Methods and kits for detecting adeno-associated virus

Abstract

The present disclosure provides methods and kits for detecting adeno-associated virus. In some embodiments, the adeno-associated virus is Anc80. Detection methods include HPLC and ELISA-based methods.

Description

[Technical Field]

[0001] The present disclosure provides methods and kits for detecting adeno-associated virus. In some embodiments, the adeno-associated virus is Anc80. Detection methods include HPLC and ELISA-based methods. [Background technology]

[0002] Adeno-associated virus (AAV) has emerged as an important tool for gene therapy. Generally, AAV for gene therapy is an assembled viral particle that lacks native viral genes and contains only a sequence of interest, such as a gene of interest (GOI), resulting in a recombinant AAV (rAAV). The gene of interest is then delivered into host cells by the rAAV particle. A typical process for producing rAAV particles involves delivering at least two plasmids into producer cells: a first plasmid (ITR / GOI plasmid) containing the gene of interest flanked by inverted terminal repeat (ITR) sequences found in the native AAV genome; a second plasmid (rep / cap plasmid) containing the AAV rep / cap genes expressed in trans for viral assembly; and a third plasmid (helper plasmid) often containing helper genes from either adenovirus or herpesvirus. The producer cells then produce assembled rAAV particles containing the ITR / GOI plasmid (i.e., "packaged" rAAV), which can then be isolated and purified from the producer cells.

[0003] Purification of rAAV typically involves harvesting producer cells, lysing the producer cells, subjecting the cell lysate to gradient purification (e.g., using an iodixanol gradient) and / or fast protein liquid chromatography (FPLC) (which can be ion exchange, size exclusion, and / or affinity-based), and buffer exchange and concentration (e.g., using tangential flow filtration). After purification, the rAAV titer is typically measured. Recombinant AAV and production and purification methods are further described, for example, in Naso et al., BioDrugs 31(4):317-334(2017) and WO2018 / 150269.

[0004] Purification of AAV, e.g., rAAV, from producer cells can be a time-consuming, labor-intensive process. Because viral titration is typically not performed until after a substantially purified sample is obtained, e.g., after most or all of the purification steps, low-yield batches (e.g., low virus particle counts and / or low full / empty capsid ratios) are not detected until a significant amount of time and reagents has been expended, which greatly increases costs and reduces production efficiency.

[0005] An additional challenge associated with AAV production is developing optimized processes for different viral vectors at large scale. Iterative optimization of both upstream and downstream processes utilizes analytical tools to characterize and quantify viral products on in-process samples. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2018 / 150269 [Non-patent literature]

[0007] [Non-Patent Document 1] Naso et al.,BioDrugs 31(4):317-334(2017) Summary of the Invention

[0008] In some embodiments, the present disclosure provides a method for detecting adeno-associated virus serotype Anc80 in a sample, the method comprising: (a) contacting the sample with (i) a capture reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8), and (ii) a detection reagent that binds to adeno-associated virus (AAVx); (b) forming a binding complex comprising the capture reagent, Anc80, and the detection reagent; and (c) detecting the binding complex, thereby detecting Anc80 in the sample.

[0009] In an additional embodiment, the present disclosure provides a kit comprising: (a) a capture reagent that binds to an adeno-associated virus (AAVx); and (b) a detection reagent that binds to an adeno-associated virus (AAVx).

[0010] In a further embodiment, the present disclosure provides a method for determining the capsid titer of an adeno-associated virus in a cell suspension or cell lysate, the method comprising: (a) subjecting the cell suspension or cell lysate to high performance liquid chromatography (HPLC), wherein the HPLC is performed using a resin that binds to adeno-associated virus viral particles (VP); and (b) detecting VP in the cell suspension or cell lysate, thereby determining the capsid titer of the adeno-associated virus. [Brief explanation of the drawings]

[0011] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present invention.

[0012] [Figure 1]1 shows an exemplary AAV purification process according to embodiments herein. AAV producer cells are grown in a bioreactor and harvested by depth filtration. AAV is isolated and purified by chromatography. [Figure 2A] 2A, 2B, and 2C show the results of the HPLC chromatography experiments described in Example 1. Figures 2A, 2B, and 2C show standard curves generated from samples of Anc80.CMV.eGFP, AAV2.CMV.eGFP, and AAV8.CMV.eGFP, respectively, using the same HPLC column. [Figure 2B] 2A, 2B, and 2C show the results of the HPLC chromatography experiments described in Example 1. Figures 2A, 2B, and 2C show standard curves generated from samples of Anc80.CMV.eGFP, AAV2.CMV.eGFP, and AAV8.CMV.eGFP, respectively, using the same HPLC column. [Figure 2C] 2A, 2B, and 2C show the results of the HPLC chromatography experiments described in Example 1. Figures 2A, 2B, and 2C show standard curves generated from samples of Anc80.CMV.eGFP, AAV2.CMV.eGFP, and AAV8.CMV.eGFP, respectively, using the same HPLC column. [Figure 3] Figure 3 shows the results of the HPLC chromatography experiments described in Example 1. Figure 3 shows HPLC chromatograms of samples obtained during or after various steps of the AAV purification process: cell suspension, cell lysate, lysate clarification, tangential flow filtration (TFF) retentate, and final filtration. [Figure 4]

[0023] Figure 1 shows the results of the AAV titer calculation described in Example 1. The full / empty capsid ratio was determined using the capsid titer measured by HPLC (calculated from the results in Figure 3) and the genome titer measured by ddPCR. [Figure 5] 1 shows a standard curve for an ELISA performed according to Example 2 using samples of Anc80.CMV.eGFP. [Figure 6]6 shows a four-parameter logistic regression calculation to determine the curve fit of the standard curve shown in FIG. 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure relates to methods and kits for detecting adeno-associated virus.

[0014] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0015] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, "a" or "an" may mean one or more. As used herein, the words "a" or "an," when used in conjunction with the word "comprise," may mean one or more than one. As used herein, "another" or "further" may mean at least a second or more.

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value or the variation that exists between study subjects. Typically, the term "about" is meant to encompass approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less, depending on the context. In some embodiments, one of ordinary skill in the art will understand the level of variation implied by the term "about" depending on the context in which it is used herein. It should also be understood that use of the term "about" includes the specifically recited value.

[0017] Although use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0018] As used herein, the terms "comprising" (and any variant or variation thereof, such as "comprise" and "comprises"), "having" (and any variant or variation thereof, such as "have" and "has"), "including" (and any variant or variation thereof, such as "includes" and "include"), or "containing" (and any variant or variation thereof, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method and / or kit of the disclosure.

[0019] The use of the term "for example" and its corresponding abbreviation "eg," unless expressly stated otherwise, means that the particular term listed is representative of examples and embodiments of the present disclosure that are not intended to be limited to the particular example referenced or cited.

[0020] As used herein, "between" is a range that includes the endpoints of the range. For example, a number between x and y explicitly includes the numbers x and y, as well as any number that falls within the range between x and y.

[0021] As used herein, "adeno-associated virus" or "AAV" refers to a small, replication-deficient, non-enveloped virus or virus particle containing single-stranded DNA of the Parvoviridae family and the Dependoparvovirus genus. Adeno-associated viruses also include ancestral AAVs (Anc AAVs). Non-limiting examples of AAV serotypes include Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2. i8, AAV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3 .1, AAV.7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC 7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 , AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM These serotypes include AAV.4-1, AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126 and Anc127.In addition to these serotypes, AAV pseudotypes have been developed.AAV pseudotypes contain the capsid of one serotype and the genome of a second serotype (for example, pseudotype AAV2 / 5 corresponds to the AAV with the genome of serotype AAV2 and the capsid of AAV5). Methods for producing derivatives, modifications, and / or pseudotypes of AAV are described, for example, in Asokan et al., Mol. Ther. 20(4):699-708 (2012).

[0022] As used herein, the "rep" gene refers to the art-recognized region of the AAV genome that encodes the replication proteins of AAV, which are collectively required to replicate the viral genome. Rep also refers to functional homologs of AAV genes, such as the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV DNA replication. In some embodiments, the AAVs described herein encode the rep coding region.

[0023] As used herein, the "cap" gene refers to the art-recognized region of the AAV genome that encodes the capsid proteins of AAV. Illustrative and non-limiting examples of capsid proteins are the AAV capsid proteins VP1, VP2, and VP3. AAV capsid proteins interact to form the AAV capsid. The Cap gene used in this disclosure can refer to cap genes from any AAV serotype or combination of serotypes.

[0024] When used in the context of AAV, the term "recombinant" means that the AAV is the product of one or more procedures that result in an AAV that is different from naturally occurring AAV. Recombinant AAV, or rAAV, refers to a virus or viral particle that contains at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector that contains a heterologous polynucleotide (i.e., a polynucleotide other than the naturally occurring AAV genome (e.g., a transgene or gene of interest to be delivered into a cell, such as a mammalian cell)). rAAV particles can be of any serotype, pseudotype, or derivative described herein (e.g., Anc80, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or derivatives, modifications, and / or pseudotypes thereof). rAAV particles can also include a combination of serotypes.

[0025] As used herein, the term "titer" or "viral titer" refers to the concentration of a virus, for example, an AAV provided herein. Viral titer can be expressed as the number of viral particles or infectious units per mL. In some embodiments, viral titer is determined by measuring the concentration of viral components, for example, viral capsid or genome.

[0026] As described herein, purification of AAV, e.g., rAAV, from producer cells can be a time-consuming, labor-intensive process. An exemplary process for rAAV production and purification is shown in Figure 1. In Figure 1, producer cells are grown in a bioreactor to a desired cell density. The cells are collected by depth filtration and subjected to chromatography to obtain purified AAV. Because viral titration is typically not performed until after a substantially purified sample has been obtained, e.g., after most or all of the purification steps, low-yield batches (e.g., low virus particle counts and / or low full / empty capsid ratios) are not detected until a significant amount of time and reagents have been expended, which greatly increases costs and reduces production efficiency.

[0027] In some embodiments, the present disclosure provides analytical methods for determining the titer of AAV produced by producer cells. In some embodiments, the method includes measuring AAV capsid titer. In some embodiments, the method includes measuring AAV genome titer. In some embodiments, the same method is used to detect different AAV serotypes, thereby providing an efficient and simplified procedure over current methods that require serotype-specific reagents, which may have different protocols. For example, for manufacturers of AAV of different serotypes, being able to utilize a single, streamlined process for detecting any AAV serotype can reduce the number of instruments and their maintenance, allow instruments to be utilized more efficiently, lower reagent, processing, and labor costs, simplify personnel training, reduce the amount of different certifications, improve manufacturability and scaling, etc.

[0028] In some embodiments, the methods can detect and / or titer AAV (e.g., Anc80) during the AAV purification process from producer cells, as described herein. The methods advantageously provide sensitive and specific detection and / or titering of AAV (e.g., Anc80) even in crude, unpurified samples that may contain interfering components, such as producer cell proteins and cell debris, thereby providing a simple and efficient in-process AAV test. For example, the methods can be performed in 90 minutes or less, 75 minutes or less, 60 minutes or less, or 45 minutes or less. In some embodiments, the methods can detect and / or titer AAV (e.g., Anc80) in cell lysates. In some embodiments, the methods can detect and / or titer AAV (e.g., Anc80) in partially purified cell lysates. A "partially purified" cell lysate refers to a cell lysate that has been subjected to one or more downstream purification process steps after cell lysis, such as centrifugation, chromatography, buffer exchange, or filtration. In some embodiments, the methods are capable of detecting and / or titrating AAV (e.g., Anc80) in a purified sample, e.g., a sample that has been purified from AAV producer cells as described herein. In some embodiments, the purified sample is free or substantially free of non-AAV particles.

[0029] Chromatography Methods In some embodiments, the present disclosure provides analytical methods for determining the titer of AAV produced by producer cells. In some embodiments, the methods are used to determine AAV titer at different time points during the production process, for example, during producer cell growth and / or during the AAV isolation and purification process described herein. In some embodiments, the methods can detect and / or titer AAV without lysing the producer cells. In some embodiments, the methods include measuring AAV capsid titer.

[0030] In some embodiments, the present disclosure provides methods for determining adeno-associated virus capsid titer in a cell suspension or cell lysate, the method comprising: (a) subjecting the cell suspension or cell lysate to high performance liquid chromatography (HPLC), where the HPLC is performed using a resin that binds adeno-associated virus viral particles (VP); and (b) detecting VP in the cell suspension or cell lysate, thereby determining the adeno-associated virus capsid titer.

[0031] In embodiments, the resins described herein are capable of binding to the capsid protein of a viral particle.

[0032] In exemplary embodiments, detection of VP is performed spectrophotometrically (e.g., including by using absorbance measurements). Devices for measuring the absorbance of a post-HPLC solution are well known in the art and are easily adapted for use either as part of an HPLC apparatus or separately from an HPLC column. In embodiments, detection of adeno-associated virus VP is performed by obtaining the absorbance of the sample at about 270-290 nm, more preferably at about 280 nm, or particularly at 280 nm. This measurement can then be compared (either manually or as part of an instrumented measurement) to a calibration curve to provide an indication of the amount of viral capsid in the sample.

[0033] In some embodiments, the cell suspension comprises producer cells suspended in growth medium and / or buffer. In some embodiments, the cell lysate is an unpurified cell lysate, i.e., a cell lysate that has not been subjected to any downstream purification processes after cell lysis. In other embodiments, the cell lysate is a partially purified cell lysate, e.g., a cell lysate that has been subjected to one or more downstream purification processes after cell lysis, e.g., as described herein. In some embodiments, the cell lysate comprises one or more impurities, which may include proteins, nucleic acids, polysaccharides, lipids, or other cellular components of the producer cells. In some embodiments, the impurities comprise viral components that are not part of assembled AAV particles. In some embodiments, the impurities comprise partially assembled AAV particles. In some embodiments, the impurities comprise misfolded viral proteins or malformed viral particles.

[0034] As described herein, in embodiments, the AAV comprises a fluorescent moiety. In some embodiments, the fluorescent moiety is a fluorescent dye. In some embodiments, the fluorescent moiety is incorporated into the AAV particle, e.g., incorporated into the capsid. In other embodiments, the fluorescent moiety is incorporated into a nucleic acid in the AAV. In further embodiments, the fluorescent moiety is incorporated into a protein or polypeptide in the AAV. In some embodiments, the fluorescent moiety is a fluorescent protein. In some embodiments, the AAV expresses a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), mCherry, mApple, mTurquoise, mVenus, mKO2, mKate2, or any variant or derivative thereof (e.g., eGFP). Additional fluorescent moieties and tags are known in the art and include, for example, red fluorescent proteins such as TagRFP, mKate2, mRuby2, and Fusion Red, as well as other fluorescent proteins, including, for example, monomerized (V206K) superfolder GFP, mTurquoise, Cerulean3, enhanced blue FP2 (EBFP2), TagBFP, mNeonGreen, and monomerized Venus(A206K). In embodiments in which a fluorescent moiety or tag is utilized with an adeno-associated virus, detection preferably involves fluorescence detection. Devices for such fluorescence detection are known in the art and can be easily combined with HPLC devices and instruments. Fluorescent moieties are further described, for example, in Jensen, The Anatomical Record 295(12):2031-2036 (2012). In some embodiments, AAV is detected using fluorescence, for example, via a fluorescence detector attached to an HPLC. Fluorescence detection (e.g., of VP) has higher sensitivity compared to detection of absorbance at 260 nm and / or 280 nm. Fluorescence detection can also provide a simpler, more understandable output compared to A260 / A280 measurements, which require ratio calculations.In some embodiments, the detection limit of the methods described herein utilizing fluorescence detection or absorbance measurements is about 10. 6 , about 10 7 , about 10 8 , about 10 9 , or about 10 10 It is a virus particle.

[0035] In some embodiments, HPLC is performed using a resin contained in a column having a volume of about 0.1 mL to about 5.0 mL, about 0.1 mL to about 4.0 mL, about 0.1 mL to about 3.0 mL, about 0.1 mL to about 2.0 mL, about 0.1 mL to about 1.0 mL, about 0.1 mL to about 0.9 mL, about 0.2 mL to about 0.8 mL, about 0.3 mL to about 0.7 mL, or about 0.4 mL to about 0.6 mL. In some embodiments, HPLC is performed using a resin contained in a column having a volume of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.5 mL, about 2.0 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, about 4.0 mL, about 4.5 mL, or about 5.0 mL.

[0036] In some embodiments, the resin is an affinity resin. Suitably, the resin comprises an antibody or variant thereof, including an antigen / epitope-binding portion thereof, an antibody fragment or derivative, an antibody analog, an engineered antibody, or a substance that binds to an antigen in a manner similar to an antibody. In embodiments, the resin comprises at least one heavy or light chain complementarity-determining region (CDR) of an antibody. In some embodiments, the resin comprises at least two CDRs from one or more antibodies. In exemplary embodiments, the resin comprises an antibody or antigen-binding fragment thereof.

[0037] In some embodiments, the resin binds to adeno-associated viruses of any serotype or a wide range of AAV serotypes. As described herein, AAVx refers to such a wide range of AAV serotypes. In some embodiments, the resin binds to Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AAV 2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, AAV.7 m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8 , AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, A AV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.RHM15 The resin can specifically bind to any of AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127. In preferred embodiments, the AAV comprises Anc80. In some embodiments, the resin binds to the capsid protein of the AAV viral particle (VP). In embodiments, the resin binds to assembled AAV particles, and preferably, the resin binds to a conformational epitope on the AAV capsid.

[0038] In some embodiments, the AAV titer (e.g., Anc80 titer) is determined by measuring the peak area on an HPLC chromatogram. In exemplary embodiments, the method further comprises measuring the AAV genome titer (e.g., Anc80 titer) by digital droplet PCR (ddPCR). Titering of a virus, e.g., AAV, by ddPCR is described in Furuta-Hanawa et al., Hum. Gene Ther. Methods 30(4):127-136 (2019). In some embodiments, the method further comprises determining the ratio of complete to empty capsids in the cell suspension or cell lysate based on the results of the AAV titer measured by HPLC and ddPCR.

[0039] As described herein, it has surprisingly been discovered that HPLC methods can be utilized to determine capsid titer or AAV serotype (including Anc80) without the need to first purify the cell sample, and with the use of small sample sizes (e.g., 10s to 100s mL of sample), allowing for a rapid and simple method of direct viral titer determination during processing, which allows for rapid decisions regarding downstream processing.

[0040] The HPLC methods described herein can be utilized in conjunction with any bioreactor and bioreactor process of any volume. Exemplary reactors include, but are not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, "reactor" can include a fermenter or fermentation unit or any other reaction vessel, and the term "reactor" is used synonymously with "fermentor." The term fermenter or fermentation refers to both microbial and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit may perform one or more, or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflowing and outflowing fermentation or cell culture media, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitating (e.g., stirring), and / or cleaning / sterilizing. An exemplary reactor unit, such as a fermentation unit, may include multiple reactors within the unit; for example, a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility may include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactors may have a volume of from about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. 10,000 liters, 15,000 liters, 20,000 liters, 25,000 liters, 30,000 liters, 35,000 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters in volume. Furthermore, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0041] Immunoassay methods In some embodiments, the present disclosure provides immunoassay methods for detecting and / or titering adeno-associated virus (AAV) in a sample. In some embodiments, the methods are used to determine AAV titer at different time points during the production process, for example, during producer cell growth and / or during the AAV isolation and purification process described herein. In some embodiments, the methods can detect and / or titer AAV without lysing producer cells. In some embodiments, the methods include measuring AAV capsid titer.

[0042] In some embodiments, the method is an immunoassay (e.g., enzyme-linked immunosorbent assay or ELISA) for detecting and / or titrating AAV (e.g., Anc80) in a sample. Immunoassays offer many advantages, such as high specificity and sensitivity, and can also be performed in a high-throughput format to evaluate multiple samples in a single experiment. In some embodiments, the immunoassay can detect and / or titer multiple AAV serotypes using the same capture and / or detection reagents. For example, the immunoassay can be performed in a multiwell plate, with each well (or group of wells) corresponding to a different AAV serotype.

[0043] In some embodiments, the sample is a cell suspension, i.e., comprises producer cells suspended in growth medium and / or buffer. In some embodiments, the sample is an unpurified cell lysate, i.e., a cell lysate that has not been subjected to any purification processes after cell lysis. In some embodiments, the sample is a partially purified cell lysate, e.g., a cell lysate that has been subjected to one or more purification processes after cell lysis, as described herein. In some embodiments, the sample comprises one or more impurities. In some embodiments, the impurities comprise proteins, nucleic acids, polysaccharides, lipids, or other cellular components of the producer cells. In embodiments, the impurities comprise viral components that are not part of assembled AAV particles. In other embodiments, the impurities comprise partially assembled AAV particles. In some embodiments, the impurities comprise misfolded viral proteins or malformed viral particles.

[0044] In exemplary embodiments, the present disclosure provides a method for detecting adeno-associated virus (AAV) in a sample, the method comprising: (a) contacting the sample with (i) a capture reagent that binds to the adeno-associated virus (AAVx), and (ii) a detection reagent that binds to the adeno-associated virus (AAVx); (b) forming a binding complex comprising the capture reagent, the AAV, and the detection reagent; and (c) detecting the binding complex, thereby detecting the AAV in the sample.

[0045] In some embodiments, the AAV is any of the serotypes provided herein. In some embodiments, the AAV is any of the serotypes Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AAV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, A AV.7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AA V.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV. HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, A In some embodiments, the AAV is Anc80.

[0046] In additional embodiments, the present disclosure provides a method for detecting adeno-associated virus serotype Anc80 in a sample, the method comprising: (a) contacting the sample with (i) a capture reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8), and (ii) a detection reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8); (b) forming a binding complex comprising the capture reagent, Anc80, and the detection reagent; and (c) detecting the binding complex, thereby detecting Anc80 in the sample.

[0047] Anc80 is the predicted ancestor of AAV serotypes 1, 2, 8, and 9 and was reconstructed by Zinn et al. as a potent gene therapy vector (see Zinn et al., Cell Reports 12:1056-1068 (2015) and Landegger et al., Nature, Biotechnol. 35:280-284 (2017)). Anc80 clones include, for example, Anc80L27, Anc80L65, and Anc80L121. In some embodiments, the methods provided herein are capable of detecting Anc80L27, Anc80L65, and Anc80L121.

[0048] Capture and detection reagents In some embodiments, the capture reagent is an antibody or variant thereof, including an antigen / epitope-binding portion thereof, an antibody fragment or derivative, an antibody analog, an engineered antibody, or a substance that binds to an antigen in a manner similar to an antibody. In some embodiments, the capture reagent comprises at least one heavy or light chain complementarity-determining region (CDR) of an antibody. In some embodiments, the capture reagent comprises at least two CDRs from one or more antibodies. In some embodiments, the capture reagent is an antibody or antigen-binding fragment thereof. In some embodiments, the capture reagent is a monoclonal antibody.

[0049] In exemplary embodiments, the capture reagent is immobilized on a surface. Suitable surfaces include, for example, particles (such as beads) (including columns prepared from such particles) and plastic substrates such as multiwell plates. In some embodiments, the surface comprises a multiwell plate, and the capture reagent is immobilized in a well of the multiwell plate. In some embodiments, the surface comprises particles, and the capture reagent is immobilized on the particles. In some embodiments, the capture reagent comprises a conjugation moiety capable of reacting with its corresponding conjugation partner on the surface. Exemplary conjugation moiety-conjugation partner pairs include, but are not limited to, receptor-ligand pairs, complementary oligonucleotides, or cross-reactive moieties such as thiols and maleimides or iodoacetamides, aldehydes and hydrazides, or azides and alkynes or cycloalkynes. In some embodiments, the capture reagent comprises biotin, and the surface comprises avidin or streptavidin.

[0050] In some embodiments, the capture reagent binds to any serotype of adeno-associated virus (AAVx), including Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AA V2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, AAV. 7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC 8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.RHM15 In some embodiments, the capture reagent can specifically bind to any of AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127. In some embodiments, the capture reagent binds to Anc80. In some embodiments, the capture reagent binds to the capsid protein (VP) of Anc80. In some embodiments, the capture reagent binds to assembled AAV particles. In some embodiments, the capture reagent binds to a conformational epitope on the AAV capsid.

[0051] In additional embodiments, the capture reagent binds to AAV8. In some embodiments, the capture reagent is capable of binding to AAV8 and Anc80.

[0052] In some embodiments, the capture reagent binds to the AAV8 viral particle (VP) capsid protein or the Anc80 protein. Preferably, the capture reagent binds to the AAV8 VP3 protein or the VP3 protein Anc80. In embodiments, the capture reagent binds to an epitope within residues 575-610, or within residues 580-600, or within residues 580-595 of the AAV8 VP3 or Anc80 VP3 protein. In preferred embodiments, the capture reagent binds to an epitope within residues 586-591 of the AAV8 VP3 protein. In other embodiments, the capture reagent binds to an epitope within residues 589-594 of the Anc80 VP3 protein. In some embodiments, the capture reagent binds to the sequence LQSANT (SEQ ID NO: 1). In other embodiments, the capture reagent binds to the sequence LQQQNT (SEQ ID NO: 2). In some embodiments, the capture reagent is the AAV8 antibody clone ADK8.

[0053] Preferably, the detection reagent is an antibody or variant thereof, including an antigen / epitope-binding portion thereof, an antibody fragment or derivative, an antibody analog, an engineered antibody, or a substance that binds to an antigen in a manner similar to an antibody. In some embodiments, the detection reagent comprises at least one heavy or light chain complementarity-determining region (CDR) of an antibody. In some embodiments, the detection reagent comprises at least two CDRs from one or more antibodies. In exemplary embodiments, the detection reagent is an antibody or antigen-binding fragment thereof. In some embodiments, the detection reagent is a monoclonal antibody.

[0054] Preferably, the detection reagent binds to any serotype of adeno-associated virus (AAVx). In some embodiments, the detection reagent binds to any serotype of adeno-associated virus (AAVx), including Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AAV1.1, AAV2.5, AAV2i8, AAV1.1, AAV2.5, AAV2i9, AAV1.1, AAV2.5, AAV2i9, AAV2i1, AAV2i1, AAV2i2, AAV2i3, AAV2i4, AAV2i5, AAV2i6, AAV2i7, AAV2i8, AAV2i9, AAV2i1, AAV2i1, AAV2i2, AAV2i3, AAV2i4, AAV2i5, AAV2i6, AAV2i8, AAV2i9, AAV2i1 ...1, AAV2i2, AAV2i3, AAV2i4, AAV2i5, AV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, AAV .7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.H AAV.HSC15-1, AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127. In some embodiments, the detection reagent binds to Anc80. In some embodiments, the detection reagent binds to the VP capsid protein of Anc80. In some embodiments, the detection reagent binds to assembled AAV particles. In some embodiments, the detection reagent binds to a conformational epitope on the AAV capsid.

[0055] In further embodiments, the detection reagent binds to AAV8. In some embodiments, the detection reagent is capable of binding to AAV8 and Anc80. In embodiments, the detection reagent binds to the AAV8 viral particle (VP) capsid protein or the Anc80 protein, preferably the detection reagent binds to the AAV8 VP3 protein or the VP3 protein Anc80. In some embodiments, the detection reagent binds to an epitope within residues 575-610, or within residues 580-600, or within residues 580-595 of the AAV8 VP3 or Anc80 VP3 protein. In some embodiments, the detection reagent binds to an epitope within residues 586-591 of the AAV8 VP3 protein. In some embodiments, the detection reagent binds to an epitope within residues 589-594 of the Anc80 VP3 protein. In some embodiments, the detection reagent binds to the sequence LQSANT (SEQ ID NO: 1). In some embodiments, the detection reagent binds to the sequence LQQQNT (SEQ ID NO: 2). In some embodiments, the detection reagent is the AAV8 antibody clone ADK8.

[0056] In additional embodiments, the capture reagent binds to AAV8 and the detection reagent binds to AAVx. In some embodiments, the capture reagent binds to AAVx and the detection reagent binds to AAV8. In some embodiments, both the capture reagent and the detection reagent bind to AAVx. In exemplary embodiments, both the capture reagent and the detection reagent bind to Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV AV2i8, AAV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3 .1, AAV.7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV. HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127.

[0057] Binding complex formation In some embodiments, a binding complex comprising a capture reagent, AAV (e.g., Anc80), and a detection reagent is formed in a single step. In other embodiments, a binding complex comprising a capture reagent, AAV (e.g., Anc80), and a detection reagent is formed in one or more steps. In some embodiments, a binding complex is formed in solution and then immobilized on a surface. In some embodiments, a binding complex is formed by binding AAV (e.g., Anc80) to a capture reagent immobilized on a surface, and then binding a detection reagent to AAV (e.g., Anc80) to form a binding complex on the surface. In some embodiments, a binding complex is formed by simultaneous binding of AAV (e.g., Anc80) to a capture reagent immobilized on a surface and to a detection reagent. In other embodiments, a binding complex is formed by binding AAV (e.g., Anc80) to a detection reagent in solution, and then binding the AAV-detection reagent complex to the capture reagent on the surface. In additional embodiments, the binding complex is formed by binding AAV (e.g., Anc80) to a capture reagent and a detection reagent in solution, and then immobilizing the capture reagent to a surface as described herein.

[0058] detection In embodiments, the binding complex is detected via a detectable moiety. For example, the detectable moiety is measured by spectrophotometry (color change), light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In embodiments, the AAV titer (e.g., Anc80 titer) is determined by measuring the detectable moiety. In some embodiments, the detectable moiety is detectable in the presence of a substrate. Preferably, the detectable moiety is an enzyme that cleaves the substrate, and detecting comprises detecting the cleaved substrate. In some embodiments, the detectable moiety comprises horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GO), or beta-galactosidase (BGAL or β-gal). Non-limiting examples of HRP substrates include, for example, 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), AMPLEX® Red, 3-amino-9-ethylcarbazole (AEC), homovanillic acid, luminol, and SUPERSIGNAL™ ELISA, QUANTABLU™, and QUANTARED™ substrates from ThermoFisher. Non-limiting examples of AP substrates include, for example, p-nitrophenyl phosphate (PNPP), and CDP-STAR™ and DYNALIGHT™ substrates from ThermoFisher. GO substrates include, for example, glucose. BGAL substrates include, for example, o-nitrophenyl-β-D-galactopyranoside (ONPG).

[0059] In some embodiments, the detection reagent comprises a detectable moiety, and detecting the binding complex comprises measuring the detectable moiety of the detection reagent. In embodiments, the detection reagent comprises a binding partner of the detectable moiety, and detecting the binding complex comprises contacting the detection reagent with the detectable moiety and measuring the detectable moiety bound to the detection reagent. In additional embodiments, the detection reagent and the detectable moiety each comprise a binding partner in a binding pair. Suitable binding pairs are known in the art and may include, but are not limited to, receptor-ligand pairs, complementary oligonucleotides, or cross-reactive moieties, such as, but not limited to, thiols and maleimides or iodoacetamides, aldehydes and hydrazides, or azides and alkynes or cycloalkynes. In some embodiments, the detection reagent comprises biotin, and the detectable moiety comprises avidin or streptavidin. In some embodiments, the detection reagent comprises biotin, and the detectable moiety comprises horseradish peroxidase (HRP) conjugated to avidin or streptavidin. Preferably, detecting the bound complex comprises binding the bound complex to HRP (e.g., via a detection reagent and a biotin-avidin / streptavidin interaction on the HRP), contacting the HRP with a chromogenic or chemiluminescent HRP substrate, and detecting a change in color or chemiluminescent signal. In some embodiments, the AAV titer (e.g., Anc80 titer) is determined by measuring the change in color and / or chemiluminescent signal.

[0060] kit In some embodiments, the present disclosure provides kits for carrying out the methods described herein, e.g., immunoassay kits. In some embodiments, the present disclosure provides kits for detecting and / or titering adeno-associated virus (AAV), the kit comprising: (a) a capture reagent that binds to the adeno-associated virus (AAVx); and (b) a detection reagent that binds to the adeno-associated virus (AAVx). The kits described herein also preferably include instructions for carrying out the various methods, including the ELISA-based methods, described herein.

[0061] In some embodiments, the kits include any AAV serotype described herein, e.g., Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1 , AAV2.5, AAV2i8, AAV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1 , AAV6.3.1, AAV.7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.H SC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AA V.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV. The AAV is used to detect and / or titer RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, or Anc127. In some embodiments, the AAV is Anc80. Anc80 is further described herein.

[0062] In some embodiments, the present disclosure provides a kit comprising: (a) a capture reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8); and (b) a detection reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8).

[0063] Capture and detection reagents are further described herein. In some embodiments, the capture reagent is an antibody or antigen-binding fragment thereof. In some embodiments, the detection reagent is an antibody or antigen-binding fragment thereof.

[0064] In some embodiments, the kit includes a capture surface. The capture surface can be any of the surfaces described herein. In some embodiments, the capture surface includes particles. In some embodiments, the capture surface includes a plastic substrate such as a multiwell plate. In some embodiments, the capture reagent is immobilized on the capture surface. In some embodiments, the capture surface is a multiwell plate and the capture reagent is immobilized on a well of the multiwell plate.

[0065] In some embodiments, the capture reagent and the capture surface are provided separately, and the kit further includes a reagent for immobilizing the capture reagent to the capture surface. In some embodiments, the capture reagent includes a conjugation moiety, and the capture surface includes its conjugation partner. Exemplary conjugation moiety-conjugation partner pairs include, but are not limited to, receptor-ligand pairs, complementary oligonucleotides, or cross-reactive moieties, such as a thiol and maleimide or iodoacetamide, an aldehyde and hydrazide, or an azide and alkyne or cycloalkyne. In some embodiments, the capture reagent includes biotin, and the capture surface includes avidin or streptavidin attached thereto.

[0066] In some embodiments, the capture reagent binds to any serotype of adeno-associated virus (AAVx), including Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AA V2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, AAV. 7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC 8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.RHM15 In some embodiments, the capture reagent can specifically bind to any of AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127. In some embodiments, the capture reagent binds to Anc80. In some embodiments, the capture reagent binds to the capsid protein (VP) of Anc80. In some embodiments, the capture reagent binds to assembled AAV particles. In some embodiments, the capture reagent binds to a conformational epitope on the AAV capsid.

[0067] In some embodiments, the capture reagent binds to AAV8. In some embodiments, the capture reagent can bind to AAV8 and Anc80. In some embodiments, the capture reagent binds to the viral particle (VP) capsid protein of AAV8 or the capsid protein of Anc80. In some embodiments, the capture reagent binds to the AAV8 VP3 protein or the VP3 protein Anc80. In some embodiments, the capture reagent binds to an epitope within residues 575-610, or within residues 580-600, or within residues 580-595 of the AAV8 VP3 or Anc80 VP3 protein. In some embodiments, the capture reagent binds to an epitope within residues 586-591 of the AAV8 VP3 protein. In some embodiments, the capture reagent binds to an epitope within residues 589-594 of the Anc80 VP3 protein. In some embodiments, the capture reagent binds to the sequence LQSANT (SEQ ID NO: 1). In some embodiments, the capture reagent binds to the sequence LQQQNT (SEQ ID NO: 2). In some embodiments, the capture reagent is the AAV8 antibody clone ADK8.

[0068] In some embodiments, the detection reagent binds to any serotype of adeno-associated virus (AAVx). In some embodiments, the detection reagent binds to any serotype of adeno-associated virus (AAVx), including Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AAV1.1, AAV2.5, AAV2i8, AAV1.1, AAV2.5, AAV2i9, AAV2i1, AAV2i1, AAV2i2, AAV2i3, AAV2i4, AAV2i5, AAV2i6, AAV2i7, AAV2i8, AAV2i9, AAV2i1, AAV2i1, AAV2i2, AAV2i3, AAV2i4, AAV2i5, AAV2i6 ... AV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6.3.1, AAV .7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.H AAV.HSC15-1, AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127. In some embodiments, the detection reagent binds to Anc80. In some embodiments, the detection reagent binds to a capsid protein (VP) of Anc80. In some embodiments, the detection reagent binds to assembled AAV particles. In some embodiments, the detection reagent binds to a conformational epitope on the AAV capsid.

[0069] In some embodiments, the detection reagent binds to AAV8. In some embodiments, the detection reagent can bind to AAV8 and Anc80. In some embodiments, the detection reagent binds to the AAV8 viral particle (VP) capsid protein or the Anc80 capsid protein. In some embodiments, the detection reagent binds to the AAV8 VP3 protein or the VP3 protein Anc80. In some embodiments, the detection reagent binds to an epitope within residues 575-610, or within residues 580-600, or within residues 580-595 of the AAV8 VP3 or Anc80 VP3 protein. In some embodiments, the detection reagent binds to an epitope within residues 586-591 of the AAV8 VP3 protein. In some embodiments, the detection reagent binds to an epitope within residues 589-594 of the Anc80 VP3 protein. In some embodiments, the detection reagent binds to the sequence LQSANT (SEQ ID NO: 1). In some embodiments, the detection reagent binds to the sequence LQQQNT (SEQ ID NO: 2). In some embodiments, the detection reagent is the AAV8 antibody clone ADK8.

[0070] In some embodiments, the capture reagent binds to AAV8 and the detection reagent binds to AAVx. In some embodiments, the capture reagent binds to AAVx and the detection reagent binds to AAV8. In some embodiments, both the capture reagent and the detection reagent bind to AAVx. In some embodiments, both the capture reagent and the detection reagent bind to Anc80, Anc80L27, Anc80L65, Anc80L121, AAV1, AAV-2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV1.1, AAV2.5, AAV2i8, AAV2G9, AAV2tYF, AAV2-TT, AAV2-TT-S312N, AAV3B, AAV3B-S312N, AAV-LK3, AAV6.1, AAV6. 3.1, AAV.7m8, AAV9.45, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV. HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh32, AAV.rh33, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV AAV.RHM15-1, AAV.RHM15-2, AAV.RHM15-3, AAV.RHM15-4, AAV.RHM15-5, AAV.RHM15-6, AAV.cy10, AAV.dj, AAV.po4, AAV.po6, AAV.hu26, AAV.hu37, AAV.PHP.B, Anc126, and Anc127.

[0071] In some embodiments, the detection reagent comprises a detectable moiety that can be measured, for example, by spectrophotometry (color change), light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In some embodiments, the detection reagent comprises a binding partner of the detectable moiety. In some embodiments, the detection reagent and the detectable moiety each comprise binding partners in a binding pair, e.g., a receptor-ligand pair, complementary oligonucleotides, or cross-reactive moieties, such as a thiol and maleimide or iodoacetamide, an aldehyde and hydrazide, or an azide and alkyne or cycloalkyne. In some embodiments, the detection reagent comprises biotin, and the detectable moiety comprises avidin or streptavidin. In some embodiments, the detectable moiety comprises horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GO), or beta-galactosidase (BGAL or β-gal). In some embodiments, the detection reagent comprises biotin and the detectable moiety comprises HRP conjugated to avidin or streptavidin.

[0072] In some embodiments, the capture reagent is lyophilized. In some embodiments, the capture reagent is provided in solution. In some embodiments, the detection reagent is lyophilized. In some embodiments, the detection reagent is provided in solution. In embodiments where the capture and / or detection reagent is lyophilized, the kit preferably further comprises a buffer for reconstituting or resuspending the lyophilized reagent. In some embodiments, the capture and / or detection reagent is provided separately from the other components of the kit, e.g., according to their optimal shipping and / or storage temperatures.

[0073] In some embodiments, the kit further comprises one or more of a buffer, an assay stop solution, a calibration reagent, a detectable moiety capable of binding to the detection reagent, and a detectable substrate.

[0074] In some embodiments, the kit includes one or more of an assay buffer, a blocking buffer, a coating buffer, a wash buffer, a reconstitution or resuspension buffer, and a storage buffer. In some embodiments, the assay buffer includes phosphate buffered saline, sodium carbonate, sodium bicarbonate, Tris, NaCl, TWEEN, or a combination thereof. In some embodiments, the kit includes an assay stop solution. In some embodiments, the assay stop solution includes sulfuric acid.

[0075] In some embodiments, the kit includes a calibration reagent. In some embodiments, the calibration reagent includes a known amount of AAV (e.g., Anc80). In some embodiments, the kit includes multiple calibration reagents including a range of concentrations of AAV (e.g., Anc80). In some embodiments, the multiple calibration reagents include AAV (e.g., Anc80) at concentrations near the upper and lower limits of quantification for the method performed using the kit. In some embodiments, the multiple calibration concentrations of the calibration reagent span the entire dynamic range of the method. In some embodiments, the multiple calibration reagents include multiple AAV serotypes, for example, to calibrate the method for detecting different AAV serotypes. In some embodiments, the calibration reagent is a positive control reagent. In some embodiments, the calibration reagent is a negative control reagent. In some embodiments, the calibration reagent is lyophilized. In some embodiments, the calibration reagent is provided in a solution.

[0076] In some embodiments, the kit includes a detectable moiety capable of binding to a detection reagent. Detectable moieties are further described herein. In some embodiments, the detectable moiety includes horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GO), or beta-galactosidase (BGAL or β-gal). In some embodiments, the kit further includes a substrate for the detectable moiety. Exemplary substrates for the detectable moiety are provided herein. In some embodiments, the substrate is 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), AMPLEX® Red, 3-amino-9-ethylcarbazole (AEC), homovanillic acid, luminol, and SUPERSIGNAL™ ELISA, QUANTABLU™, and QUANTARED™ substrates from ThermoFisher. In some embodiments, the substrate is p-nitrophenyl phosphate (PNPP), and CDP-STAR™ and DYNALIGHT™ substrates from ThermoFisher. In some embodiments, the substrate is glucose. In some embodiments, the substrate is o-nitrophenyl-β-D-galactopyranoside (ONPG).

[0077] In some embodiments, the kit further comprises one or more of assay consumables, e.g., assay modules, vials, tubes, liquid handling and transfer devices, e.g., pipette tips, covers and seals, racks, and labels. In some embodiments, the kit further comprises instructions for performing an immunoassay to detect and / or titer AAV (e.g., Anc80). In some embodiments, the immunoassay comprises a method described herein.

[0078] Illustrative Embodiments Embodiment 1 is a method for detecting adeno-associated virus serotype Anc80 in a sample, the method comprising contacting the sample with (i) a capture reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8), and (ii) a detection reagent that binds to adeno-associated virus (AAVx), forming a binding complex comprising the capture reagent, Anc80, and the detection reagent, and detecting the binding complex, thereby detecting Anc80 in the sample.

[0079] Embodiment 2 includes the method of embodiment 1, wherein the capture reagent and / or the detection reagent is an antibody or antigen-binding fragment thereof.

[0080] Embodiment 3 includes the method of embodiment 1 or 2, wherein the capture reagent is immobilized on a surface.

[0081] Embodiment 4 includes the method of embodiment 3, wherein the capture reagent comprises biotin and the surface comprises avidin or streptavidin.

[0082] Embodiment 5 includes the method of embodiment 3 or 4, wherein the surface is a multi-well plate and the capture reagent is immobilized in the wells of the multi-well plate.

[0083] Embodiment 6 includes the method of any of Embodiments 1-5, wherein the capture reagent binds to AAVx.

[0084] Embodiment 7 includes the method of any of Embodiments 1-5, wherein the capture reagent binds to AAV8.

[0085] Embodiment 8 includes the method of any of Embodiments 1-7, wherein the capture reagent binds to the viral particle (VP) of Anc80.

[0086] Embodiment 9 includes the method of embodiment 7, wherein the capture reagent binds to VP3.

[0087] Embodiment 10 includes the method of embodiment 9, wherein the capture reagent binds to an epitope within residues 580-600 of VP3.

[0088] Embodiment 11 includes the method of embodiment 10, wherein the capture reagent binds to LQSANT (SEQ ID NO: 1).

[0089] Embodiment 12 includes the method of any of Embodiments 1-11, wherein the detection reagent binds to AAVx.

[0090] Embodiment 13 includes the method of any of embodiments 1-11, wherein the detection reagent binds to AAV8.

[0091] Embodiment 14 includes the method of any of embodiments 1-13, wherein the detection reagent binds to a viral protein (VP) of Anc80.

[0092] Embodiment 15 includes the method of any of embodiments 1-14, wherein the detection reagent comprises a binding partner of the detectable moiety.

[0093] Embodiment 16 includes the method of embodiment 15, wherein the detection reagent comprises biotin and the detectable moiety comprises horseradish peroxidase conjugated to avidin or streptavidin.

[0094] Embodiment 17 includes the method of embodiment 1, wherein the capture reagent binds to AAV8 and the detection reagent binds to AAVx.

[0095] Embodiment 18 is a kit comprising a capture reagent that binds to adeno-associated virus (AAVx) or adeno-associated virus serotype 8 (AAV8) and a detection reagent that binds to adeno-associated virus (AAVx).

[0096] Embodiment 19 includes the kit of embodiment 18, wherein the capture and / or detection reagent is an antibody or antigen-binding fragment thereof.

[0097] Embodiment 20 includes the kit of embodiment 18 or 19, further comprising a capture surface.

[0098] Embodiment 21 includes the kit of embodiment 20, wherein the capture reagent comprises biotin and the capture surface comprises avidin or streptavidin attached thereto.

[0099] Embodiment 22 includes the kit of embodiment 20 or 21, wherein the capture surface is a multiwell plate and the capture reagent is immobilized on the wells of the multiwell plate.

[0100] Embodiment 23 includes the kit of any of embodiments 18-22, wherein the capture reagent binds to AAVx.

[0101] Embodiment 24 includes the kit of any of embodiments 18-22, wherein the capture reagent binds to AAV8.

[0102] Embodiment 25 includes the kit of any of embodiments 18-24, wherein the capture reagent binds to a viral protein (VP) of Anc80.

[0103] Embodiment 26 includes the kit of embodiment 24, wherein the capture reagent binds to VP3.

[0104] Embodiment 27 includes the kit of embodiment 26, wherein the capture reagent binds to an epitope within residues 580-600 of VP3.

[0105] Embodiment 28 includes the kit of embodiment 27, wherein the capture reagent binds to LQSANT (SEQ ID NO: 1).

[0106] Embodiment 29 includes the kit of any of embodiments 18-28, wherein the detection reagent binds to AAVx.

[0107] Embodiment 30 includes the kit of any of embodiments 18-28, wherein the detection reagent binds to AAV8.

[0108] Embodiment 31 includes the kit of any of embodiments 18-30, wherein the detection reagent binds to the capsid protein (VP) of Anc80.

[0109] Embodiment 32 includes the kit of any of embodiments 18-31, further comprising a detectable moiety, wherein the detection reagent comprises a binding partner of the detectable moiety.

[0110] Embodiment 33 includes the kit of embodiment 32, wherein the detection reagent comprises biotin and the detectable moiety comprises horseradish peroxidase conjugated to avidin or streptavidin.

[0111] Embodiment 34 includes the kit of any of embodiments 18-33, wherein the capture reagent, the detection reagent, or both, are lyophilized.

[0112] Embodiment 35 includes the kit of any of embodiments 18-33, wherein the capture reagent, the detection reagent, or both are provided in solution.

[0113] Embodiment 36 includes the kit of any of embodiments 18-35, further including one or more of a buffer, an assay stop solution, a calibration reagent, a detectable moiety capable of binding to a detection reagent, and a detectable substrate.

[0114] Embodiment 37 includes the kit of embodiment 18, wherein the capture reagent binds to AAV8 and the detection reagent binds to AAVx.

[0115] Embodiment 38 is a method for determining the capsid titer of an adeno-associated virus in a cell suspension or cell lysate, the method comprising subjecting the cell suspension or cell lysate to high performance liquid chromatography (HPLC), where the HPLC is performed using a resin that binds to a viral protein (VP) of the adeno-associated virus, and detecting the VP in the cell suspension or cell lysate, thereby determining the capsid titer of the adeno-associated virus.

[0116] Embodiment 39 includes the method of embodiment 38, wherein the detecting includes detecting the capsid protein spectrophotometrically at 280 nm.

[0117] Embodiment 40 includes the method of embodiment 38, wherein the adeno-associated virus includes a fluorescent moiety and the detecting includes fluorescent detection.

[0118] Embodiment 41 includes the method of any of embodiments 38-40, wherein the cell lysate is a crude cell lysate or a partially purified cell lysate.

[0119] Embodiment 42 includes the method of any of embodiments 38-41, wherein the HPLC is performed using a resin contained in a column having a volume of about 0.1 to about 1.0 mL.

[0120] Embodiment 43 includes the method of any of embodiments 38-42, wherein the resin comprises an antibody or antigen-binding fragment thereof.

[0121] Embodiment 44 includes the methods of any of embodiments 38-43, wherein the adeno-associated virus comprises Anc80. [Example]

[0122] Example 1. HPLC Assay A broad-spectrum AAV (AAVX) affinity column was prepared by Princeton Chromatography, Inc. by packing POROS™ CAPTURESELECT™ AAVX Affinity Resin (ThermoFisher) into a 50 x 4.6 mm HPLC column. The AAVX affinity column was evaluated using various AAV serotype samples, including Anc80.CMV.eGFP, AAV2.CMV.eGFP, and AAV8.CMV.eGFP, using an Agilent 1260 Infinity II Bio-Inert LC System equipped with a Fluorescence Detector. The response linearity, R, was measured as shown in Figures 2A-2C, which show the standard curves generated for Anc80.CMV.eGFP, AAV2.CMV.eGFP, and AAV8.CMV.eGFP, respectively. 2 >0.99 was observed for all three AAV serotypes, with a lower limit of detection of approximately 6 × 10 8 The total viral particles were

[0123] The HPLC method was further tested using samples from Anc80 during processing. A 50 L bioreactor producing Anc80.CMV.eGFP was tested using the HPLC method during various steps of the purification process. As shown in Figure 3, the Anc80.CMV.eGFP capsid peak was successfully detected in all steps tested: cell suspension, cell lysate, post-lysate clarification, post-tangential flow filtration (TFF), and post-final filtration.

[0124] The titers of the Anc80 samples during processing were determined based on linear regression analysis. Titers were also determined using ddPCR, and the full / empty capsid ratios of the samples during processing were calculated, as shown in Figure 4.

[0125] Example 2. Immunoassay An ELISA assay for Anc80 was developed using an anti-AAV8 capture antibody and CAPTURESELECT™ Biotin Anti-AAVX Conjugate (ThermoFisher) as the detection antibody. A standard curve for Anc80.CMV.eGFP was generated using a four-parameter logistic regression with R 2 >0.99 (see Figure 5, standard curve, and Figure 6, regression calculation).

[0126] The ELISA assay was also applied to in-process samples from a 50 L bioreactor producing Anc80.CMV.eGFP. The titers of the in-process samples were determined, and the results are comparable to titers determined by other methods.

[0127] All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent they have not already been cited, are incorporated herein by reference in their entirety.

Claims

1. 1. A method for detecting adeno-associated virus serotype Anc80 in a sample, comprising: (a) contacting the sample with (i) a capture reagent that specifically binds to adeno-associated virus serotype 8 (AAV8), and (ii) a detection reagent that binds to a broad range of adeno-associated virus serotypes (AAVx), wherein each of the capture reagent and the detection reagent is capable of binding to Anc80, and the capture reagent and the detection reagent are antibodies or antigen-binding fragments thereof; (b) forming a binding complex comprising the capture reagent, the Anc80, and the detection reagent; (c) detecting the binding complex, thereby detecting the Anc80 in the sample.

2. The method of claim 1 , wherein the capture reagent is immobilized on a surface.

3. The method of claim 2 , wherein the capture reagent comprises biotin and the surface comprises avidin or streptavidin.

4. 4. The method of claim 2 or 3, wherein the surface is a multi-well plate and the capture reagent is immobilized in the wells of the multi-well plate.

5. The method of any one of claims 1 to 4, wherein the capture reagent binds to the capsid protein (VP) of Anc80.

6. The method described in claim 5, wherein the capture reagent binds to VP3 of Anc80.

7. The method of claim 6, wherein the capture reagent binds to an epitope within residues 580-600 of the VP3.

8. The method of claim 7, wherein the capture reagent binds to LQSANT (SEQ ID NO: 1).

9. The method of any one of claims 1 to 8, wherein the detection reagent binds to the capsid protein (VP) of Anc80.

10. The method of any one of claims 1 to 9, wherein the detection reagent comprises a binding partner of a detectable moiety.

11. 11. The method of claim 10, wherein the detection reagent comprises biotin and the detectable moiety comprises horseradish peroxidase conjugated to avidin or streptavidin.

12. A kit for detecting adeno-associated virus serotype Anc80 in a sample, comprising: (a) a capture reagent that specifically binds to adeno-associated virus serotype 8 (AAV8); (b) a detection reagent that binds to a broad range of adeno-associated virus serotypes (AAVx); A kit, wherein the capture reagent and the detection reagent are each capable of binding to Anc80, and the capture reagent and the detection reagent are antibodies or antigen-binding fragments thereof.

13. The kit of claim 12 further comprising a capture surface.

14. The kit of claim 13, wherein the capture reagent comprises biotin and the capture surface comprises avidin or streptavidin attached thereto.

15. 15. The kit of claim 13 or 14, wherein the capture surface is a multi-well plate and the capture reagent is immobilized on a well of the multi-well plate.

16. The kit of any one of claims 12 to 15, wherein the capture reagent and the detection reagent each bind to the capsid protein (VP) of Anc80.

17. The kit described in claim 16, wherein the capture reagent binds to VP3 of Anc80.

18. 18. The kit of claim 17, wherein the capture reagent binds to an epitope within residues 580-600 of the VP3.

19. The kit of claim 18, wherein the capture reagent binds to LQSANT (sequence number 1).

20. The kit of any one of claims 12 to 19, further comprising a detectable moiety, wherein the detection reagent comprises a binding partner of the detectable moiety.

21. The kit of claim 20, wherein the detection reagent comprises biotin and the detectable moiety comprises horseradish peroxidase conjugated to avidin or streptavidin.

22. 22. The kit of any one of claims 12 to 21, wherein the capture reagent, the detection reagent, or both, are lyophilized, or the capture reagent, the detection reagent, or both, are provided in solution.

23. 23. The kit of any one of claims 12 to 22, further comprising one or more of a buffer, an assay stop solution, a calibration reagent, a detectable moiety capable of binding to the detection reagent, and a detectable substrate.

24. 1. A method for determining adeno-associated virus capsid titer in a cell suspension or cell lysate, the method comprising: (a) subjecting the cell suspension or cell lysate to high performance liquid chromatography (HPLC), wherein the HPLC is performed using a resin that binds to the adeno-associated virus viral particles (VP), and the resin is contained in a column having a volume of 0.1 to 1.0 mL; (b) detecting the VP in the cell suspension or cell lysate; (c) measuring the peak area of ​​the VP on the HPLC chromatogram, thereby determining the capsid titer of the adeno-associated virus.

25. 25. The method of claim 24, wherein the detecting comprises spectrophotometrically detecting the capsid protein at 280 nm, or wherein the adeno-associated virus comprises a fluorescent moiety and the detecting comprises fluorescent detection.

26. (i) whether the cell lysate is a crude cell lysate or a partially purified cell lysate; (ii) the resin comprises an antibody or an antigen-binding fragment thereof; (iii) the adeno-associated virus contains Anc80; or (iv) The method according to claim 24 or 25, which is any combination of (i) to (iii).

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