Separation and quantification of empty and complete virus capsid particles

Abstract

The present disclosure provides methods for the separation and quantification of empty and complete viral capsids (e.g., AAV capsids) within a viral preparation, such as a viral pharmaceutical composition or product.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Application No. 62 / 873,619, filed on Jul. 12, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background of the Invention Adeno - associated virus (AAV) vectors have emerged as one of the most popular viral vector delivery systems in gene therapy. AAV - based gene delivery vectors (recombinant AAV or rAAV) contain an AAV capsid that includes a therapeutic transgene. A characteristic of AAV vector production in cell culture is the formation of excessive “empty” capsids, which lack the vector genome and thus cannot provide a therapeutic benefit. The impact of empty capsids on clinical outcomes is unclear, but there is a major concern that they may increase the innate or acquired immune response to the vector.

[0003] The development of analytical methods for quantifying empty vector particles as impurities has received particular attention for the characterization of viral preparations, pharmaceutical compositions, and pharmaceutical products. The methods currently available for determining the percentage of empty viral particles (or the ratio of empty to full) are cumbersome, complex, low - throughput, and / or have low resolution. Attempts have been made to separate empty AAV particles and rAAV (“full”) particles and to purify rAAV particles by chromatography during the manufacturing process; however, successful baseline peak resolution between the peak corresponding to the empty particles and the peak corresponding to the AAV vector containing the target genome has not yet been achieved.

[0004] Therefore, there remains a need for new AAV - specific assays for screening during the manufacturing process, release of vector products, and / or pharmaceutical product analysis that are well - suited to the stringent requirements of high - quality standards such as current Good Manufacturing Practices and USP standards.

Summary of the Invention

[0005] The present disclosure provides a method for separating empty and full capsids (e.g., empty and full AAV capsids) in a virus preparation (e.g., an AAV preparation). The method includes passing the virus preparation and a mobile phase through an ion (e.g., anion or cation) exchange column, the mobile phase including a discontinuous elution gradient and at least one (e.g., one, two, three, four, or five) isocratic hold.

[0006] In another aspect, the present disclosure provides a method for quantifying empty and full capsids (e.g., empty and full AAV capsids) in a virus preparation (e.g., an AAV preparation). The method includes passing the virus preparation and a mobile phase through an anion or cation exchange column, the mobile phase being run under conditions including a discontinuous elution gradient and at least one (e.g., one, two, three, four, or five) isocratic hold.

[0007] In some embodiments of the methods of the invention, the virus preparation and the mobile phase are run on a high performance liquid chromatography (HPLC) system.

[0008] In some embodiments, the empty and full capsids are separated by a baseline resolution, such as a baseline resolution greater than 2.0.

[0009] In some embodiments, the anion exchange column used in the method is a strong anion exchange (SAX) column, such as a quaternary amine (Q-amine) column. In some embodiments, the anion exchange column is a monolithic column.

[0010] In some embodiments, the cation exchange column used in the method is a strong cation exchange (SCX) column such as a benzenesulfonic acid column. In some embodiments, the cation exchange column is a monolithic column.

[0011] In some embodiments, the mobile phase used in the method of the present invention contains a salt. In further embodiments, the salt is tetramethylammonium chloride (TMAC) or sodium acetate. In certain embodiments, the final gradient of the mobile phase contains 0.1 to 10 M (e.g., 0.5 to 5 M such as 1, 1.5, or 2 M) of the salt. In certain embodiments, the salt is 0.5 to 5 M (e.g., 1 M) of TAMC or sodium acetate.

[0012] In some embodiments, at least one isocratic hold is introduced before the mobile phase reaches 50% of the final gradient, e.g., before the mobile phase reaches 20%, 25%, 30%, 35%, 40%, 40%, or 45% of the final gradient.

[0013] In some embodiments, the pH of the mobile phase is from about 8 to about 10, e.g., about 9.

[0014] In some embodiments, the column has a temperature between 0°C and 50°C, e.g., between 20°C and 25°C.

[0015] Also provided are highly purified recombinant virus compositions (e.g., AAV compositions) prepared by the methods of the present invention. In some embodiments, the virus composition comprises 100% empty capsids or less than 100% (e.g., less than 95%, less than 90%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%) empty capsids. In certain embodiments, the present disclosure provides a virus preparation enriched for empty virus capsids, which is obtained by the methods described herein and, optionally, 20% or less (e.g., 15% or less, 10% or less, 5% or less, or 1% or less) of the virus capsids in the preparation are full virus capsids. In certain embodiments, the present disclosure provides a virus preparation enriched for full virus capsids, which is obtained by the methods described herein and, optionally, 20% or less (e.g., 15% or less, 10% or less, 5% or less, or 1% or less) of the virus capsids in the preparation are empty virus capsids.

[0016] In some embodiments, the AAV herein may be derived from one or more serotypes such as AAV1, AAV2, AAV3, AAV6, AAV8, and AAV9.

[0017] In some embodiments, the recombinant AAV (rAAV) herein has a recombinant genome having a size of about 20 to 9,000 bases.

[0018] Other features, objects, and advantages of the invention will be apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figures 1A and 1B are representative HPLC chromatograms of three different (stacked) samples showing peak positioning for empty AAV (peak 1) and full AAV (peak 2) particles. FFB: final formulation buffer. rAAV6-GLA3: recombinant AAV6 carrying the alpha-galactosidase A (GLA) transgene. rAAV6-GLA2: different production batches of recombinant AAV6-GLA virus. AAV6 empty: empty AAV6 capsid sample.

[0020] Figure 2 is a graph showing the comparison and correlation of the percent of full capsids calculated by HPLC versus the vector genome (VG / capsid) ratio to capsid particles.

[0021] Figure 3 is a stacked HPLC chromatogram showing the peak area of full capsids and the linearity of various injection volumes for the same sample.

[0022] Figure 4 is a graph showing the peak area of full capsids and the linearity of various injection volumes for the same sample analyzed by HPLC.

[0023] Figure 5 is a stacked HPLC chromatogram showing the peak area of full capsids and the linearity of various capsid concentrations for the same sample at a constant injection volume.

[0024] Figure 6 is a graph showing the peak area of full capsids and the linearity of variable capsid concentrations for the same sample measured by HPLC analysis at a constant injection volume.

[0025] Figure 7 is an HPLC chromatogram showing peak separation of different rAAV samples and AAV empty samples having normalized capsid concentrations. rAAV6-375-1: Recombinant AAV6 carrying the alpha-L-iduronidase (IDUA) transgene. rAAV6-375-2: A different production batch of recombinant AAV6 carrying the IDUA transgene. rAAV6-GLA1: Recombinant AAV6 carrying a GLA expression cassette different from rAAV6-GLA2 or 3.

[0026] Figure 8 is an HPLC chromatogram showing peak separation and the effect of column temperature on retention time for a Full rAAV6-GLA2 (“GLA2”) capsid sample.

[0027] Figures 9A and 9B are HPLC chromatograms comparing the empty and full peak separation and retention times of AAV and rAAV samples from different commercial vendors. Samples include Empty AAV6 Lot1, Empty AAV6 Lot2, and Empty AAV6 Lot3.

[0028] Figure 10 is an HPLC chromatogram comparing the retention times of samples rAAV6-GLA3 and AAV6-030 [Empty AAV6 Lot 030] using four different elution buffers.

[0029] Figure 11A is an HPLC chromatogram showing the HPLC results of consecutive injections of a mixed Full rAAV6-GLA3 and Empty AAV6-030 sample using an elution buffer containing TMAC run at a gradient of approximately 0 - 100%.

[0030] Figure 11B is an HPLC chromatogram showing the HPLC results of consecutive injections of a mixed Full rAAV6-GLA3 and Empty AAV6-030 sample using an elution buffer containing TMAC run at a gradient of approximately 15 - 30%.

[0031] Figure 11C is an HPLC chromatogram showing the results of consecutive injections of the Empty AAV6-030 sample using an elution buffer containing TMAC run at a gradient of approximately 15 - 30%.

[0032] Figure 12A is an HPLC chromatogram showing the peak separation and retention time results for the Full rAAV6-GLA3 and Empty AAV6-030 samples run on a monolithic HPLC column using elution buffers containing TMAC at various pH levels.

[0033] Figure 12B is an HPLC chromatogram showing the peak separation and retention time results for the Full rAAV6-GLA3 and Empty AAV6-030 samples run on a weak anion exchange (WAX) HPLC column using elution buffers containing TMAC at various pH levels.

[0034] Figure 13 is an HPLC chromatogram showing the peak separation and retention time results for the Full rAAV6-GLA3 and Empty AAV6-030 samples run on a tandem configuration of AAV monolithic and weak anion exchange (WAX) HPLC columns using elution buffers containing TMAC at various pH levels.

[0035] Figure 14 is an HPLC chromatogram showing the peak separation and retention time results for the mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using loose gradients of approximately 10 and 15 minutes (longer HPLC run times).

[0036] Figure 15 is an HPLC chromatogram showing the peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using a loose gradient of elution buffer of about 10, 15, 20, 25, and 30 minutes, compared to the results for Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer run time gradient of about 5 minutes.

[0037] Figure 16 is an HPLC chromatogram showing the peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an analytical time gradient of elution buffer of about 1, 2, 3, 4, and 5 minutes.

[0038] Figure 17 is an HPLC chromatogram showing the peak separation and retention time results for samples containing Full rAAV6-GLA3 using an elution buffer containing TMAC run at a gradient of about 15% - 30% or 15% - 35% with an isocratic hold of about 17% or 18%.

[0039] Figure 18 is an HPLC chromatogram showing the peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of about 15% - 30% or 15% - 35% with an isocratic hold of about 18% or 19%.

[0040] Figure 19 is an HPLC chromatogram showing the peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of about 0% - 40% with an isocratic hold of about 19%.

[0041] Figure 20 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 0% to 40% with an isocratic hold of approximately 19%.

[0042] Figure 21 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 0% to 40% with an isocratic hold of approximately 19% and a response time of approximately 0.031 s / 0.13 s / 0.5 s.

[0043] Figure 22 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at gradients of approximately 0% to 40%, 5% to 40%, or 10% to 40% with an isocratic hold of approximately 19%.

[0044] Figure 23 is an HPLC chromatogram showing results for peak separation and retention time for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 5% to 40% with an isocratic hold of approximately 19% and a column temperature of approximately 20°C, 25°C, 30°C, or 35°C.

[0045] Figure 24 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 5% to 50% with an isocratic hold of approximately 19% and a column temperature of approximately 25°C or 35°C.

[0046] Figure 25 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 0% to 40% with excitation at 280 nm and emission detection wavelengths set at 348 / 350 / 355 nm using an isocratic hold of approximately 19%.

[0047] Figure 26 is an HPLC chromatogram showing peak separation and retention time results for mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at a gradient of approximately 0% to 40% with excitation at 348 nm and emission detection wavelengths set at 280 / 350 / 355 nm using an isocratic hold of approximately 19%.

[0048] Figure 27 is a panel of HPLC chromatograms showing a comparison of peak separation and retention time results for Formulation Buffer (FB), Empty (E), Full (F), and Empty+Full capsid mixed composition samples using an elution buffer containing TMAC run at a gradient of approximately 5% to 40% or approximately 5% to 50% with an isocratic hold of approximately 19%.

[0049] Figures 28A and 28B are an HPLC chromatogram and a graph showing the loading linearity of peak areas for Full rAAV6-GLA3 samples at approximately 2, 4, 6, 8, 10, and 20 μl.

[0050] Figures 29A and 29B are an HPLC chromatogram and a graph showing the loading linearity of peak areas for Empty AAV6-030 samples at injection volumes of approximately 2, 4, 6, 8, and 10 μl.

[0051] Figures 30A and 30B are graphs showing the dilution linearity of peak areas for both Full rAAV6-GLA3 and Empty AAV6-030 samples.

[0052] Figure 31 is an HPLC chromatogram showing UV260 and 280 nm traces of a mixed sample demonstrating the conventional 260 / 280 switch at the peak heights of the empty and full AAV peaks.

[0053] Figure 32 is a graph showing a calibration plot of different mixtures of Full rAAV6-GLA3 and Empty AAV6-030 samples demonstrating the linear relationship between peak area and capsid concentration.

[0054] Figure 33 is a panel of HPLC chromatograms showing the results of peak separation and retention times of mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at various pH values.

[0055] Figure 34 is a panel of HPLC chromatograms showing the results of peak separation and retention times of mixed Full rAAV6-GLA3 and Empty AAV6-030 samples using an elution buffer containing TMAC run at various column temperatures.

[0056] Figures 35A and 35B are representative graphs of CryoTEM analysis of Full rAAV6-GLA3 and Empty AAV6-030 samples.

[0057] Figures 36A and 36B are representative HPLC chromatograms showing the results of peak separation and retention times for Full rAAV6-GLA3, Empty AAV6-030, and other samples used for comparison with CryoTEM.

[0058] Figures 37A and 37B are graphs showing a comparison of the percent quantification of empty (A) and full AAV capsids from different rAAV6 samples tested by AUC, HPLC, and CryoTEM methods.

[0059] Figures 38A and 38B are graphs showing the correlation of the percentages of empty (A) and full (B) AAV particles in different viral samples calculated using HPLC and CryoTEM using JMP software.

[0060] Figure 39 is a series of HPLC chromatograms showing peak separation, retention time, and percent quantification results for AAV1 Empty Lot and recombinant AAV1 samples (AAV1-CMV-GFP, 17-AAV-321, and 17-AAV-037) using an elution buffer containing TMAC run at a gradient of approximately 0% - 30% with an isocratic hold of approximately 20%.

[0061] Figure 40 is a series of HPLC chromatograms showing peak separation, retention time, and percent quantification results for empty and full AAV capsids of AAV2 Empty Lot and recombinant AAV2 samples (17-AAV-077 and 17-AAV-155) using an elution buffer containing TMAC run at a gradient of approximately 0% - 30% with an isocratic hold of approximately 12%.

[0062] Figure 41 is a series of HPLC chromatograms showing peak separation, retention time, and percent quantification results for empty and full AAV capsids for AAV3 Empty Lot and recombinant AAV3 samples (AAV3-CMV-GFP, 17-AAV-124, and 17-AAV-324) using an elution buffer containing TMAC run at a gradient of approximately 0% - 30% with an isocratic hold of approximately 15%.

[0063] Figures 42A and 42B are a series of HPLC chromatograms showing peak separation, retention time, and percent quantification results of empty and full AAV capsids for AAV8 Empty Lot (17-AAV-082) and recombinant AAV8 samples (18-AAV-070, 17-AAV-339, 17-AAV-340, 17-AAV-341, and AAV8 RSM)) using an elution buffer containing TMAC run at a gradient of about 0% - 30% with an isocratic hold of about 14%.

[0064] Figure 43 is a series of HPLC chromatograms showing peak separation, retention time, and percent quantification results of empty and full AAV9 capsids for samples PD Lot 076, PD70 Lot 19-BAV-484PD, PD76 Lot 20-BAV-027PD, and Run24 Lot 19-BAV-470 using an elution buffer containing TMAC run at a gradient of about 0% - 40% with an isocratic hold of about 5%.

[0065] Detailed Description of the Invention The methods described herein may be used to separate and quantify empty adeno-associated virus (AAV) capsids and full AAV (e.g., recombinant AAV or rAAV) capsids in AAV preparations such as AAV pharmaceutical compositions and pharmaceutical products using column chromatography. For example, high performance liquid chromatography (HPLC), also referred to as high pressure liquid chromatography, can be used to baseline separate empty AAV capsids and full AAV (rAAV) capsids in virus preparations, pharmaceutical compositions, and pharmaceutical products to an extent that the area or height of each peak can be accurately measured.

[0066] AAV is a non-enveloped single-stranded DNA virus that can be engineered to deliver DNA (e.g., a therapeutic gene or a reporter gene) to target cells. During the production of AAV vectors, the DNA is packaged into self-assembling viral particles by the action of AAV replicase (Rep) proteins. However, this DNA packaging process is often inefficient, resulting in a large number of empty particles lacking the vector genome. Depending on the production process used, in transfection-based procedures, the contamination of empty particles can be 20 to 30 times that of full particles (Lock et al., Hum. Gene Ther. (2010b) 21:1273-1285). The presence of empty particles effectively increases the dose of AAV capsid proteins administered during treatment and thus increases the likelihood of unwanted immune consequences against the vector capsid. Accordingly, the methods described herein may also be used to purify large-scale production batches of viral preparations, pharmaceutical compositions, and pharmaceutical products.

[0067] The terms "empty capsid", "empty virion particles", and "empty AAV" refer to AAV virions that are essentially the same as those of the desired product but contain an AAV protein capsid shell lacking the nucleic acid molecule packaged therein.

[0068] The terms "full capsid", "full virus particle", "rAAV", and "full rAAV" refer to AAV virions that contain an AAV protein capsid shell encapsidating the nucleotide sequence of interest.

[0069] The inventors have made the unexpected discovery that using improved HPLC parameters can achieve baseline resolution between chromatogram peaks corresponding to empty AAV capsids and full AAV capsids in a sample. Peak resolution is the distance between two peaks on a chromatogram. "Baseline separation" or "baseline resolution" means a resolution factor of at least 1.5. When the resolution exceeds 1.5, then there is less than about 1% interference between the two peaks. In some embodiments, the chromatographic peaks corresponding to empty and full vial particles (capsids) are separated by a baseline resolution greater than 2.0. In some embodiments, the peak resolution is greater than 2.0.

[0070] The HPLC systems described herein include a stationary phase and a mobile phase. For example, a monolithic strong anion exchange (SAX) column containing a poly(glycidyl methacrylate-co-ethylene glycol) support matrix may be used as the stationary phase for separating empty and full AAV capsids based on the slightly lower anion characteristics of the empty particles compared to the vector. Depending on the situation, it may be beneficial to utilize a weak anion exchange (WAX) column if the sample binding conditions permit, to achieve baseline separation of the chromatogram peaks. Depending on the situation, it may be beneficial to utilize a strong cation exchange (SCX) column or a weak cation exchange (WCX) column if the sample binding conditions permit, to achieve baseline separation of the chromatogram peaks. The retention time (RT) is desirably long enough to improve peak separation and increase the capacity factor. Thus, the pH of the mobile phase may be adjusted to achieve baseline separation. In some embodiments, a SAX, WAX, SCX, or WCX column, or multiple strong AEX, CEX, SAX, WAX, SCX, or WCX columns may be used connected in tandem such that the column length increases.

[0071] In some embodiments, the mobile phase comprises a buffer composition such as, for example, bis-tris propane (BTP). The concentration of the mobile phase buffer may be varied. For example, concentrations from about 1 mM to about 100 mM (e.g., 1 to 50 mM) may be used. In some embodiments, the elution buffer in the mobile phase may comprise one or more salts including, but not limited to, sodium chloride (NaCl), potassium chloride (KCl), tetramethylammonium chloride (TMAC), sodium acetate (NaOAc), and / or ammonium acetate (NH4OAc). In some embodiments, the salt may be at a concentration of about 0.1 M to about 10 M, or about 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.5 M, 2.0 M, 3.0 M, 3.5 M, 4 M, 4.5 M, 5.0 M, 5.5 M, 6.0 M, 6.5 M, 7 M, 7.5 M, 8.0 M, 8.5 M, 9.0 M, or 9.5 M. In an embodiment, the pH of the buffer composition is about 9.0. In some embodiments, the pH of the mobile phase buffer ranges from about 8.0 to about 9.5.

[0072] The mobile phase can be run as a gradient (i.e., gradient elution chromatography). A stable change in the mobile phase composition during chromatographic analysis is called gradient elution. For example, the elution solvent can start at a particular percentage and increase steadily over time. The gradient starts at 0% and increases to about 100%. In some embodiments, the gradient starts at about 2% - 15% and increases to about 30% - 100%, where the mobile phase comprises a 1 M salt elution solvent. In other embodiments, the gradient starts at about 10% and increases to about 30%. In still other embodiments, the gradient starts at about 15% and increases to about 30%. In still other embodiments, the gradient starts at about 15% and increases to about 35%. In other embodiments, the gradient starts at about 15% and increases to about 40%. In other embodiments, the gradient starts at about 15% and increases to about 45% or about 50%.

[0073] The mobile phase may be run as a shallow gradient (longer run time or slower increase in elution solvent strength), a steep gradient (shorter run time or faster increase in elution solvent strength), or a combination of the two. Additionally, an isocratic hold may be incorporated within the gradient mobile phase. During an isocratic hold or flow, the mobile phase composition is kept constant. The inventors have made the important discovery that incorporating an isocratic hold during gradient elution significantly improves the resolution of chromatographic peaks corresponding to empty and full virus particles, resulting in baseline separation of the peaks. This in turn increases the accuracy with which the amounts of empty and full virus particles in a sample may be measured.

[0074] In certain embodiments, an isocratic hold at about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% may be incorporated during the gradient elution of a chromatographic analysis. In some embodiments, peak symmetry is also improved by the addition of an isocratic hold adjacent to two gradients within the method. In other embodiments, peak asymmetry and tailing are less than 2.0.

[0075] In some embodiments, the run time of the mobile phase is between about 1 minute and 60 minutes, whether run as a gradient or not. In other embodiments, the run time of the mobile phase is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, or about 45 minutes.

[0076] The HPLC method described in this specification may be used with UV or fluorescence detection. In some embodiments, the wavelengths of excitation and emission are set to 280 nm ± 20 nm and 348 nm ± 20 nm, respectively.

[0077] In some embodiments, the response time of the detector is set to 0.5 seconds. In some embodiments, the response time is set to generate at least 20 data points over the entire chromatographic peak. In some embodiments, the response time is set between about 0.1 and 1.0 seconds.

[0078] Viral preparations may be obtained by any known production system such as mammalian cell AAV production systems (e.g., those based on 293T or HEK293 cells) and insect cell AAV production systems (e.g., those based on sf9 insect cells and / or those using a baculovirus helper vector). Viral preparations may be purified from cell cultures by using well-known techniques such as discontinuous cesium chloride density gradients (see, e.g., Grieger, Mol Ther Methods Clin Dev. (2016) 3:16002).

[0079] The method of the present invention can be used to purify and analyze viral preparations of various AAV serotypes such as AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAVrh10, AAV10, and AAV11, as well as variants, hybrids, chimeras or pseudotypes thereof. "Pseudotype" or "cross-packaging" means that rAAV is a recombinant AAV in which the capsid is replaced by the capsid of another AAV serotype, for example, to change the transduction efficiency or tropism profile of the virus (e.g., Balaji et al., J Surg Res. 184(1):691-8(2013)). "Chimeric" or "hybrid" rAAV means a recombinant AAV in which the capsid is assembled from capsid proteins derived from different serotypes and / or the capsid protein has a chimeric protein with sequences derived from different serotypes (e.g., serotypes 1 and 2; see Hauck et al., Mol Ther. 7(3):419-25(2003)). For example, the method of the present invention may be used to purify and analyze recombinant AAVs in which the genome such as ITR is derived from one serotype such as AAV2 and the capsid is derived from another serotype; for example, AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9. See, for example, U.S. Pat. Nos. 7,198,951 and 9,585,971.

[0080] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, the nomenclature and techniques used in connection with cardiology, medicine, pharmaceutical chemistry, and cell biology described herein are well known and commonly used in the art. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications as commonly accomplished in the art or as described herein. Further, unless the context requires otherwise, singular forms shall include pluralities and plural forms shall include singulars. Throughout this specification and the embodiments, the terms "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean including the stated integer or group of integers, but not to mean excluding any other integer or group of integers. All publications and other references cited herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not admit that any of these documents form part of the common general knowledge in the art. As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, the term refers to a range of values that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the recited reference value unless otherwise stated or otherwise apparent from the context. As used herein, a value "between" two numerical values may be one of the two numerical values.

[0081] To better understand the present invention, the following examples are shown. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The present disclosure relates to, for example, the following. [1] A method for separating empty and full capsids in a virus preparation, the method comprising passing the virus preparation and a mobile phase through an anion exchange column, wherein the mobile phase is run under conditions comprising a discontinuous elution gradient and at least one isocratic hold. [2] A method for quantifying empty and full capsids in a virus preparation, the method comprising passing the virus preparation and a mobile phase through an anion exchange column, wherein the mobile phase is run under conditions comprising a discontinuous elution gradient and at least one isocratic hold. [3] The method according to item 1 or 2 above, wherein the virus preparation and the mobile phase are run on a high performance liquid chromatography (HPLC) system. [4] The method according to any one of the preceding items, wherein the empty and full capsids are separated with baseline resolution. [5] The method according to item 4 above, wherein the baseline resolution is greater than 2.0. [6] The method according to any one of the preceding items, wherein the capsid comprises an adeno-associated virus (AAV) capsid. [7] The method according to any one of the preceding items, wherein the anion column is a strong anion exchange (SAX) column. [8] The method according to item 7 above, wherein the SAX column is a quaternary amine (Q-amine) column. [9] The method according to any one of the preceding items, wherein the anion exchange column is a monolithic column.

[10] The method according to any one of the preceding items, wherein the mobile phase comprises a salt.

[11] The method according to item 10 above, wherein the salt is tetramethylammonium chloride (TMAC) or sodium acetate.

[12] The method according to item 10 or 11 above, wherein the final gradient of the mobile phase comprises 0.5 to 5 M, optionally 1 M, of salt.

[13] The method according to any one of the preceding items, wherein at least one isocratic hold is introduced before the mobile phase reaches 50% of the final gradient.

[14] The method according to any one of the preceding items, wherein the pH of the mobile phase is from about 8 to about 10.

[15] The method according to item 14 above, wherein the pH of the mobile phase is about 9.

[16] The method according to any one of the preceding items, wherein the column has a temperature between 0°C and 50°C.

[17] The method according to item 16 above, wherein the column has a temperature between 20°C and 25°C.

[18] The method according to any one of the preceding items, wherein the AAV is derived from one or more serotypes and is optionally selected from AAV1, AAV2, AAV3, AAV6, AAV8, and AAV9.

[19] The method according to any one of the preceding paragraphs, wherein the complete capsid in the virus preparation contains a nucleic acid transgene construct between about 20 base pairs and about 9,000 base pairs.

[20] A virus preparation concentrated for empty virus capsids, the preparation being obtained by the method according to any one of paragraphs 1 to 19 above, wherein, if desired, 20% or less of the virus capsids in the preparation are complete virus capsids.

[21] A virus preparation concentrated for complete virus capsids, the preparation being obtained by the method according to any one of paragraphs 1 to 19 above, wherein, if desired, 20% or less of the virus capsids in the preparation are empty virus capsids.

Mode for Carrying Out the Invention

[0082] Examples Example 1: Anion Exchange HPLC for Separating Empty and Intact Virus Particles Anion exchange HPLC was evaluated for its ability to separate empty AAV particles and recombinant AAV (rAAV) (intact) particles within a virus preparation. Four different AAV samples were run to evaluate chromatographic peak separation and peak positioning of empty AAV capsids and intact AAV capsids (rAAV samples). Samples included: final formulation buffer (FFB); a reference sample mainly composed of full AAV capsids (Full rAAV6-GLA3) manufactured by the vendor; a sample prepared by Sangamo mainly composed of full AAV capsids (rAAV6-GLA3); and a sample prepared by Sangamo mainly composed of empty AAV capsids (Empty AAV6-030).

[0083] The Agilent 1100 HPLC system was used. Background can be reduced by using liquid chromatography - mass spectrometry (LC - MS) grade or HPLC grade reagents. In this example, LC - MS grade reagents were used to prepare the mobile phase composed of bis - tris propane and sodium chloride. The HPLC buffer was prepared by dissolving HPLC grade or high - purity compounds in LC - MS grade water. The buffer was adjusted to the appropriate pH and filtered through a 0.2 μm filter. Then the solution was transferred to an HPLC bottle. Line A of the HPLC system in this example was selected for the buffer containing a lower amount of salt, and line B was selected for the buffer with a high salt concentration. Line C was selected for HPLC grade water, and line D was used for isopropyl alcohol (IPA). Lines C and D were used only when necessary for solvent exchange or system cleaning. For separation, a monolithic AAV analytical column was used. Before starting the experiment, the solvent lines were placed in the appropriate bottles and flushed at 3 mL / min (per line) for at least 5 minutes.

[0084] The CIMac® AAV Complete / Empty - 0.1 Analytical Column (1.3 μm) anion - exchange column (BIA Separations, Slovenia) was washed with high - salt buffer at 1 mL / min for 10 - 15 minutes while monitoring the pressure (usually less than the column specification). Then the column was equilibrated to the initial conditions of the method. If it was necessary to switch the buffer, first the solvent line was flushed with water and then followed by the next buffer. For long - term storage, before shutting down the system, the solvent lines and the system were flushed with IPA. Bis - tris propane (BTP) was used as the buffer because it has two pKa values and covers the pH value range (about 6.5 - 9.5). The buffer concentration was selected to be about 20 mM to enable sufficient buffering capacity without significantly increasing the ionic strength. However, other appropriate concentrations may also be used.

[0085] Figures 1A and 1B are representative chromatograms of three different (stacked) samples showing peak positioning for empty AAV (peak 1) and full AAV (peak 2) particles. As shown in Figure 1A (30 minutes) and Figure 1B (15 minutes), all samples demonstrated a distinct peak separation pattern. Samples containing primarily full capsids showed a small peak corresponding to the empty capsid, followed by a large peak corresponding to the full capsid, with no baseline separation between them. The empty AAV sample showed a larger peak corresponding to the empty capsid, which indicates the peak positions of the empty versus full AAV particles.

[0086] Due to higher sensitivity, data from the fluorescence (Trp) mode are shown, although similar patterns of peaks are observed using UV.

[0087] All three samples showed consistent peak positions (retention times) for empty AAV and full AAV particles. Although there was no baseline resolution, peak areas were calculated using peak integration. The ratio of peak areas can be used to calculate the ratio of empty AAV to full AAV. Baseline resolution between peaks is required to achieve an assay that can be verified according to USP regulations, but this method can be used to estimate the approximate percentage of empty particles in AAV pharmaceutical products.

[0088] Example 2: Anion Exchange HPLC for Calculation of the Percentage of Empty and Full Virus Particles in Virus Samples Peak areas from multiple samples were used to calculate the percentage of full AAV particles in each sample. Additionally, the VG titer was measured using a primer / probe sequence targeting the BGH polyA region of the AAV cassette, and the capsid titer was measured using an AAV6 ELISA kit. Based on both the VG and capsid data, ratios were calculated to obtain the percentage of full AAV particles. Table 1 shows the peak areas determined by the HPLC method, VG, and capsid titers described in Example 1 (two runs).

[0089] Full rAAV6-381 contains an expression cassette for a zinc finger nuclease (ZFN) transgene and has a genome with a size of 2,629 bases. Full rAAV6-375 contains an expression cassette for the IDUA transgene and has a genome with a size of 3,077 bases. Full rAAV6-38 contains an expression cassette for the IDS transgene and has a genome with a size of 2,780 bases. Full rAAV6-GLA3 contains the expression of a GLA transgene cassette and has a genome with a size of 2,772 bases. Full rAAV6-GLA1 and Full rAAV6-GLA2 each contain an expression cassette for the GLA gene and have genomes with sizes of 2,729 bases and 3,321 bases, respectively. All samples used in this specification are samples for investigation only.

Table 1

[0090] For all samples, the percentages of complete virus particles measured by HPLC and VG / capsid were plotted and compared. The ratios for all samples except the GLA1 and GLA2 samples were similar, indicating a correlation between the results from HPLC and other accepted methods. A comparison of the methods is shown in Figure 2.

[0091] Example 3: Accuracy of anion exchange HPLC for calculating the percentage (%) of empty and / or complete virus particles in virus samples To test the accuracy of the HPLC method described in Example 1, the same samples of Full rAAV6-375 and Full rAAV6-381 were run 5 times, and the peak areas were calculated for the complete AAV particles. Samples Full rAAV6-375 and Full rAAV6-381 differed in that the viral particles within the samples contained different transgenes. According to USP guidelines, the accuracy should be less than 2%. As shown in Tables 2 and 3, the measured peak areas demonstrated an injection accuracy of less than 2%. The percent recovery was also close to 100% (calculated based on the first sample injection). Peak areas at UV260 and 280 were also obtained and used for the calculation of the 260 / 280 ratio. However, the fluorescence signal demonstrated a higher signal-to-noise ratio.

Table 2

Table 3

[0092] Example 4: Linearity of anion-exchange HPLC for varying injection volume, capsid concentration, and capsid content The linearity of injection volume as well as capsid concentration was analyzed. The HPLC procedure was carried out according to the method described above. As shown in Figures 3 and 4, it was found that the results of the peak areas for the complete AAV particles were linear when varying the volume of the same sample.

[0093] Samples were also diluted with FFB to different capsid titers and then the same volume was injected into the HPLC system for each of the diluted samples. Similarly, when varying the concentration of the capsid at a constant sample volume, it was found that the results of the peak areas for the complete AAV particles were linear. Figures 5 and 6 show the linearity between different injected capsid concentrations at a constant volume.

[0094] Different virus composition samples with similar capsid titers were analyzed according to the described method. The samples were diluted to the same capsid titer with FFB, and the same volume was injected into each diluted sample. These results indicate that the amount of capsid injected is approximately the same regardless of the type of sample. As shown in Figure 7, a consistent peak area for the normalized samples was also demonstrated.

[0095] Samples of AAV6 capsids containing GLA were used to determine peak separation at different temperatures. Figure 8 shows that there was consistent peak separation for temperatures in the range from about 20°C to about 35°C.

[0096] AAV samples from different commercial vendors containing either the GFP transgene or empty preparations showed that all samples contained a heterogeneous mixture of full and empty AAV particles. As shown in Figure 9A, the sample Full rAAV6-GLA3 had the highest percentage of full AAV particles.

[0097] Example 5: Variation of the eluent in an Agilent 1260 HPLC system Samples from previous examples were analyzed on an Agilent 1260 HPLC system, demonstrating similar results and peak separation patterns (data not shown).

[0098] Four elution salts (1 M solutions of NaCl, TAMC, sodium acetate, and ammonium acetate) were analyzed for baseline separation between chromatogram peaks corresponding to empty capsids and full capsids. Elution profiles for the full capsid sample (Full rAAV6-GLA3) and the empty capsid sample (AAV6-030) were obtained. The methods described above were used, except as indicated. Since the ionic strength and elution strength of the eluent differed, the gradient of the eluent buffer was maintained at 0 - 100% to obtain a complete elution profile. The retention times (RT) of the major peaks of Full rAAV6-GLA3 and Empty AAV6-030 were compared and used to calculate the net RT. The largest net difference results in the clearest baseline separation.

[0099] The elution profiles of the four elution salts are shown in Figure 10, and the net RT differences for the four elution salts are presented in Table 4. Sodium chloride (NaCl) demonstrated the highest elution strength (and thus the lowest RT), while ammonium acetate (NH4OAc) demonstrated the lowest elution strength (and thus a higher RT). The highest net RT difference was demonstrated using tetramethylammonium chloride (TMAC), followed by sodium acetate (NaOAc), while NaCl and NH4OAc resulted in a lower net RT difference between the two samples.

Table 4

[0100] Example 6: Mobile Phase Containing TMAC at Various Gradients To improve the baseline separation between the peaks corresponding to full and empty virus particles, various gradients of TMAC were analyzed. Except as shown, this method was carried out as described above. Buffer A contained 20 mM BTP at pH 9.0, while Buffer B contained 20 mM BTP with 1 M TMAC at pH 9.0. Previously, the gradient was run from 0 to 100%. However, to improve peak separation, a more shallow and narrower gradient was evaluated. Each gradient was run on a Full rAAV6-GLA3 sample, an Empty AAV6-030 sample, and a mixture of both samples. Five injections were used to evaluate reproducibility.

[0101] Chromatograms showing gradient separation using elution buffers containing TMAC for Full rAAV6-GLA3, Empty AAV6-030, and mixed samples are presented from Figure 11A to Figure 11C. Sequential injection of the full capsid mixed sample and the empty capsid mixed sample gave consistent peak RTs and peak areas, demonstrating the high precision of the method. Further, the empty peak of the mixed sample coincided with the major peak of the Empty AAV6-030 sample (Figures 11A - 11C). To further improve peak separation between empty and full capsids, the gradient was narrowed from 0 - 100% to approximately 15 - 30%, and multiple assays were performed to determine reproducibility. The narrower the gradient, the better the peak separation, but the peaks also became broader. The peaks of both the empty and full capsid mixed samples corresponded to the similar peaks of the Full rAAV6-GLA3 sample and the Empty AAV6-030 sample, indicating that no unwanted shift in peak RT occurred upon mixing the two samples. These results show that virus samples produced by different methods containing different payloads may contain valid amounts of both empty and full particles that can be identified and distinguished.

[0102] Example 7: Gradient Method by Weak Anion Exchange Column and Tandem Chromatography In Examples 1-6, an AAV strong anion exchange (AEX) monolithic column was used. In this study, a weak anion exchange (WAX) column (e.g., BioWAX NP3 (non-porous, 4.5 x 50 mm, 3 μm HPLC column)) was analyzed to examine its effect on the peak separation of empty and full virus particles. Tandem columns, as well as an increase in column length or a combination of two matrices (weak AEX and strong AEX), were analyzed to determine whether they affected the peak separation of empty and full virus particles. The monolithic column and the WAX column were mounted back-to-back for tandem chromatography using a connector. Samples containing rAAV6-GLA039 (recombinant AAV6 carrying the GLA transgene Lot 039; Sangamo, Richmond, CA) and Empty AAV6-030 were used. Except as indicated, the methods described in previous examples were used. For the WAX column and the monolithic column, different mobile phase pH levels were analyzed to examine their effect on the peak baseline separation of empty and full virus particle chromatograms.

[0103] The monolithic column demonstrated strong binding to the mobile phase at pH 9.0. At lower pH levels, the RT shifted to the left, indicating loss or decrease of binding and faster elution. The RT is desirably high enough to improve peak separation and increase the capacity factor. However, mobile phases containing certain pH levels did not demonstrate peak separation. The WAX column showed stronger binding (higher RT) of the samples at the same pH levels compared to the monolithic column. Less peak separation was seen with the WAX column compared to the monolithic column. Furthermore, peak asymmetry increased (wider peaks). At lower mobile phase pH levels, both columns showed lower binding of AAV, and some or most of the viral composition eluted in the void volume (between 0 and 1.5 mL) (Figures 12A and 12B).

[0104] When columns were used in tandem, peak separation was the same as for the monolith alone. Further, lowering the pH decreased binding (lower RT) and reduced the separation between the peaks of the full capsid and the empty capsid. Similarly, as shown in Figure 13, when two monolithic columns were attached for tandem chromatography, peak separation remained the same.

[0105] In combination with the methods and samples described, the monolithic column (with Q - amine) provided better peak separation than the WAX column (with diethylaminoethyl (DEAE)).

[0106] Example 8: Gentle Gradient Using Monolithic Column Gentler gradients (longer run times) were analyzed to determine improved peak resolution between the full and empty virus particle chromatography peaks. A monolithic AAV column was used and the gradient was set from 15 - 30% of buffer B. The run time was varied. In the original method, the gradient was run for more than 5 minutes. Five additional gradients were analyzed (10, 15, 20, 25, and 30 minutes). As a result, the run time for each experiment increased. A mixture of Full rAAV6 - GLA3 and Empty AAV6 - 030 was used as the virus composition test sample. Faster gradients (1, 2, 3, and 4 minutes) were also analyzed.

[0107] Looking at Figure 14, the gentler gradients did not improve peak resolution because the peaks were broader and resolved. However, comparing individual and mixed samples at a gradient time of 5 minutes, asymmetrical and sharper peaks were seen even though peak baseline resolution was not demonstrated. On the other hand, when faster gradients (1 - 4 minutes) were used, separation was not improved and the peaks became sharper. (Figures 15 and 16). A fast method is desirable, but the run time needs to be long enough to allow for efficient HPLC peak separation.

[0108] Example 9: An isocratic hold in a gradient mobile phase produces a baseline separation between peaks corresponding to empty and full virus particles The isocratic hold was incorporated into the gradient method to determine its effect on the baseline separation between chromatogram peaks corresponding to empty and full virus particles in a virus composition. rAAV6-GLA039 was used as the full sample. Isocratic holds of approximately 17%, 18%, 19%, or 20% were tested at a gradient time of 10 minutes (same run time, different gradients). Once the preferred conditions were determined, a mixed sample containing Full rAAV6-GLA3 and Empty AAV6-030 was analyzed.

[0109] Prior to the start of the experiment, the samples were freshly thawed on ice. Each sample was analyzed directly or diluted with FFB as needed. The samples were transferred to HPLC vials and capped with an appropriate slit cap. The vials were then transferred to the HPLC autosampler at the desired position and the positions within the array were identified.

[0110] As shown in FIGS. 17 to 19, including an isocratic hold as part of the gradient mobile phase resulted in a baseline separation between peaks corresponding to empty and full capsids of the same sample. An isocratic hold containing approximately 17% or 18% resulted in a clear separation of the Fabry sample (FIG. 17). When the mixed sample was analyzed, a similar pattern was shown with a clear peak baseline separation (FIG. 18). Peak separation was demonstrated at higher percentages of isocratic hold (about 19% or 20%), but the sample did not bind completely and a portion of the sample eluted at the void volume.

[0111] To obtain complete binding, a gradient of about 0-40% was used in combination with an isocratic hold. This gradient allowed the sample to bind at the lowest ionic strength and maintained peak separation using the isocratic hold. These conditions resulted in complete or almost complete binding of the sample composition with clear baseline resolution between the empty and complete peaks (Figure 19).

[0112] The addition of an isocratic hold to the gradient mobile phase resulted in baseline resolution between the peaks corresponding to empty virus particles and complete virus particles. For the quantification of components within a sample using the methods described herein, conditions that allow for complete binding of the entire sample are preferred to ensure that the sample undergoes gradient separation on the column and that the sample product elutes little or not at all at the void volume.

[0113] Example 10: Conditions for Enhanced Sample Binding, Peak Symmetry, and Baseline Separation between Peaks Corresponding to Empty and Complete Virus Particles Method conditions such as the initial gradient percentage and the isocratic hold percentage were analyzed to enhance sample binding and peak symmetry and reduce peak tailing. Additionally, the upper limit wavelength for fluorescence detection was obtained. Unless otherwise indicated, the method described in Example 5 was used. A mixture of samples of Full rAAV6-GLA3 and Empty AAV6-030 was used to further improve the gradient method. An isocratic hold of 19% was selected and the gradient start and end percentages were varied. Additionally, the excitation and emission wavelengths were varied to obtain the preferred wavelengths.

[0114] As shown in Figure 20, a 19% isocratic hold resulted in baseline separation of both the complete virus particles and the empty virus peak. The initial gradient was 0%, which increased linearly to about 15% and then gradually increased to about 19%, where the isocratic hold demonstrated separation of the empty AAV6 particles. Further gradient increases to about 40% or 50% resulted in elution of the peak corresponding to the complete virus particles. A further gradient increase to about 100% was used to wash the column during the method. It can be preferred to use a low % of buffer B at the start of the gradient to narrow the peak shape and width corresponding to the empty AAV6 particles. Thus, a 5% start gradient was selected as the initial gradient. The results showed that the 5% initial gradient improved the peak asymmetry of the empty AAV6. Further, a final gradient of about 50% improved the complete AAV6 peak symmetry. The use of a gradient of about 5 - 15 - 50% resulted in a resolution greater than 2.0 and an asymmetry and tailing less than 2.0, which meets the requirements for the USP method for HPLC analysis.

[0115] To confirm the preferred excitation / emission wavelengths, experiments were conducted by fixing one parameter and varying the other. Wavelengths for excitation and emission of 280 nm and 348 nm, respectively, demonstrated preferred peak characteristics, detection, and signal-to-noise ratios.

[0116] The response time for sample collection was also varied. It was found that a response time of about 0.5 seconds was sufficient to generate at least 20 data points over the entire peak, which is another requirement under the USP. Shorter response times can also increase the number of data points, but can slow down the analysis when the file becomes large enough.

[0117] The influence of temperature was also investigated. Higher temperatures were demonstrated to result in the addition of small peaks at high RT. These results indicate that this method can further distinguish other variants of the particles. The AAV6 column demonstrated acceptable performance up to 50 °C, with a preferred temperature of below 40 °C, for example, approximately 15 - 25 °C.

[0118] Example 11: Linearity of injection volume, injected capsid concentration, and accuracy of multiple injections Using Full rAAV6 - GLA3 and Empty AAV6 - 030 samples, the linearity of injection volume, injected capsid concentration, and accuracy of multiple injections was determined. Additionally, the linearity of different mixed samples prepared at different ratios was investigated.

[0119] Injection volumes between approximately 1 - 100 μL were analyzed to determine the loading linearity. Samples of Full rAAV6 - GLA3 and Empty AAV6 - 030 were used either mixed or independently. The peak areas of the peaks corresponding to full and empty virus particles were compared. At volumes up to approximately 10 μL, both samples showed high linearity despite significantly different capsid titers. At higher volumes, non - binding of a portion of the sample resulted in a non - linear increase in peak area.

[0120] The linearity of the injectable viral particle concentration (e.g., capsid) was determined. Samples of both Full rAAV6-GLA3 and Empty AAV6-030 were diluted 2-fold with the final formulation buffer, and 10 μL of each solution was injected. The capsid titer of Full rAAV6-GLA3 was 6.11E+12 capsids / mL, and the capsid titer of Empty AAV6-030 was 3.2E+13 capsids / mL. The area response was measured for both samples at both peaks and found to be linear. The major peak for each sample resulted in a high linearity range (6.11E+10 - 9.55E+8 capsids / injection for Full rAAV6-GLA3, 3.2E+11 - 5E9 for Empty AAV6-030), and the lowest point showed a sample-to-noise ratio greater than 100. Thus, the limit of detection (LOD) and limit of quantification (LOQ) could be much lower. These results indicate that this method is very sensitive and can analyze even fewer capsids than these amounts for the major peak.

[0121] Empty AAV capsids should have a higher detected UV280 vs 260 nm, while full AAV should have a higher detected UV260 above 280 nm (due to the DNA within the capsid). This was consistently observed, indicating that the expected peak positions for empty and full AAV are correct.

[0122] For each chromatogram, the % empty and complete analysis was obtained by calculating the % of the peaks associated with the total peak area of all peaks. To calculate the % empty and complete capsids in Full rAAV6-GLA3 and Empty AAV6-030, the loading linearity (see above) data was used. Data for multiple injected volumes showed that the % empty and complete for Full rAAV6-GLA3 was 8% and 92% respectively. On the other hand, the % of empty and complete AAV particles in Empty AAV6-030 was 6% and 94% respectively, with reasonable variation (<10%). Using these values, custom standard curves can be created by mixing at different ratios and calculating the theoretically % empty and complete in their mixtures. Using the peak areas of the obtained chromatograms, simple curves (linear or quadratic) can be created. Back-calculation can be used to obtain the % recovery of each mixture.

[0123] To confirm that the sample has both empty and complete AAV peaks and that the peak area response is linear, a standard curve consisting of a mixture of Full rAAV6-GLA3 and Empty AAV6-030 was prepared and presented in Figure 32. From the mixture, 10 μL was injected and the peak area for each peak was obtained. The peak areas were plotted against the % Empty or Full amount of injected capsids. Back-calculation of the % recovery was performed using the linear relationship and it was demonstrated to be within ±20%. This experiment also showed that the range and recovery of mixtures of samples containing both AAV peaks were very linear (r2>0.99). To further refine the curve approximation, software was used to approximate a linear or quadratic curve. It was found that the quadratic curve could be appropriately approximated even with a low residual standard deviation. However, both linear curve approximation and quadratic curve approximation may be used.

Table 5

[0124] It was also demonstrated that peak separation still existed with high resolution within the pH range of approximately 8.8 - 9.2 for both mobile phases. However, some peak tailing was observed at pH 9.2, and the peak RT shifted under extreme pH conditions (Figure 33).

[0125] In addition to pH, the influence of column temperature was also analyzed. The temperature of the column compartment was varied between approximately 15 - 50 °C, and the peak separation of empty and full AAV was analyzed. As shown in Figure 34, peak separation was not affected at low temperatures (with some tailing), while extra peaks may appear in the chromatogram at temperatures above 30 °C.

[0126] The influence of sample temperature on chromatography was also investigated by heating the sample to different temperatures. At higher temperatures (e.g., approximately 90 °C), the sample decomposed and did not show peaks during gradient elution (data not shown). Therefore, the stability of the sample can be analyzed using this method.

[0127] Example 12: Comparison of % empty and full particles quantified by HPLC compared to AUC and CryoTEM A total of 10 samples (including Full rAAV6 - GLA3, Full rAAV6 - 384, Full rAAV6 - GLA4 (recombinant AAV6 carrying the GLA transgene), and Empty AAV6 - 030) were analyzed using the HPLC method described in Example 10 to calculate the percentage of empty and full capsids. Five of the samples were sent for CryoTEM analysis to obtain the percentage of full and empty capsids.

[0128] Comparative analysis between the HPLC method and the CryoTEM method demonstrated a strong correlation, as shown in Tables 6 and 7. In the tables, rAAV6-378 has a genome containing an expression cassette for the left ZFN transgene; rAAV6-447 has a genome containing an expression cassette for the F8 (Factor VIII) transgene; rAAV6-000490 and 18-rAAV6-550 each have a genome containing an expression cassette for the CD19 transgene, but were produced in Sf9 cells and HEK293 cells, respectively.

[0129] The correlation between the methods is also shown in FIGS. 43 (% empty capsids) and 44 (% full capsids). The Pearson correlation coefficient (r) for the HPLC vs TEM correlation at 95% coverage was 0.999. These results indicate that the HPLC method described herein is accurate when compared to the TEM method for quantifying empty and full capsids within a sample. [Table 6] [Table 7]

[0130] According to a preferred embodiment, the following parameters may be utilized. For line A (buffer A), 20 mM bis-tris propane (BTP) at pH 9.0; for line B (buffer B), 20 mM bis-tris propane (BTP), 1 M salt (TMAC) at pH 9.0; for line C - LC-MS (or equivalent)-grade water; for line D isopropyl alcohol; for seal wash (SW), 10% IPA in LC-MS (or equivalent)-grade water; for needle wash (NW), 50% methanol; for UV detection setup - 260 ± 20 nm, 280 ± 20 nm; for fluorescence detection setup - excitation / emission = 280 nm / 348 nm; for sample injection volume, 10 μL; for multi-sampler parameters: needle wash - standard; for draw rate, 100 μL / min; for expel rate, 400 μL / min; for wait time after draw, 1.2 seconds; for sample flush out factor - 5; for column temperature, 20 °C; for DAD settings: signal A - 260 ± 20 nm, reference 400 ± 100 nm, response time > 0.25 seconds; signal B - 280 ± 20 nm, reference 400 ± 100 nm, response time > 0.25 seconds; for slit, 4 nm; for FLD settings: excitation - 280 nm, emission - 348 nm, response time 0.25 seconds.

[0131] According to a specific embodiment, the mobile phase gradient program presented in Table 8 may be utilized.

Table 8

[0132] Example 13: Broad utility of an isocratic hold HPLC method for the separation and quantification of empty and full particles Viral preparations of five additional different AAV serotypes, including AAV1, AAV2, AAV3, AAV8, and AAV9, were analyzed using the HPLC method described in Example 10 to calculate the percentages of empty and full capsids. For each serotype, different samples were run to evaluate chromatographic peak separation and peak positioning for empty AAV capsids and full AAV capsids. Additionally, for each serotype, the method was varied with respect to the concentration of 1M TMAC for efficient binding, isocratic hold, and linear gradients on both sides of the isocratic hold.

[0133] For AAV1 serotype, the samples included AAV1 Empty Lot and AAV1 CMV-GFP purchased from Virovek; Lot 17-AAV-321 and 17-AAV-037 produced by the SGMO Vector Core using a triple transfection process in HEK293 cells and purified by the CsCl density gradient method. For the triple transfection method, rAAV was produced by Sangamo's vector core group using a platform method. Briefly, HEK293 cells were seeded into 10-layer CellSTACK chambers (Corning, Acton, MA) and grown for 3 days until a density of 80%. The three plasmids, an AAV helper plasmid containing the Rep and Cap genes, an adenovirus helper plasmid containing the adenovirus helper gene, and a transgene plasmid containing the packaged sequence flanked by AAV2 inverted terminal repeats, were transfected into the cells using calcium phosphate (Xiao et.al. 1998). After 3 days, the cells were harvested. The cells were lysed by 3 rounds of freeze / thaw and cell debris was removed by centrifugation. rAAV was precipitated using polyethylene glycol. After resuspension, the virus was purified by ultracentrifugation overnight on a cesium chloride (CsCl) gradient. The virus was formulated by dialysis and then filter sterilized. The Sangamo samples were separated to isolate mainly purified complete AAV1 particles. The data showed that, as shown in Figure 39, a 20% isocratic hold resulted in baseline separation of both the complete virus particles and the empty virus peak. The initial gradient was 0%, which increased linearly to about 8% and then gradually increased to about 20%, where the isocratic hold demonstrated separation of the empty AAV particles. A further gradient increase to about 30% resulted in elution of the peak corresponding to the complete virus particles. A further gradient increase to about 95% was used to wash the column during the method. The data showed that the percentages of empty and complete in Empty AAV1 were 85.2% and 9.3% respectively.The percentages of empty and full in AAV1 CMV-GFP were 13.8% and 84.1% respectively. The percentages of empty and full in 17-AAV-321 were 20.6% and 79.4% respectively. The percentages of empty and full in 19-AAV-037 were 6.4% and 90.0% respectively.

[0134] For AAV2 serotype, the samples included an AAV2 Empty Lot purchased from Virovek; Lots 17-AAV-077 and 17-AAV-155 produced by the SGMO Vector Core using a triple transfection process in HEK293 cells and purified by the CsCl density gradient method. The Sangamo samples were separated to concentrate mainly full AAV2 particles. The data showed that, as shown in Figure 40, a 12% isocratic hold resulted in baseline separation of both full virus particles and empty virus peaks. The initial gradient was 0%, which increased linearly to about 8% and then gradually increased to about 12%, where the isocratic hold demonstrated separation of empty AAV particles. A further gradient increase to about 30% resulted in elution of the peak corresponding to full virus particles. A further gradient increase to about 100% was used to wash the column during the method. The data showed that the percentages of empty and full in AAV2 Empty were 72.3% and 21.4% respectively. The percentages of empty and full in 17-AAV-077 were 5.9% and 79.6% respectively. The percentages of empty and full in 17-AAV-155 were 78.5% and 8.9% respectively.

[0135] For AAV serotype 3, the samples included AAV3 Empty Lot and AAV3 CMV-GFP purchased from Virovek; Lot 17-AAV-124 and 17-AAV-324 produced by the SGMO Vector Core using a triple transfection process in HEK293 cells and purified by the CsCl density gradient method. The Sangamo samples were separated to isolate mainly purified complete AAV3 particles. The data, as shown in Figure 41, indicate that a 15% isocratic hold resulted in baseline separation of both the complete virus particles and the empty virus peak. The initial gradient was 0%, which increased linearly to about 8% and then gradually increased to about 15%, where the isocratic hold demonstrated the separation of the empty AAV particles. A further gradient increase to about 30% resulted in the elution of the peak corresponding to the complete virus particles. A further gradient increase to about 100% was used to wash the column during the method. The data show that the percentages of empty and complete in AAV3 Empty were 74.1% and 17.9% respectively. The percentages of empty and complete in AAV3 CMV-GFP were 55.2% and 41.5% respectively. The percentages of empty and complete in 17-AAV-124 were 55.4% and 44.6% respectively. The percentages of empty and complete in 17-AAV-324 were 72.9% and 27.2% respectively.

[0136] For AAV8 serotype, samples included AAV8 Empty (17-AAV-082) and lots 18-AAV-070, 17-AAV-339, 17-AAV-340, and 17-AAV-341, which were produced by the SGMO Vector Core using a triple transfection process in HEK293 cells and purified by the CsCl density gradient method. The AAV8 Empty lot was separated to concentrate empty AAV8 particles, while all other lots were separated to isolate purified full AAV8 particles. AAV8 RSM (mainly composed of full AAV8 capsids), purchased from ATCC and produced by Atlantic Gene Therapies - UMR 1089 in Nantes (France) and the Center for Animal Biotechnology and Gene Therapy (CBATEG) at the Autonomous University of Barcelona (Spain), was also tested. The data show that, as shown in Figures 42A and 42B, a 14% isocratic hold resulted in baseline separation of both full virus particles and empty virus peaks. The initial gradient was 0%, which increased linearly to about 8% and then gradually increased to about 14%, where the isocratic hold demonstrated the separation of empty AAV particles. A further gradient increase to about 30% resulted in the elution of the peak corresponding to full virus particles. A further gradient increase to about 95% was used to wash the column during the method. The data show that the percentages of empty and full in AAV8 Empty were 76.6% and 22.2% respectively. The percentages of empty and full in AAV8 Lot 18-070 were 15.2% and 81.5% respectively. The percentages of empty and full in AAV8 Lot 17-339 were 1.8% and 95.5% respectively. The percentages of empty and full in AAV8 Lot 17-340 were 28% and 70.4% respectively. The percentages of empty and full in AAV8 Lot 17-341 were 4.3% and 93.3% respectively. The percentages of empty and full in AAV8 RSM were 0% (not detected) and 100% respectively. At low concentrations of AAV8 RSM, the empty peak may fall below the limit of detection.

[0137] For AAV9 serotype, the samples included AAV9 Lots 070 and 076 that were manufactured using baculovirus-based infection in an Sf9 cell process and purified by affinity chromatography; and Lot 24 that was manufactured by the SGMO Vector Core using a triple transfection process in HEK293 cells and purified by CsCl density gradient method. These lots were separated to isolate mainly purified complete AAV9 particles. The data show that the AAV9 serotype, which is an isocratic hold of 5%, resulted in baseline separation of both complete virus particles and empty virus peaks, as shown in Figure 43. The initial gradient was 0%, which increased linearly to about 3% and then gradually increased to about 5%, where the isocratic hold demonstrated the separation of empty AAV particles. A further gradient increase to about 40% resulted in the elution of the peak corresponding to complete virus particles. A further gradient increase to about 100% was used to wash the column during the method. The data show that the percentages of empty and complete in PD Lot 076 were 53.6% and 43.3% respectively. The percentages of empty and complete in PD70 Lot 19-BAV-484PD were 5.8% and 87.8% respectively. The percentages of empty and complete in PD76 Lot 20-BAV-027PD were 54.2% and 41.1% respectively. The percentages of empty and complete in Run24 Lot 19-BAV-470 were 2.7% and 97.3% respectively.

Table 9

Claims

1. A method for separating empty and full capsids in a virus preparation, the method comprising passing the virus preparation and a mobile phase through an anion exchange column, wherein the mobile phase is run under conditions including a discontinuous elution gradient and at least one isocratic hold.

2. A method for quantifying empty and full capsids in a virus preparation, the method comprising passing the virus preparation and a mobile phase through an anion exchange column, wherein the mobile phase is run under conditions including a discontinuous elution gradient and at least one isocratic hold.

3. The method according to claim 1 or 2, wherein the virus preparation and the mobile phase are run on a high performance liquid chromatography (HPLC) system.

4. The method according to any one of claims 1 to 3, wherein the empty and full capsids are separated with baseline resolution.

5. The method according to claim 4, wherein the baseline resolution is greater than 2.

0.

6. The method according to any one of claims 1 to 5, wherein the capsid comprises an adeno-associated virus (AAV) capsid.

7. The method according to any one of claims 1 to 6, wherein the anion exchange column is a strong anion exchange (SAX) column.

8. The method according to claim 7, wherein the SAX column is a quaternary amine (Q-amine) column.

9. The method according to any one of claims 1 to 8, wherein the anion exchange column is a monolithic column.

10. The method according to any one of claims 1 to 9, wherein the mobile phase contains a salt.

11. The method according to claim 10, wherein the salt is tetramethylammonium chloride (TMAC) or sodium acetate.

12. The method according to claim 10 or 11, wherein the final gradient of the mobile phase contains 0.5 to 5 M of salt.

13. The method according to any one of claims 1 to 12, wherein at least one isocratic hold is introduced before the mobile phase reaches 50% of the final gradient.

14. The method according to any one of claims 1 to 13, wherein the pH of the mobile phase is from 8 to 10.

15. The method according to claim 14, wherein the pH of the mobile phase is 9.

16. The method according to any one of claims 1 to 15, wherein the column has a temperature between 0°C and 50°C.

17. The method according to claim 16, wherein the column has a temperature between 20 °C and 25 °C. **Claim 18** The method according to any one of claims 1 to 17, wherein the AAV is derived from one or more serotypes and is selected from AAV1, AAV2, AAV3, AAV6, AAV8, and AAV9. **Claim 19** The method according to any one of claims 1 to 18, wherein the complete capsid in the virus preparation contains a nucleic acid transduction gene construct between 20 base pairs and 9,000 base pairs. **Claim 20** A virus preparation concentrated for empty virus capsids, the preparation being obtained by the method according to any one of claims 1 to 19. **Claim 21** A method for separating empty and complete capsids in a virus preparation, the method comprising passing the virus preparation and a mobile phase through an anion exchange column, wherein the mobile phase is run under conditions including a discontinuous elution gradient and at least one isocratic hold incorporated in said gradient.

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