Methods and compositions for RAAV production

Abstract

The present disclosure provides methods of producing recombinant adeno-associated virus (rAAV) wherein rAAV production occurs under a controlled partial pressure of carbon dioxide. Also are methods of producing rAAV under air sparge conditions for modulating the partial pressure of carbon dioxide.

Description

[0001] METHODS AND COMPOSITIONS FOR RAAV PRODUCTION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 § 119(e) of U.S. Provisional Application No. 63 / 708,209, filed on October 16, 2024, entitled “RAAV PRODUCTION AND PARTICLE QUALITY BY MODULATING CULTURE LEVELS OF CARBON DIOXIDE,” and U.S. Provisional Application No. 63 / 794,778, filed on April 25, 2024, entitled “ RAAV PRODUCTION AND PARTICLE QUALITY BY MODULATING CULTURE LEVELS OF CARBON DIOXIDE,” the contents of each of which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

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

[0006] SUMMARY

[0007] This application provides methods and compositions for improving production and quality of rAAV particles by cultured cells. Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) while maintaining a desired partial pressure of carbon dioxide (pCO2). Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCO2) less than 200 mmHg (e.g., less than 100 mmHg, less than 70 mmHg, less than 40 mmHg, or less than 20 mmHg). Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCO2) from about 20 to about 70 mmHg. Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCO2) from about 20 to about 40 mmHg. Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCO2) from about 40 to about 70 mmHg. Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus

[0008] #14470686vl (rAAV) under a partial pressure of carbon dioxide (pCO2) from about 30 to about 60 mmHg. Aspects of the present disclosure provide methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCO2) of about 50 mmHg. In some embodiments, the cell culture is incubated under a pCO2from about 20 to about 70 mmHg for about 12 hours to about 48 hours. In some embodiments, the cell culture is incubated under a pCO2from about 20 to about 70 mmHg for about 72 hours.

[0009] In some embodiments, the cell culture is incubated under a pCO2from about 30 to about 60 mmHg for about 12 hours to about 48 hours. In some embodiments, the cell culture is incubated under a pCO2from about 30 to about 60 mmHg for about 72 hours. In some embodiments, the cell culture is incubated under a pCO2from about 40 to about 60 mmHg for about 12 hours to about 48 hours. In some embodiments, the cell culture is incubated under a pCO2from about 40 to about 60 mmHg for about 72 hours. In some embodiments, the cell culture is incubated under a pCO2of about 50 mmHg for about 12 hours to about 48 hours. In some embodiments, the cell culture is incubated under a pCO2of about 50 mmHg for about 72 hours.

[0010] In some embodiments, pH of the cell culture is between 7.0 and 7.5. In some embodiments, pH of the cell culture is about 7.3, 7.4, or 7.5.

[0011] In some embodiments, the culture is incubated with an air sparge rate of 0.005 VVM and then incubated with an air sparge rate of 0.015 to 0.025 VVM post-infection or transfection for at least 12 hours.

[0012] In some embodiments, the mammalian cells comprise rAAV producer cells, wherein the rAAV producer cells are stably integrated with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid encoding a gene of interest.

[0013] In some embodiments, the cell culture is infected with a helper virus and incubated at a pCO2from about 20 to about 70 mmHg for about 12 hours to about 48 hours post infection. In some embodiments, the cell culture is infected with a helper virus and incubated at a pCO2from about 30 to about 60 mmHg for about 12 hours to about 48 hours post infection. In some embodiments, the cell culture is infected with a helper virus and incubated at a pCO2from about 40 to about 70 mmHg for about 12 hours to about 48 hours post infection. In some embodiments, the cell culture is infected with a helper virus and incubated at a pCO2from about 45 to about 55 mmHg for about 12 hours to about 48 hours post infection. In some embodiments, the cell culture is infected with a helper virus and incubated at a pCO2from about 50 mmHg for about 12 hours to about 48 hours post infection.

[0014] #14470686vl In some embodiments, the culture is incubated with an air sparge rate of 0.005 VVM post-infection for at least 12 hours and then incubated with an air sparge rate of 0.015 to 0.025 VVM for at least 12 hours.

[0015] In some embodiments, the helper virus is an adenovirus comprising AAV helper genes, optionally the AAV helper genes comprise at least one of adenovirus genes E1A, E1B, E2A, VA, and E4orf6. In some embodiments, the adenovirus is a wtAd5 or wtAd2 adenovirus or a mutated Ad5 or mutated Ad2 adenovirus. In some embodiments, the adenovirus is a temperature sensitive mutant of a wtAd5. In some embodiments, the adenovirus is Ad5tsl49.

[0016] In some embodiments, the mammalian cells comprise at least one of: CHO cells, Vero cells, HeLa cells, MDCK cells, BHK cells, A549 cells, amniocyte cells, or HEK293 cells. In some embodiments, the mammalian cells comprise HeLa cells. In some embodiments, the HeLa cells are HeLaS3 cells.

[0017] In some embodiments, the mammalian cells are transiently transfected with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid comprising a gene of interest.

[0018] In some embodiments, the mammalian cells comprise HEK293 cells.

[0019] In some embodiments, the gene of interest encodes an antibody, an enzyme, a growth factor, a microRNA, or a hormone.

[0020] In some embodiments, the AAV ITRs comprise AAV2 ITRs or AAV9 ITRs. In some embodiments, the rAAV comprises a capsid protein comprising at least one of: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74 or AAV10 capsid protein or variants or chimeras or hybrids thereof.

[0021] In some embodiments, the pCCL level is maintained by modulating levels of CO2 in a bioreactor comprising the cell culture by:

[0022] 1) modulating the air sparge rate of the submerged gassing of the bioreactor VVM;

[0023] 2) modulating agitation (rpm) of the bioreactor impeller and corresponding density (P / V);

[0024] 3) mixing CO2 with air sparge with a fixed composition;

[0025] 4) mixing CO2 with air in the overlay with a fixed composition;

[0026] 5) providing a cell culture medium comprising a component that limits CO2 solubility (e.g., calcium chloride); and / or

[0027] 6) reducing the amount of sodium bicarbonate in the cell culture medium, optionally reducing the amount of sodium bicarbonate in the cell culture medium to about 0.5X the

[0028] #14470686vl concentration of sodium bicarbonate in a control medium. In some embodiments, CO2 is injected in a headspace of the bioreactor.

[0029] In some embodiments, the cell culture is maintained at a temperature of about 37 °C before infection with the helper virus and on day 3 post infection with the helper virus the temperature of the cell culture is raised to about 39 °C.

[0030] In some embodiments, the cell culture is maintained at a pH of about 7.25 ± 0.25 before infection with the helper virus and on day 3 post infection with the helper virus the pH of the cell culture is raised to about 8.15 ± 0.15.

[0031] In some embodiments, on day 3 post infection with the helper virus the osmolality of the cell culture is maintained at a level greater than or equal to 420 mOsm / kg, optionally at a level of about 440 mOsm / kg.

[0032] In some embodiments, the cell culture is terminated after about 16 hours of increasing the temperature, pH, and osmolality.

[0033] In some embodiments, the cell culture comprises a perfusion culture.

[0034] In some embodiments, the cell culture comprises a batch culture.

[0035] In some embodiments, the method further comprises monitoring the cell culture for one or more parameters selected from the group consisting of: total cell density, viable cell density, cell viability, average cell diameter, and glucose consumption. In some embodiments, the viable cell density of the cell culture is from about 0.25E6 viable cells / mL (vc / mL) to about 2E7 vc / mL (e.g., a cell density between or equivalent to 0.25E6 vc / mL and 2E7 vc / mL) at the time of infection. 0.25E6 is equivalent to 0.25xl06, and 2E7 is equivalent to 2xl07.

[0036] In some embodiments, the cell culture comprises a volume of at least about 50 L. In some embodiments, the cell culture comprises a volume of about 0.2 L to 2000 L. In some embodiments, the cell culture comprises a volume of about IL, 5L, 10L, 15L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, 200L, 250L, 300L, 350L, 400L, 450L, 500L, 550L, 600L, 650L, 700L, 750L, 800L, 850L, 900L, 950L, WOOL, 1050L, 1100L, 1150L, 1200L, 1250L, WOOL, 1350L, 1400L, 1450L, WOOL, 1550L, WOOL, 1650L, 1700L, 1750L, WOOL, 1850L, WOOL, 1950L, or about 2000L. In some embodiments, the cell culture comprises a volume of above 2000 L.

[0037] In some embodiments, the method further comprises isolating recombinant adeno- associated virus from the cell culture. In some embodiments, the titer of rAAV isolated from the cell culture comprises at least about IE 10 to about 5E12 vg / mL.

[0038] In some embodiments, the AAV titer is measured using a PCR method.

[0039] #14470686vl In some embodiments, the rAAV isolated from the cell culture comprises at least about 30% full capsids. In some embodiments, the percent full capsids is measured using analytical ultracentrifugation technique.

[0040] Aspects of the present disclosure relate to methods comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) in a cell culture medium comprising a reduced level of sodium bicarbonate relative to a control medium. In some embodiments, the level of sodium bicarbonate is reduced by at least 0.5X relative to a control medium.

[0041] BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 shows a cartoon outlining the AAV production process including pCO2 controls.

[0043] FIG. 2 shows the measured values of pCO2 in the culture at day 1 in a batch process in Ambr®250. The error bars represent standard deviation for n = 2 separate vessels (+ / -1 SD).

[0044] FIG. 3A shows pH as measured using a blood gas analyzer (BGA). The line chart shows tight control of pH with base addition within a narrow range to decouple the effect of pCO2from pH. The observed differences lie within the pH-controlled range.

[0045] FIG. 3B shows Viable Cell Density (VCD) as measured on Vi-CELL BLU for the batch process in Ambr®250. The line chart shows no significant differences in VCD as a result of variations in pCO2.

[0046] Fig. 3C shows glucose concentration in the cell culture as measured using a BioProfile® FLEX2 apparatus. The line chart shows differences in rate at which glucose concentration decreases over time in cell culture when subjected to variations in pCO2.

[0047] FIG. 4A shows the effect of pCO2 on AAV titers at Ambr®250 scale in batch process. Effect of pCO2during virus production in the cell culture on harvest AAV titer is observed in Ambr®250HT when various conditions of air sparge and overlay were tested to manipulate pCO2during virus production. At pCO2 levels > 70 mmHg, the rAAV titers are sub-optimal and such conditions are not desirable for AAV production.

[0048] FIG. 4B shows the effect of pCO2 on AAV particle quality quantified through sedimentation analysis of samples on an analytical ultracentrifuge (AUC). Harvested material from the conditions 1-3 were purified for sedimentation analysis to determine Empty / Partial / Full AAV capsids. Results show that high pCO2 levels may result in more empty and less full capsids, thus affecting the quality of the rAAV product in the cell culture harvest.

[0049] FIG. 4C shows the gel images from the Western Blot analysis performed on whole cell lysate samples collected on day 3 of the production culture. Gels were analyzed on ‘Image Lab’

[0050] #14470686vl software for measuring the intensity of each band. Numeric labels indicate vessel conditions. Numeric labels with a prime indicate replicate vessels for the same condition.

[0051] FIG. 4D shows Rep protein expression on day 3 quantified based on the intensity of the band observed in Western blot analysis.

[0052] FIG. 4E shows Cap protein expression on day 3 quantified based on the intensity of the band observed in Western blot analysis.

[0053] FIG. 5 shows three different values of pCCL that were targeted during the experiment by manipulation of submerged gassing and P / V in a perfusion process at AMBR® 250 scale. The bar graph shows the actual pCCL levels achieved at 24 HPI, as measured offline using a BGA.

[0054] FIG. 6A shows VCD as measured on Vi-CELL BLU. The line chart shows no significant differences in VCD as a result of variations in pCCL.

[0055] FIG. 6B shows glucose concentration in the cell culture as measured on

[0056] No vaB ioProfile® FLEX2. The line chart shows that glucose concentration is maintained similar across the different pCCL conditions as a result of perfusion of fresh medium in the process.

[0057] FIG. 7 shows the effect of pCCE on AAV titers at AMBR® 250 scale in perfusion process. The bar graph shows AAV titers for the three conditions tested in perfusion process. The line chart shows the corresponding pCCE levels at 24 hours post infection. At pCCE levels > 70 mmHg, the rAAV titers are sub-optimal in perfusion process and such conditions are not desirable for AAV production.

[0058] FIG. 8 shows the effect of three conditions on cell culture pCCE as determined by BGA analysis at 24 HPI. The conditions entailed varying air sparge rates utilized to provide aeration and strip-out CO2 from the bench scale (5L) bioreactors in a perfusion process. With increasing air sparge rate, a corresponding decrease in cell culture pCO2 is observed.

[0059] FIG. 9A shows variances in VCD over time in culture as measured on Vi-CELL BLU. The line chart shows no significant differences in VCD as a result of variations in pCO2.

[0060] FIG. 9B shows glucose concentration in cell culture as measured on NovaBioProfile® FLEX2. The line chart shows similar glucose concentration maintained across the three conditions in the perfusion process despite variations in pCO2.

[0061] FIG. 10 shows the effect of pCO2 on AAV harvest titer at 5L bench scale. Similar to AMBR® 250 result, low AAV titer is observed at air sparge condition that resulted in high pCO2in cell culture.

[0062] FIG. 11 demonstrates the experimental conditions for an alternate producer cell line producing a different transgene and a different AAV serotype. The bar graph shows the actual

[0063] #14470686vl BGA values of pCCh achieved at 28HPI. Experiments were run in duplicates and error bars represent + / -1 SD.

[0064] FIG. 12A shows variances in VCD over time in culture as measured on Vi-CELL BLU. The line chart shows no significant differences in VCD as a result of variations in pCCL.

[0065] FIG. 12B shows glucose consumption as measured on No vaB ioProfile® FLEX2. The line chart shows differences in glucose consumption as a result of variations in pCCL similar to Example 1.

[0066] FIG. 13A shows the effect of pCCE on AAV titers at AMBR® 250 scale in batch process for the alternate producer cell line producing a different transgene. Effect of pCCL during virus production in the cell culture on harvest AAV titer is observed in AMBR® 250HT when various conditions of air sparge and overlay were tested to manipulate pCO2 during virus production. Similar to Example 1 (FIG 4A), at pCCL levels > 70 mmHg, the rAAV titers are sub-optimal and such conditions are not desirable for AAV production.

[0067] FIG. 13B shows the effect of pCCL on AAV particle quality quantified through analytical ultracentrifugation method (AUC) for the alternate producer cell line producing a different transgene. Harvested material from the conditions 1-4 were purified for sedimentation analysis to determine Empty / Partial / Full AAV capsids. Results show that high pCCL levels may result in more empty and less full capsids, thus affecting the quality of the rAAV product in the cell culture harvest.

[0068] FIG. 14A shows the gel images from the Western Blot analysis performed on whole cell lysate samples collected on day 3 of the production culture. Gels were analyzed on ‘Image Lab’ software for measuring the intensity of each band. Numeric labels indicate vessel conditions. Numeric labels with a prime indicate replicate vessels for the same condition.

[0069] FIG. 14B shows Rep protein expression on day 3 quantified based on the intensity of the band observed in Western blot analysis for the alternate producer cell line producing a different transgene.

[0070] FIG. 14C shows Cap protein expression on day 3 quantified based on the intensity of the band observed in Western blot analysis for the alternate producer cell line producing a different transgene.

[0071] FIGs. 15A-15B show that reducing NAHCO3 levels reduces pCO2 (FIG. 15A) but also lowers AAV titer (vg / mL) (FIG. 15B).

[0072] FIG. 16 illustrates the scale-up challenge with AAV production. Larger scale cultures (benchscale and 50L) produce markedly lower AAV titers per unit volume relative to smaller cultures.

[0073] #14470686vl FIG. 17 shows that, for satellite culture infected in benchtop bioreactor and transferred to AMBR250 vessels, longer incubation in benchtop bioreactor leads to reduced AAV titers, approaching benchtop-scale AAV titers.

[0074] FIG. 18 shows big data analysis simulating higher perturbation strength in terms of air overlay improves AAV titer. VVM stands for vessel volumes per minute.

[0075] FIGs. 19A-19B show that, across two cell lines, pCCh control improves AAV titer. As used in the figures, low pCCh (condition 1) means pCCh between 20-40 mmHg, moderate pCCL (condition 2) means pCCh between 40-70mmHg, high pCCh (condition 3) means pCCL between 70-100 mmHg, and very high pCCh (condition 4) means pCCL over 100 mmHg.

[0076] FIGs. 20A-20B show that, across two cell lines, Ad5 production is not affected by pCCh.

[0077] FIGs. 21A-21B show that, across two cell lines, as pCCL rises, relative AAV titers decrease, and the percentage of empty capsids increases.

[0078] FIGs. 22A-22B show that, across two cell lines, expression of the Rep and Cap proteins decreases as pCCL increases.

[0079] FIG. 23 shows a cartoon of available methods to modulate pCCL during cell culture.

[0080] FIGs 24A-24B show that a pCCb-controlling process described herein (new process) produces higher AAV titer and a higher percentage of full capsids relative to a conventional process (old process) even at larger scales (50L). At 200L scale, the new process produces similar relative AAV titers to the new process at benchscale and 50L scale, showing its scale-up capability (FIG. 24A).

[0081] DETAILED DESCRIPTION

[0082] The present disclosure provides methods and compositions for improving production of recombinant adeno-associated virus (rAAV) in a batch or perfusion cell culture using mammalian host cells comprising transiently transfected or stable integrated copies of AAV rep, cap genes and an exogenous nucleic acid of interest flanked by AAV ITRs at a large scale in bioreactors. In some aspects, the cell culture process utilizes active control of partial pressure of carbon dioxide (pCCE) in the range of 20-70 mmHg. In some aspects, the cell culture process utilizes active control of pCCL in the range of 20-70 mmHg between 12 to 48 hours post infection by helper virus. In some aspects, the biomanufacturing process involves utilizing a mixture of air / CCL in air sparging and in the headspace of a bioreactor to increase partial pressure of carbon dioxide (pCCE) in the cell culture. In some aspects, increased air sparge rates are used to decrease pCCL in the cell culture by promoting CO2 stripping. In some aspects, increased power per volume (P / V) is used to decrease pCO2 in the cell culture in combination

[0083] #14470686vl with increased air sparge rates. In some embodiments, the P / V is about 15-25 Watts / m3. In some embodiments, the P / V is about 5 to about 50 W / m3(e.g., 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55 W / m3).

[0084] In some embodiments, methods and compositions described herein are useful for increasing rAAV yield. In some embodiments, methods and compositions described herein are useful for scaling-up cell culture to ensure that the process generates the desired rAAV at a similar titer and of similar quality across scales. In some embodiments, the methods described herein are used for manufacturing rAAVs in producer cell lines (PCLs) at large scale in bioreactors. In some embodiments, the methods described herein are used for manufacturing rAAVs from producer cell lines (PCLs) at 500 L scale. In some embodiments, the methods described herein are used for manufacturing rAAVs from PCLs at 2000 L scale. In some embodiments, methods and compositions described herein provide high rAAV harvest titers and improved AAV particle quality. In some embodiments, an improved product quality comprises an enhanced full rAAV capsid percentage relative to empty or partial capsids in an rAAV preparation prepared according to the methods described herein. In some embodiments, methods and compositions described herein are particularly useful for producing rAAVs to be used as gene therapeutic agents. In some embodiments, methods and compositions described herein are useful for increasing rAAV yield. In some embodiments, methods and compositions described herein are useful for reducing the cost of producing rAAV for therapeutic applications.

[0085] CO2 control

[0086] Without intending to be bound by theory, accumulation of excess carbon dioxide (CO2) or insufficient levels of CO2 during catabolic and anabolic reactions of biosynthesis can lead to lower total viral genome formation, which in turn leads to lower rAAV production.

[0087] In some embodiments, the present disclosure relates to methods of controlling the accumulation of excess CO2 in cell culture. In some embodiments, CO2 released by a cell culture exceeds the bioreactor’s capacity to remove it. In some embodiments, high CO2 levels during cell culture can cause pH changes, growth inhibition, metabolic changes and mitochondrial dysfunction.

[0088] The CO2 in cell culture impacts cell physiology through abovementioned effects and consequently the capacity of mammalian cells to produce high quantities and high quality of rAAV particles. In some embodiments, the CO2 in the culture is quantified by the partial pressure of carbon dioxide dissolved in the aqueous / liquid phase of the cell culture, termed as pCO2. In some embodiments, pCCL is measured using a pCO2 detector or analyzer. In some

[0089] #14470686vl embodiments, the pCCh detector or analyzer is an on-line pCO2 detector or analyzer. In some embodiments, the pCO2 detector or analyzer is an in-line pCCb detector or analyzer ). In some embodiments, the pCO2 detector or analyzer comprises a sensor (e.g., a Severinghaus sensor, an optical sensor, or an infrared sensor). In some embodiments, pCCb is measured using a CO2 specific pH probe, such as a blood gas analyzer (BGA). In some aspects, partial pressure of CO2 is measured and / or determined at an atmospheric pressure of 1 atmosphere (atm). 1 atm may also be expressed as 101,325 Pascals (Pa), 1013.25 millibars (mb) or 760 mmHg. Partial pressure of CO2 may be calculated as follows: 760 mmHg (percent CO2 / IOO) = partial pressure of CO2 (pCO2).

[0090] Aspects of the present disclosure relate to methods of incubating a cell culture under a pCO2level of 20 mmHg, 30 mmHg, 40 mmHg, 50 mmHg, 60 mmHg, 70 mmHg. In some embodiments, a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg. In some embodiments, a cell culture is incubated under pCCb levels of about 20 to 70 mmHg. In some embodiments, a cell culture is incubated under pCCh levels of about 30-60 mmHg (e.g., 50 mmHg). In some embodiments, a cell culture is incubated under pCCb levels of 20-40 mmHg, 30-60 mmHg, 40- 70 mmHg, 70-100. In some embodiments, a cell culture is incubated under conditions (e.g., air sparge rate, agitation rate, air sparge composition) expected to produce pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 45-55 mmHg, 50 to 60 mmHg, 60 to 70 mmHg. In some embodiments, a cell culture is incubated under conditions expected to produce pCO2levels of about 20 to 70 mmHg. In some embodiments, a cell culture is incubated under conditions expected to produce pCCh levels of about 30-60 mmHg (e.g., 50 mmHg). In some embodiments, a cell culture is incubated under conditions expected to produce pCCb levels of 20-40 mmHg, 30-60 mmHg, 40-70 mmHg, or 70-100 mmHg.

[0091] In some embodiments, the present disclosure relates to methods of incubating a cell culture under a CO2 concentration of about 2% to about 10%, or conditions expected to produce such a CO2 concentration. In some embodiments, the present disclosure relates to methods of incubating a cell culture under a CO2 concentration of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or about 10%. In some embodiments, the present disclosure relates to methods of incubating a cell culture under a CO2 concentration of less than 10%.

[0092] In some embodiments a cell culture is incubated under pCCb levels, or conditions expected to produce pCCb levels, ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for at least 12 hours. In some embodiments a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50

[0093] #14470686vl mmHg, 50 to 60 mmHg, 60 to 70 mmHg for at least 48 hours. In some embodiments a cell culture is incubated under pCO2levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for at least 72 hours. In some embodiments a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for up to 48 hours. In some embodiments a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for up to 72 hours. In some embodiments a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for 72 hours. In some embodiments a cell culture is incubated under pCCb levels ranging from 20 to 30 mmHg, 30 to 40 mmHg, 40 to 50 mmHg, 50 to 60 mmHg, 60 to 70 mmHg for about 12 hours to about 72 hours.

[0094] In some embodiments, the pH of the cell culture is maintained between 7.0 and 7.5. In some embodiments, the pH of the cell culture is maintained between 7.0 and 7.3, 6.9 and 7.5, 7.1 and 7.3, or 7.2 and 7.5. In some embodiments, the pH of the cell culture is maintained at about 7.3, 7.4, or 7.5. In some embodiments, the pH of the cell culture is about 7.3, 7.4, or 7.5 for at least

[0095] Air sparge conditions

[0096] In some embodiments, the present disclosure relates to methods of injecting CO2 in a bioreactor comprising the cell culture and / or by submerged gassing of the bioreactor to maintain pCO2levels. In some embodiments, a method of introducing air and / or oxygen to a cell culture medium using a gas supply is described as air sparging. In some embodiments, air and / or oxygen is introduced to a cell culture media in a bioreactor via air sparging. In some embodiments, air sparging is done using a fixed composition. In some embodiments, the fixed composition is ambient air. In some embodiments, the fixed composition is CO2 (e.g., for use when pCCh is lower than a set value, such as 20-70 mmHg). In some embodiments, a fixed composition is a composition whose components exist in a defined ratio with respect to each other, such as a set ratio of oxygen to nitrogen. In some embodiments, the fixed composition comprises 5-10% CO2. In some embodiments, the fixed composition comprises 15-20% O2. In some embodiments, the fixed composition comprises 70-75% N2. In some embodiments, the fixed composition comprises about 5-10% CO2. In some embodiments, the fixed composition comprises about 15-20% O2. In some embodiments, the fixed composition comprises about 70- 75% N2. In some embodiments, the fixed composition comprises 5-10% CO2, 15-20% O2, and 70-75% N2. In some embodiments, the fixed composition comprises about 5-10% CO2, 15-20% O2, and 70-75% N2. In some embodiments, air sparging rate is 0.001, 0.002, 0.003, 0.004,

[0097] #14470686vl 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 volume of sparged air per volume of medium per minute (VVM). In some embodiments, air sparge rate on day 0 (18-24 hours on the day of infection or transfection) is 0.005 VVM, increasing to 0.015, 0.025 and 0.03 VVM. In some embodiments, air sparge rate is at low sparge ( 0.001- 0.007 VVM) on day 0 or infection day, and the air sparge rate increases to high sparge (0.01- 0.03 VVM) on day 1 to maintain pCCb levels. In some embodiments, air sparge rate is from about 0.005 to about 0.05 VVM.

[0098] Methods of controlling pCO2 using air sparge are shown in FIG. 23. In some embodiments, a micro sparger is used. In some embodiments, a drilled-hole sparger is used. In some embodiments, air overlay is increased. In some embodiments, the sparge rate is increased. In some embodiments, the agitation rate is increased. In some embodiments, more than one method of pCO2 control is used. In some embodiments, all of the above-listed methods are used to control pCO2.

[0099] Bicarbonate

[0100] In some embodiments, the present disclosure relates to methods of modulating CO2 in a bioreactor by incubating a cell culture in a medium comprising reduced levels of bicarbonate. In some embodiments, bicarbonate is sodium bicarbonate. In some embodiments, the cell culture medium comprises a level of bicarbonate (e.g., sodium bicarbonate) that is reduced by 0.25X, 0.5X, or 0.75X relative to a standard medium. In some embodiments, the cell culture medium comprises a level of bicarbonate (e.g., sodium bicarbonate) that is reduced by about 0.25X, about 0.5X, or about 0.75X relative to a standard medium. In some embodiments, the cell culture medium comprises a level of bicarbonate (e.g., sodium bicarbonate) that is reduced by at least 0.25X, at least 0.5X, or at least 0.75X relative to a standard medium.

[0101] CO2 solubility

[0102] In some embodiments, the present disclosure provides methods of modulating CO2 in a bioreactor by incubating a cell culture in a medium comprising a compound that limits the solubility of CO2. In some embodiments, a compound that limits the solubility of CO2 comprises an ionic compound, such as sodium chloride (NaCl), potassium chloride (KC1), or calcium chloride (CaCh).

[0103] Bioreactor systems

[0104] As used herein, a “bioreactor” is a device, vessel, or system to culture various cell types on a desired scale. In some embodiments, optimal cell growth and metabolism are achieved in a bioreactor through the regulation of various chemical and physical parameters. In some

[0105] #14470686vl embodiments, a bioreactor is a laboratory scale bioreactor (0.5-10 L), or pilot scale bioreactor (50-200 L), or industrial scale bioreactor (500-5000 L).

[0106] In some embodiments, a bioreactor is designed to optimize oxygen transfer, minimize shear stress, and ensure effective mixing of a cell culture. In some embodiments, the demand for high levels of oxygen is addressed by elevating agitation speeds and air / Ch sparging rates in bioreactors. In some embodiments, maximum cell growth and productivity is achieved by mixing the culture well in bioreactors for consistent transfer of oxygen and nutrients to the cells. In some embodiments, a bioreactor contains impellers, which are agitation tools equipped with blades and are employed for mixing cell cultures. In some embodiments, the orientation of the blades, the impeller speed and diameter influence the shear forces on the bioreactor cell culture. In some embodiments, the blades of impellers are axial or radial. In some embodiments, “gassing” or addition of gasses in the bioreactor requires adding oxygen into a cell culture. In some embodiments, a bioreactor contains a headspace, which is an empty space above the liquid in a vessel. In some embodiments, gas is added into a bioreactor headspace, or sparged into a cell culture suspension. In some embodiments, the other essential parameters in bioreactors for optimal cell growth, survival and product formation are temperature, pH and metabolite concentrations.

[0107] Batch mode of cell culture

[0108] In some aspects, the present disclosure relates to methods of using a batch mode, or batch culture for rAAV production. In some embodiments, a batch culture comprises a mammalian cell culture. In some embodiments, a batch culture is a closed system of cell culture grown using a limited amount of nutrients in a fixed volume of liquid medium. In some embodiments, a batch culture production occurs over a short period of time. In some embodiments, a batch culture is inoculated in a bioreactor. In some embodiments, nutrients are provided in a batch culture only at the beginning of the process. Nutrients are gradually depleted from the cell culture over time. In some embodiments, the oxygen transfer rate is increased to enhance the availability of oxygen. In some embodiments, one or more parameters that help increase the oxygen availability are gas flow, mixing speed, the proportion of oxygen in gas mix, or pressure in a bioreactor.

[0109] Fed-batch mode of cell culture

[0110] In some aspects, the present disclosure relates to methods of using a fed-batch mode, or fed-batch culture for rAAV production. In some embodiments a fed-batch culture is a partly

[0111] #14470686vl open system comprising growing cell cultures in an environment containing constant concentration of nutrients. In some embodiments, a fed-batch culture production occurs over an extended interval. In some embodiments, a fed-batch culture produces high cell densities, prolonged cell viability and high product titers. In some embodiments, a bioreactor is inoculated with a low working volume, or a minimum working volume of cells. In some embodiments, one or more criteria comprising concentrations of glucose, and / or ammonia determine the start of feeding of a fed-batch culture in a bioreactor. In some embodiments, nutrients are added to a fed-batch culture until maximum working volume has met.

[0112] Perfusion mode of cell culture

[0113] In some aspects, the present disclosure relates to methods of producing rAAV while perfusing a cell culture, also referred to as using perfusion or under perfusion conditions. In some embodiments, perfusion comprises gradual replacement of media comprising the cell culture. In some embodiments, perfusion occurs over an extended interval. In some embodiments, perfusion comprises continuous or near-continuous replacement of media. In some embodiments, fresh medium is added and spent culture medium is removed. In some embodiments, the rate at which fresh medium is added is the same as the rate at which spent culture medium is removed. In some embodiments, perfusion cell culture comprises retaining cells in the culture using a retention device while removing waste products from the culture and / or adding nutrients. In some embodiments, a retention device comprises a centrifuge (e.g., a continuous centrifuge), a membrane filter (e.g., a tangential flow membrane filter (TFF), an alternating tangential flow filter (ATF)), a dynamic filter, a spin-filter, a hollow-fiber filter, a separator (e.g., an ultrasonic or dielectrophoretic separator), a settler (e.g., a gravity settler), or a hydrocyclone. In some embodiments, a retention device consists of a centrifuge (e.g., a continuous centrifuge), a membrane filter (e.g., a tangential flow membrane filter (TFF), an alternating tangential flow filter (ATF)), a dynamic filter, a spin-filter, a separator (e.g., an ultrasonic or dielectrophoretic separator), a settler (e.g., a gravity settler), or a hydrocyclone.

[0114] In some embodiments, perfusion removes waste products from the cell culture. In some embodiments, a waste product is a metabolite. In some embodiments, a waste product comprises lactate or ammonium (NH4+).

[0115] In some embodiments, perfusion adds one or more nutrients. In some embodiments, a nutrient comprises a carbon source (e.g., glucose or galactose), amino acid source, trace metal, vitamin, antioxidant source, and / or mineral.

[0116] #14470686vl In some embodiments, the cell culture is perfused both during growth and during rAAV production. In some embodiments, the cell culture is perfused only during rAAV production.

[0117] In some embodiments, the cell culture is cultured under rAAV production conditions for at least 24 hours, 24 hours, or about 24 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 48 hours, 48 hours, or about 48 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 72 hours, 72 hours, or about 72 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 24 hours, 24 hours, or about 24 hours after rAAV production has begun. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 48 hours, 48 hours, or about 48 hours after rAAV production has begun. In some embodiments, the cell culture is cultured for at least 96 hours, 96 hours, or about 96 hours after infection or transfection. In some embodiments, the cell culture is cultured under rAAV production conditions for at least 72 hours, 72 hours, or about 72 hours after rAAV production has begun. In some embodiments, the cell culture is cultured for at least 96 hours, 96 hours, or about 96 hours after rAAV production has begun. In some embodiments, the cell culture is perfused for at least 48 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for up to 48 hours of the at least 72 hours, 72 hours, or about 72 hours of rAAV production. In some embodiments, the cell culture is perfused for between 24 and 48 hours of the at least 72 hour, 72 hour, or about 72 hour timeframe of rAAV production. In some embodiments, the cell culture is perfused for at least 24 hours of the at least 72 hour, 72 hour, or about 72 hour timeframe of rAAV production. In some embodiments, the cell culture is perfused for up to 72 hours during rAAV production.

[0118] In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 24 hours, 24 hours, or about 24 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 48 hours, 48 hours, or about 48 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 72 hours, 72 hours, or about 72 hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 24 hours, 24 hours, or about 24 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 48 hours, 48 hours, or about 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 96 hours, 96 hours, or about 96

[0119] #14470686vl hours after infection or transfection. In some embodiments, the cell culture is perfused under conditions suitable for rAAV production for at least 72 hours, 72 hours, or about 72 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 96 hours, 96 hours, or about 96 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 48 hours after rAAV production has begun. In some embodiments, the cell culture is perfused for up to 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for between 24 and 48 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for at least 24 hours under conditions suitable for rAAV production. In some embodiments, the cell culture is perfused for up to 72 hours under conditions suitable for rAAV production.

[0120] In some embodiments, the rate of perfusion is measured as vessel volumes per day (VVD). In some embodiments, the rate of perfusion is between 0.5 and 2 vessel volume per day (VVD). In some embodiments, the rate of perfusion is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 VVD. In some embodiment, the rate of perfusion is 2 VVD or higher. In some embodiments, the rate of perfusion is 2-5 VVD, 2-4.5 VVD, 2-4 VVD, 2-3 VVD, or 2-2.5 VVD. In some embodiments, the rate of perfusion is 3-4 VVD, 3-5 VVD, 3-6 VVD, 4-5 VVD, or 4-6 VVD.

[0121] In some embodiments, perfusion is continuous. In some embodiments, continuous perfusion includes brief intervals where perfusion is stopped. In some embodiments, perfusion is stopped for less than half an hour, about half an hour, less than an hour, about an hour, less than two hours, about two hours, less than three hours, about three hours, less than four hours, about four hours, less than five hours, about five hours, less than 10 hours, about 10 hours, less than 12 hours, or about 12 hours. In some embodiments, perfusion is semi-continuous. In some embodiments, semi-continuous perfusion comprises regular intervals of perfusion and nonperfusion culture.

[0122] Media exchange

[0123] In some embodiments, semi-continuous perfusion comprises media exchange. In some embodiments, media exchange comprises rapid replacement of media comprising the cell culture. For example, a cell culture may be pelleted (e.g., by centrifugation), spent medium removed, and resuspended in fresh medium. A culture may also be separated from spent medium by other means, such as filtration, and resuspended in spent medium. In some embodiments, media exchange occurs over a short interval. In some embodiments, media

[0124] #14470686vl exchange within about an hour (e.g., from start to finish the process of media exchange takes about half an hour). In some embodiments, media exchange occurs within an hour. In some embodiments, media exchange occurs within less than an hour. In some embodiments, media exchange occurs within about half an hour.

[0125] In some embodiments, media exchange occurs one, twice, three times, four times, five time, or six times during rAAV production. In some embodiments, media exchange occurs about once every 12 hours, once a day, once every 36 hours, or once every two days during rAAV production. In some embodiments, media exchange occurs when the level of a particular nutrient or waste product reaches a threshold level.

[0126] In some embodiments, media exchange removes waste products from the cell culture. In some embodiments, a waste product is a metabolite. In some embodiments, a waste product comprises lactate or ammonium (NH4+).

[0127] In some embodiments, media exchange adds one or more nutrients. In some embodiments, a nutrient comprises a carbon source e.g., glucose or galactose), amino acid source, trace metal, vitamin, antioxidant source, and / or mineral.

[0128] Media

[0129] Aspects of the present disclosure provide methods of preparing a cell culture for rAAV production and contacting a cell culture with one or more enhancer elements.

[0130] In some aspects, the cell culture is provided in a medium. In some aspects, the cell culture is incubated in a medium during rAAV production. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production. In some aspects, the medium is a medium that supports cell growth. In some aspects, the medium provides one or more nutrients. In some aspects, the medium is a feed medium. In some aspects, the medium comprises a complex medium. In some aspects, the medium is a defined medium. In some aspects, the medium comprises Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV- MAX media (Gibco), EX-Cell media, or RPMI media. In some aspects, the medium is a serum- free medium. In some embodiments, the medium is supplemented with serum. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some aspects, the medium is an appropriate medium for AAV production. In some embodiments, perfusion or media exchange removes waste products from the media comprising the cell culture. In some

[0131] #14470686vl embodiments, perfusion or media exchange maintains nutrient levels in the media comprising the cell culture.

[0132] In some embodiments, the cell culture media (medium) comprises a balanced formulation of carbon and nitrogen sources, essential inorganic salts, trace elements, vitamins, buffering agents, and optional supplements such as lipids or growth factors. The medium may be used in batch, fed-batch, or perfusion-based processes and is suitable for use in adherent or suspension-adapted cells, such as HEK293 or HeLa cells.

[0133] In some embodiments, the cell culture medium includes one or more carbon sources selected from glucose, galactose, fructose, mannose, glycerol, or their derivatives. In some embodiments, the one or more carbon sources are present in the medium in a concentration range of about 0.5 g / L to 10 g / L (e.g., 1 g / L to 8 g / L; 2 g / L to 7 g / L; 3 g / L to 6 g / L, or 4 g / L to 5 g / L; about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or more).

[0134] In some embodiments, the nitrogen source may include amino acids e.g., glutamine, asparagine, arginine), peptides, or inorganic nitrogen such as ammonium salts (e.g., ammonium chloride or ammonium sulfate), present in total concentrations ranging from approximately 0.1 g / L to 5 g / L (e.g., 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L or more). In some embodiments, glutamine or a stabilized dipeptide form (e.g., alanyl-glutamine) may be included at 2-6 mM (e.g., 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or more) to support both energy metabolism and protein biosynthesis.

[0135] The cell culture media (medium), in some embodiments, comprises inorganic salts. Inorganic salts are provided, in some embodiments, to maintain osmolarity, membrane stability, and ionic balance. Examples of inorganic salts include, but are not limited to, sodium chloride (e.g., 1-8 g / L), potassium chloride (e.g., 0.1-1.0 g / L), calcium chloride (e.g., 0.05-0.5 g / L), magnesium sulfate (e.g., 0.05-0.5 g / L), and sodium phosphate buffers (e.g., 0.5-2.0 g / L). The pH of the cell culture medium (media) is maintained, in some embodiments, in the range of about 6.8 to 7.6 (e.g., 7.0-7.4), using a combination of HEPES (e.g., 10-25 mM), sodium bicarbonate (e.g., 1-3 g / L), or other suitable buffering agents. The osmolality of the cell culture medium, in some embodiments, is adjusted to support cell viability and viral vector production, and may range from 260 to 350 mOsm / kg, depending on the specific cell line and process conditions. In some embodiments, the osmolality is maintained at a value selected from 260, 270, 280, 290, 300, 310, 320, 330, 340, or 350 mOsm / kg. Osmolality may be adjusted using combinations of sodium chloride, potassium chloride, or other osmotically active components, and may be monitored throughout the culture process to ensure consistency and reproducibility.

[0136] #14470686vl In some embodiments, medium is introduced via perfusion or media exchange. In some embodiments, the medium introduced via perfusion or media exchange (“perfusion medium”) is an appropriate medium for cell growth, infection, transfection, and / or rAAV production. In some embodiments, the perfusion medium is a medium that supports cell growth. In some embodiments, the perfusion medium provides one or more nutrients. In some embodiments, at least one perfusion medium is a feed medium. In some embodiments, one or more of the perfusion media comprises a complex medium. In some embodiments, the perfusion medium is a defined medium. In some embodiments, one or more of the perfusion media comprises Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV-MAX media (Gibco), EX-Cell media, or RPMI media. In some embodiments, the perfusion medium is a serum-free medium. In some embodiments, the perfusion medium is supplemented with serum. In some embodiments, the perfusion medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some embodiments, the perfusion medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some embodiments, the perfusion medium is an appropriate medium for AAV production.

[0137] In some embodiments, the method comprises a first volume and a second volume, each of which comprise a medium, the second medium being a “perfusion medium.” In some embodiments, the first volume medium and the second volume medium are the same medium. In some embodiments the first volume medium and the second volume medium are different media. In some embodiments, the first volume medium is a medium appropriate for cell growth. In some embodiments, the second volume medium is a medium appropriate for rAAV production.

[0138] In some embodiments, the pH of the perfusion medium is between 6.9 and 7.5, 7 and 7.4, 7.1 and 7.3, 7 and 7.5, 6.9 and 7.4, or 7.2 and 7.5. In some embodiments, the pH of the perfusion medium is about 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. In some embodiments, the pH of the perfusion medium is 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0139] In some embodiments, the glutamine level in the perfusion medium is lmM-2mM, 2mM-3mM, 3mM-4mM, 4mM-5mM, 5mM-6mM, 6mM-7mM, 7mM-8mM, 8mM-9mM, or 9mM-10mM.

[0140] In some embodiments, the perfusion medium is a glutamine containing medium. In some embodiments, glutamine concentration in the perfusion medium is O.lmM-lmM, ImM- 2mM, 2mM-3mM, 3mM-4mM, 4mM-5mM, 5mM-6mM. In some embodiments, glutamine is maintained in the perfusion medium during the AAV production phase (0-72 hours post

[0141] #14470686vl infection) between 0.1-6.0 mM. In some embodiments, glutamine is maintained in the perfusion medium by feeding L-glutamine in cell culture in fed-batch mode. In some embodiments, glutamine is maintained in the perfusion medium by feeding other glutamine sources such as L- alanyl-L-glutamine dipeptide or similar dipeptide analogs. In some embodiments, glutamine is maintained in the perfusion medium by using a fresh medium containing L-glutamine, L-alanyl- L-glutamine dipeptide or similar dipeptide analogs. In some embodiments, the perfusion medium contains L-alanyl-L-glutamine dipeptide, or glycyl-l-glutamine.

[0142] In some embodiments, the perfusion medium is enriched relative to standard medium. In some embodiments, the perfusion medium is enriched 1.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 8X, 8X, or 10X relative to standard medium. In some embodiments, a perfusion medium is enriched relative to a standard medium when the enriched medium comprises a higher concentration of one or more vitamins, amino acids, or other nutrients relative to the standard medium. For example, a perfusion medium is 2X enriched for glutamine relative to a standard medium when the medium comprises a 2X higher concentration of glutamine relative to a standard medium.

[0143] In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production. In some embodiments, the cell culture comprises a medium that supports cell growth. In some embodiments, the cell culture comprises a medium that provides one or more nutrients. In some embodiments, the cell culture comprises a feed medium. In some embodiments, the cell culture comprises a complex medium. In some embodiments, the cell culture comprises a defined medium. In some embodiments, the cell culture comprises a medium selected from: Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV- MAX media (Gibco), EX-Cell media, or RPMI media. In some embodiments, the cell culture comprises a serum-free medium. In some embodiments, the cell culture comprises a medium supplemented with serum. In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some embodiments, the cell culture comprises an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some embodiments, the cell culture comprises an appropriate medium for AAV production.

[0144] In some embodiments, the method comprises a first volume and a second volume, each of which comprise a medium, each comprising a cell culture. In some embodiments, the first volume medium and the second volume medium are the same medium. In some embodiments

[0145] #14470686vl the first volume medium and the second volume medium are different media. In some embodiments, the first volume medium is a medium appropriate for cell growth. In some embodiments, the second volume medium is a medium appropriate for rAAV production.

[0146] In some embodiments, the pH of the cell culture is between 6.9 and 7.5, 7 and 7.4, 7.1 and 7.3, 7 and 7.5, 6.9 and 7.4, or 7.2 and 7.5. In some embodiments, the pH of the cell culture is about 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. In some embodiments, the pH of the cell culture is 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0147] In some embodiments, the glutamine level in the cell culture is O.lmM-lmM, ImM- 2mM, 2mM-3mM, 3mM-4mM, 4mM-5mM, 5mM-6mM, 6mM-7mM, 7mM-8mM, 8mM-9mM, or 9mM-10mM. In some embodiments, the glutamine level in the perfusion medium is maintained for at least 12 hours, 15 hours, 18 hours, 21 hours 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 hours, 51 hours, 54 hours, 57 hours, 60 hours, 63 hours, 66 hours, 69 hours, 72 hours, 75 hours, or 78 hours.

[0148] In some embodiments, the glutamine level in the cell culture starts at a first level and does not go below a second level. In some embodiments, the first level comprises a glutamine level of 3mM-4mM, 4mM-5mM, or 5mM-6mM. In some embodiments, the second level comprises a glutamine level of lmM-2mM, 2mM-3mM, or 3mM-4mM. In some embodiments, the glutamine level starts at 4mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 5mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 4mM and does not go below 3 mM. In some embodiments, the glutamine level starts at 6 mM and does not go below 2 mM. In some embodiments, the glutamine level starts at 4 mM and does not go below 1.5 mM. In some embodiments, the glutamine level starts at 5mM and does not go below 4 mM.

[0149] In some embodiments, the cell culture comprises a medium that is enriched relative to standard medium. In some embodiments, the cell culture comprises a medium that is enriched 1.25X, 1.5X, 1.75X, 2X, 2.25X, 2.5X, 2.75X, 3X, 3.25X, 3.5X, 3.75X, 4X, 4.25X, 4.5X, 4.75X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 8X, 8X, or 10X relative to standard medium. In some embodiments, the cell culture comprises a medium that is enriched relative to a standard medium when the enriched medium comprises a higher concentration of one or more vitamins, amino acids, or other nutrients relative to the standard medium. For example, a cell culture comprises a medium that is 2X enriched for glutamine relative to a standard medium when the medium comprises a 2X higher concentration of glutamine relative to a standard medium.

[0150] #14470686vl Cell Culture

[0151] In some aspects, the present disclosure relates to methods of scaling up and / or increasing rAAV production by a cell culture.

[0152] In some aspects, the cell culture comprises animal cells. In some aspects, the cell culture comprises mammalian cells. In some embodiments, the cell culture comprises HeLa cells. In some embodiments, the cell culture comprises human embryonic kidney (HEK) cells. In some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells. In some embodiments, the cell culture comprises Vero cells. In some embodiments, the cell culture comprises Madin-Darby canine kidney (MDCK) cells. In some embodiments, the cell culture comprises baby hamster kidney (BHK) cells. In some embodiments, the cell culture comprises A549 cells. In some embodiments, the cell culture comprises amniocyte cells.

[0153] 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 presently described methods are combined with other methods to increase production of rAAV particles. In some embodiments, combining the presently-described methods with additional improvements in, for example feeding methods, leads to a 10-fold increase in AAV particle production or productivity. In some embodiments, successful scale-up of the entire manufacturing method is demonstrated from 250 mL reactors to 500 L bioreactors. In some embodiments, the presently disclosed method improves the production of rAAV particles relative to a control method. In some embodiments, the presently disclosed method increases production of rAAV particles relative to a control method. In some embodiments, the method of this application increases production of rAAV particles by 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 -fold, 12 fold, 13 fold, 14 fold, or 15 fold relative to a control method. In some embodiments, a control method is a method that does not include pCCb regulation in a batch or perfusion culture.

[0154] In some embodiments, the method of this application improves the percent full capsids produced relative to a control method. In some embodiments, the method of this application increases production of full capsids by 1.5 fold, 2 fold, 3 fold, 4 fold, or 5 fold (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) relative to a control method.

[0155] In some embodiments, the method of this application reduces the time required to produce levels of rAAV for harvest relative to a control method. 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.

[0156] #14470686vl In some embodiments, the method of this application improves packaging efficiency at harvest relative to a control method. In some embodiments, the method of this application increases packaging efficiency by 1.5 fold, 2 fold, 3 fold, 4 fold, or 5 fold e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) relative to a control method. In some embodiments, packaging efficiency is improved due to higher capsids with total genomes in an early phase of rAAV production. In some embodiments, the method of this application increases total genomes at 24 and / or 48 hour timepoints (e.g., 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, or more). In some embodiments, the method of this application increases total genomes at 24 and / or 48 hour timepoints by 1.25 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, or 25 fold relative to a control method.

[0157] In some embodiments, the method of this application reduces the time required to produce levels of rAAV for harvest relative to a control method. In some embodiments, the method reduces the time by 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours, 28 hours, or more. In some embodiments, the method reduces the time by 1 day.

[0158] Methods

[0159] Aspects of the disclosure provide methods for producing recombinant adeno-associated viral (rAAV) particles. Aspects of the disclosure provide methods of preparing a cell culture for rAAV production.

[0160] In some aspects, the method comprises transient transfection of mammalian host cells. In some aspects, transfection comprises contacting a cell culture with one or more nucleic acids and one or more transfection reagents. In some aspects, the one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA). In some embodiments, the one or more recombinant nucleic acids comprise ribonucleic acid (RNA). In some embodiments, the one or more recombinant nucleic acids comprise one or more plasmids.

[0161] In some aspects, transient transfection comprises using a triple or dual transfection system. In some aspects, a dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein and the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a

[0162] #14470686vl second plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes. In some aspects, the triple plasmid transfection system comprises, (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein; (ii) a second plasmid comprising: the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes.

[0163] In some aspects, the transfection system comprises one or more recombinant nucleic acids encoding a gene of interest flanked by AAV inverted terminal repeats (ITRs). In some aspects, the ITRs are AAV2 or AAV9 ITRs.

[0164] In some aspects, the transient transfection comprises contacting a cell culture comprising the mammalian host cells with one or more enhancer elements. In some aspects, the cell culture is contacted with the one or more enhancer elements at the same time as the one or more recombinant nucleic acids.

[0165] In some aspects, the method further comprises contacting the cell culture comprising the mammalian host cells with a feed medium, about 4 hours to about 8 hours following the transient transfection.

[0166] In some aspects, the cell culture produces recombinant adeno-associated virus (rAAV) particles.

[0167] In some aspects, the method further comprises isolating rAAV from the cell culture. In some aspects, isolating rAAV from the cell culture comprises harvesting the cell culture. In some aspects, isolating rAAV from the cell culture comprises lysing the cells comprising the cell culture. In some aspects, isolating rAAV from the cell culture comprises removing rAAV from the cell culture medium.

[0168] In some aspects, the titer of rAAV isolated from the cell culture is at least 5x10A9, 6xlOA9, 7xlOA9, 8xlOA9, 9xlOA9, lxl0A10, lxl0A10, 3xl0A10, 4xl0A10, 5xl0A10, 6xl0A10, 7xl0A10, 8xl0A10, 9xl0A10, lxlOAl l, 2xlOAl l, 3xlOAl l, 4xl0Al l, 5xlOAl l, 6xlOAl l, 7xlOAl l, 8xl0Al l, 9xlOAl l, lxlOA12, 2xlOA12, 3xlOA12, 4 xlOA12, 5xlOA12, 6xlOA12 vg / mL.

[0169] In some embodiments, the method is carried out at a room temperature. In some embodiments the method is carried out at 10-40 C, for example 15-35 C, 15-20 C, 20-25 C, or 25-30 C.

[0170] In some embodiments, pH of the medium is monitored. In some embodiments, the method is carried out at a pH of between 6.9 and 7.5, between 7 and 7.4, between 7.1 and 7.3, between 6.9 and 7.2, between 7 and 7.3, between 7.3 and 7.5.

[0171] #14470686vl Recombinant A A Vs

[0172] Naturally occurring AAV capsid proteins can be used to produce rAAVs for gene therapy. Different naturally occurring AAVs have different characteristics (including for example different tissue tropisms) and can be used for different indications. AAVs are highly prevalent within the human population (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues J Virol. 2004. 78(12): p. 6381-8, and Boutin. S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population, implications forgone therapy using AAV vectors. Hum Gene Ther. 2010. 21(6): p. 704-12) and are useful as viral vectors. Many serotypes exist, each with different tropism for tissue types (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80), which allows specific tissues to be preferentially targeted with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rhlO, transduce the mammalian body. See Zincarelli, C., et al. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A. M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S. J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAVl and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65. AAV9 has been demonstrated to cross the blood-brain barrier (see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59- 65, and Rahim, A. A., et al., Intravenous administration of AAV2 / 9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011. 25(10): p. 3505-18) that is inaccessible to many viral vectors and biologies. Certain AAVs have a payload of 4.7-5.0 kb (including viral inverted terminal repeats (ITRs), which are required in cis for viral packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): p. 80-6 and Dong, J.

[0173] #14470686vl Y., P. D. Fan, and R. A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): p. 2101-12.

[0174] In some embodiments, rAAVs can include one or more variant AAV capsid proteins having one or more amino acid substitutions relative to a naturally occurring AAV capsid protein.

[0175] Accordingly, in some embodiments, the rAAV particles comprise AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, or AAV 12 capsid proteins, or amino acid sequence variants thereof, or chimeras, or hybrids thereof.

[0176] In some embodiments, the rAAV particles comprise an rAAV genome. In some embodiments, an rAAV genome comprises a gene of interest flanked by inverted terminal repeats (ITRs). In some embodiments, a gene of interest comprises a therapeutic molecule, such as a therapeutic protein or therapeutic RNA. In some embodiments, a therapeutic molecule is an antibody, a protein, a peptide, an enzyme, or a ribozyme. rAAV production

[0177] Recombinant adeno-associated virus (rAAV) vectors are useful in gene therapy to deliver therapeutic genes to patient cells and tissue. An rAAV particle typically comprises a recombinant nucleic acid encapsidated within rAAV capsid proteins to form an rAAV particle that can be administered to a subject. The recombinant nucleic (e.g., recombinant AAV genome) acid typically includes a heterologous gene of interest (e.g., encoding a therapeutic nucleic acid and / or protein) flanked by AAV inverted terminal repeat (ITR) sequences. In some embodiments, the rAAV capsid proteins can be naturally occurring capsids of different AAV serotypes. For example, different AAV serotypes have different tissue tropisms and can be used to target different tissue types and associated diseases. In some embodiments, the rAAV capsid proteins include one or more amino acid substitutions relative to naturally occurring capsid proteins.

[0178] Different manufacturing techniques can be used to produce rAAV particles. Typically, rAAV particles are assembled in a host cell culture (e.g., in a bioreactor or other cell culture vessel). In some embodiments, the cell culture is maintained or grown using standard methods (e.g., in a suspension culture, or on plates). The assembled rAAV is then isolated from the cell culture. In some embodiments, the cell culture comprises mammalian cells, insect cells or cells of another cell type.

[0179] #14470686vl Manufacturing rAAV comprises using a cell culture to produce and assemble the parts of rAAV particles. Without intending to be bound by theory, producing rAAV particles comprises i) expression of Rep and Cap genes, ii) providing a recombinant genome, typically comprising a gene of interest, and iii) providing the additional functions, sometime referred to as helper functions, required for rAAV replication. In some embodiments, the cell culture comprises cells that have genes encoding Rep and Cap integrated into the cells’ genome. In some embodiments, the cell culture comprises cells that have been stably transfected with a plasmid comprising the genes encoding Rep and Cap. In some embodiments, the cells further comprise a plasmid containing an rAAV genome. Without intending to be bound by theory, these cells are typically infected with a helper virus that provides additional factors necessary for rAAV replication. In some embodiments, the helper virus is an adenovirus, such as Ad5. In some embodiments, the cell culture comprises cells that are transiently transfected with plasmids encoding Rep, Cap, an rAAV genome, and helper functions. In some embodiments, two plasmids are used (double transfection). In some embodiments, three plasmids are used (triple transfection). Providing the helper virus, or transfecting with all necessary components, causes the cells to begin producing rAAV.

[0180] Aspects of the disclosure relate, at least in part, to methods and compositions for culturing a cell culture under conditions suitable for rAAV production (“production conditions”). As described herein, production conditions are conditions under which expression of one or more genes required for rAAV production, packaging, and / or growth is promoted (e.g., by addition of a helper virus, induction of an inducible promoter, or transfection with one or more vectors encoding one or more genes required for rAAV production, packaging, and / or growth).

[0181] Rep / Cap protein expression

[0182] In some embodiments, a cell culture is contacted with two or three plasmids for rAAV production. In some embodiments, plasmids encode trans elements. In some embodiments, trans elements encoded by plasmids are AAV rep and cap genes, adenoviral helper genes E2A, E4, and VA. In some embodiments, AAV rep and cap genes are required for vector packaging.

[0183] Producer cells

[0184] In some embodiments, a cell culture associated with the disclosure comprises producer cells. In some embodiments, a producer cell stably expresses rep and cap genes suitable for rAAV packaging. In some embodiments, a producer cell further comprises an rAAV genome. In some embodiments, the rAAV genome comprises a therapeutic gene.

[0185] #14470686vl In some embodiments, the cell culture comprises HeLa cells. In some embodiments, the cell culture comprises human embryonic kidney (HEK) cells. In some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells. In some embodiments, the cell culture comprises Vero cells. In some embodiments, the cell culture comprises Madin-Darby canine kidney (MDCK) cells. In some embodiments, the cell culture comprises baby hamster kidney (BHK) cells. In some embodiments, the cell culture comprises A549 cells. In some embodiments, the cell culture comprises amniocyte cells.

[0186] In some embodiments, the producer cell culture is infected with a helper virus. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5 (such as wtAd5 or a temperature sensitive mutant adenovirus, e.g., Adtsl49, also called Ad5tsl49). In some embodiments, the helper virus is wtAd2 or a mutated Ad2. In some embodiments, the producer cell culture does not require helper virus.

[0187] Transient transfection

[0188] Transient transfection is one way of introducing heterologous genetic material to cells of interest. As used herein, “transfection” means nucleic acid transfection. Briefly, transfection comprises contacting a cell culture with one or more nucleic acids and a transfection reagent, also called a “transfecting agent.” In some embodiments, the transfection reagent is a commercially available transfection reagent. In some embodiments, the transfection reagent is a chemical transfection reagent. In some embodiments, the transfection reagent is a liposomalbased transfection reagent. In some embodiments, the transfection reagent is a liposome. In some embodiments, the liposome is a positively charged or cationic liposome. In some embodiments, the transfection reagent is a non-liposomal-based transfection reagent. In some embodiments the transfection reagent is a calcium phosphate, dendrimer, polymer, nanoparticle, or non-liposomal lipid. In some embodiments, the transfection reagent is lipofectamine or a variant thereof. Transfection reagents include, but are not limited to, polyethylenimine (PEI), FectoVIR® (Polyplus), Nanofectamine (GE Healthcare), Oligofectamine (Invitrogen), RNAiMAX (Invitrogen), siPORT (ThermoFisher), DharmaFECT (Dharmacon), Endofectin@MAX (GeneCopoeia), Escort IV Liposome (Sigma- Aldrich), Arrest-In (Dharmacon), TurboFect (Thermo), Effectene (Qiagen), Attractene (Qiagen), PolyFect (Qiagen), SuperFect (Qiagen), ExpressFect (Thomas), GeneJammer (Stratagene), FuGENE (Promega), INTERFERin (Polyplus), NanoFectin (System Biosciences), X-tremeGENE (Roche), Xfect (ClonTech), Escort IV (Sigma- Aldrich), and N-TER (Sigma- Aldrich).

[0189] #14470686vl Downstream Processing

[0190] In some embodiments, rAAV is isolated from the cell culture and prepared (e.g., purified and / or sterilized) for delivery to a human subject (e.g., to deliver a therapeutic gene to the subject to assist in the treatment of a disease or condition).

[0191] In some embodiments, rAAV isolation comprises a further clarification and / or purification step. In some embodiments, rAAV isolation comprises a flocculation step. In some embodiments, a lysis agent, for example a detergent, can be used along with the flocculation agent.

[0192] In some embodiments, rAAV particles are further purified, for example using one or more affinity, ion exchange chromatography, and / or hydrophobic interaction chromatography steps, e.g., after clarification of an rAAV preparation.

[0193] In some embodiments, rAAV particles (e.g., after one or more purification steps) are added to a pharmaceutically acceptable solution.

[0194] The application also provides compositions comprising rAAV particles, and methods of administering the rAAV particles to a subject (e.g., a human subject having a condition that the therapeutic RNA and / or protein can help treat).

[0195] These and other aspects are illustrated by the following non-limiting examples.

[0196] EXAMPLES

[0197] Example 1. Effect of pCCh on AAV titers and particle quality in Batch process (Ambr®250)

[0198] AAV producer cell line (PCL) recombinant HeLa S3 cells from the pre-master well cell banks were thawed in vials and expanded in a commercially available serum free growth medium supplemented with 6mM L-Glutamine in shake flasks incubated in a humidified incubator at 37°C, 5% CO2, 80% humidity, and 100 RPM. Cells were passaged every 3 to 4 days while increasing the working volume of the culture from 25 mL to 2 L. Cell viability and doubling time were measured at each passage, with pass criteria to stay above 95% and below 30 hr respectively. Once the culture achieved a viable cell density (VCD) greater than 2.5x106cells / mL, they were used for AAV production.

[0199] Cells were inoculated directly into eight Ambr®250 modular bioreactors. The target density of the production culture was 5.0 x 105viable cells / mL. The final production media consisted of 80% fresh proprietary production media and 20% spent growth media. The fresh production media was added to the production vessel under controlled pH and temperature, about 18-24 hrs before inoculation to allow media equilibration. All other production culture

[0200] #14470686vl conditions and physical parameters such as pH setpoint and pH deadband, temperature and agitation were consistent between all eight Ambr®250 modular bioreactors. The cells were then infected by the addition of a helper virus at a multiplicity of infection of 150 DNase-resistant genomes (DRG) per cell and the production was allowed to proceed for three days in agitated bioreactors, while controlled at the PCL platform settings of 37°C, pH 7.0-7.45 and dissolved oxygen (DO) 30-100%. On day one (DI), 12-24 hr post infection (HPI), the process conditions were manipulated to inject different amounts of CO2 in the bioreactor head space and submerged gassing, as shown in FIG. 2, in order to achieve the different targeted amounts of pCO2 in the equilibrated culture. Four different conditions with targeted pCO2, as shown in Table 1, were tested in duplicates (FIG. 2).

[0201] Table 1. Targeted pCO2 values for four different conditions

[0202] The results of pCO2 controlled conditions could impact pH of the infected cell culture, in accordance with the negative correlation of pCCL with pH values, based on Henry’s Law. The variation in the culture pH was minimized by the addition of a base (sodium carbonate) to tightly control the pH within a narrow range of 7.2 ± 0.1 as indicated by the offline BGA pH trends for the various conditions (FIG. 3A).

[0203] The same samples were run on a cell counter (Vi-CELL BLU) and metabolite analyzer (BioProfile® FLEX2) as well. The variances in viable cell density (VCD) under various pCO2 conditions are illustrated in FIG. 3B. The overlapping VCD trends suggest that the cell growth and viability were not impacted by the pCO2 differences across the four conditions. Glucose serves as a primary carbon source for cells in culture to carry out several essential metabolic reactions resulting in cell growth, DNA synthesis and protein production. Evidently, cell culture glucose concentration decreases over time in batch cultures due to the consumption of glucose by the cells (FIG 3C). The relatively faster decrease in glucose concentration for Condition 1 which was maintained in the lowest pCO2 target range, and the slowest decrease for Condition 4 which was maintained at the highest pCO2 target range suggest that the glycolysis and other related metabolism involved in cell growth, protein production including AAV capsids, and

[0204] #14470686vl DNA synthesis including AAV transgene, could be influenced by the pCCh of the cell culture post helper virus infection during rAAV production.

[0205] Infected cells in the Ambr®250 modular bioreactors were shifted to a higher temperature, pH and osmolality at around 72 hours post infection to induce cell death and get AAV particles released into the culture and harvested at around 88 tolOO hours post infection. Samples were collected daily for day 2, 3 and 4 from each vessel to run PCR titer analysis to quantify AAV titer in the cell culture. The results showed that pCCL levels exceeding 70 mmHg led to sub- optimal rAAV titers, making these conditions unsuitable for AAV production (FIGs. 4A, 19A). Notably, under the same conditions Ad5 production is not significantly affected (FIG. 20A). Harvested material from the conditions 1-3 were purified for sedimentation analysis to determine Empty / Partial / Full AAV capsids. Results showed that high pCCh levels may result in more empty and less full capsids, thus affecting the quality of the rAAV product in the cell culture harvest (FIG. 4B, 21A). Rep and Cap protein expression in the whole cell lysate samples of the producer cells collected on day 3 was analyzed by Western Blot. The resulting gel images were analyzed utilizing ‘Image Lab’ software for measuring the intensity of each band (FIG. 4C). FIGs. 4D, 4E and 22A show relative intensity of Rep and Cap protein expression on day 3, suggesting that the Rep and Cap protein expression is significantly reduced at higher pCCL levels in the infected cell cultures, which in turn could be one of the several reasons that can influence rAAV productivity and particle quality.

[0206] Example 2. Effect of DCO2 on AAV titers in perfusion process (Ambr®250)

[0207] To demonstrate that pCCL levels are an important process parameter to monitor in a perfusion based intensified process, HeLa PCL cells were thawed, passaged and expanded grown to a higher cell density of around 8.0 xlO6viable cells / mL in a commercially available growth medium supplemented with 6 mM L-Glutamine and seeded in a proprietary production medium supplemented with 5 mM L-Glutamine at the target VCD of around 2.0 xlO6viable cells / mL. The cells were then infected by the addition of Ad5 helper virus. At 4 HPI, perfusion in the bioreactors was turned on at a rate of 2 vessel volumes per day (VVD) with proprietary production medium supplemented with 5mM L-Glutamine to help provide fresh nutrients and remove excess waste products. The culture conditions were also modified concurrently to achieve different levels of targeted pCO2 levels in the culture as shown in table 2. The data in FIG. 5 displays the actual recorded pCO2 levels in the culture at 24 HPI.

[0208] Table 2. Targeted pCO2 values for three different conditions at 24 HPI

[0209] #14470686vl

[0210] The variances in viable cell density (VCD) over time under various pCCh conditions are illustrated in FIG. 6A. In this example, the process was intensified by around 4-fold by seeding and infecting cells at around 2x10A6 viable cells / mL as compared to the batch process illustrated in Example 1 wherein the seeding density for infection was around 0.5xl0A6 viable cells / mL. The overlapping VCD trends across the three conditions suggest that the cell growth and viability were not impacted by the pCCL differences (FIG. 6A). Glucose concentration in cell culture was also maintained similar across the conditions due to media exchange at 2 VVD in this perfusion process (FIG. 6B).

[0211] Samples were collected on day 3 from each vessel to run PCR titer analysis to quantify AAV titer in the cell culture. The results showed that the conditions with the highest pCCL target range led to sub-optimal rAAV titer, making high pCCL conditions unsuitable for AAV production in perfusion processes (FIG. 7).

[0212] Example 3. Effect of pCCh on AAV titers in perfusion process (5L Bench top)

[0213] The producer cells were grown to a higher cell density of 8.0 xlO6cells / mL in the commercially available growth medium supplemented with 6mM L-Glutamine and seeded in proprietary production medium supplemented with 5mM L-Glutamine at the target VCD of 2.0 xlO6cells / mL. After Ad5 helper virus infection 4 hours post infection, the cells were perfused at a rate of 2VVD with proprietary production medium supplemented with 5mM L-Glutamine to help provide fresh nutrients and remove excess waste products. Unlike Ambr®250 scale, reactors at benchscale (3L-10L) or higher (50L to 2000L scale and above), have considerably lower volumetric mass transfer coefficient, also termed as kLa. Therefore, CO2 stripping is less effective at benchscale and cells tend to accumulate CO2 in culture. Consequently, a combination of air sparge rate and impeller agitation (rpm), governed by its corresponding power density (P / V), are leveraged to reduce excess CO2 through effective stripping. In this example, culture conditions with three different air sparged rates (VVM) were considered that helped achieve different ranges of targeted pCO2 levels in the culture, as shown in the Table 3. FIG. 8 shows the corresponding pCO2 levels achieved as determined by BGA at 24 HPI.

[0214] #14470686vl Table 3. Targeted pCO2values for three different conditions at 24 HPI

[0215] The variances in viable cell density (VCD) over time under various pCO2conditions in perfusion culture in bioreactors at 5L bench scale are illustrated in FIG. 9A. The overlapping VCD trends across the three conditions suggest that the cell growth and viability were not impacted by the pCO2differences (FIG. 6A). Glucose concentration in cell culture was also maintained similar across the conditions due to media exchange at 2 VVD in this perfusion process for up to 48 hours post infection (FIG. 9B).

[0216] The effect of pCO2modulation in cell culture through controlling air sparge rate in benchscale bioreactors in a perfusion process on AAV titer is shown in FIG. 10. The results showed that the condition with the lowest air sparge rate of 0.001 VVM and correspondingly highest pCO2target range led to sub-optimal rAAV titer, making high pCO2conditions unsuitable for AAV production in perfusion processes at bench scale (FIG. 10).

[0217] Example 4. Effect of pCCh on AAV production and particle quality using an alternate producer cell line (PCL) expressing a different transgene

[0218] A study, similar to example 1, was conducted in AMBR®250 bioreactors, which were seeded at around 0.5x106cells / mL using an alternative PCL expressing a different transgene and AAV serotype. Cells were infected with helper Ad5 virus within 2 hours of inoculation in replicate per condition. Table 4 shows four different conditions tested in duplicates accompanied by four different values of pCO2levels that were targeted starting day 1. FIG. 11 shows the actual achieved BGA values of pCO2at 28 HPI.

[0219] Table 4. Targeted pCO2values for four different conditions

[0220] #14470686vl

[0221] The variances in viable cell density (VCD) under various pCCh conditions over time for each condition are illustrated in FIG. 12A. Similar to Example 1, the overlapping VCD trends suggest that the cell growth and viability were not impacted by the pCCL differences across the four conditions. Also, similar to Example 1, cell culture glucose concentration decreases over time in batch cultures due to the consumption of glucose by the cells (FIG 12B). The relatively faster decrease in glucose concentration for Condition 1 which was maintained in the lowest pCO2target range, and the slowest decrease for Condition 4 which was maintained at the highest pCCE target range suggests that pCCL in cell culture impacts glycolysis and related metabolism that drives DNA synthesis and protein including capsid production (FIG. 12B).

[0222] The effect of pCCL on AAV titers was similar to Example 1, wherein the rAAV titers were sub-optimal at pCCL levels exceeding 70 mmHg, demonstrating that such conditions are not desirable for AAV production (FIG 13A, FIG. 19B). Notably, under the same conditions Ad5 production is not significantly affected (FIG. 20B). Harvested material from all four conditions were purified for sedimentation analysis to determine Empty / Partial / Full AAV capsids. Results showed that high pCCL levels may result in more empty and less full capsids, thus affecting the quality of the rAAV product in the cell culture harvest (FIG. 13B, FIG. 21B).

[0223] Rep and Cap protein expression in the whole cell lysate samples of the producer cells collected on day 3 was analyzed by Western Blot. The resulting gel image was analyzed on ‘Image Lab’ software for measuring the intensity of each band (FIG. 14A). FIGs. 14B and 14C show relative intensity of total Rep and Cap protein expression on day 3 for all four conditions, demonstrating that Rep expression stayed relatively unchanged, whereas Cap expression appeared to be dependent on pCCL levels showing decreased expression at high levels of pCCL (FIGs. 14B-14C, 22B).

[0224] Example 5. Modulating pCCh using NaHCCh buffer concentration in the production media

[0225] In this example, the production medium was modified to reduce the CO2buffering capacity by reducing the NaHCCE (sodium bicarbonate) concentration in the medium to 0.5X using PCL studied in Example 4. This modification allows to probe the effect of lower end of the pCCh (<20 mmHg) during AAV production in a PCL based process. A study was conducted in AMBR®250 bioreactors, which were seeded at around 0.5xl06cells / mL under control and

[0226] #14470686vl modified media condition in replicates. Cells were infected with helper Ad5 virus within 2 hours of inoculation in replicate per condition.

[0227] With reduced NaHCCh, the equilibrium pCCb in the culture in AMBR250 vessels were significantly reduced and average was below 20 mmHg which we consider sub-optimal for AAV production (FIG. 15A). Similar to other cases, the effect of sub-optimal pCCh is reflected on AAV titers wherein a significant reduction in AAV titer is observed for the low NaHCCh condition (FIG. 15B). The example demonstrates that media components that modulate the CO2 buffering capacity in cell culture can be used to attain the desired pCCb range (20 mmHg - 70 mmHg) for improved AAV productivity. The amount of NaHCCh or similar buffers in the medium can be varied with infection density to target the defined pCCb range.

[0228] Example 6. Investigation of Factors Contributing to Low-Efficiencv Scale-Un of AAV

[0229] It has been noted that, as AAV production is scaled up, AAV titer decreases (FIG. 16).

[0230] To assess whether this is due to culture- specific variations or condition-specific variations, AAV production was performed at 5L scale and samples (infected cell culture) were removed 2 hours and 24 hours post-infection and moved to satellite AMBR250 bioreactors. As shown in FIG. 17, AAV titer produced in the satellite bioreactor containing cells removed from 5L culture at 2 hours is similar to the AMBR250 control. However, titer produced in the satellite bioreactor comprising cells removed at 24 hours shows markedly lower titers, approaching benchtop AAV titer levels. This suggests that the longer the culture spends in the 5L bioreactor, the lower the AAV titer (per unit volume) will be. This suggests that it is not an issue with the base culture or the infection process itself, but rather it is an issue with the conditions as AAV production continues. pCO2 is one of the variables that can affect AAV production, as shown herein.

[0231] A big-data analysis was performed to identify factors likely to influence AAV titer at large scale. From most correlated to least correlated, pCO2 on day 2, pO2 on day 2, pCO2 on day 1, pH on day 1, and cell diameter on day 2 were the five factors most correlated with higher titers at large scale. Further analysis suggests that pCO2 is likely a key factor.

[0232] As shown in FIG. 18, based on big data model simulation, as the perturbation strength of the air overlay was increased, relative AAV titer increased, suggesting that pCO2 or pO2 might be affecting scale up.

[0233] Process changes including pC02-controlling measures was tested at bench scale (5L), 50L, and 200L and compared to an AMBR250 control, as well as an old process control run at benchscale and 50L. As shown in FIG. 24A, AAV titer is higher when using the new process

[0234] #14470686vl compared to the old process (at 5 and 50L). AAV titer is maintained even at 200L, showing that the new process is capable of scaling well.

[0235] As shown in FIG. 24B, the ratio of full to empty capsids using the new process at 50L is comparable to an AMBR250 control, whereas it is much lower in a 50L culture using the old method (not having CO2 controls).

[0236] EQUIVALENTS

[0237] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0238] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0239] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0240] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0241] #14470686vl The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0242] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0243] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements

[0244] #14470686vl other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0245] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0246] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0247] #14470686vl

Claims

CLAIMSWhat is claimed is:

1. A method comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) under a partial pressure of carbon dioxide (pCCh) from about 20 to about 70 mmHg.

2. The method of claim 1, wherein the cell culture is incubated under a pCCL from about 20 to about 70 mmHg for about 12 hours to about 48 hours.

3. The method of claim 1, wherein the cell culture is incubated under a pCCL from about 20 to about 70 mmHg for about 72 hours.

4. The method of any one of claims 1-3, wherein the pH of the cell culture is between 7.0 and 7.5.

5. The method of any one of claims 1-4, wherein the cell culture is incubated with an air sparge rate of 0.005VVM and then incubated with an air sparge rate of 0.015 to 0.025 VVM for at least 12 hours.

6. The method of any one of claims 1-5, wherein the mammalian cells comprise rAAV producer cells, wherein the rAAV producer cells are stably integrated with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid encoding a gene of interest.

7. The method of any one of claims 1-6, wherein the cell culture is infected with a helper virus, and incubated at a pCCL from about 20 to about 70 mmHg for about 12 hours to about 48 hours post infection.#14470686vl8. The method of claim 7, wherein the cell culture is incubated with an air sparge rate of 0.005VVM before infection and then incubated with an air sparge rate of 0.015 to 0.025 VVM for at least 12 hours post- infection.

9. The method of claim 7 or claim 8, wherein the helper virus is an adenovirus comprising AAV helper genes, optionally the AAV helper genes comprise at least one of adenovirus genes E1A, E1B, E2A, L4, L4-33K, VA, and E4orf6.

10. The method of claim 9, wherein the adenovirus is a wtAd5 or wtAd2 adenovirus or a mutated Ad5 or mutated Ad2 adenovirus.

11. The method of claim 9 or claim 10, wherein the adenovirus is a temperature sensitive mutant of a wtAd5.

12. The method of claim 11, wherein the adenovirus is Ad5tsl49.

13. The method of any one of claims 1-12, wherein the mammalian cells comprise at least one of: CHO cells, Vero cells, HeLa cells, MDCK cells, BHK cells, A549 cells, amniocyte cells, HEK293 cells, or a cell derived from a parental mammalian cell line.

14. The method of claim 13, wherein the mammalian cells comprise HeLa cells.

15. The method of claim 14, wherein the HeLa cells are HeLaS3 cells.

16. The method of any one of claims 1-5, wherein the mammalian cells are transiently transfected with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid comprising a gene of interest.

17. The method of claim 16, wherein the mammalian cells comprise HEK293 cells.#14470686vl18. The method of any one of claims 6-17, wherein the gene of interest encodes an antibody, an enzyme, a growth factor, a microRNA, or a hormone.

19. The method of any one of claims 6-18, wherein the AAV ITRs comprise AAV2 ITRs or AAV9 ITRs.

20. The method of any one of claims 1-19, wherein the rAAV comprises a capsid protein comprising at least one of: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein or variants or chimeras or hybrids thereof.

21. The method of any one of claims 1-20, wherein the pCCb level is maintained by modulating levels of CO2 in a bioreactor comprising the cell culture by:1) modulating the air sparge rate of the submerged gassing of the bioreactor VVM;2) modulating agitation (rpm) of the bioreactor impeller and corresponding density (P / V);3) mixing CO2 with air sparge with a fixed composition;4) mixing CO2 with air in the overlay with a fixed composition;5) providing a cell culture medium comprising a component that limits CO2 solubility (e.g., calcium chloride); and / or6) reducing the amount of sodium bicarbonate in the cell culture medium, optionally reducing the amount of sodium bicarbonate in the cell culture medium to about 0.5X the concentration of sodium bicarbonate in a control medium.

22. The method of claim 21, wherein CO2 is injected in a headspace of the bioreactor.

23. The method of any one of claims 4-19, wherein the cell culture is maintained at a temperature of about 37 °C before infection with the helper virus and on day 3 post infection with the helper virus the temperature of the cell culture is raised to about 39 °C.#14470686vl24. The method of any one of claims 6-15 and 18-23, wherein the cell culture is maintained at a pH of about 7.25 ± 0.25 before infection with the helper virus and on day 3 post infection with the helper virus the pH of the cell culture is raised to about 8.15 ± 0.15.

25. The method of any one of claims 6-15, wherein on day 3 post infection with the helper virus the osmolality of the cell culture is maintained at a level greater than or equal to 420 mOsm / kg, optionally at a level of about 440 mOsm / kg.

26. The method of any one of claims 23-25, wherein the cell culture is terminated after about 16 hours of increasing the temperature, pH, and osmolality.

27. The method of any one of claims 1-26, wherein the cell culture comprises a perfusion culture.

28. The method of any one of claims 1-26, wherein the cell culture comprises a batch culture.

29. The method of any one of claims 1-28, wherein the method further comprises monitoring the cell culture for one or more parameters selected from the group consisting of: total cell density, viable cell density, cell viability, average cell diameter, and glucose consumption.

30. The method of claim 29, wherein the viable cell density of the cell culture is from about 0.25 vc / mL to about 2E7 vc / mL at 48 hours post infection.

31. The method of any one of claims 1-30, wherein the cell culture comprises a volume of at least about 50 L.#14470686vl32. The method of any one of claims 1-31, wherein the method further comprises isolating recombinant adeno-associated virus from the cell culture.

33. The method of claim 32, wherein the titer of rAAV isolated from the cell culture comprises at least about 1E10 to about 5E12 vg / mL.

34. The method of claim 33, wherein the AAV titer is measured using a PCR method.

35. The method of any one of claims 32-34, wherein the rAAV isolated from the cell culture comprises at least about 30% full capsids.

36. The method of claim 35, wherein the percent full capsids is measured using analytical ultracentrifugation technique.

37. A method comprising incubating a cell culture comprising mammalian cells for producing a recombinant adeno-associated virus (rAAV) in a cell culture medium comprising a reduced level of sodium bicarbonate relative to a control medium.

38. The method of claim 37, wherein the level of sodium bicarbonate is reduced by at least 0.5X relative to a control medium.#14470686vl

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