Methods to improve rAAV production

Abstract

This disclosure relates to methods and compositions for scaling up the production of rAAV by cell cultures. In some embodiments, this disclosure relates to a method for transferring multiple transfection complexes to a bioreactor. Also provided are methods for improving the production of recombinant adeno-associated virus (rAAV) particles by adding a second medium to the cell culture after transfection, thereby improving the yield of rAAV particles and reducing the time required to produce high levels of rAAV particles.

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 462,209, filed Apr. 26, 2023, entitled "METHODS OF IMPROVING RAAV PRODUCTION", and U.S. Provisional Patent Application No. 63 / 462,212, filed Apr. 26, 2023, entitled "METHODS OF SCALING UP PRODUCTION OF RAAV", the contents of each of which are incorporated herein by reference.

Background Art

[0002] One effective method for producing viral vectors for therapeutic use, including but not limited to gene therapy, is to use cultured cells to produce recombinant viral particles. In clinical applications, large amounts of viral particles are required, creating a need to improve the quantity and quality of virus (e.g., rAAV) production.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, there is a need to improve current manufacturing methods for viral vectors used in gene therapy.

Means for Solving the Problems

[0004] This patent application provides methods and compositions for scaling up and / or improving the production of rAAV particles by cultured cells (e.g., using cell cultures). In some embodiments, the methods and compositions disclosed herein are useful for the large-scale production of rAAV and can improve the amount of rAAV particles produced by cultured cells. In some embodiments, the methods and compositions disclosed herein are useful for reducing the risk of contamination during rAAV production (e.g., by reducing the number of batches of components to be combined). In some embodiments, the methods and compositions disclosed herein are useful for reducing one or more resources required to produce rAAV particles. In some embodiments, the methods and compositions disclosed herein are useful for reducing the time required for cells to produce rAAV particles. In some embodiments, the methods and compositions disclosed herein are useful for improving the quality of rAAV produced in cell cultures (e.g., by increasing the number of complete viral particles, which is assessed by measuring the total number of viral particles and the viral genome within the total viral particles, and deriving a percentage of the total viral particles).

[0005] In some embodiments, the Disclosure provides a method comprising: (a) contacting a cell culture in a first medium with one or more recombinant nucleic acids and a transfection reagent; (b) incubating the cell culture for 1 to 20 hours (e.g., 2 to 15 hours, e.g., 3 to 10 hours, or 4 to 8 hours) under conditions sufficient for nucleic acid transfection; and (c) contacting the cell culture from (b) with a second medium to improve rAAV production. In some embodiments, the second medium is added about 4 to 8 hours after step (a). In some embodiments, the second medium is added in two parts, first about 4 to 8 hours after step (a) and then about 24 hours after step (a). In some embodiments, the cells are harvested about 48 hours after step (a). In some embodiments, the method of the Disclosure produces rAAV particles of higher titer. In some embodiments, the method of the Disclosure produces a higher percentage of complete capsids. In some embodiments, the methods disclosed herein enable a reduction in manufacturing time for rAAV production.

[0006] In some embodiments, incubating a cell culture under conditions sufficient for nucleic acid transfection includes contacting the cell culture with one or more transfection reagents.

[0007] In some embodiments, the method further includes measuring the level of metabolites during the above action (b) and providing a second medium when the level of metabolites meets or exceeds a threshold level. In some embodiments, the method further includes measuring the level of nutrients during the above action (b) and providing a second medium when the level of nutrients falls below a threshold level.

[0008] In some embodiments, the cell culture contains epithelial cells. In some embodiments, the cell culture contains mammalian cells. In some embodiments, the cell culture contains HEK cells, CHO cells, or HeLa cells. In some embodiments, the cell culture contains HEK293 cells.

[0009] In some embodiments, the method further includes adding a third culture medium after the start of action (b) (for example, 12 to 36 hours after the start of action (b), for example, 24 hours after the start of action (b)).

[0010] In some embodiments, the first culture medium and the second culture medium are the same. In some embodiments, the first culture medium is different from the second culture medium.

[0011] In some embodiments, one or more recombinant nucleic acids include deoxyribonucleic acid (DNA). In some embodiments, one or more recombinant nucleic acids include ribonucleic acid (RNA). In some embodiments, one or more recombinant nucleic acids include one or more plasmids.

[0012] In some embodiments, the cell culture produces adeno-associated virus (AAV) particles. In some embodiments, the method improves the production of AAV particles compared to a production method that does not include the above action (c). In some embodiments, the method improves the production of AAV particles by at least twofold compared to a production method that does not include action (c). In some embodiments, the method improves the production of AAV particles by at least twofold (e.g., at least threefold, or at least fourfold, e.g., four to fivefold, four to sixfold, four to sevenfold, four to eightfold, four to ninefold, or four to tenfold) compared to a production method that does not include action (c). In some embodiments, the method increases the total number of AAV particles produced compared to a production method that does not include action (c).

[0013] In some embodiments, the metabolite is lactate or lactic acid. In some embodiments, the nutrient is a carbon source (e.g., glucose or galactose), an amino acid source, trace metals, vitamins, antioxidant sources, or minerals.

[0014] In some embodiments, the cell culture comprises mammalian cells (e.g., HEK293 cells) transfected with one or more recombinant nucleic acids, provided as one or more plasmids. In some embodiments, HEK293 cells are transfected with (i) one plasmid containing nucleic acid sequences for encoding the genes of interest adjacent to the ITR, AAV Rep, and Cap proteins, and (ii) a second plasmid containing nucleic acid sequences for encoding a pAd helper plasmid, which contains nucleic acid sequences for AAV helper functions, e.g., helper genes E1A, E1B, E2A, VA, and E4orf6 functions. In some embodiments, HEK293 cells are transfected with (i) one plasmid containing the gene of interest flanking an ITR, (ii) a second plasmid containing nucleic acid sequences for encoding AAV Rep and Cap proteins, and (iii) a third plasmid containing nucleic acid sequences for AAV helper functions, e.g., helper gene E1A, E1B, E2A, VA, and E4orf6 functions, and a pAd helper plasmid encoding a pAd helper plasmid. In some embodiments, one or more recombinant nucleic acids encode a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome contains the gene of interest flanking an inverted terminal repeat (ITR). In some embodiments, the gene of interest encodes an antibody, enzyme, growth factor, or hormone. In some embodiments, the ITR includes an AAV2 ITR or an AAV9 ITR. In some embodiments, one or more recombinant nucleic acids encode a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein, or a variant, chimera, or hybrid thereof. In some embodiments, one or more recombinant nucleic acids also encode a Rep protein (e.g., a Rep protein derived from a preferred AAV serotype).

[0015] In some embodiments, the cell culture includes producer cells. In some embodiments, the producer cells include a stably integrated nucleic acid sequence encoding the heterologous gene of interest, adjacent to an inverted terminal repeat (ITR), as well as the AAV Rep and Cap proteins. In some embodiments, one or more helper functions are provided to generate rAAV particles from the producer cells. In some embodiments, one or more helper functions are provided by infecting the producer cells with a helper virus, such as an adenovirus or herpes simplex virus (HSV). In some embodiments, the helper virus encodes AAV helper virus genes, such as E1A, E1B, E2A, VA, and E4orf6 functions. In some embodiments, one or more helper functions are provided as a virus, optionally an Ad5 virus, optionally a wild-type Ad5 virus.

[0016] In some embodiments, the Disclosure provides a method comprising: a) incubating one or more recombinant nucleic acids and one or more transfection reagents in a first culture medium under conditions sufficient for complex formation; and b) transferring a volume of the first culture medium to a second culture medium, the volume of which is at least 25 L. In some embodiments, the volume of the first culture medium is transferred to the second culture medium using a pump. In some embodiments, the pump is a pump that generates a small amount of shear. In some embodiments, the pump is a low-shear pump such as a bioprocessing pump (e.g., a pump sold under the trademark Levitronix® and / or PuraLev® 100).

[0017] In some embodiments, the volume of the first medium is transferred to the second medium using pressure, the volume being at least 25 L, and the pressure being greater than 1 atmospheric pressure (atm). In some embodiments, the pressure is provided by a pump. In some embodiments, the pressure is greater than 1.5 atmospheric pressures (atm) and less than 5 atm. In some embodiments, the pressure is about 2 atm. In some embodiments, the pressure is about 2.5 atm. In some embodiments, the combined complex formation (step (a)) and transfer step (step (b)) lasts 5 to 20 minutes (e.g., 8 to 12 minutes, 9 to 11 minutes, e.g., about 10 minutes).

[0018] In some embodiments, the first medium is the same as the second medium. In some embodiments, the first medium is different from the second medium. In some embodiments, the second medium further comprises a cell culture. In some embodiments, the cell culture comprises mammalian cells. In some embodiments, the cell culture comprises HEK cells, CHO cells, or HeLa cells. In some embodiments, the cell culture comprises HEK293 cells.

[0019] In some embodiments, one or more recombinant nucleic acids involved in complex formation include plasmids. In some embodiments, one of the one or more recombinant nucleic acids in the complex encodes a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome includes a gene of interest adjacent to an inverted terminal repeat (ITR). In some embodiments, the gene of interest encodes an antibody, protein, enzyme, growth factor, miRNA, or hormone. In some embodiments, the ITR includes an AAV2 ITR. In some embodiments, one of the one or more recombinant nucleic acids in the complex encodes a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein, or a variant thereof. In some embodiments, one of the one or more recombinant nucleic acids in the complex also encodes a Rep protein. In some embodiments, the Cap and Rep proteins are encoded on a recombinant nucleic acid different from the recombinant nucleic acid encoding the rAAV genome.

[0020] In some embodiments, the cell culture in the transfer step (b) includes producer cells. In some embodiments, one or more helper functions are provided. In some embodiments, one or more helper functions are encoded on one or more recombinant nucleic acids. In some embodiments, one or more helper functions are provided as a virus, optionally as an Ad5 virus.

[0021] In some embodiments, the Disclosure envisions a method for transporting multiple transfection complexes into a bioreactor, wherein the multiple complexes are transported as a single volume, and the single volume containing the multiple transfection complexes comprises a volume of at least 25 L. In some embodiments, the capacity of the bioreactor is 25–50 L, 50 L–100 L, 100 L–500 L, 500 L–1,000 L, 1,000 L–5,000 L, or 5,000 L–10,000 L. In some embodiments, the multiple complexes are transported using a pressure greater than 1 atmospheric pressure (atm). In some embodiments, the pressure is provided by a pump. In some embodiments, the pressure is greater than 1.5 atm and less than 5 atm. In some embodiments, the pressure is about 2 atm. In some embodiments, the pressure is about 2.5 atm.

[0022] In some embodiments, the method for transferring multiple transfection complexes is carried out in about 10 minutes.

[0023] In some embodiments, the plurality of transfection complexes comprise one or more nucleic acids and one or more transfection reagents. In some embodiments, the one or more nucleic acids include one or more plasmids. In some embodiments, one of the one or more plasmids included in the plurality of transfection complexes contains elements sufficient for AAV production in cells. In some embodiments, the one or more recombinant nucleic acids encode a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome contains a gene of interest adjacent to inverted terminal repeats (ITRs). In some embodiments, the gene of interest encodes an antibody, protein, enzyme, growth factor, miRNA, or hormone. In some embodiments, the ITRs include AAV2 ITRs. In some embodiments, one of the one or more recombinant nucleic acids included in the plurality of transfection complexes encodes a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein, or a variant, chimera, or hybrid thereof. In some embodiments, one of the one or more recombinant nucleic acids included in the plurality of transfection complexes also encodes a Rep protein.

[0024] Aspects of the present disclosure are as follows: a) incubating one or more recombinant nucleic acids and one or more transfection reagents in a container in a first medium under conditions sufficient for complex formation; b) contacting a cell culture with the volume of the first medium, where the volume is at least 25 L; a) incubating the cell culture for 4 - 8 hours under conditions sufficient for nucleic acid transfection; d) contacting the cell culture of c) with a second medium.

[0025] In some embodiments, step (a) produces a plurality of transfection complexes, and the plurality of complexes are transferred as a single volume to a bioreactor containing a cell culture.

Brief Description of the Drawings

[0026] [Figure 1] An exemplary, non-limiting embodiment of method 100 for transfection complex formation is shown. [Figure 2] An exemplary, non-limiting embodiment of method 200 for improving virus production by a cell culture is shown. [Figure 3] An overview of the current method (top) of transferring transfection complexes to a bioreactor and an exemplary, non-limiting example of an improved method (bottom) are shown. [Figure 4] An exemplary, previous scale-up transfection method suitable for use in a bioreactor of 500 L or more, including a HEK293 cell culture, is shown. [Figure 5] An exemplary, non-limiting example of an updated method for scaling up transfection using pressure is shown. This method is suitable for use in a bioreactor of 500 L or more, including mammalian cell culture (e.g., HEK293) cell culture, and has fewer opportunities for contamination. [Figure 6A]The graphs show viral genome titers recovered from cells transfected using the transfection complex, either using a previous method (control) or the currently disclosed method. Across multiple replications, the currently disclosed method functions similarly to the current method. As shown in Figure 6A, the control is shaking flask transfection, and LevMixer® 1, 2, 3, and 4 show the results of independent shaking flask replication combined with LevMixer® complex formation. As shown in Figure 6B, the control is shaking flask transfection, and LevMixer® 1 and 2 show the results of shaking flasks infected with LevMixer®-formed complexes in a small-scale proof-of-concept. As shown in Figure 6C, LevMixer® complexes showing cells transfected with LevMixer®-formed complexes are compared to control transfection using a conventional transfection method. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figures 7A-7B] The scalability of this method is demonstrated. Figure 7A shows a diagram illustrating the scale-up of production from a 250 mL reactor to a 5 L reactor, a 50 L reactor, and then to a 500 L bioreactor. Figure 7B shows the increase in productivity compared to the current method ("conventional") resulting from combining the currently disclosed method with a dual plasmid system ("+dual"), an improved feeding method ("feed"), or both ("+dual+feed"). Figure 7C shows the relative genomic titers achieved by using the currently described method in a 250 mL reactor, a 3 L reactor, a 5 L reactor, a 50 L reactor, or a 500 L bioreactor. The values ​​are normalized to the genomic titers observed at 500 L. Figure 7D shows the relative capsid titers achieved using the currently described method in 250 mL, 3 L, 5 L, 50 L, or 500 L bioreactors. The values ​​are normalized to the capsid titers observed at 500 L. [Figure 7C-7D] Same as above. [Figure 8A-8B] The following are non-limiting examples of steps of the method described in this application. In some embodiments, the steps in Figure 8A occur within steps 205-210 of Figure 2. [Figure 9] A non-limiting example of a transfection method is shown. In the upper panel, three plasmids (Ad helper, RepCap, and ITR-adjacent transgenes) are transfected into HEK293 cells with a transfectant (TA). In the lower panel, two plasmids (Ad helper, and a second plasmid containing RepCap and ITR-adjacent transgenes) are transfected into HEK293 cells with a transfectant (TA). In some embodiments, each gene is expressed to aid in AAV replication and packaging. [Figure 10] This document describes non-limiting, exemplary methods for preparing, transfecting, maintaining, and recovering rAAV from HEK293 cells. [Figure 11] Embodiments of the currently disclosed method are shown. In this embodiment, a second medium (as the first source) is added 4-8 hours post-transfection (PT), and the second source is added 24 hours post-transfection. In this embodiment, rAAV is isolated and / or cells are harvested approximately 48 hours post-transfection. [Figure 12A]Figure 12A shows the increase in viral titer when non-limiting embodiments of the currently described method are used. Figure 12A shows a comparison of the steps of the conventional method with a non-limiting example of the currently described method. Figure 12B shows the change in productivity multiplier when the supply strategy of this application is applied to a triple transfection plasmid system (3 plasmids). This graph is plotted by taking the average multiplier change between technical replication experiments. The bar on the left shows the results using the triple transfection method (3 plasmids) with the conventional supply strategy ("conventional supply"). The bar on the right shows the results using the triple transfection method (3 plasmids) together with the supply strategy of this application ("novel supply"). [Figure 12B] Same as above. [Figures 13A-13B] Compared to the method in Figure 10, an improvement in viral particle production using one embodiment of the currently disclosed method is shown. This improvement was observed in both the triple plasmid system (Figure 13A) and the dual plasmid system (Figure 13B). [Figure 14A-14C] This report evaluates the timing of culture medium supply in relation to virus particle production. Supplying the culture medium 4 hours before transfection was found to be detrimental. Supplying the culture medium 4 hours after transfection resulted in a 3-fold improvement in titer compared to the control. Total capsid was found to be similar to the control. The percentage of complete capsid was found to be 3-fold compared to the control. [Figure 15] In combination with the method disclosed herein, the multiplier change in productivity (approximately 2.8 times) when using a dual transfection plasmid system (two plasmids) compared to a triple transfection plasmid system (three plasmids) (left bar, "Triple") is shown. [Figures 16A-16B]Significant improvements were observed in the productivity of viral particles and the quality of the product. Reproducible titers were observed for controls on D3 (48 hours post-transfection) and D4 (day 4, 72 hours post-transfection). Feeding cells 4 hours post-transfection resulted in a 2-fold improvement in titer compared to controls on D4. Unlike controls, 4-hour-PT feeding showed maximum productivity at D3. Expansion of the supply factor (over-feeding) did not affect productivity. Total capsid was higher at D4 than at D3, which was found to have a significant impact on product quality. Overall productivity was higher at D3 in terms of both productivity (titer) and product quality (percentage of complete capsid). 4-hour-PT feeding suggests the possibility of shortening the method by one day. [Figure 16C] Same as above. [Figure 17] This study demonstrates the benefits of medium supply at 4–8 hours post-transfection (PT) for AAV productivity. AAV production peaked when cells were supplied at 8 hours PT and D2 (24 hours post-transfection). D1 supply timing was 4–8 hours PT, and a decrease in productivity was observed after 4–8 hours PT. Supplying only at D2 was not very beneficial, highlighting the importance of 4 hours PT supply. Supplying only glucose, or medium minus glucose, was not very beneficial, indicating that the entire medium is important for AAV productivity. [Figure 18A] The effect of supply on day 2 (D2) (24 hours after transfection) compared to supply on day 3 (D3) is shown. Figure 18A shows the titer of the viral genome measured after recovery for each test condition. Figure 18B shows the total number of recovered capsids for each test condition. Figure 18C shows the percentage of recovered capsids containing the viral vector ("complete capsids") for each test condition. The results indicate that supplying at 4 hours after transfection (day 1 (D1)) and D2 is sufficient for overall productivity. Supplying at D3 does not appear to improve recovery, and therefore recovery may occur earlier than expected (at D3 rather than D4). [Figure 18B] Same as above. [Figure 18C] Same as above. [Modes for carrying out the invention]

[0027] Aspects of this disclosure provide improved methods for culturing cells for virus production. In some aspects, the method is useful for rAAV production. In some aspects, the method is useful for increasing total virus production by cells. In some aspects, the method is useful for increasing the percentage of complete viral capsid produced. In some embodiments, the method is useful for reducing the time required to recover the virus. In some aspects, this disclosure relates to providing cells with a culture medium (e.g., a supply medium). In some aspects, this disclosure intends to provide cells with a second culture medium (e.g., a supply medium) after incubating the cells under conditions sufficient for nucleic acid transfection in a first culture medium. In some embodiments, the first culture medium comprises a production medium (e.g., a medium sold under the trademark LV-MAX®). In some embodiments, the second culture medium is provided after transfection and after a set incubation time. In some embodiments, the second medium is provided 1 to 20 hours (e.g., 2 to 15 hours, e.g., 3 to 10 hours, or 4 to 8 hours) after transfection and incubation. In some embodiments, the second medium is provided when the metabolites reach or exceed a threshold concentration in the first medium. In some embodiments, the second medium is provided when the nutrients have decreased to below a threshold level in the first medium. In some embodiments, productivity increases 2 to 3 times. In some embodiments, further method updates result in a 10-fold increase in productivity. In some embodiments, the increase in productivity indicates an increase in the viral genome measured after recovery. In some embodiments, the increase in productivity indicates an increase in the recovered viral capsid. In some embodiments, the increase in productivity indicates an increase in the percentage of recovered complete capsid.

[0028] Methods for producing large quantities of rAAV particles are complex because viral (e.g., rAAV) production is time-sensitive, and combining small batches increases the likelihood of contamination. An example of a standard protocol is shown in Figure 4. Such protocols are difficult to implement on a large scale due to limitations in the mixing elements required to form transfection complexes and transfer the formed transfection complexes to the bioreactor. In contrast, in several embodiments, the methods and compositions relating to this disclosure have demonstrated successful scaling up of the entire production method (as shown in Figure 7A) from a 250 mL reactor to a 500 L reactor.

[0029] Aspects of this application relate to a method for mixing elements to form a transfection complex, and a method for transferring the formed transfection complex to a bioreactor. Non-limiting examples of the method, which can be implemented on a large scale (e.g., volumes of 25 to 500 L or more), are provided in Figures 1 and 2.

[0030] Figure 1 shows a non-limiting embodiment of method 100 for transfection complex formation. In action 101, one or more transfection reagents are combined with the culture medium. In action 105, one or more nucleic acids are combined with the culture medium. In action 110, the combinations from actions 101 and 105 are combined. In action 115, the combinations from action 110 are mixed. In action 120, the mixture from step 115 is incubated for complex formation. In action 125, the result of step 120 is brought into contact with the cell culture.

[0031] In some embodiments, the method shown in Figure 1 is used to form a transfection complex. In some embodiments, the method shown in Figure 1 is useful for scaling up rAAV production. In some embodiments, one or more of the actions shown in Figure 1 may be omitted and / or combined with other actions in Figure 1. In some embodiments, actions 110 and 115 may be combined.

[0032] In some embodiments, one or more actions shown in Figure 1 may be combined with one or more actions shown in Figure 2. For example, action 125 in Figure 1 may include contact with a cell culture as in action 201, and the complex of action 125 may include the rAAV-producing material as in action 205.

[0033] In some embodiments, act 101 includes combining one or more transfection reagents with a culture medium using any suitable technique, for example, a pipette for handling the solution, or other suitable apparatus, for combining the solutions and / or adding dry materials to the solutions.

[0034] In some embodiments, the transfection reagent includes a commercially available transfection reagent. In some embodiments, the transfection reagent includes a chemical transfection reagent. In some embodiments, the transfection reagent includes a liposome-based transfection reagent. In some embodiments, the transfection reagent includes liposomes. In some embodiments, the liposomes are positively charged or cationic liposomes. In some embodiments, the transfection reagent includes a non-liposome-based transfection reagent. In some embodiments, the transfection reagent includes calcium phosphate, dendrimers, polymers, nanoparticles, or non-liposomal lipids. In some embodiments, the transfection reagent includes lipofectamine or a variant thereof. In some embodiments, transfection is performed by electroporation nucleofection, a method using a combination of electrical and chemical factors with an apparatus (e.g., Nucleofector® by Lonza).Transfection reagents include polyethyleneimine (PEI), animal-free transfects (e.g., those sold under the trademark FectoVIR® (Polyplus)), pure lipids, or high-lipid transfects (e.g., Nanofectamine (GE Healthcare), Oligofectamine (Invitrogen), RNAiMAX (Invitrogen), siPORT (ThermoFisher), DharmaFECT). Examples include Endofectin@max (GeneCopoeia), Escort IV liposomes (Sigma-Aldrich), and mixed lipids, and / or non-lipid transfection reagents (e.g., those marketed as Arrest-In (Dharmacon), TurboFect (Thermo), Effectene (Qiagen), Attractene (Qiagen), PolyFect (Qiagen), SuperFect (Qiagen), ExpressFect (Thomas), GeneJammer (Stratagene), FuGENE (Promega), INTERFERin (Polyplus), NanoFectin (System Biosciences), X-tremeGENE (Roche), Xfect (ClonTech), Escort IV (Sigma-Aldrich), and N-TER (Sigma-Aldrich)).

[0035] In some embodiments, the culture medium (e.g., the first, second, or third medium) is a medium suitable for cell growth and / or transfection. In some embodiments, the medium is a medium that supports cell growth. In some embodiments, the medium provides one or more nutrients (e.g., trace metals, vitamins, carbon sources, amino acid sources, or antioxidant sources). In some embodiments, the carbon source is glucose or galactose. In some embodiments, the carbon source is provided in a medium at 50 to 200 grams / L. In some embodiments, the carbon source is 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, 85g / L, 90g / L, 95g / L, 100g / L, 105g / L, 110g / L, 115g / L, 120g / L, 125g / L The medium is supplied in g / L, 130 g / L, 135 g / L, 140 g / L, 145 g / L, 150 g / L, 155 g / L, 160 g / L, 165 g / L, 170 g / L, 175 g / L, 180 g / L, 185 g / L, 190 g / L, 195 g / L, 200 g / L, 205 g / L, 210 g / L, 215 g / L, or 220 g / L. In some embodiments, at least one medium is a supply medium. In some embodiments, one or more of the mediums include a compound medium. In some embodiments, the medium is a standard medium. In some embodiments, one or more of the mediums include Minimum Essential Medium (MEM), Eagle Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), LV-MAX® Medium (Gibco), EX-Cell Medium, or RPMI Medium. In some embodiments, the medium is serum-free. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is suitable for the proliferation and / or transfection of mammalian cells. In some embodiments, the medium is suitable for the proliferation and / or transfection of HEK293 cells. In some embodiments, the medium is suitable for the proliferation and / or transfection of HeLa cells. In some embodiments, the medium is suitable for AAV production.In some embodiments, the second medium further comprises one or more additional nutrients (e.g., a carbon source, an amino acid source, trace metals, an antioxidant source, and / or vitamins) in addition to the first medium. In some embodiments, the third medium further comprises one or more additional nutrients (e.g., a carbon source, an amino acid source, trace metals, an antioxidant source, and / or vitamins) in addition to the first and / or second medium.

[0036] In some embodiments, act 105 includes combining one or more nucleic acids with a culture medium. In some embodiments, the one or more nucleic acids are recombinant nucleic acids. In some embodiments, the one or more recombinant nucleic acids include deoxyribonucleic acid (DNA), and in some embodiments, the one or more recombinant nucleic acids include ribonucleic acid (RNA).

[0037] In some embodiments, one or more recombinant nucleic acids comprise one or more plasmids. In some embodiments, one or more recombinant nucleic acids are provided as one or more plasmids. In some embodiments, the plasmid comprises one or more replication elements, or regulatory elements such as promoters and / or transcriptional regulatory elements. In some embodiments, one or more nucleic acids comprise two plasmids. In some embodiments, the two plasmids comprise a dual transfection system. In some embodiments, one or more nucleic acids comprise three plasmids. In some embodiments, the three plasmids comprise a triple transfection system. In some embodiments, multiple molar ratios of plasmids have been studied. In some embodiments, in a dual transfection system, one plasmid comprises nucleic acid sequences encoding the gene of interest adjacent to the ITR, AAV Rep, and Cap protein, and the second plasmid comprises nucleic acid sequences encoding AAV helper functions, such as E1A, E1B, E2A, VA, and E4orf6 functions. In some embodiments, switching from a three-plasmid system (also referred to herein as a “triple transfection plasmid system”) to a dual-plasmid system increases AAV particle production by at least twofold. In some embodiments, AAV particle production increases by approximately threefold.

[0038] In some embodiments, one or more recombinant nucleic acids encode a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome contains a gene of interest adjacent to an inverted terminal repeat (ITR). In some embodiments, the ITR includes an AAV2 ITR or an AAV9 ITR. In some embodiments, the gene of interest encodes an antibody, enzyme, protein, growth factor, miRNA, or hormone. In some embodiments, one or more recombinant nucleic acids encode a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein, or a variant thereof. In some embodiments, one or more recombinant nucleic acids also encode a Rep protein.

[0039] In some embodiments, the culture medium used in act 101 and / or act 105 is the culture medium described above. In some embodiments, the culture medium in act 101 and act 105 is the same culture medium. In some embodiments, the culture medium in act 101 and act 105 are different culture media.

[0040] In some embodiments, acts 101 and 105 occur in a container, each. In some embodiments, the container includes a plastic, polymer, glass, or metal container. In some embodiments, the container is a single-use container. In some embodiments, acts 101 and / or 105 occur in a bottle, each. In some embodiments, acts 101 and / or 105 occur in a bag, each.

[0041] In some embodiments, act 110 includes combining the mixture of act 101 and act 105. In some embodiments, act 110 includes transferring the mixture of act 101 and act 105 to a different container (e.g., a subsequent container). In some embodiments, the subsequent container includes a plastic, polymer, glass, or metal container. In some embodiments, the subsequent container is a single-use container. In some embodiments, the container of act 110 is contained within a device capable of mixing. In some embodiments, the container of act 110 is contained within a device capable of gentle mixing, such as a bag. In some embodiments, the container of act 110 is contained within a mixer. In some embodiments, the mixer includes an impeller. In some embodiments, the mixer includes a magnetic or floating impeller (e.g., a mixer sold under the trademark LevMixer®). In some embodiments, acts 110 and 115 occur simultaneously.

[0042] In some embodiments, act 115 includes mixing the combination of acts 110. In some embodiments, act 115 includes gently mixing the combination of acts 110. In some embodiments, gentle mixing includes using a mixer having a low shear rate ("y"). In some embodiments, the low shear rate is a shear rate that is not harmful to the transfection complex. In some embodiments, the low shear rate includes shears less than 2650y, less than 2600y, less than 2550y, less than 2500y, less than 2450y, less than 2400y, less than 2300y, less than 2250y, less than 2200y, less than 2150y, less than 2100y, less than 2050y, less than 2000y, less than 1950y, less than 1900y, less than 1850y, or less than 1800y. In some embodiments, gentle mixing is carried out by a mixer including an impeller. In some embodiments, gentle mixing is carried out using a mixer including a magnetic or floating impeller (e.g., a mixer sold under the trademark LevMixe®). In some embodiments, the formed transfection complex is transferred to a subsequent container using a tube at a low shear rate. In some embodiments, the shear rate is y = 4q / πR 3 The formula is calculated as follows: in the equation, "y" is the shear rate / second and "q" is the volumetric flow rate through the pipe, cm³. 3 It is expressed as " / second", and "R" is the pipe radius.

[0043] In some embodiments, action 120 includes incubating the mixture of action 115 for complex formation. In some embodiments, action 120 includes incubating the mixture without mixing. In some embodiments, action 120 includes incubating the mixture with gentle mixing. In some embodiments, actions 120 and 125 occur within about 10 minutes. In some embodiments, actions 120 and 125 occur within 10-20 minutes, 5-15 minutes, 8-12 minutes, 9-13 minutes, 7-10 minutes, or 9-11 minutes. In some embodiments, actions 120 and 125 occur within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 25 minutes.

[0044] In some embodiments, action 125 includes bringing the incubation mixture of action 120 into contact with the cell culture. In some embodiments, the container of action 115 and / or action 120 is connected to a bioreactor via a tube, pipe, or other fluid connector. In some embodiments, the tube, pipe, or other fluid connector includes a standard-sized tube, pipe, or fluid connector (e.g., having an inner diameter of 1 to 25 mm, e.g., 2 to 15 mm (e.g., 3.18 mm to 12.7 mm)). In some embodiments, the tube, pipe, or other fluid connector is made of plastic, polymer material, glass, or metal. In some embodiments, the tube, pipe, or other fluid connector is a manufactured tube (e.g., sold under the trademark Masterflex® or C-flex®). In some embodiments, pressure is applied to the container to transfer the culture medium to the bioreactor via the fluid connector. In some embodiments, the culture medium is pumped from the container to the bioreactor through the fluid connector. In some aspects, this disclosure relates to a scale-up method. In some embodiments, the volume of the incubation mixture for Act 120 is at least 25 L. In some embodiments, the volume of the incubation mixture for Act 120 is 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, 55 L, 60 L, 65 L, 70 L, 75 L, 80 L, 85 L, 90 L, 95 L, 100 L, 110 L, 120 L, 130 L, 140 L, 150 L, 160 L, 170 L, 180 L, 190 L, 200 L, 210 L, or 220 L. In some embodiments, the volume of the incubation mixture for Act 120 is about 10% of the volume of the bioreactor.

[0045] In some embodiments, act 125 includes transporting multiple transfection complexes to a bioreactor, where the complexes are transported as a single volume. In some embodiments, the disclosure relates to a scale-up method. In some embodiments, the single volume is at least 25 L. In some embodiments, the single volume is about 25 L, about 30 L, about 35 L, about 40 L, about 45 L, about 50 L, about 55 L, about 60 L, about 65 L, about 70 L, about 75 L, about 80 L, about 85 L, about 90 L, about 95 L, about 100 L, about 110 L, about 120 L, about 130 L, about 140 L, about 150 L, about 160 L, about 170 L, about 180 L, about 190 L, about 200 L, about 210 L, or about 220 L. In some embodiments, the single volume is about 10% of the volume of the bioreactor.

[0046] In some embodiments, the containers for act 115 and / or act 120 contain 10% of the volume of the bioreactor for act 125. For example, the containers for act 115 and / or act 120 contain a volume of 50 L, and the bioreactor for act 125 contains a volume of 500 L.

[0047] In some embodiments, the method is useful for facilitating the transfer of transfection components from a container to a bioreactor, as described in Act 125. In some embodiments, transferring a volume of the first culture medium containing the transfection reagent and nucleic acids or multiple transfection complexes over a short period of time is important for the success of the method. In some embodiments, the transfer takes place over approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. In some embodiments, the transfer takes place over approximately 10–20 minutes, 5–15 minutes, 8–12 minutes, 9–13 minutes, 7–10 minutes, or 9–11 minutes. In some embodiments, the transfer takes place over approximately 10 minutes.

[0048] In some embodiments, the method is useful in reducing the opportunity for contamination. Alternative embodiments currently available involve transferring multiple smaller batches of transfection components to a bioreactor. In some embodiments, multiple transfers introduce further opportunities for contamination. In some embodiments, the possibility of contamination is reduced by transferring the transfection reagent and nucleic acids, or multiple transfection complexes, as a single volume. In some embodiments, the method is useful in simplifying the manufacturing process. In some embodiments, conventional transfection methods are cumbersome and not scalable. In some embodiments, the method described in this application is less cumbersome and more scalable than conventional methods.

[0049] In some embodiments, the methods of the present disclosure include using pressure to transfer a transfection reagent and nucleic acids, or a plurality of transfection complexes. In some embodiments, the pressure is at least 1 atmosphere (atm). In some embodiments, the pressure is about 1.5 atm, about 2 atm, about 2.5 atm, about 3 atm, about 3.5 atm, about 4 atm, about 4.5 atm, about 5 atm, about 5.5 atm, or about 6 atm. In some embodiments, the pressure is 1.5 atm to 5 atm. In some embodiments, the pressure is 1 atm to 4 atm. In some embodiments, the pressure is 2 atm to 4 atm. In some embodiments, the pressure is 2 atm to 3 atm.

[0050] In some embodiments, the pressure is pneumatic. In some embodiments, the pressure is supplied by a pump. In some embodiments, the pump is a low-shear pump, such as a bioprocessing pump (e.g., a pump sold under the trademark Levitronix® and / or PuraLev® 100). In some embodiments, the pump includes a magnetic or floating impeller. In some embodiments, the pump is part of a mixing device. In some embodiments, the mixing device includes a container. In some embodiments, the pump is part of a mixer, including a floating or magnetic impeller (e.g., a mixer sold under the trademark LevMixer®), or other device capable of gentle mixing.

[0051] A non-limiting embodiment of Method 100 is shown in Figure 5.

[0052] In some embodiments, the cell culture is the cell culture from act 201. In some embodiments, act 125 includes bringing the incubation mixture from act 115 into contact with the cells from act 201 by act 205.

[0053] In some embodiments, following act 125, the method further includes producing rAAV particles. In some embodiments, the rAAV particles are isolated and prepared for administration to a subject in need of treatment.

[0054] Figure 2 shows a non-limiting embodiment of Method 200 for improving virus production by cell culture. In step 201, a cell culture is prepared. In step 205, the cell culture is brought into contact with rAAV-producing material (e.g., one or more rAAV genomes or proteins, and / or one or more helper functions, and / or nucleic acids encoding one or more helper viruses). In step 210, the cell culture is brought into contact with a second medium (e.g., a feed medium). In step 215, the cell culture is brought into contact with a third medium. In step 220, rAAV is isolated from the cell culture.

[0055] In some embodiments, the method shown in Figure 2 is used to improve virus production by cell cultures. In some embodiments, one or more of the actions shown in Figure 2 may be omitted or alternatively repeated two or more times and / or combined with other actions. Non-limiting examples of one or more actions that may be omitted include actions 215 and 220.

[0056] In some embodiments, the method of Figure 2 is combined with the method of Figure 1, as described above. However, the method of Figure 2 can be used with any rAAV-producing material. The method of Figure 1 can be used with any cell proliferation and / or rAAV-producing method, including but not limited to the method of Figure 2.

[0057] In some embodiments, act 201 includes preparing a cell culture. In some embodiments, preparing a cell culture includes inoculating a cell culture and / or growing a cell culture to an appropriate density. In some embodiments, act 201 includes preparing a cell culture for infection and / or transfection. In some embodiments, act 201 includes preparing a cell culture in a first medium (e.g., growing a cell culture in a medium).

[0058] Many cell types can be cultured in vitro. In some embodiments, the cell culture contains animal cells. In some embodiments, the cell culture contains mammalian cells. In some embodiments, the cell culture contains human cells. In some embodiments, the cell culture contains epithelial cells. In some embodiments, the cell culture contains HEK cells, CHO cells, or HeLa cells. In some embodiments, the cell culture contains HEK293 cells. In some embodiments, the cell culture contains HeLa cells.

[0059] In some embodiments, the cell culture includes producer cells. In some embodiments, the producer cells stably express Rep and Cap genes suitable for rAAV packaging. In some embodiments, the producer cells further include an rAAV genome. In some embodiments, the rAAV genome includes therapeutic genes.

[0060] In some embodiments, act 205 includes bringing a cell culture into contact with rAAV-producing material.

[0061] In some embodiments, the rAAV production material comprises one or more recombinant nucleic acids. In some embodiments, one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA), and in some embodiments, one or more recombinant nucleic acids comprise ribonucleic acid (RNA). In some embodiments, one or more recombinant nucleic acids are delivered as part of a transfection complex formed in Method 100.

[0062] In some embodiments, one or more recombinant nucleic acids comprise one or more plasmids. In some embodiments, one or more nucleic acids comprise two plasmids. In some embodiments, two plasmids comprise a dual transfection system. In some embodiments, one or more nucleic acids comprise three plasmids. In some embodiments, three plasmids comprise a triple transfection system. In some embodiments, multiple molar ratios of plasmids have been studied. In some embodiments, switching from a three-plasmid system (also referred to herein as the “triple transfection plasmid system”) to a dual-plasmid system increases AAV particle production by at least twofold. In some embodiments, AAV particle production increases by approximately threefold.

[0063] In some embodiments, one or more recombinant nucleic acids encode a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome contains a gene of interest adjacent to an inverted terminal repeat (ITR). In some embodiments, the ITR includes an AAV2 ITR or an AAV9 ITR. In some embodiments, the gene of interest encodes an antibody, enzyme, protein, growth factor, miRNA, or hormone. In some embodiments, one or more recombinant nucleic acids encode a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein, or a variant thereof. In some embodiments, one or more recombinant nucleic acids also encode a Rep protein.

[0064] In some embodiments, the cell culture comprises producer cells, and the rAAV-producing material provides helper function. In some embodiments, the rAAV-producing material comprises a helper virus. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5.

[0065] In some embodiments, act 205 occurs under sufficient conditions for nucleic acid transfection. In some embodiments, sufficient conditions for nucleotide transfection are conditions sufficient to successfully transfect more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of cells, including cell cultures.

[0066] In some embodiments, act 205 occurs under conditions sufficient for infection by a helper virus. In some embodiments, conditions sufficient for infection by a helper virus are conditions sufficient to successfully transfect more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of cells, including cell cultures.

[0067] In some embodiments, the act 210 includes bringing the cell culture into contact with a second culture medium.

[0068] Aspects of this disclosure provide a method for increasing rAAV production by incubating a cell culture and then contacting the cell culture with a second medium (e.g., a supply medium), as described in Act 205. In some embodiments, the second medium is added at a set time during incubation. In some embodiments, the second medium is added when one or more conditions are met. In some embodiments, post-transfection supply is highly beneficial. In some embodiments, multiple time points are tested.

[0069] In some embodiments, action 210 occurs after a set incubation time. In some embodiments, action 210 occurs after 2 to 20 hours of incubation. In some embodiments, action 210 occurs after 2 to 16 hours of incubation. In some embodiments, action 210 occurs after 4 to 12 hours of incubation. In some embodiments, action 210 occurs after 4 to 16 hours of incubation. In some embodiments, action 210 occurs after 2 to 12 hours of incubation. In some embodiments, action 210 occurs after 4 to 14 hours of incubation. In some embodiments, action 210 occurs after 4 to 8 hours of incubation. In some embodiments, action 210 occurs after 2 to 8 hours of incubation. In some embodiments, action 210 occurs after 2 to 6 hours of incubation. In some embodiments, action 210 occurs after 4 to 6 hours of incubation.

[0070] In some embodiments, act 215 follows act 210, for example, after a total incubation period of 24 hours.

[0071] In some embodiments, the present disclosure relates to a method for monitoring a cell culture to determine the timing of adding a second medium (e.g., a supply medium). In some embodiments, one or more characteristics of the cell culture are monitored. In some embodiments, one or more characteristics of the cell culture include pH, CO2 emissions, oxygen levels, culture volume, lactate levels, nutrient levels, metabolite levels, or carbon source levels. In some embodiments, “monitoring” includes performing one or more measurements during incubation. In some embodiments, oxygen demand is increased using the supply strategy of the present application.

[0072] In some embodiments, the cell density of the cell culture is monitored. In some embodiments, the cell culture is brought into contact with a second medium (e.g., a supply medium) when the cell culture reaches a certain cell density. In some embodiments, the cell culture is brought into contact with the second medium when the cell density is approximately 1 × 10⁶ to approximately 1 × 10⁷. In some embodiments, the cell density is approximately 2 × 10⁶ to approximately 9 × 10⁶, approximately 3 × 10⁶ to approximately 8 × 10⁶, approximately 4 × 10⁶ to approximately 7 × 10⁶, approximately 2 × 10⁶ to approximately 8 × 10⁶, approximately 2 × 10⁶ to approximately 7 × 10⁶, approximately 2 × 10⁶ to approximately 6 × 10⁶, approximately 2 × 10⁶ to approximately 5 × 10⁶, approximately 3 × 10^6 to approximately 9 × 10^6, approximately 3 × 10^6 to approximately 7 × 10^6, approximately 3 × 10^6 to approximately 6 × 10^6, approximately 4 × 10^6 to approximately 9 × 10^6, approximately 4 × 10^6 to approximately 8 × 10^6, approximately 4 × 10^6 to approximately 6 × 10^6, approximately 5 × 10^6 to approximately 1 × 10^7, or approximately 1 × 10^6 to approximately 5 × 10^6.

[0073] In some embodiments, the osmotic pressure of the culture medium is monitored. In some embodiments, the cell culture is brought into contact with a second medium when the osmotic pressure is 260–320 milliosmoles (mOsm), 270–310 mOsm, 280–300 mOsm, 265–280 mOsm, 275–290 mOsm, 285–300 mOsm, 295–310 mOsm, or 305–320 mOsm.

[0074] In some embodiments, the pH of the culture medium is monitored. In some embodiments, the cell culture is brought into contact with the second culture medium when the pH is 6.9–7.5, 7–7.4, 7.1–7.3, 6.9–7.2, or 7–7.3.

[0075] In some embodiments, the viability of cells, including in the cell culture, is monitored. In some embodiments, the cell culture is brought into contact with a second medium when the cell viability is approximately 90% to 100%, 95% to 100%, 96% to 99%, 95% to 99%, 95% to 98%, or 95% to 98.5%.

[0076] In some embodiments, the Disclosure envisions a method for monitoring the concentration of one or more molecules in a first medium and, when the concentration of one or more molecules reaches a threshold, bringing the cell culture into contact with a second medium (e.g., a supply medium). In some embodiments, monitoring the concentration of one or more molecules includes continuous monitoring. In some embodiments, monitoring the concentration of one or more molecules includes performing periodic measurements to evaluate the concentration of one or more molecules. In some embodiments, the method further includes bringing the cell culture into contact with a third medium (e.g., a supply medium) after a 24-hour incubation, as in act 215.

[0077] In some embodiments, a second culture medium (e.g., a supply medium) is added when the concentration of metabolites reaches or exceeds a threshold. In some embodiments, “metabolites” refers to compounds produced by cells. In some embodiments, “metabolites” refers to metabolic by-products. In some embodiments, “metabolites” refers to waste products.

[0078] Examples of metabolites include, but are not limited to, glutamine, glutamate, lactate, ammonium (NH4+), and sodium ions (Na +) potassium ions (K +)、 or calcium ions (Ca2 + ) are some examples.

[0079] In some embodiments, the cell culture is brought into contact with the first feed when the glutamine concentration is 0.2 mmol / L, 0.25 mmol / L, 0.3 mmol / L, 0.35 mmol / L, 0.4 mmol / L, 0.45 mmol / L, or 0.5 mmol / L or higher. In some embodiments, the cell culture is brought into contact with the second medium when the glutamate concentration is 3.35 mmol / L, 3.40 mmol / L, 3.45 mmol / L, 3.5 mmol / L, 3.55 mmol / L, or 3.6 mmol / L or higher. In some embodiments, the cell culture is brought into contact with the second medium when the lactate concentration is 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, or 3.4 g / L or higher. In some embodiments, the cell culture is brought into contact with the second medium when the ammonium concentration is 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.35 mmol / L, or 1.4 mmol / L. In some embodiments, the cell culture is brought into contact with the second medium when the sodium ion concentration is 96.5 mmol / L, 98 mmol / L, 100 mmol / L, 105 mmol / L, 110 mmol / L, 115 mmol / L, 120 mmol / L, 125 mmol / L, or 130 mmol / L. In some embodiments, the cell culture is brought into contact with the second medium when the potassium ion concentration is 2.75 mmol / L, 2.85 mmol / L, 2.95 mmol / L, 3.05 mmol / L, 3.2 mmol / L, 3.35 mmol / L, or 3.5 mmol / L. In some embodiments, the cell culture is brought into contact with the second medium when the calcium ion concentration is 0.05 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, or 1.0 mmol / L or higher.

[0080] In some embodiments, the cell culture comes into contact with a second medium (e.g., a supply medium) when the nutrient concentration falls below a threshold.

[0081] In some embodiments, the nutrient is a carbon source. In some embodiments, the carbon source includes sugars. In some embodiments, the sugars are glucose, galactose, fructose, mannose, lactose, sucrose, maltose, or trehalose. In some embodiments, the carbon source includes pyruvate.

[0082] In some embodiments, the cell culture is brought into contact with the second medium when the glucose concentration is 0.1 g / L, 0.075 g / L, 0.05 g / L, 0.025 g / L, or 0.01 g / L or less.

[0083] In some embodiments, the nutrient is a nitrogen source.

[0084] In some embodiments, the nutrient is an amino acid. In some embodiments, the amino acid is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the amino acid is an essential amino acid. In some embodiments, essential amino acids include, but are not limited to, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

[0085] In some embodiments, the nutrient is a source of antioxidants. In some embodiments, the antioxidant nutrient is L-cysteine. In some embodiments, the antioxidant nutrient is N-acetyl-L-cysteine.

[0086] In some embodiments, the nutrient is lipid. In some embodiments, the lipid is cholesterol. In some embodiments, the nutrient is salt. In some embodiments, the salt is sodium, potassium, calcium, manganese, or magnesium. In some embodiments, the nutrient is vitamin. In some embodiments, the vitamin is B vitamin. In some embodiments, the vitamin is riboflavin, thiamine, or biotin. In some embodiments, the vitamin is vitamin A or vitamin E. In some embodiments, the nutrient is mineral.

[0087] In some embodiments, act 215 includes bringing the cell culture into contact with a third culture medium.

[0088] In some embodiments, a third medium (e.g., a supply medium) is added when the concentration of the metabolite reaches or exceeds a threshold. In some embodiments, the metabolite measured to determine the timing of action 215 is the same as the metabolite measured to determine the timing of action 210. In some embodiments, the metabolite measured to determine the timing of action 215 is a different metabolite from the metabolite measured to determine the timing of action 210.

[0089] In some embodiments, a third medium (e.g., a supply medium) is added when the nutrient concentration falls below a threshold. In some embodiments, the nutrient measured to determine the timing of action 215 is the same as the nutrient measured to determine the timing of action 210. In some embodiments, the nutrient measured to determine the timing of action 215 is a different nutrient from the nutrient measured to determine the timing of action 210.

[0090] Figures 8A-8B and 9 illustrate non-limiting embodiments of Method 200. In one embodiment, as shown in Figure 8A, a cell culture in a first medium is brought into contact with one or more recombinant nucleic acids. The cell culture is then incubated under conditions sufficient for nucleic acid transfection. The cell culture is then brought into contact with a second medium. In one embodiment, as shown in Figure 8B, preparing the cell culture involves thawing frozen cells and following a seed train protocol. The prepared cells are then transfected. The cells are then fed as described in this application. Once rAAV production is complete, the cells are lysed and the rAAV is recovered.

[0091] Figure 9 shows a non-limiting embodiment of the transfection method. In the upper panel, three plasmids (Ad helper, RepCap, and ITR-adjacent transgenes) are transfected into HEK293 cells with the transfectant (TA). In the lower panel, two plasmids (Ad helper, and a second plasmid containing RepCap and ITR-adjacent transgenes) are transfected into HEK293 cells with the transfectant (TA). In some embodiments, each gene is expressed to aid in AAV replication and packaging.

[0092] In some embodiments, act 220 includes isolating rAAV from a cell culture. In some embodiments, isolating rAAV from a cell culture includes recovering the cell culture. In some embodiments, isolating rAAV from a cell culture includes lysing the cells containing the cell culture. In some embodiments, isolating rAAV from a cell culture includes removing rAAV from the cell culture medium. In some embodiments, act 220 includes preparing the isolated rAAV for delivery to a subject. In some embodiments, act 220 includes purifying and / or sterilizing the isolated rAAV. In some embodiments, act 220 includes preparing rAAV for delivery to a human subject (e.g., to deliver a therapeutic gene to a subject to help treat a disease or condition).

[0093] AAV production Recombinant adeno-associated virus (rAAV) vectors are useful in gene therapy for delivering therapeutic genes to patient cells and tissues. rAAV particles typically contain recombinant nucleic acids encapsulated within an AAV capsid protein to form rAAV particles that can be administered to a target. The recombinant nucleic acid (e.g., recombinant AAV genome) typically contains a heterologous gene of interest (e.g., encoding a therapeutic nucleic acid and / or protein) adjacent to an AAV inverted terminal repeat (ITR) sequence. In some embodiments, the AAV capsid protein may be a naturally occurring capsid of a different AAV serotype. For example, different AAV serotypes may have different tissue tropisms and can be used to target different tissue types and associated diseases. In some embodiments, the AAV capsid protein contains one or more amino acid substitutions compared to a naturally occurring capsid protein.

[0094] Various manufacturing techniques can be used to produce rAAV particles. Typically, rAAV particles are assembled in host cells in a culture medium (e.g., a cell culture in a bioreactor or other cell culture vessel). Recombinant AAV genome, AAV capsid protein, and / or one or more nucleic acids encoding one or more Rep and helper genes are expressed in the host cells. The host cells are grown in a culture medium (e.g., in a suspension culture or on a plate). The assembled rAAV is then isolated from the cell culture. The host cells may be mammalian cells, insect cells, or other cell types. In some embodiments, the host cells are producer cells.

[0095] Transient transfection Transient transfection is a method for introducing heterologous genetic material into target cells. As used herein, “transfection” means nucleic acid transfection. Briefly, transfection involves contacting a cell culture with one or more nucleic acids and a transfection reagent, also called a “transfectant.”

[0096] Culture medium Aspects of this disclosure provide a method for bringing a cell culture in a first medium into contact with a second medium (e.g., a supply medium). In some embodiments, bringing a cell culture into contact with a second medium is also referred to as “supplying” the cells. In some embodiments, the medium is referred to as the “supply.” In some embodiments, the first medium is the same as the second medium (e.g., the supply medium). In some embodiments, the first medium is different from the second medium.

[0097] In some embodiments, the cell culture is brought into contact with a third medium (e.g., supply medium) after 24 hours of incubation. In some embodiments, the first, second, and third media are the same medium. In some embodiments, the first and third media are the same medium. In some embodiments, the first and second media are the same medium. In some embodiments, the second and third media are the same medium. In some embodiments, the first medium, the second medium (e.g., the second medium), and the third medium (e.g., the third medium) are each different media.

[0098] cell culture In some embodiments, the present disclosure relates to methods for scaling up and / or increasing rAAV production using cell cultures.

[0099] In some embodiments, the cell culture produces adeno-associated virus (AAV) particles. In some embodiments, the cell culture produces recombinant AAV (rAAV) particles. In some embodiments, the method currently described is combined with other methods to increase the production of rAAV particles. In some embodiments, the production or productivity of AAV particles is increased tenfold by combining the method currently described with, for example, further improvements to the supply method. In some embodiments, the successful scaling up of the entire production method is demonstrated from a 250 mL reactor to a 500 L bioreactor. In some embodiments, the method disclosed hereof improves the production of rAAV particles compared to current methods. In some embodiments, the method disclosed hereof increases the production of rAAV particles compared to current methods. In some embodiments, the method of this application increases the production of rAAV particles by 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to current methods.

[0100] In some embodiments, the method of the present application improves the percentage of complete capsid produced compared to current methods. In some embodiments, the method of the present application increases the production of complete capsid by 1.5, 2, 3, 4, or 5 times compared to current methods.

[0101] In some embodiments, the method of this application reduces the time required to generate a level of rAAV for recovery compared to current methods. In some embodiments, the method reduces the time by 6 hours, 12 hours, 18 hours, 24 hours, or 28 hours. In some embodiments, the method reduces the time by 1 day.

[0102] Recombinant AAV Naturally occurring AAV capsid proteins can be used to produce rAAV for gene therapy. Different naturally occurring AAVs have different characteristics (e.g., different tissue tropisms) and can be used for different indications. AAVs are highly prevalent in human populations (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues J Virol. 2004. 78(12): p. 6381-8, and Boutin, S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population, applications forgone therapy using AAV vectors. Hum Gene Ther. 2010. 21(6): p. 704-12), making them useful as viral vectors. Many serotypes exist, each with different histological orientations (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p.1073-80), which allows specific tissues to be preferentially targeted through appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rh10, transduce into mammalian bodies.See Zincarelli, C., et al. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A.M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S.J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K.D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65.AAV9 has been demonstrated to cross the blood-brain barrier, which is inaccessible to many viral vectors and biological agents (see Foust, KD, et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009.27(1):p.59-65, and Rahim, AA, et al., Intravenous administration of AAV2 / 9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011.25(10):p.3505-18). Certain AAVs have a payload of 4.7–5.0 kb (including viral inverted terminal repeat sequences (ITRs) required in cis for viral packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010.18(1):p.80-6 and Dong, JY, PDFan, and RA Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996.7(17):p.2101-12.

[0103] In some embodiments, rAAV may comprise one or more variant AAV capsid proteins having one or more amino acid substitutions compared to naturally occurring AAV capsid proteins.

[0104] Therefore, in some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins, or their amino acid sequence variants, chimeras, or hybrids thereof.

[0105] In some embodiments, the rAAV particle contains an rAAV genome. In some embodiments, the rAAV genome contains a gene of interest adjacent to an inverted terminal repeat (ITR). In some embodiments, the gene of interest contains a therapeutic molecule, such as a therapeutic protein or therapeutic RNA. In some embodiments, the therapeutic molecule is an antibody, protein, peptide, enzyme, or ribozyme.

[0106] Downstream processing In some embodiments, rAAV isolation includes a further clarification and / or purification step. In some embodiments, rAAV isolation includes an agglutination step. In some embodiments, a solvent, such as a washing agent, may be used together with the agglutinator.

[0107] In some embodiments, the rAAV particles are further purified, for example, after clarification of the rAAV preparation, using one or more affinity chromatography, ion exchange chromatography, and / or hydrophobic interaction chromatography steps.

[0108] In some embodiments, the rAAV particles are added to a pharmaceutically acceptable solution (for example, after one or more purification steps).

[0109] This application also provides compositions comprising rAAV particles and methods for administering rAAV particles to a subject (e.g., a human subject with a disease condition in which therapeutic RNA and / or proteins may aid in treatment).

[0110] These and other embodiments are illustrated by the following non-limiting embodiments. [Examples]

[0111] Example 1. Scaling up using an industrial mixer. An industrial mixer was used to facilitate the use of larger transfection volumes without requiring multiple transfers of the transfection complex to the bioreactor containing the cell culture. Transfection complexes were generated by mixing the plasmid and transfection reagent in the first medium using a LevMixer® (Pall). HEK293 cells were transfected using these transfection complexes. Replications using transfection complexes formed with the LevMixer® were compared to controls using transfection complexes formed using the current method. The results are shown in Figures 6A–6C. Across multiple replications, transfection complexes formed in a bag compatible with the LevMixer® resulted in improved AAV titers compared to transfection using the previous method, which did not involve transfection complex formation in the bag.

[0112] Example 2. pH monitoring of cell cultures presents a remarkable opportunity to enhance virus production. Using a standard feeding procedure, HEK 293 pH and CO2 levels were measured over time in the cell culture (see Figure 10). The pH levels were interpreted to indicate whether the cells were producing or consuming lactate. Surprisingly, the measurements showed that the cells were consuming lactate within hours of transfection (TFx). Cells consume lactate when other carbon sources are limited in the growth medium. This suggested that providing additional medium during this period could improve viral particle production. This experiment was then repeated, with a second medium provided 4 hours after transfection.

[0113] Example 3: rAAV production increases by supplying 4 hours after transfection. HEK293 cells were grown and transfected using the current method (see Figure 11), and the method further included providing the cells with culture medium 4 hours post-transfection. Three culture volumes were tested: 250 mL, 5 L, and 50 L. For the dual plasmid system, 500 L was also tested. The results are shown in Figures 13A and 13B. Both the triple plasmid system (Figure 13A) and the dual plasmid system (Figure 13B), where a second medium was provided 4 hours post-transfection, show improvement over the current method.

[0114] Example 4: Adding the second medium 4 hours before transfection is counterproductive. To evaluate whether providing additional medium at a different time point yields the same benefits as feeding 4 hours after transfection, 6 x 10^6 HEK293 cells were grown in a 15 mL reactor. Cells were either a) in contact with medium according to the current method (see Figure 10), b) provided with additional medium 4 hours before transfection, or c) provided 4 hours after transfection. Cultures were harvested 72 hours after transfection. Viral genomes were measured by digital droplet PCR (ddPCR). The number of capsids and the percentage of complete capsids were also measured using ELISA. The results are shown in Figures 14A–14C. As shown in Figure 14A, providing additional medium 4 hours before transfection reduces viral particle production. As shown in Figures 14A–14C, providing additional medium 4 hours after transfection increases genomic titer, total capsids, and the percentage of complete capsids compared to the current method.

[0115] Example 5. By providing a second culture medium 4 hours after transfection, it becomes possible to harvest the product one day earlier. To further investigate the effect of adding the second medium 4 hours after transfection, HEK293 cells were grown in a 250 mL reactor according to the current method (control, or "Con"), and single doses of the second medium were provided at 4 hours (4 HR PT(3.5)), 24 hours, and 48 hours PT after transfection, double doses were provided at 4 hours (4 HR PT(7.0)) after transfection, and single doses were provided at 24 hours and 48 hours PT after transfection, or double doses were provided at 4 hours, 24 hours, and 48 hours PT. Virus particles were collected at 48 hours (D3) and 72 hours (D4) after transfection. Viral genome titer, percentage of complete capsid, and total capsid were evaluated as described in Example 3. The results are shown in Figures 16A-16C. Interestingly, providing two doses of the second medium did not significantly improve genomic titer, the percentage of complete capsids, or total capsids compared to single-dose conditions. Furthermore, while harvesting at 72 hours post-transfection increased the total number of capsids with the same genomic titer, the percentage of complete capsids was higher at 48 hours post-transfection compared to the control.

[0116] Similar experiments were conducted to evaluate the effects of omitting the supply steps at 24 hours post-transfection (D2) and 48 hours post-transfection (D3). The results are shown in Figures 18A-18C. Omitting the D3 supply resulted in comparable titer and complete capsid percentage compared to the control. Omitting both the D2 and D3 supplies resulted in decreased titer compared to the control. Adding glucose instead of the medium in the D2 supply was nearly as productive as adding the medium at D2. Providing two doses of medium at 4 hours post-transfection and omitting the D2 supply resulted in comparable genomic titer compared to cell cultures supplied with single doses at both 4 hours post-transfection and D2, but showed a decreased percentage of complete capsid.

[0117] Example 6. Providing a second culture medium between 4 and 8 hours offers the greatest advantage. To determine the effects of feeding at other time points after transfection, HEK293 cells were grown in a 250 mL reactor and brought into contact with a second medium at 4 hours, 8 hours, 12 hours, 16 hours, and 20 hours post-transfection. The cell culture volume was 180–220 mL. Cells were additionally provided with a third medium at 24 hours post-transfection (D2). HEK293 cells in a 250 mL reactor were also provided with glucose only at 4 hours post-transfection, glucose-free second medium at 4 hours post-transfection, and only two doses of the second medium at 24 hours post-transfection (D2). Viral particles were harvested at 48 hours post-transfection (D3). As shown in Figure 17, the best viral genome titers were obtained by providing the second medium at 4 hours or 8 hours post-transfection. Providing a second medium 12 hours after transfection was beneficial, but not as beneficial as providing it 4 or 8 hours after transfection.

[0118] Example 7. Scalability of the combined method The effects of the mixing and feeding method combinations described herein on rAAV production and scalability were tested. Figure 7A shows a diagram illustrating the scale-up of production from a 250 mL reactor to a 5 L reactor, a 50 L reactor, and then a 500 L bioreactor. Figure 7B shows the increase in productivity compared to the standard method ("conventional"), resulting from combining the currently disclosed method with a dual plasmid system ("+dual"), an improved feeding method ("feed"), or both ("+dual+feed"). Figure 7C shows the relative genomic titers achieved by using the currently described method in a 250 mL reactor, a 3 L reactor, a 5 L reactor, a 5 L reactor, a 50 L reactor, or a 500 L bioreactor. The values ​​are normalized to the genomic titers observed at 500 L. Figure 7D shows the relative capsid titers achieved using the currently described method in 250 mL, 3 L, 5 L, 50 L, or 500 L bioreactors. The values ​​are normalized to the capsid titers observed at 500 L.

[0119] The advantages associated with the method disclosed herein were further demonstrated in multiple GMP runs (production runs manufactured according to cGMP guidelines) using product quantity and quality as attributes. As shown in Table 1, using a dual transfection system, a transfection complex mixing strategy and a delivery timing of approximately 4–8 hours (e.g., 6 hours) post-transfection yielded high AAV titers and an improved percentage of complete capsids. This demonstrated that the use of LexMixer® ensured the formation of the DNA complex and its successful delivery to a 500L bioreactor. The data show that post-transfection cell performance, high GMP productivity, and maintenance of product quality persist from cell culture to product recovery after cell lysis and downstream purification using chromatographic purification (e.g., anion exchange chromatography-based purification). DS corresponds to the drug substance, including the AAV vector containing the transgene of interest. Packing percentage was evaluated using a vector, an exemplary technique called mass spectrometry (MP).

[0120] [Table 1]

[0121] Equal parts While several embodiments of the present invention are described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for carrying out the function and / or obtaining one or more of the results and / or benefits described herein, and each of such modifications and / or alterations will be considered within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are intended to be illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on one or more specific applications in which the teachings of the present invention are used. Those skilled in the art will be able to recognize or confirm many equivalents to specific embodiments of the present invention described herein by mere conventional experimentation. Therefore, it should be understood that the above embodiments are presented only as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be carried out in ways other than those specifically described and claimed. The embodiments of the present invention in this disclosure are intended for each individual feature, system, article, material, kit and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention as provided that they are not inconsistent with each other.

[0122] All definitions defined and used herein should be understood to govern dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meanings of the terms defined.

[0123] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they cite, and in some cases may encompass the entire document.

[0124] As used herein and in the claims, the indefinite articles “a” and “an” should be understood to mean “at least one” unless explicitly stated otherwise.

[0125] The phrase "and / or," as used herein and in the claims, means "either or both" of the elements thus combined, that is, elements that exist in some cases as a combination and in other cases as separate. Multiple elements listed using "and / or" should be interpreted in the same way, that is, "one or more" of the combined elements. Other elements other than those specifically identified by the "and / or" phrase may exist optionally, whether related to or unrelated to those identified elements. As a non-restrictive example, with respect to "A, and / or B," when used in combination with open-ended language such as "includes," for example, in one embodiment it may refer only to A (optionally including elements other than B); in another embodiment it may refer only to B (optionally including elements other than A); and in yet another embodiment it may refer to both A and B (optionally including other elements).

[0126] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including not just one but more of the elements or the list, and optionally including additional unlisted items. Only clearly indicated terms such as “one of” or “exactly one of” or, where used in a claim, “consisting of” refer to including one element of a number or list of elements. In general, where used herein, the term “or” should be interpreted to indicate an exclusive alternative (i.e., “one or the other, but not both”) only when preceded by an exclusive term such as “either,” “one of,” “only one,” or “exactly one.” Where used in the claims, “essentially consisting of” should have its usual meaning where used in the field of patent law.

[0127] As used herein and in the claims, the phrase “at least one” means, with respect to a list of one or more elements, at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically enumerated in the list of elements, and not excluding combinations of elements in the list of elements. This definition also allows for the existence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether related to the specifically identified elements or not, at the discretion of the system. Therefore, as a non-restrictive example, “A and at least one of B” (or equivalently, “A or at least one of B” or equivalently, “A and / or at least one of B”) may, in one embodiment, refer to at least one and optionally include two or more A's in which B is absent (and optionally include elements other than B); in another embodiment, refer to at least one and optionally include two or more B's in which A is absent (and optionally include elements other than A); and in yet another embodiment, refer to at least one and optionally include two or more A's, and at least one and optionally include two or more B's (and optionally include other elements).

[0128] Unless explicitly stated otherwise, in a method comprising multiple steps or acts claimed herein, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are described.

[0129] In the claims and the above specification, all transport phrases such as “include,” “encompass,” “possess,” “have,” “contain,” “accompany,” “hold,” and “compose of” should be understood as open-ended, meaning they include but are not limited to. Only the transport phrases “consist of” and “essentially consist of” are closed or semi-closed transport phrases, respectively, as described in Section 2111.03 of the U.S. Patent and Trademark Examination Procedure Manual. Embodiments described herein using a non-restrictive transport phrase (e.g., “include”) should also be understood as construing, in alternative embodiments, the features described by the non-restrictive transport phrase as “consist of” and “essentially consist of.” For example, where this disclosure describes “a composition comprising A and B,” this disclosure also construes alternative embodiments “a composition comprising A and B,” and “a composition essentially consisting of A and B.”

Claims

1. b) Contacting the cell culture in the first medium with one or more recombinant nucleic acids, c) Incubate the cell culture for 4 to 8 hours under conditions sufficient for nucleic acid transfection, c) The cell cultures described in b) are brought into contact with the second culture medium. Methods that include...

2. The method according to claim 1, wherein one of the one or more recombinant nucleic acids encodes a recombinant AAV (rAAV) genome.

3. The method according to claim 2, wherein the rAAV genome includes a gene of interest adjacent to an inverted terminal repeat (ITR).

4. The method according to claim 3, wherein the target gene encodes an antibody, enzyme, growth factor, or hormone.

5. The method according to claim 3, wherein the ITR includes an AAV2 ITR or an AAV9 ITR.

6. The method according to any one of claims 1 to 5, wherein one of the one or more recombinant nucleic acids encodes a capsid (Cap) protein.

7. The method according to claim 6, wherein the capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein, or a variant, chimera, or hybrid thereof.

8. The method according to any one of claims 1 to 7, wherein one of the one or more recombinant nucleic acids encodes a Rep protein.

9. The method according to any one of claims 1 to 8, wherein the one or more recombinant nucleic acids further comprises a nucleic acid sequence encoding an AAV helper gene, and optionally the AAV helper gene comprises at least one of the adenovirus genes E1A, E1B, E2A, VA, and E4orf6.

10. The method according to any one of claims 1 to 9, wherein incubating the cell culture under conditions sufficient for nucleic acid transfection comprises contacting the cell culture with one or more transfection reagents.

11. The method according to any one of claims 1 to 10, further comprising measuring the level of the metabolite and providing the second culture medium when the level of the metabolite meets or exceeds a threshold level.

12. The method according to any one of claims 1 to 10, further comprising measuring the level of a nutrient and providing the second culture medium when the level of the nutrient falls below a threshold level, wherein the threshold level is optionally about 0 to 1 g / L.

13. The method according to any one of claims 1 to 12, wherein the cell culture comprises epithelial cells.

14. The method according to any one of claims 1 to 13, wherein the cell culture comprises HEK cells.

15. The method according to any one of claims 1 to 14, wherein the cell culture comprises HEK293 cells.

16. The method according to any one of claims 1 to 15, further comprising bringing the cell culture into contact with a third culture medium 24 hours after the start of (b).

17. The method according to any one of claims 1 to 16, wherein the first culture medium and the second culture medium are the same culture medium.

18. The method according to any one of claims 1 to 16, wherein the first culture medium is different from the second culture medium.

19. The method according to any one of claims 1 to 18, wherein the one or more recombinant nucleic acids include deoxyribonucleic acid (DNA).

20. The method according to claim 1, wherein the one or more recombinant nucleic acids include ribonucleic acid (RNA).

21. The method according to any one of claims 1 to 20, wherein the one or more recombinant nucleic acids comprise one or more plasmids.

22. The method according to any one of claims 1 to 21, wherein the cell culture produces adeno-associated virus (AAV) particles.

23. The method according to claim 22, wherein the method improves the production of AAV particles compared to a manufacturing method that does not include (c).

24. The method according to claim 22 or 23, wherein the method improves the production of AAV particles by at least twofold compared to a manufacturing method that does not include (c).

25. The method according to any one of claims 22 to 24, wherein the method improves the production of AAV particles by at least four times compared to a manufacturing method that does not include (c).

26. The method according to claim 22, wherein the method increases the total number of AAV particles produced compared to a manufacturing method that does not include (c).

27. The method according to claim 11, wherein the metabolite is lactate or lactic acid.

28. The method according to claim 12, wherein the nutrient is an amino acid, a carbohydrate, a vitamin, or a mineral.

29. The method according to claim 1, wherein the cell culture comprises producer cells.

30. The method according to claim 29, wherein one or more helper functions are provided.

31. The method according to claim 30, wherein the one or more helper functions are provided as a helper virus, optionally as an adenovirus, optionally as an Ad5 virus.

32. The method according to claim 21, wherein the one or more recombinant nucleic acids include a dual plasmid transfection system.

33. The method according to claim 21, wherein the one or more recombinant nucleic acids include a triple plasmid transfection system.

34. a) Incubating one or more recombinant nucleic acids and one or more transfection reagents in a container in a first culture medium under conditions sufficient for complex formation, b) Bringing the cell culture into contact with the volume of the first culture medium using pressure. Includes, The volume is at least 25 L, and the pressure is greater than 1 atmosphere (atm). method.

35. The method according to claim 34, wherein step a) includes inflating the container with air.

36. The method according to claim 34 or 35, wherein step b) includes using a pump to transfer the volume of the first culture medium from the container to the bioreactor.

37. The method according to claim 36, wherein the pressure is provided by a pump.

38. The method according to any one of claims 34 to 37, wherein the pressure is greater than 1.5 atmospheres (atm) and less than 5 atm.

39. The method according to any one of claims 34 to 38, wherein the pressure is approximately 2 atm.

40. The method according to any one of claims 34 to 39, wherein the pressure is approximately 2.5 atm.

41. The method according to any one of claims 34 to 40, wherein steps (a) and (b) together last for about 10 minutes.

42. The method according to any one of claims 34 to 41, wherein the first culture medium is the same as the second culture medium.

43. The method according to any one of claims 34 to 41, wherein the first culture medium is different from the second culture medium.

44. The method according to any one of claims 34 to 43, wherein the one or more recombinant nucleic acids and the one or more transfection reagents are mixed separately with the culture medium before being combined in the container.

45. The method according to any one of claims 34 to 44, wherein the one or more recombinant nucleic acids are provided in one or more plasmids.

46. The method according to any one of claims 34 to 45, wherein one of the one or more recombinant nucleic acids encodes a recombinant AAV (rAAV) genome.

47. The method according to claim 46, wherein the rAAV genome includes a gene of interest adjacent to an inverted terminal repeat (ITR).

48. The method according to claim 47, wherein the target gene encodes an antibody, enzyme, growth factor, or hormone.

49. The method according to claim 47 or 48, wherein the ITR includes an AAV2 ITR.

50. The method according to any one of claims 34 to 49, wherein one of the one or more recombinant nucleic acids encodes a capsid (Cap) protein.

51. The method according to claim 50, wherein the capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein.

52. The method according to any one of claims 34 to 51, wherein one of the one or more recombinant nucleic acids encodes a Rep protein.

53. The method according to any one of claims 34 to 52, wherein the cell culture comprises mammalian cells.

54. The method according to any one of claims 34 to 53, wherein the cell culture comprises HEK293 cells.

55. The method according to any one of claims 34 to 54, wherein the cell culture comprises producer cells.

56. The method according to any one of claims 34 to 55, wherein one or more helper functions are provided.

57. The method according to claim 56, wherein the one or more helper functions are encoded by the one or more recombinant nucleic acids.

58. The method according to claim 56, wherein the one or more helper functions are provided as a virus, optionally as an Ad5 virus.

59. A method for transferring multiple transfection complexes to a bioreactor, wherein the multiple complexes are transferred as a single volume, and the single volume containing the multiple transfection complexes comprises a volume of at least 25 L.

60. The method according to claim 59, wherein the plurality of complexes comprises one or more recombinant nucleic acids, and one of the one or more recombinant nucleic acids encodes a recombinant AAV (rAAV) genome.

61. The method according to claim 60, wherein the rAAV genome includes a gene of interest adjacent to an inverted terminal repeat (ITR).

62. The method according to claim 61, wherein the target gene encodes an antibody, enzyme, growth factor, or hormone.

63. The method according to claim 61 or 62, wherein the ITR includes an AAV2 ITR.

64. The method according to any one of claims 59 to 63, wherein one of the one or more recombinant nucleic acids encodes a capsid (Cap) protein.

65. The method according to claim 64, wherein the capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein.

66. The method according to any one of claims 59 to 65, wherein one of the one or more recombinant nucleic acids encodes a Rep protein.

67. The method according to any one of claims 59 to 66, wherein the plurality of composites are transported using a pressure greater than 1 atmosphere (atm).

68. The method according to any one of claims 59 to 67, wherein the pressure is provided by a pump.

69. The method according to any one of claims 59 to 68, wherein the pressure is greater than 1.5 atm and less than 5 atm.

70. The method according to any one of claims 59 to 69, wherein the pressure is approximately 2 atm.

71. The method according to any one of claims 59 to 70, wherein the pressure is approximately 2.5 atm.

72. The method according to any one of claims 59 to 71, wherein the method is carried out over a period of approximately 10 minutes.

73. The method according to any one of claims 59 to 72, wherein the plurality of transfection complexes comprises one or more nucleic acids and one or more transfection reagents.

74. The method according to claim 73, wherein the one or more nucleic acids comprise one or more plasmids.

75. The method according to claim 74, wherein one or more plasmids contain elements sufficient for intracellular AAV production.

76. The method according to any one of claims 34 to 75, wherein the method further comprises one or more steps of the method according to any one of claims 1 to 33.

77. The method according to any one of claims 1 to 76, further comprising isolating rAAV from the cell culture after step (b) or step (c).

78. a) Incubating one or more recombinant nucleic acids and one or more transfection reagents in a container in a first culture medium under conditions sufficient for complex formation, b) Contacting the cell culture with the volume of the first culture medium, wherein the volume is at least 25 L. d) Incubate the cell culture for 4 to 8 hours under conditions sufficient for nucleic acid transfection, d) The cell cultures from c) are brought into contact with the second culture medium, Methods that include...

79. The method according to claim 78, wherein (a) produces a plurality of transfection complexes, and the plurality of complexes are transferred to a bioreactor containing the cell culture as a single volume.

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