Improved batch AAV production systems and methods

Abstract

The present application provides improved batch systems and methods using perfusion (e.g., enhanced perfusion) to increase productivity and / or cell density in viral particle production to achieve high yields of viral particles, e.g., AAV.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,298, filed March 7, 2022, U.S. Provisional Patent Application No. 63 / 398,355, filed August 16, 2022, and U.S. Provisional Patent Application No. 63 / 481,539, filed January 25, 2023, the disclosures of each of which are incorporated by reference in their entirety herein for all purposes.

[0002] The present disclosure relates to improved batch (e.g., enhanced perfusion) systems and methods for producing viral vectors, such as adeno-associated viral (AAV) vectors, that can increase the productivity of clinical and commercial grade AAV production. [Background technology]

[0003] Adeno-associated virus (AAV) is a representative platform for gene delivery for the treatment of numerous human diseases, but the high dosing requirements for AAV-based therapies and low batch yields in AAV production result in significantly high production costs and limited supply to meet patient demand. Despite advances in AAV manufacturing, including the development of mammalian producer cell lines that can be used for suspension culture, there remains a critical need to improve existing AAV manufacturing technologies to achieve a robust, high-yielding, scalable, and cost-effective process to meet patient supply needs and to reduce the overall cost of treatment using AAV-based therapies. Increasing cell density only in batch mode is not sufficient to increase the volumetric yield of AAV, and in fact can result in a greater than 100-fold decrease in yield when using, for example, a four-fold increase in seeding density for AAV production. Thus, new systems and methods are needed to enable high-yield AAV production to meet clinical and therapeutic needs as well as reduce the costs of AAV therapeutic development and clinical and commercial manufacturing.

[0004] The present disclosure addresses these needs by providing a process and system for cost-effective, scalable AAV production that is compatible with mammalian producer cell lines in suspension culture, reducing the cost of goods associated with traditional AAV manufacturing platforms. Summary of the Invention

[0005] The present disclosure provides improved batch (e.g., enhanced perfusion) systems and methods for AAV production using perfusion in AAV-producing cell cultures (e.g., suspension cultures of AAV-producing cells).

[0006] The present disclosure provides improved batch systems and methods using perfusion for AAV production. In some aspects, the present disclosure provides systems and methods for producing recombinant adeno-associated virus (rAAV), comprising: (a) culturing AAV-producing host cells to a target cell density in a propagation vessel using perfusion to replenish the culture with fresh nutrients and / or remove waste products, the AAV-producing host cells comprising genetic material encoding one or more AAV components; (b) initiating expression of one or more helper virus functions in the AAV-producing host cells of step (a) to initiate rAAV production; and (c) culturing the AAV-producing host cells of step (b) in a production vessel using perfusion to replenish the culture with fresh nutrients and / or remove waste products, thereby producing rAAV.

[0007] In some embodiments of the systems and methods described herein, the AAV-producing host cell is an insect cell or a mammalian cell.In some embodiments, the mammalian cell is a HeLa cell, a Cos-7 cell, a HEK293 cell, an A549 cell, a BHK cell, a Vero cell, a RD cell, or an ARPE-19 cell.

[0008] In some embodiments of the systems and methods described herein, the AAV-producing host cell comprises an AAV-producing cell line (PCL) (e.g., a mammalian or insect PCL) that comprises genetic material encoding one or more AAV components stably integrated into the AAV-producing host cell genome. In some embodiments, the AAV PCL may comprise one or more genetic modifications that reduce the expression and / or activity of one or more genes and / or proteins to reduce lactate and / or ammonia production or accumulation.

[0009] In some embodiments of the systems and methods described herein, the target cell density in step (a) is at least about 1E06 viable cells / mL (vc / mL). For example, the target cell density in step (a) may be between 1E06vc / mL and 5E07vc / mL. In some embodiments, the target cell density in step (a) is about 1E06vc / mL, about 2E06vc / mL, about 3E06vc / mL, about 4E06vc / mL, about 5E06vc / mL, about 6E06vc / mL, about 7E06vc / mL, about 8E06vc / mL, about 9E06vc / mL, about 1E07vc / mL, about 1.1E07vc / mL, about 1.2E07vc / mL, about 1.3E07vc / mL, about 1.4E07vc / mL, about 1.5E07vc / mL, about 1.6E07vc / mL, about 1.7E07vc / mL, about 1.8E07vc / mL, about 1.9E07vc / mL, or about 2E07vc / mL. In some embodiments, the target cell density in step (a) is about 3.15E06vc / mL or about 5.0E06vc / mL.

[0010] In some embodiments of the systems and methods described herein, the production vessel has a volume between 1 L and 12000 L. For example, the production vessel can have a volume of 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 100 L, 150 L, 200 L, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 5500 L, 6000 L, or 12000 L.

[0011] In some embodiments of the systems and methods described herein, the growth vessel and the production vessel comprise a combined growth / production vessel.

[0012] In some embodiments of the systems and methods described herein, the one or more AAV components include a therapeutic payload or transgene, one inverted terminal repeat (ITR) or two ITRs, one or more AAV replication and / or packaging proteins encoded by the AAV rep gene, and / or one or more AAV structural capsid proteins encoded by the AAV cap gene.

[0013] In some embodiments of the systems and methods described herein, step (b) comprises infecting the cells of step (a) with a helper virus. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5.

[0014] In some embodiments of the systems and methods described herein, step (b) includes inducing expression of one or more helper virus functions encoded by the genetic material in the AAV-producing host cell.

[0015] In some embodiments of the systems and methods described herein, perfusion is performed at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber. For example, perfusion is performed at a flow rate of about 3 mL / min / fiber, about 4 mL / min / fiber, about 5 mL / min / fiber, about 6 mL / min / fiber, about 7 mL / min / fiber, about 8 mL / min / fiber, about 9 mL / min / fiber, about 10 mL / min / fiber, about 11 mL / min / fiber, about 12 mL / min / fiber, about 13 mL / min / fiber, about 14 mL / min / fiber, or about 15 mL / min / fiber.

[0016] In some embodiments of the systems and methods described herein, perfusion is performed at a medium exchange rate of about 0.5 vessel volumes per day (VVD) to about 10 VVD. For example, perfusion can be performed at a medium exchange rate of about 0.5 VVD, about 1 VVD, about 1.5 VVD, about 2 VVD, about 2.5 VVD, about 3 VVD, about 3.5 VVD, about 4 VVD, about 4.5 VVD, about 5 VVD, about 5.5 VVD, about 6 VVD, about 6.5 VVD, about 7 VVD, about 7.5 VVD, about 8 VVD, about 8.5 VVD, about 9 VVD, about 9.5 VVD, or about 10 VVD. In some embodiments of the systems and methods described herein, perfusion is performed at a medium exchange rate of less than 1 VVD. For example, perfusion can be performed at a medium exchange rate of about 0.1 VVD, about 0.15 VVD, about 0.2 VVD, about 0.25 VVD, about 0.3 VVD, about 0.35 VVD, about 0.4 VVD, about 0.45 VVD, about 0.5 VVD, about 0.55 VVD, about 0.6 VVD, about 0.65 VVD, about 0.7 VVD, about 0.75 VVD, about 0.8 VVD, about 0.85 VVD, about 0.9 VVD, or about 0.95 VVD. In some embodiments of the systems and methods described herein, perfusion is performed at a cell-specific perfusion rate (CSPR) of about 150 pL / cell / day to about 2000 pL / cell / day, e.g., about 150 pL / cell / day to about 750 pL / cell / day. In some embodiments, the perfusion is performed at a CSPR of greater than 750 pL / cell / day.

[0017] In some embodiments of the systems and methods described herein, perfusion can be performed using a variety of systems, including Xcellerex Automated Perfusion System (APS) (Cytiva), KrosFlo® KPS TFF systems (Repligen), XCell™ ATF systems in 2, 4, 6, 8, or 10 ATF unit formats (Repligen), GEA Kytero® Single Use Pharma Separator systems (GEA), Prostak™ Microfiltration Modules systems (Millipore), Alfa Laval CultureOne™ systems (Alfa Laval), CARR® Centritech Separation Systems CARR UniFuge® Pilot or Centritech CELL 8® systems (Pneumatic Scale Angelus), Ksep® 6000S System (Sartorius), microfluidic cell retention devices, SciLog® SciPure™ systems (Parker), acoustic sedimentation systems (acoustic Perfusion is performed using a perfusion system selected from among the following: Electrocardiogram settler systems, and EDO Electro systems (American Piezo).

[0018] In some embodiments of the systems and methods described herein, step (a) comprises culturing the AAV-producing host cells in a growth medium, or step (a) and step (c) comprise culturing the AAV-producing host cells in a growth medium, or step (a) comprises culturing the AAV-producing host cells in a production medium, or step (a) and step (c) comprise culturing the AAV-producing host cells in a production medium. In some embodiments of the systems and methods described herein, step (a) comprises culturing the AAV-producing host cells in a growth medium, and step (c) comprises culturing the AAV-producing host cells in a production medium. In some embodiments, the production medium is perfused after infection of the AAV-producing host cells with a helper virus. In some embodiments, perfusion of the production medium is initiated 1 hour to 6 hours (e.g., about 1 hour to 2 hours, about 2 hours to 3 hours, about 3 hours to 4 hours, about 4 hours to 5 hours, or about 5 hours to 6 hours) after infection of the cells with the helper virus.

[0019] In some embodiments of the systems and methods described herein, steps (a) and (c) comprise culturing the AAV-producing host cells in a combination of growth medium and production medium. For example, the combination of growth medium and production medium can be a 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20, or 90 / 10 combination of growth medium and production medium.

[0020] In some embodiments of the systems and methods described herein, step (a) comprises culturing the AAV-producing host cells in growth medium until about 24 hours prior to step (b), followed by transition to production medium. The growth medium may be perfused at a rate of about 0.5 VVD to about 1 VVD until at least about 24 hours (e.g., about 96 hours, about 72 hours, about 48 hours, or about 24 hours) prior to step (b). The production medium may be perfused at a rate of about 1.5 VVD to about 2.5 VVD beginning within about 24 hours prior to step (b) (e.g., no more than 24 hours prior to step (b), including up to about 1 hour prior to step (b) and 0 hours prior to step (b)).

[0021] In some embodiments of the systems and methods described herein, the culture of the AAV-producing host cells in the growth and / or production stage culture involves culturing for about 48 hours and / or about 2 to about 14 days. For example, the culture of the AAV-producing host cells in the production stage involves culturing for a period of about 48 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0022] Perfusion may be stopped after a production phase period of about 48 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0023] The system and method described herein can allow rAAV to be released from AAV-producing host cells into the supernatant of production medium.Such release can be continuous throughout the production phase.In some embodiments, rAAV is mostly released starting about 48 hours after the infection of cells with helper virus.That is, the majority of the rAAV produced can be released into the supernatant about 48 hours after infection.

[0024] In some embodiments, the rAAV begins to be largely released about 48 hours after perfusion of the production medium. That is, the majority of the rAAV produced can be released into the supernatant about 48 hours after perfusion in the production medium. In some embodiments, perfusion is stopped to retain the rAAV in the supernatant. In some embodiments, perfusion is stopped about 48 hours after infection.

[0025] Some embodiments of the systems and methods described herein further include recovery of rAAV (e.g., under batch mode conditions, such as in the absence of perfusion) and / or downstream processing of the rAAV. For example, the systems and methods may further include purifying, clarifying, and / or concentrating the rAAV produced according to the methods described herein. Downstream processing may include filtering cellular debris, colloids, or aggregates. In some embodiments, filtering removes particles of cellular debris, colloids, and / or aggregates greater than 1 μm in size. Some embodiments include purifying the rAAV using anion exchange (AEX) chromatography.

[0026] In another aspect, the disclosure provides a method of producing a recombinant adeno-associated virus (rAAV), comprising culturing AAV-producing host cells using a perfusion system, wherein the culture of AAV-producing host cells has a target cell density of between 1E06vc / mL and 5E07vc / mL.

[0027] In some embodiments, the AAV production host cell is an insect cell or a mammalian cell.For example, the mammalian cell can be a HeLa cell, a Cos-7 cell, a HEK293 cell, an A549 cell, a BHK cell, a Vero cell, a RD cell, or an ARPE-19 cell.

[0028] In some embodiments, the AAV-producing host cell comprises an AAV-producing cell line (PCL) that comprises genetic material encoding one or more AAV components stably integrated into the AAV-producing host cell genome. In some embodiments, the AAV PCL may comprise one or more genetic modifications that reduce the expression and / or activity of one or more genes and / or proteins to reduce lactate and / or ammonia production or accumulation.

[0029] In some embodiments, the target cell density is about 5E06vc / mL to about 5E07vc / mL. For example, the target cell density can be about 1E06vc / mL, about 2E06vc / mL, about 3E06vc / mL, about 4E06vc / mL, about 5E06vc / mL, about 6E06vc / mL, about 7E06vc / mL, about 8E06vc / mL, about 9E06vc / mL, about 1E07vc / mL, about 1.1E07vc / mL, about 1.2E07vc / mL, about 1.3E07vc / mL, about 1.4E07vc / mL, about 1.5E07vc / mL, about 1.6E07vc / mL, about 1.7E07vc / mL, about 1.8E07vc / mL, about 1.9E07vc / mL, or about 2E07vc / mL. In certain embodiments, the target cell density is about 3.15E06vc / mL or about 5.0E06vc / mL.

[0030] In some embodiments, the method is carried out in a production vessel having a volume between 1 L and 12000 L. For example, the production vessel can have a volume of 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 100 L, 150 L, 200 L, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 5500 L, 6000 L, or 12000 L.

[0031] In some embodiments, the method further comprises growing the AAV-producing host cells to a target cell density in a combined growth / production vessel using a perfusion system.

[0032] In some embodiments, the one or more AAV components include a therapeutic payload or transgene, an inverted terminal repeat (ITR) or two ITRs, one or more AAV replication and / or packaging proteins encoded by the AAV rep gene, and / or one or more AAV structural capsid proteins encoded by the AAV cap gene.

[0033] In some embodiments, the AAV-producing host cells are cultured to a target cell density under conditions suitable for the production of rAAV. Conditions suitable for AAV production may include infection with a helper virus to initiate AAV production, such as adenovirus (e.g., Ad5), triple transfection with AAV-producing plasmids, and / or induction of inducible producer cells with stable integration of components required for the production of AAV. In some embodiments, the method includes initiating rAAV production by initiating expression of one or more helper virus functions in the AAV-producing host cells. In some embodiments, initiating expression of one or more helper virus functions includes infecting the AAV-producing host cells with a helper virus. In some embodiments, initiating expression of one or more helper virus functions includes inducing expression of one or more helper virus functions encoded by genetic material in the AAV-producing host cells. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5.

[0034] In some embodiments, the perfusion system operates at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber. For example, the perfusion system may operate at a flow rate of about 3 mL / min / fiber, about 4 mL / min / fiber, about 5 mL / min / fiber, about 6 mL / min / fiber, about 7 mL / min / fiber, about 8 mL / min / fiber, about 9 mL / min / fiber, about 10 mL / min / fiber, about 11 mL / min / fiber, about 12 mL / min / fiber, about 13 mL / min / fiber, about 14 mL / min / fiber, or about 15 mL / min / fiber.

[0035] In some embodiments, the perfusion system operates at a medium exchange rate of about 0.5 vessel volumes per day (VVD) to about 10 VVD. For example, the perfusion system may operate at a medium exchange rate of about 0.5 VVD, about 1 VVD, about 1.5 VVD, about 2 VVD, about 2.5 VVD, about 3 VVD, about 3.5 VVD, about 4 VVD, about 4.5 VVD, about 5 VVD, about 5.5 VVD, about 6 VVD, about 6.5 VVD, about 7 VVD, about 7.5 VVD, about 8 VVD, about 8.5 VVD, about 9 VVD, about 9.5 VVD, or about 10 VVD. In some embodiments of the systems and methods described herein, perfusion is performed at a medium exchange rate of less than 1 VVD. For example, perfusion may be performed at a medium exchange rate of about 0.1 VVD, about 0.15 VVD, about 0.2 VVD, about 0.25 VVD, about 0.3 VVD, about 0.35 VVD, about 0.4 VVD, about 0.45 VVD, about 0.5 VVD, about 0.55 VVD, about 0.6 VVD, about 0.65 VVD, about 0.7 VVD, about 0.75 VVD, about 0.8 VVD, about 0.85 VVD, about 0.9 VVD, or about 0.95 VVD.

[0036] In some embodiments of the systems and methods described herein, perfusion is performed at a cell-specific perfusion rate (CSPR) of about 150 pL / cell / day to about 2000 pL / cell / day, e.g., about 150 pL / cell / day to about 750 pL / cell / day. In some embodiments, perfusion is performed at a CSPR of greater than 750 pL / cell / day.

[0037] In some embodiments, the perfusion system is a system such as Xcellerex Automated Perfusion System (APS) (Cytiva), KrosFlo® KPS TFF systems (Repligen), XCell™ ATF systems in 2, 4, 6, 8, or 10 ATF unit formats (Repligen), GEA Kytero® Single Use Pharma Separator systems (GEA), Prostak™ Microfiltration Modules systems (Millipore), Alfa Laval CultureOne™ systems (Alfa Laval), CARR® Centritech Separation Systems CARR UniFuge® Pilot or Centritech CELL 8® systems (Pneumatic Scale Angelus), Ksep® 6000S System (Sartorius), microfluidic cell retention devices, SciLog® SciPure™ systems (Parker), acoustic settler systems, and EDO Electro systems (American Piezo).

[0038] In some embodiments, the AAV-producing host cells are cultured in growth medium and then production medium. The transition to production medium can occur within about 0 hours to about 48 hours prior to infection of the AAV-producing host cells with a helper virus, or about 24 hours prior to infection. The growth medium can be perfused at a rate of about 0.5 VVD to about 1 VVD about 24 hours prior to infection of the AAV-producing host cells with the helper virus (e.g., up to about 1 hour prior, and up to 24 hours prior to infection, including 0 hours prior). The production medium can be perfused at a rate of about 1.5 VVD to about 2.5 VVD starting about 24 hours prior to infection of the AAV-producing host cells with the helper virus. The AAV-producing host cells can be infected with a helper virus (e.g., Ad5) after culture in the growth medium and prior to culture in the production medium. In some embodiments, the production medium is perfused 1 hour to 6 hours (e.g., about 1 hour to 2 hours, about 2 hours to 3 hours, about 3 hours to 4 hours, about 4 hours to 5 hours, about 5 hours to 6 hours) after infection of the cells with the helper virus.

[0039] In some embodiments, the AAV host producer cells are cultured to a target cell density in a combination of growth medium and production medium. For example, the combination of growth medium and production medium can be a 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20, or 90 / 10 combination of growth medium and production medium.

[0040] In some embodiments, the method includes culturing the AAV-producing host cells at the target cell density for about 48 hours and / or for about 2 to about 14 days. For example, the method can include culturing the AAV-producing host cells at the target cell density for a period of about 48 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0041] The system and method described herein can allow rAAV to be released from AAV-producing host cells into the supernatant of production medium.Such release can be continuous throughout the production phase.In some embodiments, rAAV starts to be mostly released about 48 hours after the infection of cells with helper virus.That is, the majority of the rAAV produced can be released into the supernatant about 48 hours after the infection of cells with helper virus.

[0042] In some embodiments, the rAAV begins to be largely released about 48 hours after perfusion of the production medium. That is, the majority of the rAAV produced can be released into the supernatant about 48 hours after perfusion in the production medium. In some embodiments, perfusion is stopped to retain the rAAV in the supernatant. In some embodiments, perfusion is stopped at about 48 hours. In some embodiments, perfusion is stopped about 48 hours after infection.

[0043] In some embodiments, the method further comprises recovering the rAAV (e.g., under batch mode conditions, such as in the absence of perfusion) and / or downstream processing of the rAAV. For example, downstream processing may comprise purifying, clarifying, and / or concentrating the rAAV. Downstream processing may comprise filtering cellular debris, colloids, or aggregates. In some embodiments, filtration removes particles of cellular debris, colloids, and / or aggregates greater than 1 μm in size. Some embodiments comprise purifying the rAAV using anion exchange (AEX) chromatography.

[0044] These and other aspects and features of the present disclosure are set forth in the following sections of the application. [Brief description of the drawings]

[0045] The foregoing and other objects, features, and advantages of the disclosed system and method will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings, in which like referenced elements identify common features in corresponding drawings.

[0046] [Figure 1] 1 is a schematic example of a batch mode process for AAV production (i.e., the "batch mode control process"). [Diagram 2] FIG. 1 is a flow diagram of an example process for AAV production according to the improved batch systems and methods described herein using perfusion. [Diagram 3] 1 is a schematic example of a process for AAV production according to the improved batch system and method described herein using perfusion. [Figure 4] 1 is a bar graph depicting volumetric rAAV yield in a batch mode control process. The bar graph shows a decrease in volumetric rAAV yield with increasing seeding density in a batch mode control process. [Diagram 5] 1 is a bar graph showing cell specific productivity in a batch mode control process. The bar graph shows the decrease in cell specific productivity with increasing seeding density in a batch mode control process. [Figure 6] 1 depicts a bar graph showing the effect of pH on volumetric rAAV yield in a batch mode control process. The bar graph shows the effect of pH on volumetric rAAV yield at 1.3E06 (shown as "1.3" in the legend) and 3.15E06 (shown as "3.15" in the legend) seeding densities in a batch mode control process. [Figure 7] The effect of pH on cell specific productivity in the batch mode control process is shown in a bar graph. The bar graph shows the effect of pH on cell specific productivity at 1.3E06 (shown as "1.3" in the legend) and 3.15E06 (shown as "3.15" in the legend) seeding densities in the batch mode control process. [Figure 8]1 is a graph showing the effect of pH on lactate accumulation at seeding densities of 1.3E06 and 3.15E06 in a batch mode control process. (Abbreviations used in legend: 1.3E06 seeding density and pH 7.4 is represented as "1.3, 7.4", 1.3E06 seeding density and pH 7.6 is represented as "1.3, 7.6", 1.3E06 seeding density and pH 7.8 is represented as "1.3, 7.8", 3.15E06 seeding density and pH 7.4 is represented as "3.15, 7.4", 3.15E06 seeding density and pH 7.6 is represented as "3.15, 7.6", and 3.15E06 seeding density and pH 7.8 is represented as "1.3, 7.8"). [Figure 9A] 1 is a bar graph showing the percent virus release (AAV and helper virus) over time in the batch mode control process. [Figure 9B] 1 is a bar graph showing rAAV release into the supernatant after infection. [Figure 10] 1 is a graph showing the percentage of virus release (AAV and helper virus) versus the percentage of dead cells in the culture in the batch mode control process. [Figure 11A] 1 is a bar graph depicting the volumetric yield of rAAV in the improved batch AAV manufacturing process and the batch mode control process. The graph shows the increase in volumetric yield of rAAV with increasing VVD in the improved batch AAV manufacturing process compared to the batch mode control process. [Figure 11B] rAAV volumetric yields are depicted in a bar graph comparing low and high perfusion rate processes. The graph shows a further increase in rAAV volumetric yield with further increases in VVD and cell density. [Figure 12] 1 is a bar graph showing cell-specific productivity in an improved batch AAV manufacturing process. The graph shows the increase in cell-specific productivity with increasing VVD in the improved batch AAV manufacturing process. [Figure 13]1 is a bar graph showing waste (lactate and ammonium, top right and bottom right panels) and nutrient (glucose and glutamine, top left and bottom left panels) levels in the improved batch process for medium exchange rates of 0, 1, and 2.5 VVD. [Figure 14] 1 is a bar graph showing volumetric rAAV yields using the improved batch process at 2L and scaled culture volumes compared to the 2L batch mode control process. [Figure 15] 1 is a bar graph showing cell specific productivity using the improved batch process at 2L and scaled culture volumes compared to the 2L batch mode control process. [Figure 16] 1 is a graph showing viable cell density (VCD) using the improved batch process at 2L and scaled culture volumes compared to the 2L batch mode control process. [Figure 17] 1 is a graph showing cell viability using the improved batch process at 2L and scaled culture volumes compared to a 2L batch mode control process. [Figure 18] Graphs showing nutrient levels (glucose (left graph) and glutamine (right graph)) in the 2L improved batch process and scaled culture volume compared to the 2L batch mode control process. [Figure 19] Graphs showing waste levels (lactate (left graph) and ammonium (right graph)) in the improved batch process at 2L and scaled culture volumes compared to the 2L batch mode control process. [Figure 20A] 1 is a graph showing the percentage of intact AAV capsids as assessed by analytical ultracentrifugation (AUC) analysis in the improved batch process at 2L and scaled culture volumes compared to the 2L batch mode control process. [Figure 20B] 1 is a graph showing the ratio of intermediate to complete AAV particles under various vessel volume per day (VVD) conditions. [Figure 21A] The use of the improved batch process with different media compositions, capsid serotypes, and vector genomes / genes of interest is shown. Figure 21A is a graph showing normalized volumetric rAAV yield (left graph) and specific productivity (right graph) using a batch mode control process ("Batch") versus an improved batch process ("Perf") under various program, media, and capsid conditions (A and B). The legends in both graphs indicate rAAV titers at 1E6GC / mL (y-axis of left graph starts from 0.00GC / mL to 3.50E6GC / mL, y-axis of right graph starts from 0.00GC / mL to 1.40E06GC / mL). [Figure 21B] The use of the improved batch process with different media compositions, capsid serotypes, and vector genomes / genes of interest is shown. Figure 21B is a graph showing the recovery yield between batch mode and the improved batch process (abbreviated as "HDI" in the figure) using additional program, media, and capsid conditions ("Process C"). [Figure 22] 1 is a graph showing normalized rAAV recovery yields, which show that rAAV recovery yields are reduced by the presence of lactate and / or ammonia during AAV production. [Figure 23] Graph showing R2 analysis of AAV cell specific yield (GC / cell) at cell specific perfusion rates (CSPR(pL / cell / day)) up to about 750 pL / cell / day. [Figure 24A] FIG. 24A shows that the improved batch process described herein (e.g., enhanced perfusion) reduces the cost of goods (COG) projections for rAAV production. FIG. 24A is a graph showing the approximate projected COG (cost of goods sold per patient dose) required per AAV yield in genome copies per milliliter (GC / mL). Diamonds or circles represent the improved batch systems described herein (e.g., enhanced perfusion) and squares represent batch operating conditions. [Figure 24B]Figure 24B shows that the improved batch process described herein (e.g., enhanced perfusion) reduces the cost of goods (COG) projections for rAAV production. Figure 24B is a graph showing the approximate number of production batches required per AAV yield (GC / mL). Diamonds or circles represent the improved batch systems described herein (e.g., enhanced perfusion) and squares represent batch operating conditions. [Figure 24C] Figure 24C shows that the improved batch process (e.g., enhanced perfusion) described herein reduces the projected cost of goods (COG) of rAAV production. Figure 24C is a graph showing the approximate projected COG required per number of production batches. Diamonds or circles represent the improved batch systems (e.g., enhanced perfusion) described herein, and squares represent batch operating conditions. [Diagram 25] 1 is a graph showing a comparison of rAAV titers using formulated media versus scheduled media transfer. The solid line in the graph represents rAAV released into the supernatant, and the dashed line in the graph represents intracellular rAAV. [Figure 26] 1 is a graph showing a comparison of recovery titers under different planned medium transfer conditions (VVD conditions 1 to 4). The graph shows that the comparison of recovery titers was maintained while reducing the medium consumption rate under different planned medium transfer conditions. [Figure 27] 1 is a graph depicting recovered rAAV yields for batch and HDI processes. The graph compares recovered rAAV yields in 2L in batch mode with 2L (denoted in the figure as MB-2L), 50L (denoted in the figure as MB-50L), and 250L (denoted in the figure as MB-250L) using the improved batch process (abbreviated as "HDI" in the figure title). [Figure 28] 1 is a graph depicting cell-specific rAAV productivity for batch and HDI processes. The graph compares cell-specific rAAV productivity in 2L in batch mode with 2L (denoted in the figure as MB-2L), 50L (denoted in the figure as MB-50L), and 250L (denoted in the figure as MB-250L) using the improved batch process (abbreviated as "HDI" in the figure caption). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Despite recent progress in improving the productivity and scalability of AAV manufacturing processes, there remains a significant need to improve existing AAV manufacturing platforms to achieve robust, high-yield, scalable, and cost-effective processes. AAV production in suspension cell culture faces various challenges to efficient clinical and commercial scale implementation. For example, maintaining high cell-specific productivity of viral vector products, especially in high cell density batch culture, has proven to be ambiguous. The present system and method address these challenges by using perfusion (e.g., enhanced perfusion) in an improved batch process to improve AAV production efficiency and reduce overall cost of goods.

[0048] The present disclosure is directed to a perfusion-based AAV production system and method that allows for high volumetric productivity while reducing the high cost of goods typically associated with AAV platforms that operate in batch mode, and the disclosed systems and methods are referred to throughout the description as "improved batch" systems and methods. In certain embodiments, the improved batch system is an enhanced perfusion system and is used for AAV production. Improvements to existing platform processes are described herein to demonstrate process intensification and achieve a several-fold increase in overall volumetric AAV yield. Without wishing to be bound by theory, the improved batch (e.g., enhanced perfusion) process described herein ensures sufficient nutrient availability and clearance of cell culture waste by-products to maintain high cell density, allowing for a several-fold increase in AAV productivity, e.g., to 3E11 viral genome copies per milliliter (GC / mL) or more (3E14 GC / L or more). These improvements, in addition to downstream process optimization, can deliver a high purity AAV product suitable for human therapeutic use.

[0049] Those skilled in the art of AAV production will understand that various aspects of the systems and methods described herein may be modified without departing from the inventive subject matter of the present disclosure. Aspects that may be modified or varied according to the knowledge possessed by those skilled in the art without departing from the presently disclosed invention include, but are not limited to, the following: AAV production scale (e.g., AAV production volume in liters), perfusion rate, AAV production cell density, growth medium, production medium, production feed, medium ratio, cell type, infection and / or transfection parameters or reagents, operational parameters such as pH, temperature, dissolved oxygen (DO), timing of process steps, duration of process steps, and medium exchange and / or perfusion means (e.g., ATF, Wave Lilypad perfusion, centrifugation, TFF, KTF, Gea Kytero, Krosflow KPS system, etc.).

[0050] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.Unless otherwise specified, technical terms are used according to conventional usage by those skilled in the art.Definitions of common molecular biology terms can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0051] It should be noted that unless otherwise clear from the context, the term "a" or "an" entity refers to one or more of that entity, e.g., "an amino acid" is understood to represent one or more amino acids. Thus, "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0052] Furthermore, the term "and / or" should be interpreted as a specific disclosure of each of two or more specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0053] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms used throughout the disclosure are provided below.

[0054] AAV titer, etc.: The term "AAV titer" as used herein refers to the number of viral genome copies per milliliter (GC / mL) or per cell (GC / cell). In certain embodiments, GC / mL can be determined by standard methods, including but not limited to, direct quantitative PCR (qPCR) of purified vector particles, fluorescence activated cell sorting (FACS), silver staining, etc. In certain embodiments, GC / cell can be determined by standard methods, including but not limited to, for example, dot blot, qPCR or droplet digital PCR (ddPCR), spectroscopy, or fluorescence measurement.

[0055] About: As used herein, the term "about" or "approximately" when applied to one or more values ​​of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in either direction (greater or smaller), unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0056] Helper virus functions, etc.: The term "helper virus functions" as used herein encompasses genes, components, or functions typically provided in trans that allow for the production of AAV in a host cell. Helper virus functions can be provided by infection with a helper virus, such as, for example, a herpes virus or an adenovirus, e.g., an adenovirus serotype 5 (Ad5) virus (e.g., a wild-type or recombinantly engineered Ad5 helper virus). Alternatively, helper virus functions can be provided in cis, e.g., by integration of genetic material encoding the helper virus functions in the host cell. Helper virus genes / functions include, but are not limited to, E1a, E1b, E2a, E40rf6, and VA RNA genes / functions.

[0057] Adeno-associated virus (AAV): A small, replication-deficient, non-enveloped virus that infects humans and several other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. There are at least 13 recognized serotypes of AAV (AAV1-AAV13).

[0058] Administer / Administer: To provide or expose a subject or a target tissue of a subject to an agent, such as a therapeutic agent (e.g., a recombinant AAV), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0059] Amino Acid: The term "amino acid" as used herein refers to any of the 20 standard amino acids, namely, glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid, single stereoisomers thereof, and racemic mixtures thereof. The term "amino acid" can also refer to known non-standard amino acids, such as any dipeptide, such as alanine-glutamine dipeptide, 4-hydroxyproline, eV V A-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, g-carboxyglutamate, eV-acetyllysine, co-V-methylarginine, A-acetylserine, WA'-trimethylalanine, A-formylmethionine, g-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, b-cyanoalanine, homocysteine, azaserine, and S-adenosylmethionine. In some embodiments, the amino acid is glutamate, glutamine, lysine, tyrosine, or valine. In some embodiments, the amino acid is glutamate or glutamine.

[0060] Batch culture: As used herein, the term "batch culture" refers to a method of culturing cells in which all components that will ultimately be used in culturing the cells are provided at the beginning of the culture process, including the medium as well as the cells themselves. Batch cultures are typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0061] Bioreactor: The term "bioreactor" or "culture vessel" as used herein refers to any vessel used for the growth of cell cultures (e.g., mammalian cell cultures). Bioreactors can be of any size, so long as they are useful for culturing cells (e.g., mammalian cells).

[0062] Cell Culture: As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of eukaryotic cells, either surface-attached or in suspension, that are maintained or grown in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, these terms as used herein may refer to a combination that includes a eukaryotic cell population and the medium in which the population is suspended, maintained, and / or grown.

[0063] Cell density: As used herein, the term "cell density" refers to the number of cells present in a given volume of medium. As used herein, the term "target cell density" refers to the number of cells present in a volume of medium achieved during the growth stage of the culture and used in the production stage for the production of rAAV. As used herein, the term "AAV-producing host cells" refers to cells used for the production of rAAV.

[0064] Cell viability: As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. As used herein, the term also refers to the proportion of cells that are viable at a particular time point in relation to the total number of live and dead cells in culture at that time point.

[0065] Coding sequence: "Coding sequence" refers to a nucleotide sequence that encodes a polypeptide in vitro or in vivo when operably linked to appropriate regulatory sequences. A coding sequence may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) and 3' untranslated (3'UTR) sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0066] Codon optimization: A "codon optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been altered to be optimal for expression in a particular system, such as a particular species or group of species. For example, a nucleic acid sequence can be optimized for expression in a cell, such as a mammalian cell, or a particular species, such as a human cell. Codon optimization does not change the amino acid sequence of the encoded protein.

[0067] Encode: As used herein, the term "encode" and similar terms including "encoding" and "coding" refer to the ability of a nucleic acid molecule to carry genetic information, i.e., protein coding information and / or biological function. Thus, a nucleic acid encoding a therapeutic payload may contain a genetic sequence that can be translated into a peptide therapeutic and / or may carry information to regulate the expression (transcription or translation) of a peptide therapeutic and / or may result in a nucleic acid that has a biological function apart from encoding peptides such as non-coding RNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), aptamers, long non-coding RNA (IncRNA), and any other type of non-protein-coding nucleic acid with biological activity.

[0068] Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

[0069] Fed-batch culture: The term "fed-batch culture" as used herein refers to a method of culturing cells in which additional components are provided to the culture some time after the start of the culturing process. Fed-batch cultures can be initiated with a basal medium. A culture medium in which additional components are provided to the culture at some time after the start of the culturing process is a feed medium. The components provided typically include nutrient supplements for the cells that are depleted during the culturing process. Fed-batch cultures are typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0070] Growth phase: The "growth phase" or "growth stage" of a cell culture refers to the period of exponential cell growth during which the cells are generally rapidly dividing (log phase). During this phase, the cells are cultured for a period of time, e.g., up to 14 days, and under conditions such that cell growth is maximized. The determination of the growth cycle for a host cell can be determined for the particular host cell envisioned without undue experimentation. "For a period of time and under conditions such that cell growth is maximized" and the like refer to culture conditions determined to be optimal for cell growth and cell division for a particular cell line. In some embodiments, during the growth phase, the cells are cultured in a nutrient medium containing the necessary additives, in a humidified and controlled atmosphere, generally at about 25°C to 40°C, such that optimal growth is achieved for a particular cell line. In certain embodiments, the cells are maintained in the growth phase for a period of about 1 day to 7 days, e.g., 2 days to 6 days, e.g., 6 days. The length of the growth phase for a particular cell can be determined without undue experimentation. For example, the length of the growth phase is a time sufficient to allow a particular cell to regenerate to a maximum possible viable cell density ("vcd") in the range of about 20%-80% of the vcd if the culture is maintained under growth conditions. In some embodiments, "maximum growth rate" refers to the growth rate of a particular cell line / clone measured during its exponential growth phase while the cells are in fresh culture medium (e.g., measured at a point in the culture where nutrients are sufficient and there is no significant inhibition of growth from any component of the culture).

[0071] intron: a stretch of DNA within a gene that does not contain protein-coding information. The RNA transcript is processed by the cellular machinery to remove the introns and produce the mature, processed messenger RNA.

[0072] Inverted terminal repeat (ITR): Symmetrical nucleic acid sequences in the genome of adeno-associated virus that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as replication origins for viral DNA synthesis and are essential cis elements to generate AAV integrating vectors.

[0073] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from other biological components, such as other chromosomal and extrachromosomal DNA and RNA, proteins and cells, in the cells or tissues of an organism in which the component naturally occurs, or in the organism itself. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0074] Culture medium: The terms "media", "medium", "cell culture medium", "culture medium", "tissue culture medium", "tissue culture media", and "growth medium" as used herein refer to a solution containing nutrients that nourish growing cultured eukaryotic cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. The solutions may also contain components that enhance growth and / or survival beyond a minimal rate, including hormones and growth factors. The solutions are formulated to an optimal pH and salt concentration for cell survival and growth. Media may also be "defined media" or "chemically defined media" - serum-free media that do not contain animal-derived components, hydrolysates, or components of unknown composition, and contain only components of known chemical structure or characterization. Chemically defined media are free of animal-derived components, and all components have a known chemical structure. One of skill in the art will appreciate that chemically defined media may contain recombinant glycoproteins or proteins, including, but not limited to, hormones, cytokines, interleukins and other signaling molecules.

[0075] N-stage, etc.: As used herein, the terms "N-stage," "N culture vessel," "second culture vessel," "production vessel," "production culture vessel," "N vessel," "N bioreactor," "second bioreactor," or "production bioreactor" refer to a bioreactor following the N-1 bioreactor and is used for the production of AAV. Alternatively, this may be referred to as a "production stage" culture throughout this disclosure. As used herein, AAV-producing cells in an "N-stage" culture are actively producing AAV product.

[0076] N-stage, etc.: The terms "N-" and "N-stage" as used herein refer to any AAV producing cell culture stage prior to the initiation of N-stage production.

[0077] N-1 stage, etc.: The terms "N-1 stage", "first culture vessel", "N-1 culture vessel", "N-1 seed train culture vessel", "N-1 vessel", "first bioreactor", "N-1 bioreactor", "expansion vessel", or "N-1 seed train bioreactor" as used herein refer to a culture vessel immediately preceding the N stage culture vessel (production culture vessel) and used to grow cell cultures to high viable cell densities for inoculation into the subsequent N (production stage) culture vessel. The cell culture to be grown in the N-1 culture vessel can be obtained after culturing cells in several vessels before the N-1 culture vessel, such as N-4, N-3, and N-2 vessels. Alternatively, the N-1 stage can be referred to as a "expansion stage" culture throughout the description herein. As used herein, the AAV producing cells in the "N-1 stage" culture can be in the expansion phase.

[0078] Nutrient medium, feed medium, etc.: As used herein, the terms "nutrient medium", "feed medium", "feed", "total feed", and "total nutrient medium" can be used interchangeably and include "complete" media used to grow, reproduce, and add biomass to a cell line. Nutrient media are distinguished from substances or simple media that are not sufficient in themselves to grow and reproduce a cell line. Thus, for example, glucose or simple sugars are not in themselves nutrient media, since in the absence of other necessary nutrients, they are not sufficient to grow and reproduce a cell line. Those skilled in the art can recognize that a cell may continue to grow, survive, and reproduce in the presence of an incomplete medium, but may become unstable and / or greatly reduce its growth rate. Thus, in some embodiments, the term "nutrient medium" includes medium sufficient to grow, propagate, and add biomass to a cell line without loss of stability, growth rate, or any other indicator of cell health for a period of at least 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, or 18 weeks. In some embodiments, the term "nutrient medium" includes a medium that may be deficient in one or more essential nutrients, but that is capable of continuing to grow, propagate, and add biomass to a cell line without loss of stability, growth rate, or any other indicator of cell health for a period of at least 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, or 18 weeks. In some embodiments, the nutrient medium is a cell culture medium. Optimal cell culture medium composition varies depending on the type of cell culture being propagated. In some embodiments, the nutrient medium is a commercially available medium.In some embodiments, the nutrient medium contains, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., compositions containing albumin, growth factors, and growth inhibitors, such as fetal bovine serum, newborn calf serum, and horse serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins derived from plant or animal sources), and combinations thereof. Examples of nutrient media include, but are not limited to, basal media (e.g., Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Glasgow Minimum Essential Medium GMEM), complex media RPMI medium (e.g., RPMI 1640), Iscove's DMEM, Leibovitz's L-15, Leibovitz's L-15, TC 100), serum-free media (e.g., Chinese Hamster Ovary (CHO) medium, Ham's F10 and derivatives, Ham's F12, DMEM / F12). Common buffers found in nutrient media include phosphate buffered saline (PBS), Hank's balanced salt solution (BSS), Earle's salts (Earle's salt solution), Dulbecco's phosphate buffered saline (DPBS), Hank's BSS (HBSS), and Earle's BSS (EBSS). Media for culturing mammalian cells are well known in the art and are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO), HyClone (Logan, UT), Invitrogen Corporation (Carlsbad, CA), Cambrex Corporation (East Rutherford, NJ), Irvine Scientific (Santa Ana, CA), Gibco / ThermoFisher Scientific, etc.Other components found in the nutrient medium may include ascorbate, citrate, cysteine / cystine, glutamine, folic acid, glutathione, linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, pyridoxal / pyridoxine, riboflavin, selenium, thiamine, and transferrin. Those skilled in the art will recognize that there are modifications to the nutrient medium that fall within the scope of the disclosed systems and methods.

[0079] Perfusion culture: As used herein, the terms "perfusion culture," "perfusion system," "perfusion," and the like refer to a method of culturing cells in which additional components are continuously or semi-continuously provided to the culture after the start of the culturing process. The components provided typically include nutrient supplements for the cells that are depleted during the culturing process. A portion of the cells and / or components in the medium are typically harvested and optionally purified on a continuous or semi-continuous basis.

[0080] Pharmaceutically acceptable carrier: The pharma- ceutically acceptable carrier (vehicle) useful in this disclosure may be one that is conventionally used in the art. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., l5th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules, or agents. In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutical and physiologically acceptable liquids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a vehicle. For solid compositions, such as in the form of powders, pills, tablets, or capsules, conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0081] Polypeptide, Protein: The term "polypeptide" or "protein" as used herein refers to a chain of amino acids linked together via peptide bonds. Although the term is used to refer to an amino acid chain of any length, one of skill in the art will understand that the term is not limited to long chains, but can refer to a minimal chain that includes two amino acids linked together via peptide bonds. When a single polypeptide is a separate functional unit and requires a permanent physical association with other polypeptides to form a separate functional unit, the terms "polypeptide" and "protein" as used herein are used interchangeably. When a separate functional unit is composed of multiple polypeptides that physically associate with each other, the term "protein" as used herein refers to multiple polypeptides that are physically associated and function together as a separate unit. The term "protein" as used herein is intended to encompass a singular "protein" as well as multiple "proteins." Thus, as used herein, terms including but not limited to "peptide," "polypeptide," "amino acid chain," or any other term used to refer to a chain or chains of amino acids are included in the definition of "protein," and the term "protein" may be used in place of or interchangeably with any of these terms. The term further includes proteins that have been post-translationally modified, e.g., glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with unnatural amino acids. Additionally, proteins also include polypeptides that form multimers, e.g., dimers, trimers, etc. The term protein also includes fusion proteins, e.g., proteins produced via a gene fusion process in which a protein (or a fragment of a protein) is attached to an antibody (or a fragment of an antibody).

[0082] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter comprises a necessary nucleic acid sequence near the start site of transcription. Many promoter sequences are known to those skilled in the art, and even the combination of different promoter sequences in an artificial nucleic acid molecule is possible. As used herein, a gene-specific endogenous promoter refers to the native promoter element that regulates the expression of an endogenous gene of interest. In an exemplary embodiment, the therapeutic payload is human, and the promoter is a gene-specific endogenous promoter that regulates the expression of a human gene in its natural context.

[0083] Purified: The term "purified" does not require absolute purity, but is rather intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is wholly or partially isolated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparations and subjected to fractionation (e.g., filtration) to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0084] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separated segments of one or more sequences. This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, for example by genetic engineering techniques. Similarly, a recombinant virus is a virus that contains a sequence (such as a genomic sequence) that does not occur in nature or that is created by the artificial combination of at least two sequences of different origins. The term "recombinant" also includes nucleic acids, proteins, and viruses that are changed only by the addition, substitution, or deletion of a portion of a naturally occurring nucleic acid molecule, protein, or virus. As used herein, "recombinant AAV" or "rAAV" can refer to an AAV particle in which a recombinant nucleic acid molecule, such as a recombinant nucleic acid molecule encoding a therapeutic payload, is packaged.

[0085] Seeding: The term "seeding" as used herein refers to the process of providing a cell culture to a bioreactor or another vessel (e.g., a growth vessel or a production vessel). In one embodiment, the cells have been pre-propagated in another bioreactor or vessel (e.g., a growth vessel or a production vessel). In another embodiment, the cells are frozen and thawed immediately prior to providing them to the bioreactor or vessel (e.g., a growth vessel or a production vessel). The term refers to any number of cells, including a single cell.

[0086] Serotype: a group of closely related microorganisms (such as viruses) that are distinguished by a characteristic set of antigens.

[0087] Stuffer sequence: refers to a sequence of nucleotides contained within a larger nucleic acid molecule (such as a vector) that is typically used to create spacing between two nucleic acid features (such as between a promoter and a coding sequence) or to extend a nucleic acid molecule so that it is of a desired length. Stuffer sequences do not contain protein coding information, may be of unknown / synthetic origin, and / or may be unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

[0088] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human presenting to a health care provider for diagnosis or treatment of a disease. Unless the context requires otherwise, the term "subject" is used interchangeably with "individual" or "patient" throughout this specification. A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0089] Untranslated region (UTR): A typical mRNA contains a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR) upstream and downstream of the coding region, respectively (see Mignone, F., Gissi, C., Liuni, S. et al. Untranslated regions of mRNAs. Genome Biol 3, reviews0004.1 (2002)).

[0090] Vector: A vector is a nucleic acid molecule that allows the insertion of foreign nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of the inserted gene. In some embodiments herein, the vector is an AAV vector. Depending on the context, a broader definition of the term "vector" may apply, which means a carrier or transmitter, i.e., a biological carrier, usually of a biological agent.

[0091] It should be further understood that all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acid or polypeptide are approximate and are provided for illustration.Methods and materials similar or equivalent to those described herein can be used to carry out or test this disclosure, and suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including explanations of terms, will take precedence.In addition, materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0092] II. Overview The present disclosure is directed to a perfusion-based AAV production system and method that allows high volumetric productivity while reducing the high cost of goods typically associated with comparable AAV platforms (e.g., non-enhanced AAV platforms). The systems and methods of the present disclosure may be used in the manufacture of any viral particles, e.g., adeno-associated viral particles, for example, for therapeutic applications. The systems and methods described herein can be incorporated into procedures that implement a producer cell line (e.g., a mammalian producer cell line) for the production of viral particles, e.g., a producer cell line in suspension, or alternatively, a multiple, e.g., triple transfection approach to generate viral particle-producing cells. In some embodiments, the systems and methods disclosed herein incorporate a transfection master mix, e.g., according to that described in U.S. Patent Application Publication No. US20200124505A1.

[0093] Thus, systems and methods for producing viral particles (e.g., AAV) are provided herein. The improved batch (e.g., enhanced perfusion) systems and methods described herein alleviate the challenge in the bioreactor production field of maintaining high specific productivity in batch mode and other (e.g., non-enhanced) perfusion culture systems, which is accentuated as cell density increases, e.g., about 1E06 cells / mL or higher. The systems and methods described herein improve the efficiency of AAV production with lower cost of goods by achieving up to 5-fold or more increase in volumetric productivity compared to existing methods for AAV production.

[0094] The present disclosure provides various systems and methods used in AAV manufacturing. The present disclosure demonstrates the use of improved batch systems (e.g., enhanced perfusion systems) and methods, as well as methods to increase AAV production by the optional use of increased cell density in the growth stage vessel under conditions to concentrate the medium and remove inhibitory metabolites during the production stage. In particular, systems and methods are disclosed that allow the cultivation of AAV-producing cells in a single vessel (e.g., a bioreactor) from the growth stage to the AAV production stage while maintaining a high cell density, e.g., 1E06 cells / mL or higher, in some examples. The systems and methods described herein are unique from other systems that utilize perfusion in the growth stage prior to the production stage of gene therapy viruses to achieve high-density culture and then transfer that high cell population to a production vessel for batch mode operation. In contrast, the systems and methods described herein allow perfusion of the growth and production stages, including in the same vessel, in some embodiments, and allow cell culture medium exchange after infection with helper virus or transfection with helper virus components to concentrate the culture medium and remove inhibitory waste products from the culture medium, thereby improving AAV productivity. When compared to batch mode operation at high cell densities (e.g., greater than 1E06 cells / mL) as well as batch mode operation at low cell densities, the systems and methods described herein result in a significant, several-fold increase in overall volumetric AAV productivity while maintaining cell-specific productivities.

[0095] In a batch mode AAV manufacturing process, cell expansion generally follows thawing and expansion of a production cell line (e.g., a mammalian production cell line) in shake flasks. The expanded production cells are transferred to an intermediate scale, e.g., 250 L scale (e.g., 10 times smaller than the final production stage) bioreactor before being transferred to an N-1 growth vessel. Systems useful for N-1 growth stage culture are commercially available, such as Repligen Xcell ATF™ 6 single-use alternating tangential flow filtration system and equivalent systems. Typically, these systems can achieve cell densities of greater than 13E06 viable cells per milliliter (vc / mL). The cell population is then split approximately 1:10 from N- (e.g., N-1) in growth medium to N-stage production, e.g., 2000 L scale, at approximately 1.3E06 vc / mL. Typically, this requires a new production stage vessel separate from the growth stage vessel, requiring different production medium and feed. After transfer to N-stage or production stage culture, AAV production is initiated by infection with a helper virus, e.g., adenovirus, such as adenovirus serotype 5 (Ad5), in a 90:10 combination of production medium and growth medium, and occurs in the production culture for about 4 days. Finally, harvesting operations, such as nuclease addition and pretreatment activities, are performed in the bioreactor, followed by downstream filtration clarification, AAV purification, and helper virus removal processes.

[0096] In the improved batch (e.g., enhanced perfusion) systems and methods described herein, the cell seed train cell population is thawed and delivered into shake flasks in the same manner as in the batch mode AAV manufacturing process. The bioreactor expansion stage includes additional vessels, e.g., at 250 L scale (without the use of alternating tangential flow filtration). Importantly, in some embodiments, the subsequent growth and production stages can, but do not have to, be performed in the same, e.g., 2000 L vessel (bioreactor), without the need to transfer the high cell density seed train to a new vessel for production stage culture. For the growth stage at 2000 L scale, a final high cell density target, e.g., 5E06vc / mL to 50E06vc / mL, can be achieved using a formulation of growth and production media. Once the desired cell population reaches the high cell density target (e.g., 5E06vc / mL or higher), the perfusion-mediated or other medium exchange can be stopped. AAV production is then initiated about 1 to about 2 hours later by infection with a helper virus, e.g., adenovirus, or transfection with helper virus components, and feed addition, after which perfusion-mediated or other medium exchange is resumed again with appropriate production medium and feed. In some embodiments of the systems and methods described herein, alternating tangential flow (ATF) perfusion is utilized for the production stage culture. The ATF operation may remain active for about 1 to about 2 days, e.g., after infection with a helper virus, e.g., adenovirus, or transfection with helper virus components, to concentrate the medium and eliminate accumulation of waste products. After about 1 to about 2 days, the perfusion-mediated or other medium exchange is terminated to allow accumulation of AAV particles in the culture supernatant. In some embodiments, the ATF operation may remain active for more than 2 days after infection with a helper virus or transfection with helper virus components, e.g., for up to about 14 days of induction of AAV production. Release of AAV particles into the supernatant occurs after, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours, depending on, for example, the producer cell line used.A total of about 4 to about 6 days after infection with helper virus or transfection with helper virus components, AAV recovery procedures are performed for subsequent downstream process purification, such as filtration clarification, AAV purification, and removal of helper virus, e.g., adenovirus, such as Ad5. In some embodiments, the helper virus removal process includes a heat inactivation process, such as that described in US Patent Application Publication No. US20190083554A1. In some embodiments, tangential flow depth filtration can be performed to integrate the AAV recovery and clarification processes into a single step (see Mendes, Joao P., et al. "AAV process intensification by perfusion bioreaction and integrated clarification." Frontiers in Bioengineering and Biotechnology 10 (2022)).

[0097] In addition to utilizing the improved batch systems (e.g., enhanced perfusion) and methods using perfusion described herein, additional approaches for increasing AAV production and / or AAV titer can be implemented in the disclosed systems and methods. For example, the disclosed systems and methods can be further improved to include: the use of glucocorticoid analogs, e.g., as described in U.S. Patent Application Publication No. US20190290710A1, the use of a compound selected from the group consisting of niacinamide, niacin, methyl nicotinate, nicotinyl alcohol, and any combination thereof, e.g., as described in PCT Publication No. WO / 2021 / 188449, an optimized downstream clarification process, e.g., as described in U.S. Patent Application Publication No. US20200048641A1, the use of tonicifying agents, e.g., as described in U.S. Patent Application Publication No. US20210277416, and / or co-delivery of Rep mRNA or HDAC inhibitors, e.g., as described in U.S. Patent Application Publication No. US20200032221A1.

[0098] In some embodiments, the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein increase infection density by about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold from optimal batch mode or other conditions (e.g., non-enhanced), resulting in an increase in AAV productivity, e.g., to greater than or equal to 3E11 genome copies per milliliter (GC / mL), i.e., 3E14 GC / L or greater.

[0099] The described systems and methods demonstrate perfusion during the growth phase to achieve high density culture for transfer to a production vessel for batch mode operation, which differs from conventional methods. Others have demonstrated perfusion capabilities during AAV production in transient transfection systems and HEK-based cell systems, but none have demonstrated the use of perfusion during AAV production using, for example, HeLa production cells, e.g., HeLa production cells utilizing infection with a helper virus such as adenovirus. Some embodiments of the systems and methods disclosed herein increase overall volumetric productivity by enabling production of high cell densities utilizing perfusion equipment to combine growth and production phase production into a single process in the same vessel. In some embodiments, the use of perfusion technology allows for high production cell densities (e.g., HeLa PCL densities) in the production vessel. Subsequent changes to the production medium by medium exchange after infection with a helper virus concentrate the culture medium, remove inhibitory waste products, and result in increased AAV productivity. For example, when compared to batch mode operation at lower cell densities, as well as batch mode operation at higher densities (i.e., greater than about 1E06 cells / mL), the effect of perfusion after the initiation of AAV production results in a significant fold increase in overall volumetric AAV productivity while maintaining similar cell-specific productivity to that seen in batch mode operation.

[0100] The systems, methods, reagents, and their components and features are described in more detail in the following sections.

[0101] III. Viral Vectors This section generally describes viral vectors, including AAV vectors, which can be produced by the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein. In some aspects, the present disclosure provides systems and methods for the production of adeno-associated virus (AAV). In some embodiments, the present disclosure provides systems and methods for the production of recombinant adeno-associated virus (rAAV), which includes an adeno-associated virus (AAV) capsid and a recombinant vector genome packaged therein.

[0102] The produced AAV, e.g., rAAV, may contain a packaged vector genome, e.g., including AAV 5'-ITR, a promoter sequence, a partial or complete coding sequence of a therapeutic payload, and an AAV 3'-ITR. In some embodiments, the packaged vector genome may further include an enhancer sequence, an intron sequence, a consensus Kozak sequence, and / or a polyadenylation signal sequence. In some embodiments, the packaged vector genome may further include one or more stuffer nucleic acid sequences. In some embodiments, the stuffer nucleic acid sequence is located between the intron and the partial or complete coding sequence of a therapeutic payload.

[0103] In some embodiments, AAV is a recombinant AAV vector (rAAV). For simplicity, terms such as "AAV vector" as used herein include the subgenus rAAV, such that reference to AAV vector encompasses AAV vector and rAAV. The AAV vector produced by the system and method of the present disclosure can include capsids of any AAV serotype. For example, the system and method of the present disclosure can be used to produce AAV that includes capsids of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVhu37), and any one of over 100 variants isolated from human and non-human primate tissues. See, e.g., Choi et al., 2005, Curr Gene Ther. 5: 299-310, 2005; Gao et al., 2005, Curr Gene Ther. 5: 285-297; and Drouin et al, Future Virol. 2013 Dec; 8(12):1183-1199. In addition to the capsids described above, the systems and methods of the present disclosure can be used to produce AAVs that contain mutant AAV capsids engineered to have one or more beneficial therapeutic properties (e.g., improved targeting to select tissues, increased ability to evade immune responses, reduced stimulation of neutralizing antibodies, etc.).Non-limiting examples of such engineered mutant capsids include those described in U.S. Patent Nos. 9,506,083, 9,585,971, 9,587,282, 9,611,302, 9,725,485, 9,856,539, 9,909,142, 9,920,097, 10,011,640, 10,081,659, 10,179,176, 10,202,657, 10,214,5 66, 10,214,785, 10,266,845, 10,294,281, 10,301,648, 10,385,320, and 10,392,632, and PCT Publication Nos. WO / 2017 / 165859, WO / 2018 / 022905, WO / 2018 / 156654, WO / 2018 / 222503, and WO / 2018 / 226602. These and other AAV serotype variants can be similarly produced by the AAV production system and method herein. The selection of the AAV serotype and / or variant to be produced depends in part on the cell type to be targeted by the AAV vector, for example, in gene therapy applications.

[0104] A. Vector Genome Components In some embodiments, the AAV vectors produced by the systems and methods described herein comprise a vector genome packaged within the AAV vector.

[0105] In some embodiments, the AAV vectors produced by the systems and methods described herein contain a vector genome that includes AAV ITR sequences that function as both vector DNA origins of replication and packaging signals for the vector genome, i.e., where AAV and adenovirus helper virus functions are provided in trans. Additionally, the ITRs can serve as targets for single-stranded endonuclease nicking by the large Rep protein to separate individual genomes from replicative intermediates.

[0106] In some embodiments, the AAV vectors produced by the systems and methods described herein include a vector genome that includes a promoter sequence that helps drive and regulate transgene expression, e.g., expression of a therapeutic payload. The promoter sequence can be (or can be derived from) a ubiquitous promoter or a tissue-specific promoter sequence. In some embodiments, the promoter sequence can be a chicken beta (β)-actin (CBA) promoter sequence, a cytomegalovirus (CMV) immediate early gene promoter sequence, a transthyretin (TTR) promoter sequence, a thyroxine-binding globulin (TBG) promoter sequence, an alpha-1 antitrypsin (a1 AT) promoter sequence, a CMV early enhancer / chicken β-actin (CAG) promoter sequence, or a therapeutic payload gene-specific endogenous promoter. In some embodiments, the promoter sequence is located downstream of an enhancer sequence. In some embodiments, the promoter sequence is located upstream of an intron sequence. In some embodiments, the promoter sequence is located between the selected 5'-ITR sequence and the therapeutic payload coding sequence, including any 5'-untranslated region (UTR).

[0107] In addition to the promoter, the packaged genome may contain other appropriate transcription initiation, transcription termination, enhancer sequences, and efficient RNA processing signals. As described in more detail below, such sequences include splicing and polyadenylation (polyA) signals, regulatory elements that enhance expression (i.e., WPRE), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), and sequences that enhance protein stability.

[0108] In some embodiments, the packaged vector genome further comprises a consensus Kozak sequence. In some embodiments, the consensus Kozak sequence is located downstream of the intron sequence. In one embodiment, the consensus Kozak sequence is GCCGCC. As will be understood by those skilled in the art, the consensus Kozak sequence is typically located immediately upstream of a coding sequence, such as the sequence that codes for a therapeutic payload. As will be recognized by those skilled in the art, the consensus Kozak sequence can be considered to share the ATG residue that corresponds to the start codon of the therapeutic payload polypeptide.

[0109] In some embodiments, the packaged vector genome further comprises a 5'-untranslated region (UTR). The 5'UTR can be derived from an endogenous gene-specific mRNA with desired expression characteristics. It is known that the 5'UTR plays an important role in optimizing transgene production by competing with cellular transcripts for translation initiation factors and ribosomes, increasing mRNA half-life by minimizing mRNA decay or post-transcriptional gene silencing, and avoiding deleterious interactions with regulatory proteins or inhibitory RNA secondary structures (see Chiba, Y., and Green, P. (2009). J. Plant Biol. 52, 114-124; Moore, MJ, and Proudfoot, NJ (2009). Cell 136, 688-700; and Jackson, RJ, et. al. (2010). Nat. Rev. Mol. Cell Biol. 11, 113-127).

[0110] Similarly, the packaged vector genome can further comprise a 3'UTR. The 3'UTR can be located downstream of the coding sequence for the therapeutic payload. The 3'UTR has been shown to be involved in a number of regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

[0111] In some embodiments, the coding sequence of the therapeutic payload is a partial or complete coding sequence of a therapeutic polypeptide or polynucleotide. Exemplary therapeutic polypeptides can be found in PCT Publication No. WO / 2021 / 067598, such as ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase, factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit a (PCCA) and propionyl-CoA carboxylase subunit b (PCCB), survival of motor neuron (SMN)), iduronate-2-sulfatase (IDS), alpha-1-iduronidase (IDUA), tripeptidyl peptidase 1 (TPP1), low density lipoprotein receptor. (LDLR), myotubularin 1, acid alpha-glucosidase (GAA), myotonic dystrophy protein kinase (DMPK), N-sulfoglucosamine sulfohydrolase (SGSH), fibroblast growth factor-4 (FGF-4), rab escort protein 1 (REP1), carbamoyl synthetase 1 (CPS1), argininosuccinate lyase (ASL), arginase, fumarylacetoacetate hydrolase, alpha-1 antitrypsin, methylmalonyl CoA mutase, cystic fibrosis transmembrane conductance control factor (CFTR) protein, and dystrophin gene products (e.g., mini-dystrophin or micro-dystrophin), which may be useful in the treatment of mammals, e.g., humans. A non-limiting list of therapeutic payloads may also be found in PCTWO / 2019 / 168961. The therapeutic payload can be a wild-type coding sequence for a protein or polypeptide, a codon-optimized coding sequence for a protein or polypeptide, or a sequence encoding a functional nucleotide species such as a non-coding RNA, or a combination thereof.Examples of functional nucleotide species suitable as therapeutic payloads include non-coding RNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, long non-coding RNAs (IncRNAs), and any other species of non-protein-coding nucleic acids with biological activity associated with a disease state. For example, the therapeutic payload can be a morpholino, a peptide-linked morpholino, an antisense oligonucleotide (ASO), a phosphorodiamidate morpholino oligomer (PMO), a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a peptide-linked PMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both a CRISPR-Cas protein and a guide RNA, a ribonucleoprotein comprising a CRISPR-Cas system molecule, a therapeutic transgene RNA or other genetically modified or therapeutic RNA and / or protein, or any combination thereof. As used herein, the term "wild type" refers to a biopolymer (e.g., a polypeptide sequence or a polynucleotide sequence) that is the same as a naturally occurring biopolymer (e.g., a polypeptide sequence or a polynucleotide sequence).

[0112] In some embodiments, the packaged vector genome further comprises one or more enhancer sequences, which in some embodiments are (or are derived from) a cytomegalovirus immediate early gene (CMV) enhancer sequence, a transthyretin enhancer (enTTR) sequence, a chicken beta (β)-actin (CBA) enhancer sequence, an En34 enhancer sequence, or an apolipoprotein E (ApoE) enhancer sequence.

[0113] In some embodiments, the packaged vector genome further comprises one or more intron sequences, which may be (or may be derived from) an SV40 small T intron sequence, a rabbit hemoglobin subunit beta (rHBB) intron sequence, a human beta globin IVS2 intron sequence, a Promega chimeric intron sequence (a [3-globin / IgG chimeric intron sequence), or an hFIX intron sequence.

[0114] In some embodiments, the packaged vector genome further comprises a polyadenylation signal sequence, which in some embodiments is (or is derived from) the bovine growth hormone (BGH) polyadenylation signal sequence, the SV40 polyadenylation signal sequence, the rabbit beta globin polyadenylation signal sequence, a gene-specific endogenous polyadenylation signal sequence.

[0115] B. AAV Capsid The present disclosure provides a system and method for producing AAV particles comprising AAV capsid of any AAV serotype.In some embodiments, the AAV capsid is derived from AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, or hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVhu37).In exemplary embodiments, the AAV capsid is derived from AAV serotype 9 (AAV9) vector, AAV9 variant vector, AAV serotype 8 (AAV8) vector, AAV serotype 2 (AAV2) vector, or any other known AAV serotype vector or variant. International Patent Application Publication No. WO2021041485A1, at paragraphs

[0115] to

[0131] , provides a list of known AAV serotypes, variants, mutations, and derivatives thereof, any of which may be suitable for AAV production using the systems and methods described herein.

[0116] C. Pharmaceutical Compositions The systems and methods for AAV production provided herein can be used to generate pharmaceutical compositions comprising AAV vectors. Pharmaceutical compositions comprising AAV vectors produced according to the methods disclosed herein can further comprise a pharma- ceutically acceptable carrier. Pharmaceutical compositions comprising AAV vectors produced according to the methods disclosed herein can further be formulated for administration to a subject. Suitable pharmaceutical compositions formulated for administration of AAV can be found, for example, in US Patent Application Publication No. 2012 / 0219528. Pharmaceutically acceptable carriers (vehicles) useful for AAV compositions are conventional. Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, Pa., l5th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules, or agents, including, for example, AAV produced according to the systems and methods disclosed herein.

[0117] The present disclosure relates in some aspects to a pharmaceutical composition comprising the AAV produced according to the disclosed method. In some embodiments, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0118] In some embodiments, the AAV is formulated in a buffer / carrier suitable for infusion in a human subject. The buffer / carrier may include components that prevent the AAV from adhering to the infusion tubing but do not interfere with in vivo AAV binding activity. Various suitable solutions may include one or more of a buffered saline solution, a surfactant, a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to, for example, about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration. The pH of the formulated pharmaceutical composition may be in the range of 6.5-8.5, 7-8.5, or 7.5-8. A suitable surfactant, or combination of surfactants, may be selected from among poloxamers, i.e. non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene 10 (polypropylene oxide) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide), SOLUTOL HS 15 (Macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), Polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and / or polyethylene glycol.

[0119] IV. AAV Production Methods The present disclosure provides systems and methods for the production of adeno-associated viruses (AAVs), which can be used as therapeutic payload (e.g., gene) delivery vehicles. In some embodiments, the AAVs produced by the systems and methods provided herein comprise wild-type or engineered (mutant or variant) AAV capsids packaged with an engineered vector genome, e.g., containing a transgene (e.g., a therapeutic payload, e.g., a transgene encoding a therapeutic protein). In some embodiments, such engineered AAVs can be administered to a subject in need of a therapeutic payload, and upon administration, the therapeutic payload can be expressed in the subject's own cells. The systems and methods described herein are applicable to AAVs containing any therapeutic payload.

[0120] A. AAV-producing host cells The systems and methods provided herein utilize host cells for the production of AAV vectors, including rAAV.In some embodiments, the host cell comprises a recombinant nucleic acid molecule disclosed herein, a viral vector, such as an AAV vector, or a rAAV.In certain embodiments, the host cell is suitable for the propagation of AAV.In certain embodiments, the host cell is suitable for the production of AAV.In certain embodiments, the host cell is suitable for the propagation and / or production of AAV.

[0121] Any known AAV-producing host cell can be used. Examples of known host cell types include bacterial cells, yeast, insect cells (such as Sf9 cells), and mammalian cells. In some embodiments, the host cell can be a cell (or cell line) suitable for producing AAV (e.g., rAAV), such as HeLa cells, Cos-7 cells, HEK293 cells (and HEK293 derived cell lines), A549 cells, BHK cells, vero cells, RD cells, or ARPE-19 cells. In some embodiments, stable inducible AAV-producing cell lines, such as those by doxycycline-inducible CAP cells and HEK293 cells described in WO2022 / 112218A1, are utilized in the systems and methods described herein. Additional host cell types are described elsewhere herein.

[0122] In some embodiments, recombinant nucleic acid molecule or vector can be delivered to host cell culture using any suitable method known in the art (e.g., transfection-based method).In some embodiments, a stable host cell line that inserts recombinant nucleic acid molecule or vector into its genome by transfection is utilized in the system and method provided herein.After transfection of AAV vector into host cell, the integration of AAV vector into host cell genome can be assayed by various methods such as antibiotic selection, fluorescence-activated cell sorting, Southern blot, PCR-based detection, fluorescence in situ hybridization, and are described in Nakai et al, Nature Genetics (2003) 34, 297-302, Philpott et al, Journal of Virology (2002) 76(11): 5411-5421, and Howden et al, J Gene Med 2008; 10:42-50. Additionally, stable cell lines can be established and utilized according to protocols known in the art, such as those described in Clark, Kidney International Vol 61(2002):S9-S15 and Yuan et al, Human Gene Therapy 2011 May;22(5):613-24.

[0123] In some embodiments, the host cells utilized for AAV production are engineered to be AAV producer cells. AAV producer cell lines (PCLs) can be utilized in the systems and methods described herein.

[0124] Various aspects of culturing AAV PCL in the system and method of the present disclosure are described herein.For example, the parameters of PCL culture conditions, including medium exchange technique, perfusion, perfusion rate, cell density, medium, medium supplement, cell viability, growth rate, pH, culture period, culture volume and seeding density, can be selected or utilized in the system and method of the present disclosure are described.

[0125] Various cellular / viral components are required for AAV production. For example, components for AAV replication, vector genome packaging, and structural components of capsid must all be produced to generate AAV particles. The AAV Rep gene encodes four proteins involved in packaging and replication, and the cap gene encodes three structural capsid proteins (called VP1, VP2, and VP3). Wild-type AAV is replication-deficient and requires co-infection of cells with a helper virus, such as a herpes virus, or an adenovirus, such as Ad5 virus, to replicate. For example, Ad5 virus provides Ad5 helper virus functional factors / genes, such as E1a, E1b, E40rf6, E2a, and / or virus-associated (VA) RNA, which mediate AAV replication. See, for example, Nayak et al. J Virol. 81.5(2007):2205-12. Recombinant AAV vector allows the incorporation of a gene of interest or a transgene into a viral vector, so that the transgene is transferred, encoded, and / or expressed by viral machinery.In some cases, recombinant AAV vector comprises an inverted terminal repeat (ITR) that serves as an origin of replication and / or packaging.In some embodiments, recombinant AAV vector comprises a transgene flanked by ITRs (one ITR on each side of the transgene).See, for example, Carter B. Adeno- Associated Virus and AA V Vectors for Gene Delivery, in Gene and Cell Therapy, 4th Edition, NS Templeton, Editor. 2015, CRC Press.

[0126] AAV PCLs can be generated that contain one or more of these components required for AAV production. An exemplary list of AAV components includes: (a) a nucleic acid sequence comprising a transgene, e.g., a therapeutic payload; (b) a nucleic acid sequence comprising an inverted terminal repeat (ITR), e.g., one or two ITRs, e.g., where the two ITRs are flanked by one or more additional AAV components (e.g., a transgene); (c) a nucleic acid sequence encoding one or more AAV replication and / or packaging proteins (e.g., encoded by the AAV rep gene); (d) a nucleic acid sequence encoding one or more AAV structural capsid proteins (e.g., VP1, VP2, or VP3 proteins, encoded by the AAV cap gene); (e) one or more AAV replication and / or packaging proteins; (f) one or more AAV structural capsid proteins; and / or (g) one or more helper virus components, e.g., Ad5 helper virus components (e.g., E1a, E1b, E40rf6, E2a, and / or VA RNA, or Ad5 helper virus). As used herein, an "AAV PCL" or "AAV producing cell line" refers to a cell, e.g., a cell described herein, that comprises one or more of (a)-(g) above and that, when supplied with any necessary helper virus components, is capable of producing AAV under appropriate production conditions well known in the art.

[0127] In some embodiments, any combination of the AAV components (e.g., (a)-(d) or (g) above) are provided on the same nucleic acid molecule or on separate nucleic acid molecules (e.g., on one, two, three, or more separate nucleic acid molecules). In some embodiments, the transgene is flanked on both sides by ITRs.

[0128] In some embodiments, the PCL comprises any combination of (a)-(g) above. In some embodiments, the PCL may comprise, for example, a recombinant AAV vector and / or Rep and / or cap genes stably integrated into a permissive host cell. In some embodiments, the PCL comprises a nucleic acid sequence encoding a transgene and a nucleic acid sequence comprising one or more ITRs, for example, the transgene flanked on either side by one ITR. In other embodiments, the PCL comprises a packaging cell line comprising the rep and / or cap genes but no vector / transgene (the vector / transgene can be provided by a separate virus, e.g., recombinant adenovirus, e.g., Ad5). PCLs can be made that comprise other combinations of AAV components. Any such PCL and other variations of PCLs can be used in conjunction with the systems and methods described herein. In some examples, AAV production can be induced by infection with a helper virus, such as Ad5, for example in N cultures.

[0129] Any cell line suitable for use in AAV production, including suitable mammalian and insect cell lines (including Sf9 cells), can be used as AAV production host cell using the systems and methods described herein.For example, any mammalian cell line, such as PCL derived from any mammalian cell line, that contains one or more components required for AAV production can be used in any of the systems and methods described herein.In some embodiments, the systems and methods described herein include culturing mammalian cells, such as human cells or non-human mammalian cells, such as mammalian PCL, such as human PCL, non-human mammalian PCL.Exemplary cell types include BALB / c mouse myeloma line (NSO / 1, ECACC number: 85110503), human retinoblastoma cells (PER.C6 (CruCell, Leiden, The Netherlands)), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)), and mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)). (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HeLa, ATCC CCL 2, or HeLa S3, ECACC catalog number 87110901), baby hamster kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), FS4 cells, human hepatocellular carcinoma line (Hep G2), and / or PCL versions of any of the cell types described herein. In some embodiments, the PCL versions of the cell types described herein include cell types engineered to have one or more components necessary for AAV production.

[0130] In some embodiments, the systems and methods described herein include culturing HeLa cells (e.g., HeLa PCLs), CHO cells (e.g., CHO PCLs), or HEK cells (e.g., HEK PCLs). In some embodiments, the systems and methods described herein include culturing HeLa cells, e.g., HeLa PCLs.

[0131] As mentioned above, in some cases, cells are selected or engineered (e.g., engineered into PCL) to contain one or more components required for AAV production.Alternatively or additionally, engineered PCL can contain genetic modifications to reduce the expression and / or activity of one or more genes and / or proteins, which modifications increase AAV titer.Exemplary engineered PCL useful in the systems and methods of the present disclosure include those described in US Patent Application Publication No. US20200325455A1.

[0132] In some embodiments, engineered PCL may include genetic modifications to reduce the expression and / or activity of one or more genes and / or proteins, which modifications reduce the production or accumulation of waste by-products in the PCL. As an example of a target gene to mitigate waste by-product accumulation, Soo Min Noh et al. showed that shRNA treatment of CHO production cells to reduce expression of lactate dehydrogenase-A improved the production and quality of the monoclonal antibodies produced. (See Noh, Soo Min, et al. "Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells." Applied microbiology and biotechnology 101(3) (2017): 1035-1045, which is incorporated herein by reference in its entirety.) also described waste by-product levels and their impact on Ad5 production in Shen, Chun Fang, et al. “Reassessing culture media and critical metabolites that affect adenovirus production” Biotechnology progress 26(1) (2010):200-207, showing that knockdown of LDH-A or LDH-B can reduce lactate production in Khajah et al. “Lactate Dehydrogenase A or B Knockdown Reduces Lactate Production and Inhibits Breast Cancer Cell Motility in vitro” Front Pharmacol. 2021, 12:747001, and showing that knockdown of PFKP inhibits lactate production in Prasad et al. “Reduced production and uptake of lactate are essential for the ability of WNT5A signaling to inhibit breast cancer cell migration and invasion” Oncotarget. 2017 Sep 22; 8(42):71471-71488. Each of Shen et al., Khajah et al., and Prasad et al. is incorporated herein by reference in its entirety. Thus, the present disclosure contemplates AAV PCLs engineered to upregulate or downregulate expression of metabolic enzymes that can reduce the formation or accumulation of waste by-products such as lactic acid or ammonia.

[0133] For example, the present disclosure contemplates engineered AAV PCLs that express glutamine synthetase to reduce lactate and ammonia accumulation. Such AAV PCLs can express exogenous glutamine synthetase (GS). The expression of glutamine synthetase can be constitutive or inducible. The expression of glutamine synthetase can be driven by promoters, enhancers, and / or other expression regulatory elements that are optimized for appropriate levels of expression to reduce the accumulation of waste by-products during AAV production.

[0134] Additionally, the present disclosure contemplates engineered AAV PCLs with reduced expression of lactate dehydrogenase to reduce lactate accumulation. In some embodiments, PCLs may reduce expression of lactate dehydrogenase-A (LDH-A). In some embodiments, PCLs may reduce expression of lactate dehydrogenase-B (LDH-B). In some embodiments, PCLs may reduce expression of the glycolytic enzyme phosphofructokinase platelet type (PFKP).

[0135] In some embodiments, PCL can increase the expression of glutamine synthetase and decrease the expression of lactate dehydrogenase.

[0136] Different methodologies for regulating gene expression in PCLs are recognized by those skilled in the art, and include, for example, the approach described in US Patent Application Publication No. US20200325455A1. Examples of methodologies that can be utilized to regulate the expression of genes or gene products in PCLs engineered to reduce waste production or accumulation include the use of nucleases, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotides (ASO). Examples of nucleases include zinc finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins.

[0137] Alternative approaches to reduce lactate and / or ammonia accumulation in PCL cultures include those described by Freund and Croughan (Int. J. Sci. 2018 Feb;19(2): 385). These include pH shifts, clonal selection of lactate-consuming phenotypes, and various media supplementation approaches. Based on the observation that EAA supplementation in fed-batch cultures shifts the metabolic profile of AAV-producing cells, specifically reducing lactate accumulation, it is further contemplated herein that media supplementation with essential amino acids (EAA) may promote rAAV production by reducing lactate and other waste accumulation and promoting protein production (data not shown).

[0138] One of skill in the art will understand that different cell lines may have different nutritional requirements and / or may require different culture conditions for optimal growth, and one of skill in the art will be able to modify the conditions as necessary.

[0139] B. Methods of AAV Production Various aspects of AAV production are described herein, including system and method aspects.

[0140] Batch mode process for i.AAV production Generally, in batch mode AAV production, AAV PCL is cultured in N-1 culture vessel to achieve high viable cell density.Then, N-1 culture cells are used to inoculate N culture vessel at a certain seeding density.The cells inoculated in N culture vessel can be cultured under conditions that allow AAV production.

[0141] Figure 1 shows an exemplary process for low-density batch (LDB) mode AAV production, i.e., an exemplary batch mode control process. For example, an AAV producer cell line, such as a HeLa producer cell line (PCL), is thawed, grown in a shake flask, transferred to an intermediate-scale bioreactor, and then transferred to an N-1 perfusion growth vessel, for example at a 250L scale, prior to a production vessel at a production stage, for example at a 2000L scale. As shown in Figure 1, the process begins with pumping growth medium into the 250L N-1 stage perfusion growth vessel (step 1), followed by seeding the N-2 stage producer cell inoculum preceding the N-1 stage perfusion growth vessel (step 2). Growth medium is perfusion exchanged (step 3) for, e.g., 4-6 days through the use of an alternating tangential flow (ATF) filtration system or similar medium exchange system (e.g., Repligen Xcell ATF™ 6 uses a single alternating tangential flow filtration system at 250 L scale) to achieve a target cell density of 13E06vc / mL (viable cells per milliliter) or greater (step 4). For the production stage in batch mode operation, a separate 2000 L vessel is primed with production medium at approximately 90% of the final volume (step 5) and AAV producing cells are transferred from the N-1 to the production stage reactor at a split ratio of approximately 1:10 to allow for sufficient exchange of growth medium into production medium (step 6). In FIG. 1, the solid line separating the N-1 stage growth reactor and the N-stage production reactor indicates that these two stages are carried out in separate vessels. In this manner, a 2000 L production vessel is inoculated with high concentration cell culture (i.e., greater than 13E06vc / mL) targeting a final concentration of greater than about 1.3E06vc / mL. After inoculation, AAV production is initiated by helper virus (e.g., adenovirus, Ad5) infection (step 7), followed by addition of a bolus production feed to the bioreactor vessel (step 8). Approximately 4-6 days post infection ("dpi"), AAV product is harvested from the culture supernatant for subsequent purification and helper virus removal procedures (step 9). For example, Ad5 removal processes include those described, for example, in U.S. Patent Application Publication No. US20190083554A1.

[0142] ii. Improved batch systems and methods for AAV production using perfusion (e.g., enhanced perfusion) Described herein is an improved batch process for AAV production using perfusion (e.g., enhanced perfusion). An exemplary process is shown in FIG. 2. In general, the improved batch process includes preparing a vessel with growth medium for the growth stage culture of AAV producing cells. In some embodiments, the vessel can be a single vessel intended to culture both the growth stage culture and the production stage culture, i.e., a "combined growth / production vessel." Such a vessel is subsequently inoculated with AAV producing cells. Perfusion may be initiated in the inoculated growth or combined growth / production vessel to initiate AAV producing cell growth. Once the final cell density target is achieved, the production stage culture can be initiated. In some embodiments, the production stage initiation includes infecting the growth stage culture with a helper virus to initiate AAV production. AAV production may be initiated by alternative means that do not rely on AAV helper virus infection. Such alternatives include, for example, the use of a triple transfection approach, or the use of producer cells with stable integration of the components required to produce AAV, such as adenovirus helper virus functions, AAV replicase, and capsid genes (e.g., ELEVECTA® (CEVEC) doxycycline-inducible AAV producer cells, and similar systems). The AAV production stage can be maintained, for example, by providing feed, exchanging medium with production medium, and / or initiating perfusion to maintain production. Thus, the produced AAV can be recovered using any suitable separation technique and purified using standard downstream processing steps known to those skilled in the art.

[0143] Figure 3 shows an exemplary process of the AAV production system and method described herein. In some embodiments, the preliminary steps of the improved batch (e.g., enhanced perfusion) process can be similar to the conventional steps used in batch mode AAV production processes. For example, the cell seed train cell population can be thawed and transported into shake flasks in the same manner as in batch mode operation.

[0144] To scale up to a growth / production culture, a bioreactor expansion stage may be utilized. Any suitable scale may be employed or adapted to the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein. In some embodiments, the bioreactor expansion stage uses, for example, a 250 L scale vessel. In some embodiments, the bioreactor expansion stage uses sub-liter culture volumes. In some embodiments, the bioreactor expansion stage uses any suitable culture volume from sub-liter to 6000 L scale or larger (including up to 12000 L scale). Thus, the present system and method can utilize sub-liter, 1L, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessels for bioreactor expansion stage cultivation. In some embodiments, the bioreactor expansion stage uses vessels with a capacity of more than 6000L. Those skilled in the art will understand that larger culture volumes (such as 6000L or more) may require specialized media handling systems / equipment. Thus, the bioreactor expansion stage may utilize an appropriate perfusion device, such as an alternating tangential flow (ATF) filtration system. The bioreactor growth stage culture may include a perfusion device such as the Repligen Xcell ATF™ 6 or other perfusion system appropriate for the culture volume selected for the bioreactor expansion stage. For larger culture volumes (such as 6000 L or more), multiple such devices can be used, for example, multiple Repligen Xcell ATF™ 6 or ATF™ 10 systems. In some embodiments, the bioreactor expansion stage does not utilize an ATF filtration system.

[0145] In some embodiments, the final perfusion production stage is carried out in a production vessel, for example at a 2000 L scale. The production vessel can be a vessel suitable for any culture volume for AAV production at any scale. For example, the production vessel can be a sub-liter, 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 100 L, 150 L, 200 L, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 5500 L, 6000 L, 6500 L, 7000 L, 7500 L, 8000 L, 8500 L, 9000 L, 9500 L, 10000 L, 10500 L, 11000 L, 11500 L, 12000 L vessel, or more.

[0146] In some embodiments, the final perfusion growth and production stages are combined in the same vessel, referred to herein as a combined growth / production vessel, for example at the 2000 L scale. The combined growth / production vessel can be any suitable culture vessel for AAV production at any scale. For example, the combined growth / production vessel can be a sub-liter, 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 100 L, 150 L, 200 L, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 5500, 6000 L, 6500 L, 7000 L, 7500 L, 8000 L, 8500 L, 9000 L, 9500 L, 10000 L, 10500 L, 11000 L, 11500 L, 12000 L vessel, or more. In some embodiments, the combined growth / production vessel uses a vessel having a volume of more than 6000 L.

[0147] Those skilled in the art will appreciate that larger culture volumes (e.g., 6000 L or more) may require specialized media handling systems / apparatus. Thus, the growth, production, and / or combined growth / production vessels may utilize appropriate perfusion devices, such as alternating tangential flow (ATF) filtration systems. The growth, production, and / or combined growth / production vessel cultures may include perfusion devices, such as Repligen Xcell ATF™ 6, or other perfusion systems appropriate for the culture volume selected for the bioreactor stage. In larger culture volumes (e.g., 6000 L or more), multiple such devices may be used, e.g., multiple Repligen Xcell ATF™ 6, ATF™ 10 systems, or flat sheet alternating tangential flow filtration systems. For example, Repligen Xcell ATF™ 6 or equivalent systems may be used at, e.g., 50 L to 500 L scale, while ATF™ 10 or equivalent systems may be used at larger scales. In some embodiments, multiple ATF™ 10 systems, etc., can be implemented to support increased cell density. In some embodiments, the growth, production, and / or combined growth / production vessels do not utilize an ATF filtration system.

[0148] In some embodiments, a perfusion system, such as an ATF filtration system, such as the Repligen Xcell ATF™ 10, is implemented during the growth and / or production phases in a combined growth / production vessel. Alternative perfusion technologies suitable for use in combined growth / production vessels include the Xcellerex Automated Perfusion System (APS) (Cytiva), KrosFlo® KPS TFF systems, e.g., KPS 700, or XCell® ATF6 (Repligen), GEA Kytero® Single Use Pharma Separator Systems (GEA), Prostak® Microfiltration Modules (Millipore), Alfa Laval CultureOne™ (Alfa Laval), CARR® Centritech Separation Systems such as the CARR UniFuge® Pilot or Centritech CELL 8® (Pneumatic Scale Angelus), the Ksep® 6000S system (Sartorius), microfluidic cell retention devices (described in Kwon T. et al. Sci Rep 7, 6703 (2017)), SciLog® Perfusion techniques include, but are not limited to, the SciPure™ system, the Acoustic Settler system (described in Coronel J. et al. Front. Bioeng. Biotechnol., 02 July 2020) (Sonosep), and / or the EDO Electro system (described in Wang, Zhaowei, “Two Approaches for Cell Retention in Perfusion Culture Systems” (2009). ETD Archive. 304) (American Piezo). Various formats may be available from vendors for the aforementioned perfusion techniques and may be suitable for use in the systems and methods described herein.For example, the Repligen ATF system can be implemented in 2, 4, 6, or 10 filter unit formats depending on the desired culture scale and cell density. Small-scale perfusion technologies such as the WAVE bioreactor (Cytiva) can also be utilized. Alternative medium exchange techniques can be implemented, including tangential flow filtration (TFF) and / or centrifugation-based techniques.

[0149] In some embodiments, N-phase expansion cultures can be targeted to a final density of at least 5E06vc / mL. N-phase expansion can be targeted to any final density suitable for seeding a production stage AAV bioreactor, including, for example, 1E06, 2E06, 3E06, 4E06, 5E06, 6E06, 7E06, 8E06, 9E06, 10E06, 20E06, 30E06, 40E06, or up to 50E6vc / mL, or more.

[0150] In some embodiments, the growth stage culture may be targeted to a final density using, for example, a combination of growth medium and production medium that is perfused, for example, for 2-14 days. In some embodiments, only growth medium is used in the growth stage culture. In some embodiments, only production medium is used in the growth stage culture. In some embodiments, a 50 / 50 combination of growth medium and production medium is used in the growth stage culture. In other embodiments, a 10 / 90, 20 / 80, 30 / 70, or 40 / 60 combination of growth medium and production medium is used in the growth stage culture. In still other embodiments, a 90 / 10, 80 / 20, 70 / 30, or 60 / 40 combination of growth medium and production medium is used in the growth stage culture. The growth medium, production medium, or combination of growth medium and production medium may be perfused, for example, for 2-14 days. In some embodiments, only growth medium is used until the final density target is achieved, at which point only production medium is used throughout the remainder of the production stage.

[0151] Referring to the exemplary process depicted in Figure 3, when the target cell density is reached in the expansion stage, the growth medium is primed in the combined growth / production vessel (step 1). Any suitable PCL, e.g., AAV production cells such as HeLa cells (e.g., HeLa PCL), are inoculated from the bioreactor expansion stage described above at the target final density for seeding (step 2). The perfusion rate is then increased with the cell population to maintain a constant cell-specific perfusion rate (CSPR) for a sustained period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or more than 6 days (step 3). In some embodiments, the perfusion rate is increased with cell mass to maintain a constant CSPR for a duration of about 2 to about 6 days, e.g., about 2 to about 3 days, about 2 to about 4 days, about 2 to about 5 days, about 2 to about 6 days, about 3 to about 4 days, about 3 to about 5 days, about 3 to about 6 days, about 4 to about 5 days, about 4 to about 6 days, or about 5 to about 6 days. In some embodiments, maintaining a constant CSPR minimizes nutrient depletion and waste accumulation.

[0152] In some embodiments, a fixed volume perfusion rate is utilized regardless of cell density.

[0153] Once the target cell density for AAV production (e.g., 5E06vc / mL) is reached (step 4), the growth perfusion exchange may be stopped. AAV production can then be initiated by helper virus (e.g., adenovirus Ad5) infection (step 5). In FIG. 3, the dashed line separating the growth stage reactor and the production stage reactor indicates that both stages can, but need not, be carried out in the same vessel, e.g., a 2000 L production stage bioreactor. In some embodiments, after helper virus infection, a bolus feed is added to support production stage activity (step 6). In some embodiments, the growth medium is then exchanged with a production medium / feed mixture (step 7), e.g., in an ATF-10 setup. In some embodiments, perfusion is resumed about 1 hour to about 2 hours post infection (hpi) (step 8). Perfusion can be maintained for 1 to 2 dpi to concentrate the medium, reduce waste accumulation, and retain the AAV product intracellularly. In some embodiments, the AAV particles remain cell-associated / intracellular until about 48 hpi. Therefore, perfusion can be disabled before this time to minimize product loss to the waste permeate stream. After about 1-2 dpi, perfusion is disabled to allow retention and accumulation of AAV particles in the culture supernatant (step 9). After about 48 hours, most AAV product is produced but remains intracellular, but from 2-6 dpi, AAV particles are preferentially released into the cell culture supernatant and can be collected (step 10). In some embodiments, AAV product can be collected from about 2 dpi, about 3 dpi, about 4 dpi, about 5 dpi, about 6 dpi, or later than 6 dpi. For example, AAV products can be harvested at about 2 dpi to about 3 dpi, about 2 dpi to about 4 dpi, about 2 dpi to about 5 dpi, about 2 dpi to about 6 dpi, about 3 dpi to about 4 dpi, about 3 dpi to about 5 dpi, about 3 dpi to about 6 dpi, about 4 dpi to about 5 dpi, about 4 dpi to about 6 dpi, or about 5 dpi to about 6 dpi for subsequent downstream purification.

[0154] In some embodiments, Ad5 removal processes include, for example, those described in U.S. Patent Application Publication No. US20190083554A1.

[0155] Those skilled in the art will appreciate that in both batch mode operation and improved batch mode operation (e.g., enhanced perfusion) described herein, it is important to optimize growth and production culture conditions to improve AAV production yield. Temperature, pH, osmolality, salt concentration, medium and feed composition, oxygen saturation, agitation rate, starting glucose / glutamine concentration, waste carryover level, split ratio, culture age, state of cell growth / synchronization, helper virus type or mutation / attenuation, and multiplicity of infection may each affect AAV productivity in a particular system or process. In addition, waste or metabolite production / consumption rates and medium exchange rates may need to be optimized for high-yield, high-cell-density AAV production processes.

[0156] The Examples section of the application demonstrates exemplary embodiments of improved batch systems and methods using perfusion (e.g., enhanced perfusion) as described herein that consistently increase infection density during AAV production by approximately 4-fold from existing optimal low-density batch (LDB) processes, achieving a 2.5-3-fold increase in AAV volumetric productivity of greater than 3E11 GC / mL (genome copies per milliliter) or greater than 3E14 GC / L.

[0157] The improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein were evaluated in the context of two separate HeLa producer cell systems utilizing different growth media, different genes of interest in the therapeutic payload, and different capsids, and achieved similar fold improvements in productivity compared to the corresponding low-density batch mode operating conditions. These results demonstrate that the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein can be implemented to achieve AAV yield benefits across multiple AAV producer cell systems, under different media conditions, with different genes of interest, and with different AAV capsids.

[0158] Sections a.-j. below provide examples of various AAV culture parameters that may be utilized in various embodiments of the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein.

[0159] a. Medium exchange and perfusion In some embodiments, culturing AAV PCLs in improved batch systems and methods using perfusion (e.g., enhanced perfusion) disclosed herein includes removing waste products from the culture medium and / or medium that replenishes nutrients, for example, by using perfusion, e.g., alternating tangential flow (ATF) perfusion in the vessels of the growth stage and / or production stage culture. Perfusion can be utilized, for example, to build cell populations in growth stage cultures and / or to maintain cell populations in production stage cultures.

[0160] ATF employs a filtration system that provides a method for continuous or semi-continuous exchange of cell culture medium, for example, by continuously / semi-continuously replenishing the culture with fresh medium through a number of hollow fibers while removing waste products. The ATF flow rate indicates how fast the cell culture circulates through each hollow fiber. In some embodiments, the diameter of each fiber is about 0.5 mm to about 1.5 mm, for example, about 0.5 mm, about 1 mm, or about 1.5 mm. Without wishing to be bound by theory, it is believed that higher flow rates tend to increase the amount of shear stress on the cells. Also, a flow rate that is too low can (1) increase the risk of exposing the cell culture to a low-oxygen environment for an extended period of time, and (2) increase the likelihood of filter fouling due to lack of backflushing. The methods described herein include performing the ATF perfusion at a flow rate that minimizes shear stress on the cells but provides sufficient oxygen to the cells. In some embodiments, the ATF perfusion is performed at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber, for example, about 5 mL / min / fiber to about 10 mL / min / fiber, about 5 mL / min / fiber to about 8 mL / min / fiber, about 7 mL / min / fiber to about 10 mL / min / fiber, or about 8 mL / min / fiber to about 10 mL / min / fiber. In some embodiments, the ATF perfusion is performed at a flow rate of about 3 mL / min / fiber, about 4 mL / min / fiber, about 5 mL / min / fiber, about 6 mL / min / fiber, about 7 mL / min / fiber, about 8 mL / min / fiber, about 9 mL / min / fiber, about 10 mL / min / fiber, about 11 mL / min / fiber, about 12 mL / min / fiber, about 13 mL / min / fiber, about 14 mL / min / fiber, or about 15 mL / min / fiber. In other embodiments, the ATF perfusion is performed at a flow rate per fiber of about 50 meters / second to about 320 meters / second, e.g., about 50 meters / second to about 250 meters / second, about 50 meters / second to about 200 meters / second, about 100 meters / second to about 320 meters / second, about 100 meters / second to about 250 meters / second, about 200 meters / second to about 320 meters / second, or about 200 meters / second to about 275 meters / second.

[0161] In some embodiments, perfusion rate can be optimized to maintain shear rate and increase AAV product release.Shear stress on cells introduced by perfusion can cause cell rupture and / or release of AAV particles.Thus, perfusion rate can be optimized to maintain shear rate that causes sustained release of AAV product in perfusion culture.The AAV released in this manner into culture can be collected at various times or continuously during production stage, for example, from the permeate when free AAV particles pass through the perfusion membrane.

[0162] In the first 12, 24, 36, 48, 60, 72, 84, or 96 hours of AAV production, medium exchange can be accomplished using any suitable perfusion-based or other medium exchange technique, including ATF as described above. Alternative perfusion technologies suitable for use in various embodiments of the systems and methods described herein include the Xcellerex Automated Perfusion System (APS) (Cytiva), KrosFlo® KPS TFF systems, e.g., KPS 700, or XCell® ATF6 (Repligen), GEA Kytero® Single Use Pharma Separator Systems (GEA), Prostak® Microfiltration Modules (Millipore), Alfa Laval CultureOne™ (Alfa Laval), CARR® Centritech Separation Systems such as CARR UniFuge® Pilot Centritech CELL 8® (Pneumatic Scale Angelus), Ksep® 6000S system (Sartorius), microfluidic cell retention devices (described in Kwon T. et al. Sci Rep 7, 6703 (2017)), SciLog® Examples include the SciPure™ system, the Acoustic Sedimentation System (described in Coronel J. et al. Front. Bioeng. Biotechnol., 02 July 2020) (Sonosep), and / or the EDO Electro System (described in Wang, Zhaowei, “Two Approaches for Cell Retention in Perfusion Culture Systems” (2009). ETD Archive. 304) (American Piezo). For example, depending on the scale and cell density, the Repligen ATF system in 2, 4, 6, or 10 filter unit format can be implemented. Alternative media exchange techniques can be performed, including tangential flow filtration (TFF) and / or centrifugation-based techniques.

[0163] Higher cell densities may require higher perfusion rates, medium exchange rates, and / or waste removal rates to maintain a particular cell growth rate or viability. Cell density may vary over time in cell cultures. Thus, a cell-specific perfusion rate, a parameter that quantifies the perfusion rate based on the cell density of the culture, may be used in the systems and methods described herein. In some embodiments, improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein include performing perfusion in the growth stage and / or production stage culture at a cell-specific perfusion rate (CSPR) of about 0.02 nL / cell / day to about 0.1 nL / cell / day, e.g., about 0.02 nL / cell / day to about 0.08 nL / cell / day, about 0.02 nL / cell / day to about 0.06 nL / cell / day, about 0.03 nL / cell / day to about 0.08 nL / cell / day, about 0.03 nL / cell / day to about 0.06 nL / cell / day, e.g., about 0.03 nL / cell / day, about 0.04 nL / cell / day, about 0.05 nL / cell / day, or about 0.06 nL / cell / day. In some embodiments, improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein include performing perfusion in growth stage and / or production stage cultures with a CSPR of about 100 pL / cell / day to about 2000 pL / cell / day, the range including all integers between 100 pL / cell / day and 2000 pL / cell / day. For example, the CSPR can be from about 100 pL / cell / day to about 1500 pL / cell / day, from about 100 pL / cell / day to about 1000 pL / cell / day, from about 100 pL / cell / day to about 900 pL / cell / day, from about 100 pL / cell / day to about 800 pL / cell / day, from about 100 pL / cell / day to about 900 pL / cell / day, from about 100 pL / cell / day to about 600 pL / cell / day, from about 100 pL / cell / day to about 500 pL / cell / day, from about 100 pL / cell / day to about 400 pL / cell / day, from about 100 pL / cell / day to about 300 pL / cell / day, or from about 100 pL / cell / day to about 200 pL / cell / day.In some embodiments, the CSPR can be about 150 pL / cell / day to about 750 pL / cell / day, for example, about 150 pL / cell / day, about 200 pL / cell / day, about 250 pL / cell / day, about 300 pL / cell / day, about 350 pL / cell / day, about 400 pL / cell / day, about 450 pL / cell / day, about 500 pL / cell / day, about 550 pL / cell / day, about 600 pL / cell / day, about 650 pL / cell / day, about 700 pL / cell / day, or about 750 pL / cell / day. It will be appreciated that the CSPR in a given system will vary depending on various parameters, including, for example, cell density.

[0164] In some embodiments, the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein include performing perfusion in a growth stage and / or production stage culture at a constant perfusion rate regardless of cell density.

[0165] b. Cell Media and Supplements The cell cultures described herein can be prepared in any medium suitable for the particular cells to be cultured. In some embodiments, the medium contains, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., compositions containing albumin, growth factors, and growth inhibitors, such as fetal bovine serum, newborn calf serum, and horse serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins from plant or animal sources), and combinations thereof. Exemplary suitable cell culture media include Minimum Essential Medium (MEM), such as Eagle's Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Alpha (MEM-alpha), Mesenchymal Cell Basal Medium (MSCBM), Ham's F-12 Medium and Ham's F-10 Medium, DMEM / F12 Medium, William's Medium E, RPMI-1640 Medium, MCDB Medium, Medium 199, Fisher's Medium, Iscove's Modified Dulbecco's Medium (IMDM), and McCoy's Modified Medium. In addition, any of the media described in Ham R., McKeehan W., (1979) Meth. Enzymol, 58:44-93, Barnes and Sato, (1980) Anal. Biochem., 102 (2):255-70, U.S. Pat. Nos. 4,657,866, 4,767,704, 4,927,762, 5,122,469, and 4,560,655, or International Publication Nos. WO90 / 03430 and WO87 / 00195 can be used as a culture medium in the systems and methods of the present disclosure.Any of these media can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), lipids (such as linoleic acid or other fatty acids) and their appropriate carriers, as well as glucose or an equivalent energy source. In some embodiments, the nutrient medium is a serum-free medium, a protein-free medium, or a chemically defined medium. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art.

[0166] In some embodiments, culturing AAV PCLs in the growth and / or production stage culture vessel (or combined growth / production vessel) includes supplementing nutrients, for example, using a fed-batch process, including periodically supplementing the culture with supplements (e.g., fresh medium, amino acids, and / or glucose). In some embodiments, supplementation is performed at least once every two days, for example, once a day, twice a day, three times a day, four times a day, or more. In further embodiments, supplementation is performed about once a day. In some embodiments, the supplement comprises an amino acid, for example, a mixture of one or more amino acids. In some embodiments, the supplement comprises glutamine. In some embodiments, the supplement comprises glucose. In some embodiments, the supplement comprises glucose and glutamine. In some embodiments, the concentration of the supplement added each time is determined based on the viable cell density, for example, the viable cell density determined at or just before the supplement is added. In some embodiments, the amount (e.g., volume and / or concentration) of the supplement is determined, for example, based on the integrated cell growth (ICG) in the growth and / or production stage culture. ICG can be calculated, for example, as described in International Patent Application Publication No. W02020 / 154607.

[0167] In some embodiments, the supplement comprises a total amino acid concentration of about 50 mM to about 2 M. In certain embodiments, the supplement comprises a total amino acid concentration of at least 50 mM. In some embodiments, the supplement comprises a total amino acid concentration of about 75 mM to about 500 mM. In some embodiments, the supplement comprises a total amino acid concentration of about 50 mM to about 1 M, about 50 mM to about 500 mM, about 50 mM to about 100 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 500 mM, about 750 mM, about 800 mM, about 1 M, about 1.5 M, or about 2 M.

[0168] In some embodiments, nutrient concentrations are maintained at desired levels and / or waste products are maintained at low levels, e.g., during the growth stage culture, e.g., during any day of the growth stage culture, or during the production stage culture. In some aspects, the concentrations of glucose, glutamine, glutamate, total amino acids, lactate, and / or ammonia in the N-1 culture are maintained at levels within 10-fold (e.g., within 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or less) of the concentrations on day 0 of the growth stage and / or production stage culture. In some embodiments, the concentrations of any of these molecules are measured, e.g., using standard methods, e.g., on any day of the culture.

[0169] c. Container volume per day In some embodiments, the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein include culturing cells during the growth and / or production stages at a medium exchange rate expressed in terms of vessel volumes of medium per day (VVD). Growth stage cultures can be performed at a medium exchange rate of about 1 VVD to about 10 VVD. For example, growth stage cultures can be performed at a medium exchange rate of 1 VVD, 1.5 VVD, 2 VVD, 2.5 VVD, 3 VVD, 3.5 VVD, 4 VVD, 4.5 VVD, 5 VVD, 5.5 VVD, 6 VVD, 6.5 VVD, 7 VVD, 7.5 WD, 8 VVD, 8.5 VVD, 9 VVD, 9.5 VVD, or 10 VVD. Similarly, production stage cultures can be performed at a medium exchange rate of about 1 VVD to about 10 VVD. For example, the production stage culture can be performed at a medium exchange rate of 1VVD, 1.5VVD, 2VVD, 2.5VVD, 3VVD, 3.5WD, 4VVD, 4.5VVD, 5VVD, 5.5VVD, 6VVD, 6.5VVD, 7VVD, 7.5VVD, 8VVD, 8.5VVD, 9VVD, 9.5VVD, or 10VVD. In some embodiments, the growth and production stage cultures are performed at a medium exchange rate of between 1VVD, 1.5VVD, 2VVD, 2.5VVD, 3VVD, 3.5VVD, 4WD, 4.5VVD, 5WD, 5.5VVD, 6VVD, 6.5VVD, 7VVD, 7.5VVD, 8VVD, 8.5VVD, 9VVD, 9.5VVD, or 10VVD.

[0170] d. Cell density and viability In some embodiments, the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein include culturing cells to a target cell density (e.g., expressed in terms of viable cell density) of at least 1E06 viable cells / mL (vc / mL), at least 1E07vc / mL, at least about 1E08vc / mL, or at least about 1E09vc / mL. In some embodiments, the methods described herein include culturing cells in an expansion stage culture to a target cell density of about 1E06vc / mL to about 50E06vc / mL, e.g., about 1E06vc / mL, about 2E06vc / mL, about 3E06vc / mL, about 4E06vc / mL, about 5E06vc / mL, 6E06vc / mL, 7E06vc / mL, 8E06vc / mL, 9E06vc / mL, 10E06vc / mL, 20E06vc / mL, 30E06vc / mL, 40E06vc / mL, or 50E06vc / mL.In some embodiments, the methods described herein provide for the administration of a medicament containing a medicament containing at least about 1E06vc / mL, at least about 1.1E06vc / mL, at least about 1.2E06vc / mL, at least about 1.3E06vc / mL, at least about 1.4E06vc / mL, at least about 1.5E06vc / mL, at least about 1.6E06vc / mL, at least about 1.7E06vc / mL, at least about 1.8E06vc / mL, at least about 1.9E06vc / mL, at least about 2E06vc / mL, at least about 2.1E06vc / mL, at least about 2.2E06vc / mL, at least about 2.3E06vc / mL, at least about 2.4E06vc / mL, at least about 2.5E06vc / mL, at least about 2.6E06vc / mL, at least about 2.7E06vc / mL, at least about 2.8E06vc / mL, at least about 2.9E06vc / mL, at least about 3E06vc / mL, at least about 3.1E06vc / mL c / mL, about 3.2E06vc / mL, about 3.3E06vc / mL, about 3.4E06vc / mL, about 3.5E06vc / mL, about 3.6E06vc / mL, about 3.7E06vc / mL, about 3.8E06vc / mL, about 3.9E06vc / mL, about 4E06vc / mL, about 4.1E06vc / mL, about 4.2E06vc / mL, about 4.3E06vc / mL, about 4.4E06vc / mL, about 4.5E06vc / mL, about 4.6E06vc / mL, about 4.7E06vc / mL, about 4.8E06vc / mL, about 4.9E06vc / mL, about 5E06vc / mL, or to a target cell density of greater than 5E06vc / mL. In some embodiments, the methods described herein include culturing cells in an expansion stage culture to a target cell density of about 1E06vc / mL to about 1E07vc / mL, about 1E07vc / mL to about 1E08vc / mL, or about 1E08vc / mL to about 1E09vc / mL. In some embodiments, the methods described herein include culturing cells in an expansion stage culture to a target cell density of about 1E06vc / mL to about 1.5E06vc / mL, about 1.5E06vc / mL to about 2E06vc / mL, about 2E06vc / mL to about 2.5E06vc / mL, about 2.5E06vc / mL to about 3E06vc / mL, about 3E06vc / mL to about 3.5E06vc / mL, about 3.5E06vc / mL to about 4E06vc / mL, about 4E06vc / mL to about 4.5E06vc / mL, or about 4.5E06vc / mL to about 5E06vc / mL.

[0171] In some embodiments, the methods described herein include determining the viable cell density periodically, e.g., at least once every 3 days, once every 2 days, once a day, twice a day, or more frequently, e.g., once per minute, twice per minute, three times per minute, 4-10 times per minute, once per hour, twice per hour, three times per hour, 4-10 times per hour, once per second, twice per second, three times per second, four times per second, five times per second, six times per second, seven times per second, eight times per second, nine times per second, or ten times per second.

[0172] e. Cell proliferation rate In some embodiments, the cell growth rate is maintained at a growth rate close to the maximum growth rate of the cultured cell line / clone. The systems and methods described herein can achieve a growth rate within 15% (e.g., within 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, 1%, or less) of the maximum growth rate of the cultured cell line / clone.

[0173] In some aspects, the maximum growth rate is the growth rate of a particular cell line / clone measured in fresh culture medium and during its exponential growth phase (e.g., measured at a time point when nutrients are sufficient and components in the culture do not cause significant growth inhibition). In some embodiments, the overall growth rate depends on the particular cell type / clone being cultured. In some embodiments, the overall growth rate of the cells is about 0.2 / day to about 1 / day, e.g., about 0.3 / day to about 0.8 / day, about 0.4 / day to about 0.7 / day, about 0.5 / day to about 0.7 / day, about 0.4 / day to about 0.6 / day, about 0.2 / day to about 0.8 / day, or about 0.5 / day to about 1 / day. In some embodiments, the overall growth rate is determined based on the cell density on day 0 of the culture.

[0174] f. pH In some embodiments of the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein, the pH of the culture during the growth and / or production phase is maintained at, for example, 5 to about 9, e.g., about 6.4 to about 8.2. For example, in some embodiments, the pH of the culture during the growth phase is maintained at about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, or about 8.2. In some embodiments, the pH of the production stage culture is maintained at about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, or about 8.2.

[0175] g. Dissolved oxygen In some embodiments of the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein, dissolved oxygen (DO) levels are maintained for optimal growth and / or AAV production in the growth and / or production stage cultures. For example, DO levels can be maintained between 10% and 100%, e.g., at a set point of 50%.

[0176] h.Culture period In some embodiments, AAV producer cells are cultured in growth and / or production cultures for 4 to 12 days, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days. In some embodiments, production cultures are maintained for 2 or more days, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0177] In some embodiments, the production stage culture is maintained under conditions that allow AAV production. In some embodiments, one or more components of AAV are added to the production stage culture so that the cells in the production stage culture are replication-competent and can produce AAV. In some embodiments, a helper virus, such as an adenovirus (e.g., Ad5 helper virus) or a herpes virus, is added to the production stage culture.

[0178] In certain embodiments, the method comprises culturing the cells in a production stage culture for less than 1 week, e.g., less than 7 days, e.g., less than 6 days, e.g., 5 days, 4 days, 3 days, 2 days, 1 day or less, e.g., 2 to 5 days, e.g., 2, 3, 4, or 5 days.

[0179] i.Culture volume In some embodiments, the growth stage culture is carried out at any suitable scale for the AAV production cell growth phase. For example, the growth stage culture can be carried out at a sub-liter to 6000L scale. Thus, the present system and method can utilize sub-liter, IL, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessels in the bioreactor for the growth stage culture.

[0180] In some embodiments, any pre-production culture is carried out at any suitable scale for the AAV production cell growth phase. For example, the pre-production culture can be carried out at a sub-liter to 6000L scale. Thus, the present system and method can utilize sub-liter, IL, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessels in bioreactors for pre-production or growth phase culture.

[0181] In some embodiments, the production stage culture is carried out at any suitable scale for AAV production. For example, the production stage culture can be carried out at a sub-liter to 6000L scale. Thus, the present system and method can utilize sub-liter, IL, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessels in the bioreactor for the production stage culture.

[0182] In some embodiments, the growth and production stage cultures are performed in a combined growth / production vessel at any suitable scale for the growth and production of AAV. For example, the growth / production stage cultures can be performed at sub-liter to 6000 L scale in the same combined growth / production vessel. Thus, the present systems and methods can utilize sub-liter, IL, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessels in the bioreactor for the production stage culture.

[0183] j. Downstream process The method described herein further comprises harvesting or recovering the AAV product from the production stage culture.For example, standard methods of harvesting or separating viral particles can be used, including but not limited to filtration or centrifugation.Furthermore, downstream purification processes can be used to purify the harvested AAV product.

[0184] According to the method described herein, for example, AAV titer in N culture can be determined, for example, on any one or more days of N culture. An exemplary AAV titer measurement is vector genome copies per cell (GC / cell). GC / cell can be determined by standard methods in the art, including, but not limited to, dot blot, quantitative PCR or ddPCR, spectroscopy, or fluorescence measurement. See, for example, Dorange et al. Cell Gene Therapy Insights 4.2(2018): 119-129.

[0185] In some embodiments, the methods described herein can provide, for example, a production stage culture of at least about 1E09vg / mL, at least about 2E09vg / mL, at least about 3E09vg / mL, at least about 4E09vg / mL, at least about 5E09vg / mL, at least about 6E09vg / mL, at least about 7E09vg / mL, at least about 8E09vg / mL, at least about 9E09vg / mL, at least about 1E10vg / mL, at least about 2E10vg / mL, at least about 3E10vg / mL, at least about 4E10vg / mL, at least about 5E10vg / mL, at least about 6E10vg / mL, at least about 7E10vg / mL, AAV titers of 10vg / mL, at least about 8E10vg / mL, at least about 9E10vg / mL, at least about 1E11vg / mL, at least about 2E11vg / mL, at least about 3E11vg / mL, at least about 4E11vg / mL, at least about 5E11vg / mL, at least about 6E11vg / mL, at least about 7E11vg / mL, at least about 8E11vg / mL, at least about 9E11vg / mL, at least about 1E12vg / mL, at least about 2E12vg / mL, at least about 3E12vg / mL, at least about 4E12vg / mL, at least about 5E12vg / mL, or more can be produced.

[0186] In this disclosure, when an element or component is said to be included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group consisting of two or more of the listed elements or components.

[0187] Furthermore, it should be understood that the elements and / or features of the compositions, systems, or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present disclosure. For example, when a particular compound or system component is mentioned, that compound or system component can be used in various embodiments of the disclosed compositions / systems and / or methods, unless otherwise understood from the context. In other words, within this disclosure, although embodiments have been described and depicted to enable a clear and concise disclosure to be written and depicted, it is intended and understood that the embodiments may be variously combined or separated without departing from the present teachings. For example, it should be understood that all features described and depicted herein may be applicable to all aspects of the invention described and depicted herein.

[0188] The phrase "at least one of" should be understood to include each of the listed objects individually and various combinations of two or more of the listed objects following the phrase, unless otherwise understood from context and usage. The phrase "and / or" in reference to two or more listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0189] Use of the terms "include," "includes," "including," "have," "having," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should generally be understood to be open-ended and non-limiting, e.g., not excluding additional, unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0190] It should be understood that the order of steps or order for performing certain actions in a system or process is not important so long as the objectives of the disclosure remain operable. Moreover, two or more steps or actions may occur simultaneously in many embodiments of the systems and methods described herein.

[0191] The use of any and all examples or exemplary language herein, such as "such as" or "including," is intended merely to better describe the invention of the present disclosure and does not limit the scope of the invention unless claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosed systems and methods for AAV production. EXAMPLES

[0192] The systems and methods of the present invention, having been generally described herein, will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to limit the disclosure.

[0193] material and method Metabolites (glucose, L-glutamine, ammonia, and lactate) were assessed in AAV-producing cell cultures by NovaBio-profile Flex2 (Nova Biomedical). Cell density was assessed in AAV-producing cell cultures by Vi-Cell XR (Beckman-Coulter). AAV titers were quantified by DNase-resistant particle (DRP) analysis by quantitative DNA polymerase chain reaction (qPCR) for specific AAV gene therapy products. Additionally, adenoviral by-products were assessed by DRP-qPCR for specific adenoviral genes, such as E2A.

[0194] Example 1: Increasing cell density in batch mode alone is not sufficient to increase the volumetric yield of AAV

[0195] High cell density production was tested in batch AAV production mode to determine whether increasing cell density could improve AAV volumetric yield. It was determined that increasing cell density in batch mode operation had a detrimental effect on AAV productivity.

[0196] HeLa producer cells were inoculated into bioreactors (2L wv) at 1.3E06 viable cells per milliliter (vc / mL), 3.15E06vc / mL, or 5.0E06vc / mL and infected with adenovirus type 5 (Ad5) to initiate AAV production for gene therapy products in batch mode operation. The AAV capsid was clade E capsid, AAV hu37. Batch mode conditions in this process utilized a target seeding density of 1.3E06vc / mL. Increasing the cell density by 2-fold and 4-fold (to 3.15E06 and 5E06 vc / mL) resulted in approximately 10-fold and 100-fold decreases in volumetric rAAV yield (Figure 4) and cell-specific rAAV productivity (Figure 5), respectively.

[0197] We observed rapid depletion of nutrients such as glucose and L-glutamine, and waste by-products (such as lactate and ammonia) increased significantly. Therefore, we hypothesized that nutrient depletion and / or waste accumulation led to a decline in AAV productivity (data not shown).

[0198] This example demonstrates that batch AAV production methods cannot support AAV production at higher cell densities. Indeed, increasing cell density in batch mode resulted in a greater than 100-fold decrease in AAV yield with a four-fold increase in seeding density for AAV production.

[0199] Example 2: Reducing pH at higher cell densities in batch mode increases AAV productivity Attempts were made to improve AAV productivity at higher densities in batch mode (e.g., above about 1E06vc / mL). It was determined that lowering the pH at higher cell densities would increase AAV productivity and reduce waste accumulation in batch mode. HeLa producer cells were inoculated into bioreactors (2L wv) at 1.3E06vc / mL or 3.15E06vc / mL and infected with Ad5 to initiate AAV production in batch mode operation at pH set points of pH 7.4, 7.6, or 7.8. In batch mode at 1.3E06vc / mL, a pH of 7.6 to 7.8 showed the highest rAAV yields (although not significantly different from the yields seen at pH 7.4) (Figures 6 and 7, solid bars), however, at a cell density of 3.15E06vc / mL, a pH of 7.4 showed higher AAV volumetric (Figure 6, empty bars) and specific productivity (Figure 7, empty bars) yields. At higher cell density (3.15E06vc / mL) and lower pH (7.4), AAV productivity increased, which was accompanied by a decrease in waste by-product accumulation (lactate) (Figure 8). This suggests that AAV productivity may be inhibited by waste by-product accumulation. Process and medium / feed modifications to reduce waste by-product accumulation (e.g., lowering pH to reduce lactate levels) and / or increased nutrient utilization may lead to improved AAV production capacity in higher cell density cultures.

[0200] Example 3: AAV and adenovirus cell release kinetics enable perfusion capacity AAV and Ad5 titers and cell viability were monitored over time in several 2L bioreactors (AAV, n=17 and Ad5, n=25) batch mode runs following infection with adenovirus. Cell infection with adenovirus is known to induce apoptosis through E1A, E3, and E4 genes, resulting in cell lysis (see, e.g., Jiang, Hong, et al. J. Virology 85.10 (2011): 4720-4729 and Chinnadurai, G. Seminars in VIROLOGY. Vol. 8. No. 5. Academic Press, 1998). When plotting % virus release against days post-infection, it was observed that the majority of AAV and adenovirus remained associated with the intracellular fraction during the first two days post-infection (Figure 9A). From day 2 to day 3 to day 4, an approximately 30% increase in non-cell-associated (i.e., free) AAV and adenovirus particles per day was observed. A plot of rAAV titers versus days post-infection (Figure 9B) showed the onset of rAAV release into the supernatant post-infection. Furthermore, a strong correlation of AAV and Ad5 release with cell death during production was observed (Figure 10), suggesting that virus release results from cell lysis. AAV and adenovirus release kinetics are important to understand when to start and how long perfusion should be maintained to maximize product yield and recovery. Based on the above findings from HeLa producer cells (Examples 1 and 2), we identified that the optimal perfusion initiation timing based on adenovirus cell uptake at different densities was approximately between 1-2 hpi (data not shown). Furthermore, because the majority of AAV remains intracellular for the first 2 days after helper virus infection, perfusion was maintained until 48 hpi to avoid significant product loss due to perfusion medium exchange.

[0201] These results also demonstrate that optimal recovery can be extended beyond 4 days to support maximum product recovery from culture supernatants, as only 70%-80% of AAV is released 4 days after infection and it may be important to extend recovery to 5, 6, 7 days, etc., or even longer, depending on the specific optimized culture AAV kinetics.

[0202] Example 4: Perfusion process optimization of medium exchange rate In batch mode operation, the cell specific perfusion rate (CSPR) at 3E06vc / mL and 5E06vc / mL was found to be 0.5nL / cell / day to 0.6nL / cell / day based on nutrient consumption and waste accumulation rates (data not shown). To evaluate the perfusion mode operation for improved batch AAV production at increased cell density, HeLa producer cells were inoculated at 5E06vc / mL in a 2L bioreactor equipped with an XCell Alternating Tangential Flow 2 (Repligen, ATF™ 2) system and infected with adenovirus helper virus to initiate AAV production. Perfusion was initiated 48 hours after infection with perfusion medium exchange rates of either 0, 1 vessel volume / day, or 2.5 vessel volumes / day (VVD) of production medium / feed mixture 2 hours after infection and compared to the batch mode control process. An increase in rAAV volumetric yield (Figure 11A) and specific productivity (Figure 12) with increasing VVD was observed. The highest levels were observed at 2.5VVD, where volumetric yield increased approximately 3-fold and cell-specific productivity was approximately 60% compared to the batch mode control process. Therefore, higher VVD conditions were tested up to 10VVD. Specifically, volumetric productivity was evaluated at 10VVD at cell densities of 12E06 cells / mL to 13E06 cells / mL. Compared to the 2.5VVD condition, volumetric productivity increased under conditions of increased perfusion rate and cell density (Figure 11B), and an increase in extracellular rAAV product was observed compared to the 2.5VVD condition on each of days 2 to 5.

[0203] Glucose and glutamine levels were rapidly depleted in high cell density cultures with either 0 or 1 VVD, but were maintained at 2.5 VVD over 48 h during perfusion exchange, similar to the batch mode control process (Figure 13, left two graph panels). Lactate levels increased significantly in the 0 VVD condition, while the 1 and 2.5 VVD conditions were consistent with the batch mode control (Figure 13, top right graph panel). All ammonium concentrations were higher in the high density perfusion condition, but remained lower in the 2.5 VVD condition compared to the 0 and 1 VVD conditions (Figure 13, bottom right graph panel), indicating that perfusion medium exchange reduces waste accumulation and nutrient depletion from 2 to 48 hpi. All ammonium concentrations were higher in the high-density perfusion conditions but remained lower in the 2.5 VVD condition compared to the 0 and 1 VVD conditions (Figure 13, bottom right graph panel), indicating that ammonium concentrations were reduced by reducing waste accumulation and nutrient depletion due to perfusion medium exchange from 2 to 48 hpi.

[0204] These data indicate that increasing the perfusion medium exchange rate improved cell-specific AAV productivity at high density and increased rAAV volumetric yield.

[0205] Example 5: Scale-up of the improved batch process to 50 L AAV production was performed using the improved batch process at 2 L scale using ATF2 and at 50 L scale using ATF6 with scaled operating parameters and productivity was compared to the batch mode control process at 2 L scale. AAV volumetric productivity was maintained at 2 L and 50 L scale using the improved batch process, achieving an approximately 2.3-fold improvement in volumetric yield over the batch mode control (FIG. 14) (3.3E11 GC / mL improved batch process vs. 1.4E11 GC / mL batch mode process) and cell specific productivity was approximately 60% compared to the batch mode process (FIG. 15).

[0206] These data demonstrate that the improved batch process for AAV production is scalable and consistently results in fold-increases in volumetric yield despite a modest decrease in specific productivities.

[0207] As shown in Figure 16, the viable cell density (VCD) increases in the improved batch process during the production phase compared to the batch mode control process at 1, 2, 3, and 4 dpi. The cell viability profile is similar in the 2L and 50L improved batch processes compared to the batch mode control process (Figure 17).

[0208] Metabolite analysis demonstrates that the improved batch process maintains similar nutrient and waste levels compared to the batch mode control process. For example, glucose and L-glutamine levels are maintained at similar levels through 2 dpi compared to the batch process (Figure 18), and lactate and ammonium accumulation is comparable to the batch process (Figure 19). These data demonstrate that the improved batch process allows for increased viable cell density in a scalable manner compared to the batch mode control process by maintaining critical nutrient consumption and waste accumulation rates, resulting in higher AAV productivity per volume.

[0209] Furthermore, as shown in Figure 20A, the percentage of total capsids determined to contain complete vector genomes as assessed by AUC analysis was found to be consistent in the 2L and scaled-up improved batch processes, indicating that the improved batch process does not impair AAV packaging efficiency. Indeed, further analysis demonstrated that the packaging efficiency utilizing the improved batch process described herein was improved with increased VVD. As shown in Figure 20B, the ratio of intermediate to complete AAV particles decreased with increasing VVD using the improved batch process compared to the control batch production process. The observed enrichment in complete species and decrease in intermediate species as WD increased suggested that further waste removal / nutrient enrichment would be beneficial to the quality of the AAV product.

[0210] Example 6: The improved batch AAV production process is universal and can be performed with different media, vector genomes / genes of interest, and AAV capsid serotypes. An improved batch process was developed and optimized using Program A, media composition A, and capsid A (clade E capsid, AAV hu37) and compared to a different capsid serotype B (clade E, AAV8), and a different gene of interest (Program B) with a different media composition B. In both Processes A and B, we observed a 2.3- to 3-fold increase in AAV volumetric productivity (Figure 21A, left graph panel), indicating that our technology can be universally applied to other AAV processes, media, and culture conditions, as well as other capsid serotypes. In Process A, we observed approximately 60% cell-specific productivity compared to the batch mode control, and in Process B, we observed approximately 80% cell-specific productivity (Figure 21A, right graph panel), indicating that optimization of process parameters and media composition can result in higher-yielding production conditions that more closely match batch mode AAV cell-specific production rates.

[0211] The improved batch process was further tested using a different program (Program C) with a different gene of interest than Programs A and B, a different media composition than Programs A and B, and a different capsid (clade F capsid, AAV9) than Programs A and B, and the results were compared to the batch mode process. The improved batch process increased the initial cell density of infection from 1.17E6 cells / mL to 4.5E6 cells / mL. At harvest, using the improved batch process described herein, the yield increased from 7.1E10 GC / mL to 3.1E11 GC / mL, an approximately four-fold increase (Figure 21B). Cell health and metabolite profiles (data not shown) were comparable to those seen in other runs using the improved batch process.

[0212] These data indicate that the improved batch method (e.g., enhanced perfusion) is applicable to a wide range of products, processes, and production conditions with a variety of media conditions, vector genomes / genes of interest, and AAV capsid serotypes.

[0213] Example 7: Lactate and ammonia waste by-products inhibit AAV production As described in Example 1, high cell density in batch mode AAV production resulted in a significant increase in waste by-product formation, including lactate and ammonia / ammonium. To determine the effect of these waste by-products on AAV production, production was tested in batch AAV production mode with varying levels of ammonia, lactate, or both. Figure 22 showed that increased lactate (30 mM) (shown as "High Lac" in Figure 22) or increased ammonia (3 mM) (shown as "High NH4" in Figure 22) reduced rAAV yield to approximately 60% of the control rAAV recovery yield condition, while the addition of both increased lactate and ammonia (shown as "Both" in Figure 22) reduced rAAV yield to approximately 35% of the control rAAV recovery yield condition. This example confirms that high cell density AAV production methods require effective waste by-product removal or mitigation to achieve optimal yield.

[0214] Example 8: Specific productivity increases with increasing CSPR As shown in Figure 23, increasing the cell-specific perfusion rate from 150 pL / cell / day to 750 pL / cell / day increased specific productivity (measured in genome copies (GC) per cell). A strong linear correlation of AAV cell-specific yield (GC / cell) with CSPR (pL / cell / day) was observed up to approximately 750 pL / cell / day (R 2 =0.91). No clear limit (or plateau) in cell-specific productivity was observed with increasing perfusion rate, suggesting that further increases in CSPR rate beyond 750 pL / cell / day would continue to increase AAV productivity within our system. These data demonstrate that the conditions tested to date have not revealed a plateau in cell-specific productivity, and illustrate that the improved batch process described herein may achieve further increases in cell-specific productivity, possibly beyond those seen using batch mode AAV production processes.

[0215] Example 9: Improved batch AAV manufacturing process can significantly reduce cost of goods Cost of goods (COG) projections demonstrate the potential to reduce costs associated with AAV production by using the improved batch (e.g., enhanced perfusion) production process described herein. As shown in Figures 24A-C, given the higher initial cost per AAV production batch, the gain in productivity per volume is projected to be greater than the increase in material and operating costs required to run the improved batch process. As a result, fewer production batches, shorter time frames to generate material, and lower overall COG can be achieved using the improved batch process described herein. Figure 24A shows that the approximate predicted COG required per AAV yield is reduced in the improved batch process compared to batch production. Figure 24B shows that the approximate number of production batches required per AAV yield is reduced in the improved batch process compared to batch production. Figure 24C shows that the approximate predicted COG required per number of production batches is reduced in the improved batch process compared to batch production. In Figures 24A-24C, the diamonds or circles represent the improved batch systems described herein and the squares represent the batch operating conditions.

[0216] Example 10: Transitional perfusion schedule for replacing formulated growth medium Growth and production formulated media were used in the N growth stage to increase cell density and balance the growth and production needs of the cells. However, the use of formulated media has drawbacks, including the lack of stability data for the formulated media, the time required for preparation and general availability of the formulated media, and higher media consumption during the growth stage. To determine whether a timed transition from growth to production media could avoid the need for formulated media, production media was perfused into the reactor 24 hours prior to infection with 2.5VVD. No significant differences in rAAV titers were observed between the transition to production media and the use of formulated media for either released or intracellular rAAV (Figure 25), indicating that the transition to perfusion media prior to infection can replace the need for formulated media.

[0217] The large amount of medium required for perfusion-based production processes remains a major limitation despite increased rAAV productivity. For example, perfusion-based production can require 8.5 or more vessel volumes of perfusion medium per run, or 17,000 L of medium at a 2,000 L production scale. Therefore, to investigate approaches to limit medium consumption, a reduction in the perfusion rate during the growth phase was tested using a transitional perfusion schedule. Figure 26 shows that the perfusion rate during the growth phase can be reduced to about 0.5 VVD (from about 1 VVD) and the perfusion rate during the transition phase can be reduced to about 1.5 VVD (from about 2.5 VVD), representing a saving of about 3,000 L of medium per batch without losing titer. In Figure 26, VVD condition 1 is 1 VVD during the proliferation phase and 2.5 VVD during the transition phase, condition 2 is 1 VVD during the proliferation phase and 1.5 VVD during the transition phase, condition 3 is 0.5 VVD during the proliferation phase and 2.5 VVD during the transition phase, and condition 4 is 0.5 VVD during the proliferation phase and 1.5 VVD during the transition phase.

[0218] Example 11: Scaling up of the improved batch process to 250 L scale Example 5 shows the successful scale-up of the improved batch process to 50L scale. To further evaluate the scalability of the improved batch rAAV production process, a test pilot scale run was conducted at 250L using ATF6 with scaled operating parameters, and productivity was compared to the batch mode control process at 2L scale (shown as "Batch" in FIG. 27), the improved batch process at 2L (shown as "MB-2L" in FIG. 27), the improved batch process at 50L (shown as "MB-50L" in FIG. 27), and the improved batch process at 250L (shown as "MB-250L" in FIG. 27). In the 50L run, a blended growth medium and production medium were used in the growth phase. In the 250L run, a 1VVD perfusion rate was used for the growth phase and 2.5VVD was used for the transition phase. rAAV titers at harvest indicated that an approximately 3-4 fold increase in volumetric yield was achieved with an approximately 3-4 fold increase in viable cell density (VCD). As shown in Figure 27, the improved batch process (abbreviated as "MB" in Figures 27 and 28) maintained yield increases at the 250 L scale (approximately 5E11 GC / mL) comparable to the 2 L scale versus the batch process (less than 2E11 GC / mL). As shown in Figure 28, cell-specific productivities were maintained at the 250 L scale comparable to those seen at the 50 L and 2 L scales.

[0219] These data further confirm that the improved batch process for AAV production is scalable and consistently results in fold-increases in volumetric yield despite a modest decrease in specific productivities.

Claims

1. 1. A method for producing a recombinant adeno-associated virus (rAAV), comprising: (a) culturing AAV-producing host cells in a growth vessel to a target cell density of at least about 1E06 viable cells / mL (vc / mL) using perfusion to replenish the culture with fresh nutrients and / or remove waste products, wherein the AAV-producing host cells comprise genetic material encoding one or more AAV components; (b) initiating expression of one or more helper virus functions in the AAV-producing host cells of step (a) to initiate rAAV production; (c) culturing the AAV-producing host cells of step (b) in a production vessel using perfusion to replenish the culture with fresh nutrients and / or remove waste products; thereby producing said rAAV.

2. The method described in claim 1, wherein the AAV-producing host cells are HeLa cells, Cos-7 cells, HEK293 cells, A549 cells, BHK cells, Vero cells, RD cells, or ARPE-19 cells.

3. The method described in claim 1, wherein the AAV-producing host cell comprises an AAV-producing cell line (PCL) containing genetic material encoding one or more AAV components stably integrated into the AAV-producing host cell genome.

4. The method described in claim 3, wherein the AAV PCL comprises a genetic modification to reduce expression of lactate dehydrogenase.

5. The method described in claim 3, wherein the AAV PCL comprises a genetic modification to increase expression of glutamine synthetase.

6. A method described in any one of claims 1 to 5, wherein the production vessel has a volume of 1 L to 12,000 L.

7. The method of claim 1, wherein the growth vessel and the production vessel are a combined growth / production vessel.

8. The method of any one of claims 3 to 5, wherein the one or more AAV components comprise a therapeutic payload or transgene, one inverted terminal repeat (ITR) or two ITRs, one or more AAV replication and / or packaging proteins encoded by the AAV rep gene, and / or one or more AAV structural capsid proteins encoded by the AAV cap gene.

9. Step (b) (i) infecting the AAV-producing host cells of step (a) with a helper virus; or (ii) inducing expression of one or more helper virus functions encoded by the genetic material in said AAV-producing host cell. The method according to any one of claims 1 to 5, comprising:

10. A method according to any one of claims 1 to 5, wherein the perfusion is carried out at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber.

11. The perfusion (i) a medium exchange rate of about 0.5 vessel volumes per day (VVD) to about 10 VVD; or (ii) a cell-specific perfusion rate (CSPR) of about 150 pL / cell / day to about 2000 pL / cell / day, or greater than 750 pL / cell / day; The method according to any one of claims 1 to 5, wherein the method is carried out in 12. (i) step (a) comprises culturing the AAV-producing host cells in a growth medium or a combination of a growth medium and a production medium; (ii) step (c) comprises culturing the AAV-producing host cells in a production medium or a combination of a growth medium and a production medium; and (iii) The method of any one of claims 1 to 5, wherein the perfusion in step (c) is initiated about 1 hour to 6 hours after step (b).

13. A method described in any one of claims 1 to 5, comprising a step of culturing the AAV-producing host cells for about 48 hours and / or about 2 days to about 14 days during the growth and / or production stage.

14. The method of claim 13, comprising culturing the AAV-producing host cells during the production stage for a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days, and then stopping perfusion after the production stage period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

15. The method of claim 14, wherein the perfusion in step (c) is stopped approximately 48 hours after step (b).

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