RU2785961C2 - Methods for determination of activity of adeno-associated viral preparations - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to methods for measurement of quantitative signs of gene-therapeutic vector preparations. A method for quantitative determination of a dose of AAV preparation is disclosed, providing quantitative determination of the total number of AAV capsids in AAV preparation or AAV fraction used for obtainment of AAV preparation, using AAV-specific ELISA analysis, and assessment of percentage or ratio of full and empty (full:empty) AAV capsids in AAV preparation or AAV fraction, using cryogenic translucent electronic microscopy (hereinafter – CryoTEM). A method for measurement of a concentration of hollow AAV capsids in AAV preparation, methods for administration of AAV preparation to a subject who needs it, and a method for the production of AAV preparation are also disclosed, each of which includes stages of AAV-specific ELISA analysis and CryoTEM.

EFFECT: group of inventions provides highly reliable and more accurate determination of a concentration, dose, and/or activity of AAV preparation.

44 cl, 16 dwg, 9 tbl, 4 ex

Description

Related Applications

This application claims priority under U.S. Provisional Application No. 62/467,045, filed March 3, 2017, which is incorporated herein by reference in its entirety.

Background of the Invention

Adeno-associated virus (AAV) is a small non-enveloped virus that packages the genome as a linear single-stranded DNA. AAV belongs to the family Parvoviridae and the genus Dependovirus because productive infection with AAV occurs only in the presence of a helper virus such as, for example, adenovirus or herpes virus. Even in the absence of a helper virus, AAV (serotype 2) can achieve latency by integrating into chromosome 19q13.4 of the human host genome. It is the only mammalian DNA virus known to be capable of site-specific integration (Daya and Berns, Clinical Microbiology Reviews, pages 583-593 (2008)).

For safe clinical use of AAV, AAV has been genetically modified at several positions in its genome. For example, the Rep gene, which is required for viral replication, and an element required for site-specific integration, have been removed from the AAV genome in many viral vectors. This recombinant AAV (rAAV) exists in an extrachromosomal state and is characterized by a very low efficiency of integration into genomic DNA. Thus, the likelihood of rAAV-induced random mutagenesis occurring in the host cell is reduced, if not completely eliminated. Because of these properties and lack of pathogenicity, rAAV has shown great potential as a gene therapy vector in many aspects of preclinical and clinical applications. The clinic is testing new serotypes and independent vectors. Along with these ongoing vector developments, ongoing efforts have been focused on variable manufacturing processes that can efficiently produce high titer quantities of rAAV vectors with high purity and potency.

While efforts to develop efficient, large-scale methods for purifying an AAV product suitable for human administration have progressed, there is still a need for analytical methods to measure the performance of recombinant rAAV gene therapy vector preparations for product release and accurate clinical dosing. For example, current methods of producing AAV in cell culture lead to the formation of "empty" capsids that lack the vector genome and have been shown to lead to T-cell mediated immune responses. In addition, empty capsids are unable to provide a therapeutic benefit associated with transgene production, and there is the potential to enhance innate or adaptive immune responses to the vector (eg, T-cell mediated), which then makes empty capsids a problem in the context of gene therapy. Wright, Molecular Therapy 22:1-2 (2014).

Classically, the use of quantitative PCR (qPCR) is the most widely accepted and preferred method of dosing AAV drugs because it provides a measure of vector genomes (eg, vector genome (vg)/kg subject). See Halbert CL et al., J Virol 1997; 71:5932-5941; Samulski RJ et al., J Virol 1989; 63:3822-3828; Clark KR et al., Hum Gene Ther 1999; 10:1031-1039 and Wright JF, Hum Gene Ther 2011; 22:519-521. However, the use of qPCR is limited by the fact that only total DNA is measured, which can lead to significant problems. For example, qPCR reading can be affected by problems such as the use of inefficient primers, problems with calibration standards, or tertiary DNA structures (eg, hairpin, circular, or supercoiled structures). Thus, the use of qPCR may lead to inaccurate determination of the dose and, ultimately, the activity of the final AAV preparation due to the inclusion in the dose calculation of capsids containing more than one copy of DNA and/or destroyed capsids. This is because the activity of an AAV preparation has two parts: 1) the ability of the AAV capsid to transduce cells, and 2) the ability of the DNA in the AAV to produce the desired product. Non-functioning capsids that would not be captured by qPCR do not efficiently transduce cells. In addition, qPCR is also limited due to its inherent variability (i.e., high variability up to 0.5 log steps), which can lead to inaccurate dose determination of the final AAV drug. The inherent variability can also be exacerbated by the inter-assay variability observed with qPCR due to sample preparation that involves several steps including capsid protein digestion, DNA isolation, and DNA purification. If the dose and/or activity is not precisely defined, patients may receive the wrong dose and/or widely varying doses from treatment to treatment. Thus, an accurate tool is needed to measure the dose and, ultimately, the activity of AAV drugs, as well as to monitor the quality and activity of the gene therapy vector.

Brief summary of the present invention

This document provides methods for measuring quantitative traits of gene therapy vector preparations. In certain embodiments, the gene therapy vector preparations are adeno-associated virus (AAV) preparations. The methods disclosed herein can be used at any purification point. In some embodiments, the methods may be used prior to preparing or packaging AAV capsids for use. The methods disclosed herein can be used to improve determination of the concentration, dose and/or potency of an AAV preparation.

A peculiarity of obtaining the AAV vector in cell culture is the formation of an excess of "empty" capsids, in which the vector genome is absent. In some embodiments, the number of empty capsids is evaluated to accurately determine the concentration, dose, and/or potency of the AAV preparation. Empty capsids can also be considered as product related impurities which should be reduced during purification. In certain embodiments, it is important to determine the total number of empty capsids in order to reduce the innate and/or adaptive immune response against the capsid antigen.

Classically, the concentration, dose, and/or potency of an AAV drug is dependent on quantitation by quantitative PCR (qPCR). Surprisingly, the present inventors have found that AAV drug quantification can only rely on an AAV-specific enzyme-linked immunosorbent assay (ELISA), and its use can improve concentration and/or dose measurement of AAV drug and, ultimately, activity. The methods of the present invention are advantageous over those known in the art because ELISA antigen detection provides very reliable results, surprisingly more accurate than qPCR. In certain embodiments, the method does not include the step of measuring concentration, dose, and/or activity by qPCR. In some embodiments, the concentration, dose, and/or activity may be determined by the concentration of AAV capsids in the AAV formulation. In some embodiments, the ELISA methods disclosed herein result in an AAV preparation that has at least about 10% less variability in concentration, dose, and/or activity compared to a qPCR method. In some embodiments, the ELISA methods disclosed herein result in an AAV preparation that has at least about 20% less variability in concentration, dose, and/or activity compared to a qPCR method. In some embodiments, the ELISA methods disclosed herein result in an AAV preparation that has approximately 10-80% less variability in concentration, dose, and/or activity compared to the qPCR method. In some embodiments, the ELISA methods disclosed herein result in an AAV preparation that has approximately 15-50% less variability in concentration, dose, and/or activity compared to the qPCR method. In some embodiments, the ELISA methods disclosed herein result in an AAV preparation that has approximately 20-33% less variability in concentration, dose, and/or activity compared to the qPCR method.

In some aspects, the methods of the present invention quantify the dose and/or potency of an AAV preparation. In some embodiments, the method comprises examining an AAV preparation using an AAV-specific ELISA to quantify the total amount of AAV capsids in the AAV preparation. The number of capsids can then be used to determine the dose to be administered to the subject. Dose can be used to determine potency (ie dose or concentration required to achieve a particular effect (eg EC ₅₀ )). In certain embodiments, the method does not include the step of measuring dose and/or activity by qPCR.

In some aspects, the methods of the present invention include measuring the concentration of AAV capsids in an AAV fraction or preparation. In some embodiments, the method involves examining an AAV fraction or preparation using an AAV-specific ELISA to quantify the total amount of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration with qPCR.

In some aspects, the methods of the present invention comprise measuring the concentration of total AAV capsids in an AAV fraction or preparation. In some embodiments, the method comprises examining an AAV fraction or preparation using an AAV-specific ELISA assay to quantify the total AAV capsids in the AAV fraction or preparation and estimate the percentage of full and empty AAV capsids in the AAV preparation (e.g., using a cryogenic transmission electron microscopy (CryoTEM)). In certain embodiments, the method does not include the step of measuring the concentration using qPCR.

In some embodiments, empty capsids are removed from the AAV fraction or preparation, resulting in an AAV fraction or preparation with a certain concentration of full capsids and/or a certain ratio of full:empty capsids. In some embodiments, the concentration or ratio between batches is constant such that an AAV-specific ELISA is sufficient to determine the concentration, dose, and/or activity of an AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration, dose, and/or activity by qPCR.

In some aspects, the methods of the present invention quantify the dose and/or potency of an AAV preparation. In some embodiments, the method comprises examining an AAV preparation using an AAV-specific ELISA in combination with a method that evaluates or confirms the percentage or ratio of full to empty (full:empty) AAV capsids in the AAV preparation. In certain embodiments, the method does not include the step of measuring dose and/or activity by qPCR.

In some embodiments, the method comprises measuring the concentration of AAV capsids in an AAV fraction or preparation using an AAV-specific ELISA in combination with a method that evaluates or confirms the percentage or ratio of full:empty AAV capsids in the AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration with qPCR.

In certain embodiments, a method for quantifying the dose and/or potency of an AAV formulation comprises confirming the biological activity of the AAV formulation. In certain embodiments, the method comprises confirming the biological activity of an AAV preparation using an in vitro method. In some embodiments, the method comprises confirming the biological activity of an AAV preparation using an in vivo method. In some embodiments, the methods of the present invention do not require confirmation using a biological assay if an AAV-specific ELISA is combined with a method for confirming product quality (eg, by measuring full:empty ratios). For example, in certain embodiments, the methods of the present invention do not require confirmation using a biological assay if an AAV-specific ELISA assay is combined with CryoTEM. In certain embodiments, the method does not include the step of measuring dose and/or activity by qPCR.

In certain aspects, the methods of the present invention provide for an AAV preparation comprising: (i) transfecting host cells with at least one plasmid containing a gene of interest; (ii) collecting the supernatant or cell suspension of the cell culture containing AAV capsids to create an AAV fraction; (iii) quantifying the total AAV capsids in the AAV fraction using an AAV-specific ELISA assay; and (iv) obtaining an AAV preparation with the desired AAV capsid concentration based on the total AAV capsids determined in step (iii). In certain embodiments, the method involves concentrating the AAV fraction. In certain embodiments, the method includes removing at least a portion of the empty capsids from the AAV fraction. In some embodiments, at least a portion of the empty capsids are removed to create an AAV fraction or preparation with a certain concentration of full capsids and/or a certain ratio of full:empty capsids. In some embodiments, the method includes evaluating or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration, dose, and/or activity by qPCR. In certain embodiments, the method further comprises lyophilizing the AAV fraction or preparation.

In certain aspects, the methods of the present invention provide for administering an AAV preparation to a subject in need thereof, comprising (i) providing a purified AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay; (iii) administering to the subject a therapeutically effective amount of the purified AAV preparation. In some embodiments, the AAV preparation in steps (i) and (ii) is an AAV fraction that can be used to produce a final AAV preparation in which the AAV fraction is purified or diluted to form the AAV preparation of steps (i), (ii) and/or (iii). In some embodiments, the dose and/or activity of the purified AAV fraction or preparation is measured by quantifying the total amount of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method includes evaluating or confirming the percentage or ratio of full:empty AAV capsids in an AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration, dose, and/or activity by qPCR.

In certain aspects, the methods of the present invention comprise administering a specific dose of an AAV preparation to a subject in need thereof, comprising (i) providing a purified AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay; (iii) administering a specific dose of the AAV preparation to the subject. In some embodiments, the AAV preparation in steps (i) and (ii) is an AAV fraction that can be used to produce a final AAV preparation in which the AAV fraction is purified or diluted to form the AAV preparation of steps (i), (ii) and/or (iii). In some embodiments, the dose of the purified AAV fraction or preparation is measured by quantifying the total amount of AAV capsids in the AAV fraction or preparation. In certain embodiments, the method includes evaluating or confirming the percentage or ratio of full:empty AAV capsids in an AAV fraction or preparation. In certain embodiments, the method does not include the step of measuring concentration, dose, and/or activity by qPCR.

In certain embodiments, the ELISA assay may be a sandwich ELISA, a direct ELISA, an indirect ELISA, or a competitive ELISA. In certain embodiments of the methods disclosed herein, the AAV-specific ELISA is a sandwich ELISA specific for AAV antigen. In some embodiments, the AAV antigen is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 antigen, or a chimeric AAV antigen. In certain embodiments, the AAV antigen is an AAV8 antigen. In certain embodiments, the AAV antigen is derived from recombinant AAV (rAAV). In some embodiments, the AAV antigen is derived from a genetically engineered AAV or a chemically modified AAV, or both.

In certain embodiments, the ELISA provides for an antibody specific for an epitope of the AAV antigen. In some embodiments, the AAV epitope is a conformational epitope present on the assembled AAV capsids. In some embodiments, the AAV epitope is a linear epitope present on the assembled AAV capsids. Examples of such epitopes include, without limitation, the VP1, VP2 and/or VP3 virion capsid proteins. In some embodiments, AAVs are genetically engineered to express additional virion proteins on the surface of the capsid, and these engineered proteins can be used/detected in an ELISA assay. In some embodiments, the AAVs are chemically modified to express virion protein variants that can be used/detected in an ELISA assay (eg, VP1', VP2', VP3', etc.). In certain embodiments, the antibodies identify serotype-specific capsid proteins of the virion.

In some embodiments, for the methods disclosed herein, the method for assessing or confirming the percentage or ratio of full:empty AAV capsids in an AAV fraction or preparation can be CryoTEM, negative staining TEM, capillary electrophoresis, analytical ultracentrifugation, or combinations thereof. In some embodiments, a method for assessing or confirming the percentage of full and empty (full:empty) AAV capsids in an AAV fraction or preparation is CryoTEM. In some embodiments, the assessment involves quantifying the percentage or ratio of full:empty AAV capsids.

In some embodiments, the CryoTEM step comprises the following: (i) immersing the AAV fraction or preparation in the substrate into an inert support; (ii) flash freezing the submerged fraction or AAV preparation; (iii) visualization of the submerged AAV fraction or preparation using cryogenic transmission electron microscopy; and (iv) quantification of the percentage of full AAV capsids compared to empty AAV capsids in the AAV fraction or preparation. In certain embodiments, the substrate is amorphous non-crystalline ice. In certain embodiments, the AAV fraction or preparation does not contain cellular debris. For example, vector samples do not contain undefined particles >2%, do not contain broken or oligomerized particles >2%.

The use of CryoTEM in combination with an AAV-specific ELISA assay is advantageous over those known in the art because CryoTEM provides a direct measurement of full:empty capsids, as opposed to quantitative PCR (qPCR). In certain embodiments, the method does not include the step of measuring concentration or dose with qPCR.

In certain embodiments, for the methods disclosed herein, an ELISA assay is performed prior to quantification of full:empty AAV capsids. In certain embodiments, the ELISA assay is performed prior to CryoTEM. In some embodiments, an ELISA assay is performed to dilute the total number of AAV capsids to a validated range (eg, aq/mL ranges as described herein). In some embodiments, the ELISA assay is performed after the quantitation of full:empty AAV capsids. In certain embodiments, the ELISA assay is performed after CryoTEM. In some embodiments, an ELISA assay is performed to dilute the total number of AAV complete capsids to a validated range. In certain embodiments, the full:blank quantification is performed only once. For example, the CryoTEM step can be used once to verify that a batch has a constant empty:full ratio, as in previous batches.

In certain embodiments for the methods disclosed herein, the use of both ELISA and CryoTEM results in a method that can be used for all AAV serotypes, regardless of vector DNA. This is beneficial as it allows analysis across all platforms.

In certain embodiments, for the methods, fractions, or preparations disclosed herein, the percentage of full AAV capsids in the AAV fraction or preparation, compared to empty capsids, is from about 40% to about 100%. In a particular embodiment, the percentage of complete AAV capsids is from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods disclosed herein are used to produce AAV fractions or preparations with constant amounts of total capsids between batches of AAV fractions or preparations.

In certain embodiments, for the methods and formulations disclosed herein, the concentration of AAV formulations is between about 1×10¹⁰ k.h./ml up to approximately 1×10^twenty k.h./ml. In some embodiments, for the methods and preparations disclosed herein, the concentration of AAV preparations is from about 1x10^eleven k.h./ml up to approximately 1×10^nineteen k.h./ml, from approximately 1×10¹² k.h./ml up to approximately 1×10^eighteenk.h./ml, from approximately 1×10¹³ k.h./ml up to approximately 1×10¹⁷ k.h./ml or from about 1×10^fourteen k.h./ml up to approximately 1×10¹⁶ k.h./ml. In some embodiments, for the methods and preparations disclosed herein, the concentration of AAV preparations is from about 1x10¹² k.h./ml up to approximately 1×10^fifteen k.h./ml, from approximately 1×10¹³ k.h./ml up to approximately 1×10^fifteenk.h./ml or from about 1×10¹² k.h./ml up to approximately 1×10¹³ k.h./ml. In some embodiments, for the methods and preparations disclosed herein, the concentration of AAV preparations is from about 1x10^fourteen k.h./ml up to approximately 5×10^fourteen k.h./ml, from approximately 2×10^fourteen k.h./ml up to approximately 3×10^fourteen k.h./ml or from about 3.5×10^fourteen k.h./ml up to approximately 5×10^fifteen k.h./ml. In some embodiments, the q/ml can be all capsids per ml or complete capsids per ml. In some embodiments, c.h./ml is all capsids per ml.

In certain embodiments, for the methods or formulations disclosed herein, the dose of the AAV formulation is from about 1x10 ⁵ ch/kg to about 1x10 ²⁵ ch/kg. In certain embodiments, for the methods, fractions, or preparations disclosed herein, the dose of the AAV fraction or preparation is from about 1x10 ¹⁰ ch/kg to about 1x10 ²⁰ ch/kg. In certain embodiments, for the methods, fractions, or preparations disclosed herein, the dose of the AAV fraction or preparation is from about 1×10 ¹⁰ q/kg to about 1×10 ¹⁶ q/kg. In some embodiments, q.h./kg can be all capsids per kg of subject or full capsids per kg of subject. In certain embodiments, c.h./kg is all capsids per kg of subject.

In certain embodiments, for all of the methods disclosed herein, the subject is a human or non-human animal (e.g., chimpanzees and other monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous, ducks or geese). In certain embodiments, the subject is a human.

In some embodiments, for all methods disclosed herein, AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or an AAV chimeric vector. In certain embodiments, the AAV is AAV8. In certain embodiments, the AAV is AAV9. In certain embodiments, the AAV is recombinant AAV (rAAV). In certain embodiments, the AAV is genetically engineered, chemically modified, or both.

In certain aspects, the present invention provides an AAV fraction or preparation, wherein the concentration, dose and/or activity of the fraction or preparation is measured by an AAV-specific ELISA assay. In certain aspects, the present invention provides an AAV fraction or preparation, wherein the concentration, dose, and/or activity of the fraction or preparation is measured by an AAV-specific ELISA assay in combination with an assay that measures the percentage or ratio of full versus empty AAV capsids in the fraction or preparation. AAV. In certain aspects, the present invention provides an AAV fraction or preparation, wherein the concentration, dose and/or activity of the fraction or preparation is measured using an AAV-specific ELISA in combination with CryoTEM. In some embodiments, the concentration, dose, and/or activity of the AAV drug is not measured by qPCR.

Brief description of graphic materials

Fig. 1 is a graph comparing percent errors for measuring AAV vector amount and vector genome content using antigen ELISA or qPCR, respectively. ELISA AAV (QC ("Quality Control")): n=60; ELISA AAV (R&D ("Research and Development")): n=83; ITR-qPCR (R&D): n=64; FIX-qPCR (QC): n=40; FIX-qPCR (R&D): n=135.

Fig. 2 is the result of an agarose gel electrophoresis study investigating the ability of ELISA antigen and qPCR to determine the dose of AAV capsids immersed in agarose gel (2.0 x 10 ¹⁰ capsids/lane).

Fig. 3 is a graph showing the use of an ELISA method antigen to determine the dose of an AAV drug administered in an in vivo bioassay using a mouse model (n=7).

Fig. 4 is a plot comparing lot-to-lot variation of FAVIII AAV8 single-stranded DNA using AAV-specific ELISA versus ITR-qPCR. AAV8: antigen - median: 3.03 k.h./ml ± 0.66 k.h./ml. ITR-qPCR median: 9.11 vg/ml ± 3.01 vg/ml.

Fig. 5A-5B: FIG. 5A is a graph showing the ratio of AAV vector genomes (v.g.) per AAV8 antigen ELISA capsid particles (c.h.). Fig. 5B is a graph demonstrating the high consistency of "full" capsids in an AAV formulation (for example, see the process described in International Application Publication No. PCT/US2017/059967 incorporated herein in its entirety for all purposes).

Fig. 6A-6C: FIG. 6A is a CryoTEM image of one full AAV8 capsid and one empty AAV8 capsid. Fig. 6B is a CryoTEM image of a single oligomerized AAV8 capsid (i.e., the AAV capsid forms a trimeric structure that may be empty or complete). Fig. 6C is a CryoTEM image of one broken AAV8 capsid.

Fig. 7 is a graph of the analysis of the AAV8 fraction using CryoTEM. CryoTEM images were taken at 38k magnification and subjected to internal density analysis. Principal component analysis to create clusters of the radial density profile of each AAV particle revealed clusters of packing levels: complete (left), blue (right), indeterminate (middle) (darker marks indicate overlapping data points); dotted rings: 99% confidence intervals. 5 images analyzed, 1568 particles.

Fig. 8 is a graph confirming the use of CryoTEM to measure the percentage of complete AAV8 vector capsids.

Fig. 9 is a graph showing the correlation of vector genomes with capsid particles with the percentage of complete capsids.

Fig. 10 is a graph showing the effect of AAV8 percent complete capsid particles on in vivo activity in a factor IX knockout mouse model. Plasma samples were taken after 14 days and human factor IX clotting was measured.

Fig. 11 is a graph showing the effect of the percentage of total AAV8 capsid particles on in vitro biological activity. Biological activity was measured in a cellular assay associated with c.h. and plotted against percentage of total particles.

Fig. 12 is a plot comparing lot-to-lot variation of FAVIII AAV8 single-stranded DNA using AAV-specific ELISA versus ITR-qPCR for lots 17004-17013 (see Table 7).

Fig. 13 is a graph demonstrating the high consistency of "full" capsids in an AAV preparation (for example, see the process described in International Application Publication No. PCT/US2017/059967) for Lots 17004-17013 (see Table 7).

The additional accompanying figures are only for additional illumination of the present disclosure and do not limit the scope of the present disclosure, unless otherwise indicated.

Detailed description of the present invention

This document provides methods for measuring qualitative and/or quantitative traits of gene therapy vector preparations. In certain embodiments, the gene therapy vector preparations are AAV preparations, such as AAV8 or AAV9. All of the methods disclosed herein can be used to prepare an AAV preparation.

The use of gene therapy vectors depends on at least two biological activities for its activity: 1) the ability to transfer the DNA sequence into the cell and 2) the biological effect of the expressed genetic sequence. Achieving these activities requires the correct concentration or dose of AAV capsids containing the genomic sequence. This is due to the fact that "empty" capsids are not able to provide the therapeutic benefit associated with the production of the transgene. Thus, it is important to accurately measure the concentration or dose of an AAV preparation in order to ensure the delivery of an appropriate and/or consistent amount of complete AAV vector particles. This is particularly true because for recombinant proteins, the appropriate inter-batch/dose dispersion coefficient is ≦10% to 20%, with ≦10% being more ideal. Correct dose determination is important for determining the potency of an AAV drug. This is because activity is determined by graduated or quantitative dose-response curves. If the dose is inaccurate, the inaccuracy will be translated into measured activity. Inaccurate activity can lead to under- or over-treatment of a patient population.

As used herein, the terms "capsid", "capsid particle" and "particle" are used interchangeably and refer to an AAV particle consisting of at least one intact AAV capsid shell.

As used herein, the term "empty" in relation to AAVs or AAV capsids refers to those lacking a complete vector genome. Empty AAVs or empty AAV capsids or empty AAV particles cannot provide therapeutic benefit. As used herein, the term "complete AAV capsids" in relation to AAVs or AAV capsids or AAV particles refers to those containing the majority of the complete vector genome. Complete AAV capsids may provide therapeutic benefit to recipient patients. In some embodiments, "complete" may also include "incomplete vector DNA" or "truncated vector DNA". In certain embodiments, complete vs. incomplete and/or truncated vector DNA can be differentiated using additional analytical methods. Such methods include, but are not limited to, DNA sizing by capillary electrophoresis, AUC (analytical ultracentrifugation), % DNA in agarose gel (native or alkaline), Southern blot, dot blot hybridization, UV spectrophotometry, weak anion exchange chromatography, and mass -spectrometry (see Resolving Adeno-Associated Viral Particle Diversity with Charge Detection Mass Spectrometry Elizabeth E. Piersonet.al Anal. Chem., 2016, 88 (13), pp 6718-6725, which is incorporated herein in its entirety for all purposes) .

According to FDA guidelines published in January 2011 (Industry Guidance: Activity Study for Cellular and Gene Therapy Products), there are three ways to measure activity, such as: 1) in vivo biological assays; 2) non-biological in vitro assays; and 3) multiple studies. Biological assays, however, result in great variability due to dosage, assay, and readout variability. Non-biological assays also have problems. For example, quantitative PCR (qPCR) only measures the gene of interest. Thus, the use of qPCR may lead to an inaccurate determination of the activity of the final AAV preparation due to the inclusion of non-functional capsids, such as those that are oligomerized, disrupted, and/or containing more than one copy of DNA, in the dose calculation. Nonfunctional capsids cannot effectively transduce cells. In addition, qPCR is also limited due to its high intrinsic and inter-assay variability (ie, high variability up to 0.5 log step), which can lead to inaccurate determination of activity. If the concentration, dose and/or activity is not precisely defined, patients may receive widely varying doses from batch to batch and/or treatment to treatment. Overestimation of the dose or activity may lead to inadequate treatment of the disease and/or disorder. Insufficient assessment of dose or activity may result in side effects (eg, overcorrection (eg, blood clots) or an immune response caused by a large number of capsid particles in the body).

In some embodiments, the present invention provides methods for quantifying the concentration, dose, and/or activity of an AAV preparation, comprising quantifying the total amount of AAV capsids in the AAV preparation. In some embodiments, the method includes a) quantifying total AAV capsids, and optionally b) estimating and/or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in the AAV preparation.

In some embodiments, the quantification step involves determining the total amount of AAV capsids using ELISA, SPR (Surface Plasmon Resonance), differential scanning fluorometry technology, immunoassay methods, or combinations thereof. In certain embodiments, the quantitation step involves determining the total amount of AAV capsids by means of an AAV-specific ELISA. In some embodiments, the ELISA assay may be a sandwich ELISA, direct ELISA, indirect ELISA, or competitive ELISA. In certain embodiments, for the methods disclosed herein, the AAV-specific ELISA is a sandwich ELISA specific for AAV antigen.

In some embodiments, a method for assessing or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV fraction or preparation may be cryogenic transmission electron microscopy (CryoTEM), TEM with negative staining, chromatographic separation of full and empty capsids ( SEC, IEX, HIC, etc.), capillary electrophoresis, analytical ultracentrifugation, or combinations thereof. In some embodiments, a method for evaluating or validating the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV fraction or preparation may be CryoTEM. In certain embodiments, the methods include both AAV-specific ELISA and CryoTEM.

The methods disclosed herein accurately measure activity over this narrow range of variability. In certain embodiments, the disclosed methods result in a coefficient of variation from about 30% to about 1%, from about 27% to about 2%, from about 25% to about 5%, from about 22% to about 7%, from about 20% to about 10%, or from about 17% to about 12%. In some embodiments, the disclosed methods result in a coefficient of variation of ≤ about 30%, ≤ about 29%, ≤ about 28%, ≤ about 27%, ≤ about 26%, ≤ about 25%, ≤ about 25%, ≤ 24% ≤ approximately 23% ≤ approximately 22% ≤ approximately 21% ≤ approximately 20% ≤ approximately 19% ≤ approximately 18% ≤ approximately 17% ≤ approximately 16% ≤ approximately 16% ≤ 15% ≤ approximately 14% ≤ approximately 14% ≤ approximately 13% ≤ approximately 12% ≤ approximately 11% ≤ approximately 10% ≤ approximately 9% ≤ approximately 8% ≤ approximately 7% ≤ approximately 6%, ≤ approximately 6%, ≤ approximately 5%, ≤ approximately 4%, ≤ approximately 4%, ≤ approximately 3%, ≤ approximately 2%, ≤ 1%, but in all cases ≥ 0.

In some embodiments, the methods disclosed herein, including ELISA, result in an AAV preparation with a variability in concentration, dose, and/or potency from about 10% to about 80%, compared to a qPCR method. In some embodiments, the methods disclosed herein, including ELISA, result in an AAV preparation with less variability in concentration, dose, and/or activity from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 65%, about 15% to about 60%, about 15% to about 55%, about 20% to about 50%, about 20% to about 48%, about 20% to about 46 %, from about 20% to about 44%, from about 20% to about 42%, from about 20% to about 40%, from about 20% to about 38%, from about 20% to about 36%, from about 20 % to about 34%, or from about 20% to about 32%, compared to the method that uses qPCR. In some embodiments, the methods disclosed herein, including ELISA, result in an AAV preparation with approximately 20-33% less concentration, dose, and/or potency variability compared to a qPCR method. In some embodiments, the methods disclosed herein, including ELISA, result in an AAV preparation with less concentration, dose, and/or potency variability of at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18% %, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least e by about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42 %, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, or at least about 50%, compared to the method that uses qPCR.

In certain embodiments, the methods involve testing an AAV preparation using an AAV-specific ELISA. In some embodiments, an AAV-specific ELISA is sufficient to provide a representative reading of the dose or activity of the AAV preparation, since most of the capsids in the AAV preparation are complete capsids. For example, the percentage of AAV complete capsids is in the majority from about 50% to about 100%. In a particular embodiment, the percentage of complete AAV capsids is from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 55% to about 50%, from about 60% to about 80%, or from about 65% to about 75%. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 % or at least about 100% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In some embodiments, the AAV-specific ELISA is sufficient to provide a representative reading of the activity of the AAV preparation because the ratio of full to empty capsids in the AAV preparation is constant from lot to lot. In some embodiments, the term "constant" refers to a coefficient of variation (CV) from about 0% to about 15%, from about 2% to about 12%, from about 5% to about 10%, from about 7% to about 9% . In some embodiments, the term "permanent" refers to a CV of about 15% or less, about 12% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. In certain embodiments, "permanent" refers to a CV of about 1% or less. In some embodiments, an AAV-specific ELISA in combination with CryoTEM is required to study the activity of an AAV preparation.

In certain embodiments, the methods involve examining an AAV fraction or preparation using CryoTEM. In certain embodiments, the CryoTEM is sufficient to provide a representative indication of the activity of the AAV preparation. In some embodiments, CryoTEM in combination with an AAV-specific ELISA is required to study the activity of an AAV preparation.

In certain embodiments, an AAV preparation produced and/or measured using the methods of the present invention is suitable for administration to a subject. Applicable AAV vectors are described in more detail below. In certain embodiments, the AAV preparation produced by the methods of the present invention is sterile and/or Good Manufacturing Practice (GMP) grade. In some embodiments, an AAV preparation made using the methods of this disclosure complies with the requirements set forth in USP Chapter 1046 or the European Pharmacopoeia for Gene Therapy Medicines, or as prescribed by the US Food and Drug Administration (USFDA) or the European Pharmacopoeia. medicines agency (EMA).

The methods used herein can be used for an AAV fraction and/or an AAV preparation. The terms "preparation" and "fraction" can be used interchangeably in this application. In some embodiments, the term "fraction" may mean a sample or batch of AAV that can be used to create a final AAV "preparation" to be administered to a subject. For example, the AAV fraction may be further processed, purified, or volume adjusted before forming the final AAV preparation. Quantitative and/or qualitative methods

The methods of the present invention include one or more quality control steps, e.g., steps to measure the concentration, dose, and/or activity of fractions or AAV preparations obtained after one or more steps (e.g., after each step) of the purification process, including a final preparation step. drug for administration.

The methods of the present invention may include an ELISA specific for AAV (eg, AAV antigen) to quantify the amount of AAV capsids. In some embodiments, the ELISA assay may include, without limitation, immunosorbent assay, direct ELISA, indirect ELISA, sandwich ELISA, and/or competitive ELISA. In certain embodiments, the ELISA is a sandwich ELISA. A non-limiting example of an ELISA assay is provided in Example 1.

In certain embodiments, the AAV antigen is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 antigen, or a chimeric AAV antigen. In certain embodiments, the AAV antigen is an AAV8 antigen. In certain embodiments, the AAV antigen is derived from recombinant AAV (rAAV). In some embodiments, the AAV antigen is derived from a genetically engineered AAV or a chemically modified AAV, or both.

In certain embodiments, the ELISA contains an antibody specific for an AAV epitope. In some embodiments, the AAV epitope is a conformational epitope present on the assembled AAV capsids. In some embodiments, the AAV epitope is a linear epitope present on the assembled AAV capsids. Examples of such epitopes include, without limitation, the VP1, VP2 and/or VP3 virion capsid proteins. In some embodiments, AAVs are genetically engineered to express additional virion proteins on the surface of the capsid, and these engineered proteins can be used/detected in an ELISA assay. In some embodiments, the AAVs are chemically modified to express virion protein variants that can be used/detected in an ELISA assay (eg, VP1', VP2', VP3', etc.). In certain embodiments, the antibodies identify serotype-specific proteins of the capsid virion.

ELISA can replace qPCR as a method for determining the dose and/or activity of an AAV fraction or preparation. As shown in FIG. 1, the ELISA method of the present invention has significantly less variability than the qPCR method. In some embodiments, the methods of the present invention involve evaluating an AAV fraction or preparation using an AAV-specific ELISA. In certain embodiments, all of the methods disclosed herein do not include a qPCR step.

In some embodiments, the methods of the present invention involve testing the ultracentrifuged AAV fraction or preparation with an AAV-specific ELISA to determine the amount of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention include the analysis of the fraction or preparation of AAV obtained after deep filtration, using AAV-specific ELISA to determine the number of AAV capsids in the fraction or preparation of AAV. In some embodiments, the methods of the present invention involve examining an AAV fraction or preparation obtained after concentration of the AAV fraction or preparation using an AAV-specific ELISA ultra-/diafiltration system to determine the amount of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve testing an AAV fraction or preparation obtained after a tangential flow filtration (TFF) step using an AAV-specific ELISA to determine the amount of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present disclosure involve examining a negative anion exchange chromatography (AEX) AAV fraction or preparation with an AAV-specific ELISA to determine the amount of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve examining the AAV fraction or preparation obtained after the final purification step using an AAV-specific ELISA to determine the amount of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve testing a purified AAV preparation with an AAV-specific ELISA to determine the amount of AAV capsids in the AAV preparation.

Additional methods for quantifying AAV capsids include, but are not limited to, surface plasmon resonance (SPR) (e.g., BIACORE, OCTET), differential scanning fluorometry (e.g., Prometheus NT48, Nanotemper), magnetic immunoassay (MIA), and cloned enzyme donor immunoassay. (CEDIA). These methods can be used in addition to or instead of quantitative ELISA analysis.

In some embodiments, the methods of the present disclosure comprise measuring full and empty AAV capsids (eg, percentage of full vs. empty AAV capsids). A method for evaluating or confirming the percentage or ratio of full:empty AAV capsids in an AAV fraction or preparation includes, but is not limited to, cryogenic transmission electron microscopy (CryoTEM), negative staining TEM, capillary electrophoresis, analytical ultracentrifugation, or combinations thereof.

In some embodiments, the method for measuring full and empty AAV capsids is CryoTEM (see FIG. 6). In certain embodiments, the method involves the use of both CryoTEM and AAV-specific ELISA as described above. In certain embodiments, the ELISA is a sandwich ELISA. In some embodiments, the sandwich ELISA contains an antibody specific for an AAV epitope. In some embodiments, the AAV epitope is a conformational epitope present on the assembled AAV capsids. A non-limiting method for measuring the number of full and empty capsids using CryoTEM is described herein as Example 2.

One advantage of using CryoTEM is that there is no need for negative staining. CryoTEM also provides the ability to quantify the amount of complete AAV capsids. In some embodiments, the use of CryoTEM results in no false positive overestimation of AAV full capsids.

The methods of the present disclosure include a method assessing the amount or percentage of full and empty AAV capsids. In certain embodiments, the methods include CryoTEM. In certain embodiments, the methods comprise: (i) immersing the AAV fraction or preparation in a substrate into an inert support; (ii) flash freezing the submerged fraction or AAV preparation; (iii) visualization of the submerged fraction or AAV preparation using cryogenic transmission electron microscopy; and (iv) quantification of the percentage of full AAV capsids compared to empty AAV capsids.

For step (i), examples of suitable substrates for use in the process include, without limitation, amorphous non-crystalline ice. Examples of suitable inert support for use in the process include, but are not limited to, carbon film, thermoplastic resins, and polyvinyl formals (e.g., polymers formed from polyvinyl alcohol and formaldehyde as copolymers with polyvinyl acetate, such as, but not limited to, Formvar or Vinylec , carbon-stabilized polyvinyl formaldehyde, silicon monoxide on polyvinyl formaldehyde, pure carbon film, type A carbon: carbon carrier film (for example, removable polyvinyl formal on the opposite side of the grid), type B carbon: polyvinyl formal film (for example, coated with a heavier layer of carbon) and silicon monoxide on carbon type A). In some embodiments, step (i) above involves depositing a sample onto a thin support film of carbon in a temperature (eg, (-196° C.) or lower) and humidity controlled environment. In certain embodiments, the humidity level may be relative humidity. In step (ii) the sample may be flash frozen. In some embodiments, flash freezing may involve the use of liquid ethane, liquid nitrogen, liquid propane, or helium near liquid nitrogen temperature. For example, a container of liquid ethane, liquid nitrogen, liquid propane, or helium is surrounded by liquid nitrogen.

In some embodiments, the AAV fraction or preparation is frozen so rapidly (eg, at 104-106 K per second) that ice crystals cannot form. In some embodiments, amorphous ice is produced either by rapidly cooling liquid water or by compressing regular ice at low temperatures. In some embodiments, after removing excess AAV fraction or preparation, leaving some samples stuck, the mesh is vitrified in liquid ethane and then stored in liquid nitrogen. For a discussion of additional steps for performing CryoTEM, see Cabra and Samso, J. Visualized Experiments, (2015) 95(e52311): 1-11, incorporated herein by reference in its entirety for all purposes.

CryoTEM analysis of AAV particles can be used to assess the overall morphology of a sample, i.e. the presence of various AAV morphologies (generally including spherical and deformed AAV particles, subunit structures, and larger structurally less defined morphologies). In FIG. 6A is an example of how a "full" from an "empty" AAV particle can be visually distinguished. For example, a full capsid displays internal density without a clear boundary between the shell and the nucleus. AAV capsids that have a distinctly distinct shell and minimal internal density are classified as empty capsids.

CryoTEM can also be used to evaluate undefined capsids. In FIG. 6B and 6C are examples of deformed/indeterminate capsids that will not be included as full capsids. For example, CryoTEM can be used to enumerate "full", "empty", and "indeterminate" capsids.

CryoTEM can also be used to determine particle packing levels by manual particle classification or using automated image analysis methodology.

In some embodiments, the methods of the present invention involve examining an AAV fraction or preparation obtained after CryoTEM ultracentrifugation to determine the quantity and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present disclosure involve examining an AAV fraction or preparation obtained after deep filtration with CryoTEM to determine the amount and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention examine an AAV fraction or preparation obtained after concentration of an AAV fraction or preparation using an ultra-/diafiltration system through CryoTEM to determine the quantity and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present disclosure involve examining an AAV fraction or preparation obtained from a tangential flow filtration (TFF) step with CryoTEM to determine the amount and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve examining a negative anion exchange chromatography (AEX) AAV fraction or preparation using CryoTEM to determine the quantity and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve examining an AAV fraction or preparation obtained from a CryoTEM final purification step to determine the quantity and/or quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention include CryoTEM examination of a purified AAV preparation to determine the quantity and/or quality of AAV capsids in an AAV fraction or preparation.

In some embodiments, the methods of the present invention involve testing the ultracentrifuged AAV fraction or preparation using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present disclosure involve examining the AAV fraction or preparation obtained after deep filtration using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present disclosure involve testing an AAV fraction or preparation obtained after concentrating an AAV fraction or preparation using an ultra-/diafiltration system using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. . In some embodiments, the methods of the present disclosure involve examining an AAV fraction or preparation obtained after a tangential flow filtration (TFF) step using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention include testing an AAV fraction or preparation obtained after negative anion exchange chromatography (AEX) using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve examining the AAV fraction or preparation obtained from the final purification step using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in the AAV fraction or preparation. In some embodiments, the methods of the present invention involve testing a purified AAV preparation using an AAV-specific ELISA and CryoTEM to determine the quantity and quality of AAV capsids in an AAV fraction or preparation.

Analytical ultracentrifugation (AUC) is able to separate proteins according to sedimentation ratio. For identical proteins, the sedimentation coefficient can be related to the state of aggregation. Briefly, samples are diluted with an appropriate sample buffer and then transferred to cells with built-in quartz windows and loaded into a rotor which then rotates at a constant speed. Protein molecules of different sizes migrate at different settling rates to the bottom of the cell and are continuously monitored during centrifugation by UV detection at 280 nm. The collected dataset allows for a computational analysis that leads to a deconvolution of the sedimentation and diffusion processes, resulting in a differential distribution of the sedimentation coefficient c(s). It allows different kinds of samples and represents their s-values and populations. The distribution of sedimentation coefficients is integrated, and relative area percentages as well as S-values of peak maxima are given as the results of the analysis.

AAV drug

An AAV preparation obtained by the method of the present invention is further provided herein. In certain embodiments, a method for producing an AAV preparation comprises the following: (i) transfecting host cells with at least one plasmid containing a gene of interest; (ii) collecting the supernatant or cell suspension of the cell culture containing AAV capsids to create an AAV fraction or preparation; (iii) quantifying the total AAV capsids in the AAV fraction or preparation using an AAV-specific ELISA assay; and (iv) obtaining an AAV preparation at the desired concentration based on the total AAV capsids determined in step (iii). In certain embodiments, the method involves concentrating the AAV fraction. In certain embodiments, the method includes removing at least a portion of the empty capsids from the AAV fraction. In some embodiments, the number of empty capsids is removed to create an AAV fraction or preparation with a specific concentration of full capsids and/or a specific ratio of full:empty capsids. In a certain embodiment, the AAV fraction or preparation is diluted with an appropriate buffer to the desired dose.

In some embodiments, the method includes evaluating or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV fraction or preparation. In some embodiments, a method comprising estimating or confirming a percentage or ratio of full to empty (full:empty) AAV capsids using CryoTEM. In some embodiments, dose and/or activity are not determined by qPCR. In some embodiments, the percentage of complete AAV capsids is from about 40% to about 100%. In a particular embodiment, the percentage of complete AAV capsids is from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In some embodiments, the percentage of complete AAV capsids is from about 60% to about 80%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods disclosed herein are used to obtain AAV fractions or preparations with constant amounts of total capsids between AAV fractions or preparation.

The method of administration of the AAV preparation obtained by the method of the present invention is further presented in this document. In certain embodiments, a method of administering an AAV preparation to a subject in need comprises the following: (i) obtaining a purified AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay; (iii) administering to the subject a therapeutically effective amount of the purified AAV preparation. In some embodiments, the formulation in steps (i) and (ii) is an AAV fraction that can be used to produce a final AAV formulation in which the AAV fraction is further purified or diluted to form the AAV formulation for steps (i), (ii) and /or (iii). In some embodiments, the concentration, dose, and/or potency of the purified AAV preparation is measured by quantifying the total amount of AAV capsids in the AAV preparation. In some embodiments, the method includes evaluating or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV preparation. In some embodiments, the method for assessing or confirming the percentage or ratio of full to empty (full:empty) AAV capsids is CryoTEM. In some embodiments, the concentration, dose, and/or activity is not determined by qPCR. In some embodiments, the percentage of complete AAV capsids is from about 40% to about 100%. In a particular embodiment, the percentage of complete AAV capsids is from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In some embodiments, the percentage of complete AAV capsids is from about 60% to about 80%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids.

The method of administration of the AAV preparation obtained by the method of the present invention is further presented in this document. In certain aspects, the methods of the present invention provide for administering a dose of an AAV preparation to a subject in need thereof, comprising: (i) obtaining a purified AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay; (iii) administering a specific dose of the AAV preparation to the subject. In some embodiments, the formulation in steps (i) and (ii) is an AAV fraction that can be used to produce a final AAV formulation in which the AAV fraction is further purified or diluted to form the AAV formulation for steps (i), (ii) and /or (iii). In some embodiments, the method includes evaluating or confirming the percentage or ratio of full to empty (full:empty) AAV capsids in an AAV preparation. In some embodiments, the method for assessing or confirming the percentage or ratio of full to empty (full:empty) AAV capsids is CryoTEM. In some embodiments, the concentration, dose, and/or activity is not determined by qPCR. In some embodiments, the percentage of complete AAV capsids is from about 40% to about 100%. In a particular embodiment, the percentage of complete AAV capsids is from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 45% to about 75%, about 50% to about 70%, or about 55% to about 65%. In some embodiments, the percentage of complete AAV capsids is from about 60% to about 80%. In certain embodiments, at least about 60% of the AAV capsids are complete AAV capsids. In certain embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 %, at least about 98%, at least about 99%, or at least about 100% of the AAV capsids are complete AAV capsids.

In certain embodiments, the activity of the AAV preparation can be further confirmed by in vitro and/or in vivo assay of biological activity. For example, the steps may include: 1) determining the content of AAV in the preparation (for example, using ELISA); 2) determination of the percentage of complete capsids (eg, using CryoTEM); and 3) confirmation of biological activity (eg, using quantitative analysis of biological activity in vivo and/or in vitro).

In certain embodiments, for the methods, fractions, or preparations disclosed herein, the concentration of AAV preparations is from about 1×10 ¹⁰ q/ml to about 1×10 ²⁰ q/ml. In certain embodiments, for the methods, fractions, or formulations disclosed herein, the concentration of AAV formulations is from about 1×10 ¹¹ q/ml to about 1×10 ¹⁹ q/ml, from about 1×10 ¹² k .h/ml up to about 1×10 ¹⁸ q/ml, from about 1×10 ¹³ q/ml to 1×10 ¹⁷ q/ml or from 1×10 ¹⁴ q/ml ./ml up to 1×10 ¹⁶ k.h./ml. In some embodiments, for the methods and formulations disclosed herein, the AAV drug concentration is from about 1×10 ¹² q/ml to about 1×10 ¹⁵ q/ml, from about 1×10 13 q/ml, from about 1×10 ¹³ q/ml ./ml to about 1×10 ¹⁵ q/ml or from about 1×10 ¹² q/ml to about 1×10 ¹³ q/ml. In certain embodiments, for the methods and formulations disclosed herein, the AAV drug concentration is from about 1×10 ¹⁴ q/ml to about 5×10 ¹⁴ q/ml, from about 2×10 14 q/ml, from about 2×10 ¹⁴ q/ml ./ml to about 3×10 ¹⁴ q/ml or from about 3.5×10 ¹⁴ q/ml to about 5×10 ¹⁵ q/ml. In some embodiments, the q.h./ml can be all capsids per ml or complete capsids per ml. In some embodiments, c.h./ml is all capsids per ml.

In some embodiments, the AAV formulation contains at least about 10 ¹² viral particles (v.p.) derived from about 1000 L of starting material (e.g., cell culture), or at least about 10 ¹³ viral particles (b.p. ) obtained from approximately 1000 liters of starting material (eg cell culture).

In certain embodiments, for the methods or preparations disclosed herein, the dose of an AAV preparation is from about 1x10^five c.h./kg up to approximately 1×10²⁵ k.h./kg. In certain embodiments, for the methods or preparations disclosed herein, the dose of an AAV preparation is from about 1x10⁶ c.h./kg up to approximately 1×10²⁴k.h./kg, from approximately 1×10⁷ c.h./kg up to approximately 1×10²³ k.h./kg, from approximately 1×10^eight c.h./kg up to approximately 1×10²² k.h./kg, from approximately 1×10^nine c.h./kg up to approximately 1×10²¹ k.h./kg, from approximately 1×10¹⁰ c.h./kg up to approximately 1×10^twenty k.h./kg, from approximately 1×10^eleven c.h./kg up to approximately 1×10^nineteenk.h./kg, from approximately 1×10¹² c.h./kg up to approximately 1×10^eighteen k.h./kg, from approximately 1x10¹³ c.h./kg up to approximately 1×10¹⁷ k.h./kg or from about 1×10^fourteen k.h./kg up to 1×10¹⁶ k.h./kg. In certain embodiments, for the methods and formulations disclosed herein, the dose of an AAV formulation is from about 1×10¹² c.h./kg up to approximately 1×10^twenty k.h./kg, from approximately 1×10¹³ c.h./kg up to approximately 1×10^nineteen k.h./kg, from approximately 1×10^fourteen c.h./kg up to approximately 1×10^eighteen k.h./kg or from about 1×10^fifteen c.h./kg up to approximately 1×10¹⁷ k.h./kg. In certain embodiments, for the methods and formulations disclosed herein, the dose of an AAV formulation is from about 1×10¹⁰ c.h./kg up to approximately 1×10¹⁶ k.h./kg. In certain embodiments, for the methods and formulations disclosed herein, the dose of an AAV formulation is at least about 1×10⁶ k.h./kg, at least approximately 1×10⁷ k.h./kg, at least approximately 1×10^eight k.h./kg, at least approximately 1×10^nine k.h./kg, at least approximately 1×10¹⁰k.h./kg, at least approximately 1×10^eleven k.h./kg, at least approximately 1×10¹² k.h./kg, at least approximately 1×10¹³ k.h./kg, at least approximately 1×10^fourteen k.h./kg, at least approximately 1×10^fifteen k.h./kg, at least approximately 1×10¹⁶ k.h./kg, at least approximately 1×10¹⁷ k.h./kg, at least approximately 1×10^eighteen k.h./kg, at least approximately 1×10^nineteenk.h./kg, at least approximately 1×10^twenty k.h./kg, at least approximately 1×10²¹ k.h./kg, at least approximately 1×10²² k.h./kg, at least approximately 1×10²³ k.h./kg, at least approximately 1×10²⁴ k.h./kg or at least approximately 1×10²⁵ k.h./kg. In some embodiments, q.h./ml can be all capsids per kg of subject or complete capsids per kg of subject. In some embodiments, c.h./ml is all capsids per kg of subject.

In certain embodiments, the AAV formulation of the present invention is highly pure, highly potent, and suitable for clinical use in a subject. In certain embodiments, the AAV preparation contains AAV capsid particles of a homogeneous population and high purity. In certain embodiments, the AAV preparation contains full-length vector DNA. In exemplary embodiments, the AAV preparation is substantially free of unwanted contaminants, including, but not limited to, AAV capsid particles containing truncated or incomplete vector DNA, incomplete proteinaceous AAV particles with oligomerized structures, or contaminating viruses, e.g., non-AAV, lipid-enveloped viruses. In exemplary embodiments, the AAV preparation contains a large amount of the cDNA coding for the protein of interest. In certain embodiments, the AAV formulation of the present invention is suitable for administration to a subject. In certain embodiments, the AAV preparation is sterile and/or Good Manufacturing Practice (GMP) grade. In some embodiments, the AAV formulation meets the requirements set forth in USP Chapter 1046 or the European Pharmacopoeia for Gene Therapy Medicines, or meets the requirements of the US Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). In certain embodiments, the AAV formulation is a ready-to-use formulation for direct administration to a subject with little or no processing.

The terms "patient" and "subject" are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administering the composition of the present invention, and includes both humans and animals other than humans. Examples of subjects include, without limitation, humans, chimpanzees, and other primate species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and hunting birds, such as chickens, turkeys and other chickens, ducks, geese, etc. The term does not denote a specific age. Thus, adults, minors and newborns are of interest.

AAV Source

With respect to the methods of the present disclosure, the AAV may be of any AAV serotype. In certain embodiments, the AAV described herein is characterized by AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, or is an AAV chimeric vector. In certain embodiments, the AAV is wild-type AAV. In certain embodiments, the AAV is recombinant AAV (rAAV). In certain embodiments, the AAV is genetically engineered and/or chemically modified. In certain embodiments, the AAV contains a modified capsid, such as a genetically engineered or chemically modified AAV capsid. In certain embodiments, AAV is characterized by the AAV8 serotype. In certain embodiments, AAV is characterized by the AAV9 serotype.

With respect to the methods of the present invention, the AAV fraction according to illustrative aspects is a concentrated AAV fraction. In some embodiments, the AAV fraction contains at least about 1x10 ¹⁰ , about 1x10 ¹¹ , about 1x10 ¹² , about 1x10 ¹³ , about 1x10 ¹⁴ , about 1x10 ¹⁵ , or about 1x10 ¹⁶ total AAV capsids per ml. In certain embodiments, the AAV fraction contains at least about 1×10 ¹² total AAV capsids per ml. AAV capsids may include AAV empty capsids and AAV full capsids.

In certain embodiments, the AAV is a fraction or preparation of AAV produced by the transfected host cells. In some embodiments, the AAV fraction or preparation is a harvested supernatant or cell suspension from a cell culture containing host cells transfected with a triple plasmid system, wherein one plasmid of the system contains a gene or cDNA of interest, one plasmid encodes a VP1 capsid protein, a VP2 capsid protein and/or VP3 capsid protein. In some embodiments, VP1, VP2 and/or VP3 are VP1, VP2 and/or VP3 of AAV8. Triple plasmid transfection to produce rAAV is known in the art. See, for example, Qu et al., 2015, supra, and Mizukami et al., "A Protocol for AAV vector production and purification." PhD dissertation, Division of Genetic Therapeutics, Center for Molecular Medicine, 1998 and Kotin et al., Hum Mol Genet 20(R1): R2-R6 (2011). In some embodiments, transfection can be performed using inorganic compounds, such as calcium phosphate, or organic compounds, polyethyleneimine (PEI), or non-chemical means, such as electroporation.

In some embodiments, the host cells are adherent cells. In certain embodiments, the host cells are suspension cells. In some embodiments, the host cells are HEK293 cells or Sf9 cells (eg, Sf9 cells infected with baculovirus) or HeLa or BHK (herpes virus system). In certain embodiments, the cell culture contains a culture medium that is free of serum and protein. In some embodiments, the medium is chemically defined and does not contain animal derived components, such as hydrolysates.

In certain embodiments, the fraction containing rAAV particles is a fraction containing HEK293 cells transfected with a triple plasmid system. In some embodiments, the fraction containing AAV particles is the harvest fraction about 2-7 days after transfection of HEK293 cells, or when the cell culture has a cell density greater than or about 5×10 ⁶ cells/mL and cell viability is greater than or about fifty%.

In some embodiments, AAV is obtained by triple plasmid transfection followed by harvest 1-7 days later. In certain embodiments, AAV is obtained from cell disruption.

In some embodiments, AAV is produced as follows: HEK293 cells are adherent and grown in a commercially available culture medium, which may be chemically defined and may be free of animal components such as serum and proteins. The cells are cultured to a cell density of about 3x10 ⁶ to about 12x10 ⁶ cells/ml, eg about 6x10 ⁶ to about 10x10 ⁶ cells/ml. The cells are then diluted at a ratio of approximately 1:2 so that the cell density is approximately 3-5×10 ⁶ cells/ml. Cells can then be transfected with three plasmids that include (1) a helper plasmid capable of providing one or more of the viral helper functions required for AAV production, (2) a plasmid that encodes one or more genes involved in capsid production , replication and packaging of the virus, and (3) a plasmid containing a gene of interest (GOI) to be packaged into the resulting rAAV particle. For example, the GOI may be a vector DNA containing human clotting factor IX Padua in single stranded self-complementary form with the vector DNA. As another example, the GOI may be a vector DNA containing human Padua clotting factor IX in a double-stranded self-complementary form, the vector DNA having a total length of 4.8 kb. As another example, the GOI may be a vector DNA containing human coagulation factor VIII with the B domain deleted in a single-stranded self-complementary form, the vector DNA having a total length of 4.8 kb. Other GOIs may be used. Transfection can be carried out in a transient manner, for example using cationic polymers. Prior to eluting, the HEK293 cell line may be cultured for at least about 1 day, eg 3-5 days, prior to harvest.

In some embodiments, the fraction containing AAV particles is described in US Provisional Application No. 62/417775 and International Application Publication No. PCT/US2017/059967, each of which is incorporated herein in its entirety.

Purification stages

The methods of the present disclosure include any combination of the steps disclosed herein and may optionally be combined with one or more additional steps.

In exemplary embodiments, the methods of the present invention include an ultracentrifugation step during which a density gradient is formed. Without wishing to be bound by theory, it is believed that the ultracentrifugation step separates full AAV capsids from empty AAV capsids. Examples of ultracentrifugation protocols can be found, for example, in WO 2008135229 and PCT/US17/59967, each of which is incorporated herein in its entirety for all purposes.

The methods of the present invention may include still other additional steps that may further improve the purity of the AAV and remove other unwanted components and/or concentrate the fraction and/or prepare the fraction for the next step.

In certain embodiments, the method includes a deep filtration step. In some embodiments, the method involves subjecting the supernatant fraction of the transfected HEK293 cell culture to deep filtration using a filter containing cellulose and perlite and having a minimum permeability of approximately 500 l/m ² . In certain embodiments, the method further comprises using a filter with a minimum pore size of approximately 0.2 microns. In some embodiments, depth filtration is followed by filtration through a filter with a minimum pore size of approximately 0.2 microns. In some embodiments, one or both of the depth filter and the filter with a minimum pore size of approximately 0.2 µm are washed and the washes are collected. In some embodiments, the washes are combined and combined with the filtrate obtained from deep filtration and filtration with a filter having a minimum pore size of approximately 0.2 microns. In certain embodiments, the deep filtration step and the other filtration step occur prior to the ultracentrifugation step described herein.

In certain embodiments, the methods of the present invention include one or more chromatography steps. In some embodiments, the methods include a negative chromatography step in which unwanted components are bound to the chromatographic resin and the desired AAV is not bound to the chromatographic resin. In some embodiments, the methods include a negative anion exchange chromatography (AEX) step or an AEX chromatography step in "no binding mode". The advantages of "no-binding mode" include the relative ease of procedure and subsequent analysis. Accordingly, in some embodiments, methods for purifying AAV particles comprise performing negative anion exchange chromatography (AEX) on a fraction containing AAV particles by applying the fraction to an AEX chromatography column or membrane under conditions that allow the AAV to pass through the AEX chromatography column or membrane, and collecting AAV particles. In some embodiments, the fraction is applied to an AEX chromatography column or membrane with a loading buffer containing from about 100 mM to about 150 mM salt, such as NaCl, optionally, the pH of the loading buffer is from about 8 to about 9. In some embodiments, the loading buffer the buffer contains from about 115 mM to about 130 mM salt, eg NaCl, optionally, the loading buffer contains from about 120 mM to about 125 mM salt, eg NaCl. In certain embodiments, the negative AEX step occurs prior to the ultracentrifugation step described herein.

In certain embodiments, the methods of the present disclosure involve concentrating the AAV fraction using an ultra/diafiltration system. In certain embodiments, the methods of the present disclosure include one or more tangential flow filtration (TFF) stages. In certain embodiments, the AAV fraction is subjected to ultra-/diafiltration. In some embodiments, the AAV fraction is concentrated in the ultra/diafiltration system before the negative AEX chromatography step, after the negative AEX chromatography step, or before and after the negative AEX chromatography.

In some embodiments, the methods of the present invention involve treating a fraction containing AAV particles with a soluble detergent to inactivate lipid-enveloped viruses.

In certain embodiments, the methods of the present invention filter the fraction containing rAAV particles to remove viruses larger than the rAAV particles in the fraction. In some embodiments, the method of the present invention includes filtering the AAV-containing fraction to remove viruses of 35 nm or greater. In some embodiments, the pore size of the filter is in the nanometer range, and in some embodiments, the method involves nanofiltration. In some embodiments, the method of the present invention involves the use of a nanofilter with a pore size in the range of 35 nm ± 2 nm, as determined by the water flow method. Filter type classification depends on membrane structure, material and supplier.

According to certain embodiments, a differential pressure across the filter is maintained during the filtration step. In some embodiments, the pressure (pressure drop across the filter) is from about 0.02 MPa to about 0.1 MPa. In some embodiments, the pressure (eg, pressure drop across the filter) is from about 0.02 MPa to about 0.08 MPa. In the event that the filter is in dead-end mode, the pressure drop can be influenced by the supply pressure of the sample being fed (i.e. by setting the pump to a specific flow that affects the supply pressure).

In certain embodiments, the filtration step to remove viruses larger than rAAV particles occurs once during the process of the present invention. In certain embodiments, the filtration step occurs twice during the process. In certain embodiments, the filtration step to remove viruses larger than rAAV particles occurs after the ultracentrifugation step described herein. In some embodiments, the filtration step to remove viruses larger than rAAV particles occurs after the final purification step.

In some embodiments, the methods of the present invention include a final purification step that involves performing AEX chromatography, optionally with a tentacle gel column.

All references, including publications, patent applications, and patents cited herein, are hereby incorporated by reference to the same extent as if each reference were individually and specifically cited for inclusion by reference and are set forth herein in their entirety.

The use of the singular and similar references in the context of the description of the present disclosure (especially in the context of the following claims) is to be construed to include both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. The terms "comprising", "having", and "including" are to be construed as open terms (ie, meaning "including, without limitation"), unless otherwise indicated.

The listing of ranges of values in this document is simply intended to serve as a concise way of individually referring to each individual value falling within the range and each endpoint, unless otherwise noted herein, and each individual value and endpoint is included in the description as if it were individually stated in this document.

All methods described herein can be performed in any suitable order, unless otherwise specified herein or otherwise clearly contradicts the context. The use of any and all examples or exemplary language (eg, "such as") provided herein is merely intended to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure unless otherwise stated. No mention in the description should be construed as pointing to any unclaimed element as essential to the practice of this disclosure.

Embodiments of the present disclosure are described herein. Variations to these embodiments may become apparent to those skilled in the art upon reading the foregoing description. The present inventors expect those skilled in the art to make use of such variations as the case may be, and the present inventors expect the present disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter set forth in the appended claims of the present invention, as permitted by applicable law. In addition, any combination of the above elements in all their possible variations is covered by this disclosure, unless otherwise specified herein or otherwise clearly contradicts the context.

The following examples are given merely to illustrate the present invention and not to limit its scope.

EXAMPLES

Example 1

This example demonstrates an AAV-specific ELISA. ELISA determines the number of AAV8 particles present in the investigated fraction or preparation.

AAV-specific ELISA was performed to identify AAV-containing fractions. A conformation-specific monoclonal antibody is used to detect an epitope repeatedly expressed on assembled AAV-8 particles.

ELISA for AAV8 was performed using the AAV-8 titration ELISA kit (art. no. PRAAV8; Progen (Heidelberg, Germany)) on a TECAN Roboter system. Briefly, a monoclonal antibody specific for a conformational epitope on assembled AAV8 capsids (ADK8) was applied to microtiter strips and used to capture AAV8 particles from the AAV fraction. The capture of AAV8 particles was detected in two steps. In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 antibody was bound to an immune complex (ADK8 and ADK8 antibodies). Streptavidin peroxidase conjugates were added to the immune complexes associated with the biotin-conjugated monoclonal antibody, and the streptavidin peroxidase conjugates were reacted with biotin. A preparation for AAV2/8 particles, WL217S, containing labeled AAV-8 particles at specified concentrations was used as a standard. The peroxidase substrate solution was added resulting in a color reaction proportional to the amount of bound AAV capsid particles. The color reaction was measured photometrically at 450 nm.

The pre-coated plate was filled with 100 μl/well dilution buffer. Then a series of dilutions for the assay standard, assay control and samples were prepared directly on the plate. The diluted assay standard was diluted in duplicate six times 1+1 directly on the plate by mixing 100 µl with the 100 µl dilution buffer already present in the wells, while the assay control was diluted 1/200 before the six serial dilutions were prepared directly on the plate. Two serial dilutions were prepared for the samples in independent duplicates. The plate was then incubated for 60±10 minutes at +37°C (set point) on a microplate shaker, rotating the plate 180° after 30±5 minutes. After the wash step (wash buffer: phosphate buffered saline (PBS) with Tween 20, 0.8% NaCl, 0.02% KCl, 0.02% KH ₂ PO ₄ , 0.126% Na ₂ HPO ₄ ×2H ₂ O , 0.05% Tween 20, pH neutral (tested with indicator paper)) 100 µl/well of detection antibody was added and the plate was incubated 30 ± 5 min after addition. This incubation was terminated by a washing step. Then 100 μl/well of tetramethylbenzidine substrate was added. The color reaction was allowed to proceed at room temperature for 25±5 minutes before it was stopped by adding a stopping solution. The plate was measured for 30 minutes using an ELISA reader at 450 nm and 620 nm as the reference wavelength. A calibration curve was then obtained after a double logarithmic fit.

Example 2

This example demonstrates cryogenic transmission electron microscopy (CryoTEM).

CryoTEM provides visualization of nano-sized particles such as AAV. The technique involves applying an AAV preparation to a thin carrier film of carbon at controlled temperature and humidity. After removing excess AAV preparation, leaving some adherent AAV preparation, the mesh was vitrified in liquid ethane and then stored in liquid nitrogen. CryoTEM analysis of AAV capsid particles was used to assess the overall morphology of the sample, i.e. the presence of different AAV morphologies (generally including spherical and deformed AAV particles, subunit structures and larger structurally less defined morphologies), and the level of particle packing using automated image analysis methodology.

The AAV fraction or preparation was kept close to its original state by immersing it in amorphous non-crystalline ice on an inert support by flash freezing under cryogenic conditions. Image analysis was performed using an automated objective analysis method using the Vironova Analyzer (VAS) software.

Capsid AAV particles showing internal density without a clear boundary between shell and core are classified as filled particles (FIG. 6A). AAV capsid particles having a clear outer shell and minimal internal density are classified as void particles (FIG. 6A). Indeterminate particles include AAV capsids in the form of a trimeric structure (FIG. 6B), which may be empty or full, and disrupted AAV capsids (FIG. 6C).

CryoTEM determines the number of completely full and empty capsids in AAV preparations.

Example 3

This example demonstrates that using the AAV-specific ELISA results in less variability than using the qPCR method.

AAV8 ELISA: Total AAV8 capsid particle titer was measured using a commercial kit (Progen) calibrated against AAV8 ATCC® reference standard material for capsid particle concentrations (cp) as described in Example 1.

qPCR: A quantitative PCR assay based on Taqman® chemistry was developed to measure the copy concentration of vector genomic transgenic DNA. For FVIII ITR-qPCR, vector genome titer (v.g.) per milliliter (mL) was determined using TaqMan-based qPCR with primers and a fluorescently labeled probe detecting the sequence in the ITR sequences of the vector genome. To identify the ITR-specific sequence in the AAV particle, the samples were treated with DNase I and, after a subsequent proteinase K step, the scAAV genome was released from the capsid. Finally, restriction enzyme digestion was performed to cleave the AAV ITR T-shaped structures.

The plasmid used as a reference was linearized with a single cut restriction enzyme and further purified from an agarose gel. The A260 UV absorption of the gel-extracted DNA was measured three times on a UV spectrophotometer, and the average DNA concentration was calculated in μg per ml.

To determine the number of copies per ml for the linearized standard plasmid, the molecular weight of double-stranded DNA was calculated in grams per mol, taking into account the exact mass for each individual nucleotide of the main sequence.

The titer of the vector genome in the test article (in vector genomes per ml) was calculated by fitting a standard plasmid curve with linear regression.

CryoTEM: Samples were kept close to their original state by immersing them in amorphous non-crystalline ice on an inert support via cryogenic flash freezing as described in Example 2.

Gel Electrophoresis: Electrophoresis was performed as described (Fagone et al., 2012). Briefly, AAV samples were treated for 10 minutes at 75°C in the presence of 0.5% SDS (sodium dodecyl sulfate) and then cooled to room temperature. Samples (approximately 1.5E10 vector genomes (vg)/lane) were then loaded onto a 1% 1X TAE agarose gel and separated in an electric field (60 min at 7 V/cm gel length). The gel was then stained in 2x GelRed (Biotium) solution and visualized with ChemiDocTMMP (Biorad).

In vivo bioactivity: An AAV-specific in vivo bioavailability assay was performed using an AAV8 preparation assessed using an ELISA antigen assay to determine capsid particles per ml - q/ml. Samples were sent to the test facility (frozen, ≤-60°C), thawed and diluted with preparation buffer to 4×10 ¹¹ q/ml. The in vivo bioavailability assay included up to eight FVIII knockout male mice per group ((B6; 129S4-F8 tm2Kaz); Charles River GmbH, Sulzfeld, Germany). Animals received a single intravenous bolus injection of 4×10 ¹² k.h./kg of the studied or control samples shortly after thawing of the samples. The studied samples were AAV8-FVIII gene therapy vectors. The collected mouse citrate plasma is used to assess FVIII activity using an FVIII activity assay. Analysis of FVIII activity is carried out in the presence of calcium ions and phospholipids. Factor X is activated to factor Xa by factor IXa. This activation is stimulated by factor VIII, which acts as a cofactor in this reaction. When using optimal amounts of Ca2+, phospholipid and factor IXa and excess factor X, the rate of factor X activation is linearly related to the amount of factor VIII. Factor Xa hydrolyzes the chromogenic substrate, releasing the chromophore group. The color is then read photometrically at 405 nm. The factor Xa produced, and therefore the intensity of the color, is proportional to the factor VIII activity in the sample.

In Vitro Biological Activity: An AAV-specific quantitative in vitro biological activity assay was performed using an AAV8 preparation assessed using an ELISA antigen assay to determine capsid particles per ml - q/ml. The AAV8 viral vector containing the Factor VIII (FVIII) gene of interest (GOI) infected a target liver cell line, which subsequently secreted a functional and measurable FVIII protein into the cell culture medium. Factor VIII activity of the cell culture supernatant was directly measured by FVIII chromogenic assay. AAV8-FVIII sample measurement is given as a percentage relative to the control material. The method allows to quantify the biological function of the AAV8-FVIII gene therapy vector. The same assay was used as described above for in vivo biological activity.

As shown in FIG. 1, the use of qPCR methods resulted in greater variability compared to the AAV-specific ELISA method. For example, the ITR-based qPCR method resulted in a dispersion coefficient of ≦43%, while the dispersion coefficient for the AAV8-specific ELISA was ≦10%. The ELISA method was 20-33% less variable than the qPCR method. This surprising difference allows the dose of the AAV fraction to be determined only by an AAV-specific ELISA assay. This approach not only provides a more accurate way to dose the AAV fraction, but is also more economical and time efficient.

To investigate whether the ELISA method or the qPCR method leads to an accurate method for measuring capsid content in an AAV preparation, AAV preparations were prepared and evaluated using ELISA and qPCR alone. The results of these measurements were used to prepare a sample for loading into each lane (2.0×10 ¹⁰ capsids/lane). As shown in FIG. 2, samples assessed by ELISA were consistently less variable than those assessed by qPCR because the signal for samples associated with ELISA was more consistent compared to those associated with qPCR results, even if the drugs were from those the same batches, for both types of analyses. Thus, ELISA dosing was more reliable than qPCR-related dosing.

All results from ELISA and ITR-qPCR AAV8 capsid ELISA and GMP bulk drug substance (BDS) and FDP series release studies are shown (Tables 1-4), as well as vector genome ratios per total the number of capsid particles (table 5). For comparison, AAV8 ELISA values plotted against ITR-qPCR values are also shown (FIG. 4). In FIG. 5, the ratio of AAV vector genomes (v.g.) to capsid particles (c.p.) of the AAV8 antigen ELISA is estimated. In FIG. 5A shows the variability of the qPCR method based on the ratio of vector genomes per AAV antigen. In FIG. 5B shows a high constant percentage of total AAV obtained during manufacture according to PCT/US2017/059967 ultracentrifugation using method 50-55-60. The ultracentrifugation process ensures a consistent full to empty capsid profile from batch to batch. As such, inter-assay variability should be consistent (eg, Figure 5B). When qPCR is, however, used to calculate dose, there is high inter-batch variability (eg, Figure 5A).

The mice were then injected with the AAV preparation in an in vivo biological activity assay using an ELISA method to measure the activity. In FIG. 3 confirms the use of an ELISA antigen method to determine the activity of an AAV preparation using an in vivo bioassay assay using a mouse model. As shown in FIG. 3, the biological analysis confirmed that there was biological activity in all lots and that the activities of the lots were comparable and consistent. As stated above, in vivo bioassay assay evaluates both the ability of AAV to transduce cells and to produce a protein or gene of interest.

Table 1: Lots of Bulk Drug Substances (BDS): AAV8 capsid particle total titers measured by ELISA and vector genome titers measured by ITR-qPCR and their ratios. Means, standard deviations, coefficients of variation (CV) and 3 standard deviation limits of the ratios are also shown. The ITR-qPCR/ELISA AAV8 ratio provides the "specific activity" of the AAV drug. The ratio or "specific activity" can provide information/number of complete capsids, but the variation is very large as it is driven by both qPCR and ELISA. The differences stem primarily from qPCR, which demonstrates why the AAV-ELISA or AAV-ELISA/TEM method is superior to the qPCR method.

Table 2: Good manufacturing practices (GMP) of BDS lots: AAV8 capsid particle total titers measured by ELISA and vector genome titers measured by ITR-qPCR and their ratios. Means, standard deviations, coefficients of variation (CV) and 3 standard deviation limits of the ratios are also shown.

Table 3: Finished drug product (FDP) batches: total AAV8 capsid particle titers measured by ELISA and vector genome titers measured by ITR-qPCR and their ratios. Means, standard deviations, coefficients of variation (CV) and 3 standard deviation limits of the ratios are also shown.

Table 4: GMP FDP lots: AAV8 capsid particle total titers measured by ELISA and vector genome titers measured by ITR-qPCR and their ratios. Means, standard deviations, coefficients of variation (CV) and 3 standard deviation limits of the ratios are also shown.

Table 5: Ratio of vector genomes to capsid particles for all batches of BDS and FDP shown.

Table 6: In vitro and in vivo bioactivity data from production lots of AAV8 containing FVIII-deleted B-domain vector DNA. AAV8 dosing was based on the AAV8 antigen ELISA and % complete capsids were determined using CryoTEM. Batch-to-batch consistency in terms of % total capsids was demonstrated with a CV of 2.7%.

In FIG. 12 and 13 show ELISA, qPCR and CryoTEM results for lots 17004-17014. Lots 17004-17013 (Table 6) have the same lot size, ranging from 2x 500 liter lots. These ten batches contain a comparable amount of AAV8 capsids in the final product. Analysis of batches 17004-17013 is presented in Table 7.

Table 7: In vitro and in vivo bioactivity data for production lots of AAV8 containing FVIII-deleted B-domain vector DNA for lots 17004-17013.

Example 4

This example validates CryoTEM as a tool to measure the number of full and empty capsids in an AAV preparation.

A combination of robust analytical methods is required to measure the qualitative features of recombinant AAV8 (rAAV8) based gene therapy vector products to ensure product release and accurate clinical dosing. CryoTEM was designed to quantify full, containing vector genome, and empty vector particles in AAV8 gene therapy vector preparations, and to determine the significance of this parameter for activity using orthogonal methods.

CryoTEM was tested for full/empty quantitation using rAAV8 vector preparations encoding human coagulation factor IX (FIX) with 40-80% total particles obtained by mixing vector preparations or by selective enrichment of vector fractions by ultracentrifugation. Validation resulted in intermediate and inter-assay accuracy below a relative standard deviation of 1%. The linearity of six independent experiments was achieved with each r2 above 0.996. Vector preparations derived from the same mass of vectors showed a correlation of full to empty ratio with measured activity in an in vitro cell-based assay and in an in vivo model.

Methods and materials

CryoTEM: vector samples were kept close to their original state by immersing them in amorphous non-crystalline ice on an inert support by flash freezing under cryogenic conditions. Image analysis was performed using an automatic, objective analysis method using the Vironova Analyzer (VAS) software as described in Example 2.

AAV8 ELISA: Total AAV8 capsid particle titer was measured using a commercial kit (Progen) calibrated against AAV8 ATCC® reference standard material for capsid particle concentrations (cp) as described in Example 1.

In Vitro Biological Activity: The chromogenic activity of FIX in the supernatant of FTX-AAV8 infected HepG2 cells was measured as an arbitrary unit of biological activity relative to a reference preparation and normalized to capsid particles. In an in vitro biological activity assay, the AAV8 viral vector infected a target hepatic cell line, which subsequently secreted a functional, measurable encoded protein into the medium. In the first step, HepG2 target cells are transduced infected with AAV8. During incubation, the encoded protein was released into the supernatant. In the second step, the activity of the encoded protein secreted into the supernatant was directly measured by an activity assay (enzymatic assay - ROX FIX (Rossix) was used to assay biological activity in vitro). The AAV8 sample measurement is given as a percentage relative to the reference material. The method allows to quantify the biological function of the AAV8 gene therapy vector. The reference is a WHO standard or reference preparation calibrated against the WHO standard for human FIX.

In vivo biological activity: Human FIX encoding AAV8 particles were administered intravenously to eight FIX knockout mice (Charles River Germany (CRO in Sulzfeld, Germany)). The AAV8 preparation was evaluated by antigen ELISA to determine capsid particles per ml - c.h./ml. Samples were sent to the test facility (frozen, ≤-60°C), thawed and diluted with preparation buffer to 4.95E+10 c.h./ml. Animals received a single intravenous bolus injection of 2.47E+11 k.h./kg of test or control samples shortly after the samples were thawed. Plasma FIX activity was regularly monitored by FIX clotting activity measured by the one-step APTT clotting assay against a human plasma standard calibrated to an international standard. The FIX containing sample was diluted to range within the calibration curve in imidazole buffer+1% albumin. 50 µl of the diluted sample is added to 50 µl of FIX-deficient plasma and brought into contact with 50 µl of phospholipid suspension (Pathromtin SL, Siemens) after a 240 second incubation, coagulation is started by adding 50 µl of 25 mM CaCl ₂ solution, time for clot formation is measured at the BCS XP station (Siemens) or ACL 500 (Werfen).

qPCR: A quantitative PCR assay based on Taqman® chemistry was developed to measure the concentration of vector genome copies in transgenic DNA. For the FIX DNA vector, two FIX-specific qPCR methods were used, one for the double-stranded DNA AAV vector and the other for the single-stranded AAV vector.

Vector genome titer (vg) per milliliter (ml) was determined using TaqMan-based qPCR with primers and a fluorescently labeled probe detecting the sequence in the FIX sequence and a polyadenylation signal. To reveal the FIX-specific sequence, it was treated with DNase I and, after a subsequent proteinase K step, the scAAV genome was released from the capsid. Finally, restriction enzyme digestion was performed to separate the AAV ITR T-shaped structures.

The plasmid used as a reference material was linearized with a single cut restriction endonuclease and further purified from the agarose gel. The UV absorption of the A260 gel-extracted DNA was measured three times in a UV spectrophotometer and the average DNA concentration in μg per ml was calculated.

To determine the number of copies per ml for the linearized standard plasmid, the molecular weight of double-stranded DNA was calculated in grams per mol, taking into account the exact mass for each individual nucleotide of the main sequence.

The titer of the vector genome in the test article (in vector genomes per ml) was calculated by plotting a standard plasmid curve with linear regression.

results

In FIG. 6 shows that CryoTEM can be used to image full and empty capsids. Fig. 7 is an example of sample analysis. CryoTEM images taken at 38k magnification were subjected to intrinsic density analysis. Principal component analysis of each AAV particle radial density profile revealed clusters of packing levels: full (red; left), blue (empty; right), indeterminate (green; middle); dotted rings: 99% confidence intervals. See Table 8 for a summary of the results.

Table 8: Data obtained from 5 images analyzing a total of 1568 particles.

Fig. 8 represents the validation of CryoTEM according to ICH Q2 (R1) (i.e. International Conference on Harmonization of Specifications for Registration of Pharmaceuticals for Human Use), including specificity, repeatability, intermediate accuracy, linearity, dilution accuracy and reliability. Linearity results from six independent experiments are presented. In FIG. 8 shows the difference between theoretical and measured levels of total AAV8 particles. Preparations of rAAV8 containing almost 80% complete capsids and another preparation with empty capsids in the bulk were mixed to obtain 4 samples with a percentage of complete capsids from 40 to 80%. These preparations were measured on two grids each, with the x-axis representing the theoretical percentage of "complete" capsids and the y-axis representing measured values using CryoTEM. One data point represents the average of two repetitions. All showed good correlation between theoretical and experimental values, with correlation coefficients above 0.996.

In FIG. 9 deals with preparations with increasing degrees of complete capsids that have been purified from ultracentrifugation fractions (see International Application Publication PCT/US2017/059967). In particular, in FIG. 9 shows the increase in qPCR titer as a function of the percentage of "complete" capsids. The x-axis is the percentage of "complete" capsids measured by CryoTEM and the y-axis is the measured qPCR value. The higher the percentage of "complete" capsids, the higher the qPCR titer. The qPCR ratio for the AAV8 antigen in FIG. 9 shows how this ratio increases with the number of "complete" particles - the higher the percentage of complete capsids, the higher the ratio of qPCR to AAV8 antigens [Y: w.g./c.h. on X: percentage of complete capsids (CryoTEM)]. With a higher percentage of complete capsids, qPCR variability is higher, making it less accurate. This is especially important given that the product provided to the subject will be in the higher full capsid range. Vector genomes for capsid particle titers were calculated from qPCR and ELISA results of AAV8 capsid particles and correlated with CryoTEM data.

In FIG. 10 and 11 show the effect of % total FIX-AAV8 capsid particles on in vivo and in vitro biological activity. In FIG. 10 is the same AAV vector preparation used in FIG. 8 were administered intravenously to factor IX knockout mice (n=8). Plasma samples were taken after 14 days and the clotting activity of huFIX was measured. For FIG. 11 preparations with varying degrees of complete capsids (same as in FIG. 9) were prepared by isolating ultracentrifugation fractions and subsequent purification. Biological activity was measured in a cellular assay associated with c.h. and plotted against % total particles. Biological activity in vitro increases with increasing degree of complete capsids.

% complete capsids were measured using CryoTEM. In this case, AAV8 containing double-stranded DNA encoding Padua FIX was used. For the results of this in vivo experiment, samples with different contents of full and empty particles were prepared by mixing the full capsid containing BDS product with the empty capsid BDS product as shown in FIG. 10. Animals received a single intravenous bolus injection on day 0 and blood was collected on days 7, 14 and 28. FIX activity was analyzed in the FIX stage of the clotting assay (APTT-based factor analysis is widely used to measure factors VIII, IX, XI and XI). The sample was brought into contact with plasma and factor IX deficient phospholipids, incubated, and coagulation started with a calcium chloride solution and the coagulation time was measured. Coagulation time has a clear mathematical relationship with the concentration of coagulation factors.

findings

CryoTEM is a valuable tool to quantify the activity of an AAV preparation, which will allow the release of a gene therapy vector preparation for therapeutic use. It can serve as an orthogonal method to measure the number of full and empty capsid particles, or the titer of the vector genome to quantify vectors. The advantage of CryoTEM is that it allows you to control the quality and activity of the gene therapy vector.

CryoTEM is superior to other electron microscopy-based methods because the principle of sample preparation largely preserves the original state of the sample. Moreover, there is no need to rely on coloring agents.

Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications should also fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications and non-patent literature, is incorporated herein by reference in its entirety.

Claims (61)

1. A method for quantifying the dose of an AAV preparation, comprising quantifying the total number of AAV capsids in an AAV preparation or the fraction of AAV used to obtain an AAV preparation using an AAV-specific ELISA and evaluating the percentage or ratio of full and empty (full:empty) capsids AAV in AAV preparation or AAV fraction by cryogenic transmission electron microscopy (CryoTEM). 2. A method for measuring the concentration of AAV complete capsids in an AAV preparation, comprising quantifying the total number of AAV capsids in an AAV preparation using an AAV-specific ELISA assay and estimating the percentage of AAV complete and empty capsids in the AAV preparation or the fraction of AAV used to obtain the AAV preparation , using cryogenic transmission electron microscopy (CryoTEM). 3. The method of claim 1 or 2, wherein the ELISA assay is a sandwich ELISA, direct ELISA, indirect ELISA, or competitive ELISA specific for AAV antigen. 4. The method according to any one of paragraphs. 1-3, wherein the ELISA is a sandwich ELISA specific for the AAV antigen. 5. The method according to any one of paragraphs. 1-4, in which the evaluation step is performed once. 6. The method according to claim 1 or 2, wherein the evaluation step is carried out prior to the ELISA analysis. 7. The method according to claim 1 or 2, wherein the evaluation step is carried out after the ELISA assay. 8. The method according to any one of paragraphs. 1-4, 6 or 7, in which the evaluation step further comprises determining the percentage or ratio of full:empty AAV capsids using cryogenic transmission electron microscopy (CryoTEM), TEM with negative staining, capillary electrophoresis, analytical ultracentrifugation, native agarose gel, alkaline agarose gel, Southern blot, dot blot hybridization, UV spectrophotometry, weak anion exchange chromatography or mass spectrometry. 9. The method according to any one of paragraphs. 1-8, and the method does not involve quantitative PCR. 10. A method of administering an AAV preparation to a subject in need thereof, comprising: (i) obtaining a purified AAV preparation or an AAV fraction used to obtain an AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation or AAV fraction using an AAV-specific ELISA and assessing the percentage or ratio of full to empty (full:empty) AAV capsids in the AAV preparation or AAV fraction, where the evaluation step involves the use of CryoTEM ; (iii) obtaining an AAV preparation with a desired concentration based on the total number of AAV capsids; and (iv) administering to the subject a therapeutically effective amount of the purified AAV preparation. 11. The method of claim 10, wherein the ELISA is a sandwich ELISA, direct ELISA, indirect ELISA, or competitive ELISA specific for the AAV antigen. 12. The method of claim 10 or 11, wherein the ELISA is a sandwich ELISA specific for the AAV antigen. 13. The method according to any one of paragraphs. 10-12, wherein the evaluation step is performed before or after the ELISA assay. 14. The method according to any one of paragraphs. 10-13, in which the concentration of the purified AAV preparation is from 1×10 ¹⁰ q/ml to 1×10 ²⁰ q/ml. 15. The method according to any one of paragraphs. 10-14, in which the therapeutically effective amount of the AAV preparation is a dose of from 1×10 ¹⁰ k.h./kg to 1×10 ¹⁶ k.h./kg. 16. The method according to any one of paragraphs. 10-15, in which the method does not include quantitative PCR. 17. The method according to any one of paragraphs. 10-16, wherein the subject is a human. 18. A method of administering a dose of AAV to a subject in need, comprising (i) obtaining a purified AAV preparation; (ii) measuring the concentration of AAV capsids in the purified AAV preparation using an AAV-specific ELISA assay and evaluating the percentage or ratio of full to empty (full:empty) AAV capsids in the AAV preparation or the fraction of AAV used to obtain the AAV preparation, where the evaluation step provides for the use of CryoTEM; and (iii) administering a specific dose of the purified AAV preparation to the subject. 19. The method of claim 18, wherein the ELISA is a sandwich ELISA, direct ELISA, indirect ELISA, or competitive ELISA specific for AAV antigen. 20. The method of claim 18 or 19, wherein the ELISA is a sandwich ELISA specific for AAV antigen. 21. The method according to any one of paragraphs. 18-20, wherein the evaluation step is performed before or after the ELISA assay. 22. The method according to any one of paragraphs. 18-21, in which the method does not involve quantitative PCR. 23. The method according to any one of paragraphs. 18-23, in which the concentration of the purified AAV preparation is from 1×10 ¹⁰ q/ml to 1×10 ²⁰ q/ml. 24. The method according to any one of paragraphs. 18-24, in which the therapeutically effective amount of the AAV preparation is a dose of from 1×10 ¹⁰ k.h./kg to 1×10 ¹⁶ k.h./kg. 25. The method according to any one of paragraphs. 18-25, wherein the subject is a human. 26. A method for producing an AAV preparation, comprising: (i) transfecting the host cells with at least one plasmid containing the gene of interest; (ii) collecting the supernatant or cell suspension of the cell culture containing AAV capsids to create an AAV fraction; (iii) quantify the total amount of AAV capsids in the AAV fraction using an AAV-specific ELISA assay; (iv) obtaining an AAV preparation with a desired concentration based on the total number of AAV capsids; as well as an estimate of the percentage or ratio of full to empty (full:empty) AAV capsids in the AAV preparation, and where the evaluation stage involves the use of CryoTEM. 27. The method of claim 26, further comprising concentrating the AAV fraction. 28. The method of claim 26 or 27, further comprising removing at least a portion of the empty capsids from the AAV fraction. 29. The method according to any one of paragraphs. 26-28, wherein the ELISA is a sandwich ELISA, direct ELISA, indirect ELISA, or competitive ELISA specific for AAV antigen. 30. The method according to any one of paragraphs. 26-29, wherein the ELISA is a sandwich ELISA specific for the AAV antigen. 31. The method according to any one of paragraphs. 26-30, wherein the evaluation step is performed before or after the ELISA assay. 32. The method according to any one of paragraphs. 26-31, and the method does not involve quantitative PCR. 33. The method according to any one of paragraphs. 26-32, in which the percentage of complete AAV capsids is from 40% to 100%. 34. The method according to any one of paragraphs. 26-33, wherein at least 60% of the AAV capsids are complete AAV capsids. 35. The method according to any one of paragraphs. 26-34, at which the concentration is from 1×10 ¹⁰ q/ml to 1×10 ²⁰ q/ml. 36. The method according to any one of paragraphs. 26-35, wherein the method further comprises lyophilizing the AAV preparation. 37. The method according to any one of paragraphs. 1-36, in which the CryoTEM step includes: (i) immersing the AAV preparation or AAV fraction used to prepare the AAV preparation into a substrate in an inert support; (ii) flash freezing the submerged AAV preparation or AAV fraction; (iii) visualization of the immersed AAV preparation or AAV fraction using cryogenic transmission electron microscopy; as well as (iv) quantifying the percentage of full AAV capsids versus empty AAV capsids in the AAV preparation or AAV fraction. 38. The method of claim 37 wherein the substrate is amorphous non-crystalline ice. 39. The method of claim 37, wherein the AAV preparation does not contain cellular debris. 40. The method according to any of the preceding claims, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or an AAV chimeric vector. 41. The method of claim 40, wherein the AAV is AAV8. 42. The method of claim 40 wherein the AAV is AAV9. 43. The method of any one of the preceding claims, wherein the AAV is a genetically engineered AAV, a chemically modified AAV, or both. 44. The method according to any one of paragraphs. 3-9, 11-17, 19-25 and 29-36, wherein the AAV antigen is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV chimeric, AAV genetically engineered, or chemically modified AAV.

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