2 AAV2 binding AAV2 capsids in Q buffer 1800 1000

[0073] At the end of the run, each nanometer (NM) shift was determined and the V value (ratio of NM shift at 60°C / NM shift at RT) was calculated. The results are shown in Table 3.

[0074] Table 3 .

[0075]

[0076] The results in Table 3 show that the V values ​​of empty capsids remain relatively constant around 1 (1.01-1.07) using 9.0E10, 3.0E10 and 1.0E10 vp / mL capsids. For intact capsids, the V values ​​also remain relatively constant (0.31-0.38), regardless of the input capsid concentration.

[0077] Example 3. Standard curve and test samples

[0078] Standards of AAV2 samples with 0%, 15%, 30%, 45%, 60%, 75%, 100% intact capsids were prepared by mixing various amounts of 0% intact (empty) and 100% intact (intact) capsids. The test samples in this example were prepared by mixing equal amounts of 0% intact (empty) and 100% intact (intact) capsids to produce a 50% intact sample.

[0079] AAV2 capsid (Sirion Biotech) was diluted to 1.8E10 vp / mL in Q buffer (PBS with 0.2% BSA, 0.02% Tween-20 and 0.02% Proclin-30). For each standard and test sample, half of the sample was kept at room temperature and the other half was heated at 60°C for 30 minutes. Then, 100 μL of each RT sample and each heated sample was pipetted into a 384-well black plate (Greiner) with anti-AAV2 coated probes inserted and run on a GatorPlus instrument to determine their wavelength shift according to the same protocol of Table 1.

[0080] At the end of the run, each nanometer (NM) shift was determined and the V value was calculated (ratio of NM shift at 60°C / NM shift at RT). The results are shown in Table 4 and plotted to generate the following table: Figure 4 Standard curve shown. For all capsid content percentages tested (0%, 15%, 30%, 45%, 60%, 75% and 100% intact), the %CV was less than 20%.

[0081] Table 4. V value

[0082] %whole Rep 1 gep 2 Rep 3 Ave CV 0 0.967 0.842 1.07 0.961 12％ 15 0.822 0.677 0.78 0.759 10％ 30 0.636 0.529 0.56 0.574 10％ 45 0.486 0.441 0.44 0.455 6％ 60 0.433 0.403 0.39 0.408 6％ 75 3.373 0.329 0.35 3.349 6％ 100 0.213 0.266 0.20 0.225 16％

[0083] The V value of the test sample (50% intact) was quantified against the standard curve ( Figure 4 ) to determine its concentration. The results are shown in Table 5.

[0084] Table 5. Quantification of test samples

[0085] Rep 1 Rep 2 Rep 3 Ave Actual (% complete) 50.0 50.0 50.0 50.0 Calculation (% complete) 52.1 42.0 64.6 52.9 Difference (% complete) 2.1 8.0 14.6 8.2

[0086] Table 5 shows that the average difference between the calculated capsid content and the known capsid content in three independent experiments was 8.2%.

[0087] The invention and the manner and process of making and using the same are now described in full, clear, concise and precise terms to enable any person skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. In order to particularly point out and distinctly claim the subject matter regarded as inventive, the specification ends with the following claims.

Claims

1. A method for determining the percentage of intact capsids of an adeno-associated virus (AAV) in a sample comprising the AAV, the method comprising the following steps: (a) obtaining a first probe and a second probe, wherein the first probe and the second probe have the same immobilized amount of the same anti-AAV antibody immobilized on the tips of the probes; (b) heating an aliquot of the sample at 2°C-18°C below the melting temperature of the AAV; (c) immersing the first probe tip in the heated aliquot of (b) under binding conditions for a period of time to capture the AAV on the probe, and measuring a first wavelength shift due to optical interference; (d) immersing the second probe tip in an unheated aliquot from the sample for the same period of time under the same binding conditions as in step (c) to capture AAV on the second probe, and measuring a second wavelength shift for the second period of time due to optical interference; as well as (e) determining a ratio of the first wavelength shift relative to the second wavelength and quantifying the ratio against a calibration curve to determine the percentage of intact capsids of AAV in the sample.

2. The method of claim 1, wherein the AAV is AAV2.

3. The method of claim 2, wherein the aliquot of (b) is heated at 50°C-65°C for 5 minutes to 1 hour.

4. The method of claim 1, wherein the AAV is AAV1 and the aliquot of (b) is heated at 65°C-80°C.

5. The method of claim 1, wherein the AAV is AAV3 and the aliquot of (b) is heated at 55°C-70°C.

6. The method of claim 1, wherein the AAV is AAV4 and the aliquot of (b) is heated at 55°C-72°C.

7. The method of claim 1, wherein the AAV is AAV5 and the aliquot of (b) is heated at 70°C-85°C.

8. The method of claim 1, wherein the AAV is AAV6 and the aliquot of (b) is heated at 60°C-75°C.

9. The method of claim 1, wherein the AAV is AAV7 and the aliquot of (b) is heated at 60°C-75°C.

10. The method of claim 1, wherein the AAV is AAV8 and the aliquot of (b) is heated at 55°C-70°C.

11. The method of claim 1, wherein the AAV is AAV9 and the aliquot of (b) is heated at 60°C-75°C.

12. The method of claim 1, wherein the AAV is AAV10 and the aliquot of (b) is heated at 60°C-75°C.

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