Improved batched AAV generation system and method

Abstract

The present application provides improved batch systems and methods that use perfusion (e.g., enhanced perfusion) to increase the production rate and / or cell density in viral particle production to achieve high viral particle (e.g., AAV yield).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 317,298, filed on March 7, 2022; U.S. Provisional Patent Application No. 63 / 398,355, filed on August 16, 2022; and U.S. Provisional Patent Application No. 63 / 481,539, filed on January 25, 2023, the disclosures of each of which are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure relates to improved batch (e.g., intensified perfusion) systems and methods for manufacturing viral vectors, such as adeno-associated viral (AAV) vectors, which can increase clinical and commercial-grade AAV manufacturing yields. Background Art

[0004] Adeno-associated virus (AAV) is a leading platform for gene delivery for a variety of human disease therapies; however, the high dose requirements of AAV-based therapies and the low batch yields in AAV production have resulted in significantly high manufacturing costs and limited supply that cannot meet patient needs. Despite advances in AAV manufacturing, including the development of mammalian producer cell lines that can be used for suspension culture, there is still a significant need to improve existing AAV manufacturing technologies to achieve robust, high-yield, scalable and cost-effective processes to meet patient supply needs and reduce the total cost of treatment using AAV-based therapies. Increasing cell density in batch mode alone is not enough to increase AAV volume production, and in fact, for example, when a 4-fold increased inoculation density is used for AAV production, it may result in a more than 100-fold reduction in production. Therefore, new systems and methods are needed to achieve higher yields of AAV production to meet clinical and therapeutic needs, as well as to reduce the cost of AAV therapy development and clinical and commercial manufacturing.

[0005] The present disclosure addresses these needs by providing a cost-effective, scalable AAV production process and system that is compatible with mammalian producer cell lines in suspension culture that reduces the cost of goods associated with existing AAV manufacturing platforms. SUMMARY OF THE INVENTION

[0007] The present disclosure provides improved batch (eg, intensified perfusion) systems and methods for AAV manufacturing using perfusion in AAV producer cell cultures (eg, AAV producer cell suspension cultures).

[0008] The present disclosure provides improved batch systems and methods for AAV production using perfusion. In some aspects, the present disclosure provides systems and methods for producing recombinant adeno-associated virus (rAAV), the systems and methods comprising the following steps: (a) using perfusion to replenish the culture with fresh nutrients and / or remove waste products to cultivate AAV production host cells in a growth container to a target cell density, wherein the AAV production host cells contain genetic material encoding one or more AAV components; (b) initiating expression of one or more helper virus functions in the AAV production host cells of step (a) to initiate rAAV production; and (c) using perfusion to replenish the culture with fresh nutrients and / or remove waste products to cultivate the AAV production host cells of step (b) in a production container, thereby producing rAAV.

[0009] In some embodiments of the systems and methods described herein, the AAV production 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.

[0010] In some embodiments of the systems and methods described herein, the AAV producing host cell comprises an AAV producer cell line (PCL) (e.g., a mammalian or insect PCL) comprising genetic material encoding one or more AAV components stably integrated into the genome of the AAV producing host cell. 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 the production or accumulation of lactate and / or ammonia.

[0011] 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) can be between 1E06 vc / mL and 5E07 vc / mL. In some embodiments, the target cell density in step (a) is about 1E06 vc / mL, about 2E06 vc / mL, about 3E06 vc / mL, about 4E06 vc / mL, about 5E06 vc / mL, about 6E06 vc / mL, about 7E06 vc / mL, about 8E06 vc / mL, about 9E06 vc / mL, about 1E07 vc / mL, about 1.1E07 vc / mL, about 1.2E07 vc / mL, about 1.3E07 vc / mL, about 1.4E07 vc / mL, about 1.5E07 vc / mL, about 1.6E07 vc / mL, about 1.7E07 vc / mL, about 1.8E07 vc / mL, about 1.9E07 vc / mL, or about 2E07 vc / mL. In some embodiments, the target cell density in step (a) is about 3.15E06 vc / mL or about 5.0E06 vc / mL.

[0012] 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, a volume of 2 L, a volume of 5 L, a volume of 10 L, a volume of 20 L, a volume of 50 L, a volume of 100 L, a volume of 150 L, a volume of 200 L, a volume of 250 L, a volume of 500 L, a volume of 1000 L, a volume of 1500 L, a volume of 2000 L, a volume of 2500 L, a volume of 3000 L, a volume of 3500 L, a volume of 4000 L, a volume of 4500 L, a volume of 5000 L, a volume of 5500 L, a volume of 6000 L, or a volume of 12000 L.

[0013] In some embodiments of the systems and methods described herein, the growth vessel and the production vessel comprise a combined growth / production vessel.

[0014] 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.

[0015] 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.

[0016] In some embodiments of the systems and methods described herein, step (b) comprises inducing expression of one or more helper virus functions encoded by the genetic material in the AAV-producing host cell.

[0017] 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 can be 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.

[0018] In some embodiments of systems and methods described herein, perfusion is carried out with a culture medium exchange rate of about 0.5 container volume / day (VVD) to about 10VVD. For example, perfusion can be carried out with a culture medium exchange rate of about 0.5VVD, about 1VVD, about 1.5VVD, about 2VVD, about 2.5VVD, about 3VVD, about 3.5VVD, about 4VVD, about 4.5VVD, about 5VVD, about 5.5VVD, about 6VVD, about 6.5VVD, about 7VVD, about 7.5VVD, about 8VVD, about 8.5VVD, about 9VVD, about 9.5VVD or about 10VVD. In some embodiments of systems and methods described herein, perfusion is carried out with a culture medium exchange rate less than 1VVD. 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, such as 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.

[0019] In some embodiments of the systems and methods described herein, perfusion is performed using a perfusion system selected from: Xcellerex Automated Perfusion System (APS) (Cytiva); KPS TFF System (Repligen); XCell with 2, 4, 6, 8 or 10 ATF cells TM ATF System (Repligen); GEA Disposable Drug Separator System (GEA); Prostak TM Microfiltration module system (Millipore); Alfa Laval CultureOne TM System (AlfaLaval); Centritech Separation System CARR Pilot or Centritech CELL System (Pneumatic Scale Angelus); 6000S system (Sartorius); microfluidic cell retention device; SciPure TM system (Parker); acoustic settler system; and EDO Electro system (American Piezo).

[0020] In some embodiments of the systems and methods described herein, step (a) includes culturing AAV to produce host cells in a growth medium; step (a) and step (c) include culturing AAV to produce host cells in a growth medium; step (a) includes culturing AAV to produce host cells in a production medium; or step (a) and step (c) include culturing AAV to produce host cells in a production medium. In some embodiments of the systems and methods described herein, step (a) includes culturing AAV to produce host cells in a growth medium, and step (c) includes culturing AAV to produce host cells in a production medium. In some embodiments, the production medium is perfused after the AAV production host cells are infected with a helper virus. In some embodiments, the perfusion of the production medium is started 1 hour-6 hours (e.g., about 1 hour-2 hours, about 2 hours-3 hours, about 3 hours-4 hours, about 4 hours-5 hours, or about 5 hours-6 hours) after the cells are infected with a helper virus.

[0021] In some embodiments of the systems and methods described herein, steps (a) and (c) comprise culturing the AAV producing host cells in a blend of a growth medium and a production medium. For example, the blend of the growth medium and the production medium can be a 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20, or 90 / 10 blend of growth medium and production medium.

[0022] In some embodiments of the systems and methods described herein, step (a) includes culturing the AAV producing host cells in a growth medium until about 24 hours before step (b), and then transitioning to a production medium. The growth medium can 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) before step (b). The production medium can be perfused at a rate of about 1.5 VVD to about 2.5 VVD within about 24 hours before step (b) (e.g., 24 hours or less before step (b), including up to about 1 hour before step (b) and 0 hours before step (b)).

[0023] In some embodiments of the systems and methods described herein, the AAV producing host cells are cultured in a growth phase culture and / or a production phase culture for about 48 hours and / or about 2 days to about 14 days. For example, the AAV producing host cells are cultured in the production phase for a duration 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.

[0024] Perfusion can be stopped after a production phase duration 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.

[0025] The systems and methods described herein can allow rAAV to be released from AAV producing host cells into the supernatant of the production culture medium. Such release can be continuous throughout the production period. In some embodiments, rAAV begins to be released mainly after about 48 hours of infection of cells with helper virus. That is, most of the rAAV produced can be released into the supernatant after about 48 hours of infection.

[0026] In some embodiments, rAAV begins to be released primarily after about 48 hours of perfusion in the production medium. That is, most of the rAAV produced can be released into the supernatant after about 48 hours of perfusion in the production medium. In some embodiments, perfusion is stopped to retain rAAV in the supernatant. In some embodiments, perfusion is stopped about 48 hours after infection.

[0027] Some embodiments of the systems and methods described herein also include harvesting rAAV (e.g., under batch mode conditions, such as in the absence of perfusion) and / or downstream processing of rAAV. For example, the systems and methods may also include purifying, clarifying and / or concentrating the rAAV produced according to the methods described herein. Downstream processing may include filtering cell debris, colloids, or aggregates. In some embodiments, filtration removes particles of cell debris, colloids, or aggregates greater than 1 μm in size. Some embodiments include purifying rAAV using anion exchange (AEX) chromatography.

[0028] In other aspects, the present disclosure provides methods of producing recombinant adeno-associated virus (rAAV) comprising culturing AAV-producing host cells using a perfusion system, wherein the AAV-producing host cell culture has a target cell density between 1E06 vc / mL and 5E07 vc / mL.

[0029] 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.

[0030] In some embodiments, the AAV producing host cell comprises an AAV producer cell line (PCL) comprising 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 the production or accumulation of lactate and / or ammonia.

[0031] In some embodiments, the target cell density is about 5E06 vc / mL to about 5E07 vc / mL. For example, the target cell density can be about 1E06 vc / mL, about 2E06 vc / mL, about 3E06 vc / mL, about 4E06 vc / mL, about 5E06 vc / mL, about 6E06 vc / mL, about 7E06 vc / mL, about 8E06 vc / mL, about 9E06 vc / mL, about 1E07 vc / mL, about 1.1E07 vc / mL, about 1.2E07 vc / mL, about 1.3E07 vc / mL, about 1.4E07 vc / mL, about 1.5E07 vc / mL, about 1.6E07 vc / mL, about 1.7E07 vc / mL, about 1.8E07 vc / mL, about 1.9E07 vc / mL, or about 2E07 vc / mL. In certain embodiments, the target cell density is about 3.15E06 vc / mL or about 5.0E06 vc / mL.

[0032] In some embodiments, the method can be performed 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, a volume of 2 L, a volume of 5 L, a volume of 10 L, a volume of 20 L, a volume of 50 L, a volume of 100 L, a volume of 150 L, a volume of 200 L, a volume of 250 L, a volume of 500 L, a volume of 1000 L, a volume of 1500 L, a volume of 2000 L, a volume of 2500 L, a volume of 3000 L, a volume of 3500 L, a volume of 4000 L, a volume of 4500 L, a volume of 5000 L, a volume of 5500 L, a volume of 6000 L, or a volume of 12000 L.

[0033] In some embodiments, the method further comprises growing the AAV-producing host cells to a target cell density in the combined growth / production vessel using a perfusion system.

[0034] In some embodiments, 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.

[0035] In some embodiments, AAV produces host cells and is cultured at a target cell density under conditions suitable for producing rAAV. Suitable conditions for AAV production can include infection with a helper virus such as adenovirus (e.g., Ad5) to start AAV production, triple transfection with AAV to produce plasmids, and / or induction of inducible producer cells with stable integration of components required for producing AAV. In some embodiments, the method includes starting the expression of one or more helper virus functions in AAV production host cells, thereby starting rAAV production. In some embodiments, starting the expression of one or more helper virus functions includes infecting AAV production host cells with helper viruses. In some embodiments, starting the expression of one or more helper virus functions includes inducing the expression of one or more helper virus functions encoded by genetic material in AAV production host cells. In some embodiments, the helper virus is adenovirus. In some embodiments, the helper virus is Ad5.

[0036] In some embodiments, the perfusion system is operated at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber. For example, the perfusion system can be operated 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.

[0037] In some embodiments, the perfusion system is operated with a culture medium exchange rate of about 0.5 container volume / day (VVD) to about 10VVD. For example, the perfusion system can be operated with a culture medium exchange rate of about 0.5VVD, about 1VVD, about 1.5VVD, about 2VVD, about 2.5VVD, about 3VVD, about 3.5VVD, about 4VVD, about 4.5VVD, about 5VVD, about 5.5VVD, about 6VVD, about 6.5VVD, about 7VVD, about 7.5VVD, about 8VVD, about 8.5VVD, about 9VVD, about 9.5VVD or about 10VVD. In some embodiments of systems and methods described herein, perfusion is carried out with a culture medium exchange rate less than 1VVD. 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.

[0038] 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, such as about 150 pL / cell / day to about 750 pL / cell / day. In some embodiments, perfusion is performed at a CSPR greater than 750 pL / cell / day.

[0039] In some embodiments, the perfusion system can be one selected from the following: Xcellerex Automated Perfusion System (APS) (Cytiva); KPS TFF System (Repligen); XCell with 2, 4, 6, 8 or 10 ATF cells TM ATF System (Repligen); GEA Disposable Drug Separator System (GEA); Prostak TM Microfiltration module system (Millipore); Alfa Laval CultureOne TM System (Alfa Laval); Centritech Separation System CARR Pilot or Centritech CELL System (Pneumatic Scale Angelus); 6000S system (Sartorius); microfluidic cell retention device; SciPure TM system (Parker); acoustic sedimentation system; and EDO Electro system (American Piezo).

[0040] In some embodiments, AAV produces host cells and is cultivated in growth medium, and then is cultivated in production medium. Transition to production medium can occur in about 0 hours to about 48 hours before AAV produces host cells with helper virus infection or about 24 hours before AAV produces host cells with helper virus infection. Growth medium can be perfused with a rate of about 0.5VVD to about 1VVD, until about 24 hours (for example, 24 hours before or less hours, including about 1 hour before and 0 hours before) before AAV produces host cells with helper virus infection. Production medium can start to perfuse with a rate of about 1.5VVD to about 2.5VVD in about 24 hours before AAV produces host cells with helper virus infection. AAV produces host cells and can be infected with helper virus (such as Ad5) before cultivation in growth medium and before cultivation in 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, or about 5 hours to 6 hours) after the cells are infected with the helper virus.

[0041] In some embodiments, the AAV host producer cells are cultured at a target cell density in a blend of growth medium and production medium. For example, the blend 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 blend of growth medium and production medium.

[0042] In some embodiments, the method comprises culturing the AAV-producing host cells at the target cell density for about 48 hours and / or about 2 days to about 14 days. For example, the method may comprise culturing the AAV-producing host cells at the target cell density for a duration 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.

[0043] The systems and methods described herein can allow rAAV to be released from AAV producing host cells into the supernatant of the production culture medium. Such release can be continuous throughout the production period. In some embodiments, rAAV begins to be released mainly after about 48 hours of infection of cells with helper virus. That is, most of the rAAV produced can be released into the supernatant after about 48 hours of infection with helper virus.

[0044] In some embodiments, rAAV begins to be released primarily after about 48 hours of perfusion in the production medium. That is, most of the rAAV produced can be released into the supernatant after about 48 hours of perfusion in the production medium. In some embodiments, perfusion is stopped to retain rAAV in the supernatant. In some embodiments, perfusion is stopped at about 48 hours. In some embodiments, perfusion is stopped at about 48 hours after infection.

[0045] In some embodiments, the method further comprises harvesting rAAV (e.g., under batch mode conditions, such as in the absence of perfusion) and / or downstream processing rAAV. For example, downstream processing may include purifying, clarifying and / or concentrating rAAV. Downstream processing may include filtering cell debris, colloids, or aggregates. In some embodiments, filtering removes particles of cell debris, colloids, or aggregates greater than 1 μm in size. Some embodiments include purifying rAAV using anion exchange (AEX) chromatography.

[0046] These and other aspects and features of the present disclosure are described in the following sections of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The foregoing and other objects, features and advantages of the systems and methods disclosed herein will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like referenced elements identify common features in corresponding drawings.

[0049] Figure 1 is a schematic diagram of an exemplary batch-mode process for AAV production (ie, a "batch-mode control process").

[0050] Figure 2 is a process flow diagram of an exemplary process for AAV production using the improved batch system and methods of perfusion according to the present invention.

[0051] Figure 3 is a schematic diagram of an exemplary process for AAV production according to the improved batch system and methods using perfusion described herein.

[0052] Figure 4 is a bar graph representation of volumetric rAAV production in a batch mode control process. The bar graph shows that in a batch mode control process, as the seeding density increases, the volumetric rAAV production decreases.

[0053] Figure 5 is a bar graph representation of the cell specific productivity in a batch mode control process. The bar graph shows that in a batch mode control process, as the seeding density increases, the cell specific productivity decreases.

[0054] Figure 6 is a bar graph representation of 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) inoculation densities in a batch mode control process.

[0055] Figure 7 is a bar graph representation of the effect of pH on cell specific productivity in a batch mode control process. 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) inoculation densities in a batch mode control process.

[0056] Figure 8 is a graph showing the effect of pH on lactic acid accumulation at 1.3E06 and 3.15E06 inoculation densities in a batch mode control process. (Abbreviations used in the legend: 1.3E06 inoculation density and pH 7.4 are represented as "1.3, 7.4"; 1.3E06 inoculation density and pH 7.6 are represented as "1.3, 7.6"; 1.3E06 inoculation density and pH 7.8 are represented as "1.3, 7.8"; 3.15E06 inoculation density and pH 7.4 are represented as "3.15, 7.4"; 3.15E06 inoculation density and pH 7.6 are represented as "3.15, 7.6"; 3.15E06 inoculation density and pH 7.8 are represented as "1.3, 7.8").

[0057] Fig. 9A is a bar graph showing the percentage of virus release (AAV and helper virus) over time in a batch mode control process. Fig. 9B is a bar graph showing the release of rAAV into the supernatant after infection.

[0058] Fig.10 is a graph showing the percentage of virus release (AAV and helper virus) relative to the percentage of dead cells in culture in a batch mode control process.

[0059] Fig.11A is a bar graph representation of rAAV volumetric yield in an improved batch AAV manufacturing process and a batch-mode control process. The graph shows that rAAV volumetric yield increases with increasing VVD in the improved batch AAV manufacturing process compared to the batch-mode control process. Fig. 11B is a bar graph representation comparing rAAV volumetric yields of low perfusion rate versus high perfusion rate processes. The graph shows that as VVD and cell density are further increased, rAAV volumetric yields further increase.

[0060] Fig.12 is a bar graph representation of cell specific productivity in an improved batch AAV manufacturing process. The graph shows that in the improved batch AAV manufacturing process, as VVD increases, the cell specific productivity increases.

[0061] Fig.13 are bar graphs showing nutrient levels (glucose and glutamine, upper and lower left panels) and waste product levels (lactic acid and ammonium, upper and lower right panels) in the modified batch process at medium exchange rates of 0, 1 and 2.5 VVD.

[0062] Fig.14 is a bar graph showing volumetric rAAV yield using a modified batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0063] Fig.15 is a bar graph showing the cell specific productivity using the modified batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0064] Fig.16 is a graph showing the viable cell density (VCD) using the modified batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0065] Fig.17 is a graph showing cell viability using the improved batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0066] Fig.18 are graphs showing nutrient levels (glucose, left panel and glutamine, right panel) in the modified batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0067] Fig.19 are graphs showing waste product levels (lactic acid, left panel and ammonium, right panel) in the modified batch process at 2 L and scaled culture volumes compared to a 2 L batch mode control process.

[0068] Fig. 20A is a graph showing the percentage of intact AAV capsids assessed by analytical ultracentrifugation (AUC) analysis in the modified batch process at 2L and scaled culture volumes compared to a 2L batch mode control process. Fig. 20B is a graph showing the ratio of intermediate AAV particles to complete AAV particles under different vessel volumes / day (VVD) conditions.

[0069] Fig.21A and Fig. 21BThe use of a modified batch process with different media compositions, capsid serotypes, and vector genomes / genes of interest is shown. Fig.21A Graphs showing normalized volumetric rAAV yield (left graph) and specific productivity (right graph) using a batch mode control process ("batch") compared to a modified batch process ("perfusion") under different procedures, media and capsid conditions (A and B). The legends in both graphs represent rAAV titer in 1E6 GC / mL (for the left graph, the y-axis starts at 0.00 GC / mL to 3.50E6 GC / mL, and for the right graph, the y-axis starts at 0.00 GC / mL to 1.40E06 GC / mL). Fig. 21B is a graph showing harvest yields between batch mode and a modified batch process (abbreviated as "HDI" in the figure) using alternative procedures, media and capsid conditions ("Process C").

[0070] Fig. 22 is a graph showing normalized rAAV harvest yield. The graph shows that rAAV harvest yield is reduced by the presence of lactate and / or ammonia in AAV production.

[0071] Fig.23 is an R showing AAV cell specific production (GC / cell) at cell specific perfusion rates (CSPR (pL / cell / day)) up to approximately 750 pL / cell / day 2 Analyzed graph.

[0072] FIG. 24A to FIG. 24C Projections for reducing rAAV production cost of goods (COGs) through the improved batch processes described herein (eg, enhanced perfusion) are shown. Fig.24A is a graph showing the approximate estimated COGs (cost of goods sold per patient dose) required for each AAV production volume in genome copies / mL (GC / mL). Fig. 24B is a graph showing the approximate number of production batches required for each AAV yield (GC / mL). Fig.24C is a graph showing the approximate estimated COGs required per batch size. FIG. 24A to FIG. 24C In the figures, diamonds or circles represent the modified batch systems described herein (eg, enhanced perfusion), while squares represent batch operating conditions.

[0073] Fig.25 is a graph showing a comparison of rAAV titers using a blended medium versus a predetermined medium transition. The solid line in the graph represents rAAV released into the supernatant, while the dashed line in the graph represents intracellular rAAV.

[0074] Fig.26is a graph showing a comparison of harvest titers under different predetermined medium transition conditions (VVD conditions 1-4). The graph shows a comparison of harvest titers maintained while reducing the medium consumption rate under different predetermined medium transition conditions.

[0075] Fig. 27 is a graphical representation of harvest rAAV yields for batch and HDI processes. The figure compares harvest rAAV yields for 2L in batch mode with 2L (denoted MB-2L in the figure), 50L (denoted MB-50L in the figure), and 250L (denoted MB-250L in the figure) using an improved batch process (abbreviated "HDI" in the figure title).

[0076] Fig.28 is a graphical representation of the cell specific rAAV production rates of batch and HDI processes. The figure compares the cell specific rAAV production rates of 2L in batch mode with 2L (denoted as MB-2L in the figure), 50L (denoted as MB-50L in the figure), and 250L (denoted as MB-250L in the figure) using a modified batch process (abbreviated as "HDI" in the figure title). DETAILED DESCRIPTION OF THE INVENTION

[0078] Despite recent progress in improving the productivity and scalability of AAV manufacturing processes, there is still 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 for effective clinical and commercial scale implementation. For example, maintaining high cell-to-cell productivity of viral vector products, and particularly in high cell density batch cultures, has proven difficult to circumvent. The present systems and methods use improved perfusion (e.g., enhanced perfusion) in batch processes to address these challenges to improve AAV production efficiency and reduce the overall cost of goods.

[0079] The present disclosure relates to perfusion-based AAV production systems and methods, which are capable of achieving high volume production rates, reducing the high cost of commodities typically associated with AAV platforms operating in batch mode; the systems and methods of the present disclosure are referred to as "improved batch" systems and methods throughout the description. In certain embodiments, the improved batch system is an enhanced perfusion system and is used for AAV production. This paper describes an improvement in existing platform processes, wherein process enhancement is shown to achieve a multiple increase in total volume AAV production. Without wishing to be bound by theory, the improved batch (e.g., enhanced perfusion) process described herein ensures nutrient availability and cell culture waste byproduct removal sufficient to maintain high cell density, and enables AAV production rates to be increased multiple times, for example, increased to greater than or equal to 3E11 viral genome copies per milliliter (GC / mL) (greater than or equal to 3E14 GC / L). These improvements and optimized downstream processes can deliver high-purity AAV products suitable for human therapeutic use.

[0080] Those skilled in the art of AAV manufacturing will appreciate that various aspects of the systems and methods described herein may be varied without departing from the inventive subject matter of the present disclosure. Aspects of the invention that may be altered or varied based on the knowledge possessed by those skilled in the art without departing from the inventive disclosure of the present invention include, but are not limited to: 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, operating parameters such as pH, temperature, dissolved oxygen (DO), etc., time of process steps, duration of process steps, and medium exchange and / or perfusion methods (e.g., ATF, Wave Lilypad perfusion, centrifugation methods, TFF, KTF, Gea Kytero, Krosflow KPS systems, etc.), and others.

[0081] I. Definitions

[0082] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure relates. Unless otherwise noted, technical terms are used according to conventional usage by those skilled in the art. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes V, 1994 (ISBN 0-19-854287-9) published by Oxford University Press; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, 1994 (ISBN 0-632-02182-9) published by Blackwell Science Ltd.; and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, 1995 (ISBN 1-56081-569-8) published by VCH Publishers, Inc.

[0083] It should be noted that, unless otherwise clear from the context, the term "a" or "an" entity refers to one or more of the entity; for example, "an amino acid" should be understood to mean one or more amino acids. Therefore, the terms "a" (or "an"), "a or more" and "at least one" can be used interchangeably herein.

[0084] In addition, the term "and / or" should be regarded as a specific disclosure of each of two or more specified features or components with or without one or more others. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone) in this article. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover 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 (alone); B (alone) and C (alone).

[0085] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms as used throughout the disclosure are provided below.

[0086] AAV titer, etc.: As used herein, the term "AAV titer" refers to the number of viral genome copies per milliliter (GC / mL) or the number of viral genome copies 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, for example, including but not limited to dot blot, qPCR or droplet digital PCR (ddPCR), spectroscopy or fluorescence.

[0087] 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 fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a stated reference value in either direction (greater or less than), unless otherwise stated or otherwise obvious from the context (unless such a number would exceed 100% of the possible value).

[0088] Helper virus function, etc.: As used herein, the term "helper virus function" encompasses genes, components or functions that are capable of producing AAV in a host cell, which are typically provided in trans. Helper virus function can be provided, for example, by infection with a helper virus such as a herpes virus or an adenovirus, for example, an adenovirus serotype 5 (Ad5) virus (e.g., a wild-type or recombinantly engineered Ad5 helper virus). Alternatively, helper virus function can also be provided in cis, for example, by incorporating genetic material encoding helper virus function into a host cell. Helper virus genes / functions include, but are not limited to, E1a, E1b, E2a, E40rf6 and VA RNA genes / functions.

[0089] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primates. AAV is known to cause no disease and to elicit a very mild immune response. Gene therapy vectors using AAV can infect both dividing and resting cells and can persist in an extrachromosomal state without integrating into the host cell's genome. These features make AAV an attractive viral vector for gene therapy. There are at least 13 recognized AAV serotypes (AAV1-AAV13).

[0090] Administration / administer: Providing or otherwise exposing an agent, such as a therapeutic agent (e.g., a recombinant AAV), to a subject or a targeted tissue of a subject 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.

[0091] Amino acid: As used herein, the term "amino acid" refers to any of the twenty 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, their single stereoisomers and their racemic mixtures. The term "amino acid" can also refer to known non-standard amino acids, for example any dipeptide, such as alanine-glutamine dipeptide, 4-hydroxyproline, eV V A-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, g-carboxyglutamic acid, eV-acetylysine, co-V-methylarginine, A-acetylserine, W A '-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 glutamic acid, glutamine, lysine, tyrosine or valine. In some embodiments, the amino acid is glutamic acid or glutamine.

[0092] 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 to culture the cells (including culture medium as well as the cells themselves) are provided at the beginning of the culture process. Batch culture is usually stopped at some point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0093] Bioreactor: As used herein, the term "bioreactor" or "culture vessel" refers to any container used for growing cell cultures (eg, mammalian cell cultures). A bioreactor can be of any size as long as it can be used to culture cells (eg, mammalian cells).

[0094] Cell culture: As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of surface-attached or suspended eukaryotic cells maintained or cultured in a culture medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of ordinary skill in the art, these terms as used herein may refer to a combination comprising a population of eukaryotic cells and a culture medium in which the population is suspended, maintained and / or cultured.

[0095] Cell density: As used herein, the term "cell density" refers to the number of cells present in a given volume of culture medium. As used herein, the term "target cell density" refers to the number of cells present in the volume of culture medium achieved during the growth phase of the culture and used in the production phase for producing rAAV. As used herein, the term one or more "AAV production host cells" refers to cells used to produce rAAV.

[0096] 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. The term as used herein also refers to the fraction of cells that survive at a particular time relative to the total number of live and dead cells in the culture at that time.

[0097] Coding sequence: "Coding sequence" means a nucleotide sequence that encodes a polypeptide in vitro or in vivo when operably linked to appropriate regulatory sequences. The 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).

[0098] Codon optimized: A "codon optimized" nucleic acid refers to a nucleic acid sequence that has been altered so that the codons are optimal for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in a cell (e.g., a mammal) or a particular species (such as a human cell). Codon optimization does not change the amino acid sequence of the encoded protein.

[0099] 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 that regulates the expression (transcription or translation) of a peptide therapeutic and / or produce a nucleic acid that has a biological function in addition to encoding a peptide, such as non-coding RNA, antisense oligonucleotides (ASOs), small interfering RNA (siRNA), micro RNA (miRNA), aptamers, long non-coding RNA (lncRNA), and any other type of non-protein coding nucleic acid with biological activity.

[0100] Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

[0101] Fed-batch culture: As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culturing process. A fed-batch culture can be started with a basal medium. A 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 nutritional supplements for the cells that have been depleted during the culturing process. A fed-batch culture is typically stopped at some point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0102] Growth phase: The "growth phase" or "growth phase" of cell culture refers to the time period (logarithmic phase) of exponential cell growth in which cells usually divide rapidly. During this phase, cells are cultured for a certain period of time, such as up to 14 days, and under such conditions that cell growth is maximized. The determination of the growth cycle of host cells can be determined for the specific host cells envisioned without excessive experiments. "The time period for maximizing cell growth and under such conditions that cell growth is maximized" etc. refer to those culture conditions that are determined to be optimal for cell growth and cell division for a specific cell line. In some embodiments, during the growth phase, cells are usually cultured in a nutrient medium containing necessary additives at about 25°C-40°C, in a humid controlled atmosphere, so that a specific cell line achieves optimal growth. In certain embodiments, cells are maintained between about 1 day and 7 days, such as between 2 days and 6 days, such as a time period of 6 days during the growth phase. The length of the growth phase of a specific cell can be determined without excessive experiments. For example, if the culture is maintained under growth conditions, the length of the growth phase will be a period of time sufficient to allow the particular cells to proliferate to a viable cell density ("vcd") in the range of about 20%-80% of the maximum possible vcd. In some embodiments, "maximum growth rate" refers to the growth rate of a particular cell line / clone measured during its exponential growth phase when the cells are in fresh culture medium (e.g., measured during the culture period when nutrients are sufficient and there is no significant inhibition of growth by any component of the culture).

[0103] Intron: A section of DNA within a gene that does not contain protein-coding information. The RNA transcript is processed by the cellular machinery to remove introns to produce mature, processed messenger RNA.

[0104] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences required for efficient replication in the adeno-associated virus genome. 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 components for the production of AAV integration vectors.

[0105] 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 the organism in which the component naturally occurs, or from the organism itself. Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acid molecules and proteins.

[0106] Media, Medium: As used herein, the terms "media," "medium," "cell culture medium," "culture medium," "tissue culture medium," "tissue culture media," and "growth medium" refer to solutions containing nutrients that nourish growing eukaryotic cells in culture. Typically, these solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimum growth and / or survival of the cells. The solution may also contain components that enhance growth and / or survival above a minimum rate, including hormones and growth factors. The solution is formulated to a pH and salt concentration that is optimal for cell survival and proliferation. The culture medium may also be a "defined medium" or a "chemically defined medium" - a serum-free medium that does not contain components of animal origin, hydrolysates, or components of unknown composition and contains only components of known chemical structure or characteristics. A chemically defined medium contains no components of animal origin, and all components have known chemical structures. It is understood by those skilled in the art that chemically defined media may contain recombinant glycoproteins or proteins, such as but not limited to hormones, cytokines, interleukins and other signaling molecules.

[0107] N stage, etc.: As used herein, the terms "N stage," "N culture vessel," "secondary culture vessel," "production vessel," "production culture vessel," "N vessel," "N bioreactor," "secondary bioreactor," or "production bioreactor" refer to the bioreactor that follows the N-1 bioreactor and is used to produce AAV. This is alternatively referred to throughout this specification as a "production stage" culture. As used herein, AAV producer cells in "N stage" culture are actively producing AAV product.

[0108] N-stage, etc.: As used herein, the terms "N-" and "N-stage" refer to any AAV production cell culture stage prior to the initiation of N-stage production.

[0109] N-1 stage, etc.: As used herein, 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", "growth vessel" or "N-1 seed-train bioreactor" refer to the culture vessel that immediately precedes the N-stage culture vessel (production culture vessel) and is used to grow the cell culture to a high viable cell density for subsequent inoculation into the 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 containers preceding the N-1 culture vessel, such as N-4, N-3, and N-2 containers. Alternatively, the N-1 stage may be referred to as the "growth stage" culture throughout this specification. As used herein, the AAV producing cells in the "N-1 stage" culture may be in the growth phase.

[0110] 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 a "complete" medium for growing, proliferating, and adding biomass to a cell line. A nutrient medium is different from a substance or simple medium that is not sufficient by itself to grow and proliferate a cell line. Thus, for example, glucose or a monosaccharide are not nutrient media by themselves because, in the absence of other required nutrients, they will not be sufficient to grow and proliferate a cell line. It will be appreciated by those skilled in the art that cells can continue to grow, survive, and proliferate in the presence of an incomplete medium, but become unstable and / or have their growth rate greatly reduced. Thus, in some embodiments, the term "nutrient medium" includes medium sufficient to grow, proliferate, and add biomass to a cell line 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 without losing stability, growth rate, or reducing any other indicator of cell health. In some embodiments, the term "nutrient medium" includes a medium that may lack one or more essential nutrients, but which can continue to grow, propagate, and add biomass to a cell line without loss of stability, growth rate, or reduction in any other cell health indicator over 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. The 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 culture 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., a composition comprising 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), hydrolyzates (hydrolyzed proteins derived from plant or animal sources), hydrolyzates (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 media (e.g., RPMI 1640), Iscoves DMEM, Leibovitz L-15, Leibovitz L-15, TC100), serum-free media (e.g., Chinese hamster ovary (CHO) medium, Ham's F10 and derivatives, Ham's F12, DMEM / F12). Common buffers present 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). Substrates for culturing mammalian cells are well known in the art and are available from, for example, Sigma-Aldrich Corporation (St. Louis, MO), HyClone (Logan, UT), Invitrogen Corporation (Carlsbad, CA), Cambrex Corporation (E. Rutherford, NJ), Irvine Scientific (Santa Ana, CA), Gibco / Thermo Fisher Scientific, etc. Other components present 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 will fall within the scope of the systems and methods disclosed herein.

[0111] 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 culturing process has begun. The components provided typically include nutritional supplements for the cells that have been depleted during the culturing process. A portion of the cells and / or components in the culture medium are typically harvested on a continuous or semi-continuous basis and optionally purified.

[0112] Pharmaceutically acceptable carrier: pharmaceutically acceptable carriers (vehicles) that can be used for the present disclosure can be those conventionally used in the art. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 15th edition (1975) by EW Martin describes compositions and preparations suitable for drug delivery of one or more therapeutic compounds, molecules or agents. Generally, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally contain injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, dextrose aqueous solutions, glycerol, etc. as vehicles. For solid compositions, such as powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered can contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0113] Polypeptide, protein: As used herein, the term "polypeptide" or "protein" refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to amino acid chains of any length, but it will be understood by those of ordinary skill in the art that the term is not limited to long chains and may refer to the smallest chain containing two amino acids linked together via peptide bonds. If a single polypeptide is a discrete functional unit and does require permanent physical association with other polypeptides to form a discrete functional unit, the terms "polypeptide" and "protein" as used herein are interchangeable. If a discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" as used herein refers to multiple polypeptides that are physically coupled and function together as discrete units. As used herein, the term "protein" is intended to cover the singular "protein" as well as the plural "protein". Therefore, as used herein, terms including but not limited to "peptide", "polypeptide", "amino acid chain" or any other term used to refer to one or more chains of amino acids are included in the definition of "protein", and the term "protein" can replace any of these terms or be used interchangeably with any of these terms. The term also includes proteins that have undergone post-translational modifications, such as glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Proteins also include polypeptides that form multimers (e.g., dimers, trimers, etc.). The term protein also includes fusion proteins, such as 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).

[0114] Promoter: A DNA region that directs / starts transcription of a nucleic acid (e.g., a gene). Promoters include essential nucleic acid sequences near the transcription start site. Many promoter sequences are known to those skilled in the art, and even combinations of different promoter sequences in artificial nucleic acid molecules are possible. As used herein, a gene-specific endogenous promoter refers to a natural 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 a natural environment.

[0115] Purified: The term "purified" does not require absolute purity, but is intended as a relative term. Thus, for example, a purified peptide, protein, virus or other active compound is one that is completely or partially separated 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 separated from cells, cell culture media 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).

[0116] Recombination: A recombinant nucleic acid molecule is a nucleic acid molecule having a non-naturally occurring sequence or a sequence prepared by an artificial combination of two originally separated segments of one or more sequences. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of the separated segments of the nucleic acid molecule, such as by genetic engineering techniques. Similarly, a recombinant virus is a virus containing a sequence (such as a genomic sequence) that is non-naturally occurring or prepared by an artificial combination of sequences from at least two different sources. The term "recombination" also includes nucleic acids, proteins and viruses that are changed only by adding, replacing or deleting a portion of a natural nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" or "rAAV" may refer to AAV particles in which recombinant nucleic acid molecules such as recombinant nucleic acid molecules encoding therapeutic payloads have been packaged.

[0117] Inoculation: As used herein, the term "inoculation" refers to the process of providing a cell culture to a bioreactor or another container (e.g., a growth container or a production container). In one embodiment, the cells have previously been propagated in another bioreactor or container (e.g., a growth container or a production container). In another embodiment, the cells are frozen and thawed immediately before providing the cells to a bioreactor or container (e.g., a growth container or a production container). The term refers to any number of cells, including a single cell.

[0118] Serotype: A group of closely related microorganisms (such as viruses) distinguished by a set of characteristic antigens.

[0119] Stuffer sequence: refers to a nucleotide sequence contained in a larger nucleic acid molecule (such as a vector) that is usually used to create a desired interval between two nucleic acid features (such as between a promoter and a coding sequence) or to extend a nucleic acid molecule to a desired length. The stuffer sequence does not contain protein coding information and may be of unknown / synthetic origin and / or unrelated to other nucleic acid sequences in the larger nucleic acid molecule.

[0120] 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). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a human being who presents to a medical provider for diagnosis or treatment of a disease. Unless the context requires otherwise, the term "subject" is used interchangeably herein with "individual" or "patient." A subject may have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0121] 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, reviews 0004.1 (2002)).

[0122] Vector: A vector is a nucleic acid molecule that allows the insertion of exogenous nucleic acid without destroying the ability of the vector to replicate and / or integrate in a host cell. The vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector containing regulatory sequences necessary for transcription and translation of one or more genes that allow insertion. In some embodiments herein, the vector is an AAV vector. Depending on the context, a broader definition of the term "vector" may be applied, which means a vector or transmitter, i.e., a biological vector of a biological agent, typically.

[0123] It should also be understood that all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​provided for nucleic acids or polypeptides are approximate and are provided for description. Although methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, the interpretation of the term should be determined by this specification. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0124] II. Overview

[0125] The present disclosure relates to perfusion-based AAV production systems and methods, which are capable of achieving high volume production rates, reducing the high commodity costs typically associated with comparable AAV platforms (e.g., non-enhanced AAV platforms). The systems and methods of the present disclosure can be used to manufacture any viral particles, such as adeno-associated viral particles, for example, for therapeutic applications. The systems and methods described herein can be incorporated into a procedure for implementing a production cell line (e.g., a mammalian producer cell line) (e.g., a producer cell line in a suspension) for producing viral particles, or alternatively incorporated into a procedure for implementing a multiple (e.g., triple) transfection method to produce viral particle-producing cells. In some embodiments, the systems and methods disclosed herein are incorporated into a transfection master mixture according to those described in, for example, U.S. Patent Application Publication No. US20200124505A1.

[0126] Therefore, there is provided herein a system and method for producing viral particles (e.g., AAV). Improved batch (e.g., enhanced perfusion) systems and methods described herein mitigate the challenge of maintaining high specific production rates in batch mode and other perfusion culture systems (e.g., non-enhanced) in the field of bioreactor production, which is outstanding at improved cell densities (e.g., about 1E06 cells / mL or higher density). Compared to existing methods for AAV production, systems and methods described herein achieve up to five times or greater increases in volume production rates, thereby reducing commodity costs and improving the efficiency of AAV production.

[0127] The present disclosure provides various systems and methods for AAV manufacturing. The present disclosure shows how to increase AAV production by using an improved batch system (e.g., an enhanced perfusion system) and method and optionally using an increased cell density under conditions of enriching the medium and removing inhibitory metabolites in the growth phase container during the production phase. In particular, systems and methods are disclosed that can culture AAV production cells from the growth phase to the AAV production phase in a single container (e.g., a bioreactor), while maintaining a high cell density, such as at or greater than 1E06 cells / mL in some cases. The systems and methods described herein are different from other systems, which utilize perfusion in the growth phase before the production phase of the gene therapy virus to achieve high-density culture, and then transfer the high cell mass to the production container for batch mode operation. In contrast, the systems and methods described in the present invention can achieve perfusion in the growth phase and the production phase, included in the same container in some embodiments, and allow the exchange of cell culture medium after infection with a helper virus or after transfection with a helper virus component to enrich the culture medium and remove inhibitory waste from the culture medium, thereby improving the AAV production rate. The systems and methods described herein result in significant and multi-fold increases in total volumetric AAV production rates when compared to batch mode operation at low cell density and to batch mode operation at higher cell density (e.g., greater than 1E06 cells / mL) while maintaining cell specific production rates.

[0128] In a batch mode AAV manufacturing process, cell expansion is typically performed after the production cell line (e.g., a mammalian producer cell line) is thawed and expanded in a shake flask. The expanded producer cells are transferred to a mid-scale bioreactor and then transferred to an N-1 growth vessel at, for example, a 250L scale (e.g., 10× smaller than the final production stage). Systems that can be used for N-1 growth stage culture are commercially available and include, for example, the Repligen Xcell ATF TM6 disposable alternating tangential flow filtration systems and similar systems. Typically, these systems can achieve a cell density of>13E06 viable cells / ml (vc / mL). Next, the cell mass is separated from the N- (e.g., N-1) in the growth medium to the N stage production at approximately 1:10, for example, at a 2000L scale, with approximately 1.3E06 vc / mL. Typically, this requires a new production phase container separated from the growth phase container, and requires different production mediums and feeds. After being transferred to the N stage or production phase culture, AAV production is started by helper virus (e.g., adenovirus such as adenovirus serotype 5 (Ad5)), infected in a 90:10 production medium and growth medium mixture, and carried for about 4 days in the production culture. Finally, harvest operations are performed in the bioreactor, such as nuclease addition and pretreatment activities, and subsequent downstream filter clarification, AAV purification and helper virus removal processes are performed.

[0129] In the improved batch (e.g., enhanced perfusion) system and method described herein, the cell seed queue cell mass is thawed and placed in a shake flask in the same manner as in the batch mode AAV manufacturing process. The bioreactor expansion stage includes, for example, another container on a 250L scale (without using alternating tangential flow filtration). Importantly, in some embodiments, the subsequent growth phase and production phase can be but need not be carried out in the same, for example, 2000L container (bioreactor), without the need to transfer the high cell density seed queue to a new container for production phase culture. For the growth phase on a 2000L scale, for example, a final high cell density target of 5E06 vc / mL-50E06 vc / mL is achievable using a blend of growth medium and production medium. After the desired cell mass reaches a high cell density target (e.g., 5E06 vc / mL or higher), perfusion-mediated or other culture medium exchanges can be stopped. Then AAV production is started by infection with helper virus (e.g., adenovirus) or transfection with helper virus components and adding feed after about 1 hour to about 2 hours, and thereafter perfusion-mediated or other culture medium exchanges are started again with appropriate production culture medium and feed. In some embodiments of the systems and methods described herein, alternating tangential flow (ATF) perfusion is utilized in production phase culture. ATF operation can maintain activity for example about 1 day to about 2 days to enrich culture medium and eliminate waste product accumulation after infection with helper virus (e.g., adenovirus) or transfection with helper virus components. After about 1 day to about 2 days, perfusion-mediated or other culture medium exchanges are terminated to allow AAV particles to accumulate in culture supernatant. In some embodiments, ATF operation can maintain activity for more than 2 days after infection with helper virus or transfection with helper virus components, for example, inducing AAV to produce up to about 14 days. AAV particles are released into the supernatant, for example, after about 12 hours, after about 24 hours, after about 36 hours, after about 48 hours, after about 60 hours, after about 72 hours, after about 84 hours, or after about 96 hours, depending on, for example, the producer cell line used. After infection with helper virus or transfection with helper virus components for a total of about 4 to about 6 days, AAV harvesting procedures are performed for subsequent downstream process purification, such as filter clarification, AAV purification, and helper virus (e.g., adenovirus such as Ad5) removal. In some embodiments, the helper virus removal process includes a heat inactivation process, such as those described in, for example, U.S. Patent Application Publication No. US20190083554A1. In some embodiments, tangential flow depth filtration can be implemented to integrate AAV harvesting and clarification processes into a single step (see Mendes, P., et al. "AAV processintensification by perfusion bioreaction and integrated clarification." Frontiers in Bioengineering and Biotechnology10 (2022)).

[0130] In addition to using the improved batch system (e.g., enhanced perfusion) and method using perfusion described herein, additional methods 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 refined to include: the use of glucocorticoid analogs as described in, for example, U.S. Patent Application Publication No. US20190290710A1; the use of compounds selected from the group consisting of: nicotinamide, nicotinic acid, methyl nicotinate, nicotinic alcohol, and any combination thereof as described in, for example, PCT Publication No. WO / 2021 / 188449; the optimized downstream clarification process as described in, for example, U.S. Patent Application Publication No. US20200048641A1; the use of tension agents as described in, for example, U.S. Patent Application Publication No. US20210277416; and / or the co-delivery of Rep mRNA or HDAC inhibitors as described in, for example, U.S. Patent Application Publication No. US20200032221A1.

[0131] In some embodiments, the improved batch systems and methods described herein using perfusion (e.g., enhanced perfusion) increase the infection density by about 2-fold, about 3-fold, about 4-fold, about 5-fold, or greater than 5-fold from optimal batch mode or other conditions (e.g., non-enhanced), resulting in an increase in AAV production rate to greater than or equal to, for example, 3E11 genome copies / milliliter (GC / mL), i.e., greater than or equal to 3E14 GC / L.

[0132] The system and method described in the present invention are different from previous methods. The system and method currently described are shown in the growth phase perfusion to achieve high-density culture to be transferred to the production container for batch mode operation. Although others have shown the perfusion capability during the AAV production phase in the transient transfection system and during the AAV production in the HEK-based cell system, no one has shown the use of perfusion during the AAV production using, for example, HeLa producer cells (e.g., HeLa producer cells infected with helper viruses such as adenovirus). Some embodiments of the systems and methods disclosed herein can utilize perfusion equipment to combine the growth phase and the production phase production into a single step in the same container to achieve high cell density production, thereby increasing the total volume production rate. In some embodiments, the use of perfusion technology can achieve high producer cell density (e.g., HeLa PCL density) in the production container. Subsequently, the change of the production medium by medium exchange after infection with the helper virus can enrich the medium and remove inhibitory waste, resulting in an increase in the AAV production rate. Perfusion behavior following initiation of AAV production resulted in a significant fold increase in total volumetric AAV production rate when compared to batch mode operation (e.g., at lower cell densities) as well as batch mode operation at higher densities (i.e., greater than about 1E06 cells / mL), while maintaining similar cell specific production rates as observed in batch mode operation.

[0133] The systems, methods, reagents and components and their features will now be described in more detail in the following sections.

[0134] III. Viral Vectors

[0135] This section generally describes viral vectors, including AAV vectors, which can be produced by improved batch systems and methods using perfusion (e.g., enhanced perfusion) as described herein. In some aspects, the present disclosure provides systems and methods for producing adeno-associated viruses (AAV). In some embodiments, the present disclosure provides systems and methods for producing recombinant adeno-associated viruses (rAAV), comprising an adeno-associated virus (AAV) capsid and a recombinant vector genome packaged therein.

[0136] The AAV (e.g., rAAV) to be produced can contain a packaged vector genome comprising, for example, an 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 can also include an enhancer sequence, an intron sequence, a consensus Kozak sequence, and / or a polyadenylation signal sequence. In some embodiments, the packaged vector genome can also include one or more filler nucleic acid sequences. In some embodiments, the filler nucleic acid sequence is located between an intron and a partial or complete coding sequence of a therapeutic payload.

[0137] In some embodiments, the AAV vector is a recombinant AAV vector (rAAV). For simplicity, the term "AAV vector" as used herein includes the rAAV subgenus, so that the reference to the AAV vector covers the AAV vector and rAAV. The AAV vector produced by the system and method of the present disclosure can include the capsid of any AAV serotype. For example, the disclosed system and method can be used to produce an AAV comprising 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 of the capsids of more than 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 mentioned above, the disclosed systems and methods can be used to produce AAVs comprising variant AAV capsids that have been engineered to have one or more beneficial therapeutic properties (e.g., improved targeting to selected tissues, increased ability to evade immune responses, reduced stimulation of neutralizing antibodies, etc.). Non-limiting examples of such engineered variant capsids are described in U.S. Pat. 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, 10,202,65 7, 10,214,566, 10,214,785, 10,266,845, 10,294,281, 10,301,648, 10,385,320, and 10,392,632, and PCT Publication Nos. WO / 2018 / 022905, WO / 2018 / 156654, WO / 2018 / 222503, and WO / 2018 / 226602. These and other AAV serotype variants can also be produced by the AAV production systems and methods herein. The selection of AAV serotypes and / or variants to be produced will depend in part on one or more cell types to be targeted by the AAV vector. For example, in gene therapy applications.

[0138] A. Vector genome components

[0139] In some embodiments, the AAV vectors produced by the systems and methods described herein comprise a vector genome packaged within an AAV vector.

[0140] In some embodiments, the AAV vectors produced by the systems and methods described herein comprise vector genomes containing AAV ITR sequences that function both as an origin of vector DNA replication and as a packaging signal for the vector genome, i.e., when the AAV and adenovirus helper virus functions are provided in trans. Additionally, the ITRs can serve as targets for single-stranded endonucleolytic nicking by the large Rep protein, thereby disassembling individual genomes from replication intermediates.

[0141] In some embodiments, the AAV vector produced by the systems and methods described herein comprises a vector genome containing a promoter sequence, which helps drive and regulate transgene expression (e.g., expression of a therapeutic payload). The promoter sequence can be (or 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) early gene promoter sequence, a thyroxine transporter protein (TTR) promoter sequence, a thyroxine binding globulin (TBG) promoter sequence, an alpha-1 antitrypsin (A1AT) promoter sequence, a CMV early enhancer / chicken beta actin (CAG) promoter sequence, or a therapeutic payload gene-specific endogenous promoter sequence. In some embodiments, the promoter sequence is located downstream of the enhancer sequence. In some embodiments, the promoter sequence is located upstream of the 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).

[0142] In addition to the promoter, the packaged genome may also contain other appropriate transcription initiation, termination, enhancer sequences and effective RNA processing signals. As described in further detail below, such sequences include splicing and polyadenylation (poly A) 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.

[0143] 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 appreciated by those skilled in the art, the consensus Kozak sequence is typically located immediately upstream of a coding sequence such as a sequence encoding a therapeutic payload. As will be appreciated by those skilled in the art, the consensus Kozak sequence can be considered to have a consensus of the ATG residue corresponding to the start codon of the therapeutic payload polypeptide.

[0144] In some embodiments, the packaged vector genome also includes a 5'-untranslated region (UTR). 5'UTR can be derived from an endogenous gene-specific mRNA with a desirable expression profile. It is known that 5'UTR increases mRNA half-life by competing with cell transcripts for translation initiation factors and ribosomes, by minimizing mRNA decay or post-transcriptional gene silencing, and avoiding harmful interactions with regulatory proteins or inhibitory RNA secondary structures, and plays an important role in optimizing transgenic production (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).

[0145] Similarly, the packaged vector genome may also include a 3'UTR. The 3'UTR may be located downstream of the coding sequence of the therapeutic payload. The 3'UTR has been shown to be involved in many regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization (see Barrett, LW, et al. (2012). Cell Mol Life Sci. Nov; 69(21): 3613-3634).

[0146] 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, and include, for example, polypeptides that can be used to treat mammals (e.g., humans), including ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase), factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), arginine succinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit a (PCCA) and propionyl-CoA carboxylase subunit b (PCCB), survival motor neurons (SMN), iduronate-2-sulfatase (IDS), alpha-1-iduronate (IDUA), tripeptidyl peptidase 1 (TPP1), low-density lipoprotein receptor (LDLR), myotubularin 1 (myotubularin 1), acid alpha-glucosidase (GAA), dystrophic myotonic 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, fumaryl acetate hydrolase, alpha-1 antitrypsin, methylmalonyl CoA mutase, cystic fibrosis transmembrane conductance regulator (CFTR) protein, and dystrophin gene products (e.g., mini-dystrophin or microdystrophin). A non-limiting list of therapeutic payloads can also be found in PCT Publication No. WO / 2019 / 168961. The therapeutic payload can be a wild-type coding sequence of a protein or polypeptide, a codon-optimized coding sequence of 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 (lncRNAs), and any other species of non-protein-coding nucleic acids having 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-conjugated 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 transgenic RNA or other genetic modification or therapeutic RNA and / or protein, or any combination thereof.As used herein, the term "wild type" refers to a biopolymer (eg, a polypeptide sequence or a polynucleotide sequence) that is identical to a biopolymer (eg, a polypeptide sequence or a polynucleotide sequence) that occurs in nature.

[0147] In some embodiments, the packaged vector genome further comprises one or more enhancer sequences. In some embodiments, the enhancer sequence is (or is derived from) a cytomegalovirus 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.

[0148] In some embodiments, the packaged vector genome further comprises one or more intron sequences. The intron sequence may be (or may be derived from) an SV40 small T intron sequence, a rabbit hemoglobin subunit β (rHBB) intron sequence, a human β-globin IVS2 intron sequence, a Promega chimeric intron sequence (β-globin / IgG chimeric intron sequence) or an hFIX intron sequence.

[0149] In some embodiments, the packaged vector genome further comprises a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence is (or is derived from) a bovine growth hormone (BGH) polyadenylation signal sequence, an SV40 polyadenylation signal sequence, a rabbit beta globin polyadenylation signal sequence, a gene-specific endogenous polyadenylation signal sequence.

[0150] B. AAV capsid

[0151] The present disclosure provides systems and methods for producing AAV particles comprising an AAV capsid of any AAV serotype. In some embodiments, the AAV capsid is from an 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, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10 or AAVhu37). In an exemplary embodiment, the AAV capsid is from an AAV serotype 9 (AAV9) vector, an AAV9 variant vector, an AAV serotype 8 (AAV8) vector, an AAV serotype 2 (AAV2) vector, or any other known AAV serotype vector or variant. International Patent Application Publication No. WO2021041485A1 provides a list of known AAV serotypes, variants, mutations, and derivatives thereof in paragraphs

[0115] -

[0131] , any of which may be suitable for AAV production using the systems and methods described herein.

[0152] C. Pharmaceutical Compositions

[0153] The system and method for AAV production provided herein can be used to produce a pharmaceutical composition comprising an AAV vector. The pharmaceutical composition comprising an AAV vector produced according to the method disclosed herein can also include a pharmaceutically acceptable carrier. The pharmaceutical composition comprising an AAV vector produced according to the method disclosed herein can also be formulated for administration to a subject. Suitable pharmaceutical compositions formulated for administration of AAV can be found in, for example, U.S. Patent Application Publication No. 2012 / 0219528. Pharmaceutically acceptable carriers (vehicles) that can be used for AAV compositions are conventional. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 15th edition (1975) by EWMartin describes compositions and preparations suitable for drug delivery of one or more therapeutic compounds, molecules or agents (including, for example, AAV produced according to the system and method of the present disclosure).

[0154] In some aspects, the disclosure relates to pharmaceutical compositions comprising AAV produced according to the methods disclosed herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration, intramuscular administration, intradermal administration, intraperitoneal administration, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0155] 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 tube but do not interfere with the binding activity of the AAV in vivo. Various suitable solutions may include one or more of: a buffered saline, a surfactant, and 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 to 8.5, or 7 to 8.5, or 7.5 to 8. Suitable surfactants or combinations of surfactants may be selected from the following: poloxamers, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene 10 (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylylglyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and / or polyethylene glycol.

[0156] IV. Methods for Producing AAV

[0157] The present disclosure provides systems and methods for producing adeno-associated viruses (AAV), which can be used as therapeutic payload (e.g., gene) delivery vehicles. In some embodiments, the AAV produced by the systems and methods provided herein comprises a wild-type or engineered (mutant or variant) AAV capsid packaged with an engineered vector genome, and the engineered vector genome comprises, for example, a transgenic, such as a transgenic encoding a therapeutic payload such as a therapeutic protein. In some embodiments, such an engineered AAV can be applied to a subject with a therapeutic payload need, and after application, the therapeutic payload can be expressed in the subject's own cells. The systems and methods described herein are applicable to AAVs comprising any therapeutic payload.

[0158] A. AAV production host cells

[0159] The systems and methods provided herein utilize host cells to produce AAV vectors (including rAAV). In some embodiments, the host cell comprises a recombinant nucleic acid molecule disclosed herein, a viral vector (e.g., an AAV vector) or rAAV. In specific embodiments, the host cell is suitable for the propagation of AAV. In specific embodiments, the host cell is suitable for the production of AAV. In specific embodiments, the host cell is suitable for the propagation and / or production of AAV.

[0160] Any known AAV can be used to produce host cells. Examples of known host cell types include bacterial cells, yeast cells, insect cells (such as Sf9 cells) and mammalian cells, etc. 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 produces cell lines, such as those of the doxycycline-inducible CAP cells and HEK293 cells described in WO 2022 / 112218 A1 for use in the systems and methods described herein. Other host cell types are described elsewhere herein.

[0161] In some embodiments, the recombinant nucleic acid molecule or vector can be delivered to the host cell culture using any suitable method known in the art (e.g., a method based on transfection). In some embodiments, a stable host cell line having a recombinant nucleic acid molecule or vector inserted into its genome by transfection is used in the system and method provided herein. After the AAV vector is transfected into the host cell, the integration of the AAV vector into the host cell genome can be determined by various methods, such as antibiotic selection, fluorescence activated cell sorting, Southern blotting, PCR-based detection, fluorescence in situ hybridization, as described below: 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. Furthermore, 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.

[0162] In some embodiments, host cells for AAV production are engineered into AAV producer cells. Thus, AAV producer cell lines (PCLs) can be used in the systems and methods described herein.

[0163] Various aspects of culturing AAV PCLs in the systems and methods of the present disclosure are described herein. For example, parameters of PCL culture conditions that can be selected or used in the systems and methods of the present disclosure are described, including medium exchange techniques, perfusion, perfusion rate, cell density, culture medium, medium supplementation, cell viability, growth rate, pH, culture duration, culture volume, and seeding density, among others.

[0164] AAV production requires various cell / virus components. For example, components for AAV replication, vector genome packaging and structural components of capsids must be produced to produce 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-defective, and requires cells to be co-infected by helper viruses (e.g., herpes virus or adenovirus (e.g., Ad5 virus)) for replication. For example, Ad5 virus provides Ad5 helper virus functional factors / genes that mediate AAV replication, such as Ela, Elb, E40rf6, E2a and / or virus-related (VA) RNA. See, for example, Nayak et al. J Virol. 81.5 (2007): 2205-12. Recombinant AAV vectors allow genes of interest or transgenes to be integrated into viral vectors so that transgenes are delivered, encoded and / or expressed by viral mechanisms. In some cases, recombinant AAV vectors include terminal inverted repeats (ITRs) used as replication and / or packaging starting points. In some embodiments, the recombinant AAV vector comprises a transgene flanked by ITRs (one ITR on either side of the transgene). See, e.g., Carter B. Adeno-Associated Virus and AAV Vectors for Gene Delivery, in Gene and Cell Therapy, 4th edition, NS Teston, ed. 2015, CRC Press.

[0165] AAV PCLs containing one or more of these components required for AAV production can be produced. 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., wherein two ITRs flank 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 an AAV rep gene); (d) a nucleic acid sequence encoding one or more AAV structural capsid proteins (e.g., encoded by an AAV cap gene, such as VP1, VP2, or VP3 proteins); (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, such as Ad5 helper virus components (e.g., E1a, E1b, E40rf6, E2a, and / or VA RNA; or Ad5 helper virus). As used herein, "AAV PCL" or "AAV producer cell line" refers to cells that contain one or more of (a)-(g) above and, when provided with any necessary helper virus components, are capable of producing AAV under appropriate production conditions well known in the art, such as the cells described herein.

[0166] In some embodiments, any combination of 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 four or more separate nucleic acid molecules). In some embodiments, the transgene is flanked on both sides by ITRs.

[0167] In some embodiments, the PCL comprises any combination of (a)-(g) above. In some embodiments, the PCL may comprise, for example, one or more recombinant AAV vectors 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, wherein the transgene is flanked by an ITR on either side. In other embodiments, the PCL comprises a packaging cell line comprising rep and / or cap genes but not a vector / transgene (and the vector / transgene may be provided by a separate virus, such as a recombinant adenovirus, such as Ad5). PCLs comprising combinations of other AAV components may be prepared. Any such PCL and other variants of the PCL may be used in conjunction with the systems and methods described herein. In some instances, AAV production may be induced by infection with a helper virus such as Ad5, for example, in N culture.

[0168] Any cell line suitable for use in AAV production can be used as an AAV production host cell for the systems and methods described herein, including suitable mammalian and insect cell lines (including Sf9 cells). For example, a mammalian cell line comprising one or more components required for AAV production, such as PCL derived from any mammalian cell line, 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 or non-human mammalian PCL. Exemplary cell types include, but are not limited to, BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblasts (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 suspension culture growth, 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)); 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); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (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 hepatoma cell line (Hep G2); and / or a PCL form of any cell type described herein. In some embodiments, a PCL form of a cell type described herein comprises a cell type that has been engineered to possess one or more components required for AAV production.

[0169] In some embodiments, the systems and methods described herein include culturing HeLa cells (e.g., HeLa PCL), CHO cells (e.g., CHO PCL), or HEK cells (e.g., HEK PCL). In some embodiments, the systems and methods described herein include culturing HeLa cells, e.g., HeLa PCL.

[0170] As noted above, in some cases, the cells will be selected or engineered to contain one or more components necessary for AAV production (e.g., engineered to PCL). Alternatively or additionally, the engineered PCL may comprise a genetic modification to reduce the expression and / or activity of one or more genes and / or proteins, which modification increases AAV titer. Exemplary engineered PCLs that can be used in the systems and methods of the present disclosure include those described in U.S. Patent Application Publication No. US20200325455A1.

[0171] In some embodiments, the engineered PCL may include genetic modifications to reduce the expression and / or activity of one or more genes and / or proteins, which reduce the generation or accumulation of waste byproducts in the PCL. As an example of genes targeted to reduce the accumulation of waste byproducts, Soo Min Noh et al. showed that shRNA treatment of CHO producer cells to reduce the expression of lactate dehydrogenase A increased the yield and quality of the monoclonal antibodies produced. (See Noh, Soo Min, et al. "Reduction of ammonia and lactate through the coupling of glutaminesynthetase selection and downregulation of lactate dehydrogenase-A in CHO cells." Applied microbiology and biotechnology 101(3)(2017): 1035-1045, incorporated herein by reference in its entirety). See also: Shen, Chun Fang, et al. "Reassessing culture media and critical metabolites that affect adenovirus production." Biotechnology progress 26(1)(2010):200-207, which describes the levels of waste byproducts and their effects on Ad5 production; Khajah et al. "Lactate Dehydrogenase A or B Knockdown Reduces Lactate Productionand Inhibits Breast Cancer Cell Motility in vitro." Front Pharmacol. 2021, 12:747001, which shows that knocking down LDH-A or LDH-B can reduce lactate production; and 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, which shows that PFKP knockdown inhibits lactate production.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 that are engineered to upregulate or downregulate the expression of metabolic enzymes that can reduce the formation or accumulation of waste byproducts such as lactate or ammonia.

[0172] 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 can be inducible. The expression of glutamine synthetase can be driven by promoters, enhancers, and / or other expression regulatory elements optimized for appropriate expression levels to mitigate the accumulation of waste byproducts during AAV production.

[0173] The present disclosure also contemplates engineered AAVPCLs with reduced lactate dehydrogenase expression to reduce lactate accumulation. In some embodiments, the PCL may have reduced expression of lactate dehydrogenase-A (LDH-A). In some embodiments, the PCL may have reduced expression of lactate dehydrogenase-B (LDH-B). In some embodiments, the PCL may have reduced expression of the glycolytic enzyme phosphofructokinase platelet type (PFKP).

[0174] In some embodiments, PCL may have increased expression of glutamine synthetase and decreased expression of lactate dehydrogenase.

[0175] Those skilled in the art will recognize different methods for regulating gene expression in PCL, and the different methods include, for example, the methods set forth in U.S. Patent Application Publication No. US20200325455A1. Examples of methods that can be used to regulate the expression of genes or gene products in PCL engineered to reduce the production or accumulation of waste byproducts include the use of nucleases, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotides (ASOs). Examples of nucleases include zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins.

[0176] Alternative methods to reduce lactate and / or ammonia accumulation in PCL cultures include those described by Freund and Crougan (Int. J. Sci. 2018 Feb; 19(2): 85). These include changes in pH, clonal selection of lactate consumption phenotypes, and various media supplementation methods. Based on the observation that EAA supplementation in fed-batch culture alters the metabolic profile of AAV-producing cells and, in particular, reduces lactate accumulation (data not shown), it is now further considered that supplementation of the culture medium with essential amino acids (EAA) can reduce lactate and other waste product accumulation and promote rAAV production by promoting protein production.

[0177] Those skilled in the art will appreciate that different cell lines may have different nutrient requirements and / or may require different culture conditions for optimal growth; those skilled in the art will be able to modify the conditions as necessary.

[0178] B. Methods for AAV Production

[0179] Various aspects of AAV manufacturing are described herein, including aspects of described systems and methods.

[0180] i. Batch Mode Process for AAV Production

[0181] Typically, in batch mode AAV production, AAV PCLs are cultured in an N-1 culture vessel to achieve a high viable cell density. The N-1 culture cells are then used to inoculate an N culture vessel at a specific inoculation density. The inoculated cells in the N culture vessel can be cultured under conditions that allow the production of AAV.

[0182] Figure 1 An exemplary process for low density batch (LDB) mode AAV production, i.e., an exemplary batch mode control process, is shown. For example, an AAV producer cell line such as a HeLa producer cell line (PCL) is thawed and expanded in a shake flask, transferred to a mid-scale bioreactor, then transferred to an N-1 perfusion growth vessel at, for example, a 250L scale, and then transferred to a production stage production vessel at, for example, a 2000L scale. Figure 1 As shown in FIG. 1 , the process begins by pumping growth medium into a 250 L N-1 stage perfusion growth vessel (step 1), followed by inoculation of the N-1 stage perfusion growth vessel with the previous N-2 stage producer cell inoculum (step 2). The growth medium is then filtered through an alternating tangential flow (ATF) filtration system or a similar medium exchange system (e.g., Repligen Xcell ATF at 250 L scale). TM6 disposable alternating tangential flow filtration system) for perfusion exchange (step 3) for, e.g., 4-6 days to achieve a target cell density of greater than or equal to 13E06 vc / mL (viable cells / mL) (step 4). For the production phase operated in batch mode, a separate 2000 L vessel is primed with approximately 90% of the final volume of production medium (step 5), and the AAV producer cells are transferred from N-1 to the production phase reactor at a split ratio of approximately 1:10 to enable sufficient exchange of growth medium for production medium (step 6). Figure 1 In the figure, the solid line separating the N-1 stage growth reactor and the N-stage production reactor indicates that the two stages are carried out in separate containers. In this way, the 2000L production container is inoculated with a high concentration of cell culture (i.e., greater than 13E06 vc / mL), and the target final concentration is approximately greater than 1.3E06vc / mL. After inoculation, AAV production is initiated by infection with a helper virus (e.g., adenovirus Ad5) (step 7), and then a push injection is added to the bioreactor container to produce the feed (step 8). After about 4-6 days ("dpi") after infection, the AAV product is harvested from the culture supernatant (step 9) for subsequent purification and helper virus removal operations. For example, Ad5 removal processes include those described in, for example, U.S. Patent Application Publication No. US20190083554A1.

[0183] ii. Improved batch systems and methods for AAV production using perfusion (eg, enhanced perfusion)

[0184] Described herein are improved batch processes for AAV production using perfusion (eg, enhanced perfusion). Figure 2 An exemplary process is shown in . In general, the improved batch process involves preparing a container with a growth medium for the growth phase culture of AAV producing cells. In some embodiments, the container can be a single container intended to culture both growth and production phase cultures, i.e., a "combined growth / production container". Such a container is then inoculated with AAV producing cells. Perfusion can be initiated in the inoculated growth container or combined growth / production container to initiate the growth of AAV producing cells. After achieving the final cell density target, the production phase culture can be initiated. In some embodiments, the production phase initiation involves infecting the growth phase culture with a helper virus to initiate AAV production. AAV production can be initiated by alternative means that do not rely on AAV helper virus infection. Such alternative means include, for example, the use of a triple transfection method or the use of producer cells (e.g., (CEVEC) doxycycline-inducible AAV production cells and similar systems). The AAV production phase can be maintained, for example, by providing feed, exchanging the culture medium for production medium, and / or initiating perfusion to maintain production. The AAV thus produced can be harvested using any suitable separation technique and purified using standard downstream processing steps known to those skilled in the art.

[0185] Figure 3 An exemplary process for the AAV production systems and methods described herein is shown. In some embodiments, the preparatory steps of the improved batch (e.g., enhanced perfusion) process can be similar to the conventional steps used in the batch mode AAV production process. For example, the cell seed queue cell mass can be thawed and placed into a shake flask in the same manner as in batch mode operation.

[0186] The bioreactor expansion stage can be used for scale-up to growth / production culture.Any suitable scale can be adopted or adapted to the improved batch system and method of perfusion (e.g., intensive perfusion) described herein.In some embodiments, the bioreactor expansion stage uses a container of, for example, 250L scale.In some embodiments, the bioreactor expansion stage uses a subliter culture volume.In some embodiments, the bioreactor expansion stage uses from subliter to 6000L scale or larger, including any suitable culture volume up to 12000L scale.Therefore, the system and method of the present invention can utilize subliter, 1L, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L or 6000L container in the bioreactor expansion stage culture. In some embodiments, the bioreactor expansion stage uses a vessel with a capacity greater than 6000 L. Those skilled in the art will appreciate that larger culture volumes (such as 6000 L or greater) may require specialized media handling systems / equipment. Therefore, the bioreactor expansion stage may utilize appropriate perfusion equipment, such as an alternating tangential flow (ATF) filtration system. The bioreactor expansion stage culture may involve perfusion equipment, such as the Repligen Xcell ATF TM 6 or other perfusion system appropriate for the culture volume selected for the bioreactor expansion phase. At larger culture volumes (such as 6000 L or greater), multiple such devices may be employed, such as multiple Repligen Xcell ATF TM 6 or ATF TM 10 system. In some embodiments, the bioreactor expansion stage does not utilize an ATF filtration system.

[0187] In some embodiments, the final perfusion production stage is performed in a production vessel at, for example, a 2000 L scale. The production vessel can be a container of any culture volume suitable for use in AAV production at any scale. For example, the production vessel can be a subliter, 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 container or larger.

[0188] In some embodiments, the final perfusion growth and production stages are combined into the same vessel at, for example, a 2000 L scale, referred to herein as a combined growth / production vessel. A combined growth / production vessel can be a vessel suitable for any culture volume 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 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 container or larger. In some embodiments, the combined growth / production vessel uses a container having a capacity greater than 6000 L.

[0189] Those skilled in the art will appreciate that larger culture volumes (such as 6000 L or greater) may require specialized media handling systems / equipment. Thus, one or more growth, production, and / or combined growth / production vessels may utilize appropriate perfusion equipment, such as an alternating tangential flow (ATF) filtration system. One or more growth, production, and / or combined growth / production vessel cultures may involve perfusion equipment, such as the Repligen Xcell ATF TM 6 or other perfusion system suitable for the culture volume selected for the bioreactor stage. At larger culture volumes (such as 6000 L or greater), multiple such devices may be employed, such as multiple Repligen Xcell ATF TM 6 or ATF TM 10 system or flat plate tangential flow filtration system. For example, Repligen Xcell ATFTM 6 or similar comparable systems can be used at a scale of, for example, 50L-500L, while ATF TM 10 or similar systems can be used on a larger scale. In some embodiments, multiple ATFs can be implemented to support increased cell density. TM 10 systems, etc. In some embodiments, the growth, production and / or combined growth / production vessels do not utilize an ATF filtration system.

[0190] In some embodiments, a perfusion system such as an ATF filtration system such as the Repligen Xcell ATF TM 10 is implemented during the growth and / or production phase in a combined growth / production vessel. Alternative perfusion technologies suitable for use in a combined growth / production vessel include, but are not limited to, the Xcellerex Automated Perfusion System (APS) (Cytiva), KPS TFF systems, such as KPS 700 or XCell TM ATF 6 (Repligen), GEA Disposable Drug Separator System (GEA), Prostak TM Microfiltration module (Millipore), Alfa Laval CultureOne TM (Alfa Laval), Centritech separation systems such as CARR Pilot or Centritech CELL (Pneumatic ScaleAngelus), 6000S System (Sartorius), microfluidic cell retention device (as described in Kwon T. et al. SciRep 7, 6703 (2017)), SciPure TMSystem (Parker), acoustic sedimentation system (as described in Coronel J. et al. Front. Bioeng. Biotechnol., 02July 2020) (Sonosep) and / or EDO Electro system (as described in Wang, Zhaowei, "Two Approaches for Cell Retention in Perfusion Culture Systems" (2009). ETD Archive. 304) (American Piezo). Various formats are available from suppliers for the aforementioned perfusion technologies and can be adapted for use in the systems and methods described herein. For example, depending on the desired culture scale and cell density, the Repligen ATF system can be implemented in 2, 4, 6 or 10 filter unit formats. Smaller-scale perfusion technologies, such as WAVE bioreactors (Cytiva) can also be utilized. Alternative culture medium exchange technologies can be implemented, including tangential flow filtration (TFF) and / or centrifugation-based technologies.

[0191] In some embodiments, the N-stage growth culture can target a final density of at least 5E06 vc / m. The N-stage growth can target any final density suitable for inoculating a production stage AAV bioreactor, including, for example, 1E06, 2E06, 3E06, 4E06, 5E06, 6E06, 7E06, 8E06, 9E06, 10E06, 20E06, 30E06, 40E06, or up to 50E6 vc / mL or greater.

[0192] In some embodiments, the growth phase culture can use a blend of growth and production culture medium, for example, perfused during the process of, for example, 2-14 days, to target final density. In some embodiments, only growth medium is used in the growth phase culture. In some embodiments, only production medium is used in the growth phase culture. In some embodiments, a 50 / 50 blend of growth and production culture medium is used in the growth phase culture. In other embodiments, a 10 / 90, 20 / 80, 30 / 70 or 40 / 60 blend of growth and production culture medium is used in the growth phase culture. In still other embodiments, a 90 / 10, 80 / 20, 70 / 30 or 60 / 40 blend of growth and production culture medium is used in the growth phase culture. Growth medium, production medium or a blend of growth and production culture medium can be perfused during the process of, for example, 2-14 days. In some embodiments, a single growth medium is used until the final density target is achieved, at which point a single production culture medium is used to run through the remainder of the production phase.

[0193] refer to Figure 3In the example process described in, when the target cell density is reached in one or more growth stages, the growth medium is triggered in the combined growth / production container (step 1). AAV producer cells, such as any suitable PCL, for example HeLa cells (e.g., HeLa PCL) are inoculated from the previous bioreactor expansion stage with the target final density for inoculation (step 2). Then the perfusion rate is increased with the cell mass to maintain a constant cell specific perfusion rate (CSPR) for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or a duration greater than 6 days (step 3). In some embodiments, the perfusion rate is increased with the cell mass to maintain a constant CSPR for about 2 days to about 6 days, such as about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 4 days to about 5 days, about 4 days to about 6 days or about 5 days to about 6 days. In some embodiments, maintaining a constant CSPR minimizes nutrient depletion and waste accumulation.

[0194] In some embodiments, a fixed volume perfusion rate is utilized regardless of cell density.

[0195] After reaching the target cell density for AAV production (e.g., 5E06 vc / mL) (step 4), the growth perfusion exchange can be stopped. AAV production can then be initiated by infection with a helper virus (e.g., adenovirus Ad5) (step 5). Figure 3In the figure, the dotted line separating the growth phase and the production phase reactor indicates that the two phases can but do not need to be carried out in the same container (e.g., a 2000L production phase bioreactor). In some embodiments, helper virus infection is followed by push feed addition to support production phase activities (step 6). In some embodiments, the growth medium is then replaced with a production medium / feed mixture in, for example, an ATF-10 setting (step 7). In some embodiments, perfusion is restarted at about 1 hour to about 2 hours (hpi) after infection (step 8). Perfusion can be maintained for 1-2dpi to enrich the culture medium, reduce the accumulation of waste, and retain the AAV product in the cell. In some embodiments, the AAV particles remain cell-associated / inside the cell until about 48hpi. Therefore, in order to minimize the loss of product to the waste permeate flow, perfusion can be disabled before this time. After about 1-2dpi, perfusion is disabled to allow AAV particles to remain and accumulate in the culture supernatant (step 9). After about 48 hours, most AAV products are produced but retained in the cells; however, starting from 2-6 dpi, AAV particles are preferentially released into the cell culture supernatant and can be harvested (step 10). In some embodiments, AAV products can be harvested starting at 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.

[0196] In some embodiments, Ad5 removal processes include those described, for example, in U.S. Patent Application Publication No. US20190083554A1.

[0197] Those skilled in the art will appreciate that it will be important to optimize growth and production culture conditions to increase AAV production yields, both in batch mode operation and modified batch mode operation (e.g., enhanced perfusion) as described herein. Each of temperature, pH, osmotic pressure, salt concentration, medium and feed composition, oxygen saturation, agitation rate, initial 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 can affect the AAV production rate 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.

[0198] The Examples section of this application shows exemplary embodiments of the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein, which continuously increase the infection density during AAV production from the existing best low density batch (LDB) process by about 4 times to achieve a 2.5-fold to 3-fold increase in AAV volumetric production rate, greater than or equal to 3E11 GC / mL (genome copies / mL) or greater than or equal to 3E14 GC / mL.

[0199] The improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein have been evaluated in the context of two separate HeLa producer cell line systems utilizing different growth media, different genes of interest for therapeutic payloads, and different capsids, and have achieved similar fold improvements in production rates relative to corresponding low-density batch mode operating conditions. These results show that the improved batch systems and methods using perfusion (e.g., enhanced perfusion) described herein can be implemented to achieve AAV yield benefits in multiple AAV producer cell systems under different media conditions, with different genes of interest and different AAV capsids.

[0200] Sections a.-j. below provide examples of various AAV culture parameters that can be used in various embodiments of the improved batch systems and methods described herein that use perfusion (eg, enhanced perfusion).

[0201] a. Medium exchange and perfusion

[0202] In some embodiments, the improved batch systems and methods disclosed herein using perfusion (e.g., enhanced perfusion) for culturing AAV PCLs include, for example, removing waste from the culture medium and / or replenishing the culture medium with nutrients by using perfusion, such as alternating tangential flow (ATF) perfusion, in the growth phase and / or production phase culture vessels. Perfusion can be used, for example, to build cell clusters in growth phase culture, and / or to maintain cell clusters in production phase culture.

[0203] ATF uses a filtration system that provides a continuous or semi-continuous way to exchange cell culture media, such as by continuously / semi-continuously supplementing the culture with fresh culture media while, for example, removing waste by a large number of hollow fibers. How fast the ATF flow rate indicates that the cell culture circulates in each hollow fiber. In some embodiments, the diameter of each fiber is about 0.5 mm to about 1.5 mm, such as 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 cells. In addition, too low flow rates may increase the risk of (1) cell culture exposure to a hypoxic environment for a long period of time and (2) the possibility of filter contamination due to insufficient backwashing. The method described herein includes ATF perfusion at a flow rate that minimizes the shear stress on the cells but provides enough oxygen to the cells. In some embodiments, ATF perfusion is carried out at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber, such as 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, ATF perfusion is carried out 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, ATF infusion is performed at a flow rate of about 50 m / s to about 320 m / s per fiber, e.g., about 50 m / s to about 250 m / s, about 50 m / s to about 200 m / s, about 100 m / s to about 320 m / s, about 100 m / s to about 250 m / s, about 200 m / s to about 320 m / s, or about 200 m / s to about 275 m / s.

[0204] In some embodiments, the perfusion rate can be optimized to maintain a shear rate to increase AAV product release. The shear stress on the cells introduced by perfusion can lead to cell rupture and / or release of AAV particles. Therefore, the perfusion rate can be optimized to maintain a shear rate that leads to constant release of AAV products in perfusion culture. The AAV released in the culture in this way can be continuously collected at different time points or during the production stage, for example, from the permeate when free AAV particles pass through the perfusion membrane.

[0205] During the first 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 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 techniques suitable for use in various embodiments of the systems and methods described herein include the Xcellerex Automated Perfusion System (APS) (Cytiva), KPS TFF systems, such as KPS 700 or XCell TM ATF 6 (Repligen), GEA Disposable Drug Separator System (GEA), Prostak TM Microfiltration module (Millipore), Alfa Laval CultureOne TM (Alfa Laval), Centritech separation systems such as CARR Pilot or Centritech CELL (Pneumatic Scale Angelus), 6000S System (Sartorius), microfluidic cell retention device (as described in Kwon T. et al. Sci Rep 7, 6703 (2017)), SciPure TM System (Parker), acoustic sedimentation system (as described in Coronel J. et al. Front. Bioeng. Biotechnol., 02July 2020) (Sonosep) and / or EDO Electro system (as 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 can be implemented in 2, 4, 6 or 10 filter unit formats. Alternative culture medium exchange techniques can be implemented, including tangential flow filtration (TFF) and / or centrifugation-based techniques.

[0206] Higher cell densities may require higher perfusion, medium exchange, and / or waste removal rates in order to maintain a certain cell growth rate or viability. In cell culture, cell density can change over time. Therefore, cell specific perfusion rate (a parameter that quantifies perfusion rate based on the cell density of the culture) can be used in the systems and methods described herein. In some embodiments, the improved batch systems and methods described herein using perfusion (e.g., enhanced perfusion) include perfusing in the growth and / or production phase 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, the improved batch systems and methods described herein using perfusion (e.g., intensified perfusion) include perfusing at a CSPR of about 100 pL / cell / day to about 2000 pL / cell / day in the growth phase and / or production phase culture, the range including all integers between and in between 100 pL / cell / day and 2000 pL / cell / day. For example, the CSPR can be about 100 pL / cell / day to about 1500 pL / cell / day, about 100 pL / cell / day to about 1000 pL / cell / day, about 100 pL / cell / day to about 900 pL / cell / day, about 100 pL / cell / day to about 800 pL / cell / day, about 100 pL / cell / day to about 700 pL / cell / day, about 100 pL / cell / day to about 600 pL / cell / day, about 100 pL / cell / day to about 500 pL / cell / day, about 100 pL / cell / day to about 400 pL / cell / day, about 100 pL / cell / day to about 300 pL / cell / day, 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, such as 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 should be understood that the CSPR in a given system will vary according to various parameters including, for example, cell density.

[0207] In some embodiments, the improved batch systems and methods described herein that use perfusion (eg, intensified perfusion) include perfusing at a constant perfusion rate during growth and / or production phase cultures, regardless of cell density.

[0208] b. Cell Culture Media and Supplements

[0209] One or more cell cultures described herein can be prepared in any culture medium suitable for the specific cells being cultured. In some embodiments, the culture 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 comprising 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), hydrolyzates (hydrolyzed proteins derived from plant or animal sources), hydrolyzates (hydrolyzed proteins derived 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-α), 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 culture medium 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. WO 90 / 03430 and WO 87 / 00195 can be used as the culture medium in the systems and methods disclosed herein. Any of these culture media can be supplemented 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 suitable carriers thereof and glucose or an equivalent energy source as required. 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.

[0210] In some embodiments, culturing AAV PCL in a growth phase and / or production phase culture vessel (or a combined growth / production vessel) includes supplementing with nutrients, such as using a fed-batch culture process, which includes regularly supplementing the culture with supplements (e.g., fresh culture medium, amino acids, and / or glucose). In some embodiments, supplementation occurs at least once every two days, such as once a day, twice a day, three times a day, four times a day, or more. In other embodiments, supplementation occurs about once a day. In some embodiments, the supplement includes amino acids, such as a mixture of one or more amino acids. In some embodiments, the supplement includes glutamine. In some embodiments, the supplement includes glucose. In some embodiments, the supplement includes glucose and glutamine. In some embodiments, the concentration of each supplement added is determined based on viable cell density, for example, at the time of adding the supplement or at the viable cell density determined just before the supplement is added. In some embodiments, the amount (e.g., volume and / or concentration) of the supplement is determined based on, for example, integrated cell growth (ICG) in the growth phase and / or production phase culture. ICG can be calculated as described, for example, in International Patent Application Publication No. WO2020 / 154607.

[0211] 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 includes 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.

[0212] In some embodiments, for example, during the growth phase culture period, for example, during any day of the growth phase culture period, or during the production phase culture period, nutrient concentration is maintained at a desired level and / or waste is maintained at a low level. In some embodiments, the concentration of glucose, glutamine, glutamate, total amino acids, lactic acid and / or ammonia in the N-1 culture is maintained at a level within 10 times (for example, within 10 times, within 9 times, within 8 times, within 7 times, within 6 times, within 5 times, within 4 times, within 3 times, within 2 times or less) of the concentration on day 0 of the growth phase and / or production phase culture. In some embodiments, the concentration of any one of these molecules is, for example, measured using standard methods on any day of cultivation.

[0213] c. Daily container volume

[0214] In some embodiments, the improved batch system and method of perfusion (for example, intensive perfusion) described herein are included in the growth phase and / or the production phase during the culture medium exchange rate culture cells, and the culture medium exchange rate is expressed by the container volume (VVD) of culture medium every day. The growth phase culture can be carried out with the culture medium exchange rate of about 1VVD to about 10VVD. For example, the growth phase culture can be carried out with the culture medium exchange rate of 1VVD, 1.5VVD, 2VVD, 2.5VVD, 3VVD, 3.5VVD, 4VVD, 4.5VVD, 5VVD, 5.5VVD, 6VVD, 6.5VVD, 7VVD, 7.5VVD, 8VVD, 8.5VVD, 9VVD, 9.5VVD or 10VVD. Similarly, the production phase culture can be carried out with the culture medium exchange rate of about 1VVD to about 10VVD. For example, the production phase culture can be carried out at a medium exchange rate of 1 VVD, 1.5 VVD, 2VVD, 2.5 VVD, 3VVD, 3.5 VVD, 4VVD, 4.5 VVD, 5VVD, 5.5 VVD, 6VVD, 6.5 VVD, 7VVD, 7.5 VVD, 8VVD, 8.5 VVD, 9VVD, 9.5 VVD or 10 VVD. In some embodiments, one or more of the growth and production phase cultures are performed with a media exchange rate of between 1 VVD, 1.5 VVD, 2VVD, 2.5 VVD, 3VVD, 3.5 VVD, 4VVD, 4.5 VVD, 5VVD, 5.5 VVD, 6VVD, 6.5 VVD, 7VVD, 7.5 VVD, 8VVD, 8.5 VVD, 9VVD, 9.5 VVD, or 10 VVD.

[0215] d. Cell density and viability

[0216] In some embodiments, the improved batch systems and methods described herein using perfusion (e.g., intensified perfusion) include culturing cells to a target cell density (e.g., expressed as viable cell density) of at least 1E06 viable cells / mL (vc / mL), at least 1E07 vc / mL, at least about 1E08 vc / mL, or at least about 1E09 vc / mL. In some embodiments, the methods described herein include culturing cells in growth phase culture to a target cell density of about 1E06 vc / mL to about 50E06 vc / mL, e.g., about 1E06 vc / mL, about 2E06 vc / mL, about 3E06 vc / mL, about 4E06 vc / mL, about 5E06 vc / mL, 6E06 vc / mL, 7E06 vc / mL, 8E06 vc / mL, 9E06 vc / mL, 10E06 vc / mL, 20E06 vc / mL, 30E06 vc / mL, 40E06 vc / mL, or 50E06 vc / mL. In some embodiments, the methods described herein include culturing the cells in growth phase culture to a target cell density of about 1E06 vc / mL, about 1.1E06 vc / mL, about 1.2E06 vc / mL, about 1.3E06 vc / mL, about 1.4E06 vc / mL, about 1.5E06 vc / mL, about 1.6E06 vc / mL, about 1.7E06 vc / mL, about 1.8E06 vc / mL, about 1.9E06 vc / mL, about 2E06 vc / mL, about 2.1E06 vc / mL, about 2.2E06 vc / mL, about 2.3E06 vc / mL, about 2.4E06 vc / mL, about 2.5E06 vc / mL, about 2.6E06 vc / mL, about 2.7E06 vc / mL, about 2.8E06 vc / mL, about vc / mL, about 2.9E06 vc / mL, about 3E06 vc / mL, about 3.1E06 vc / mL, about 3.2E06 vc / mL, about 3.3E06 vc / mL, about 3.4E06 vc / mL, about 3.5E06 vc / mL, about 3.6E06 vc / mL, about 3.7E06 vc / mL, about 3.8E06 vc / mL, about 3.9E06 vc / mL, about 4E06 vc / mL, about 4.1E06 vc / mL, about 4.2E06 vc / mL, about 4.3E06 vc / mL, about 4.4E06 vc / mL, about 4.5E06 vc / mL, about 4.6E06 vc / mL, about 4.7E06 vc / mL, about 4.8E06 vc / mL vc / mL, about 4.9E06 vc / mL, about 5E06 vc / mL, or greater than 5E06 vc / mL.In some embodiments, the methods described herein include culturing cells in growth phase culture to a target cell density of about 1E06 vc / mL to about 1E07 vc / mL, about 1E07 vc / mL to about 1E08 vc / mL, or about 1E08 vc / mL to about 1E09 vc / mL. In some embodiments, the methods described herein include culturing cells in growth phase culture to a target cell density of about 1E06 vc / mL to about 1.5E06 vc / mL, about 1.5E06 vc / mL to about 2E06 vc / mL, about 2E06 vc / mL to about 2.5E06 vc / mL, about 2.5E06 vc / mL to about 3E06 vc / mL, about 3E06 vc / mL to about 3.5E06 vc / mL, about 3.5E06 vc / mL to about 4E06 vc / mL, about 4E06 vc / mL to about 4.5E06 vc / mL, or about 4.5E06 vc / mL to about 5E06 vc / mL.

[0217] In some embodiments, the methods described herein include determining the viable cell density periodically, such as at least once every 3 days, once every 2 days, once a day, twice a day, or more frequently, such as once a minute, twice a minute, three times a minute, four to ten times a minute, once an hour, twice an hour, three times an hour, four to ten times an hour, once a second, twice a second, three times a second, four times a second, five times a second, six times a second, seven times a second, eight times a second, nine times a second, or ten times a second.

[0218] e. Cell growth rate

[0219] 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.

[0220] In some embodiments, the maximum growth rate is the growth rate of a specific cell line / clone measured in fresh medium and during its exponential growth phase (e.g., measured at a time point when nutrients are sufficient and no component in the culture causes significant growth inhibition). In some embodiments, the total growth rate depends on the specific cell type / clone cultivated. In some embodiments, the total growth rate of the cell is about 0.2 / day to about 1 / day, such as 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 total growth rate is determined based on the cell density at the 0th day of cultivation.

[0221] f. pH

[0222] In some embodiments of the improved batch systems and methods described herein using perfusion (e.g., intensified perfusion), the pH of the growth phase and / or production phase culture is maintained, for example, between 5 and about 9, such as from about 6.4 to about 8.2. For example, in some embodiments, the pH of the growth phase 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. In some embodiments, the pH of the production phase 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.

[0223] g. Dissolved oxygen

[0224] In some embodiments of the improved batch systems and methods described herein using perfusion (e.g., enhanced perfusion), dissolved oxygen (DO) levels are maintained for optimal growth and / or AAV production in the growth phase and / or production phase cultures. For example, the DO level can be maintained at 10% to 100%, such as at a 50% set point.

[0225] h. Cultivation duration

[0226] In some embodiments, the AAV producer cells are cultured in a growth phase and / or production phase culture for 4 to 12 days, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days. In some embodiments, the production phase culture is maintained for a duration of 2 or more days, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0227] In some embodiments, the production phase culture is maintained under conditions that allow the production of AAV. In some embodiments, one or more components of AAV are added to the production phase culture so that the cells in the production phase culture are replication competent and can produce AAV. In some embodiments, a helper virus, such as an adenovirus (e.g., an Ad5 helper virus) or a herpes virus is added to the production phase culture.

[0228] In certain embodiments, the method comprises culturing the cells in production phase culture for less than a 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., 2-5 days, e.g., 2, 3, 4 or 5 days.

[0229] i. Culture volume

[0230] In some embodiments, the growth phase culture is performed at any suitable scale for the AAV producer cell growth phase. For example, the growth phase culture can be performed at a subliter to 6000 L scale. Thus, the present systems and methods can utilize subliter, 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, or 6000 L vessels for growth phase culture in a bioreactor.

[0231] In some embodiments, any pre-production stage culture is performed at any suitable scale for the AAV producer cell growth phase. For example, any pre-production stage culture can be performed at a sub-liter to 6000 L scale. Thus, the present systems and methods can utilize 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, or 6000 L vessels in a bioreactor for pre-production or growth phase cultures.

[0232] In some embodiments, the production phase culture is performed at any suitable scale for AAV production. For example, the production phase culture can be performed at a subliter to 6000 L scale. Thus, the present systems and methods can utilize subliter, 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, or 6000 L vessels for production phase culture in a bioreactor.

[0233] In some embodiments, one or more growth and production phase cultures are performed in a combined growth / production vessel at any suitable scale for AAV growth and production. For example, one or more growth / production phase cultures can be performed in the same combined growth / production vessel at a subliter to 6000L scale. Thus, the systems and methods of the present invention can utilize subliter, 1L, 2L, 5L, 10L, 20L, 50L, 100L, 150L, ​​200L, 250L, 500L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L containers in a bioreactor for production phase cultures.

[0234] j. Downstream Process

[0235] In some embodiments, the methods described herein further include collecting or harvesting the AAV product from the production stage culture. Standard methods for collecting or isolating viral particles can be used, for example, including but not limited to filtration or centrifugation. Further downstream purification processes can be used to purify the collected AAV product.

[0236] According to the methods described herein, for example, the AAV titer in N culture can be determined, for example, on any one or more days of N culture. An exemplary AAV titer measurement result is vector genome copies / cell (GC / cell). GC / cell can be determined by standard methods in the art, for example, including but not limited to dot blot, quantitative PCR or ddPCR, spectroscopy or fluorescence. See, for example, Dorange et al. Cell Gene Therapy Insights 4.2 (2018): 119-129.

[0237] In some embodiments, the methods described herein are capable of producing, for example, in a production phase culture, an AAV titer of at least about 1E09 vg / mL, at least about 2E09 vg / mL, at least about 3E09 vg / mL, at least about 4E09 vg / mL, at least about 5E09 vg / mL, at least about 6E09 vg / mL, at least about 7E09 vg / mL, at least about 8E09 vg / mL, at least about 9E09 vg / mL, at least about 1E10 vg / mL, at least about 2E10 vg / mL, at least about 3E10 vg / mL, at least about 4E10 vg / mL, at least about 5E10 vg / mL, at least about 6E10 vg / mL, at least about 7E10 vg / mL, at least about 8E10 vg / mL, at least about 9E10 vg / mL, at least about 1E11 vg / mL, at least about 2E11 vg / mL, vg / mL, at least about 3E11 vg / mL, at least about 4E11 vg / mL, at least about 5E11 vg / mL, at least about 6E11 vg / mL, at least about 7E11 vg / mL, at least about 8E11 vg / mL, at least about 9E11 vg / mL, at least about 1E12 vg / mL, at least about 2E12 vg / mL, at least about 3E12 vg / mL, at least about 4E12 vg / mL, at least about 5E12 vg / mL or more.

[0238] In the present disclosure, when an element or component is referred to as being included in a list of elements or components and / or selected from a list of elements or components, it should be understood that the element or component can be any one of the elements or components, or the element or component can be selected from two or more of the elements or components.

[0239] In addition, it should be understood that the elements and / or features of the compositions, systems or methods described herein can be combined in various ways without departing from the spirit and scope of the present disclosure, whether explicitly or implicitly in this article. For example, unless the context otherwise understands, otherwise in the case of referring to a specific compound or system component, the compound or system component can be used in the various embodiments of the compositions / systems of the present disclosure and / or the methods of the present disclosure. In other words, in the present disclosure, embodiments have been described and depicted in a manner that enables writing and drawing clear and concise disclosures, but it is intended and will be understood that embodiments can be combined or separated in various ways without departing from this teaching. For example, it should be understood that all features described and depicted herein can be suitable for all aspects of one or more inventions described and depicted herein.

[0240] It should be understood that, unless the context and usage indicate otherwise, the expression "at least one of..." includes each of the objects following the expression individually and various combinations of two or more of the objects. Unless the context indicates otherwise, the expression "and / or" related to two or more of the objects should be understood to have the same meaning.

[0241] Unless the context specifically states or understands otherwise, the use of the terms "include", "includes", "including", "have", "has", "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.

[0242] It should be understood that the order of steps in a system or process or the order in which certain actions are performed is not important as long as the purpose of the present disclosure remains operable. In addition, in many embodiments of the systems and methods described herein, two or more steps or actions may be performed simultaneously.

[0243] The use of any and all examples or exemplary language (e.g., "such as" or "including") herein is intended merely to better illustrate the invention disclosed herein and does not constitute a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the systems and methods for AAV production disclosed herein. Example

[0244] Having now generally described the systems and methods of the present invention, the same will be more readily understood by reference to the following examples, which are intended merely to illustrate certain aspects and embodiments of the present disclosure and are not intended to limit the present disclosure.

[0245] Materials and methods

[0246] Metabolites (glucose, L-glutamine, ammonia, and lactate) were evaluated in AAV producer cell cultures by Nova Bio-profile Flex2 (Nova Biomedical). Cell density was evaluated in AAV producer cell cultures by Vi-Cell XR (Beckman-Coulter). For one or more specific AAV gene therapy products, AAV titers were quantified by DNase-resistant particles (DRP) analysis by quantitative DNA polymerase chain reaction (qPCR). One or more specific adenoviral genes of adenoviral byproducts, such as E2A, were also evaluated by DRP-qPCR.

[0247] Example 1: Increasing cell density in batch mode alone is not sufficient to increase AAV volume production

[0248] High cell density production was tested in batch AAV production mode to determine whether increasing cell density could improve AAV volumetric production. It was determined that increasing cell density in batch mode operation had a negative impact on AAV production rate.

[0249] HeLa producer cells were inoculated at 1.3E06 viable cells / ml (vc / mL), 3.15E06 vc / mL, or 5.0E06 vc / mL in a bioreactor (2L wv) and infected with adenovirus type 5 (Ad5) to initiate gene therapy product AAV production in batch mode operation. The AAV capsid was the clade E capsid AAV hu37. Batch mode conditions in this process utilized a target inoculation density of 1.3E06 vc / mL. Increasing the cell density by 2-fold and 4-fold (to 3.15E06 and 5E06 vc / mL) resulted in volumetric rAAV yields of ( Figure 4 ) and cell-specific rAAV production rate ( Figure 5 ) are reduced by approximately 10 and 100 times.

[0250] Rapid depletion of nutrients such as glucose and L-glutamine was observed, accompanied by a significant increase in waste byproducts such as lactate and ammonia. Therefore, it was hypothesized that nutrient depletion and / or waste accumulation led to a decrease in AAV production rate (data not shown).

[0251] This example shows that batch AAV production methods cannot support AAV production at higher cell densities. In fact, increasing cell density in batch mode resulted in a more than 100-fold decrease in AAV yield, accompanied by a 4-fold increase in the inoculum density for AAV production.

[0252] Example 2: Lowering pH increases AAV production rates at higher cell densities in batch mode

[0253] Attempts were made to increase AAV production rates at higher densities (e.g., above about 1E06 vc / mL) in batch mode. It was determined that lowering the pH at higher cell densities in batch mode increased AAV production rates and reduced waste accumulation. HeLa producer cells were inoculated in a bioreactor (2 L wv) at 1.3E06 vc / mL or 3.15E06 vc / mL and infected with Ad5 to initiate AAV production in batch mode operation at a pH set point of pH 7.4, 7.6, or 7.8. At 1.3E06 vc / mL in batch mode, a pH of 7.6 to 7.8 showed the highest rAAV yield (although not significantly different from the yield observed at pH 7.4) ( Figure 6 and Figure 7 , solid bars); however, at a cell density of 3.15E06 vc / mL, a pH of 7.4 showed a higher AAV volumetric yield ( Figure 6 , open bars) and specific production rate ( Figure 7 , open bars) yield. At higher cell density (3.15E06 vc / mL) and low pH (7.4), AAV production increased, consistent with decreased accumulation of waste byproducts (lactate) ( Figure 8 ). This suggests that AAV production rates may be inhibited by the accumulation of waste byproducts. Process and media / feed improvements to reduce the accumulation of waste byproducts (e.g., lower pH to reduce lactate levels) and / or increase nutrient utilization may lead to increased AAV production capacity in higher cell density cultures.

[0254] Example 3: AAV and adenovirus cellular release kinetics to achieve perfusion capability

[0255] After infection with adenovirus, AAV and Ad5 titers and cell viability were monitored over time in several 2L bioreactor (AAV, n=17 and Ad5, n=25) batch mode runs. Adenovirus infection of cells is known to induce apoptosis through the E1A, E3 and E4 genes, leading to 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). Plotting the % viral release versus days post infection, it was observed that for the first 2 days post infection, most of the AAV and adenovirus remained associated with the intracellular fraction ( Fig. 9A From day 2 to day 3 to day 4, an approximately 30% increase per day in non-cell associated (i.e., free) AAV and adenovirus particles was observed. rAAV titers were plotted versus days post infection ( Fig. 9B) illustrates the onset of rAAV release into the supernatant after infection. In addition, a strong correlation between AAV and Ad5 release and cell death during production was observed ( Fig.10 ), indicating that viral release is caused by cell lysis. The kinetics of AAV and adenovirus release are important for understanding when to initiate perfusion and how long to maintain perfusion to maximize product yield and recovery. Based on the above findings from HeLa producer cells (Examples 1 and 2), the optimal perfusion start time was identified to be approximately between 1 and 2 hpi based on adenovirus cellular uptake at different densities (data not shown). In addition, perfusion was maintained up to 48 hpi to avoid significant product loss due to perfusion medium exchange, because most of the AAV remained intracellular during the first 2 days after infection with helper virus.

[0256] These results also show that optimal harvest can be extended beyond 4 days to support maximal product recovery from the culture supernatant, as only 70%-80% of AAV is released after 4 days post-infection, and that extending harvest to 5, 6, 7, etc. days or longer may be important, depending on the specific optimized culture AAV kinetics.

[0257] Example 4: Perfusion process optimization of medium exchange rate

[0258] In batch mode operation, the cell specific perfusion rate (CSPR) at 3E06 vc / mL and 5E06 vc / mL was found to be between 0.5 nL / cell / day and 0.6 nL / cell / day based on nutrient consumption and waste accumulation rates (data not shown). To evaluate the improved perfusion mode operation of batch AAV production at increased cell density, HeLa producer cells were seeded at 5E06 vc / mL in a flow cytometer with XCell Alternating Tangential Flow 2 (Repligen, ATF TM 2) system in a 2L bioreactor and infected with adenoviral helper virus to initiate AAV production. 2 hours after infection, perfusion was initiated with a production medium / feed mixture perfusion medium exchange rate of 0, 1 vessel volume / day, or 2.5 vessel volumes / day (VVD), which was continued for 48 hours after infection and compared with a batch mode control process. rAAV volume production ( Fig.11A ) and specific production rate ( Fig.12) increased with increasing VVD. The highest level was observed at 2.5VVD, with an approximately 3-fold increase in volumetric productivity and an approximately 60% increase in cell specific productivity compared to the batch mode control process. Therefore, higher VVD conditions up to 10VVD were tested. Specifically, volumetric productivity was evaluated at 10VVD with cell densities of 12E06 cells / mL to 13E06 cells / mL. Compared to the 2.5VVD condition, volumetric productivity increased ( Fig. 11B ), on each day from day 2 to day 5, an increase in extracellular rAAV production was observed compared to the 2.5VVD condition.

[0259] Glucose and glutamine levels were rapidly depleted in high cell density cultures with 0 or 1 VVD, but were maintained at 2.5 VVD for 48 h during perfusion exchange, similar to the batch mode control process ( Fig.13 , left two panels). Lactate levels increased significantly under the 0 VVD condition, but the 1 and 2.5 VVD conditions matched the batch mode control ( Fig.13 , upper right panel). All ammonium concentrations were higher under high-density perfusion conditions compared with 0 and 1 VVD conditions, but remained lower under 2.5 VVD conditions ( Fig.13 , lower right panel), demonstrating that an improved batch process can achieve high cell density production conditions in an AAV production system using HeLa producer cells and helper virus by reducing waste accumulation and nutrient depletion through perfusion medium exchange between 2-48 hpi.

[0260] These data show that increasing the perfusion medium exchange rate improves cell-specific AAV production rates at high density and increases rAAV volumetric production.

[0261] Example 5: Improved batch process scale-up to 50 L

[0262] AAV production was performed using a modified batch process at 2L scale, using ATF2, and at 50L scale, using ATF6, with scaled operating parameters, and the production rates were compared to a batch-mode control process at 2L scale. AAV volumetric production rates were maintained at both 2L and 50L scales using the modified batch process, and an approximately 2.3-fold improvement in volumetric yield was achieved compared to the batch-mode control ( Fig.14 ) (3.3E11 GC / mL with the modified batch process vs. 1.4E11 GC / mL with the batch mode process), the cell specific productivity was approximately 60% compared to the batch mode process ( Fig.15 ).

[0263] These data show that the improved batch process for AAV production is scalable and consistently produces several-fold improvements in volumetric yield, despite a slight reduction in specific production rate.

[0264] like Fig.16 As shown in Figure 5, at 1, 2, 3 and 4 dpi, the viable cell density (VCD) increased in the modified batch process during the production phase compared to the batch mode control process. The cell viability profiles in the 2L and 50L modified batch processes were similar compared to the batch mode control process ( Fig.17 ).

[0265] Metabolite analysis showed that the modified batch process maintained similar nutrient and waste levels compared to the batch mode control process. For example, glucose and L-glutamine levels were maintained at similar levels up to 2 dpi compared to the batch process ( Fig.18 ), and the accumulation of lactic acid and ammonium was comparable to that of the batch process ( Fig.19 ). These data show that the improved batch process is able to increase viable cell density in a scalable manner by maintaining key nutrient consumption and waste accumulation rates, resulting in higher AAV production rates per volume, compared to a batch-mode control process.

[0266] In addition, if Fig. 20A As shown in Figure 1, 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 improved batch processes, indicating that the improved batch process does not compromise AAV packaging efficiency. In fact, further analysis showed that packaging efficiency using the improved batch process described herein increased with increasing VVD. Fig. 20B As shown in , the ratio of intermediate AAV particles to complete AAV particles using the improved batch process decreased with increasing VVD compared to the control batch production process. The observed enrichment of complete species and reduction of intermediate species with increasing VVD suggests that additional waste removal / nutrient enrichment is beneficial to AAV product quality.

[0267] Example 6: The improved batch AAV production process is generic and can be implemented with different culture media, vector genomes / genes of interest, and AAV capsid serotypes

[0268] An improved batch process was developed and optimized using procedure A, medium composition A, and capsid A (clade E capsid, AAV hu37) and compared to a different gene of interest (procedure B) with a different capsid serotype B (clade E, AAV8) and different medium composition B. A 2.3-fold to 3-fold improvement in AAV volumetric productivity was observed in both processes A and B ( Fig.21A, left), indicating that our technology can be generally applied to other AAV processes, media and culture conditions and other capsid serotypes. For process A, approximately 60% cell-to-cell productivity was observed compared to the batch mode control, and for process B, approximately 80% cell-to-cell productivity was observed ( Fig.21A , right), indicating that optimization of process parameters and media composition can lead to higher yield production conditions that more closely match batch-mode AAV cell specific production rates.

[0269] The improved batch process was further tested using a different program (Procedure C) with a different gene of interest from Program A and Program B, a different medium composition from Program A and Program B, and a different capsid (clade F capsid, AAV9) from Program A and Program B; the results were compared to the batch mode process. For the improved batch process, the starting cell density for infection increased from 1.17E6 cells / mL to 4.5E6 cells / mL. At harvest, the yield increased from 7.1E10 GC / mL to 3.1E11 GC / mL using the improved batch process described herein, an approximately 4-fold increase ( Fig. 21B ). Cell health and metabolite profiles (data not shown) were comparable to those observed in other runs using the modified batch process.

[0270] These data show that the improved batch method (e.g., intensified perfusion) is suitable for a wide range of products, processes, and production conditions with different media conditions, vector genomes / genes of interest, and AAV capsid serotypes.

[0271] Example 7: Lactic acid and ammonia waste byproducts inhibit AAV production

[0272] As described in Example 1, high cell density in batch mode AAV production leads to a significant increase in waste byproduct formation, including lactate and ammonia / ammonium. To determine the impact of these waste byproducts on AAV production, production was tested in batch AAV production mode with varying levels of ammonia, lactate, or both. Fig. 22 shows increased lactate (30 mM) (in Fig. 22 indicated as "High Lac" in Fig. 22 NH4” in the Figure 5) reduced rAAV yield to approximately 60% of the control rAAV harvest yield condition, while increased lactate and ammonia (in Fig. 22 The increase of 20% NA (indicated as "both" in Figure 1) reduced rAAV yield to approximately 35% of the control rAAV harvest yield condition. This example confirms that high cell density AAV production methods require effective waste byproduct removal or mitigation to achieve optimal yields.

[0273] Example 8: Specific production rate increases with increasing CSPR

[0274] like Fig.23 As shown in Figure 1, increasing the cell specific perfusion rate from 150 pL / cell / day to 750 pL / cell / day resulted in an increase in specific production rate (measured as genome copies (GC) / cell). A strong linear correlation was observed between AAV cell specific production (GC / cell) and CSPR (pL / cell / day) (up to approximately 750 pL / cell / day), R 2 =0.91. As the perfusion rate increased, no obvious limitation (or plateau) was observed in the cell specific production rate, indicating that additional increases in CSPR rates greater than 750 pL / cell / day will continue to increase the AAV production rate within our system. These data show that the conditions tested so far have not revealed a plateau in the cell specific production rate, indicating that the improved batch process described herein may be able to achieve further increases in the cell specific production rate, possibly exceeding that observed using a batch mode AAV production process.

[0275] Example 9: Improved batch AAV production process can significantly reduce the cost of goods

[0276] Cost of Goods (COGs) estimates show the potential to reduce costs associated with AAV production by using the improved batch (e.g., intensified perfusion) production processes described herein. Figure 24A-24C As shown in , even taking into account the higher up-front costs per AAV production batch, the gains per volume production rate are expected to outweigh the increased material and operating costs required to operate the improved batch process. Thus, using the improved batch process described herein, fewer production batches, shorter time frames for producing materials, and lower overall COGs can be achieved. Fig.24A The approximate expected COG reduction required per AAV yield in the improved batch process compared to batch production is shown. Fig. 24B It is shown that the approximate number of production batches required per AAV yield is reduced in the improved batch process compared to batch production. Fig.24C The approximate expected reduction in COGs required per batch quantity produced in the improved batch process is shown compared to batch production. FIG. 24A to FIG. 24C In the figures, diamonds or circles represent the improved batch systems described herein, while squares represent batch operating conditions.

[0277] Example 10: Transition Perfusion Schedule for Alternative Blended Growth Media

[0278] Blended growth and production medium are used in the N-growth phase to expand cell density and balance the growth and production requirements of the cells. However, the use of blended medium has disadvantages, including the lack of stability data for blended medium, the time required for the preparation of blended medium and the general availability, and the higher medium consumption during the growth phase. In order to determine whether the timed transition from growth medium to production medium can avoid the need for blended medium, the production medium was perfused into the reactor at 2.5VVD 24 hours before infection. With respect to released or intracellular rAAV, no significant difference in rAAV titer was observed between the transition to production medium and the use of blended medium ( Fig.25 ), suggesting that transitioning to perfusion medium prior to infection can replace the need for blended culture medium.

[0279] Despite the gains in rAAV production rates, the large volumes of culture medium required for perfusion-based production processes remain a major limitation. For example, perfusion-based production may require 8.5 or more vessel volumes of perfusion medium per run, or 17,000 L of culture medium at a 2,000 L production scale. Therefore, to investigate methods to limit culture medium consumption, a reduction in perfusion rate during the growth phase was tested using a transitional perfusion schedule. Fig.26 It is shown 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 loss of titer. Fig.26 , VVD condition 1 is 1VVD in the growth stage, followed by 2.5VVD in the transition stage; condition 2 is 1VVD in the growth stage and 1.5VVD in the transition stage; condition 3 is 0.5VVD in the growth stage and 2.5VVD in the transition stage; and condition 4 is 0.5VVD in the growth stage and 1.5VVD in the transition stage.

[0280] Example 11: Improved batch process scale-up to 250 L scale

[0281] Example 5 shows that the improved batch process was successfully scaled up to 50 L scale. To further evaluate the scalability of the improved batch rAAV production process, a test pilot scale run was performed at 250 L using ATF6 with scaled operating parameters and the production rate was compared with a batch mode control process at 2 L scale (at Fig. 27 Indicated as "batch" in the 2L modified batch process (in Fig. 27 In the modified batch process (indicated as "MB-2L") at 50 L Fig. 27 In the modified batch process (indicated as "MB-50L" in Fig. 27 In the 50L run, a blended growth and production medium was used during the growth phase. In the 250L run, a 1 VVD perfusion rate was used for the growth phase and a 2.5 VVD perfusion rate was used for the transition phase. The rAAV titers at harvest showed that an approximately 3- to 4-fold increase in volumetric yield was achieved using an approximately 3- to 4-fold increase in viable cell density (VCD). Fig. 27 As shown in , the improved batch process (in Fig. 27 and Fig.28 The yield gain of the 250L scale (abbreviated as "MB") was maintained at 250L scale (approximately 5E11 GC / mL) comparable to that of the 2L scale. Fig.28 As shown in , cell specific productivity comparable to that observed at the 50 L and 2 L scales was maintained at the 250 L scale.

[0282] These data further confirm that, despite a slight reduction in specific productivity, the improved batch process for AAV production is scalable and consistently produces several-fold improvements in volumetric yield.

Claims

1. A method for producing a recombinant adeno-associated virus (rAAV), comprising: (a) cultivating AAV-producing host cells to a target cell density in a growth vessel 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 cell 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, the rAAV is produced.

2. The method of claim 1, wherein the AAV-producing host cell is an insect cell or a mammalian cell.

3. The method of claim 2, wherein the AAV producing host cell is a mammalian cell.

4. The method according to claim 3, wherein 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.

5. The method according to any one of claims 1-4, wherein the AAV producing host cell comprises an AAV producer cell line (PCL), which contains genetic material encoding one or more AAV components stably integrated into the genome of the AAV producing host cell.

6. The method of any one of claims 1-5, wherein the AAV PCL comprises one or more genetic modifications that reduce the expression and / or activity of one or more genes and / or proteins to reduce the production or accumulation of lactate and / or ammonia.

7. The method of any one of claims 1-6, wherein the target cell density in step (a) is at least about 1E06 viable cells / mL (vc / mL).

8. The method according to any one of claims 1-7, wherein the target cell density in step (a) is between 1E06 vc / mL and 5E07 vc / mL.

9. The method of any one of claims 1-7, wherein the target cell density in step (a) is about 1E06 vc / mL, about 2E06 vc / mL, about 3E06 vc / mL, about 4E06 vc / mL, about 5E06 vc / mL, about 6E06 vc / mL, about 7E06 vc / mL, about 8E06 vc / mL, about 9E06 vc / mL, about 1E07 vc / mL, about 1.1E07 vc / mL, about 1.2E07 vc / mL, about 1.3E07 vc / mL, about 1.4E07 vc / mL, about 1.5E07 vc / mL, about 1.6E07 vc / mL, about 1.7E07 vc / mL, about 1.8E07 vc / mL, about 1.9E07 vc / mL, or about 2E07 vc / mL.

10. The method of any one of claims 1-9, wherein the target cell density in step (a) is about 3.15E06 vc / mL or about 5.0E06 vc / mL.

11. The method according to any one of claims 1-10, wherein the production vessel has a volume between 1 L and 12000 L.

12. The method of any one of claims 1-11, wherein the production container has 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.

13. The method of any one of claims 1-12, wherein the growth vessel and the production vessel are a combined growth / production vessel.

14. The method of any one of claims 1-13, wherein the one or more AAV components comprise a therapeutic payload or a transgene, one inverted terminal repeat (ITR) or two ITRs, one or more AAV replication and / or packaging proteins encoded by an AAV rep gene, and / or one or more AAV structural capsid proteins encoded by an AAV cap gene.

15. The method of any one of claims 1-14, wherein step (b) comprises infecting the cells of step (a) with a helper virus.

16. The method of any one of claims 1-14, wherein step (b) comprises inducing expression of one or more helper virus functions encoded by genetic material in the AAV-producing host cell.

17. The method of claim 15, wherein the helper virus is an adenovirus.

18. The method of claim 17, wherein the helper virus is Ad5.

19. The method of any one of claims 1-18, wherein the perfusing is performed at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber.

20. The method of any one of claims 1-19, wherein the 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.

21. The method of any one of claims 1-20, wherein the perfusion is performed at a medium exchange rate of about 0.5 vessel volumes per day (VVD) to about 10 VVD.

22. The method of any one of claims 1-21, wherein the perfusion is 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.

23. The method of any one of claims 1-22, wherein the perfusion is performed at a cell specific perfusion rate (CSPR) of about 150 pL / cell / day to about 2000 pL / cell / day.

24. The method of any one of claims 1-22, wherein the perfusion is performed at a CSPR of about 150 pL / cell / day to about 750 pL / cell / day.

25. The method of any one of claims 1-22, wherein the perfusion is performed at a CSPR of greater than 750 pL / cell / day.

26. The method of any one of claims 1-25, wherein the perfusion is performed using a perfusion system selected from the group consisting of: Xcellerex Automated Perfusion System (APS) (Cytiva); KPS TFF System (Repligen); XCell with 2, 4, 6, 8 or 10 ATF cells TM ATF System (Repligen); GEA Disposable Drug Separator System (GEA); Prostak TM Microfiltration module system (Millipore); Alfa Laval CultureOne TM System (AlfaLaval); Centritech Separation System CARR Pilot or Centritech CELL System (Pneumatic Scale Angelus); 6000S system (Sartorius); microfluidic cell retention device; SciPure TM system (Parker); acoustic sedimentation system; and EDO Electro system (American Piezo).

27. The method of any one of claims 1-26, wherein step (a) comprises culturing the AAV-producing host cell in a growth medium.

28. The method of any one of claims 1-27, wherein steps (a) and (c) comprise culturing the AAV-producing host cells in a growth medium.

29. The method of any one of claims 1-26, wherein step (a) comprises culturing the AAV-producing host cell in a production medium.

30. The method of claim 29, wherein steps (a) and (c) comprise culturing the AAV-producing host cells in a production medium.

31. The method of any one of claims 15-26, wherein 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.

32. The method of claim 31, wherein perfusion of the production medium is initiated about 1 hour to 6 hours after infection of the cells with the helper virus.

33. The method of any one of claims 1-26, wherein steps (a) and (c) comprise culturing the AAV-producing host cells in a blend of growth medium and production medium.

34. The method of claim 33, wherein the blend of growth medium and production medium is a 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20 or 90 / 10 blend of growth medium and production medium.

35. The method of any one of claims 1-26, wherein step (a) comprises culturing the AAV producing host cells in a growth medium until about 24 hours prior to step (b) and then transitioning to a production medium.

36. The method of claim 35, wherein the growth medium is perfused at a rate of about 0.5 VVD to about 1 VVD up to about 24 hours prior to step (b).

37. The method of claim 35 or 36, wherein the production medium is perfused at a rate of about 1.5 VVD to about 2.5 VVD beginning within about 24 hours prior to step (b).

38. The method of any one of claims 1-37, comprising culturing the AAV producing host cell in a growth phase culture and / or a production phase culture for about 48 hours and / or about 2 days to about 14 days.

39. The method of claim 38, comprising culturing the AAV producing host cell in the production phase for a duration 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.

40. The method of claim 38 or 39, further comprising stopping perfusion after a production phase duration 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.

41. The method of any one of claims 38-40, wherein the rAAV is released from the AAV-producing host cell into the supernatant of the production medium.

42. The method of claim 41, wherein the rAAV begins to be released primarily about 48 hours after infection of the cells with the helper virus.

43. The method of claim 42, wherein the perfusion is stopped about 48 hours after infection.

44. The method of claim 43, further comprising harvesting the rAAV.

45. The method of claim 44, wherein harvesting the rAAV is performed under batch mode conditions.

46. ​​The method of any one of claims 1-45, further comprising downstream processing of the rAAV.

47. The method of claim 46, wherein the downstream processing comprises purifying, clarifying and / or concentrating the rAAV.

48. The method of claim 47, wherein the downstream processing comprises filtering out cell debris, colloids or aggregates.

49. The method of claim 48, wherein the filtering removes particles of cell debris, colloids or aggregates having a size greater than 1 μm.

50. The method of any one of claims 46-49, comprising purifying the rAAV using anion exchange (AEX) chromatography.

51. A method of producing a recombinant adeno-associated virus (rAAV), comprising culturing AAV-producing host cells using a perfusion system, wherein the AAV-producing host cell culture has a target cell density between 1E06 vc / mL and 5E07 vc / mL.

52. The method of claim 51, wherein the AAV producing host cell is an insect cell or a mammalian cell.

53. The method of claim 52, wherein the AAV producing host cell is a mammalian cell.

54. The method of claim 53, wherein 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.

55. The method of any one of claims 51-54, wherein the AAV producing host cell comprises an AAV producer cell line (PCL), which comprises genetic material encoding one or more AAV components stably integrated into the genome of the AAV producing host cell.

56. The method of any one of claims 51-55, wherein the AAV PCL comprises one or more genetic modifications that reduce the expression and / or activity of one or more genes and / or proteins to reduce the production or accumulation of lactate and / or ammonia.

57. The method of any one of claims 51-56, wherein the target cell density is about 5E06 vc / mL to about 5E07 vc / mL.

58. The method of any one of claims 51-57, wherein the target cell density is about 1E06, about 2E06, about 3E06, about 4E06, about 5E06, about 6E06, about 7E06, about 8E06, about 9E06, about 1E07, about 1.1E07, about 1.2E07, about 1.3E07, about 1.4E07, about 1.5E07, about 1.6E07, about 1.7E07, about 1.8E07, about 1.9E07, or about 2E07 vc / mL.

59. The method of any one of claims 51-58, wherein the target cell density is about 3.15E06 vc / mL or about 5.0E06 vc / mL.

60. The method of any one of claims 51-59, wherein the method is performed in a production vessel having a volume between 1 L and 12000 L.

61. The method of any one of claims 51-60, wherein the method is carried out in a production vessel having 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.

62. The method of any one of claims 51-61, further comprising growing the AAV producing host cells to the target cell density in a combined growth / production vessel using the perfusion system.

63. The method of any one of claims 51-62, wherein the one or more AAV components comprise a therapeutic payload or a transgene, one inverted terminal repeat (ITR) or two ITRs, one or more AAV replication and / or packaging proteins encoded by an AAV rep gene, and / or one or more AAV structural capsid proteins encoded by an AAV cap gene.

64. The method of any one of claims 51-63, further comprising initiating expression of one or more helper virus functions in the AAV-producing host cell, thereby initiating rAAV production.

65. The method of claim 64, wherein initiating expression of one or more helper virus functions comprises infecting the AAV producing host cell with a helper virus.

66. The method of claim 64, wherein initiating expression of one or more helper virus functions comprises inducing expression of one or more helper virus functions encoded by genetic material in the AAV-producing host cell.

67. The method of claim 65, wherein the helper virus is an adenovirus.

68. The method of claim 67, wherein the helper virus is Ad5.

69. The method of any one of claims 51-68, wherein the AAV producing host cells are cultured at the target cell density under conditions suitable for producing rAAV.

70. The method of any one of claims 51-68, wherein the perfusion system operates at a flow rate of about 3 mL / min / fiber to about 15 mL / min / fiber.

71. The method of any one of claims 51-70, wherein the perfusion system operates 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.

72. The method of any one of claims 51-71, wherein the perfusion system operates at a media exchange rate of about 0.5 vessel volumes per day (VVD) to about 10 VVD.

73. The method of any one of claims 51-72, wherein the perfusion system operates 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.

74. The method of any one of claims 51-73, wherein the perfusion is performed at a cell specific perfusion rate (CSPR) of about 150 pL / cell / day to about 2000 pL / cell / day.

75. The method of any one of claims 51-73, wherein the perfusion is performed at a CSPR of about 150 pL / cell / day to about 750 pL / cell / day.

76. The method of any one of claims 51-73, wherein the perfusion is performed at a CSPR of greater than 750 pL / cell / day.

77. The method of any one of claims 51-76, wherein the perfusion system is selected from: Xcellerex Automated Perfusion System (APS) (Cytiva); KPS TFF System (Repligen); XCell with 2, 4, 6, 8 or 10 ATF cells TM ATF System (Repligen); GEA Disposable Drug Separator System (GEA); Prostak TM Microfiltration module system (Millipore); Alfa Laval CultureOne TM System (Alfa Laval); Centritech Separation System CARR Pilot or Centritech CELL System (Pneumatic Scale Angelus); 6000S system (Sartorius); microfluidic cell retention device; SciPure TM system (Parker); acoustic sedimentation system; and EDO Electro system (American Piezo).

78. The method of any one of claims 51-77, wherein the AAV producing host cell is cultured in a growth medium and subsequently cultured in a production medium.

79. The method of claim 78, wherein the transition to the production medium occurs about 24 hours prior to infection of the AAV production host cells with the helper virus.

80. The method of claim 78 or 79, wherein the growth medium is perfused at a rate of about 0.5 VVD to about 1 VVD until within about 24 hours prior to infection of the AAV-producing host cells with the helper virus.

81. The method of any one of claims 78-80, wherein the production medium is perfused at a rate of about 1.5 VVD to about 2.5 VVD beginning about 24 hours prior to infection of the AAV production host cells with the helper virus.

82. The method of claim 78, wherein the AAV producing host cells are infected with a helper virus after being cultured in the growth medium and before being cultured in the production medium, wherein the production medium is perfused 1 hour to 6 hours after infection of the cells with the helper virus.

83. The method of any one of claims 51-77, wherein the AAV host producer cells are cultured at the target cell density in a blend of growth medium and production medium.

84. The method of claim 83, wherein the blend of growth medium and production medium is a 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20 or 90 / 10 blend of growth medium and production medium.

85. The method of any one of claims 51-84, comprising culturing the AAV-producing host cells at the target cell density for about 48 hours and / or about 2 days to about 14 days.

86. The method of claim 85, comprising culturing the AAV producing host cells at the target cell density for a duration 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.

87. The method of claim 85 or 86, wherein the rAAV is released from the AAV-producing host cell into the supernatant of the production medium.

88. The method of claim 82, wherein the rAAV begins to be released primarily about 48 hours after the cells are infected with the helper virus.

89. The method of claim 88, wherein the perfusion is stopped about 48 hours after infection.

90. The method of any one of claims 51-89, further comprising harvesting the rAAV.

91. The method of claim 90, wherein harvesting the rAAV is performed under batch mode conditions.

92. The method of any one of claims 51-91, further comprising downstream processing of the rAAV.

93. The method of claim 92, wherein the downstream processing comprises purifying, clarifying and / or concentrating the rAAV.

94. The method of claim 93, wherein the downstream processing comprises filtering cell debris, colloids or aggregates.

95. The method of claim 94, wherein the filtering removes particles of cell debris, colloids or aggregates having a size greater than 1 μm.

96. The method of any one of claims 93-95, comprising purifying the rAAV using anion exchange (AEX) chromatography.

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