Perfusion in production bioreactors

Abstract

The present disclosure provides methods of producing recombinant adeno-associated virus (rAAV) wherein rAAV production occurs under perfusion conditions. Also contemplated are methods of producing rAAV under media exchange conditions.

Description

[0001] PERFUSION IN PRODUCTION BIOREACTORS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 § 119(e) of U.S. Provisional Application No. 63 / 666,472, filed on July 1, 2024, entitled “PERFUSION IN PRODUCTION BIOREACTOR,” and U.S. Provisional Application No. 63 / 788,653, filed on April 14, 2025, entitled “PERFUSION IN PRODUCTION BIOREACTOR,” the contents of each of which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] During typical gene therapy manufacturing, viral vectors are produced in cell cultures. For clinical applications, large quantities of viral particles are required, creating a need to improve quantity and quality of rAAV production. Therefore, there is a need to improve current manufacturing processes for viral vectors used in gene therapy.

[0006] SUMMARY

[0007] This application provides methods and compositions for improving production of rAAV particles by cultured cells. In some aspects, cultured cells are perfused or grown under media exchange conditions. In some embodiments, perfusing or exchanging media removes waste products and / or provides nutrients. In some embodiments, perfusing or exchanging media improves rAAV production yield and / or quality.

[0008] In some aspects, the present disclosure provides a method of producing recombinant adeno associated virus (rAAV) particles wherein the cell culture is perfused under conditions suitable for rAAV production. In some aspects, the method comprises perfusing a cell culture in a glutamine-containing medium. In some embodiments, the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture is perfused under conditions suitable for rAAV production. In some embodiments, the cell culture comprises producer cells. In some embodiments, the producer cells are HeLa cells.

[0009] In some aspects, the present disclosure provides a method of producing recombinant adeno associated virus (rAAV) particles, comprising perfusing a cell culture with a medium, wherein the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture produces rAAV particles during perfusion.

[0010] In some embodiments, the method further comprising contacting the cell culture with one or more rAAV-encoding recombinant nucleic acids.

[0011] In some embodiments, the pH of the medium is at or between 7-7.4. In some embodiaments, the cell culture is cultured for at least 72 hours after AAV production has begun, and wherein the cell culture is perfused for up to 48 hours of the at least 72 hours. In some embodiments, the cell culture is perfused for at least 24 hours after AAV production has begun.

[0012] In some embodiments, the glutamine levels are above 2 mM.

[0013] In some embodiments, the rate of perfusion is between 0.5 and 2 vessel volume per day (VVD). In some embodiments, the rate of perfusion is 2 VVD or higher.

[0014] In some embodiments, the medium is enriched at least 2X relative to a standard medium.

[0015] In some embodiments, the cell culture is infected with a helper virus. In some embodiments, the helper virus is Ad5.

[0016] In some embodiments, the glutamine level of the media starts at or above 4mM and does not go below 2 mM.

[0017] In some embodiments, the method further comprises isolating rAAV particles from the cell culture.

[0018] In some aspects, the present disclosure provides a method of producing recombinant adeno associated virus (rAAV) particles, comprising: a) infecting a cell culture in a first volume in a first reactor; b) perfusing the cell culture in a second volume, wherein rAAV particle production occurs during perfusion, and wherein the first volume is smaller than the second volume.

[0019] In some embodiments, the first volume is a quarter of the volume of the first reactor. In some embodiments, the first volume is less than half the volume of the second volume.

[0020] In some embodiments, the cell culture of (a) is transferred to a second reactor along with additional medium to obtain the second volume. In some embodiments, the cell culture of (a) is maintained in the first reactor and additional medium is added to obtain the second volume.

[0021] In some embodiments, the glutamine level of the second volume is kept at or above 2 mM. In some embodiments, the second volume is perfused with a medium comprising a glutamine level at or above 2 mM.

[0022] In some embodiments, the cell culture in the first volume comprises at least 5 million, at least 6 million, at least 7 million, at least 8 million, at least 9 million, at least 10 million, at least

[0023] 11 million, at least 12 million, at least 13 million, at least 14 million, at least 15 million, at least

[0024] 16 million, at least 17 million , at least 18 million, at least 19 million, at least 20 million, at least 21 million, at least 22 million, at least 23 million, at least 24 million, at least 25 million, at least

[0025] 26 million, at least 27 million, at least 28 million, at least 29 million, at least 30 million, at least

[0026] 31 million, at least 32 million, or at least 33 million cells / mL. In some embodiments, the second volume comprises more than 2.5 times the volume of the first volume. In some embodiments, the second volume comprises about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 5.5X, about 6X, about 6.5X, about 7X, about 7.5X, about 8X, about 8.5X, about 9X, about 9.5X, about 10X, about 10.5X, about 11X, about 11.5X, or about 12X the volume of the first volume. In some embodiments, the cell culture comprises 7 million cells / mL or less, 6 million cells / mL or less, 5 million cells / mL or less, 4 million cells / mL or less, 3 million cells / mL or less, 2 million cells / mL or less, 1.5 million cells / mL or less, or 1 million cells / mL. In some embodiments, after transferring the cell culture to the second volume, the concentration of cells is reduced by about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 5.5X, about 6X, about 6.5X, about 7X, about 7.5X, about 8X, about 8.5X, about 9X, about 9.5X, about 10X, about 10.5X, about 11X, about 11.5X, or about 12X.

[0027] In some embodiments, the cell culture comprises producer cells. In some embodiments, the producer cells are HeLa cells.

[0028] In some embodiments, the method further comprises, contacting the cell culture with one or more rAAV-encoding recombinant nucleic acids.

[0029] In some embodiments, the cell culture is comprised in a medium and the pH of the medium is at or between 7.0-7.4.

[0030] In some embodiments, the cell culture is perfused for at least 48 hours after AAV production has begun. In some embodiments, the cell culture is perfused for at least 24 hours after AAV production has begun.

[0031] In some embodiments, the glutamine level is maintained above 2 mM.

[0032] In some embodiments, the rate of perfusion is between 0.5 and 2 vessel volumes per day (VVD). In some embodiments, the rate of perfusion is between 0.5 and 3 vessel volume per day. In some embodiments, the rate of perfusion is 0.6VVD. In some embodiments, the rate of perfusion is 2VVD or higher.

[0033] In some embodiments, the second volume comprises a medium and the medium is enriched at least 2X relative to a standard medium.

[0034] In some embodiments, the cell culture is infected with a helper virus. In some embodiments, the helper virus is Ad5.

[0035] In some embodiments, the glutamine level of the cell culture in the second volume starts at or above 4mM and does not go below 2 mM.

[0036] In some embodiments, the method further comprises rAAV particles from the cell culture. Aspects of the present disclosure provide a method comprising a) culturing rAAV- producing cells in a small-scale system (e.g., an Ambrl5 system), including agitation, b) allowing the cells to settle, c) removing a supernatant from a vessel of the small-scale system, d) adding a new medium to the vessel of the small-scale system, and e) resuming agitation, wherein steps (b)-(e) are performed at least three times. In some embodiments, the method further comprises isolating rAAV from the cells.

[0037] In some embodiments, the method further comprises comparing the rAAV produced by cells in a method of the present disclosure to rAAV produced by batch-cultured cells.

[0038] BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 shows a schematic of an example mock perfusion process conducted in spinner tubes for rAAV production. Culture from N-l is added to 50mL spinner tube to a working volume of lOmL and then infected with wild type Ad5. Media exchanges happen on Day 1 or Day 1 and Day 2.

[0040] FIG. 2 shows a schematic of example batch (1) and perfusion (2,3,4) processes for rAAV production in bioreactors. Scenario 2 has a transfer of up to 25% final volume from N-l reactor and infection followed by perfusion. Scenario 3 has infection in N-l which is converted to N via perfusion of production media. Scenario 4 is an N-l at very high cell density and low volume. Post infection this culture can then be added to a production reactor with production media already in it, thus diluting the culture or the infected culture can have production media added to it to dilute the culture.

[0041] FIG. 3 shows a bar graph representing normalized volumetric rAAV titer in a mock perfusion experiment. Abbreviations: #E6 = seed cell density (millions of cells per mL); RV = number of media exchanges per day; X = number of days of media exchanges, starting on day 1 (e.g. 2x = media exchanged on days 1 & 2); (X) = number of instances condition was independently run (e.g. x3 = 3 spinner tubes). The control condition was conducted in batch mode at low density (0.5E6 c / mL). The graph shows an increase in rAAV titer at higher seed densities and with increased number of media exchanges.

[0042] FIG. 4A shows a bar graph representing normalized volumetric rAAV titer in 5L batch and perfusion production bioreactors. The labels describe the seed density (4E6 = 4E6 c / mL) and perfusion rate (2 RV = 2 reactor volumes / day for 2 days) post infection. The graph shows an increase in titer with the high-density perfusion process with two different cell lines. FIG. 4B shows a bar graph representing normalized volumetric rAAV titer in 5L batch culture with increasing cell density at time of infection. This shows that without perfusion, increased cell density does not equate to increased productivity.

[0043] FIG. 5A shows a bar graph representing normalized volumetric rAAV titer in Ambr250 production bioreactors. All vessels were seeded at the same density (3E6 c / mL) and operated in either batch or perfusion mode at different perfusion rates and media compositions. Abbreviations: X = enrichment of amino acids and vitamins in production media; VVD = perfusion rate in vessel volumes / day; N = number of biological replicates. Error bar represents 1 standard deviation across biological and assay replicates. The graph shows an increase in titer with perfusion vs batch process, and similar titers across different perfusion rates and media formulations. FIG. 5B shows graphs of glutamine, ammonia, glucose, and lactate concentrations over the culture duration.

[0044] FIG. 6 shows a bar graph representing normalized volumetric rAAV titer in 5L (left) and Ambr250 (right) perfusion production bioreactors. The 5L bioreactor run modeled the process shown in FIGs. 1-4B, and the Ambr250 bioreactor run modeled the process shown in FIG. 1-4A. In both cases, rAAV titer improved with infection in growth media and subsequent dilution before initiation of perfusion during production.

[0045] FIG. 7 shows a bar graph representing normalized volumetric rAAV titer in Ambr250 perfusion production bioreactors. Different combinations of production media formulation and perfusion rates were tested. X = enrichment of amino acids and vitamins in production media; VVD = perfusion rate in vessel volumes per day. The graph shows an increase in titer with both increased media enrichment and perfusion rate.

[0046] FIG. 8A shows a bar graph representing normalized volumetric rAAV titer in Ambr250 production bioreactors seeded at different densities. The graph shows an increase in rAAV titer with increased seed density. Error bar represents 1 standard deviations across biological (N) and assay replicates. FIG. 8B shows a line graph representing the level of glutamine in culture for the conditions shown in FIG. 8A over the culture duration.

[0047] FIG. 9 shows a bar graph representing normalized volumetric rAAV titer in 5L perfusion production bioreactors seeded at different densities. The graph shows an increase in rAAV titer as seed density was increased from 2E6 to 10E6 c / mL. FIG. 10 shows a bar graph representing normalized volumetric rAAV titer in Ambr250 perfusion production bioreactors. Different combinations of perfusion rates (VVD) and seed densities were tested. The graph shows an increase in titer as perfusion rate was increased at high seed density but not at low seed density.

[0048] FIG. 11 shows a bar graph representing normalized volumetric rAAV titer in Ambr250 perfusion production bioreactors. Different timings of perfusion were tested (hpi = hours post infection). The graph shows similar titers across vessels where perfusion was initiated on day 0, and lower titer when perfusion was initiated on day 1.

[0049] FIG. 12 shows a bar graph representing normalized volumetric rAAV titer in Ambr250 production bioreactors. Production bioreactors were seeded from N-l cultures that were grown to different densities. The extent of culture dilution transferring from N-l to production cultures is shown. The graph shows similar titers across perfusion cultures, and lower titer in the batch culture control.

[0050] FIG. 13A shows a bar graph representing normalized volumetric titer in Ambr250 perfusion production bioreactors. Cultures were controlled at different pH setpoints during production. The graph shows higher titers at higher pH setpoints. Error bar represents 1 standard deviation across biological (N) and assay replicates. FIG. 13B shows graphs of viable cell density, pH, glutamine, and glucose concentrations over the culture duration, averaged across replicate pH conditions (error bar = 1 standard deviation). The graphs show higher glucose and glutamine consumption at higher pH.

[0051] FIG. 14 shows the results of a bridging study comparing rAAV titer from cells cultured under Ambrl5 mock perfusion conditions (mock perfusion), cells cultured under Ambrl5 batch culture conditions, and under shake-flask batch culture (at 0.5 million cells / mL cell density)(“screen”). As shown in FIG. 14, rAAV titer was highest for cells grown under Ambrl5 mock perfusion conditions than the other two conditions.

[0052] FIGs. 15A-15C show the correlation between rAAV titer obtained with small-scale screening (Ambrl5 mock perfusion, and three other screening methods) and rAAV titer obtained from larger-scale culture (Ambr250 mock perfusion). Each of FIGs. 15A-15C show the results for a single program. As shown in these FIGs., rAAV titer from small-scale Ambrl5 mock perfusion showed a higher correlation to rAAV titer from Ambr250 mock perfusion conditions than other rAAV clone screening methods. FIG. 16 shows an example of an Ambrl5 mock perfusion protocol. The inset shows a detailed view of the mock perfusion procedure.

[0053] FIG. 17 shows AAV titer as measured by qPCR in two cell lines. AAV titer is shown normalized to batch culture. Cells of each cell line were grown under batch culture conditions (450L), perfusion culture conditions (45L), or perfusion culture conditions (200L). The results show that, for both cell lines, perfusion culture conditions result in higher AAV titers, even for large-scale production (200L).

[0054] DETAILED DESCRIPTION

[0055] The present disclosure provides methods and compositions for cell culture production of rAAV. Without intending to be bound by theory, cell culture conditions may affect production of rAAV, for example, if cells are limited for nutrients or exposed to waste products from growth. In some aspects, the present disclosure relates to methods of perfusing cultured cells while the cultured cells produce rAAV (during rAAV production). In some aspects, the present disclosure relates to methods of culturing cells producing rAAV that include media exchange during the production of rAAV. In some embodiments, perfusion or media exchange keeps waste products at or below a threshold value. In some embodiments, perfusion or media exchange keeps one or more nutrients (e.g., glutamine) at or above a threshold value. In some embodiments, during perfusion or media exchange the glutamine level is maintained at or above 2mM during rAAV production. In some embodiments, the cell culture is infected in a smaller volume and then diluted for rAAV production. In some embodiments, the cell culture is infected in a larger volume and the cell culture is perfused under conditions suitable for rAAV production.

[0056] In some aspects, the method comprises perfusing a cell culture in a glutamine-containing medium. In some embodiments, the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture is perfused under conditions suitable for rAAV production. In such an embodiment, the medium used for perfusing the culture (perfusion medium) may have whatever concentration of glutamine is necessary for maintaining a culture medium glutamine level at or above 2 mM (e.g., 2 mM, 2.5 mM, 3 mM,3.5 mM, 4 mM, 4.5 mM, 5 mM). In some embodiments, the glutamine level is at or above 2 mM for at least 24 hours during AAV production. In some embodiments, the glutamine level is at or above 2 mM for at least 36 hours during AAV production. In some embodiments, the glutamine level is at or above 2 mM for at least 48 hours during AAV production. In some embodiments, the glutamine level is at or above 2 mM for at least 60 hours during AAV production. In some embodiments, the glutamine level is at or above 2 mM for at least 72 hours during AAV production. In some embodiments, the glutamine level is at or above 2 mM for up to 94 hours during AAV production.

[0057] In some aspects, the present disclosure provides a method of producing recombinant adeno associated virus (rAAV) particles, comprising perfusing a cell culture with a medium, wherein the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture produces rAAV particles during perfusion. In such embodiments, the perfusion medium comprises a glutamine level at or above 2 mM, while the culture medium may comprise a different glutamine level, either transiently or for a portion of AAV production.

[0058] In some embodiments, methods and compositions described herein are useful for increasing rAAV yield. In some embodiments, methods and compositions described herein are useful for increasing the percentage of full capsids produced. In some embodiments, methods and compositions described herein are useful for reducing the cost of producing rAAV for therapeutic applications.

[0059] FIG. 2 shows non-limiting examples of cell-based production methods. Scenario 1 shows perfusion during cell growth (N-l perfusion), 10% carryover to a second reactor and infection in the second reactor, and production under batch conditions. Scenario 2 shows perfusion during cell growth (N-l perfusion), 25% carryover to a second reactor and infection in the second reactor, and production under perfusion conditions. In some embodiments, perfusing the cell culture during production allows for larger carryover between a cell culture in the first reactor and the second reactor. Scenario 3 shows perfusion during cell growth (N-l perfusion), infection in growth medium in the same reactor, and production under perfusion conditions. In some embodiments, scenario 3-type processes comprise using perfusion to remove growth medium and add production medium after infection or transfection. Scenario 4 shows growth of high- density starter cultures (N-l cultures). In scenario 4A, a high-density N-l culture is added to a second bioreactor and diluted to low density with perfusion medium. In scenario 4B, production medium is added to the high-density culture in the first reactor, diluting the high-density culture to low density. In both scenario 4A and 4B, production occurs under perfusion conditions. As described herein, a “high-density culture” has a density greater than 10e6 cells / mL. In some embodiments, a high-density cell culture has a density of between 10e6 cell / mL and 50e6 cells / mL. In some embodiments, a high-density cell culture has a density of between 50e6 cell / mL and 75e6 cells / mL. In some embodiments, a high-density cell culture has a density of between 75e6 cell / mL and 100e6 cells / mL. In some embodiments, a high-density cell culture has a density of between 100e6 cell / mL and 150e6 cells / mL. In some embodiments, a high- density cell culture has a density of between 150e6 cell / mL and 200e6 cells / mL. In some embodiments, a high-density culture has a density of 100e6 cells / mL or greater.

[0060] In some embodiments, the methods shown in FIG. 2 allow a shorter N-l phase relative to standard methods. In some embodiments, the methods shown in Scenario 3 and Scenario 4 comprise a shorter N-l phase relative to standard methods. In some embodiments, the N-l phase lasts until the target cell density is reached, followed immediately by infection with a helper virus or transfection.

[0061] In some embodiments, an N-l phase comprises the phase of cell growth immediately before infection or transfection. In some embodiments, an N-l phase comprises the phase of cell growth immediately before introduction of a production medium.

[0062] FIG. 2 depicts cell-based production methods using producer cells. Wherein “infection” comprises contacting the culture with a helper virus. The cell-based production methods shown in FIG. 2 could also be used with transiently-transfected cells or any other cells used for cellbased production of products of interest.

[0063] Perfusion

[0064] In some aspects, the present disclosure relates to methods of producing rAAV while perfusing a cell culture, also referred to as using perfusion or under perfusion conditions. In some embodiments, perfusion comprises gradual replacement of media comprising the cell culture. In some embodiments, perfusion occurs over an extended interval. In some embodiments, perfusion comprises continuous or near-continuous replacement of media. In some embodiments, fresh medium is added and spent culture medium is removed. In some embodiments, the rate at which fresh medium is added is the same as the rate at which spent culture medium is removed. In some embodiments, perfusion cell culture comprises retaining cells in the culture using a retention device while removing waste products from the culture and / or adding nutrients. In some embodiments, a retention device comprises a centrifuge (e.g., a continuous centrifuge), a membrane filter (e.g., a tangential flow membrane filter (TFF), an alternating tangential flow filter (ATF)), a dynamic filter, a spin-filter, a hollow-fiber filter, a separator (e.g., an ultrasonic or dielectrophoretic separator), a settler (e.g., a gravity settler), or a hydrocyclone. In some embodiments, a retention device consists of a centrifuge (e.g., a continuous centrifuge), a membrane filter (e.g., a tangential flow membrane filter (TFF), an alternating tangential flow filter (ATF)), a dynamic filter, a spin-filter, a separator (e.g., an ultrasonic or dielectrophoretic separator), a settler (e.g., a gravity settler), or a hydrocyclone. In some embodiments, perfusion removes waste products from the cell culture. In some embodiments, a waste product is a metabolite. In some embodiments, a waste product comprises lactate or ammonium (NH4+).

[0065] In some embodiments, perfusion adds one or more nutrients. In some embodiments, a nutrient comprises a carbon source (e.g., glucose or galactose), amino acid source, trace metal, vitamin, antioxidant source, and / or mineral.

[0066] In some embodiments, the cell culture is perfused both during growth and during rAAV production. In some embodiments, the cell culture is perfused only during rAAV production.

[0067] In some embodiments, the cell culture is cultured under rAAV production conditions for at least 24 hours, 24 hours, or about 24 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 48 hours, 48 hours, or about 48 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 72 hours, 72 hours, or about 72 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 24 hours, 24 hours, or about 24 hours after rAAV production has begun. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 48 hours, 48 hours, or about 48 hours after rAAV production has begun. In some embodiments, the cell culture is cultured for at least 96 hours, 96 hours, or about 96 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 72 hours, 72 hours, or about 72 hours after rAAV production has begun. In some embodiments, the cell culture is cultured for at least 96 hours, 96 hours, or about 96 hours after rAAV production has begun. In some embodiments, the cell culture is perfused for at least 48 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for up to 48 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for between 24 and 48 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for at least 24 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for up to 72 hours during rAAV production.

[0068] In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 24 hours, 24 hours, or about 24 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 48 hours, 48 hours, or about 48 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 72 hours, 72 hours, or about 72 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 24 hours, 24 hours, or about 24 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 48 hours, 48 hours, or about 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 96 hours, 96 hours, or about 96 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 72 hours, 72 hours, or about 72 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 96 hours, 96 hours, or about 96 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 48 hours after rAAV production has begun. In some embodiments, the cell culture is perfused for up to 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for between 24 and 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 24 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for up to 72 under conditions suitable for rAAV production.

[0069] In some embodiments, the rate of perfusion is measured as vessel volumes per day (VVD). In some embodiments, the rate of perfusion is between 0.5 and 2 vessel volume per day (VVD). In some embodiments, the rate of perfusion is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 VVD. In some embodiment, the rate of perfusion is 2 VVD or higher. In some embodiments, the rate of perfusion is 2-5 VVD, 2-4.5 VVD, 2-4 VVD, 2-3 VVD, or 2-2.5 VVD. In some embodiments, the rate of perfusion is 3-4 VVD, 3-5 VVD, 3-6 VVD, 4-5 VVD, or 4-6 VVD.

[0070] In some embodiments, perfusion is continuous. In some embodiments, continuous perfusion includes brief intervals where perfusion is stopped. In some embodiments, perfusion is stopped for less than half an hour, about half an hour, less than an hour, about an hour, less than two hours, about two hours, less than three hours, about three hours, less than four hours, about four hours, less than five hours, about five hours, less than 10 hours, about 10 hours, less than 12 hours, or about 12 hours. In some embodiments, perfusion is semi-continuous. In some embodiments, semi-continuous perfusion comprises regular intervals of perfusion and nonperfusion culture. Media Exchange

[0071] In some embodiments, semi-continuous perfusion comprises media exchange. In some embodiments, media exchange comprises rapid replacement of media comprising the cell culture. For example, a cell culture may be pelleted (e.g., by centrifugation), spent medium removed, and resuspended in fresh medium. A culture may also be separated from spent medium by other means, such as filtration, and resuspended in spent medium. In some embodiments, media exchange occurs over a short interval. In some embodiments, media exchange within about an hour (e.g., from start to finish the process of media exchange takes about half an hour). In some embodiments, media exchange occurs within an hour. In some embodiments, media exchange occurs within less than an hour. In some embodiments, media exchange occurs within about half an hour.

[0072] In some embodiments, media exchange occurs one, twice, three times, four times, five time, or six times during rAAV production. In some embodiments, media exchange occurs about once every 12 hours, once a day, once every 36 hours, or once every two days during rAAV production. In some embodiments, media exchange occurs when the level of a particular nutrient or waste product reaches a threshold level.

[0073] In some embodiments, media exchange removes waste products from the cell culture. In some embodiments, a waste product is a metabolite. In some embodiments, a waste product comprises lactate or ammonium (NH4+)

[0074] In some embodiments, media exchange adds one or more nutrients. In some embodiments, a nutrient comprises a carbon source (e.g., glucose or galactose), amino acid source, trace metal, vitamin, antioxidant source, and / or mineral.

[0075] Small-Scale Perfusion

[0076] Aspects of the present disclosure provide small-scale perfusion methods for predicting which rAAV clones will perform well under perfusion conditions. In some embodiments, a small-scale perfusion method allows high throughput testing of rAAV candidates. In some embodiments, a small-scale perfusion method comprises culturing cells in a small-scale system. In some embodiments, a small-scale system comprises a miniature bioreactor. In some embodiments, the miniature bioreactor is an automated bioreactor. In some embodiments, the miniature bioreactor is an automated microbioreactor (e.g., Ambrl5 (Sartorius)). In some embodiments, rAAV clone candidates are cultured under mock perfusion conditions in the small-scale system. In some embodiments, rAAV clones are evaluated for growth, rAAV titer, and / or percent full capsids during mock perfusion in the small-scale system. In some embodiments, small-scale perfusion (e.g., a small-scale perfusion method) comprises mock perfusion (e.g., media exchange). In some embodiments, small-scale perfusion comprises culturing cells in about 5 mLs, about 10 mLs, about 15 mLs, about 20 mLs, about 25 mLs, about 30 mLs, about 35 mLs, about 40 mLs, about 45 mLs, or about 50 mLs. In some embodiments, small-scale perfusion comprises culturing cells in less than 5 mLs, less than 10 mLs, less than 15 mLs, less than 20 mLs, less than 25 mLs, less than 30 mLs, less than 35 mLs, less than 40 mLs, less than 45 mLs, less than 50 mLs, less than 60 mLs, less than 70 mLs, less than 80 mLs, less than 90mL, or less than 100 mLs.

[0077] In some embodiments small-scale perfusion predicts rAAV clone performance in a larger-scale perfusion method. In some embodiments, the larger-scale perfusion method comprises culturing cells in 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, 100 times 200 times, 500 times, 750 times, 1000 times or more the volume of the small-scale perfusion method. In some embodiments, the larger-scale perfusion method comprises culturing cells in at least 100 mLs, at least 250 mLs, at least 500 mLs, at least 750 mLs, at least 1 L, at least 5 L, at least 10 L, at least 15 L, at least 25 L, at least 50L, at least 100 L, at least 200 L, at least 400 L, at least 500 L, at least 1000 L, at least 1500 L or at least 2000 L.

[0078] In some embodiments, cell sedimentation is used as the cell retention mechanism. In some embodiments, cell sedimentation comprises allowing the cells to settle for a set time. In some embodiments, the cells are allowed to settle for about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, cell settling time is changed based on the rAAV vector being assessed. In some embodiments, centrifugation is used as the cell retention mechanism.

[0079] In some embodiments, small-scale perfusion is performed at low cell density (e.g., about 0.5 million cells / mL). In some embodiments, small-scale perfusion is performed at high cell density (e.g. 2 million cells / mL, 4 million cells / mL, 10 million cells / mL, or higher). In some embodiments, small-scale perfusion is performed at the same or a similar cell density to larger- scale perfusion as described herein (e.g., a density greater than 10 million cells / mL, a density of between 10 million cells / mL and 50 million cells / mL, a density of between 50 million cells / mL and 75 million cells / mL, a density of between 75 million cells / mL and 100 million cells / mL, a density of between 100 million cells / mL and 150 million cells / mL, a density of between 150 million cells / mL and 200 million cells / mL, or a density of 100 million cells / mL or greater). In some embodiments, small-scale perfusion comprising culturing rAAV producing cells with agitation (e.g., vessel shaking) in an Ambrl5 system. In some embodiments, the cells are allowed to settle (e.g., by pausing vessel shaking). In some embodiments, once the cells have settled, a supernatant is removed from a vessel of the Ambrl5 system. In some embodiments, the removed supernatant is replaced with fresh medium. In some embodiments, agitation is subsequently restarted. In some embodiments, at least two, three, four, five, six, seven, eight, nine, or at least 10 cycles of settling, media removal, fresh media addition, and shaking resumption occur before small-scale perfusion is stopped. In some embodiments, small-scale perfusion is followed by isolation of rAAV. In some embodiments, the isolated rAAV is analyzed for titer and / or percent full capsids.

[0080] In some embodiments, an rAAV clone selected from testing in small-scale perfusion conditions performs better under large-scale perfusion conditions than an rAAV clone selected from testing in batch-culture conditions. In some embodiments, an rAAV clone selected from testing in small-scale perfusion growth produces a higher percentage of full capsids and / or a higher titer of rAAV particles relative to an rAAV clones selected from testing in batch-culture conditions.

[0081] Media

[0082] In some embodiments, perfusion or media exchange removes waste products from the media comprising the cell culture. In some embodiments, perfusion or media exchange maintains nutrient levels in the media comprising the cell culture.

[0083] In some embodiments, the medium introduced via perfusion or media exchange (“perfusion medium”) is an appropriate medium for cell growth, infection, transfection, and / or rAAV production. In some embodiments, the perfusion medium is a medium that supports cell growth. In some embodiments, the perfusion medium provides one or more nutrients. In some embodiments, at least one perfusion medium is a feed medium. In some embodiments, one or more of the perfusion media comprises a complex medium. In some embodiments, the perfusion medium is a defined medium. In some embodiments, one or more of the perfusion media comprises Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV-MAX media (Gibco), EX-Cell media, or RPMI media. In some embodiments, the perfusion medium is a serum- free medium. In some embodiments, the perfusion medium is supplemented with serum. In some embodiments, the perfusion medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some embodiments, the perfusion medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some embodiments, the perfusion medium is an appropriate medium for AAV production.

[0084] In some embodiments, the method comprises a first volume and a second volume, each of which comprise a medium, the second medium being a “perfusion medium”. In some embodiments, the first volume medium and the second volume medium are the same medium. In some embodiments the first volume medium and the second volume medium are different media. In some embodiments, the first volume medium is a medium appropriate for cell growth. In some embodiments, the second volume medium is a medium appropriate for rAAV production.

[0085] In some embodiments, the growth media is changed at the time of infection or transfection such that the cells undergo osmotic shock. In some embodiments, the osmolality of the growth media is raised at the time of infection to the osmolality of the AAV production medium.

[0086] In some embodiments, the pH of the perfusion medium is between 6.9 and 7.5, 7 and 7.4, 7.1 and 7.3, 7 and 7.5, 6.9 and 7.4, or 7.2 and 7.5. In some embodiments, the pH of the perfusion medium is about 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. In some embodiments, the pH of the perfusion medium is 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0087] In some embodiments, the glutamine level in the perfusion medium is lmM-2mM, 2mM-3mM, 3mM-4mM, 4mM-5mM, 5mM-6mM, 6mM-7mM, 7mM-8mM, 8mM-9mM, or 9mM-10mM.

[0088] In some embodiments, the perfusion medium is enriched relative to standard medium. In some embodiments, the perfusion medium is enriched 1.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 8X, 8X, or 10X relative to standard medium. In some embodiments, a perfusion medium is enriched relative to a standard medium when the enriched medium comprises a higher concentration of one or more vitamins, amino acids, or other nutrients relative to the standard medium. For example, a perfusion medium is 2X enriched for glutamine relative to a standard medium when the medium comprises a 2X higher concentration of glutamine relative to a standard medium.

[0089] In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production. In some embodiments, the cell culture comprises a medium that supports cell growth. In some embodiments, the cell culture comprises a medium that provides one or more nutrients. In some embodiments, the cell culture comprises a feed medium. In some embodiments, the cell culture comprises a complex medium. In some embodiments, the cell culture comprises a defined medium. In some embodiments, the cell culture comprises a medium selected from: Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV- MAX media (Gibco), EX-Cell media, or RPMI media. In some embodiments, the cell culture comprises a serum-free medium. In some embodiments, the cell culture comprises a medium supplemented with serum. In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some embodiments, the cell culture comprises an appropriate medium for AAV production.

[0090] In some embodiments, the method comprises a first volume and a second volume, each of which comprise a medium, each comprising a cell culture. In some embodiments, the first volume medium and the second volume medium are the same medium. In some embodiments the first volume medium and the second volume medium are different media. In some embodiments, the first volume medium is a medium appropriate for cell growth. In some embodiments, the second volume medium is a medium appropriate for rAAV production.

[0091] In some embodiments, the pH of the cell culture is between 6.9 and 7.5, 7 and 7.4, 7.1 and 7.3, 7 and 7.5, 6.9 and 7.4, 7.1 and 7.4, or 7.2 and 7.5. In some embodiments, the pH of the cell culture is about 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. In some embodiments, the pH of the cell culture is 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0092] In some embodiments, the glutamine level in the cell culture is lmM-2mM, 2mM-3mM, 3mM-4mM, 4mM-5mM, 5mM-6mM, 6mM-7mM, 7mM-8mM, 8mM-9mM, or 9mM-10mM. In some embodiments, the glutamine level in the perfusion medium is maintained for at least 12 hours, 15 hours, 18 hours, 21 hours 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, or 48 hours.

[0093] In some embodiments, the glutamine level in the cell culture starts at a first level and does not go below a second level. In some embodiments, the first level comprises a glutamine level of 3mM-4mM, 4mM-5mM, or 5mM-6mM. In some embodiments, the second level comprises a glutamine level of lmM-2mM, 2mM-3mM, or 3mM-4mM. In some embodiments, the glutamine level starts at 4mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 5mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 4mM and does not go below 3 mM. In some embodiments, the glutamine level starts at 6 mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 4 mM and does not go below 1.5 mM. In some embodiments, the glutamine level starts at 5mM and does not go below 4 mM.

[0094] In some embodiments, the cell culture comprises a medium that is enriched relative to standard medium. In some embodiments, the cell culture comprises a medium that is enriched

[0095] I.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 8X, 8X, or 10X relative to standard medium. In some embodiments, the cell culture comprises a medium that is enriched relative to a standard medium when the enriched medium comprises a higher concentration of one or more vitamins, amino acids, or other nutrients relative to the standard medium. For example, a cell culture comprises as medium that is 2X enriched for glutamine relative to a standard medium when the medium comprises a 2X higher concentration of glutamine relative to a standard medium.

[0096] Dilution

[0097] In some embodiments, the cell culture is infected in a smaller volume (first volume) and rAAV production occurs in a larger volume (second volume). In some embodiments, the second volume is 1.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 7.25X, 7.5X, 7.75X, 8X, 8.25X, 8.5X, 8.75X, 9X, 9.25X, 9.5X 9.75X, 10X, 10.25X, 10.5X, 10.75X, 11X, 11.25X,

[0098] I I.5X, 11.75X, 12X, 12.25X, 12.5X, or 12.75X larger than the first volume. For example, in an embodiment, the cell culture is infected in 12.5L and rAAV production occurs in 50L.

[0099] In some embodiments, the first volume is comprised in a reactor. In some embodiments, the first volume is less than half or less than one third of the volume of the reactor. In some embodiments, the first volume is between one quarter and one third the volume of the reactor. In some embodiments, the first volume is between one third and one half of the volume of the reactor. In some embodiments, the first volume is between one half and two thirds of the volume of the reactor.

[0100] In some embodiments, the first volume and the second volume are comprised in the same reactor (infection and production occur in the same reactor). In some embodiments, the first volume and the second volume are comprised in different reactors.

[0101] In some embodiments the first volume and the second volume comprise the same media. In some embodiments, the first volume and the second volume comprise different media. In some embodiments, the second volume comprises media that is enriched relative to a standard medium. In some embodiments, the second volume medium is enriched 1.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 8X, 8X, or 10X relative to standard medium. rAAV production

[0102] Recombinant adeno-associated virus (rAAV) vectors are useful in gene therapy to deliver therapeutic genes to patient cells and tissue. An rAAV particle typically comprises a recombinant nucleic acid encapsidated within rAAV capsid proteins to form an rAAV particle that can be administered to a subject. The recombinant nucleic (e.g., recombinant AAV genome) acid typically includes a heterologous gene of interest (e.g., encoding a therapeutic nucleic acid and / or protein) flanked by AAV inverted terminal repeat (ITR) sequences. In some embodiments, the rAAV capsid proteins can be naturally occurring capsids of different AAV serotypes. For example, different AAV serotypes have different tissue tropisms and can be used to target different tissue types and associated diseases. In some embodiments, the rAAV capsid proteins include one or more amino acid substitutions relative to naturally occurring capsid proteins.

[0103] Different manufacturing techniques can be used to produce rAAV particles. Typically, rAAV particles are assembled in a host cell culture (e.g., in a bioreactor or other cell culture vessel). In some embodiments, the cell culture is maintained or grown using standard methods (e.g., in a suspension culture, or on plates). The assembled rAAV is then isolated from the cell culture. In some embodiments, the cell culture comprises mammalian cells, insect cells or cells of another cell type.

[0104] In some embodiments, a bioreactor is a perfusion bioreactor. In some embodiments, a perfusion bioreactor is a bioreactor configured to allow medium to be added or removed while maintaining the cell culture within the bioreactor.

[0105] In some embodiments, a bioreactor (e.g., a perfusion bioreactor) is a small-scale bioreactor. In some embodiments, a small-scale bioreactor can hold about 5 mLs, about 10 mLs, about 15 mLs, about 20 mLs, about 25 mLs, about 30 mLs, about 35 mLs, about 40 mLs, about 45 mLs, or about 50 mLs. In some embodiments, a small-scale bioreactor can hold less than 5 mLs, less than 10 mLs, less than 15 mLs, less than 20 mLs, less than 25 mLs, less than 30 mLs, less than 35 mLs, less than 40 mLs, less than 45 mLs, less than 50 mLs, less than 60 mLs, less than 70 mLs, less than 80 mLs, less than 90mL, or less than 100 mLs.

[0106] In some embodiments, a bioreactor (e.g., a perfusion bioreactor) is a large-scale bioreactor. In some embodiments, a large-scale bioreactor can hold at least 100 mLs, at least 250 mLs, at least 500 mLs, at least 750 mLs, at least 1 L, at least 5 L, at least 10 L, at least 15 L, at least 25 L, at least 50L, at least 100 L, at least 200 L, at least 400 L, at least 500 L, at least 1000 L, at least 1500 L or at least 2000 L. In some embodiments, a large-scale bioreactor can hold up to 50000 L.

[0107] Manufacturing rAAV comprises using a cell culture to produce and assemble the parts of rAAV particles. Without intending to be bound by theory, producing rAAV particles comprises i) expression of Rep and Cap genes, ii) providing a recombinant genome, typically comprising a gene of interest, and iii) providing the additional functions, sometime referred to as helper functions, required for rAAV replication. In some embodiments, the cell culture comprises cells that have genes encoding Rep and Cap integrated into the cells’ genome. In some embodiments, the cell culture comprises cells that have been stably transfected with a plasmid comprising the genes encoding Rep and Cap. In some embodiments, the cells further comprise a plasmid containing an rAAV genome. Without intending to be bound by theory, these cells are typically infected with a helper virus that provides additional factors necessary for rAAV replication. In some embodiments, the helper virus is an adenovirus, such as Ad5. In some embodiments, the Ad5 virus is a wildtype Ad5 virus. In some embodiments, the Ad5 virus is a mutated Ad5 virus. In some embodiments, the cell culture comprises cells that are transiently transfected with plasmids encoding Rep, Cap, an rAAV genome, and helper functions. In some embodiments, two plasmids are used (double transfection). In some embodiments, three plasmids are used (triple transfection). Providing the helper virus, or transfecting with all necessary components, causes the cells to begin producing rAAV.

[0108] Aspects of the disclosure relate, at least in part, to methods and compositions for culturing a cell culture under conditions suitable for rAAV production (“production conditions”). As described herein, production conditions are conditions under which expression of one or more genes required for rAAV production, packaging, and / or growth is promoted (e.g., by addition of a helper virus, induction of an inducible promoter, or transfection with one or more vectors encoding one or more genes required for rAAV production, packaging, and / or growth).

[0109] Producer cells

[0110] In some embodiments, a cell culture associated with the disclosure comprises producer cells. In some embodiments, a producer cell stably expresses rep and cap genes suitable for rAAV packaging. In some embodiments, a producer cell further comprises an rAAV genome. In some embodiments, the rAAV genome comprises a therapeutic gene. In some embodiments, the cell culture comprises HeLa cells. In some embodiments, the cell culture comprises human embryonic kidney (HEK) cells. In some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells. In some embodiments, the cell culture comprises Vero cells. In some embodiments, the cell culture comprises Madin-Darby canine kidney (MDCK) cells. In some embodiments, the cell culture comprises baby hamster kidney (BHK) cells. In some embodiments, the cell culture comprises A549 cells. In some embodiments, the cell culture comprises amniocyte cells.

[0111] In some embodiments, the producer cell culture is infected with a helper virus. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5. In some embodiments, the producer cell culture does not require helper virus.

[0112] Transient transfection

[0113] Transient transfection is one way of introducing heterologous genetic material to cells of interest. As used herein, “transfection” means nucleic acid transfection. Briefly, transfection comprises contacting a cell culture with one or more nucleic acids and a transfection reagent, also called a “transfecting agent.” In some embodiments, the transfection reagent is a commercially available transfection reagent. In some embodiments, the transfection reagent is a chemical transfection reagent. In some embodiments, the transfection reagent is a liposomalbased transfection reagent. In some embodiments, the transfection reagent is a liposome. In some embodiments, the liposome is a positively charged or cationic liposome. In some embodiments, the transfection reagent is a non-liposomal-based transfection reagent. In some embodiments the transfection reagent is a calcium phosphate, dendrimer, polymer, nanoparticle, or non-liposomal lipid. In some embodiments, the transfection reagent is lipofectamine or a variant thereof. Transfection reagents include, but are not limited to, polyethylenimine (PEI), FectoVIR® (Polyplus), Nanofectamine (GE Healthcare), Oligofectamine (Invitrogen), RNAiMAX (Invitrogen), siPORT (ThermoFisher), DharmaFECT (Dharmacon), Endofectin@MAX (GeneCopoeia), Escort IV Liposome (Sigma- Aldrich), Arrest-In (Dharmacon), TurboFect (Thermo), Effectene (Qiagen), Attractene (Qiagen), PolyFect (Qiagen), SuperFect (Qiagen), ExpressFect (Thomas), Genejammer (Stratagene), FuGENE (Promega), INTERFERin (Polyplus), NanoFectin (System Biosciences), X-tremeGENE (Roche), Xfect (ClonTech), Escort IV (Sigma- Aldrich), and N-TER (Sigma- Aldrich). Recombinant A A Vs

[0114] Naturally occurring AAV capsid proteins can be used to produce rAAVs for gene therapy. Different naturally occurring rAAVs have different characteristics (including for example different tissue tropisms) and can be used for different indications. rAAVs are highly prevalent within the human population (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues J Virol. 2004. 78(12): p. 6381-8, and Boutin. S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population, implications forgone therapy using AAV vectors. Hum Gene Ther. 2010. 21(6): p. 704-12) and are useful as viral vectors. Many serotypes exist, each with different tropism for tissue types (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80), which allows specific tissues to be preferentially targeted with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rhlO, transduce the mammalian body. See Zincarelli, C., et al. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A. M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S. J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAVl and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65. AAV9 has been demonstrated to cross the blood-brain barrier (see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65, and Rahim, A. A., et al., Intravenous administration of AAV2 / 9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011. 25(10): p. 3505-18) that is inaccessible to many viral vectors and biologies. Certain rAAVs have a payload of 4.7-5.0 kb (including viral inverted terminal repeats (ITRs), which are required in cis for viral packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): p. 80-6 and Dong, J. Y., P. D. Fan, and R. A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): p. 2101-12. In some embodiments, rAAVs can include one or more variant AAV capsid proteins having one or more amino acid substitutions relative to a naturally occurring AAV capsid protein.

[0115] Accordingly, in some embodiments, the rAAV particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins, or amino acid sequence variants thereof.

[0116] In some embodiments, rAAV is isolated from the cell culture and prepared (e.g., purified and / or sterilized) for delivery to a human subject (e.g., to deliver a therapeutic gene to the subject to assist in the treatment of a disease or condition).

[0117] In some embodiments, rAAV isolation comprises a further clarification and / or purification step. In some embodiments, rAAV isolation comprises a flocculation step. In some embodiments, a lysis agent, for example a detergent, can be used along with the flocculation agent.

[0118] In some embodiments, rAAV particles are further purified, for example using one or more affinity, ion exchange chromatography, and / or hydrophobic interaction chromatography steps, e.g., after clarification of an rAAV preparation.

[0119] In some embodiments, rAAV particles (e.g., after one or more purification steps) are added to a pharmaceutically acceptable solution.

[0120] The disclosure also provides compositions comprising rAAV particles, and methods of administering the rAAV particles to a subject (e.g., a human subject having a condition that the therapeutic RNA and / or protein can help treat).

[0121] These and other aspects are illustrated by the following non-limiting examples.

[0122] EXAMPLES

[0123] Example 1. Mock perfusion improves rAAV yield

[0124] Cells were grown using standard methods in preparation for rAAV production. Spinner tubes were seeded at 0.5, 1, 2, and 4E6 c / mL and infected with Ad5 on day 0. The production media was manually exchanged once or twice daily (1 VVD and 2 VVD, respectively) 1 and 2 days after infection. Results show an increase in rAAV titer at higher seed densities and with increased number of media exchanges, and considerably higher titers compared to the control low-density batch condition (FIG. 3). Example 2. Perfusion in bioreactors improves rAAV yield across different cell lines and AAV serotypes

[0125] Cells were grown as usual in preparation for rAAV production. 5L bioreactors were inoculated and infected with Ad5 at 0.5E6 c / mE and continued in batch mode (FIG. 4A) or inoculated and infected at 4E6 c / mE and perfused at 2 reactor volumes / day for 2 days (FIG. 4A). Significantly higher titers were observed with the high-density perfusion process with two different cell lines and rAAV serotypes. As shown in FIG. 4B, cells grown under batch culture conditions produce lower rAAV titers per unit volume at higher cell densities (3E6 cells / mE and 4E6 cells / mE) compared to lower cell densities (0.5E6 cells / mE). This demonstrates that perfusion culture conditions can produce higher rAAV titer than even favorable batch culture conditions.

[0126] In another example, Ambr250 bioreactors were inoculated and infected with Ad5 at 3E6 c / mE and operated in either batch or perfusion mode at different perfusion rates (VVD) and media compositions (IX or 2X, denoting enrichment of amino acids and vitamins) (FIG. 5A). All perfusion cultures were perfused from day 0 to day 2. The batch cultures were supplemented with glutamine and glucose to avoid depletion. Results show perfusing the cultures improves titer compared to the batch cultures at the same density, and that increasing the perfusion rate improves titer. FIG. 5B shows glutamine was maintained > 2 mM during perfusion. Eactate and ammonia, which are waste products from cell metabolism, accumulated to high concentrations in the batch cultures and may have contributed to the lower titers.

[0127] Example 3. Infecting high density growth cultures before dilution and perfusion improves rAAV yield

[0128] Cells were grown as usual in preparation for rAAV production. Production cultures were seeded in 5E bioreactors (FIG. 6, left) and Ambr250 bioreactors (FIG. 6, right). The 5E bioreactor run modeled the process shown in FIG. 2, scenario 4B, and the Ambr250 bioreactor run modeled the process shown in FIG. 2, scenario 4A. In both cases, rAAV titer improved with infection in growth media and subsequent dilution before initiation of perfusion during production.

[0129] Example 4. Enriching production media improves rAAV yield at higher densities

[0130] Ambr250 production cultures were seeded at about 5E6 c / mE and infected with Ad5 on day 0. The conditions are shown in FIG. 7. The cultures were perfused at different rates (VVD) for 2 days after infection and with different media formulations (X = enrichment of amino acids and vitamins in production media). Results show an increase in titer with both increased media enrichment at the same perfusion rates, and with increased perfusion rate using the same media formulation. The results suggest that a combination of media enrichment and perfusion to remove waste products are beneficial to rAAV production for this cell type.

[0131] Example 5. Increasing seed density improves overall yield but reduces cell-specific productivity

[0132] Ambr250 production bioreactors were seeded at different densities (0.5, 1.5, 3.0, 6.0E6 c / mL) and infected with Ad5 on day 0. The low-density culture was operated in batch mode, and the three higher densities operated in perfusion mode (2 VVD, 2-48 hours post infection). Results show an increase in rAAV titer with increased seed density, however the cell-specific productivity decreases with cell density (FIG. 8A). Glutamine was maintained at > 2mM for all cultures (FIG. 8B).

[0133] In another example, 5L production bioreactors were seeded at 2E6 and 10E6 c / mL and infected with Ad5. The cultures were perfused at 2 VVD for 2 days after infection. Results show an increase in overall titer, but a decrease in cell-specific productivity (FIG. 9).

[0134] Example 6. Increasing perfusion rate improves rAAV yield at higher densities

[0135] A study examining increasing perfusion rates was conducted in Ambr250 bioreactors. Different combinations of perfusion rates (VVD) and seed densities were tested, with perfusion enabled for 2 days after Ad5 infection (FIG. 10). Results show an increase in titer as perfusion rate was increased at high seed density but not at low seed density.

[0136] Example 7. Perfusion timing impacts rAAV yield

[0137] Ambr250 production bioreactors were seeded at 3E6 c / mL and infected with Ad5 on day 0. Different timings of perfusion were tested (hpi = hours post infection). Results shows similar rAAV titers across vessels where perfusion was initiated on day 0, and lower titer when perfusion was initiated on day 1 (FIG. 11).

[0138] Example 8. Production cultures may be seeded from a range of N-l densities

[0139] Ambr250 N-l cultures were grown with perfusion to different densities (7, 21, and 33e6 c / mL). The cultures were used to seed production bioreactors at 3-4E6 c / mL, therefore the cultures were diluted between 2 and 1 IX from N-l to production. The experimental details are shown in Table 1. The cultures were infected with Ad5 and perfused for 2 or 3 days after infection. One low-density batch culture was also included. Results show similar titers across perfusion cultures, and lower titer in the batch culture control (FIG. 12). This suggests the N-l culture density and the dilution does not impact productivity, within the ranges tested. Table 1. Experimental details of production in Ambr250 vessels.

[0140] Example 9. Low pH inhibits rAAV production

[0141] A study was conducted in Ambr250 bioreactors, which were seeded at 3E6 c / mL and perfused for 2 days after Ad5 infection. Production culture pH (6.95, 7.15, and 7.35) and other factors were tested, however only pH had a statistically significant effect on titer, therefore results shown were averaged across bioreactors with the same pH setpoint (FIG. 13A). Results show higher titers at higher pH setpoints. FIG. 13B shows higher glutamine and glucose consumption at higher pH at the same cell densities, suggesting these more productive cells were more metabolically active (higher rate of glycolysis).

[0142] Example 10. Small-Scale Perfusion

[0143] To assess the ability of small-scale perfusion methods to predict success of rAAV clones in larger- scale perfusion culture, rAAV clones were tested in rAAV-producing cells cultured in an Ambrl5 system under mock perfusion conditions and in batch culture conditions. Cell cultures were about 12 mLs in volume. Mock perfusion was performed as shown in FIG. 16.

[0144] As shown in FIG. 14, Ambrl5 mock perfusion outperformed batch culture and low- density batch culture in shake flasks (“screen”) for many clones tested.

[0145] Clones selected from screening under Ambrl5 mock perfusion conditions, batch culture conditions, and other non-perfusion screening conditions were tested in an Ambr250-scale rAAV production process. rAAV titer was tested for each condition. As shown in FIGs. 15A- 15C and Table 2 below Ambrl5 mock perfusion rAAV titer levels showed a higher correlation (R2= 0.905, and 0.996) to Ambr250 mock perfusion rAAV titer levels relative to other screening methods for two programs.

[0146] Table 2. Correlation Coefficients showing correlation between rAAV titer levels obtained from four rAAV clone screening processes and rAAV titer levels obtained from Ambr250 culture.

[0147] Example 10. Large-Scale Perfusion

[0148] To assess the effect of perfusion growth conditions on scaled-up (large-scale) AAV production relative to batch culture conditions, two rAAV-producing cell lines were grown under batch culture conditions at large scale (450L), under perfusion culture conditions at bench scale (45L), and under perfusion culture conditions at large scale (200L).

[0149] As shown in FIG. 17, cell cultures grown under perfusion conditions produced higher AAV titers, both at 45L scale and 200L scale. This demonstrates that perfusion culture can maintain AAV production in scaled- up manufacturing conditions.

[0150] EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0151] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0152] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0153] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0154] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0155] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0156] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0157] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0158] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMSWhat is claimed is:

1. A method of producing recombinant adeno associated virus (rAAV) particles, comprising perfusing a cell culture in a medium, wherein the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture produces rAAV particles during perfusion.

2. A method of producing recombinant adeno associated virus (rAAV) particles, comprising perfusing a cell culture with a medium, wherein the medium comprises a glutamine level at or above 2 mM, and wherein the cell culture produces rAAV particles during perfusion.

3. The method of claim 1 or claim 2, wherein the cell culture comprises producer cells.

4. The method of claim 3, wherein the producer cells are HeLa cells.

5. The method of any one of claims 1-4, further comprising contacting the cell culture with one or more rAAV-encoding recombinant nucleic acids.

6. The method of any one of claims 1-5, wherein the pH of the medium is at or between 7-7.4.

7. The method of any one of claims 1-6, wherein the cell culture is cultured for at least 72 hours after AAV production has begun, and wherein the cell culture is perfused for up to 48 hours of the at least 72 hours.

8. The method of any one of claims 1-7, wherein the cell culture is perfused for at least 24 hours after AAV production has begun.

9. The method of any one of claims 1-8, wherein the glutamine levels are at or above 2 mM for at least 24 hours during AAV production.

10. The method of any one of claims 1-9, wherein the rate of perfusion is between 0.5 and 2 vessel volume per day (VVD).

11. The method of any one of claims 1-9, wherein the rate of perfusion is 2 VVD or higher.

12. The method of any one of claims 1-11, wherein the medium is enriched at least 2X relative to a standard medium.

13. The method of any one of claims 3-12, wherein the cell culture is infected with a helper virus.

14. The method of claim 13, wherein the helper virus is Ad5.

15. The method of claim 14, wherein the Ad5 virus is a wildtype Ad5 virus.

16. The method of claim 14, wherein the Ad5 virus is a mutated Ad5 virus.

17. The method of any one of claims 1-16, wherein the glutamine level of the media starts at or above and does not go below 2 mM.

18. The method of any one of claims 1-17, further comprising isolating rAAV particles from the cell culture.

19. A method of producing recombinant adeno associated virus (rAAV) particles, comprising: a) infecting a cell culture in a first volume in a first reactor; b) perfusing the cell culture in a second volume, wherein rAAV particle production occurs during perfusion, and wherein the first volume is smaller than the second volume.

20. The method of claim 19, wherein the first volume is a quarter of the volume of the first reactor.

21. The method of claim 19, wherein the first volume is less than half the volume of the second volume.

22. The method of any one of claims 19-21, wherein the cell culture of (a) is transferred to a second reactor along with additional medium to obtain the second volume.

23. The method of any one of claims 19-21, wherein the cell culture of (a) is maintained in the first reactor and additional medium is added to obtain the second volume.

24. The method of any one of claims 19-23, wherein the glutamine level of the second volume is kept at or above 2 mM.

25. The method of any one of claims 19-24, wherein the cell culture comprises at least 10 million, 12 million, 14 million, or 16 million cells / mL.

26. The method of any one of claims 19-25, wherein the second volume comprises more than 2.5 times the volume of the first volume.

27. The method of any one of claims 19-26, wherein the cell culture comprises 4 million cells / mL or less, or 3 million cells / mL or less, 2 million cells / mL or less, or 1 million cells / mL.

28. The method of any one of claims 19-27, wherein after transferring the cell culture to the second volume, the concentration of cells is reduced by about 2.5X, 3X, 4X or 4.5X.

29. The method of any one of claims 19-28, wherein the cell culture comprises producer cells.

30. The method of claim 29, wherein the producer cells are HeLa cells.

31. The method of any one of claims 19-30, further comprising contacting the cell culture with one or more rAAV-encoding recombinant nucleic acids.

32. The method of any one of claims 19-31, wherein the cell culture is comprised in a medium and the pH of the medium is at or between 7.0-7.4.

33. The method of claim 32, wherein the pH of the medium is at or between 7.1 and 7.4.

34. The method of any one of claims 19-33, wherein the cell culture is perfused for at least 48 hours after AAV production has begun.

35. The method of any one of claims 19-34, wherein the cell culture is perfused for at least 24 hours after AAV production has begun.

36. The method of any one of claims 19-35, wherein the glutamine level is maintained above 4 mM.

37. The method of any one of claims 19-36, wherein the rate of perfusion is between 0.5 and 2 vessel volume per day (VVD).

38. The method of claim 37, wherein the rate of perfusion is 0.6VVD.

39. The method of any one of claims 19-36, wherein the rate of perfusion is 2VVD or higher.

40. The method of any one of claims 19-39, wherein the second volume comprises a medium and the medium is enriched at least 2X relative to a standard medium.

41. The method of any one of claims 19-40, wherein the cell culture is infected with a helper virus.

42. The method of claim 41, wherein the helper virus is Ad5.

43. The method of claim 42, wherein the Ad5 virus is a wildtype Ad5 virus.

44. The method of claim 42, wherein the Ad5 virus is a mutated Ad5 virus.

45. The method of any one of claims 19-44, wherein the glutamine level of the cell culture in the second volume starts at or above 4mM and does not go below 2 mM.

46. The method of any one of claims 19-45, further comprising isolating rAAV particles from the cell culture.

47. A method comprising: a) culturing rAAV-producing cells in a small-scale system, including agitation, b) allowing the cells to settle, c) removing a supernatant from a vessel of the small-scale system, d) adding a new medium to the vessel of the small-scale system, and e) resuming agitation,wherein steps (b)-(e) are performed at least three times.

48. The method of claim 47, further comprising isolating rAAV from the cells.

49. The method of claim 48, further comprising comparing the rAAV produced by cells in the method of claim 47 to rAAV produced by batch-cultured cells.

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