RU2773406C2 - Methods for purification of adeno-associated viruses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for the purification of full capsids of an adeno-associated virus (hereinafter – AAV) from a concentrated AAV fraction containing empty AAV capsids and full AAV capsids provides for: (i) loading to a zonal rotor of a) the concentrated AAV fraction and b) at least two sugar solutions, each of which is characterized by different sugar concentration, and each of which contains sugar at a concentration equivalent to sucrose concentration in the range from about 45% (in mass terms) to about 65% (in mass terms) of sucrose, (ii) operation of an ultracentrifuge containing a zonal rotor in a periodic mode, after which sugar gradient is formed, (iii) production of a sugar gradient fraction for the production of AAV fraction, wherein at least or about 60% of AAV particles in AAV fraction are full AAV capsids, wherein the total volume of sugar solutions and the concentrated AAV fraction is less than or equal to the volume of the zonal rotor, and a ratio of the volume of sugar solutions to the volume of the concentrated AAV fraction is less than or equal to one.

EFFECT: method allows for obtaining a high-purity effective product suitable for clinical use.

64 cl, 22 dwg, 19 tbl, 16 ex

Description

Related Applications

This application claims priority under U.S. Provisional Application No. 62/417,775, filed Nov. 4, 2016, which is incorporated herein by reference in its entirety.

The field of technology to which the present invention relates

The present invention relates to materials and methods for purifying adeno-associated virus (AAV).

Background of the Invention

Adeno-associated virus (AAV) is a small, unenveloped virus that packages a linear, single-stranded DNA genome. 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 locations 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. These recombinant AAVs (rAAVs) exist in an extrachromosomal state and are characterized by a very low efficiency of integration into genomic DNA. Thus, the likelihood of random mutagenesis occurring in the host cell by rAAV 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 self-complementary vectors. Along with these ongoing vector developments, ongoing efforts have been focused on large scale manufacturing processes that can efficiently produce large numbers of titers of rAAV vectors with high purity and potency.

Although efforts to develop efficient, large-scale methods for purifying an AAV product suitable for human administration have been significant, there still remains a need for more efficient methods for purifying AAV. For example, current methods of producing AAV in cell culture lead to the formation of "empty" capsids, which have been shown to lead to T-cell mediated immune responses against the capsid antigen, resulting in a low degree of hepatotoxicity and partial loss of expression (Wright, Molec Therapy 22(1): 1-2 (2014)). Therefore, there is a need for AAV purification methods that include the steps of removing empty AAV capsids from the final AAV product.

Brief summary of the present invention

A feature of obtaining the AAV vector in cell culture is the formation of an excess of "empty" capsids, in which the vector genome is absent. Such empty capsids are unable to provide the therapeutic benefit associated with the production of the transgene. The effect of empty capsids on clinical outcome is unclear. However, there is the potential to enhance innate or adaptive immune responses to the vector, which then causes empty capsid problems in the context of gene therapy. Wright, Molecular Therapy 22:1-2 (2014).

This document provides methods for preparing an adeno-associated virus (AAV) product, methods for purifying AAV, and methods for purifying complete AAV capsids from a concentrated fraction of AAV containing empty AAV capsids and complete AAV capsids. The methods of the present invention are advantageous over those known in the art because the methods provided herein are suitable for large-scale production of AAV and provide a highly pure, effective product suitable for clinical use. In exemplary aspects, the methods described herein provide an AAV product containing AAV particles of a homogeneous population and high purity. In illustrative aspects, the methods described herein provide an AAV product containing full length vector DNA. In exemplary embodiments, the methods described herein provide an AAV product that is substantially free of unwanted contaminants, including, but not limited to, empty AAV particles (including those containing truncated or incomplete vector DNA), AAV particles with incomplete protein composition, and oligomerized structures or contaminating viruses, eg non-AAV lipid-enveloped viruses. In exemplary embodiments, the methods described herein provide an AAV product containing a large amount of DNA (cDNA) encoding a protein of interest.

In exemplary embodiments, the methods of the present invention include an ultracentrifugation step to separate full AAV capsids from empty AAV capsids. In exemplary aspects, the methods include (i) loading a rotor with a concentrated AAV fraction with at least two sugar solutions each having a different sugar concentration, (ii) operating an ultracentrifuge containing the loaded rotor in batch mode to form a sugar gradient, and (iii) ) obtaining a sugar gradient fraction to obtain an AAV fraction containing complete AAV capsids. In illustrative aspects, the rotor is a zonal rotor.

In exemplary aspects, each of the at least two sugar solutions contains sugar at a concentration equivalent to a sucrose concentration ranging from about 45% (w/w) to about 65% (w/w) sucrose. In illustrative aspects, one sugar solution contains sugar at a concentration equivalent to a sucrose concentration in the range of from about 52% (w/w) to about 58% (w/w) sucrose, and the other sugar solution contains sugar at a concentration equivalent to a sucrose concentration in the range from about 57% (w/w) to about 63% (w/w) sucrose. Optionally, there is another sugar solution containing sugar at a concentration equivalent to a sucrose concentration ranging from about 47% (w/w) to about 53% (w/w) sucrose. When loading to the bottom of the rotor or compartment, the order of loading may be: first, a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose in the range from about 47% (w/w) to about 53% (w/w) sucrose, if present, then a sugar solution containing sugar at a concentration equivalent to a sucrose concentration ranging from about 52% (w/w) to about 58% (w/w) sucrose, and then a sugar solution containing sugar at a concentration equivalent to a sucrose concentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.

In certain embodiments, the processed sample is added prior to the addition of the sugar solutions. In some embodiments, ultracentrifugation is performed in continuous flow mode and the sample is loaded after a density gradient has been reached during centrifugation.

In some embodiments, the methods of the present invention include an ultracentrifugation step to separate full AAV capsids from empty AAV capsids. In exemplary aspects, the methods include (i) loading a rotor with a concentrated AAV fraction with at least two sugar solutions, each having a different sugar concentration and each containing sugar at a concentration equivalent to a sucrose concentration ranging from about 45% (w/w ratio) to about 65% (w/w) sucrose, (ii) operating the ultracentrifuge containing the loaded rotor in batch mode to form a sugar gradient, and (iii) obtaining a fraction of the sugar gradient to obtain an AAV fraction, wherein at least or about 60 % AAV particles in the AAV fraction are complete AAV capsids. In illustrative aspects, the rotor is a zonal rotor. In certain embodiments, the volume of the sugar solutions is greater than or about 50% of the volume of the zonal rotor. In some embodiments, the total volume of the sugar solutions and the AAV fraction is less than or equal to the volume of the zonal rotor. In some embodiments, the ratio of the volume of sugar solutions to the volume of the AAV fraction is less than or equal to one.

In certain embodiments, each sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 50% (w/w) to about 60% (w/w) sucrose. In some embodiments, each sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 55% (w/w) to about 60% (w/w) sucrose. In certain embodiments, at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration greater than about 50% (w/w) sucrose. In certain embodiments, at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration greater than about 55% (w/w) sucrose. In certain embodiments, at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration ranging from about 60% (w/w) to about 65% (w/w) sucrose.

In some embodiments, one sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 52% (w/w) to about 58% (w/w) sucrose, wherein the second sugar solution contains sugar at a concentration equivalent to a concentration of sucrose in range from about 57% (w/w) to about 63% (w/w) sucrose, and optionally, the third sugar solution contains sugar at a concentration equivalent to a concentration of sucrose in the range from about 47% (w/w) to about 53% (w/w) sucrose.

In certain aspects, two sugar solutions are loaded into a zone rotor. In certain embodiments, two sugar solutions are loaded into a zone rotor, wherein one sugar solution contains sugar at a concentration equivalent to a sucrose concentration in the range of about 52% (w/w) to about 58% (w/w) sucrose, and a second sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.

In certain aspects, at least three sugar solutions are loaded into the zonal rotor. In certain embodiments, three sugar solutions are loaded into the zonal rotor. In certain embodiments, three sugar solutions are loaded into the zonal rotor, wherein one sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 47% (w/w) to about 53% (w/w) sucrose, the second sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 52% (w/w) to about 58% (w/w) sucrose, and the third sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 57% (w/w) ratio) to about 63% (w/w) sucrose.

In some embodiments, the concentrated AAV fraction is loaded before a sugar solution containing sugar at a concentration equivalent to a sucrose concentration in the range of about 52% (w/w) to about 58% (w/w) sucrose, and wherein the sugar solution containing sugar in a concentration equivalent to a sucrose concentration in the range of about 52% (w/w) to about 58% (w/w) sucrose is added before a sugar solution containing sugar at a concentration equivalent to a sucrose concentration in the range of about 57% (w/w) ) to about 63% (w/w) sucrose.

In certain embodiments, the concentrated AAV fraction is loaded before a sugar solution containing sugar at a concentration equivalent to a sucrose concentration in the range of from about 47% (w/w) to about 53% (w/w) sucrose, wherein the sugar solution containing sugar at a concentration of , equivalent to a concentration of sucrose in the range from about 47% (w/w) to about 53% (w/w) sucrose, is loaded before a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose in the range from about 52% (w/w) to about 58% (w/w) sucrose, and wherein a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose in the range from about 52% (w/w) to about 58% (w/w) sucrose is loaded before the sugar solution containing sugar in a concentration equivalent to con sucrose concentrations ranging from about 57% (w/w) to about 63% (w/w) sucrose.

In certain embodiments, each sugar solution is loaded into the zone rotor in equal volumes. In some embodiments, the sugar solution with the lowest concentration of sugar is loaded into the zone rotor in a volume that is twice the volume of at least one of the other sugar solutions in the zone rotor. In certain embodiments, the sugar solution with the lowest sugar concentration is loaded into the zone rotor in a volume that is at least equal to the volume of all other sugar solutions combined in the zone rotor. In some embodiments, the lowest sugar concentration sugar solution is loaded into the zone rotor in a volume that is at least twice the volume of the highest sugar concentration sugar solution, where the volume of the highest sugar concentration sugar solution is equal to the volume of the intermediate sugar concentration sugar solution. In certain embodiments, at least two sugar solutions are loaded into the zone rotor, wherein the sugar solution with the lowest sugar concentration is loaded into the zone rotor in a volume that is at least twice the volume of at least one other sugar solution in the zone rotor. In some embodiments, at least two sugar solutions are loaded into the zone rotor, wherein the sugar solution with the lowest sugar concentration is loaded into the zone rotor in a volume equal to the volume of at least one other sugar solution in the zone rotor. In some embodiments, the sugar solution with the lowest sugar concentration is loaded into the zone rotor in a volume that is half the volume of the concentrated AAV fraction. In certain embodiments, the ratio of the volume of the total sugar gradient to the volume of the AAV fraction loaded into the zonal rotor is from about 1:1 to about 1:5.

In some aspects, the AAV fraction contains a buffer solution. In some embodiments, the buffer solution includes, without limitation, phosphate buffers, histidine (e.g., L-histidine), sodium citrate, HEPES, tris, bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycylglycine, lysine, arginine, sodium phosphate and mixtures thereof. In certain embodiments, the AAV fraction includes Tris-HCl and NaCl. In certain embodiments, the concentration of Tris-HCl is from about 20 to about 50 mM and the concentration of NaCl is from about 150 to about 900 mM. In certain embodiments, NaCl is present at a concentration of from about 500 mM to about 750 mM. In certain embodiments, the buffer solution has a pH of about 7.4 to about 9.0. In some embodiments, the buffer contains approximately 50 mM Tris-HCl and approximately 500 mM NaCl at pH 8.5. In some embodiments, the buffer contains about 50 mM Tris-HCl and about 750 mM NaCl at pH 8.0. In certain embodiments, the buffer solution contains 45-55% (w/w) ethylene glycol.

In certain embodiments, each sugar solution contains a disaccharide or trisaccharide. In certain embodiments, the disaccharide comprises sucrose, maltose, lactose, and combinations thereof. In certain embodiments, each sugar solution contains sucrose. In certain embodiments, each of the sugar solutions further comprises Tris-HCl and NaCl. In certain embodiments, the Tris-HCl concentration is from about 20 to about 50 mM and the NaCl concentration is from about 150 to about 500 mM. In certain embodiments, the buffer solution has a pH of about 7.4 to about 8.5. In some embodiments, the buffer contains 20 mM Tris-HCl and 8 g/L NaCl pH 7.4.

In certain aspects, the methods include operating the ultracentrifuge at a first speed of less than 10,000 rpm for less than 60 minutes and at a second speed of about 30,000 to about 40,000 rpm for at least 4 hours.

In some aspects, the methods include operating the ultracentrifuge at a first speed of less than 10,000 rpm for less than 60 minutes and at a second speed of about 30,000 to about 40,000 rpm for at least 12 hours.

In certain embodiments, the first rotation speed is from about 3000 to about 6000 rpm, optionally about 4000 rpm. In some embodiments, the second rotation speed is about 35,000 rpm and optionally maintained for about 4 to about 6 hours. In some embodiments, the second rotation speed is maintained for at least about 16 hours, or at least 20 hours. In some embodiments, the second rotation speed is about 35,000 rpm and optionally maintained for about 16 to about 20 hours.

In certain embodiments, the concentrated AAV fraction loaded into the zonal rotor contains at least 1×10 ¹² AAV capsids per ml. In some embodiments, the methods involve collecting supernatant from a cell culture containing HEK293 cells transfected with a triple plasmid system. In certain embodiments, the methods comprise (i) harvesting the supernatant about 3 to 5 days after transfection of HEK293 cells, or (ii) when the cell culture has a cell density greater than or about 5x10 ⁶ cells/mL and has a cell viability greater than 50 %. In certain embodiments, the methods include filtering the collected supernatant using depth filtration. In some embodiments, the methods involve filtering the collected supernatant through a filter containing cellulose and perlite and having a minimum permeability of approximately 500 l/m ² . In certain embodiments, the methods involve filtering the collected supernatant through a filter having a minimum pore size of approximately 0.2 microns. In certain embodiments, the methods involve concentrating the AAV fraction using an ultra/diafiltration system. In some embodiments, the methods involve concentrating the AAV fraction using an ultra/diafiltration system before, after, or before and after the step of loading the AAV fraction onto an anion exchange (AEX) chromatography column under conditions that allow AAV to flow through the AEX chromatography column. In certain embodiments, the methods involve inactivating lipid-enveloped AAV fraction viruses with a solvent-detergent. In certain embodiments, the methods involve nanofiltration of the AAV fraction to remove viruses larger than 35 nm. In some embodiments, the methods include a post-chromatographic purification step that involves performing AEX chromatography on a column containing a tentacle gel.

In some embodiments, the methods include (i) introducing an AAV fraction into an anion exchange (AEX) chromatography column under conditions that allow the AAV to flow through the AEX chromatography column, and (ii) collecting the flow material containing the AAV. In some embodiments, the AAV fraction is applied to an AEX column chromatography with a loading buffer containing from about 100 mM to about 150 mM NaCl, optionally, the pH of the loading buffer is from about 8 to about 9. In certain embodiments, the loading buffer contains from about 115 up to about 130 mm NaCl, optionally, the loading buffer contains from about 120 to about 125 mm NaCl.

In certain embodiments, host cell proteins are removed. In certain embodiments, the host cell proteins are HSP70 and/or LDH.

In exemplary aspects, at least or about 55% of the AAV particles in the sugar gradient fraction are complete AAV capsids. In exemplary aspects, greater than or about 60% of the AAV particles in the sugar gradient fraction are complete AAV capsids. According to illustrative aspects, more than or about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the AAV particles in the fraction obtained from the sugar gradient are complete AAV capsids.

In certain embodiments, the methods involve testing the AAV fraction with an AAV-specific ELISA. In certain embodiments, the AAV-specific ELISA is an AAV-specific sandwich ELISA. In certain embodiments, the methods do not include the step of measuring activity by quantitative PCR.

In certain embodiments, AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In certain embodiments, the AAV is AAV8.

This document also presents AAV products obtained by the methods described above and herein.

Brief description of graphic materials

Fig. 1 is a diagram of an exemplary method of the present disclosure.

Fig. 2 is a graph of the number of virus particles (grams of virus) per ml of transfected cell culture supernatant plotted over a period of time (days). Increase in AAV8 in HEK293 cell cultures under established standard conditions (e.g., pH, temperature, agitation speed) on a 2 L scale (4 parallel runs with two different lots of PEI): titer increase in cell culture supernatant over time as measured by specific for FIX qPCR (T01 = transfection day 1, T02 = transfection day 2, etc.). The titer was measured by FIX-specific qPCR in 3×2 L scale bioreactors.

Fig. 3 is a graph of the number of virus particles (grams of virus) per ml of transfected cell culture supernatant plotted over time (days). Increase in AAV8 in HEK293 cell cultures under established standard conditions (e.g., pH, temperature, agitation speed) on a 2 L scale (4 parallel cycles with two different lots of PEI): titer increase in cell culture supernatant over time as measured by AAV -specific ELISA (T01 = day 1 of transfection, T02 = day 2 of transfection, etc.). The titer of the vector genome was measured using AAV-specific ELISA in 3×2 L bioreactors.

Fig. 4 is a graph of pressure and flow rate in the depth filtration stage measured continuously with two pressure transducers (pressure 1 before depth filtration, pressure 2 before membrane filter after depth filter; flow = flow rate measured with a flow meter).

Fig. 5 is a flow chart of the Mustang Q AEX chromatography product in negative (non-binding) mode.

Fig. 6 is a silver stain (left) and western blot (right) of indicated fractions.

Fig. 7 is silver staining (left) and Western blot (right) of indicated fractions.

Fig. 8 is an ultracentrifugation plot of the elution profile separating empty from full capsids. Each fraction was 50 ml.

Fig. 9 is silver staining (left) and western blot (right) of indicated fractions.

Fig. 10 is the final purification of AAV8 on Fractogel TMAE.

Fig. 11 is a diagram of an exemplary method of the present disclosure.

Fig. 12 is a curve showing ultracentrifugation of separated full and empty AAV8 particles using a 50-55-60% protocol in aqueous solution, including particles containing different DNA sequence variants. VectorDNA is human Padua factor IX, double stranded, total length of approximately 4.8 kb. Separate fractions, as indicated in the UC profile, were run on a 4 ml AEX-TMAE column and pooled to obtain the final product. Each of the composed fractions was analyzed. Fractions 10 to 14 were pooled and represent a homologous end product that contains AAV8 with full length vector DNA. Fractions 15 to 21 and additionally fractions 25 and 26 are processed separately and show decreasing length of vector DNA encapsulated in AAV8 particles. This is shown in xDNA-Agarose gels in FIG. 14 and 15 and in the analytical ultracentrifugation (AUC) data shown in FIG. 16.

Fig. 13 is a silver stain (left) and a western blot (right) of the indicated fractions.

Fig. 14 is a photograph of a 1% agarose gel of the following samples taken from the ultracentrifugation of AAV8 particles described in Example 9: lane 1 = marker; lane 2 = PV007 (AAV8 derived from the original Chatham process); lane 3 = fraction 15; lane 4 = fraction 16; lane 5 = fraction 17; lane 6 = fraction 18; lane 7 = fraction 19; lane 8 = fraction 20; lane 9 = fraction 21; lane 10 = fraction 25; lane 11 = fraction 26; lane 12 = AAV8 clinical product and lane 13 = marker. BDS = bulk drug substance (final diluted TMAE eluate). This figure shows that the pooled fractions 10-14 of the UV peak, obtained at higher density of sucrose in ultracentrifugation, contain a highly pure single band of DNA without any additional indication of smaller DNA variants of the vector DNA.

Fig. 15 is a photograph of a 0.8% alkaline agarose gel of the following samples taken from the ultracentrifugation of AAV8 particles described in Example 9: lane 1 = marker; lane 2 = PV007 (AAV8 obtained from previous manufacturing processes); lane 3 = fraction 15; lane 4 = fraction 16; lane 5 = fraction 17; lane 6 = fraction 18; lane 7 = fraction 19; lane 8 = fraction 20; lane 9 = fraction 21; lane 10 = fraction 25; lane 11 = fraction 26; lane 12 = AAV8 clinical product and lane 13 = marker. BDS = volume of drug substance (final diluted TMAE eluate). This figure shows that the pooled fractions 10-14 of the UV peak, obtained at higher density of sucrose in ultracentrifugation, contain a highly pure single band of DNA without any additional indication of smaller DNA variants of the vector DNA.

Fig. 16A-16B are Fractogel TMAE elution profiles obtained from the indicated analytical ultracentrifugation (AUC) fractions as starting material. Full and empty capsids are marked with arrows. A subpopulation of capsids containing incomplete vector DNA (also defined herein as empty) has also been identified. The data is also presented in Table 17. See FIG. 12 regarding fractions.

Fig. 17 is a graph of an ultracentrifugation elution profile in which AAV8 in 50% (w/w) ethylene glycol buffered in Tris/NaCl is ultracentrifuged for 20 hours at 35,000 rpm using a sucrose gradient with 55% and 60% sucrose solutions. The loading ratio of AAV8 sample to sucrose gradient is 1:1. The DNA vector is human coagulation factor IX Padua, single stranded, self complementary, full length (2.6 kb).

Fig. 18 is a graph of the separation of AAV8 containing Padua human coagulation factor IX single strand vector DNA (2.6 kb) using ultracentrifugation with a 55-60% sugar layer protocol where the AAV8 sample is loaded at 50% (in mass ratio) buffered solution of ethylene glycol in Tris/NaCl. Each fraction is examined with: AAV8 qPCR ITR, AAV8 antigen, WAX (weak anion exchange resin - complete capsids), DNA/AAV ratio is the ratio of ITR qPCR vector genomes (µg/ml)/AAV8 capsid antigen antigen (cP/ml). The data was normalized so that the graphs could be presented on the same axes.

Fig. 19 is a graph of an ultracentrifugation elution profile in which AAV8 in 50% (w/w) ethylene glycol in Tris/NaCl buffer is ultracentrifuged for 20 hours at 35,000 rpm in a gradient of 55% and 60% sucrose solutions. The ratio of AAV8 sample loaded to sucrose gradient is 1:1 with a core volume of 3200 ml. The DNA vector is human clotting factor VIII, full length (~4.8 kb).

Fig. 20 is an overlay of sedimentation coefficient plots from fractions 8-10 (see FIG. 19) demonstrating subspecies separation. The arrow indicates a shift towards lower mass AAV8 from higher density fractions to lower density in the UC gradient. Fraction 8 > fraction 9 > fraction 10.

Fig. 21 is a graph of the separation of AAV8 containing Padua human factor IX single strand vector DNA (2.6 kb) using ultracentrifugation with a 50-55-60% sugar layer protocol where the AAV8 sample is loaded into buffered tris-HCl/NaCl solution. Each fraction is examined using: qPCR ITR AAV8 and ELISA. The DNA/AAV ratio is the ratio of the ITR qPCR vector genomes (vg/ml) to the AAV8 ELISA capsid antigen (sr/ml). The data was normalized so that the graphs could be presented on the same axes.

Fig. 22 is a graph of an ultracentrifugation elution profile in which AAV8 in 50% (w/w) ethylene glycol in Tris/NaCl buffer is ultracentrifuged for 20 hours at 35,000 rpm in a sucrose gradient of 55% and 60% sucrose solutions. The ratio of AAV8 sample loaded to sucrose gradient is 1:1 with a core volume of 7700 ml. The vector DNA is human coagulation factor IX Padua, single-stranded self-complementary, full-length (~2.6 kb).

Detailed description of the present invention

Provided herein are methods for producing an adeno-associated virus (AAV) product, methods for purifying AAV, and methods for purifying complete AAV capsids from a concentrated fraction of AAV containing empty AAV capsids and complete AAV capsids.

Advantageously, the methods are scaled up to large volumes of starting material, such as cell culture. In certain embodiments, the methods provided herein are large scale methods capable of purifying AAV from volumes of at least or about 150 L, at least or about 250 L, at least or about 500 L, at least or about 600 L , at least or about 700 liters, at least or about 800 liters, at least or about 900 liters, or at least or about 1000 liters. In some embodiments, the methods are scaled up to a minimum starting material (e.g., cell culture) volume of at least or about 1250 L, at least or about 1500 L, at least or about 2000 L, at least or about 2500 L , at least or about 3000 liters, at least or about 4000 liters, at least or about 5000 liters, at least or about 6000 liters, at least or about 7000 liters, at least or about 8000 liters, according to at least or about 9000 liters, at least or about 10000 liters or more. For example, the methods are carried out with a minimum volume of about 1000 L or about 10000 L or 25000 L or more of AAV producing cell culture.

The methods for producing and purifying AAV described herein are also preferred because the methods result in high titer AAV. In some embodiments, an AAV product containing at least about 10 ¹⁰ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹¹ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹² viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹³ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁴ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁵ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 2×10 ¹⁵ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 5×10 ¹⁵ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture).

Methods of the present disclosure that provide high yields of AAV include, in some embodiments, a nanofiltration step to remove viruses that exceed the exclusion specification of the filter used in the nanofiltration step. This filtration step allows the final AAV product to be made safer for human administration than those AAV products prepared by other methods. The nanofiltration step is an effective step to reduce the number of viruses above the exclusion specification of the filter used in the nanofiltration step, which in some embodiments means that the method is capable of removing more than 4 logs of contaminating viruses from an AAV product via nanofiltration, as further described herein.

Another advantage of the methods described herein is that these methods produce a highly pure AAV product. In some embodiments, the AAV product produced by the methods of the present invention is substantially free of one or more of the following contaminants: host cell proteins, host cell nucleic acids (e.g., host cell DNA), plasmid DNA, empty viral vectors (including containing truncated or incomplete vector DNA), AAV particles with incomplete protein composition and oligomerized structures or infecting viruses, for example, non-AAV lipid-enveloped viruses, heat shock protein 70 (HSP70), lactate dehydrogenase (LDH), proteasomes that contaminate non-AAV viruses (eg, lipid-enveloped viruses), host cell culture components (eg, peptides, antibiotics), process-related components (eg, 0.3% tri-n-butyl phosphate; 1.0% Triton X100, polyethyleneimine) , mycoplasma, pyrogens, bacterial endotoxins and incidental agents. AAV products can be determined to be substantially free if they appear to be free of impurities as determined by standard assay methods such as, but not limited to, thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), enzyme immunoassay (ELISA) or ITR qPCR used by those skilled in the art to assess such purity, or pure enough that further purification does not reveal visible changes in physical and chemical properties, such as enzymatic and biological activity of the substance. In one embodiment, the term "substantially free of impurities" encompasses AAV formulations containing less than about 50% (by dry weight), 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10 %, 5%, 4%, 3%, 2%, or 1% incomplete AAV capsid material. In another embodiment, the term "substantially free of impurities" encompasses AAV formulations in which the impurity is less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or equal to the volume of the AAV product.

In exemplary embodiments, the methods of the present invention provide a purified AAV product that removes at least or about 50% of a contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of the present invention provide a purified AAV product that removes at least or about 60% of a contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of the present invention provide a purified AAV product that removes at least or about 70% of a contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of the present invention provide a purified AAV product that removes at least or about 80% of a contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of the present invention provide a purified AAV product that removes at least or about 90% of a contaminant found in the starting material (eg, cell culture).

In certain embodiments, an AAV product made by the methods of the present invention is suitable for administration to a human. In certain embodiments, the AAV is recombinant AAV (rAAV). In certain embodiments, the AAV product made by the methods of the present invention is sterile and/or Good Manufacturing Practice (GMP) grade. In some embodiments, an AAV product 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).

In addition, the AAV products obtained by the methods described herein are highly effective. The activity of an AAV product, eg an AAV8 product, can be described in terms of (1) in vivo bioactivity (eg active protein production in mice) which is given in units (FIX or FVIII) per ml of mouse plasma; or (2) in vitro bioactivity. The in vitro bioactivity assay measures the ability of AAV vectors to transduce cells, eg HepG2 cells, that express and secrete a protein of interest into the medium and quantify by ELISA and/or enzyme activity. Suitable methods for measuring in vivo and in vitro bioactivity are known in the art and are also described herein as Example 12.

In other embodiments, an AAV product made using the methods described herein exhibits an excellent DNA/AAV ratio. In exemplary embodiments, an AAV product prepared using the methods described herein exhibits an excellent ratio of vector genomes per µg of AAV, demonstrating that the AAV product contains a high number of complete virus particles. In some embodiments, the methods of the present invention involve testing the AAV fraction with an AAV-specific ELISA. In some embodiments, the AAV-specific ELISA is sufficient to provide a representative reading of AAV fraction activity because most of the capsids in the AAV fraction are complete capsids.

Sugar density gradient ultracentrifugation

In exemplary embodiments, the methods of the present disclosure comprise an ultracentrifugation step that generates a density gradient. Without wishing to be bound by theory, it is believed that the ultracentrifugation step separates full AAV capsids from empty AAV capsids. As used herein, the term "complete AAV capsids" in relation to AAV or AAV capsids or AAV particles refers to those containing the complete vector genome. Complete AAV capsids may provide therapeutic benefit to recipient patients. As used herein, the term "empty" in relation to AAVs or AAV capsids or AAV particles refers to those lacking an entire (ie, complete) vector genome. In some embodiments, "blank" may also include "incomplete vector DNA" or "truncated vector DNA". Such empty AAVs or empty AAV capsids or empty AAV particles may not have a vector genome in part or in full, i.e. they may be partially empty or completely empty and as such not be able to provide a therapeutic benefit.

Accordingly, the present disclosure provides a method for separating full AAV capsids from empty AAV capsids, or a method for purifying full AAV capsids from a concentrated fraction of AAV containing full AAV capsids and empty AAV capsids. According to illustrative aspects, the methods of the present disclosure include (i) loading an AAV fraction (e.g., a solution containing AAV) into a rotor with at least two sugar solutions, each sugar solution having a different sugar concentration, (ii) operating an ultracentrifuge containing a loaded rotor, batchwise to form a sugar gradient; and (iii) obtaining a sugar gradient fraction to obtain an AAV fraction containing complete AAV capsids. In certain embodiments, at least three, at least four, at least five, or at least six sugar solutions are loaded. In certain embodiments, two sugar solutions are loaded. In certain embodiments, three sugar solutions are loaded.

The load order may be AAV fraction first (eg, a solution containing AAV), then the lowest concentration of sugar solution, then the next, more concentrated sugar solution, and so on.

According to exemplary aspects, the method involves loading the rotor with an AAV fraction (e.g., a solution containing AAV) with at least two sugar solutions, each sugar solution (a) containing a different concentration of sugar and (b) containing sugar at a concentration equivalent to the concentration of sucrose in range from about 45% (w/w) to about 65% (w/w) sucrose. In some embodiments, the method provides sugar at a concentration equivalent to a concentration of sucrose in the range of from about 50% (w/w) to about 60% (w/w) sucrose, or from about 55% (w/w) to about 60% (w/w). mass ratio) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 52 to 58% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 57 to 63% ( (w/w) sucrose, and optionally another sugar solution contains sugar at a concentration equivalent to a sucrose concentration of 47 to 53% (w/w) sucrose.

In certain embodiments, at least one sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 54 to 56% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 59 to 61% ( (w/w) sucrose, and optionally the other sugar solution contains sugar at a concentration equivalent to a sucrose concentration of 49 to 51% (w/w) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a sucrose concentration greater than about 54% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a sucrose concentration greater than about 59% ( (w/w) sucrose, and optionally another sugar solution at a concentration equivalent to a sucrose concentration greater than about 49% (w/w) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a sucrose concentration equal to or greater than 55% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a sucrose concentration equal to or greater than about 60% (w/w) sucrose, and optionally another sugar solution at a concentration equivalent to that of sucrose equal to or greater than about 50% (w/w) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a sucrose concentration of about 55% (w/w) sucrose, and at least another sugar solution contains sugar at a concentration equivalent to a sucrose concentration of about 60 % (w/w) sucrose, and optionally another sugar solution at a concentration equivalent to a sucrose concentration of about 50% (w/w) sucrose.

The loading sequence may be AAV fraction first (e.g. a solution containing AAV), then the lowest concentration of sugar solution, followed by the next more concentrated sugar solution (e.g., first a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 47 to 53 % (w/w) sucrose, if present, then a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 52 to 58% (w/w) sucrose, and then a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 57 up to 63% (w/w) sucrose).

An advantage of embodiments with at least one sugar solution containing sugar at a concentration equivalent to a sucrose concentration of 52-58% (w/w) sucrose, 53-57% (w/w) sucrose, or about 55% (w/w) ) sucrose, and at least another sugar solution containing sugar at a concentration equivalent to a sucrose concentration of 57-63% (w/w) sucrose, 58-62% (w/w) sucrose, or about 60% (w/w) sucrose , is that gradients formed from such sugar solutions can provide improved separation of AAV capsids.

According to exemplary aspects, the method involves loading the rotor with an AAV fraction (e.g., a solution containing AAV) with at least three sugar solutions, each sugar solution (a) having a different sugar concentration and (b) containing sugar at a concentration equivalent to the sucrose concentration in range from about 45% (w/w) to about 65% (w/w) sucrose. In some embodiments, the method comprises sugar at a concentration equivalent to a sucrose concentration ranging from about 50% (w/w) to about 60% (w/w) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 47 to 53% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 52 to 58% ( by weight) of sucrose, and at least the other sugar solution contains sugar at a concentration equivalent to a sucrose concentration of 57 to 63% (by weight) of sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 49 to 51% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a concentration of sucrose from 54 to 56% ( sucrose, and at least the other sugar solution contains sugar at a concentration equivalent to a sucrose concentration of 59 to 61% (w/w) sucrose.

In certain embodiments, at least one sugar solution contains sugar at a concentration equivalent to a sucrose concentration greater than about 49% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a sucrose concentration greater than about 54% ( by weight) of sucrose, and at least one sugar solution at a concentration equivalent to a sucrose concentration greater than about 49% (by weight) of sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to a sucrose concentration equal to or greater than about 50% (w/w) sucrose, at least another sugar solution contains sugar at a concentration equivalent to a sucrose concentration equal to or greater than about 55% (w/w) sucrose, and at least one sugar solution at a concentration equivalent to a concentration of sucrose equal to or greater than about 60% (w/w) sucrose.

In some embodiments, at least one sugar solution contains sugar at a concentration equivalent to about 50% (w/w) sucrose concentration of sucrose, at least one sugar solution contains sugar at a concentration equivalent to about 55% (w/w) sucrose concentration sucrose, and at least another sugar solution contains sugar at a concentration equivalent to a concentration of sucrose of approximately 60% (w/w).

In some embodiments, the loading order may be as follows: first, a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 47 to 53% (w/w) sucrose, then a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 52 to 58 % (w/w) sucrose, and then a sugar solution containing sugar at a concentration equivalent to a sucrose concentration of 57 to 63% (w/w) sucrose.

The loading sequence can be first the AAV fraction (e.g. containing AAV solution), followed by the lowest concentration of sugar solution, then the next more concentrated sugar solution (e.g., first sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 47 to 53% (w/w) sucrose, then a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 52 to 58% (w/w) sucrose, and then a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose from 57 to 63 % (w/w) sucrose).

In exemplary aspects, each of the AAV fraction and/or sugar solutions may contain additional components such as buffers, salts, and the like. In certain embodiments, each of the AAV fractions and/or sugar solutions contains, individually, any buffering agent or combinations thereof to stabilize the pH from about 5.5 to about 8.5. Examples of suitable buffering agents are well known in the art and include, but are not limited to, phosphate buffers, histidine (e.g., L-histidine), sodium citrate, HEPES, tris, bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate , glycylglycine, lysine, arginine, sodium phosphate and mixtures thereof. In certain embodiments, the buffer is Tris-HCl or Tris-HCl/NaCl. The pH of the buffer/fraction/solution may be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6, 5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 7.5, 7.6, 7.7, 7 .8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0 . The pH of the buffer/fraction/solution may be 7.0 to 9.0, 7.1 to 8.9, 7.2 to 9.0, 7.0 to 8.8, 7.2 to 8 .8, 7.1 to 8.6, 7.3 to 8.9, 7.4 to 9.0, or 7.4 to 8.5. In certain embodiments, buffer, AAV fractions, and/or sugar solutions each have a pH of approximately 7.4. In certain embodiments, buffer, AAV fractions, and/or sugar solutions each have a pH of approximately 8.0. In some embodiments, each of the buffer, AAV fraction, and/or sugar solutions individually has a pH of about 8.5.

In certain embodiments, the buffer may be Tris-HCl/NaCl. In some embodiments, Tris-HCl is at a concentration of about 10 mM to about 100 mM, or about 20 to about 50 mM. In some embodiments, Tris-HCl is at a concentration of about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mm or about 100 mm. In some embodiments, the NaCl is at a concentration of about 100 to about 1 mM, about 150 to about 750 mM, about 150 to about 500 mM, or about 500 to about 750 mM. In some embodiments, the NaCl concentration is about 100 mM, about 120 mM, about 125 mM, about 130 mM, about 136 mM, about 140 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM , approximately 275 mM, approximately 300 mM, approximately 325 mM, approximately 350 mM, approximately 375 mM, approximately 400 mM, approximately 425 mM, approximately 450 mM, approximately 475 mM, approximately 500 mM, approximately 525 mM, approximately 550 mM, approximately 575 mM, ca. 600 mM, ca. 625 mM, ca. 650 mM, ca. 675 mM, ca. 700 mM, ca. 725 mM, ca. 750 mM, ca. 775 mM, ca. , approximately 900 mM, approximately 925 mM, approximately 950 mM, approximately 975 mM or approx. of about 1 M. In some embodiments, Tris-HCl is at a concentration of about 10 mM to about 100 mM, or about 20 mM to about 50 mM, and NaCl at a concentration of about 100 to about 1 mM, about 150 to about 750 mM , from about 150 to about 500 mm or from about 500 to about 750 mm. In certain embodiments, the Tris-HCl concentration is about 50 mM and the NaCl concentration is about 500 mM. In certain embodiments, the Tris-HCl concentration is about 20 mM and the NaCl concentration is about 136 mM. In certain embodiments, the Tris-HCl concentration is about 50 mM and the NaCl concentration is about 750 mM. In some embodiments, the buffer is Tris-HCl/NaCl buffer pH 7.4 or about 7.4. In some embodiments, the buffer is Tris-HCl/NaCl buffer pH 8.0 or about 8.0. In certain embodiments, the buffer is Tris-HCl/NaCl buffer pH 8.5 or about 8.5.

In certain embodiments, the AAV fraction solution contains approximately 50 mM Tris-HCl and 500 mM NaCl at pH 8.5. In some embodiments, the AAV fraction solution contains approximately 50 mM Tris-HCl and 750 mM NaCl at pH 8.0. In certain embodiments, the sugar solutions contain approximately 20 mM Tris-HCl and 136 mM NaCl at pH 7.4.

In exemplary aspects, the method involves loading the rotor with an AAV fraction (e.g., an AAV-containing solution) containing a buffered ethylene glycol solution and at least two sugar solutions, each sugar solution (a) having a different concentration of sugar and (b) containing sugar at a concentration , equivalent to a concentration of sucrose in the range from about 45% (w/w) to about 65% (w/w) sucrose. In some embodiments, the method comprises sugar at a concentration equivalent to a sucrose concentration in the range of from about 50% (w/w) to about 60% (w/w) sucrose, or from about 55% (w/w) to about 60%. (in mass ratio) sucrose. The range of sugar concentrations is the same as above. The advantage of using a buffered ethylene glycol solution is that small gradients can be made. Without wishing to be bound by any theory, the ethylene glycol solution may alter the viscosity and/or density properties of the ultracentrifugation matrix. Surprisingly, it was found that the addition of ethylene glycol, combined with an increase in processing time, creates a shallow gradient that allows you to separate full capsids from empty capsids containing less vector DNA (for example, ≤3 kb or ≤5 kb ). As used herein, the term "shallow gradient" is one in which the density of the solution or the concentration of the solution forming the gradient changes (eg, sucrose gradient) gradually with distance. This is the opposite of when the density of the solution or the concentration of the gradient-forming solution changes rapidly with distance. For example, AAV with a DNA vector containing from about 2.5 to about 5.0 kb can be separated more efficiently when the loading buffer contains from about 45% to about 55% ethylene glycol solution. In some embodiments, the use of ethylene glycol in the AAV fraction solution allows separation of a DNA vector containing from about 2.5 to about 3.0 kb. In some embodiments, the method provides greater resolution of a DNA vector containing about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3, 2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4, 2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0 kb. In some embodiments, improved resolution occurs with a sugar solution with at least three different sugar concentrations. In some embodiments, improved resolution occurs with a sugar solution with at least two different sugar concentrations. In some embodiments, improved resolution occurs with a sugar solution with two different sugar concentrations. In some embodiments, improved resolution is observed when using a single sugar solution at a concentration equivalent to a sucrose concentration of 52-58% (w/w) sucrose, 53-57% (w/w) sucrose, or approximately 55% (w/w) sucrose, and at least another sugar solution containing sugar at a concentration equivalent to a sucrose concentration of 57-63% (w/w) sucrose, 58-62% (w/w) sucrose, or about 60% (w/w) sucrose.

According to exemplary aspects, the method involves loading an AAV fraction (e.g., an AAV-containing solution) into the rotor with an ethylene glycol buffer solution and at least three sugar solutions, each sugar solution of which (a) has a different sugar concentration and (b) contains sugar at a concentration, an equivalent concentration of sucrose in the range of from about 45% (w/w) to about 65% (w/w) sucrose. In some embodiments, the method comprises sugar at a concentration equivalent to a sucrose concentration ranging from about 50% (w/w) to about 60% (w/w) sucrose. The range of sugar concentrations is the same as above.

The buffered ethylene glycol solution may contain any buffering agent or combinations thereof to stabilize the pH from about 5.5 to about 8.5. Examples of suitable buffering agents are well known in the art and include, but are not limited to, phosphate buffers, histidine, sodium citrate, HEPES, tris, bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycylglycine, lysine, arginine, sodium phosphate and their mixtures. In certain embodiments, the buffer is Tris-HCl or Tris-HCl/NaCl. The pH of the buffer/solution can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 7.6, 7.7, 7.8 , 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. The pH of the buffer/solution may be 7.0 to 9.0, 7.1 to 8.9, 7.2 to 9.0, 7.0 to 8.8, 7.2 to 8.8 , 7.1 to 8.6, 7.3 to 8.9, 7.4 to 9.0, or 7.4 to 8.5. In certain embodiments, the buffer/solution has a pH of about 7.4. In certain embodiments, the buffer/solution has a pH of about 8.0. In certain embodiments, the buffer/solution has a pH of about 8.5.

In certain embodiments, the buffered ethylene glycol solution contains Tris-HCl/NaCl. In some embodiments, Tris-HCl is at a concentration of about 10 mM to about 100 mM, or about 20 to about 50 mM. In some embodiments, Tris-HCl is at a concentration of about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mm or about 100 mm. In some embodiments, NaCl is at a concentration of about 100 mM to about 1 mM, about 150 mM to about 750 mM, or about 150 mM to about 500 mM. In some embodiments, the NaCl concentration is about 100 mM, about 120 mM, about 125 mM, about 130 mM, about 136 mM, about 140 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM , approximately 275 mM, approximately 300 mM, approximately 325 mM, approximately 350 mM, approximately 375 mM, approximately 400 mM, approximately 425 mM, approximately 450 mM, approximately 475 mM, approximately 500 mM, approximately 525 mM, approximately 550 mM, approximately 575 mM, ca. 600 mM, ca. 625 mM, ca. 650 mM, ca. 675 mM, ca. 700 mM, ca. 725 mM, ca. 750 mM, ca. 775 mM, ca. , approximately 900 mM, approximately 925 mM, approximately 950 mM, approximately 975 mM or approx. of about 1 M. In some embodiments, Tris-HCl is at a concentration of about 10 mM to about 100 mM, or about 20 mM to about 50 mM, and NaCl at a concentration of about 100 mM to about 1 mM, about 150 mM to about 750 mM, from about 150 mM to about 500 mM, or from about 500 mM to about 750 mM. In certain embodiments, the Tris-HCl concentration is about 50 mM and the NaCl concentration is about 500 mM. In certain embodiments, the Tris-HCl concentration is about 20 mM and the NaCl concentration is about 136 mM. In certain embodiments, the Tris-HCl concentration is about 50 mM and the NaCl concentration is about 750 mM. In some embodiments, the buffer is Tris-HCl/NaCl buffer pH 7.4 or about 7.4. In some embodiments, the buffer is Tris-HCl/NaCl buffer pH 8.0 or about 8.0. In certain embodiments, the buffer is Tris-HCl/NaCl buffer pH 8.5 or about 8.5.

The buffered ethylene glycol solution can be 45% to 55% (w/w), 46% to 54% (w/w), 45% to 53% (w/w), 47% to 55% (w/w) ) or 48% to 52% (w/w) buffer (eg Tris-HCl/NaCl). The buffered ethylene glycol solution may contain 40% (w/w), 41% (w/w), 42% (w/w), 43% (w/w), 44% (w/w), 45% (w/w) ratio), 46% (w/w), 47% (w/w), 48% (w/w), 49% (w/w), 50% (w/w), 51% (w/w) , 52% (w/w), 53% (w/w), 54% (w/w), 55% (w/w), 56% (w/w), 57% (w/w) 58% (w/w), 59% (w/w) or 60% (w/w) buffer (eg Tris-HCl/NaCl). The buffered ethylene glycol solution may be aqueous.

In some embodiments, the buffered ethylene glycol solution contains 45-55% (w/w) ethylene glycol, 50 mM Tris-HCl, and 750 mM NaCl at pH 8.0. In some embodiments, the buffered ethylene glycol solution contains 50% (w/w) ethylene glycol, 50 mM Tris-HCl, and 750 mM NaCl at pH 8.0.

In certain embodiments, the ultracentrifuge rotor core volume is from about 800 ml to about 9000 ml. In certain embodiments, the ultracentrifuge rotor core volume is from about 800 ml to about 7700 ml. In some embodiments, the ultracentrifuge rotor core volume is approximately 800 ml. In certain embodiments, the ultracentrifuge rotor core volume is approximately 1600 ml. In certain embodiments, the ultracentrifuge rotor core volume is approximately 3200 ml. In some embodiments, the ultracentrifuge rotor core volume is approximately 7700 ml.

In certain embodiments, the sugar solutions are loaded at a constant flow rate. In some embodiments, the constant flow rate is from about 0.25 L/hr to about 10 L/hr, or from about 1.5 L/hr to about 7 L/hr. In some embodiments, the constant flow rate is about 0.25 L/h, about 0.5 L/h, about 0.75 L/h, about 1 L/h, about 1.25 L/h, about 1.5 l/h, approximately 1.75 l/h, approximately 2 l/h, approximately 2.5 l/h, approximately 3 l/h, approximately 3.5 l/h, approximately 4 l/h, approximately 4.5 l/h, approximately 5 l/h, approximately 5.5 l/h, approximately 6 l/h, approximately 6.5 l/h, approximately 7 l/h, approximately 7.5 l/h, approximately 8 l/h hour, about 8.5 l/h, about 9 l/h, about 9.5 l/h, or about 10 l/h.

In some embodiments, the method involves loading the rotor with about 45-55% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 45-55% (w/w) of at least two sugar solutions. In exemplary embodiments, the method involves loading the rotor with about 50% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 50% (w/w) of at least two sugar solutions. In some embodiments, the ultracentrifuge rotor core volume is from about 800 ml to about 3200 ml, and about 45-55% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 45-55% ( by weight) of at least two sugar solutions. In some embodiments, the ultracentrifuge rotor core volume is from about 800 ml to about 3200 ml, and the rotor is loaded with about 50% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 50% (w/w) at least two sugar solutions in total. For example, if the rotor core is 800 ml, the AAV fraction may be 400 ml and the total sugar solution fraction may be 400 ml.

In some embodiments, the method involves loading the rotor with about 65-85% (w/w) of an AAV fraction (with or without ethylene glycol buffer) and 15-35% (w/w) of at least two sugar solutions. In some embodiments, the method involves loading the rotor with about 70-80% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 20-30% (w/w) of at least two sugar solutions. In exemplary embodiments, the method involves loading the rotor with about 75% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 25% (w/w) of at least two sugar solutions. In exemplary embodiments, the method involves loading the rotor with about 74% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 26% (w/w) of at least two sugar solutions. In some embodiments, the ultracentrifuge rotor core volume is from about 7700 ml to about 9000 ml, and about 70-80% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 20-30% ( by weight) of at least two sugar solutions. In some embodiments, the ultracentrifuge rotor core volume is from about 7700 ml to about 9000 ml and the rotor is loaded with about 75% (w/w) of the AAV fraction (with or without ethylene glycol buffer) and about 25% (w/w) at least two sugar solutions. In some embodiments, the ultracentrifuge rotor core volume is from about 7700 ml to about 9000 ml and the rotor is loaded with about 74% (w/w) AAV fraction (with or without ethylene glycol buffer) and about 26% (w/w) at least two sugar solutions. For example, if the rotor core is 7700 ml, the AAV fraction may be 6100 ml and the total sugar solution fraction may be 1600 ml.

In certain embodiments, the sugar solutions are added in approximately equal volumes. In some embodiments, the method involves loading at least two sugar solutions, and the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume equal to the volume of the other sugar solutions in the rotor. In some embodiments, the method involves loading at least two sugar solutions, and the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is twice the volume of one of the other sugar solutions in the rotor. In some embodiments, the method involves loading at least two sugar solutions, and the sugar solution with the lowest concentration of sugar is loaded into the rotor in a volume that is about 1.6 times or about 2.6 times the volume of one of the other sugar solutions in the rotor. . In some embodiments, the method involves loading at least two sugar solutions, and the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is half the volume of one of the other sugar solutions in the rotor. In some embodiments, the lowest sugar concentration sugar solution is loaded into the rotor in a volume that is twice the volume of the highest sugar concentration sugar solution, optionally, where the volume of the highest sugar concentration sugar solution is equal to the volume of the intermediate sugar concentration sugar solution. In some embodiments, the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is approximately 2.6 times the volume of the sugar solution with the highest sugar concentration, optionally, the volume of the sugar solution with an intermediate sugar concentration is approximately 1.6 times volume of sugar solution with the highest concentration of sugar. In certain embodiments, the lowest concentration sugar solution is loaded into the rotor in a volume that is equal to the volume of the highest sugar concentration sugar solution, optionally, where the volume of the highest sugar concentration sugar solution is half the volume of the intermediate sugar concentration sugar solution. In certain embodiments, the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is half the volume of the AAV fraction. In some embodiments, the sugar solution with the lowest concentration of sugar is loaded into the rotor in a volume that is three-quarters of the volume of the AAV fraction. In some embodiments, the ratio of the volume of sugar solutions to the volume of the AAV fraction is less than or equal to one.

In some embodiments, the method involves loading two sugar solutions, and the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is equal to the volume of the solution with the highest sugar concentration in the rotor. In certain embodiments, the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is half the volume of the AAV fraction. In some embodiments, the ratio of the volume of sugar solutions to the volume of the AAV fraction is less than or equal to one.

In some embodiments, the method involves loading three sugar solutions, and the sugar solution with the lowest concentration of sugar is loaded into the rotor in a volume that is at least twice the volume of one of the other two sugar solutions in the rotor. In some embodiments, the lowest sugar concentration sugar solution is loaded into the rotor in a volume that is twice the volume of the highest sugar concentration sugar solution, optionally, where the volume of the highest sugar concentration sugar solution is equal to the volume of the intermediate sugar concentration sugar solution. For example, a sugar solution with the smallest sugar solution can have a volume of from about 750 ml to about 900 ml or about 800 ml, a sugar solution with an intermediate sugar concentration can be from about 350 ml to about 450 ml or about 400 ml, and a sugar solution with the highest the sugar concentration can be characterized by a volume of from about 350 ml to about 450 ml or about 400 ml. In certain embodiments, the sugar solution with the lowest sugar concentration is loaded into the rotor in a volume that is half the volume of the AAV fraction. In some embodiments, the sugar solution with the lowest concentration of sugar is loaded into the rotor in a volume that is three-quarters of the volume of the AAV fraction. In some embodiments, the ratio of the volume of sugar solutions to the volume of the AAV fraction is less than or equal to one. In illustrative aspects, the rotor is a zonal rotor. In some embodiments, the total volume of the sugar solutions and the AAV fraction is less than or equal to the volume of the zonal rotor. In some embodiments, the volume of the total volume of the solutions in the zonal rotor is between about 800 ml and 9 L. In some embodiments, the volume of the sugar solutions is greater than or equal to about 50% of the volume of the zonal rotor. For example, the volume of the sugar solutions is greater than or equal to about 50% of the volume of a zone rotor having a volume of less than about 3200 ml, such as about 3200 ml, about 1600 ml, or about 800 ml. In some embodiments, the volume of the sugar solutions is less than or equal to about 25% of the volume of the zonal rotor, such as when using a core volume greater than 7 L, such as 7.7 L.

In some embodiments, the ratio of the volume of the total sugar gradient to the volume of the AAV fraction loaded into the zonal rotor is from about 1:1 to about 1:5. In some embodiments, the volume of the total sugar gradient to the volume of the AAV fraction loaded into the zone rotor is about 1:1, about 1:1.25, about 1:1.5, about 1:1.75, about 1:2, about 1:2.25, about 1:2.5, about 1:2.75, about 1:3, about 1:3.25, about 1:3.5, about 1:3.75, about 1: 4, about 1:4.25, about 1:4.5, about 1:4.75, or about 1:5.

In exemplary aspects, the method comprises loading a zone rotor with a concentrated AAV fraction with at least two sugar solutions, each having a different sugar concentration and each containing sugar at a concentration equivalent to a sucrose concentration ranging from about 45% (w/w) to about 65% (w/w) sucrose (ranges as above), wherein (A) the volume of sugar solutions is greater than or equal to about 50% of the volume of the zonal rotor, (B) the total volume of sugar solutions and the AAV fraction is less than or equal to the volume zone rotor, (C) the ratio of the volume of sugar solutions to the volume of the AAV fraction is less than or equal to one, or (D) a combination of (A), (B) and (C). In some embodiments, the volume of the total volume of the solutions in the zonal rotor is between about 800 ml and 9 L. In some embodiments, the volume of the sugar solutions is greater than or equal to about 50% of the volume of the zonal rotor. For example, the volume of sugar solutions is greater than or equal to about 50% of the volume of a zone rotor having a volume of less than about 3200 ml, such as about 3200 ml, about 1600 ml, or about 800 ml. In some embodiments, the volume of the sugar solutions is less than or equal to about 25% of the volume of the zonal rotor, such as when using a core volume greater than 7 L, such as 7.7 L.

In exemplary aspects, each sugar solution contains a soluble carbohydrate or mixture of carbohydrates. In certain embodiments, each sugar solution contains a disaccharide (eg, sucrose, maltose, or lactose) and/or a trisaccharide. In some embodiments, the density of sugar solutions is equal to the density of sucrose solutions with a concentration ranging from about 45% (w/w) to about 65% (w/w). In certain embodiments, at least one of the sugar solutions has a density of approximately 60% sucrose at a given temperature. In certain embodiments, the ranges are as indicated above.

For the purposes of the present invention, a sugar other than sucrose may be used, provided that the sugar in the sugar solution has a concentration equivalent to the concentration of sucrose in the specified range or in a certain amount (% (w/w)). The sucrose concentration can be determined using a refractive index measurement method that measures the sugar content of an aqueous solution in degrees Brix ("Bx"), where one degree Brix represents 1 g of sucrose per 100 g of solution. Degrees Brix represents the concentration of a solution as a percentage by mass - if the solution contains dissolved solids other than pure sucrose, then Bx only approximates the dissolved solid content. The sucrose concentration can also be determined by measuring the density in the case of pure solutions. If both identity and concentration are to be determined, a commercially available enzymatic kit can be used to determine the concentration and isolate sucrose from other disaccharides and carbohydrates. A sucrose assay kit such as the kit sold by Sigma-Aldrich (St. Louis, MO) under catalog number SCA20 can be used.

In exemplary aspects, each sugar solution contains sucrose. According to exemplary aspects, the method involves loading a rotor (e.g., a zonal rotor) with an AAV fraction (e.g., a solution containing AAV) with at least two sucrose solutions, each sucrose solution (a) having a different concentration of sucrose and (b) containing sucrose in a concentration of from about 45% (w/w) to about 65% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration of 52-58% (w/w) sucrose and at least another solution contains sucrose at a concentration of 57-63% (w/w) sucrose, and optionally at least least another solution contains sucrose at a concentration of 47-53% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration of 54-56% (w/w) sucrose, and at least another solution contains sucrose at a concentration of 59-61% (w/w) sucrose, and optionally at least At least the other solution contains sucrose in a concentration of 49 to 51% (w/w) sucrose. In certain embodiments, at least one solution contains sucrose at a concentration greater than about 54% (w/w) sucrose, and at least the other solution contains sucrose at a concentration greater than about 59% (w/w) sucrose, and optionally , at least the other solution contains sucrose at a concentration greater than about 49% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration equal to or greater than about 55% (w/w) sucrose, at least one solution contains sucrose at a concentration equal to or greater than about 60% (w/w) sucrose, and optionally, at least one solution contains sucrose at a concentration equal to or greater than about 50% (by weight) of sucrose. In certain embodiments, at least one solution contains sucrose at a concentration of about 55% (w/w) sucrose, at least one solution contains sucrose at a concentration of about 60% (w/w) sucrose, and optionally at least one solution contains sucrose at a concentration of approximately 50% (w/w) sucrose. In some embodiments, the AAV-containing solution is a buffered solution (eg, Tris-HCl/NaCl buffer). In certain embodiments, the AAV containing solution is a buffered ethylene glycol solution as described above. In certain embodiments, the buffered ethylene glycol solution is an aqueous solution. In some embodiments, the buffered ethylene glycol solution is an aqueous solution that contains 45-55% (w/w) ethylene glycol and a buffer containing Tris-HCl and NaCl. In some embodiments, Tris-HCl is at a concentration of about 20 to about 50 mM. In certain embodiments, NaCl is present at a concentration of about 100 mM to about 750 mM, or about 150 mM to about 500 mM. In some embodiments, the Tris-HCl concentration is from about 20 mM to about 50 mM and the NaCl concentration is from about 100 mM to about 750 mM, from about 150 mM to about 500 mM, or from about 500 mM to about 750 mM. In some embodiments, the pH of the AAV-containing solution is 7.4 to 8.5, 7.6 to 8.3, 7.8 to 8.5, 7.4 to 7.8, 7. 6 to 8.0, 7.8 to 8.2, or 8.0 to 8.5.

In exemplary aspects, the method involves loading a rotor (e.g., a zonal rotor) with an AAV fraction (e.g., a solution containing AAV) with at least two sucrose solutions, each sucrose solution of which (a) has a different concentration of sucrose and (b) contains sucrose at a concentration of from about 50% (w/w) to about 60% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration of 52-58% (w/w) sucrose and at least another solution contains sucrose at a concentration of 57-63% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration of 54-56% (w/w) sucrose and at least another solution contains sucrose at a concentration of 59-61% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration greater than about 54% (w/w) sucrose and at least another solution contains sucrose at a concentration greater than about 59% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration equal to or greater than about 55% (w/w) sucrose, and at least one solution contains sucrose at a concentration equal to or greater than about 60% (w/w) sucrose . In certain embodiments, at least one solution contains sucrose at a concentration of about 55% (w/w) sucrose, and at least one solution contains sucrose at a concentration of about 60% (w/w) sucrose. In some embodiments, the AAV-containing solution is a buffered solution (eg, Tris-HCl/NaCl buffer). In certain embodiments, the AAV containing solution is a buffered ethylene glycol solution as described above.

According to exemplary aspects, the method involves loading a rotor (e.g., a zonal rotor) with an AAV fraction (e.g., a solution containing AAV) with at least three sucrose solutions, each sucrose solution of which (a) has a different concentration of sucrose and (b) contains sucrose at a concentration of from about 45% (w/w) to about 65% (w/w) sucrose, optionally from about 50% (w/w) to about 60% (w/w). In some embodiments, at least one solution contains sucrose at a concentration of 47-53% (w/w) sucrose, at least another solution contains sucrose at a concentration of 52-58% (w/w) sucrose, and at least another solution contains sucrose at a concentration of 57-63% (w/w) sucrose. In some embodiments, at least one solution contains sucrose at a concentration of 49-51% (w/w) sucrose, at least another solution contains sucrose at a concentration of 54-56% (w/w) sucrose, and at least another solution contains sucrose at a concentration of 59-61% (w/w) sucrose. In certain embodiments, at least one solution contains sucrose at a concentration greater than about 49% (w/w) sucrose, at least another solution contains sucrose at a concentration greater than 54% (w/w) sucrose, and at least another the solution contains sucrose at a concentration greater than about 59% (by weight) of sucrose. In certain embodiments, at least one solution contains sucrose at a concentration equal to or greater than about 50% (w/w) sucrose, at least one solution contains sucrose at a concentration equal to or greater than about 55% (w/w) sucrose, and at least one solution contains sucrose at a concentration equal to or greater than about 60% (by weight) of sucrose. In certain embodiments, at least one solution contains sucrose at a concentration of about 50% (w/w) sucrose, at least one solution contains sucrose at a concentration of about 55% (w/w) sucrose, and at least one solution contains sucrose at a concentration of approximately 60% (by weight) of sucrose. In some embodiments, the AAV-containing solution is a buffered solution (eg, Tris-HCl/NaCl buffer). In certain embodiments, the AAV containing solution is a buffered ethylene glycol solution as described above.

In exemplary aspects, the method involves loading a rotor (e.g., a zonal rotor) with three, four, five, six or more sucrose solutions, each sucrose solution of which (a) has a different concentration of sucrose and (b) contains sucrose at a concentration of from about 45% (w/w) to about 65% (w/w) sucrose, optionally in the range of about 50% (w/w) to about 60% (w/w), or optionally in the range of about 55% (w/w) wt.) to about 60% (wt.). The full ranges of sucrose concentrations are as described above with respect to the ranges of sugar solutions. The ratio of sucrose solutions to AAV fraction and/or other sugar solutions is as above.

In illustrative aspects, the volume of the sucrose solution with the lowest sucrose concentration and the volume of the sucrose solution with the highest concentration of sucrose are from about 20% to about 30% and from about 20% to about 30% of the volume of the rotor, respectively. In some embodiments, the volume of sucrose solution with the lowest sucrose concentration, the volume of sucrose solution with an intermediate sucrose concentration, and the volume of sucrose solution with the highest sucrose concentration are each approximately 25% of the rotor volume. In exemplary aspects, the volumes of the lowest sucrose concentration sucrose solution, the intermediate sucrose concentration sucrose solution, and the highest sucrose concentration sucrose solution are from about 20% to about 30%, from about 10% to about 15%, and from about 10% to approximately 15% of the rotor volume, respectively. In some embodiments, the volume of the lowest sucrose concentration sucrose solution, the intermediate sucrose concentration sucrose solution, and the highest sucrose concentration sucrose solution are about 25%, about 12.5%, and about 12.5% of the volume of the rotor, respectively. In some embodiments, the ultracentrifuge core volume or rotor volume is in the range of about 200 ml to about 10,000 ml, in the range of about 700 ml to about 8500 ml, or in the range of about 700 ml to about 70,000 ml.

A wide variety of ultracentrifuge cores are available and known to those skilled in the art. For example, the smallest ultracentrifuge core is a 200 ml Hitachi CC40S and the largest core is an 8000 ml Hitachi CC40. CC40CT3 Core E can be used with all other continuous flow ultracentrifuges and cores from other suppliers.

In illustrative aspects, the method involves operating an ultracentrifuge containing a rotor (eg, a zonal rotor) in batch mode, after which a sugar gradient is formed. In certain embodiments, the ultracentrifuge is operated at a first rotation speed of less than 10,000 rpm. In some embodiments, the first rotation speed is from about 3000 rpm to about 6000 rpm (eg, 4000 rpm or 5000 rpm) and the first rotation speed is reached within about 15 to about 25 minutes. In certain embodiments, the ultracentrifuge is operated at a first rotational speed at a temperature of from about 2°C to about 10°C. In certain embodiments, the ultracentrifuge is operated at a first rotational speed with the goal of eventually achieving a higher rotational speed. In some embodiments, the ultracentrifuge is accelerated to a second rotation speed that is at least 2 times or at least 3 times greater than the first rotation speed. In some embodiments, the second rotation speed is achieved by accelerating the ultracentrifuge for about 5 to about 60 minutes. In certain embodiments, the ultracentrifuge is operated at a second rotation speed that is greater than or equal to about 30,000 rpm. In certain embodiments, the ultracentrifuge is operated at a second rotation speed that is greater than or equal to about 30,000 rpm and less than or equal to about 50,000 rpm. In some embodiments, the second rotation speed is from about 30,000 rpm to about 40,000 rpm, such as about 35,000 rpm. In certain embodiments, the ultracentrifuge is operated at the second rotation speed for at least or about 3 hours, at least or about 4 hours, at least or about 5 hours, or at least or about 6 hours, at least or about 10 hours, at least or about 12 hours, at least or about 14 hours, at least or about 16 hours, at least or about 18 hours, at least or about 20 hours, or at least or about 22 hours. In certain embodiments, the ultracentrifuge is operated at a second rotation speed for at least or about 4 hours. In certain embodiments, the ultracentrifuge is operated at the second rotation speed for about 16 to about 20 hours. In certain embodiments, the ultracentrifuge is operated at a second rotation speed for at least or about 16 hours. In some embodiments, the ultracentrifuge is operated at a second rotation speed at a temperature of from about 15°C to about 30°C, such as at least about 18°C, from about 20°C to about 25°C (for example, about 22°C ). In some embodiments, the ultracentrifuge is subsequently driven to reduce the rotation speed, eg, to a first rotation speed, eg, about 4000 rpm, optionally within about 5 to about 90 minutes. In some embodiments, slowdown occurs at a temperature of less than about 18°C, from about 2°C to about 10°C, or less than about 8°C. In certain embodiments, the ultracentrifuge is operated at a temperature of less than about 8° C. for at least 30 minutes before the ultracentrifuge is stopped.

In exemplary aspects, the method includes obtaining a sugar gradient fraction to obtain an AAV fraction. In certain embodiments, the resulting AAV fraction contains greater than 60% total capsids. In certain embodiments, the resulting AAV fraction contains 70-80% total capsids. In certain embodiments, one or more sugar gradient fractions are obtained, and in some embodiments, fractions are obtained from a gradient containing higher density fractions. In some embodiments, fractions equivalent to fractions 7-11 are obtained as shown in FIG. 8. In some embodiments, fractions equivalent to fractions 10-14 are obtained as described in Example 9. In certain embodiments, fractions equivalent to fractions 1-8 are obtained as described in Example 16. In some embodiments, any fraction containing complete capsids , separated from the empty capsids by their density difference as they eluted from the rotor in a density gradient. Separation control can be achieved either by fractionating in small volumes followed by an analytical study [for example, as described in example 9], or fractionation according to UV signals with linear measurement of UV signals (for example, at signals UV 254 nm / UV 280 nm and control along the line of their ratio). In certain embodiments, the AAV fraction is obtained from the higher sucrose density zone in the ultracentrifugation step. In some embodiments, the resulting fraction of AAV (e.g., AAV8) is confirmed to be substantially complete capsids (e.g., >60% complete capsids) by a ratio of [UV signal at 254 nm]/[UV signal at 280 nm] independent of the volume of the used core UC. If this ratio (OD 254 hm/OD 280 nm)>1, then the fraction is a complete capsid.

In exemplary aspects, the methods of the present disclosure produce an AAV product in which at least 50% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods of the present disclosure produce an AAV product wherein at least 55% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods of the present disclosure produce an AAV product in which at least 60% of the AAV capsids are complete AAV capsids. In certain embodiments, the methods of the present disclosure produce an AAV product wherein at least 70% of the AAV capsids are complete AAV capsids. In some embodiments, the methods of the present disclosure produce an AAV product wherein at least 80% of the AAV capsids are complete AAV capsids. In some embodiments, the methods of the present disclosure produce an AAV product wherein at least 90% of the AAV capsids are complete AAV capsids. It is understood that "at least 50%" refers to the range where 50% is the minimum percentage. The maximum percentage of such a range according to various embodiments is 100%, although sub-ranges are also considered herein where the maximum percentage is, for example, 99%, 98%, 95%, 80%, 85%, and the like. Suitable methods for measuring the amount of full capsids versus empty capsids are known in the art and include, for example, negative staining transelectron microscopy and analytical anion exchange chromatography using a detection setup that distinguishes between full and empty capsids. See, for example, Human Gene Ther Part B 23:56-64 (2012) and Qu et al., J Virol Methods 140: 183-192(2007).

In exemplary aspects, sugar solutions (eg, sucrose) vary in concentration from about 5% (w/w) to about 15% (w/w). In some embodiments, sugar solutions vary in sucrose equivalent concentration from about 5% (w/w) to about 10% (w/w), from about 4% (w/w) to about 8% (w/w) or from about 2% (w/w) to about 3% (w/w). In some embodiments, sugar solutions contain sucrose and vary in sucrose concentration from about 5% (w/w) to about 10% (w/w), from about 4% (w/w) to about 8% (w/w) or from about 2% (w/w) to about 3% (w/w).

In exemplary aspects, a method for purifying recombinant adeno-associated virus (rAAV) particles and/or a method for separating complete viral particles from empty capsids involves ultracentrifuging a fraction containing rAAV particles with two sucrose solutions, each sucrose solution containing a different concentration of sucrose ranging from about 50% (w/w) to about 65% (w/w) at a first speed less than 10,000 rpm (e.g., 4,000 rpm) for about 15 minutes to about 45 minutes, or about 17 minutes to about 25 minutes and at a second rotation speed in the range of about 30,000 to about 40,000 rpm (eg, 35,000 rpm) for at least 4 hours, or about 16 to about 20 hours. In certain embodiments, one solution contains sucrose at a concentration of about 55% (w/w) sucrose, and one solution contains sucrose at a concentration of about 60% (w/w) sucrose. In illustrative aspects, the ultracentrifugation step is carried out as described in Example 6.

In exemplary aspects, a method for purifying recombinant adeno-associated virus (rAAV) particles and/or a method for separating complete viral particles from empty capsids involves ultracentrifuging a fraction containing rAAV particles with three sucrose solutions, each sucrose solution containing a different concentration of sucrose ranging from about 45% (w/w) to about 65% (w/w) at a first speed less than 10,000 rpm (e.g., 4,000 rpm) for about 15 minutes to about 45 minutes, or about 17 minutes to about 25 minutes and at a second rotation speed in the range of about 30,000 to about 40,000 rpm (eg, 35,000 rpm) for at least 4 hours, or about 16 to about 20 hours. In some embodiments, one solution contains sucrose at a concentration of about 50% (w/w) sucrose, one solution contains sucrose at a concentration of about 55% (w/w) sucrose, and one solution contains sucrose at a concentration of about 60% (w/w). relation) sucrose. In illustrative aspects, the ultracentrifugation step is carried out as essentially described in Examples 7 or 9.

Source of rAAV particles

With respect to the methods of the present disclosure, the AAV may be an AAV of any serotype. In certain embodiments, AAV purified by the methods described herein is characterized by AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAV10 serotype. In certain embodiments, AAV particles purified by the methods described herein have the AAV8 serotype. With respect to the methods of the present invention, the AAV fraction that is loaded into the rotor is, by way of example, the concentrated AAV fraction. In certain embodiments, the AAV fraction loaded into the rotor contains at least 1×10 ¹⁰ , 1×10 ¹¹ , or 1×10 ¹² AAV capsids per ml. In some embodiments, the AAV fraction loaded into the rotor contains at least 1×10 ¹² AAV capsids per ml, where the AAV capsids include empty AAV capsids and full AAV capsids.

In some embodiments, the AAV fraction is the AAV fraction produced by the transfected host cells. In some embodiments, the AAV fraction is a supernatant collected 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 a capsid VP3 protein. In some embodiments, VP1, VP2, and/or VP3 are AAV8 VP1, AAV8 VP2, and/or AAV8 VP3. 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 Thesis, Genetic Therapeutics Division, 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 certain embodiments, the host cells are adherent cells. In certain embodiments, the host cells are suspension cells. In certain embodiments, the host cells are HEK293 cells or SP9 cells (eg, baculovirus-infected Sf9 cells). In certain embodiments, the cell culture includes a culture medium that is free of serum and protein. In some embodiments, the medium is chemically defined and free of 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 rAAV particles is the supernatant fraction collected about 3 to about 5 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 has cell viability more than or about 50%. In some embodiments, the fraction containing rAAV particles is a fraction containing HEK293 cells, as described in Example 1.

In certain embodiments, AAV is obtained by triple plasmid transfection, followed by harvest one to five days later. In certain embodiments, AAV is obtained through cell disruption.

In certain embodiments, AAVs are 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 12 cells/ml, eg, about 6x10 ⁶ to about 10 cells/ml. The cells are then separated at a ratio of approximately 1:2 so that the cell density is approximately 3-5×10 ⁶ cells/ml. After digestion, cells can 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 Padua factor IX in single stranded self-complementary form, with the vector DNA having a total length of 2.6 kb. 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 apply. 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 3 days, eg 3-5 days, prior to harvest.

Additional stages

The methods of the present disclosure comprise any combination of the steps disclosed herein and may optionally be combined with one or more additional steps. Accordingly, according to illustrative aspects, the methods of the present disclosure further comprise the step of transfecting host cells with a triple plasmid system, as described herein. In exemplary aspects, the methods of the present invention involve harvesting supernatant from a cell culture containing host cells, eg, HEK293 or Sf9 cells (eg, Sf9 cells infected with baculovirus) transfected with a triple plasmid system. In exemplary aspects, the methods of the present disclosure involve collecting the supernatant about 3-5 days after transfection of HEK293 cells, or when the cell culture has a cell density greater than or about 5x10 ⁶ cells/mL and cell viability is greater than 50%. In certain embodiments, AAV is obtained through cell disruption. In exemplary aspects, the transfection and harvest step occurs prior to the ultracentrifugation step described herein. The methods of the present invention may contain yet other additional steps that can further improve the purity of the AAV and remove other undesirable components and/or concentrate the fraction and/or prepare the fraction for the next step. Additional steps may occur before or after the ultracentrifugation step described above.

In exemplary aspects, the method includes a depth filtration step. In exemplary aspects, the method comprises subjecting the transfected HEK293 cell culture supernatant fraction to depth filtration using a filter containing cellulose and perlite and having a minimum permeability of approximately 500 l/m ² . According to illustrative aspects, the method further includes the use of a filter with a minimum pore size of approximately 0.2 microns. In exemplary aspects, depth filtration is followed by filtration through a filter having a minimum pore size of approximately 0.2 microns. In exemplary aspects, one or both of the depth filter and the filter having a minimum pore size of approximately 0.2 µm are washed and the washes are collected. In exemplary aspects, the washes are pooled together and combined with the filtrate obtained after depth filtration and filtration with a filter having a minimum pore size of approximately 0.2 microns. Example 2 provides an exemplary method for depth filtration and filtration through a filter with a minimum pore size of approximately 0.2 µm. In exemplary aspects, the depth filtration step and the other filtration step occur prior to the ultracentrifugation step described herein.

In some embodiments, the collection is performed as follows: the cell culture supernatant is collected at this time by depth filtration, for example, by filtering from about 150 to about 250 L of AAV-containing cell suspension through a depth filter element and a polyethersulfone element. The flow rate may be from about 40 kg/hour to about 280 kg/hour, or from about 60 kg/hour to 240 kg/hour. The total pressure is less than 1.7 bar. The two filters are washed with about 10 to about 30 L of Tris buffered saline (TBS) buffer. The filtered fermentation broth or harvest is collected. The depth filtration procedure can remove at least 70% of the protein and 60% of the DNA of HEK293 cells while retaining at least 60% of the 65% AAV particles.

In some embodiments, the harvest containing the AAV fraction is subjected to ultrafiltration/diafiltration (UF/DF) and TFF to concentrate and condition the harvest as follows: the harvest is concentrated about 10-20 times to a target volume of about 10 L to about 15 L using TFF. The concentrated harvest is then subjected to two stages of diafiltration to bring the concentrated harvest to a pH of about 8.3 to about 8.7 and a conductivity of about 13 mS/cm to about 17 mS/cm. Each diafiltration step can be performed with a 5-fold volume exchange with diafiltration buffer, for example, the first buffer contains 50 mM Tris and 500 mM NaCl, is characterized by a pH of 8.5±0.2 and a temperature of 25°C, and the second buffer contains 50 mM Tris and 125 mM NaCl, characterized by a pH of 8.5±0.2 and a temperature of 25°C. TFF is then taken to concentrate the retentate to a final target volume of about 8 L to about 12 L. The concentrate is then filtered through a 0.2 µm filter element. The AAV yield can be increased by washing the filter element with approximately 1.5 to 2.5 times the second diafiltration buffer.

In exemplary aspects, the methods of the present invention include one or more chromatography steps. In exemplary aspects, 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 exemplary aspects, the methods comprise a negative anion exchange (AEX) chromatography step or a "no binding mode" AEX chromatography step. Example 4 describes such a step. Accordingly, in exemplary embodiments, methods for purifying AAV particles comprise performing negative anion 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 illustrative aspects, 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, e.g., NaCl, optionally, wherein the pH of the loading buffer is from about 8 to about 9. 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 exemplary aspects, the negative AEX step occurs prior to the ultracentrifugation step described herein.

In exemplary aspects, the methods of the present disclosure involve concentrating the AAV fraction using an ultra/diafiltration system. In exemplary aspects, the methods of the present disclosure include another tangential flow filtration (TFF) step. In exemplary aspects, the AAV fraction is ultra/diafiltered. In exemplary aspects, the AAV fraction is concentrated in the ultra/diafiltration system prior to the negative AEX chromatography step, after the negative AEX chromatography step, or before and after the inclusion of negative AEX chromatography. Examples 3 and 5 describe such TFF steps. In exemplary aspects, the TFF steps occur prior to the ultracentrifugation step described herein.

In some embodiments, an additional TFF step is performed after the negative AEX chromatography as follows: The AAV-containing flow-through fraction obtained from the AEX chromatography is concentrated and diafiltered against a buffer, eg containing about 500 mM NaCl, about 50 mM Tris-HCl; at a pH of about 8.3 to about 8.7 to prepare the product for ultracentrifugation. Then the TFF step is performed.

In some embodiments, empty and full AAV particles are separated from each other by ultracentrifugation according to the "two sucrose protocol" as follows. AAV concentrate in Tris-HCl/NaCl buffer, followed by a first sucrose solution containing sucrose at a sucrose concentration of 55% (w/w) and then a second sucrose solution containing sucrose at a sucrose concentration of 60% (w/w) ). In certain embodiments, the AAV solution contains approximately 50 mM Tris-HCl and approximately 500 mM NaCl. In certain embodiments, the sucrose solution contains about 50 mM Tris-HCl and about 136 mM NaCl. Ultracentrifugation is first carried out at about 4000 rpm at a temperature of 2-10°C, the rotation speed is increased from about 33000 rpm to about 37000 rpm, the temperature is increased from about 20°C to about 25°C, and then maintained at for about 4 hours, from about 14 hours to about 20 hours, or from about 16 hours to about 20 hours. The fractions are then collected.

In some embodiments, empty and full AAV particles are separated from each other by ultracentrifugation in a "two sugar protocol" as follows. A concentrate containing AAV and a buffered ethylene glycol solution containing 45-50% (w/w) ethylene glycol in Tris-HCl/NaCl buffer, followed by a first sucrose solution containing sucrose at a sucrose concentration of 55% (w/w) and then a second sucrose solution containing sucrose at a sucrose concentration of 60% (w/w). In some embodiments, the AAV solution contains about 55% ethylene glycol, about 50 mM Tris-HCl, and about 750 mM NaCl. In certain embodiments, the sucrose solution contains about 50 mM Tris-HCl and about 136 mM NaCl. Ultracentrifugation is first carried out at about 4000 rpm at a temperature of 2-10°C, the rotation speed is increased from about 33000 rpm to about 37000 rpm, the temperature is increased from about 20°C to about 25°C, and then maintained at for about 4 hours, from about 14 hours to about 20 hours, or from about 16 hours to about 20 hours. The fractions are then collected.

In some embodiments, empty and full AAV particles are separated from each other by ultracentrifugation according to the "three sugar protocol" as follows. A concentrate containing AAV in Tris-HCl/NaCl buffer, followed by a first sucrose solution containing sucrose at a sucrose concentration of 50% (w/w), a second sucrose solution containing sucrose at a sucrose concentration of 55% (w/w), and a third sucrose solution containing sucrose at a sucrose concentration of 60% (w/w). The first sucrose solution is loaded into the bottom of the rotor; then a second sucrose solution and a third sucrose solution. In certain embodiments, the AAV solution contains approximately 50 mM Tris-HCl and approximately 500 mM NaCl. In certain embodiments, the sucrose solution contains about 50 mM Tris-HCl and about 136 mM NaCl. Ultracentrifugation is first carried out at about 4000 rpm at a temperature of 2-10°C, the rotation speed is increased from about 33000 rpm to about 37000 rpm, the temperature is increased from about 20°C to about 25°C, and then maintained at for about 3 hours to about 6 hours, from about 4 hours, from about 14 hours to about 20 hours, or from about 16 hours to about 20 hours. The fractions are then collected.

In some embodiments, empty and full AAV particles are separated from each other by ultracentrifugation according to the "three sucrose protocol" as follows. A concentrate containing AAV and a buffered ethylene glycol solution containing 45-50% (w/w) ethylene glycol in Tris-HCl/NaCl buffer, followed by a first sucrose solution containing sucrose at a sucrose concentration of 50% (w/w), a second a sucrose solution containing sucrose at a sucrose concentration of 55% (w/w); and a third sucrose solution containing sucrose at a sucrose concentration of 60% (w/w). The first sucrose solution is loaded into the bottom of the rotor; then a second sucrose solution and a third sucrose solution. In some embodiments, the AAV solution contains about 55% ethylene glycol, about 50 mM Tris-HCl, and about 750 mM NaCl. In certain embodiments, the sucrose solution contains about 50 mM Tris-HCl and about 136 mM NaCl. Ultracentrifugation is first carried out at about 4000 rpm at a temperature of 2-10°C, the rotation speed is increased from about 33000 rpm to about 37000 rpm, the temperature is increased from about 20°C to about 25°C, and then maintained at for about 3 hours to about 6 hours, from about 4 hours, from about 14 hours to about 20 hours, or from about 16 hours to about 20 hours. The fractions are then collected.

In exemplary aspects, the methods of the present disclosure involve treating a fraction containing AAV particles with a detergent solvent to inactivate lipid-enveloped viruses. Example 8 describes an exemplary method for inactivating a lipid-enveloped virus. In exemplary aspects, the solvent-detergent treatment step occurs after the ultracentrifugation step described herein.

In exemplary aspects, the methods of the present invention filter a fraction containing rAAV particles to remove viruses larger than the rAAV particles in the fraction. In exemplary aspects, the method of the present invention includes filtering the AAV-containing fraction to remove viruses larger than or about 35 nm. In exemplary aspects, the pore size of the filter is in the nanometer range, and in exemplary aspects, the method involves nanofiltration. In exemplary aspects, the method of the present disclosure 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. An exemplary nanofilter with this pore size contains bundles of microporous hollow fibers constructed from natural hydrophilic regenerated cuprammonium cellulose. Filter type classification depends on membrane structure, material and supplier. In exemplary aspects, the nanofilter is examined with an integrity leak test to confirm that the filter does not contain pinholes or large defects and is pre-washed with formulation buffer.

An exemplary nanofiltration is described herein as Example 10.

In exemplary aspects, a fraction containing rAAV particles, eg, an anion exchange eluate, is pre-diluted with elution buffer (PBS + 600 mM NaCl) to control the concentration of viral particles.

According to exemplary aspects, a differential pressure across the filter is maintained during the filtration step. In exemplary aspects, the pressure (pressure drop across the filter) is from about 0.02 MPa to about 0.1 MPa. In exemplary aspects, the pressure (eg, pressure drop across the filter) is from about 0.02 MPa to about 0.08 MPa. When the filter is in dead-end mode, the pressure drop can be influenced by the supply pressure of the sample being fed (ie, by setting the pump to a specific flow that affects the supply pressure). According to illustrative aspects, filtration is carried out at a constant pressure not exceeding 0.1 MPa, in a dead-end mode through a nanofilter. According to exemplary aspects, the filter is run in a tangential flow manner. In exemplary aspects, the yield of isolated viral particles is increased by subsequent washing of the nanofilter with a buffer (eg, drug buffer). In exemplary aspects, testing after the integrity of the nanofilter is performed with a leak test and/or a gold particle test to determine that the pore size distribution of the nanofilter does not change and that the nanofilter maintains its integrity during filtration. In exemplary aspects, more than 50 liters of solution is applied per m ² of filter area to increase sample yield.

In exemplary aspects, the filtration step to remove viruses larger than rAAV particles occurs once during the process of the present disclosure. In exemplary aspects, the filtration step occurs twice during the process. In exemplary aspects, the filtration step to remove viruses larger than rAAV particles occurs after the ultracentrifugation step described herein. In exemplary aspects, a filtration step to remove viruses larger than rAAV particles occurs after a post-purification step.

In exemplary aspects, the methods of the present invention include a post-purification step comprising performing AEX chromatography, optionally with a tentacle gel column. Example 8 describes an exemplary process involving such a post-purification step. In exemplary aspects, the post-purification step occurs after the ultracentrifugation step described herein.

In exemplary aspects, the methods of the present disclosure include one or more quality control steps, such as steps for measuring effectiveness. DNA/AAV ratio or specific activity of AAV fractions obtained after one or more steps (eg after each step) of the process. In illustrative aspects, the methods of the present disclosure include analysis for the presence of AAV in fractions using qPCR, eg qPCR ITR. The ITR qPCR is a NRP-based quantitation assay to measure the amount of AAV inverted terminal repeat (ITR) nucleic acid in a fraction. Other AAV-specific or AAV8-specific sequences can be analyzed by qPCR.

According to illustrative aspects, the methods of the present invention include the analysis of the presence of AAV in fractions using ELISA. In exemplary aspects, the ELISA is a sandwich ELISA. In exemplary aspects, the sandwich ELISA contains an antibody specific for an AAV epitope. In exemplary aspects, an AAV epitope is a conformational epitope present on assembled AAV capsids. A suitable method for testing the DNA/AAV ratio is described herein as Example 12. As discussed herein, ELISA can replace qPCR as a method for determining AAV fraction potency. According to illustrative aspects, the methods of the present disclosure provide for the analysis of the AAV fraction using an AAV-specific ELISA, and the methods do not provide a method for measuring efficiency by quantitative PCR. In exemplary aspects, the AAV-specific ELISA is sufficient to provide a representative reading of the activity of the AAV fraction, since most of the capsids in the AAV fraction are complete capsids.

In exemplary aspects, the methods of the present disclosure provide for an AAV-specific ELISA following one or more of the steps of the present disclosure. In exemplary aspects, the methods of the present disclosure involve examining the AAV fraction obtained after ultracentrifugation with an AAV-specific ELISA to determine the AAV DNA/AAV ratio in that fraction. In exemplary aspects, the methods of the present disclosure involve examining the fraction of AAV obtained after deep filtration with an AAV-specific ELISA to determine the ratio of AAV DNA/AAV in that fraction. In exemplary aspects, the methods of the present disclosure involve examining the AAV fraction obtained after concentrating the AAV fraction using an AAV-specific ELISA ultra-/diafiltration system to determine the AAV DNA/AAV ratio in that fraction. In exemplary aspects, the methods of the present disclosure involve examining a fraction of AAV obtained after a tangential flow filtration (TFF) step by means of an AAV-specific ELISA to determine the ratio of AAV DNA/AAV in that fraction. In exemplary aspects, the methods of the present disclosure involve examining a fraction of AAV obtained after negative anion exchange (AEX) chromatography with an AAV-specific ELISA to determine the ratio of AAV DNA/AAV in that fraction. In exemplary aspects, the methods of the present disclosure involve examining the fraction of AAV obtained after the post-purification step with an AAV-specific ELISA to determine the ratio of AAV DNA/AAV in that fraction.

In illustrative aspects, the method of the present disclosure includes one or a combination of the steps illustrated in FIG. 1. According to illustrative aspects, the method of the present disclosure includes all of the steps illustrated in FIG. one.

The AAV product obtained by the method of the present disclosure is further provided herein. In exemplary aspects, an AAV product contains at least about 10 ¹² virus particles (vp) derived from about 1000 L of starting material (e.g. cell culture) or at least about 10 ¹³ virus particles (vp) derived from about 1000 L starting material (eg, cell culture), and wherein at least 50% or at least 55% of the AAV capsids present in the AAV product are complete AAV capsids. In exemplary aspects, the AAV product of the present invention is highly pure, highly potent, and suitable for clinical use in humans. In exemplary aspects, the AAV product contains AAV particles of a homogeneous population and high purity. In exemplary aspects, the AAV product contains full length vector DNA. In exemplary embodiments, the AAV product is substantially free of unwanted contaminants, including, but not limited to, AAV particles containing truncated or incomplete vector DNA, AAV particles with incomplete protein composition, and oligomerized structures or contaminating viruses, e.g., non-AAV viruses with lipid sheath. In exemplary embodiments, the AAV product contains a large amount of the cDNA coding for the protein of interest. In illustrative aspects, the AAV product of the present invention is suitable for administration to a human. In exemplary aspects, the AAV product is sterile and/or has a Good Manufacturing Practice (GMP) rating. In exemplary aspects, the AAV product complies with USP Chapter 1046 or European Pharmacopeia for Gene Therapy Medicines or complies with US Food and Drug Administration (USFDA) or European Medicines Agency (EMA) requirements. In exemplary aspects, the AAV product is a ready-to-use product for direct administration to a human with little or no processing.

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

EXAMPLES

Example 1

The following example describes an exemplary method for transfecting a HEK293 cell line with a triple plasmid system to produce rAAV particles containing a nucleic acid encoding a protein of interest.

Adherent HEK293 cells are grown in suspension in a commercially available culture medium that is chemically defined and free of animal, protein and serum components. The cells are cultured to a cell density of approximately 6-10×10 ⁶ cells/ml. Prior to transfection, a separation of approximately 1:2 is performed to achieve a cell density of approximately 3-5×10 ⁶ cells/ml.

Cells are transfected with three plasmids: (1) a helper plasmid that provides accessory viral functions required for productive AAV infection, (2) a repcap plasmid that carries all the information regarding capsid formation, viral replication, and packaging, and (3) a plasmid containing the gene of interest (GOI), which is packaged in the resulting rAAV particle. For example, the GOI can be factor IX Padua vector DNA (single-stranded (~2.6 kb) or double-stranded (~4.8 kb)) or factor VIII single-stranded vector DNA (~4. 8 kb).

Transient transfection of HEK293 cells is carried out using a cationic polymer of polyethyleneimine (PEI). Briefly, PEI and plasmid DNA are incubated for more than 10 minutes at 4 to 37° C. until the transfection mixture is mixed with the cell culture so that pre-formation of PEI and plasmid DNA takes place. The preformed complexes are added to the cell culture along with the transfection mix and incubated for about 3-4 hours at 30°C to 40°C, eg about 37°C. Transfection is stopped by adding a known stop medium to the transfected culture containing, for example, a medium such as Hyclone™ CDM4HEK293, a chemically defined cell culture medium without animal components and proteins, including 0.6 g/l glutamine.

rAAV particles bearing GOI are present in the HEK293 cell line for 3-5 days after transfection. As shown in FIG. 2 and 3, the HEK293 cell culture shows high cell density (eg, greater than about 5×10 ⁶ cells/ml) with >50% viability at about 5 days post-transfection.

Example 2

The following example describes an illustrative method for collecting the supernatant of a transfected HEK293 cell culture.

As discussed in Example 1, rAAV production occurs during the first 3-5 days after transfection. After this period of time, the HEK293 cell culture shows a high cell density (eg, greater than about 5×10 ⁶ cells/ml) with >50% viability. The cell culture supernatant was collected at this time by depth filtration. AAV particles were separated from cells and cell debris by filtering approximately 200 L (±20 L) of AAV-containing cell suspension through a cellulose-based depth filter element (eg, Pall, STAX K900P; 4 m ² ) and a 0.2 μm polyethersulfone filter element. (PES) (e.g. Pall, ECV; 2" x 5" parallel). The typical flow rate through the filters was 160±100 kg/h and the total pressure was <1.7 bar. The two filters were washed with approximately 10-30 L Tris buffered saline (TBS) (20 mM Tris, 8 g/L NaCl, pH 7.4±0.2 at 25°C). Together with the filter wash, the filtered fermentation broth is collected in a 500 L bag and referred to as the collection. The parameters of the collection stage and the characteristics of the collection (output) after the collection stage are presented in tables 1 and 2, respectively. In FIG. 4 is a continuous recording of pressure and flow rate during the collection stage.

As shown in Table 2 above, more than 70% of the protein and more than 62% of the HEK DNA found in the cell suspension prior to filtration were removed at this filtration step. The amount of inverted terminal repeat (ITR) AAV nucleic acid in the pooled fraction containing the harvest and filter wash (P) measured by qPCR was approximately half that found in the cell suspension prior to filtration. In addition, according to ELISA, more than 65% of the AAV of the pre-filtration cell suspension was retained during filtration.

Example 3

The following example describes an illustrative method for concentrating and conditioning a collection using a tangential flow filtration (TFF) ultrafiltration/diafiltration (UF/DF) system.

The collection obtained in Example 2 was subjected to a UF/DF TFF system to concentrate and condition the collection. In the first step, the harvest was concentrated approximately 16-fold to a target volume of 12 L with TFF using 3 x 0.5 m ² Pall 100 kDa Omega Centramate membranes (part number: OS100T06). The concentrated harvest was then subjected to two stages of diafiltration to reduce media components and bring the concentrated harvest to pH 8.5 and a conductivity of 15 mS/cm. The first stage of diafiltration was carried out by 5-fold volume exchange with buffer 1 for diafiltration (DB1: 50 mm Tris/500 mm NaCl/pH 8.5±0.2 at 25°C). The second stage of diafiltration was carried out using a 5-fold volume exchange with buffer 2 for diafiltration (DB2: 50 mm Tris/125 mm NaCl/pH 8.5±0.2 at a temperature of 25°C). Each diafiltration step was performed using Pall Series T Omega Centramate 3 x 0.5 m ² 100 kDa membranes (part number: OS100T06). After the second diafiltration step, the retentate was concentrated to a final target volume of 10 L with TFF as described above. Then 10 L of the concentrate was filtered through a 0.2 µm filter element (5 inches, Pall ECV filter, part number: NP5LUECVP1S). To increase the yield of AAV8, the UF/DF membranes, UF/DF system, and 0.2 µm filter were washed with approximately 2 L of DB2. The retentate and wash filter were collected and mixed in a bag to prepare it for the next capture step. In FIG. 7 shows the intermediate steps of TFF in silver staining (left) and Western blotting (right). The parameters of the UF/DF TFF steps and the characterization of the resulting conditioned concentrate [UF/DF TFF steps] are shown in Tables 3 and 4, respectively.

Description of silver staining

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°С, 10 min cooling JAA.

Description of Western Blotting

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°С, 10 min cooling JAA.

1st antibody: monoclonal antibody to VP1, VP2 and VP3 AAV (adeno-associated virus)

Protein A affinity chromatography

PROGEN61058

2nd antibody: GOAT to mouse ALP

SIGMA A4656 1:2000 1 hour

As shown in Table 4, the volume of the resulting conditioned concentrate was approximately 6% of the original volume, however, the percentage of AAV in the concentrate was 74% as measured by ELISA.

Example 4

This example demonstrates an exemplary negative anion exchanger chromatography method.

After concentration and collection diafiltration with TFF as described in Example 3, the buffer containing AAV was conditioned to 125 mM NaCl/50 mM Tris-HCl/pH 8.5 for negative chromatography using a Mustang anion exchange membrane (Pall catalog # XT5000MSTGQP1 ). Under this condition, the AAV does not bind to the anion resin membrane and instead flows through the column. After loading the column, two full volumes (equal to 2x the membrane capsule) of wash buffer (125 mM NaCl/50 mM Tris-HCl; pH 8.5) were used to wash any unbound AAV8 from the column. In FIG. 5 is a chromatogram of the flow through the AEX Mustang Q chromatography product in the negative (non-binding) mode. Elution was performed to study bound proteins. Table 5 details the negative chromatography steps and Table 6 shows the yield.

As shown in Table 6, the negative chromatography step successfully removed a significant amount of total protein while retaining a significant amount of AAV. This increase in purity is also illustrated by silver staining and Western blotting in FIG. 6. The flow-through fraction was approximately 65% of the original amount of AAV, while less than 6% AAV was present in the eluted fraction. In addition, the total amount of protein in the flow fraction was significantly reduced. Only approximately 34% of the total protein remained in the FT fraction.

Description of silver staining

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°C, 10 min cooling JAA.

Description of Western Blotting

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°C, 10 min cooling JAA.

1st antibody: monoclonal antibody to VP1, VP2 and VP3 AAV (adeno-associated virus)

Protein A affinity chromatography

PROGEN61058

2nd antibody: GOAT to mouse ALP

SIGMA A4656 1:2000 1 hour

Example 5

This example demonstrates an exemplary second stage TFF method.

The AAV8-containing flow-through fraction (FT) obtained by negative chromatography (Example 4) was concentrated and diafiltered against diafiltration buffer (DB1) containing 500 mM NaCl/50 mM Tris-HCl; pH 8.5 to prepare the product for the next ultracentrifugation (UC) step (eg example 6, 7, 9, 14 or 16). The second stage TFF (TFF2) was performed using Pall T 100 kDa 4×0.1 m ² Omega Centramate membranes (4× Pall, catalog # OS100T12) and a Pall CM500 UF/DF system. After ultracentrifugation/diafiltration, the product was concentrated to a target volume of 1200 ml and discarded from the system. To increase the yield of AAV8, the membrane and the UF/DF system were then washed with approximately 400 ml of diafiltration buffer (10 min recirculation on the retentate side at 1 bar feed pressure). The membrane wash and concentrated product were combined and filtered using a 0.2 µm filter (PALL Cat. No.: KA02EAVP2S) to reduce bioburden. Table 7 details the TFF2 scheme and Table 8 details the yield of this step.

As shown in Table 8, the TFF2 step successfully reduced the volume to approximately 7.5% of the original volume (load volume) while retaining 88% of the original AAV.

Example 6

This example demonstrates an illustrative method for separating blank from full AAV particles by ultracentrifugation using 50% (w/w) ethylene glycol buffer and 50% (w/w) sucrose gradient. This step further removes host cell proteins (eg HSP70). This step also removes product-related impurities such as "empty capsids" and host cell proteins such as HSP70 and LDH.

50% (w/w) ethylene glycol buffered solution in 50 mM Tris-HCl/750 mM NaCl at pH 8.0 containing AAV8 sample containing Padua human factor IX vector DNA in single-stranded self-complementary form (2.6 kb .n.), were loaded into an ultracentrifuge (UC), followed by loading of two different sucrose solutions that differ in sucrose concentration. The sucrose solution loaded into the UC was initially characterized by a sucrose concentration of 55%. The sucrose solution loaded into the UC immediately after the first sucrose solution had a sucrose concentration of 60%. The ratio of the loaded sample and the sucrose gradient is 1:1, and the sucrose solutions were characterized by equal volumes. The total volume of the core is 1600 ml.

One kilogram of a buffered sucrose solution with a sucrose concentration of 55% was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 550.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

One kilogram of a buffered sucrose solution with a sucrose concentration of 60% was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 600.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

A density gradient is formed during the first phase of UC in which the rotation speed was set to 4000 rpm and the temperature was maintained at 2-10°C. After the first phase, the rotation speed was increased to 35,000 rpm and the temperature was increased to 22°C. During this second phase, at higher speed and temperature, full AAV particles separated from empty capsids. After 20 hours, the ultracentrifuge was stopped and fractions containing most of the total AAV particles were collected. Additional details related to the UC step performed, including details of the input parameters for gradient construction, collection of the fraction containing total particles, elution rates, etc., are provided in Table 9 below.

The results are shown in FIG. 17, in which empty capsids (in fraction 15) are separated from full capsids (in fraction 11).

In FIG. 18 shows the results of four different assays for each of the fractions shown in FIG. 17. qPCR refers to ITR AAV8 quantitative PCR (ITR-qPCR). AAV8:AG refers to an ELISA assay against the AAV8 antigen. WAX% refers to the percentage of complete capsids quantified by weak anion exchange. The DNA/AAV ratio (sometimes referred to herein as Spec. Act. or specific activity) refers to the ratio of total vector genomes (ITR-qPCR) to the total number of rAAV8 particles (AAV8:AG), which indicates both the proportion of DNA-containing capsids, as well as complete capsids. A higher DNA/AAV ratio correlates with more complete capsids. The transition from full to empty capsids is visible by a sharp decrease in Spec. act. from fraction 13 to fraction 15. Values for each assay were normalized to each other as indicated by the y-axis.

Example 7

This example demonstrates an illustrative method for separating blank from full AAV particles by ultracentrifugation using a sucrose gradient. At this stage, proteins of the host cell (eg, HSP70) were further removed. Product related impurities such as "empty capsids" (as defined above) were also removed at this stage.

A concentrated fraction containing vector DNA containing single-stranded DNA encoding human B-domain deletion coagulation factor VIII obtained with TFF2 from Example 5 was loaded into an ultracentrifuge (UC) followed by the addition of three different sucrose solutions that differ in sucrose concentration. The sucrose solution loaded into the UC was initially characterized by a sucrose concentration of 50%. The sucrose solution loaded into the UC immediately after the first sucrose solution had a sucrose concentration of 55%, and the sucrose solution loaded last into the UC had a sucrose concentration of 60%.

One kilogram of buffered sucrose solution with 50% sucrose concentration was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 500.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

One kilogram of 55% sucrose buffered sucrose solution was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 550.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

One kilogram of a buffered sucrose solution with a sucrose concentration of 60% was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 600.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

A density gradient was formed during the first UC phase in which the rotation speed was set to 4000 rpm and the temperature was maintained at 2-10°C. After the first phase, the rotation speed was increased to 35,000 rpm and the temperature was increased to 22°C. During this second phase at higher speed and temperature, full AAV particles were separated from empty capsids. Approximately 5 hours later, the ultracentrifuge was stopped and fractions containing most of the total AAV particles were collected. Additional details relating to the UC step performed, including details of the input parameters for gradient construction, collection of fraction containing total particles, elution rates, etc., are provided in Tables 11 and 12 below.

The elution scheme is shown in Fig. 8, and silver stains and western blots of the load and individual fractions are shown in FIG. 9. As shown in Table 12, 82% of the particles in the peak pool are considered full particles.

Description of silver staining

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°C, 10 min cooling JAA.

Description of Western Blotting

NuPAGE 4-12% Bis-Tris midi gel 1.0 mm, 20 wells, p/n WG1402BX10

MES SDS moving buffer, Invitrogen, catalog # NP0002

Incubation SB + DTT 10 min at 70°C, 10 min cooling JAA.

1st antibody: monoclonal antibody to VP1, VP2 and VP3 AAV (adeno-associated virus)

Protein A affinity chromatography

PROGEN61058

2nd antibody: GOAT to mouse ALP

SIGMA A4656 1:2000 1 hour

Example 8

This example demonstrates an exemplary lipid-enveloped virus inactivation method and a final purification step.

AAU8-containing fractions from the UC step in Example 7 were pooled and diluted 1:2.5 with equilibration buffer (50 mM Tris; pH 8.5±0.2). A solvent-detergent treatment was carried out (0.3% tri-n-butyl phosphate; 1.0% Triton X100; 0.3% polysorbate 80). The conductivity of the solvent was subsequently adjusted to 3.6±0.2 mS/cm with a further dilution of 1:2. The resulting solution was loaded onto a chromatographic column containing Fractogel® EMD TMAE (M) (Merck-Millipore, catalog no.: 116881), bed height 22 mm, 57 mm (Merck-Millipore Vantage VL 22×250. catalog no.: 96220250) and then washed with equilibrating buffer (50 mM Tris, pH 8.5±0.2). The second washing step was performed with buffer (8.09 mM Na ₂ HPO ₄ ; 1.47 mM KH ₂ PO ₄ ; 2.68 mM KCl; 5% sorbitol). Column elution was performed with a 12 column volume NaCl gradient from the first elution buffer containing 8.09 mM Na ₂ HPO ₄ ; 1.47 mM KH ₂ PO ₄ ; 2.68 mM KCl; 5% sorbitol to the last elution buffer containing 8.09 mm Na ₂ HPO ₄ , 1.47 mm KH ₂ PO ₄ ; 2.68 mM KCl; 5% sorbitol + 600 mM NaCl. The majority of AAV8 elutes as a distinct sharp peak and is collected as E2 (eluate 2) which is the purified AAV8 product. At the end, the column is desorbed with 2M sodium chloride, followed by a regeneration procedure. Tables 13 and 14 describe these steps in detail and the yield obtained after performing these steps. Fig. 10 is the final purification of AAV8 on Fractogel TMAE.

L Dil: represents the diluted pool after ultracentrifugation before SD exposure and final dilution; E is the eluate from the AEX column; Bulk is a 1:2 diluted eluate from the AEX column. Bulk is a formulated product prior to dilution to the final drug product (FDP).

Example 9

This example demonstrates an illustrative method for purifying AAV from crude fractions, which includes an ultracentrifugation step during which a density gradient is formed.

Fig. 11 is a diagram of steps performed in an exemplary method. The ultracentrifugation step was performed using a 3.2 L core (1.6 L product + 1.6 L gradient/TBS buffer).

With the UC at rest, a 1.6 L feed (intermediate from the previous process step = UFB) was loaded into the rotor at a constant flow rate of 6 L/h, and then 3 sucrose solutions of different densities were loaded. The sucrose solutions were loaded at a constant flow rate of 1.5 l/h in the following order: 800 ml of 50% sucrose, 400 ml of 55% sucrose and 400 ml of 60% sucrose.

The filled rotor was accelerated from 0 to 4000 rpm to achieve zoning to build a density increasing sucrose gradient followed by a second acceleration from 4000 to 35000 rpm. The UC runs at 35,000 rpm (approximately 90,000 g-force) for 5 hours. Once the final speed was reached, the temperature was changed from +4°C to +22°C. The AAV8 particles contained in the starting material move into the sucrose gradient until they reach a point where their density matches that of the surrounding gradient. Since full and empty capsids differ in their density, this function is used to separate full and empty viral capsids. One hour before the completion of the centrifugation step, the temperature was manually shifted from +22°C to +4°C. Stopping UC from 35000 to 0 rpm was done in 2 stages: from 35000 to 4000 rpm by slowing down and from 4000 rpm to 0 rpm by allowing the rotor to gradually stop.

The product was recovered at a flow rate of 6 l/h by collecting 25 ml fractions, as well as waste fractions at the end of the elution.

The collection of fractions of 25 ml each begins with fraction 7 and ends at the beginning of the waste peak. The first peak in Fig. 12 at a higher density of sucrose is a "full capsid". The second peak is "mostly empty capsids".

Fractions F10 to F14 were pooled and further purified on Fractogel® EMD TMAE (M) as AAV8 standard "full capsids" in the same manner as other fractions containing AAV8. Table 15 provides details of the TMAE chromatography step and Table 16 lists the buffers used.

Approximately 25 ml of one ultracentrifugation fraction was added separately to a 4 ml TMAE column diluted 1:2.5 with 50 mM Tris equilibration buffer, pH 8.5 ± 0.2. After solvent treatment in the presence of 0.3% tri-n-butyl phosphate, 1% Triton X-100 and 0.3% polysorbate 80, the conductivity was adjusted to 3.6±0.2 mS/cm followed by a 1:2 dilution, and containing The AAV8 solution was applied to a chromatographic column containing a Fractogel® EMD TMAE (M) (Merck-Millipore, cat. no. first wash step with 50 mM Tris equilibration buffer, pH 8.5±0.2 and second wash step with 8.09 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 5% sorbitol, pH 7.4. Elution is carried out with a gradient of 12 column volumes from 8.09 mm Na ₂ HPO ₄ , 1.47 mm KH ₂ PO ₄ , 2.68 mm KCl, 5% sorbitol, pH 7.4, to 8.09 mm Na ₂ HPO ₄ . 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 5% sorbitol + 600 mM NaCl, pH 7.4. The majority of AAV8 elutes as a distinct sharp peak and is collected as E2 (eluate 2) which is the purified AAV8 product. The obtained eluate E2 containing AAV8 (eluate 2) was diluted 1:2 with 8.09 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 5% sorbitol + 600 mM NaCl, pH 7.4 to bring to the matrix of the buffer for the composition (8.09 mm Na ₂ HPO ₄ , 1.47 mm KH ₂ PO ₄ , 2.68 mm KCl, 5% sorbitol + 350 mm NaCl, pH 7.4). Finally, the column is purified with 2 M sodium chloride, followed by a regeneration procedure. The chromatographic system was placed at ambient temperature from +18°C to +25°C.

Fractions 10 to 21 were analyzed by analytical ultracentrifugation (AUC), and DNA analysis of AAVS vectors (containing double-stranded factor IX Padua (4.8 kb)) was performed by native agarose gel electrophoresis and electrophoresis in alkali agarose gel. In FIG. 13-16 show the analytical data of individual fractions obtained with UC after final purification on TMAE. Fig. 14 is a photograph of agarose with 1% native, and FIG. 15 is a photograph of 0.8% caustic agarose. As shown in FIG. 15, pooled X10-X 14 peak UV fractions obtained at higher sucrose density by ultracentrifugation contained highly pure DNA single band without any further indication of smaller DNA variants of the vector DNA.

Analytical UC data are presented in Table 17.

Fractions No. 22, 23 and 24 were not analyzed

As shown in Table 17, fractions 10-15 contained the highest amounts of complete AAV capsids. Although significant amounts of full capsids were found in fractions 16 and 17, the numbers were slightly lower and the number of empty capsids increased compared to fractions 10-15. A further decrease in the number of full capsids and a further increase in empty capsids were observed in fractions 19 and 20.

In FIG. 16 shows data for one BDS from the indicated UC fractions. These results show a homogeneous AAV8 product from the UC 10 to 15 fraction with no detection of incomplete vector DNA content in AUC and agarose gel electrophoresis. Incomplete vector DNA emerges from fraction 17 and is found in later fractions of the "mostly empty capsids" zone. The detection of incomplete vectors was surprising because no one expected to get such a wide range of resolution. Thus, the developed separation method is not limited to separating capsids that are completely empty (i.e., no DNA) and capsids that are "full", but it unexpectedly also allows you to separate vector DNA of different lengths that cannot be separated from each other. It also indicates that there is a significant separation of "full capsids" from "empty capsids" regardless of the length of DNA in the population of "empty capsids". In other words, this method allows the depletion of empty vectors (including truncated and incomplete DNA) to produce AAVs with a high content of complete AAV capsids.

Example 10

This method describes an exemplary nanofiltration method for removing viruses larger than AAV and larger than the pore size of the applied nanofilter.

The Asahi PLANOVA 35nm filter, which contains bundles of microporous hollow fibers made from natural hydrophilic cuprammonium regenerated cellulose with a narrow pore size range of 35nm +/- 2nm, is examined with an integrity leak test to ensure that there are no holes or large defects, and pre-washed with formulation buffer. Then the anion-exchange eluate is taken off and filtered at a constant pressure not exceeding 0.1 MPa in a dead-end mode through a nanofilter. Alternatively, the filter can also be run in a tangential flow manner. The anion exchange eluate can be pre-diluted with elution buffer (PBS + 600 mM NaCl) to adjust the concentration of viral particles. To remove all virus particles, the nanofilter is washed with a buffer (eg formulation buffer). The examination of the nanofilter after the integrity examination is performed by a leak test and a gold particle test to prove that the pore size distribution of the nanofilter has not changed and the nanofilter has retained its integrity during filtration. The higher the sample loading, the higher the yield. Therefore, as a rule, more than 50 liters of solution per m ² of filter area are used.

Example 11

This method describes an exemplary nanofiltration method for removing viruses larger than AAV and larger than the pore size of the applied nanofilter.

The Asahi PLANOVA 35nm filter, which contains bundles of microporous hollow fibers engineered from natural hydrophilic cuprammonium regenerated cellulose with a narrow pore size range of 35nm +/- 2nm, was examined with an integrity leak test to ensure that no holes or large defects, and pre-washed with formulation buffer. Then 4.05 ml of pooled fractions containing 2870.98 μg/ml AAV-8 (concentration determined by ELISA method) were first diluted in 4.05 buffer 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 8, 09 mm Na ₂ HPO ₄ , 600 mm NaCl and 5% sorbitol at pH 7.4. The dilution ratio was 1:2. Then 8.10 g of the first dilution was diluted in 61.57 g of a buffer of 1.47 mm KH ₂ PO ₄ , 2.68 mm KCl, 8.09 mm Na ₂ HPO ₄ , 350 mm NaCl and 5% sorbitol at pH 7, 4 to get the boot buffer. The loading buffer dilution ratio was 1:8.6 compared to the first dilution and 1:17.2 compared to the pooled fractions.

The nanofilter was conditioned with 25 ml buffer containing 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 8.09 mM Na ₂ HPO ₄ , 350 mM NaCl and 5% sorbitol at pH 7.4. During conditioning, 10 ml of buffer was used to purge the filter without pressure and with the retentate outlet open. Approximately 15 ml of buffer was then filtered through hollow fibers, a constant pressure of 0.9 bar was applied and the outlet of the retentate was closed.

Then 9 ml of wash buffer was applied to the storage tank at a constant pressure of 0.9 bar. The wash buffer contained 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 8.09 mM Na ₂ HPO ₄ , 350 mM NaCl and 5% sorbitol at pH 7.4. Samples were taken after washing. After sampling, 60 g of the above loading buffer was then filtered at a constant pressure of 0.9 bar in a dead-end mode through a nanofilter.

Filtration data is presented in Table 18 below. The yield of AAV after filtration is at least 77%.

Example 12

The following example describes an exemplary method for testing the bioactivity of an AAV8 product.

In an in vitro bioactivity assay, the AAV8-FIX viral vector transfects a hepatic target cell line, which subsequently secretes a functional, measurable FIX protein into the medium.

In the first step, target HepG2 cells are transduced with AAV8-FIX. During incubation, FIX is released into the cell supernatant. In the second step, the factor IX activity of the cell culture supernatant is directly measured by the FIX chromogenic assay. The AAV8-FIX sample measurement is given as a percentage relative to the control material. The method allows to quantify the biological function of the AAV8-FIX gene therapy vector.

Example 13

This example demonstrates an AAV-specific ELISA.

In the examples above, an AAV-specific ELISA was performed after the process steps of the present disclosure to identify AAV-containing fractions and to calculate the DNA/AAV ratio or AAV "specific activity", which is represented by the ratio of qPCR to µg of AAV8. The DNA/AAV ratio reflects the vector DNA encapsulated in the AAV particles.

AAV8 ELISA was performed using the AAV-8 Titration ELISA Kit (Part 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 ADK8 antibody was bound to an immune complex (ADK8 and ADK8 antibody). 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. The peroxidase substrate solution was added and a color reaction occurred which is proportional to the number of bound AAV particles. The color reaction is measured photometrically at 450 nm.

ELISA determines the number of AAV8 particles present in the investigated fraction.

Example 14

This example demonstrates an illustrative method for separating blank from full AAV particles by ultracentrifugation using a buffered 50% ethylene glycol solution and a 50% sucrose gradient (ie, 1:1 ratio). This step removes product-related impurities such as "empty capsids" and host cell proteins such as HSP70 and LDH.

Buffered ethylene glycol solution (50% (w/w) in Tris-HCl/NaCl) containing AAV8 with human coagulation factor VIII (~4.8 kb total length) was loaded into an ultracentrifuge (UC) with followed by a sucrose gradient formed from two different sucrose solutions that differ in sucrose concentration. The ratio of loaded sample to sucrose gradient is 1:1. The sucrose solution loaded first into the UC had a sucrose concentration of 55%. The sucrose solution loaded into the UC immediately after the first sucrose solution had a sucrose concentration of 60%.

One kilogram of a buffered sucrose solution with a sucrose concentration of 55% was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 550.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

One kilogram of a buffered sucrose solution with a sucrose concentration of 60% was prepared by mixing 2.42 g of tris(hydroxymethyl)aminomethane (trometamol) with 8.00 g of sodium chloride and 600.00 g of sucrose. WFI was added to 1 kg using 1 M NaOH and 25% HCl to adjust the pH if necessary. WFI was then added to 1 kg.

In this example, a 3200 ml core was used, with 50% buffered ethylene glycol solution containing AAV8 being 1600 ml, 55% (w/w) sucrose solution being 800 ml, and 60% (w/w) sucrose solution being 800 ml.

A density gradient is formed during the first UC phase in which the rotation speed was set to 4000 rpm and the temperature was maintained at 2-10°C. After the first phase, the rotation speed was increased to 35,000 rpm and the temperature was increased to 22°C. During this second phase at higher speed and temperature, full AAV particles were separated from empty capsids. After 20 hours, the ultracentrifuge was stopped and fractions containing most of the total AAV particles were collected.

The results are shown in FIG. 19, in which full capsids (fractions 1-6) are separated from empty capsids (fractions 8-17).

Fig. 20 is an overlay of plots of sedimentation coefficients from fractions 8-10 showing subspecies separation (ie, incomplete vector DNA). The arrow indicates a shift towards lower mass AAV8 from higher density to lower density fractions in the UC gradient (ie density: fraction 8 > fraction 9 > fraction 10). This is another demonstration that vector DNA can be separated based on size with appropriate resolution by this protocol. See also table 19.

Example 15

This example demonstrates ultracentrifugation using a sucrose gradient using the 50-55-60 sucrose solution method. The method is the same as described in example 7, except that the AAV8 particles contained a single-stranded vector DNA of human coagulation factor IX Padua (2.6 kb). As shown in FIG. 21, the separation between full and empty capsids is not resolved as indicated by the overlay of ITR qPCR and AAV ELISA.

Example 16

This example demonstrates sucrose gradient ultracentrifugation using the 55-60 sucrose solution method as described in Example 6, except that a total core volume of 7700 ml is used. Thus, the loading volume was 6100 ml in 50% Tris-HCl buffered ethylene glycol solution, the volume of 55% sucrose solution was 800 ml, and the 60% sucrose solution was also 800 ml. The ultracentrifugation elution profile is shown in FIG. 22. There are no "waste peaks" in this document because AAV is highly purified. Complete AAV8 is collected from fractions 1-8 (50 ml each). "Empty" AAV is collected from fractions 9-15 (50 ml each). Next, fractions 15-21 are collected (100 ml each). The first peak in Fig. 22 at a higher density of sucrose is "full capsids". The second peak represents "empty capsids".

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 terms singular and plural and similar references in the context of the description of the present disclosure (especially in the context of the following claims) should be construed as embracing both the singular and the plural, unless otherwise specified herein or clearly contrary to 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 this specification. as if it were individually stated in this document.

All of the methods described herein may 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 wording in the present description should be construed as indicating any unclaimed element as essential to the practice of this disclosure.

Preferred embodiments of the present disclosure are described herein, including the best mode known to the present inventors for carrying out the present disclosure. Variations to these preferred 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 as 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.

Claims (68)

1. A method for purifying full capsids of an adeno-associated virus (AAV) from a concentrated fraction of AAV containing empty AAV capsids and complete AAV capsids, which includes: (i) loading the zone rotor with a) a concentrated AAV fraction and b) at least two sugar solutions, each having a different sugar concentration and each containing sugar at a concentration equivalent to a sucrose concentration ranging from about 45% (w/w) ratio) to about 65% (w/w) sucrose, (ii) operating the ultracentrifuge containing the zonal rotor in batch mode, after which a sugar gradient is formed, (iii) obtaining a sugar gradient fraction to obtain an AAV fraction, wherein at least or about 60% of the AAV particles in the AAV fraction are complete AAV capsids, wherein the total volume of the sugar solutions and the concentrated AAV fraction is less than or equal to the volume of the zonal rotor and the ratio of the volume of solutions sugar to the volume of the concentrated AAV fraction is less than or equal to one. 2. The method of claim 1 wherein each sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 50% (w/w) to about 60% (w/w) sucrose. 3. The method of claim 1 or claim 2, wherein each sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 55% (w/w) to about 60% (w/w) sucrose. 4. The method according to any one of paragraphs. 1-3, wherein at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration greater than about 50% (w/w) sucrose. 5. The method according to any one of paragraphs. 1-4, wherein at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration greater than about 55% (w/w) sucrose. 6. The method according to any one of paragraphs. 1-5, wherein at least one of the sugar solutions contains sugar at a concentration equivalent to a sucrose concentration ranging from about 60% (w/w) to about 65% (w/w) sucrose. 7. The method according to any one of paragraphs. 1, 2, or 4-6, wherein one sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 52% (w/w) to about 58% (w/w) sucrose, wherein the second sugar solution contains sugar in a concentration equivalent to a sucrose concentration ranging from about 57% (w/w) to about 63% (w/w) sucrose. 8. The method of claim 7, wherein the third sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 47% (w/w) to about 53% (w/w) sucrose. 9. The method according to any one of paragraphs. 1-8, in which two sugar solutions are loaded into a zone rotor. 10. The method according to any one of paragraphs. 1, 2 or 4-9, in which two sugar solutions are loaded into a zonal rotor and wherein one sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 52% (w/w) to about 58% (w/w) sucrose, and the second sugar solution contains sugar at a concentration equivalent to the concentration sucrose in the range from about 57% (w/w) to about 63% (w/w) sucrose. 11. The method according to any one of paragraphs. 1-8, wherein at least three sugar solutions are loaded into the zone rotor. 12. The method according to any one of paragraphs. 1-8 or 11, in which three sugar solutions are loaded into the zone rotor. 13. The method according to any one of paragraphs. 1, 2, 4-8, 11, or 12 wherein three sugar solutions are loaded into a zone rotor and wherein one sugar solution contains sugar at a concentration equivalent to a sucrose concentration ranging from about 47% (w/w) to about 53% (w/w) sucrose, the second sugar solution contains sugar at a concentration equivalent to a concentration of sucrose ranging from about 52% (w/w) to about 58% (w/w) sucrose, and the third sugar solution contains sugar at a concentration equivalent to sucrose concentrations ranging from about 57% (w/w) to about 63% (w/w) sucrose. 14. The method according to any one of paragraphs. 1, 2, or 4-13, wherein in step (i), the concentrated AAV fraction is loaded before a sugar solution containing sugar at a concentration equivalent to a sucrose concentration in the range of from about 52% (w/w) to about 58% (w/w ) sucrose, and wherein a sugar solution containing sugar at a concentration equivalent to a concentration of sucrose in the range from about 52% (w/w) to about 58% (w/w) sucrose is loaded before a sugar solution containing sugar at a concentration equivalent to the concentration of sucrose in the range from about 57% (w/w) to about 63% (w/w) sucrose. 15. The method according to any one of paragraphs. 1, 2, 4-8, or 11-14, wherein in step (i), the concentrated AAV fraction is loaded before a sugar solution containing sugar at a concentration equivalent to a sucrose concentration of from about 47% (w/w) to about 53% (w/w). mass ratio) of sucrose, and a sugar solution containing sugar in a concentration equivalent to the concentration of sucrose, is in the range from about 47% (w/w) to about 53% (w/w) sucrose, is loaded before the sugar solution containing sugar in a concentration , equivalent to a concentration of sucrose in the range from about 52% (w/w) to about 58% (w/w) sucrose, and moreover, a sugar solution containing sugar in a concentration equivalent to a concentration of sucrose in the range from about 52% (w/w) to approximately 58% (w/w) sucrose, is loaded before a sugar solution containing sugar at a concentration equivalent to con sucrose concentrations ranging from about 57% (w/w) to about 63% (w/w) sucrose. 16. The method according to any one of paragraphs. 1-15, in which each sugar solution is loaded into the zonal rotor in equal volumes. 17. The method according to any one of paragraphs. 1-15, in which the sugar solution with the lowest sugar concentration is loaded into the zonal rotor in a volume twice the volume of at least one of the other sugar solutions in the zonal rotor. 18. The method according to any one of paragraphs. 1-15 or 17, in which the sugar solution with the lowest concentration of sugar is loaded into the zonal rotor in a volume equal to at least the volume of all other sugar solutions combined in the zonal rotor. 19. The method according to paragraphs. 1-15, 17 or 18, in which the sugar solution with the lowest sugar concentration is loaded into the zonal rotor in a volume that is at least twice the volume of the sugar solution with the highest sugar concentration, optionally, and the volume of the sugar solution with the highest sugar concentration is equal to the volume of the solution sugar with an intermediate concentration of sugar. 20. The method according to any one of paragraphs. 1-15 or 17-19, in which at least two sugar solutions are loaded into the zonal rotor, and the sugar solution with the lowest sugar concentration is loaded into the zonal rotor in a volume that is at least twice the volume of at least one other sugar solution in zone rotor. 21. The method according to any one of paragraphs. 1-20, in which at least two sugar solutions are loaded into the zonal rotor, and the sugar solution with the lowest sugar concentration is loaded into the zonal rotor in a volume equal to the volume of at least one other sugar solution in the zonal rotor. 22. The method according to any one of paragraphs. 1-21, in which the sugar solution with the lowest sugar concentration is loaded into the zonal rotor in a volume that is half the volume of the concentrated AAV fraction. 23. The method according to any one of paragraphs. 1-22, in which the ratio of the volume of the total sugar gradient to the volume of the concentrated fraction of AAV loaded into the zonal rotor is from about 1:1 to about 1:5. 24. The method according to any one of paragraphs. 1-23, in which the concentrated AAV fraction loaded into the zonal rotor contains a buffered solution. 25. A method according to any one of the preceding claims, wherein each sugar solution contains a disaccharide or trisaccharide. 26. The method of claim 25, wherein the disaccharide contains sucrose, maltose, lactose, and combinations thereof. 27. A method according to any one of the preceding claims, wherein each sugar solution contains sucrose. 28. A method according to any one of the preceding claims, wherein each of the sugar solutions and/or AAV fractions contains Tris-HCl and NaCl. 29. The method of claim 28, wherein the concentration of Tris-HCl is from about 10 to about 100 mM and the concentration of NaCl is from about 100 mM to about 1 M. 30. The method according to p. 29, wherein the concentration of Tris-HCl is from 20 to 50 mm. 31. The method according to claim 29 or claim 30, wherein the NaCl concentration is between 150 and 750 mM. 32. The method according to any one of paragraphs. 29-31, in which the concentration of NaCl is from 150 to 500 mm. 33. The method according to any one of paragraphs. 29-32, wherein each of the sugar solutions and/or the AAV fraction has a pH of 7.0 to 9.0. 34. The method according to any one of paragraphs. 29-33, wherein each of the sugar and/or AAV fraction solutions has a pH of about 7.4 to about 8.5. 35. The method according to any one of paragraphs. 1-34, wherein each of the sugar and/or AAV fraction solutions contains 45-55% (w/w) ethylene glycol. 36. The method according to any one of paragraphs. 1-34, wherein each of the sugar solutions and/or AAV fractions contains 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% (w/w) ethylene glycol. 37. The method of any one of the preceding claims, comprising operating the ultracentrifuge at a first speed of less than 10,000 rpm for less than 60 minutes and at a second speed of about 30,000 to about 40,000 rpm for a minimum of 4 hours. 38. The method of any one of the preceding claims, comprising operating the ultracentrifuge at a first speed of less than 10,000 rpm for less than 60 minutes and a second speed of about 30,000 to about 40,000 rpm for a minimum of 12 hours. 39. The method of claim 37 or 38, wherein the first rotation speed is from about 3000 to about 6000 rpm, optionally about 4000 rpm. 40. The method of claim 37 or 39, wherein the second rotation speed is about 35,000 rpm and optionally maintained for about 4 to about 6 hours. 41. The method according to any one of paragraphs. 37-39, wherein the second rotation speed is maintained for at least about 16 hours, or at least 20 hours. 42. The method according to any one of paragraphs. 37-39 or 41 wherein the second rotation speed is about 35,000 rpm and optionally maintained for about 16 to about 20 hours. 43. The method according to any one of the preceding claims, wherein the concentrated AAV fraction loaded into the zonal rotor contains at least 1x10 ¹² AAV capsids per ml. 44. A method according to any one of the preceding claims, comprising collecting supernatant from a cell culture containing HEK293 cells transfected with a triple plasmid system. 45. The method of claim 44, comprising collecting the supernatant about 3 to about 5 days after transfection of HEK293 cells, or when the cell culture has a cell density greater than or about 5x10 ⁶ cells/mL and cell viability greater than 50%. 46. The method according to claim 44 or 45, which involves filtering the collected supernatant using deep filtration. 47. The method according to p. 46, providing for filtering the collected supernatant through a filter containing cellulose and perlite and characterized by a minimum permeability of approximately 500 l/m ² . 48. The method of claim 47, comprising filtering the collected supernatant through a filter having a minimum pore size of approximately 0.2 microns. 49. The method of any one of the preceding claims, comprising (i) introducing a concentrated AAV fraction into an anion exchange chromatography (AEX) column under conditions that allow the AAV to pass through the AEX chromatography column, and (ii) collecting the stream containing the AAV. 50. The method of claim 49, wherein the concentrated AAV fraction is applied to an AEX chromatography column with a loading buffer containing from about 100 mM to about 150 mM NaCl, optionally, the pH of the loading buffer is from about 8 to about 9. 51. The method of claim 50, wherein the loading buffer contains from about 115 mM to about 130 mM NaCl, optionally, the loading buffer contains from about 120 mM to about 125 mM NaCl. 52. A method according to any one of the preceding claims, comprising concentrating the concentrated AAV fraction using an ultra/diafiltration system. 53. The method of claim 52, comprising concentrating the concentrated AAV fraction using an ultra/diafiltration system before, after, or before and after the step of introducing the concentrated AAV fraction into an anion exchange chromatography (AEX) column under conditions that allow the AAV to flow through the chromatographic AEX column. 54. A method according to any one of the preceding claims, wherein the proteins of the host cell are removed. 55. The method of claim 54, wherein the host cell proteins are HSP70 and/or LDH. 56. A method according to any one of the preceding claims, further comprising inactivating lipid-enveloped viruses of the AAV fraction with a solvent and/or detergent. 57. A method according to any one of the preceding claims, further comprising nanofiltration of the AAV fraction to remove viruses larger than 35 nm. 58. A method according to any one of the preceding claims, further comprising a post-purification step comprising performing AEX chromatography on a column containing a tentacle gel. 59. The method according to any of the preceding paragraphs, which involves the study of the AAV fraction using AAV-specific ELISA. 60. The method of claim 59, wherein the method does not include the step of measuring activity by quantitative PCR. 61. The method of claim 59 or 60, wherein the AAV-specific ELISA is an AAV-specific sandwich ELISA. 62. A method according to any one of the preceding claims, wherein the method comprises a first sugar solution containing sugar at a concentration equivalent to about 55% sucrose, a second sugar solution containing sugar at a concentration equivalent to about 60% sucrose, and optionally a third solution. sugar containing sugar at a concentration equivalent to a concentration of sucrose of approximately 50%. 63. The method according to any one of the preceding claims, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10. 64. The method according to any one of the preceding claims, wherein the AAV is AAV8.

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