Systems and methods for gene therapy vector production

Abstract

Provided herein are systems and methods for the production of gene therapy products. The system may comprise a reactor vessel housing a fixed-bed substrate for cell growth, a recirculation mixing vessel connected to the reactor vessel via a closed-loop recirculation path, and a pump regulating the recirculation rate between the two vessels. The methods and systems provided herein provide a controlled environment for efficient production of gene therapy products, enhancing the scalability and reproducibility of the manufacturing process.

Description

WSGR Docket No. 69389-701.601SYSTEMS AND METHODS FOR GENE THERAPY VECTOR PRODUCTIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application Number 63 / 721,308 filed on November 15, 2024, and U.S. Provisional Patent Application Number 63 / 804,431 filed on May 12, 2025, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Gene therapy has emerged as a promising approach for treating a wide range of genetic disorders and diseases. This therapeutic strategy involves delivering genetic material, such as DNA or RNA, into a patient's cells to correct or modify gene expression. A critical component of gene therapy is the use of viral vectors, which serve as delivery vehicles for the therapeutic genetic material.

[0003] The production of viral vectors for gene therapy applications presents several challenges. Traditional methods often rely on adherent cell culture systems, such as roller bottles or cell stacks, which are labor-intensive, space-consuming, and difficult to scale up. These systems also suffer from limitations in terms of process control and reproducibility.

[0004] Suspension culture systems have been explored as an alternative for large-scale production of viral vectors. However, many cell lines used for gene therapy vector production do not adapt well to suspension culture conditions. Issues such as cell clumping, inefficient transfection, and increased cellular debris can complicate downstream purification processes.

[0005] Bioreactor systems offer potential advantages for viral vector production, including improved process control, scalability, and reduced labor requirements. However, existing bioreactor designs do not fully address the specific needs of gene therapy vector production. Challenges remain in achieving high cell densities, maintaining optimal culture conditions, scale up of vector production and ensuring efficient transfection and vector production.SUMMARY

[0006] There is an unmet need for improved bioreactor systems and methods that can overcome the limitations of current production approaches. Such systems should ideally support high-density cell growth, provide precise control over culture conditions, facilitate efficient transfection, and simplify downstream purification. Additionally, these systems should be scalable and adaptable to meet the increasing demand for gene therapy products as the field continues to advance. This disclosure meets this unmet need.WSGR Docket No. 69389-701.601

[0007] Described herein, in some aspects, is a method of transfecting cells in a fixed-bed bioreactor, the method comprising: providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate; and contacting the plurality of cells with a cell culture media comprising a nucleic acid payload and a transfection reagent for at least 7 hours, such that the plurality of cells are transfected with the nucleic acid payload. In some embodiments, the fixed-bed bioreactor comprises a volume of cell culture media, and the volume of cell culture media is replaced at least about 0.5 times per hour during the contacting. In some embodiments, the volume of cell culture media is replaced from about 0.5 to about 5 times per hour duringthe contacting. In some embodiments, the method further comprises adjusting the pH of the cell culture media to maintain the pH of the cell culture media within about 5% of a target pH during the contacting. In some embodiments, the adjusting comprises monitoring the pH of the cell culture media, and adjusting a perfusion rate to alter the pH of the cell culture media. In some embodiments, the adjusting comprises monitoring or detecting a level of at least one cell waste product in the cell culture media, and adjusting a perfusion rate to alter the amount of the at least one cell waste product in the cell culture media. In some embodiments, the at least one cell waste product is or comprises lactate. In some embodiments, the adjusting comprises monitoring or detecting a level of glucose in the cell culture media, and adjusting a perfusion rate to alter the amount of glucose in the cell culture media. In some embodiments, the method further comprises adjusting a perfusion rate to maintain a concentration of the transfection reagent within about 25% of a target transfection concentration. In some embodiments, the fixed-bed bioreactor is coupled to a recirculation system. In some embodiments, the fixed-bed bioreactor is coupled to the recirculation system via a closed loop. In some embodiments, the recirculation system comprises at least one pump for adjusting a perfusion rate. In some embodiments, the transfection reagent comprises calcium phosphate. In some embodiments, the fixed-bed bioreactor comprises at least one sensor. In some embodiments, the at least one sensor comprises a pH sensor. In some embodiments, the at least one sensor comprises a glucose sensor. In some embodiments, the at least one sensor comprises a lactate sensor. In some embodiments, the at least one sensor comprises a temperature sensor. In some embodiments, the at least one sensor comprises a dissolved oxygen sensor. In some embodiments, the at least one sensor comprises a pressure sensor. In some embodiments, the nucleic acid payload comprises a viral plasmid. In some embodiments, the plurality of cells comprises HEK293 cells. In some embodiments, the plurality of cells comprises Vero cells. In some embodiments, the method further comprises harvesting a plurality of viral particles from theWSGR Docket No. 69389-701.601plurality of cells. In some embodiments, the viral particles are or comprise adeno-associated viral (AAV) particles or lentiviral particles. In some embodiments, the viral particles express an antigen. In some embodiments, the viral particles comprise AAV particles and the method results in at least 2E13 vg / L AAV particles. In some embodiments, the method further comprises replacing the cell culture media after the contacting with fresh cell culture media for a period of time. In some embodiments, the harvesting occurs at least 24 hours, 48 hours, or 72 hours after the contacting. In some embodiments, the method further comprises removing empty capsids prior to the harvesting. In some embodiments, the contacting comprises continuously contacting the plurality of cells with the cell culture media during the contacting.

[0008] Described herein, in some aspects, is a method of culturing cells in a fixed-bed bioreactor, the method comprising: providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate and a volume of cell culture media in contact with the plurality of cells; and replacing the volume of cell culture media at least about 0.5 times per hour. In some embodiments, the volume of cell culture media is completely replaced from about 0.5 times to about 4 times per hour. In some embodiments, the method further comprises monitoring the pH of the cell culture media. In some embodiments, the method further comprises adjusting a perfusion rate of the cell culture media to maintain a pH within about 5% of a target pH. In some embodiments, the method further comprises monitoring or detecting one or more cell waste products in the cell culture media. In some embodiments, the method further comprises adjusting a perfusion rate to control a level of the one or more cell waste products in the cell culture media. In some embodiments, the cell waste product is or comprises lactate, glucose, or both.

[0009] Described herein, in some aspects, is a method of producing a biologic, the method comprising: providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate; continuously contacting, for a first time period, the plurality of cells with a first volume of cell culture media comprising a transfection reagent and a plasmid, such that at least one cell is transfected with the plasmid and produces a biologic; replacing the first volume of cell culture media with a second volume of cell culture media that does not contain the transfection reagent and the plasmid by continuously contacting, for a second time period, the plurality of cells with the second volume of cell culture media; replacing the second volume of cell culture media with a third volume of cell culture media by continuously contacting, for a third time period, the plurality of cells with the third volume of cell culture media; and collecting the biologic during a fourth time period. In someWSGR Docket No. 69389-701.601embodiments, the biologic is or comprises a plurality of viral particles. In some embodiments, the plasmid is or comprises at least one viral plasmid. In some embodiments, the plurality of viral particles comprise adeno-associated viral (AAV) particles. In some embodiments, the plurality of viral particles expresses an antigen. In some embodiments, the plasmid is selected from the group consisting of: a helper plasmid, a plasmid containing Rep and / or Cap genes, a transgene plasmid, and any combination there of. In some embodiments, the first time period is at least 7 hours. In some embodiments, the second time period is at least 12 hours. In some embodiments, the third time period is from about 24 hours to about 48 hours after the continuously contacting. In some embodiments, the fourth time period is from about 48 hours to about 72 hours after the continuously contacting. In some embodiments, the method further comprises contacting the plurality of cells with a lysis buffer to lyse the plurality of cells and release the AAV particles into the lysis buffer. In some embodiments, the method further comprises collecting the lysis buffer containing the AAV particles. In some embodiments, the method comprises discarding the third volume of cell culture media. In some embodiments, at least about 70% of the AAV particles in the third volume of cell culture media are empty AAV particles. In some embodiments, at least about 35% of the AAV particles collected in (e) are full AAV particles. In some embodiments, the plurality of viral particles are or comprises lentiviral particles, retroviral particles, or adenoviral particles. In some embodiments, the biologic is or comprises a protein or polypeptide. In some embodiments, the biologic is or comprises an antibody or fragment thereof. In some embodiments, the biologic is or comprises a vaccine. In some embodiments, the biologic is or comprises an antigen. In some embodiments, any one or any combination of the previous steps comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour. In some embodiments, any one or any combination of the previous steps comprises completely replacing the full bioreactor volume of cell culture media at a rate of from about 0.5 times to about 4 times per hour. In some embodiments, the method further comprises replacing the third volume of cell culture media with a fourth volume of cell culture media by continuously contacting, for a fifth time period, the plurality of cells with the fourth volume of cell culture media. In some embodiments, the fifth time period is from about 72 hours to about 96 hours after the continuously contacting. In some embodiments, the method further comprises, prior to (e) and after (f), (g) replacing the fourth volume of cell culture media with a fifth volume of cell culture media by continuously contacting, for a sixth time period, the plurality of cells with the fifth volume of cell culture media. In some embodiments, the sixth time period is greater than about 96 hoursWSGR Docket No. 69389-701.601after the continuously contacting. In some embodiments, (f), (g), or both comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour. In some embodiments, the collecting comprises continuously perfusing the bioreactor with a volume of cell culture media or lysis buffer during the course of the fourth time period. In some embodiments, the fixed-bed bioreactor is coupled to a recirculation system. In some embodiments, the recirculation system is coupled to a plurality of fixed-bed bioreactors.

[0010] Described herein, in some aspects, is a composition comprising at least 2E13 vg / L viral particles, and no amount or an undetectable amount of a lipid -based or a polymer-based transfection reagent. In some embodiments, the undetectable amount is an amount of the lipid-based or polymer-based transfection reagent that is undetectable by high-performance liquid chromatography (HPLC). In some embodiments, the polymer-based transfection reagent is a polyethylenimine (PEI) transfection reagent. In some embodiments, the composition further comprises an amount of calcium detectable by HPLC. In some embodiments, the viral particles comprise AAV particles.

[0011] Described herein, in some aspects, is a bioreactor system for producing gene therapy products, comprising: a reactor vessel containing a fixed -bed substrate for cell growth; a recirculation mixing vessel fluidically connected to the reactor vessel; a recirculation path forming a closed loop between the reactor vessel and the recirculation mixing vessel; a pump configured to control a recirculation rate between the recirculation mixing vessel and the reactor vessel; and a gas supply connected to the system to provide gases for cell growth and metabolism. In some embodiments, the fixed-bed substrate comprises a plurality of layers of material. In some embodiments, the plurality of layers of material comprise at least one cell immobilization layer and at least one flow distribution layer. In some embodiments, the at least one cell immobilization layer comprises a fibrous structure for cell attachment. In some embodiments, the pump is configured to control the recirculation rate to achieve an exchange rate of between 0.5 and 5 reactor volumes per hour. In some embodiments, the bioreactor further comprises a pH control system configured to maintain pH within a predetermined range during cell growth, transfection, and production. In some embodiments, the pH control system comprises pH sensor, wherein in response to detecting an out-of-range pH, the pH control system is configured to release a solution into the recirculation mixing vessel. In some embodiments, the pH control system comprises a base pump connected to the recirculation path for adjusting pH of media circulating through the system.WSGR Docket No. 69389-701.601

[0012] Described herein, in some aspects, is a method for producing a biologic in a bioreactor system, the method comprising: seeding cells on a fixed -bed substrate in a reactor vessel; circulating media between the reactor vessel and a recirculation mixing vessel through a closed loop recirculation path or a uni -directional perfusion; adjusting pH in the reactor vessel using the recirculation path; introducing a transfection mixture into the system; incubating the cells with the transfection mixture; and harvesting the biologic from the system. In some embodiments, seeding the cells comprises seeding at a density of about lxl02-5xl06cells / cm2on the fixed-bed substrate. In some embodiments, seeding the cells comprises seeding at the density of about 1X103-5X105cells / cm2on the fixed-bed substrate. In some embodiments, seedingthe cells comprises seeding at the density atabout IxlO3, 5xl03, 7.5xl03, or lx 104cells / cm2on the fixed-bed substrate. In some embodiments, adjusting the pH comprises maintaining a pHbetween 7.0 and 7.8 during cell growth and transfection. In some embodiments, adjusting the pH comprises maintaining a pH of approximately 7.6 during a pre-transfection phase and a pH of approximately 7.4 during a transfection phase. In some embodiments, circulating the media comprises controlling a recirculation rate, via the pump, to achieve an exchange rate of between 0.5 and 4 reactor volumes per hour. In some embodiments, the transfection mixture comprises 8 - 20% of an overall working volume of the bioreactor system. In some embodiments, harvesting the biologic comprises: obtaining a supernatant from the reactor vessel; performing at least one rinse or wash of the fixed -bed substrate; and lysing cells remaining on the fixed-bed substrate. In some embodiments, the transfection mix containing media is exchanged with serum-free media between 12 - 36 hours after transfection, and the serum-free media is recirculated through the recirculation path utilizing an exchange rate of 0.5 and 5 reactor vessel volumes per hour via pump. In some embodiments, the recirculation mixing vessel monitors and controls temperature, pH, glucose, and lactate. In some embodiments, the method further comprises modulating recirculation speed using the recirculation mixing vessel to control at least one of pH, temperature, glucose, and lactate in the reactor vessel. In some embodiments, the biologic comprises a gene therapy product. In some embodiments, the biologic comprises a virus. In some embodiments, the virus expresses antigen. In some embodiments, the biologic comprises a vaccine. In some embodiments, the cells grow from the density of about IxlO3-5xl05cells / cm2to a second density of about IxlOMxlO6cells / cm2. In some embodiments, the cells grow to the second density of about l.lxlO5, 1.2xl05, 1.3xl05, 1.4xl05, or 1.2xl05cells / cm2. In some embodiments, the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density. In someWSGR Docket No. 69389-701.601embodiments, the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density. In some embodiments, the cells grow to the second density at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding. In some embodiments, the cells do not exhibit decreased cell growth at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding. In some embodiments, the cells do not exhibit increased cell death atabout24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding.

[0013] Described herein, in some aspects, is a method of monitoring cells cultured on the fixed-bed substrate of any one of the preceding, comprising monitoring the cells for a plurality of days. In some embodiments, the cells are transfected before or after of said monitoring. In some embodiments, the cells are cultured on the fixed-bed substrate for at least one, two, three, four, five, six, seven, eight, nine, 10, 11, or 12 days. In some embodiments, the cells are monitored without lysis. In some embodiments, the monitoring results in decreased toxicity on the cells. In some embodiments, the method allows the cells to generate progeny cells. In some embodiments, the cells and the progeny cells produce a biologic. In some embodiments, the cells or the progeny cells can be seeded on a second fixed-bed substrate. In some embodiments, the cells or the progeny ells produce the biologic when seeded on the second fixed-bed progeny. In some embodiments, the monitoring comprises monitoring the cells or the biologic. In some embodiments, the monitoring comprises monitoring a viability of the cells or the progeny cells. In some embodiments, the monitoring comprising monitoring a quality of the biologic produced by the cells or the progeny cells. In some embodiments, the biologic comprises antibody, antigen, viral particle, viral capsid, vector, or a combination thereof. In some embodiments, the monitoring results in increased quality of the biologic produced by the cells or the progeny cells. In some embodiments, the cells or the progeny cells can be transfected or re -transfected to produce the biologic. In some embodiments, the cells or the progeny cells can be reseeded or transfected more than one time.

[0014] In one aspect, provided herein is a method of transfecting cells in a fixed-bed bioreactor, the method comprising: (a) providing or obtaining a fixed -bed bioreactor comprising a plurality of cells attached to a substrate; and (b) contacting the plurality of cells with a cell culture media comprising a nucleic acid payload and a transfection reagent for at least 7 hours, such that the plurality of cells are transfected with the nucleic acid payload. In some cases, the fixed-bed bioreactor comprises a volume of cell culture media, and theWSGR Docket No. 69389-701.601volume of cell culture media is replaced at least about 0.5 times per hour during the contacting. In some cases, the volume of cell culture media is replaced from about 0.5 to about 5 times per hour during the contacting. In some cases, the method further comprises adjusting the pH of the cell culture media to maintain the pH of the cell culture media within about 5% of a target pH during the contacting. In some cases, the adjusting comprises monitoring the pH of the cell culture media, and adjusting a perfusion rate to alter the pH of the cell culture media. In some cases, the adjusting comprises monitoring or detecting a level of at least one cell waste product in the cell culture media, and adjusting a perfusion rate to alter the amount of the at least one cell waste product in the cell culture media. In some cases, the at least one cell waste product is lactate. In some cases, the adjusting comprises monitoring or detecting a level of glucose in the cell culture media, and adjusting a perfusion rate to alter the amount of glucose in the cell culture media. In some cases, the method further comprises adjusting a perfusion rate to maintain a concentration of the transfection reagent within about 25% of a target transfection concentration. In some cases, the fixed-bed bioreactor is coupled to a recirculation system. In some cases, the fixed-bed bioreactor is coupled to the recirculation system via a closed loop. In some cases, the recirculation system comprises at least one pump for adjusting a perfusion rate. In some cases, the transfection reagent comprises calcium phosphate. In some cases, the fixed-bed bioreactor comprises at least one sensor. In some cases, the at least one sensor comprises a pH sensor. In some cases, the at least one sensor comprises a glucose sensor. In some cases, the at least one sensor comprises a lactate sensor. In some cases, the at least one sensor comprises a temperature sensor. In some cases, the at least one sensor comprises a dissolved oxygen sensor. In some cases, the at least one sensor comprises a pressure sensor. In some cases, the nucleic acid payload comprises a viral plasmid. In some cases, the plurality of cells comprise HEK293 cells. In some cases, the method further comprises harvesting a plurality of viral particles from the plurality of cells. In some cases, the viral particles are adeno-associated viral (AAV) particles or lentiviral particles. In some cases, the viral particles are AAV particles and the method results in at least 2E13 vg / L AAV particles. In some cases, the method further comprises replacing the cell culture media after the contacting with fresh cell culture media for a period of time. In some cases, the harvesting occurs at least 24 hours, 48 hours, or 72 hours after the contacting of (b). In some cases, the method further comprises removing empty capsids prior to the harvesting. In some cases, the contacting of (b) comprises continuously contacting the plurality of cells with the cell culture media during the contacting.WSGR Docket No. 69389-701.601

[0015] In another aspect, a method of culturing cells in a fixed-bed bioreactor is provided, the method comprising: (a) providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate and a volume of cell culture media in contact with the plurality of cells; and (b) replacing the volume of cell culture media at least about 0.5 times per hour. In some cases, the volume of cell culture media is completely replaced from about 0.5 times to about 4 times per hour. In some cases, the method further comprises monitoring the pH of the cell culture media. In some cases, the method further comprises adjusting the perfusion rate of the cell culture media to maintain a pH within about 5% of a target pH. In some cases, the method further comprises monitoring or detecting one or more cell waste products in the cell culture media. In some cases, the method further comprises adjusting the perfusion rate to control a level of the one or more cell waste products in the cell culture media. In some cases, the cell waste product is lactate, glucose, or both.

[0016] In another aspect, a method of producing a biologic is provided, the method comprising: (a) providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate; (b) continuously contacting, for a first time period, the plurality of cells with a first volume of cell culture media comprising a transfection reagent and a plasmid, such that at least one cell is transfected with the plasmid and produces the biologic ; (c) replacing the first volume of cell culture media with a second volume of cell culture media that does not contain the transfection reagent and the plasmid by continuously contacting, for a second time period, the plurality of cells with the second volume of cell culture media; (d) replacing the second volume of cell culture media with a third volume of cell culture media by continuously contacting, for a third time period, the plurality of cells with the third volume of cell culture media; and (e) collecting the biologic during a fourth time period. In some cases, the biologic is a plurality of viral particles. In some cases, the plasmid is at least one viral plasmid. In some cases, the plurality of viral particles comprise adeno-associated viral (AAV) particles. In some cases, the plasmid is selected from the group consisting of: a helper plasmid, a plasmid containing Rep and / or Cap genes, a transfer plasmid, and any combination thereof. In some cases, the first time period is at least 7 hours. In some cases, the second time period is at least 12 hours. In some cases, the third time period is from about 24 hours to about 48 hours after (b). In some cases, the fourth time period is from about 48 hours to about 72 hours after (b). In some cases, the method further comprises contacting the plurality of cells with a lysis buffer to lyse the plurality of cells and release the AAV particles into the lysis buffer. In some cases, the method further comprises collecting the lysis buffer containing the AAV particles. In some cases, the method furtherWSGR Docket No. 69389-701.601comprises discarding the third volume of cell culture media. In some cases, at least about 70% of the AAV particles in the third volume of cell culture media are empty AAV particles. In some cases, at least about 35% ofthe AAV particles collected in (e) are full AAV particles. In some cases, the plurality of viral particles are lentiviral particles, retroviral particles, or adenoviral particles. In some cases, the biologic is a protein or polypeptide. In some cases, the biologic is an antibody or fragment thereof In some cases, any one or any combination of (b), (c), (d), or (e) comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour. In some cases, any one or any combination of (b), (c), (d), or (e) comprises completely replacing the full bioreactor volume of cell culture media at a rate of from about 0.5 times to about 4 times per hour. In some cases, the method further comprises prior to (e), (f) replacing the third volume of cell culture media with a fourth volume of cell culture media by continuously contacting, for a fifth time period, the plurality of cells with the fourth volume of cell culture media. In some cases, the fifth time period is from about 72 hours to about 96 hours after (b). In some cases, the method further comprises, prior to (e) and after (f), (g) replacing the fourth volume of cell culture media with a fifth volume of cell culture media by continuously contacting, for a sixth time period, the plurality of cells with the fifth volume of cell culture media. In some cases, the sixth time period is greater than about 96 hours after (b). In some cases, (f), (g), or both comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour. In some cases, the collecting of (e) comprises continuously perfusing the bioreactor with a volume of cell culture media or lysis buffer during the course of the fourth time period. In some cases, the fixed-bed bioreactor is coupled to a recirculation system. In some cases, the recirculation system is coupled to a plurality of fixed-bed bioreactors.

[0017] In yet another aspect, a composition is provided comprising at least 2E13 vg / L viral particles, and no amount or an undetectable amount of a lipid-based or a polymer-based transfection reagent. In some cases, the undetectable amount is an amount of the lipid-based or polymer-based transfection reagent that is undetectable by high-performance liquid chromatography (HPLC). In some cases, the polymer-based transfection reagent is a polyethylenimine (PEI) transfection reagent. In some cases, the composition further comprises an amount of calcium detectable by HPLC. In some cases, the viral particles are AAV particles.

[0018] In yet another aspect, a bioreactor system for producing gene therapy products is provided, comprising:(a) a reactor vessel containing a fixed-bed substrate for cell growth; (b)WSGR Docket No. 69389-701.601a recirculation mixing vessel fluidically connected to the reactor vessel; (c) a recirculation path forming a closed loop between the reactor vessel and the recirculation mixing vessel; (d) a pump configured to control a recirculation rate between the recirculation mixing vessel and the reactor vessel; and (e) a gas supply connected to the system to provide gases for cell growth and metabolism. In some cases, the fixed-bed substrate comprises a plurality of layers of material. In some cases, the plurality of layers of material comprise at least one cell immobilization layer and at least one flow distribution layer. In some cases, the at least one cell immobilization layer comprises a fibrous structure for cell attachment. In some cases, the pump is configured to control the recirculation rate to achieve an exchange rate of between 0.5 and 5 reactor volumes per hour. In some cases, the bioreactor system further comprises a pH control system configured to maintain pH within a predetermined range during cell growth, transfection, and production. In some cases, the pH control system comprises pH sensor, wherein in response to detecting an out-of-range pH, the pH control system is configured to release a solution into the recirculation mixing vessel. In some cases, the pH control system comprises a base pump connected to the recirculation path for adjusting pH of media circulating through the system.

[0019] In yet another aspect, a method for producing gene therapy products in a bioreactor system is provided, the method comprising: (a) seeding cells on a fixed-bed substrate in a reactor vessel; (b) circulating media between the reactor vessel and a recirculation mixing vessel through a closed loop recirculation path or uni-directional perfusion; (c) adjusting pH in the reactor vessel using the recirculation path; (d) introducing a transfection mixture into the system; (e) incubating the cells with the transfection mixture; and (f) harvesting the gene therapy products from the system. In some cases, seeding the cells comprises seeding at a density between IxlO2and 5xl06cells / cm2on the fixed-bed substrate. In some cases, adjusting the pH comprises maintaining a pH between 7.0 and 7.8 during cell growth and transfection. In some cases, adjusting the pH comprises maintaining a pH of approximately 7.6 during a pre -transfection phase and a pH of approximately 7.4 during a transfection phase. In some cases, circulating the media comprises controlling a recirculation rate, via the pump, to achieve an exchange rate of between 0.5 and 4 reactor volumes per hour. In some cases, the transfection mixture comprises 8 - 20% of an overall working volume of the bioreactor system. In some cases, harvesting the gene therapy products comprises: obtaining a supernatant from the reactor vessel; performing at least one rinse or wash of the fixed-bed substrate; and lysing cells remaining on the fixed-bed substrate. In some cases, the transfection mix containing media is exchanged with serum-free media between 12 - 36WSGR Docket No. 69389-701.601hours after transfection, and the serum-free media is recirculated through the recirculation path utilizing an exchange rate of 0.5 and 5 reactor vessel volumes per hour via pump. In some cases, the recirculation mixing vessel monitors and controls temperature, pH, glucose, and lactate. In some cases, the method further comprises modulating recirculation speed using the recirculation mixing vessel to control at least one of pH, temperature, glucose, and lactate in the reactor vessel.

[0020] Described herein, in some aspects, is a method of monitoring cells cultured on the fixed-bed substrate, comprising monitoring the cells for a plurality of days. In some embodiments, the cells are transfected before or after of said monitoring. In some embodiments, the cells are cultured on the fixed-bed substrate for at least one, two, three, four, five, six, seven, eight, nine, 10, 11, or 12 days. In some embodiments, the cells are monitored without lysis. In some embodiments, the monitoring results in decreased toxicity on the cells. In some embodiments, the method allows the cells to generate progeny cells. In some embodiments, the cells and the progeny cells produce a biologic. In some embodiments, the cells or the progeny cells can be seeded on a second fixed -bed substrate. In some embodiments, the cells or the progeny ells produce the biologic when seeded on the second fixed-bed progeny. In some embodiments, the monitoring comprises monitoring the cells or the biologic. In some embodiments, the monitoring comprises monitoring a viability of the cells or the progeny cells. In some embodiments, the monitoring comprising monitoring a quality of the biologic produced by the cells or the progeny cells. In some embodiments, the biologic comprises antibody, viral particle, viral capsid, or vector. In some embodiments, the monitoring results in increased quality of the biologic produced by the cells or the progeny cells. In some embodiments, the cells or the progeny cells can be transfected or re-transfected to produce the biologic. In some embodiments, the cells or the progeny cells can be reseeded or transfected more than one time.INCORPORATION BY REFERENCE

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure willWSGR Docket No. 69389-701.601be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0023] FIG. 1 illustrates a schematic view of a bioreactor system for producing gene therapy products, according to aspects of the present disclosure.

[0024] FIG.2 illustrates a perspective view of an enhanced bioreactor system, according to an embodiment.

[0025] FIG.3 illustrates a microscopic view of a cellular structure within a fixed -bed reactor vessel, according to aspects of the present disclosure.

[0026] FIGS. 4A-4C illustrate graphs showing cell growth across fixed-bed locations, according to an embodiment.

[0027] FIG. 5 illustrates a schematic view of a bioreactor system for gene therapy product production, according to aspects of the present disclosure.

[0028] FIG. 6 illustrates a graph showing glucose concentration over culture time for a bioreactor, according to an embodiment.

[0029] FIG. 7 illustrates a graph showing lactate concentration over culture time for a bioreactor, according to aspects of the present disclosure.

[0030] FIG. 8 illustrates a graph showing total glucose over culture time for a bioreactor, according to an embodiment.

[0031] FIG. 9 illustrates a graph showing total lactate over culture time for a bioreactor, according to aspects of the present disclosure.

[0032] FIG. 10 illustrates a bioreactor system with harvesting stages for gene therapy products, according to an embodiment.

[0033] FIGS. 11 A and 11B illustrate microscopy images of cells in a bioreactor system 48 hours post-transduction, according to aspects of the present disclosure.

[0034] FIGS. 12A and 12B illustrate microscopy images of cells in a bioreactor system 72 hours post-transduction, according to an embodiment.

[0035] FIG. 13 illustrates an output comparison between a bioreactor system and traditional roller bottles, according to aspects of the present disclosure.

[0036] FIGS. 14A and 14B illustrate timelines comparing improved and traditional processes for gene therapy production, according to an embodiment.

[0037] FIGS. 15A and 15B illustrate orthogonal views of reactor vessels at different scales, according to aspects of the present disclosure.WSGR Docket No. 69389-701.601

[0038] FIG. 16 illustrates a bioreactor system including a plurality of reactor vessels interfaced to a single recirculation mixing vessel, according to an embodiment.

[0039] FIG. 17 illustrates a recirculation mixing vessel configured for media replacement, according to an embodiment.

[0040] FIG. 18A illustrates increased productivity of the manufacturing process illustrated in Example 1. This process was a hyper-intensified and highly productive process that could consistently achieve viral genome yields of 4.00E14 vg or greater at the 2.4mA2 and 1.50E15 vg at the 10mA2 fixed bed bioreactors.

[0041] FIG. 18B illustrates design of the design of the process illustrated in Example 1. Vector kinetics were examined in 3 -Day, 5-Day, and 7-Day harvest experiments. In the 3-Day harvest experiment, supernatant was harvested each day post -transfection and replaced with fresh medium, and cells were lysed on Day 3. In the subsequent two experiments, this process was extended to 5 days and then 7 days, respectively.

[0042] FIG. 19 illustrates cumulative viral genome yield in supernatant.

[0043] FIG. 20A illustrates viral genome yields from supernatant vs. lysate.

[0044] FIG. 20B illustrates significant reduction in HCP impurity.

[0045] FIGs. 21A-21C illustrate sustained cell viability.

[0046] FIG. 21D illustrates long term host cell viability and productivity post transfection.

[0047] FIGs. 22A-22F illustrate changing AUC profile of daily harvest media over the course of 7 days.

[0048] FIG. 22G illustrates improved full capsid content.

[0049] FIG. 23 illustrates the procedure for culturing Vero cells.

[0050] FIG. 24 illustrates monitoring of glucose or lactate trends.

[0051] FIG. 25 illustrates cell density of the Vero cells.

[0052] FIG. 26 illustrates Vero cell viability.DETAILED DESCRIPTION

[0053] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0054] A capsid, as referred to herein, may be defined as the protein shell of a virus that encapsulates the viral genome. In some aspects, capsids may contain the vector, which includes the therapeutic genetic material. However, in some cases, capsids may be empty,WSGR Docket No. 69389-701.601lacking the genetic payload. These empty capsids may result from incomplete viral assembly or may be intentionally produced for various research or therapeutic applications. The presence of both full and empty capsids in gene therapy product preparations may impact the efficacy and safety of the treatment, necessitating careful consideration in production and purification processes.

[0055] The present disclosure provides systems and methods for the production of gene therapy vectors. These systems and methods may address challenges associated with traditional methods of gene therapy vector production, such as inefficiencies in cell transfection and difficulties in purification due to cellular debris.

[0056] The system may include a reactor, a recirculation system, and the use of specific transfection reagents. The recirculation system may also be configured to facilitate harvesting of a gene therapy product. The product may be harvested from media in the recirculation system at the end of and / or through a production cycle.

[0057] The reactor may be designed to promote even monolayer cell growth, optimized density for transfection and improved circulation of a buffer system. This may result in cells being equally exposed to growth media, the buffer, transfection mixture, and other elements during the production process, potentially enhancing the efficacy of transfection. The material of the reactor's coating and the evenness of this coating may also contribute to the system's performance.

[0058] The recirculation system may be configured to control parameters (such as pH, glucose and / or lactate levels, etc.) through the flow rate, which may be critical for changing buffers, improving transfection efficacy, and reducing the burden of purification. The system may also include the use of specific transfection reagents, such as calcium phosphate, which may increase transfection efficacy and potentially reduce costs associated with the use of expensive transfection reagents.

[0059] The system may also include a process for seeding cells, adjusting pH levels, adding a transfection DNA complex (e.g., calcium / DNA complex), changing media, and harvesting the product. This process may be designed to optimize various factors, such as the linear speed of circulation of media / buffer, the height of the reactor bed, even cell growth, optimized cell density and pH for efficient transfection, the flow rate of recirculation, and time harvest. In some embodiments, the flow rate maybe configured to replace a volume of the reactor in a period of time. In some aspects, a flow rate may be maintained to change at least about 0.5 reactor vessel volumes per hour. In some embodiments, the flow rate may beWSGR Docket No. 69389-701.601maintained to change from about 0.5 to about 4 reactor vessel 102 (e.g., as illustrated in FIG.1) volumes per hour.

[0060] In some aspects, the system may offer several operational advantages over traditional methods, such as an optimized, controlled environment for cell growth, less variability, increased scalability, a simplified seed train, reduced time and labor requirements, and improved physical separation of the cell and supernatant. The system may also produce fewer residual impurities, potentially resulting in fewer purification steps.

[0061] In some cases, the system may be scalable, allowing for increased production. This scalability may be achieved by, e.g., increasing the height and / or the radius of the reactor. The system may also include a filtering system for full capsid enrichment, based on the observation of preferential delayed production of full capsids in delayed harvest material.

[0062] In some aspects, the system may provide a significant improvement in the production of gene therapy vectors compared to traditional methods, potentially offering a more efficient and cost-effective solution for gene therapy vector production.

[0063] In some aspects, the gene therapy vectors may include lentiviral vectors, adenoviral vectors, AAV vectors, retroviral vectors, or other gene therapy vectors. In some aspects, the lentiviral vectors may include any plasmid transfection based products, either 3 or 4 plasmids. In some aspects, the adenoviral vectors may include any plasmid transfection based products. In some aspects AAV vectors may include any serotypes that manufactured utilizing plasmid transfection based production. In some aspects, retroviral vectors may include any plasmid transfection based products.

[0064] In some embodiments, the system or method described herein increases a yield of producing a biologic (e.g., an antibody, a virus, or a vector) by a host cell. In some embodiments, the system or method described herein allows monitoring of viability of the host cells for a prolonged period of time. In such case, the viability of the host cell can be monitored both before and after transfection or when the transfected host cells produce the highest yield or the highest quality of the biologic. Such monitoring also prevents lysing of the host cells that can still produce the biologic. For example, FIG. 19 and FIG. 20A illustrates cumulative harvest of viral particles in supernatant across seven days. Also, by not lysing the cells, overall protein burden on the host cell can be decreased (FIG. 20B). Since the host cells can be seeded and monitored for multiple days, the system or method described herein also allows the host cells to propagate and produce progeny cells that can also produce the biologic. Additionally, the prolonged culturing of the host cells also allow re-transfecting of the host cells for continued production of the biologic. In some embodiments, the hostWSGR Docket No. 69389-701.601cells can be seeded at a low density, where the host cells can immediately produce the biologic without the need of expanding the host cells. In some embodiments, the host cells can be seeded at a low density, where the host cells can immediately produce the biologic while expanding. In some embodiments, the host cells can be seeded and proliferating into at least one order of magnitude of progenitor cells, where both the host cells and the progenitors can produce the biologic. In some embodiments, the cells (e.g., host cells) can be seeded at a density of about 1X102-5X106cells / cm2on the fixed-bed substrate. In some embodiments, the cells can be seeded at the density of about 1X103-5X105cells / cm2on the fixed-bed substrate. In some embodiments, the cells can be seeded at the density at about IxlO3, 5xl03, 7.5xl03, or IxlO4cells / cm2on the fixed-bed substrate. In some embodiments, the cells grow from the density of about 1X103-5X105cells / cm2to a second density of about IxlOMxlO6cells / cm2In some embodiments, the cells grow to the second density of about 1. IxlO5, 1.2xl05, 1.3xl05, 1.4xl05, or 1.2xl05cells / cm2. In some embodiments, the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density. In some embodiments, the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density. In some embodiments, the cells grow to the second density at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding. In some embodiments, the cells do not exhibit decreased cell growth at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding. In some embodiments, the cells do not exhibit increased cell death at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding.

[0065] In some embodiments, the system or method described herein monitors the quality of the biologic produced by the host cells over a period of time (e.g., as illustrated in FIGs.22A-F). In some embodiments, the system or method identifies when the highest quality of the biologic is being produced by the host cells. For example, FIG. 22G illustrates continuous monitoring of the host cells across seven days, where the host cells produced the highest percentage of full capsid at Day 5 or Day 6 compared to Day 2, and starting on Day 3 majority of the capsid harvested in supernatant was full as opposed to empty.

[0066] In some embodiments, the system or method described herein increases yield of producing the biologic compared to other bioreactor systems. In some embodiments, the system or method described herein produces a viral genome yield of 4.00E14 vg or greater at the 2.4mA2 and 1.50E15 vg at the 10mA2 fixed bed bioreactors. In some embodiments, theWSGR Docket No. 69389-701.601system or method described herein produces a viral genome yield of 2.5E14 vg or greater at the 2.4mA2 and 1.0E15 vg at the 10mA2 fixed bed bioreactors.

[0067] In some embodiments, the system or method described herein allows low-density seeding of cells (e.g., host cells for producing biologic). In some embodiments, the seeding density can be lower than 10,00 cells / cmA2. In some embodiments, the cells, when seeded, can be incubated for a period of time before production of the biologic. In some embodiments, the cells, when seeded, can immediately produce the biologic.

[0068] Referring to FIG. 1, an example of a bioreactor system 100 for producing gene therapy products is illustrated. The bioreactor system 100 may include a reactor vessel 102, a recirculation mixing vessel 104, a recirculation path 106, a pump, and a gas supply 108.

[0069] The reactor vessel 102 may contain a fixed-bed substrate for cell growth. In some aspects, the fixed-bed substrate may be designed to promote even cell growth and improved circulation of a buffer system. This may result in cells being equally exposed to the media, transfection mixture, buffer, and any other elements circulated through the reactor vessel 102 potentially enhancing the efficacy of transfection. The material of the reactor's coating and the evenness of this coating may also contribute to the system's performance.

[0070] The recirculation mixing vessel 104 maybe fluidically connected to the reactor vessel 102. In some cases, the recirculation mixing vessel 104 maybe a distinct container connected to the reactor vessel 102 via the recirculation path 106. The recirculation path 106 may form a closed loop between the reactor vessel 102 and the recirculation mixing vessel 104, allowing for continuous circulation of media and other components.

[0071] In some aspects, the recirculation mixing vessel 104 may contain an automated means of agitating the contained media. This agitation mechanism may include a propeller, impeller, or stirring rod. In some cases, a magnetic stirring system may be employed. The agitation mechanism may help ensure thorough mixing of the media and any added components, such as nutrients or transfection reagents. In further embodiments, the reactor vessel 102 may also contain an agitation mechanism. In some embodiments, the mixing vessel can include singlemixing system. In some embodiments, the mixing vessel can include mechanical mixing system.

[0072] The propeller or impeller, if used, may be mounted on a shaft extending into the recirculation mixing vessel 104. This shaft may be connected to a motor that drives the rotation of the propeller or impeller at a controlled speed. The speed of rotation may be adjustable to optimize mixing based on the specific requirements of the cell culture process.WSGR Docket No. 69389-701.601

[0073] In some implementations, a magnetic stirring system may be utilized. This system may comprise a magnetic stir bar placed inside the recirculation mixing vessel 104 and a magnetic stirrer placed beneath the vessel. The magnetic stirrer may create a rotating magnetic field that causes the stir bar to rotate, thereby mixing the media. This approach may be particularly useful in maintaining a sterile environment as it eliminates the need for a shaft to penetrate the vessel.

[0074] The agitation mechanism may be designed to provide gentle yet effective mixing to avoid damaging the product while ensuring homogeneous distribution of nutrients and other components throughout the media. In some cases, the agitation speed or intensity may be automatically adjusted based on various parameters such as cell density, media viscosity, or the stage of the production process.

[0075] A pump may be configured to control a recirculation rate between the recirculation mixing vessel 104 and the reactor vessel 102. In some aspects, the pump may be configured to control the recirculation rate to achieve an exchange rate of at least about 0.5 reactor vessel volumes per hour. In some embodiments, the pump may be configured to control the recirculation rate to achieve an exchange rate of about 0.5 to about 4 reactor vessel volumes per hour. Various types of pumps may be used in the bioreactor system, including peristaltic pumps, diaphragm pumps, or centrifugal pumps, depending on the specific requirements of the system and the media being circulated.

[0076] The gas supply 108 may be connected to the system to provide gases for cell growth and metabolism. In some cases, the gas supply 108 may include compressed air, CO2, and O2, which are connected to the system to provide necessary gases for cell growth and metabolism. The arrangement of these components may allow for controlled cell culture conditions and efficient production of gene therapy vectors.

[0077] In some aspects, the pump may be configured to control the recirculation rate between the recirculation mixing vessel 104 and the reactor vessel 102. The pump may be designed to achieve an exchange rate of at least about 0.5 reactor vessel volumes per hour. In some embodiments, the pump maybe designed to achieve an exchange rate of about 0.5 to about 4 reactor vessel volumes per hour. This controlled recirculation rate may be critical for changing buffers and reducing the burden of purification. In some cases, the pump may be a peristaltic pump, which is sufficient for the purpose with a speed that can achieve up to 4 reactor volume exchanges per hour.

[0078] The reactor vessel 102 may have a working volume. For example, the working volume of the reactor vessel 102 may be 1.8L. In some embodiments, the reactor vessel 102WSGR Docket No. 69389-701.601may have a volume 10 to 30% of the overall system volume (e.g., including the recirculation mixing vessel 104). This volume may be optimal for the system's performance, allowing for efficient cell culture and product formation within the bioreactor system 100. In other embodiments, the working volume of the reactor vessel 102 may be different, depending on the specific requirements of the system and the media being circulated.

[0079] In some cases, the reactor vessel 102 may be cylindrical in shape and feature a top lid with multiple ports or openings. These ports or openings may allow for the introduction of media, nutrients, gases, and other components necessary for cell growth and metabolism. The reactor vessel 102 may also allow for the removal of waste products and the harvesting of the gene therapy product.

[0080] In some aspects, the reactor vessel 102 maybe designed to promote even cell growth and improved circulation of a buffer system. This may result in cells being equally exposed to the buffer, potentially enhancing the efficacy of transfection.

[0081] In some embodiments, the reactor vessel 102 may be configured to accommodate a fixed-bed substrate for cell growth. The fixed -bed substrate may serve as a substrate for cell growth and as a medium for fluid distribution within the bioreactor system 100. The layered structure of the fixed-bed substrate may facilitate efficient nutrient delivery and waste removal in a cell culture environment.

[0082] In some cases, the bioreactor system 100 may be configured to provide a controlled environment for cell growth. The recirculation path 106 may allow for the continuous exchange of media and nutrients between the reactor vessel 102 and the recirculation mixing vessel 104, while the gas supply 108 may help maintain appropriate gas concentrations.

[0083] Referring to FIG. 2, an enhanced bioreactor system 200 is depicted. The visualized enhanced bioreactor system 200 maybe an example of the system described in reference to FIG. 1. The bioreactor system 200 may include a reactor vessel and a recirculation mixing vessel, which are fluidically interfaced. In some aspects, the reactor vessel may be designed to promote even cell growth and improved circulation of a buffer system. This may result in cells being equally exposed to the buffer, potentially enhancing the efficacy of transfection. A recirculation path may form a closed loop between the reactor vessel and the recirculation mixing vessel, allowing for continuous circulation of media and other components. This fluidic interface between the reactor vessel and the recirculation mixing vessel may facilitate efficient exchange of media, nutrients, and other components necessary for cell growth and metabolism.WSGR Docket No. 69389-701.601

[0084] Referring to FIG. 3, an example microscopic view of a cellular structure generated within the fixed-bed reactor vessel, without transfection, is depicted. The view illustrates consistent, uniform cell growth within the fixed-bed reactor vessel, even without transfection.

[0085] The reactor vessel 102 of the bioreactor system 100 may include an internal fabric roll. The fabric roll may be housed within the reactor vessel 102 and may serve as a fixed-bed substrate for cell growth and / or as a medium for fluid distribution within the bioreactor system 100. The fabric roll may be designed to promote even cell growth and improved circulation of a buffer system, potentially enhancing the efficacy of transfection. The fabric roll may comprise multiple layers configured to support cell growth and fluid distribution.

[0086] The innermost layer of the fabric roll may be a first cell immobilization layer, which may have a fibrous structure for cell attachment. In some cases, the first cell immobilization layer may be designed to promote cell adhesion and growth. The fibrous structure of the first cell immobilization layer may provide a large surface area for cell attachment, potentially enhancing cell density and overall productivity of the bioreactor system 100. In some aspects, the first immobilization layer may be hydrophilic.

[0087] Adjacent to the first cell immobilization layer may be a second cell immobilization layer, which may also provide a surface for cell growth. In some aspects, the second cell immobilization layer may be designed to further enhance cell adhesion and growth. The second cell immobilization layer may work in conjunction with the first cell immobilization layer to provide a high -density cell culture environment within the bioreactor system 100. In some aspects, the second immobilization layer may be hydrophilic.

[0088] The outermost layer may be a flow distribution layer, characterized by a mesh -like structure. This flow distribution layer may facilitate the even distribution of media and nutrients throughout the fabric roll. In some cases, the flow distribution layer may be designed to promote efficient nutrient delivery and waste removal in a cell culture environment. In some aspects, the flow distribution layer may be hydrophilic.

[0089] The layered structure of the fabric roll may allow for efficient cell culture and product formation within the bioreactor system 100. In some embodiments, the fabric roll may include 30 layers. The two cell immobilization layers may be polyethylene terephthalate (PET) sheets, a thermoplastic polymer resin of the polyester family. The use of PET sheets for the cell immobilization layers may provide a durable and biocompatible surface for cell growth. The PET sheets may be configured to withstand the conditions within the bioreactor system 100, includingthe presence of various media, nutrients, and transfection reagents. In some cases, the PET sheets may be treated or coated to enhance cell adhesion and growth. InWSGR Docket No. 69389-701.601some aspects, the layers may be coated with plasma. In some aspects, the layers may be hydrophilic. The hydrophilic nature of the layers may aid in their ability to captured capsids.

[0090] In some aspects, the fabric roll may be configured to support cell growth at a high density. Cells may be seeded on the fabric roll at a density between 1x102and 1x106cells / cm2. This range of cell seeding density may be optimal for promoting efficient cell growth and transfection within the bioreactor system 100. In some cases, the cell seeding density may be adjusted based on the specific requirements of the cell culture process, such as the type of cells being cultured, the desired product yield, or the stage of the production process.

[0091] In some embodiments, the total surface area of the two cell immobilization layers may be 10 meters squared. This large surface area may provide ample space for cell attachment and growth, potentially enhancing the cell density and overall productivity of the bioreactor system 100. In some cases, the surface area of the cell immobilization layers may be adjusted based on the specific requirements of the cell culture process, such as the type of cells being cultured, the desired product yield, or the stage of the production process.

[0092] The bioreactor designs described herein are provided as examples of the reactor vessel 102. However, it should be understood that other bioreactor designs may also be utilized in the system. For example, a lattice bioreactor design may be employed as an alternative to the fabric roll configuration. The lattice bioreactor may provide a different structural arrangement for cell growth and fluid distribution, potentially offering unique advantages in certain applications. The choice of bioreactor design may depend on factors such as the specific cell type being cultured, the desired product yield, or particular process requirements. Regardless of the specific design, the bioreactor may be configured to promote even cell growth, efficient nutrient distribution, and effective transfection within the system. In some embodiments, a bioreactor may include a bed for cell growth, a source for inoculation, and monitoring sensors, as disclosed herein.

[0093] Referring to FIGS. 4A-4C, an example distribution of cell growth across different layers of the fixed-bed substrate within the reactor vessel 102 is depicted. As illustrated, the cell growth may be evenly distributed across the layers of the fixed -bed substrate. This even distribution of cell growth may be facilitated by the configuration of the fixed -bed substrate. In some cases, the even distribution of cell growth may be further enhancedby the controlled circulation of media and nutrients within the bioreactor system 100, as well as the controlled recirculation rate between the recirculation mixing vessel 104 and the reactorWSGR Docket No. 69389-701.601vessel 102. The even distribution and high density of cell growth may contribute to the efficient production of gene therapy vectors within the bioreactor system 100.

[0094] In some aspects, the bioreactor system 100 may include sensors for monitoring various parameters critical to cell growth and gene therapy product formation. These sensors may be positioned in the reactor vessel 102 and / or the recirculation mixing vessel 104 to provide real-time data on the culture conditions. For instance, pH sensors may be incorporated to continuously monitor and maintain optimal pH levels for cell growth and transfection. Gas sensors may be included to measure dissolved oxygen, carbon dioxide, and other relevant gases, ensuring appropriate gas concentrations for cellular metabolism. Temperature sensors may be integrated to maintain the culture at the ideal temperature for cell growth and gene therapy vector production.

[0095] In some cases, the bioreactor system 100 may also include sensors for detecting the presence and concentration of metabolites. These sensors may be positioned in the reactor vessel 102 and / or the recirculation mixing vessel 104. These metabolite sensors may monitor glucose consumption, lactate production, and other key metabolic indicators, providing valuable insights into cell health and productivity. The data from these sensors may be used to automatically adjust culture conditions, such as nutrient feed rates or gas flow rates, to optimize the production process. In some embodiments, the sensor data may be integrated with a control system that allows for automated feedback loops, ensuring consistent and optimal culture conditions throughout the gene therapy production process. For example, in response to detecting higher metabolic indicators system 100 may automatically increase the flow rate between the recirculation mixing vessel 104 and the reactor vessel 102 to remove excess waste.

[0096] In some aspects, the bioreactor system 100 may further comprise a pH control system configured to maintain pH within a predetermined range during cell growth and transfection. The components of the pH control system may be integrated into the reactor vessel 102 and / or the recirculation mixing vessel 104. The pH control system may be integral to the operation of the bioreactor system 100, as the pH level can significantly impact the efficiency of cell growth and transfection. In some cases, the pH control system may comprise pump control. The pump may be the main recirculation pump connected to the recirculation path 106 for adjusting the pH of media circulating through the system. Alternatively, the pump may be specifically utilized for pH control. The pump may be configured to add a base to the media in the recirculation path 106, thereby adjusting the pH of the media. The pH may beWSGR Docket No. 69389-701.601maintained within a predetermined range, which may be between 7.0 and 7.8 in some embodiments. The pH may be maintained within about 5% of a target pH level.

[0097] In some aspects, the pH control system may be configured to maintain different pH levels during different stages of the cell culture and vector production process. For example, during a pre-transfection phase, the pH control system may maintain a pH of approximately 7.6, while during a transfection phase, the pH control system may maintain a pH of approximately 7.4. These pH levels may be optimal for the transfection of cells with gene therapy vectors using calcium phosphate as a transfection reagent.

[0098] In some cases, the pH control system may be configured to adjust the pH of the media in the recirculation path 106 as soon as the system limit is reached. This rapid adjustment of pH may prevent the creation of local microenvironments within the reactor vessel 102 that could be damaging to cell growth.

[0099] In some embodiments, the bioreactor system 100 may also include a temperature control system. The temperature control system may be configured to maintain a predetermined temperature within the reactor vessel 102 and the recirculation mixing vessel 104. The temperature control system may include a heating element and / or a cooling element, which may be controlled to maintain the temperature within a predetermined range. The temperature control system may be integral to the operation of the bioreactor system 100, as the temperature can significantly impact the efficiency of cell growth and transfection. The components of the temperature control system may be integrated into the reactor vessel 102 and / or the recirculation mixing vessel 104.

[0100] In some aspects, the temperature control system may be configured to maintain different temperatures during different stages of the cell culture process. For example, during a cell growth phase, the temperature control system may maintain a higher temperature to promote cell proliferation, while during a transfection phase, the temperature control system may maintain a lower temperature to facilitate the uptake of gene therapy vectors by the cells.

[0101] In some cases, the temperature control system and the pH control system may be integrated within the reactor vessel 102, the recirculation path 106, and / or the recirculation mixing vessel 104, allowing for the continuous adjustment of temperature and pH as media and nutrients are circulated between the reactor vessel 102 and the recirculation mixing vessel 104. This integration may allow for the maintenance of optimal conditions for cell growth and transfection within the bioreactor system 100.

[0102] Referring to FIG. 5, in some aspects, the bioreactor system 100 may include a process for introducing a transfection mixture 502 into the system. The transfection mixture 502 mayWSGR Docket No. 69389-701.601be introduced via the recirculation mixing vessel 104. The transfection mixture 502 may include a combination of DNA and / or a transfection reagent, such as calcium phosphate. The DNA may contain the genetic material to be transferred into the cells, while the reagent may facilitate the uptake of the DNA by the cells.

[0103] In some cases, the transfection mixture 502 may be prepared separately and then added to the recirculation mixingvessel 104. The transfection mixture 502 maybe introduced into the recirculation mixing vessel 104 in a controlled manner, such as by using a pump or a valve. The transfection mixture 502 may then be circulated through the recirculation path 106 and into the reactor vessel 102, where it may come into contact with the cells growing on the fixed-bed substrate.

[0104] In certain aspects, the methods include transfecting cells with DNA. In some aspects transfection reagents may include chemical-based reagents (e.g., calcium phosphate), lipid-based reagents (e.g., lipofectamine), and / or polymer-based reagents (e.g., polyethylenimine).

[0105] In some embodiments, the transfection mixture 502 may be introduced into the recirculation mixing vessel 104 at a specific stage of the cell culture process. For example, the transfection mixture 502 may be introduced after the cells have been seeded and grown to a sufficient density on the fixed-bed substrate. The introduction of the transfection mixture 502 may be timed to coincide with a specific phase of the cell growth cycle, such as the exponential growth phase, to maximize the efficiency of transfection.

[0106] In further embodiments, a portions of the transfection mixture 502 may be directly introduced into the reactor vessel 102.

[0107] In some aspects, the circulation of the transfection mixture 502 through the bioreactor system 100 may be controlled to ensure efficient transfection of the cells. The recirculation rate between the recirculation mixing vessel 104 and the reactor vessel 102 may be adjusted to optimize the exposure of the cells to the transfection mixture 502. For example, the recirculation rate may be increased to ensure that the transfection mixture 502 is evenly distributed throughout the reactor vessel 102, thereby maximizing the contact between the cells and the transfection mixture 502.

[0108] In some cases, the recirculation rate may be controlled based on various parameters, such as the cell density, the concentration of the transfection mixture 502, or the stage of the production process. The recirculation rate may be adjusted to ensure that the cells are exposed to an optimal concentration of the transfection mixture 502, thereby enhancing the efficiency of transfection.WSGR Docket No. 69389-701.601

[0109] In some aspects, the transfection reaction occurs in about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours and / or up to 4 week post-seeding (e.g., depending on seeding density). In certain aspects, the transfection reaction occurs in about 72 hours post-seeding.

[0110] In some embodiments, the bioreactor system 100 may also include a process for circulating the transfection mixture 502 through the system. The transfection mixture 502 may be circulated through the recirculation path 106 and into the reactor vessel 102, where it may come into contact with the cells growing on the fixed-bed substrate. The circulation of the transfection mixture 502 may be controlled to ensure efficient transfection of the cells. The circulation of the transfection mixture may be continued for a predetermined period of time to allow for sufficient time for the cells to uptake the DNA from the transfection mixture 502. In some embodiments, the circulation of the transfection mixture is controlled such that the cells are contacted with the transfection reagent for at least 7 hours.

[0111] In some cases, the circulation of the transfection mixture 502 may be stopped after a predetermined period of time, and the media in the bioreactor system 100 may be changed to a serum -free media. The change of media may be performed to facilitate the production of the gene therapy product by the transfected cells. The gene therapy product may then be harvested from the bioreactor system 100 at a later stage of the production process.

[0112] Referring to FIG. 6, the graph 600 illustrates example glucose concentration trends over the culture period in the bioreactor system 100. The graph 600 displays four different data series representing viral and growth runs in both BRX and recirculation conditions. The x-axis of graph 600 represents the culture time in days, ranging from 0 to 4.5 days, while the y-axis represents the glucose concentration in g / L, ranging from 2 to 4.5 g / L. Consistent metabolite trends are observed in each run. These glucose concentration trends may be indicative of cell growth and metabolism within the bioreactor system 100. In some aspects, the glucose concentration trends may be used to estimate cell density within the bioreactor system 100.

[0113] Referring to FIG. 7, the graph 700 illustrates lactate concentration trends over the culture period in the bioreactor system 100. The graph 700 displays four different data series representing viral and growth runs in both BRX and recirculation conditions. The x-axis of graph 700 represents the culture time in days, ranging from 0 to 4.5 days, while the y-axis represents the lactate concentration in g / L, ranging from 0 to 1.6 g / L. Consistent metabolite trends are observed in each run. These lactate concentration trends may be indicative of cellWSGR Docket No. 69389-701.601growth and metabolism within the bioreactor system 100. In some aspects, the lactate concentration trends may be used to estimate cell density within the bioreactor system 100.

[0114] Referring to FIG. 8, the graph 800 illustrates total glucose trends over the culture period in the bioreactor system 100. The graph 800 displays two different data series representing viral and growth runs. The x-axis of graph 800 represents the culture time in days, ranging from 0 to 4.5 days, while the y-axis represents the total glucose in grams, ranging from 42 to 87 grams. Consistent metabolite trends are observed in each run. These total glucose trends may be indicative of cell growth and metabolism within the bioreactor system 100.

[0115] Referring to FIG. 9, the graph 900 illustrates total lactate trends over the culture period in the bioreactor system 100. The graph 900 displays two different data series representing viral and growth runs. The x-axis of graph 900 represents the culture time in days, ranging from 0 to 4.5 days, while the y-axis represents the total lactate in grams, ranging from 2 to 32 grams. Consistent metabolite trends are observed in each run. These total lactate trends may be indicative of cell growth and metabolism within the bioreactor system 100.

[0116] Referring to FIG. 10, the bioreactor system 1000 may provide an enhanced yield by harvesting the gene therapy products from multiple sources within the system. The harvesting process may be divided into several stages, each represented by a yield container. The supernatant yield 1002 may represent approximately 11.4 L of supernatant, accounting for approximately 48% of the total yield. In other embodiments, the supernatant yield 1002 may represent 10 to 50% of the total yield. The supernatant may be obtained from the reactor vessel 102 and / or the recirculation mixing vessel 104 after a predetermined period of cell growth and transfection. In some cases, the supernatant may be collected by drainage or by using a pump to remove the supernatant from the reactor vessel 102 and / or recirculation mixing vessel 104.

[0117] Following the collection of the supernatant yield 1002, the fixed-bed substrate within the reactor vessel 102 may be rinsed to collect additional gene therapy product. This rinse may be represented by the first rinse yield 1004, which may show about 1.4 L of rinse solution, contributing approximately 4% to the total yield. The rinse may be performed by introducing a rinse solution into the reactor vessel 102 and allowing the rinse solution to circulate through the fixed-bed substrate. The rinse solution may thenbe collected as the first rinse yield 1004. In some cases, the rinse solution may be a buffer solution or a media solution that is compatible with the cells and the gene therapy product.WSGR Docket No. 69389-701.601

[0118] In some aspects, a second rinse may be performed to collect additional gene therapy product. This second rinse may be represented by the second rinse yield 1006, which may indicate another 1.4 L of rinse solution, adding approximately 3% to the total yield. The second rinse may be performed in a similar manner to the first rinse, with a rinse solution being introduced into the reactor vessel 102, allowed to circulate through the fixed-bed substrate, and then collected as the second rinse yield 1006.

[0119] Following the rinse stages, the cells remaining on the fixed-bed substrate may be lysed to release any remaining gene therapy product. In some cases, the lysis solution may be a buffer solution containing a lysis agent, such as a detergent or an enzyme, that is capable of lysing the cells. In some embodiments, there may be additional vectors also trapped in the reactor vessel 102 as well. The buffer solution containing the lysis agent may recover some of these trapped vectors. This lysis may be represented by the lysate yield 1008, which may represent approximately 1.4 L of cell lysate, contributing approximately 40% to the total yield. The lysis may be performed by introducing a lysis solution into the reactor vessel 102 and allowing the lysis solution to circulate through the fixed-bed substrate. The lysis solution may then be collected as the lysate yield 1008. In some embodiments, lysing may be combined with a rinse.

[0120] In some embodiments, a wash may be performed to collect any remaining gene therapy product. This wash may be represented by the wash yield 1010, which may show about 1.4 L of wash solution, accounting for approximately 6% of the total yield. The wash may be performed by introducing a wash solution into the reactor vessel 102 and allowing the wash solution to circulate through the fixed-bed substrate. The wash solution may then be collected as the wash yield 1010. In some cases, the wash solution may be a buffer solution or a media solution that is compatible with the cells and the gene therapy product.

[0121] In some aspects, the rinses or wash may include circulating a rinse or wash solution through the bioreactor system 100 using the recirculation mixing vessel 104. The rinse or wash solution may be introduced into the recirculation mixing vessel 104 and then circulated through the recirculation path 106 and into the reactor vessel 102. This circulation may allow the rinse or wash solution to thoroughly permeate the fixed -bed substrate, potentially enhancing the extraction of gene therapy products. The recirculation rate may be adjusted to optimize the contact time between the rinse or wash solution and the fixed -bed substrate. In some cases, the rinse or wash solution maybe recirculated multiple times through the system to maximize the collection of gene therapy products. The use of the recirculation mixing vessel 104 in this process may allow for better control of the rinse or wash parameters, suchWSGR Docket No. 69389-701.601as temperature, pH, and flow rate, potentially improving the overall efficiency of the harvesting process.

[0122] In some aspects, a media replacement may be performed at some period during the application of the bioreactor system 100. For example, the media may be replaced 48 hours after the initiation of transfection. The media replacement may remove a portion of capsids in the system not containing the gene therapy vectors.

[0123] In some aspects, the harvesting process maybe designed to maximize the overall yield of gene therapy product from the bioreactor system 1000. The multiple stages of the harvesting process, including the collection of supernatant, the rinses, the lysis, and the wash, may each contribute to the total yield of gene therapy product. This multi-stage harvesting process may allow for the efficient and / or selective extraction of the gene therapy product from the bioreactor system 1000, potentially enhancing the productivity and costeffectiveness of the system.

[0124] Referring to FIG. 11A and FIG. 11B, these figures illustrate example microscopy images of cells in the bioreactor system 48 hours post -transduction. The images may provide a visual representation of the cells' response to the transfection process. The appearance of the cells at this stage are indicative of the efficiency of the gene therapy vector production process. For instance, the morphology, density, and distribution of the cells provide insights into the success of the transfection and the subsequent production of gene therapy vectors.

[0125] Referring to FIG. 12A and FIG. 12B, these figures illustrate example microscopy images of cells in the bioreactor system 72 hours post -transduction. The images provide a visual representation of the cells' response to the transfection process. The appearance of the cells at this stage are indicative of the peak production of gene therapy vectors within the bioreactor system. For instance, the morphology, density, and distribution of the cells provide insights into the success of the transfection and the subsequent production of gene therapy vectors. The images show a high density of cells, suggesting a successful transfection process and efficient production of gene therapy vectors.

[0126] Referring to FIG. 13, an output comparison 1300 between the yield 1302 from a bioreactor system as described herein and traditional system yield 1304 in roller bottles is illustrated. The output comparison 1300 provides a visual representation of the production capacity of two different systems usedin cell culture and gene therapy vector production. In this example, the bioreactor yield 1302 is equivalent to 150 roller bottles. This comparison highlights the increased production capacity of the bioreactor system over traditional roller bottle methods.WSGR Docket No. 69389-701.601

[0127] Referring to FIG. 14A and FIG. 14B, these figures illustrate two timelines comparing an improved process and a traditional process for gene therapy production. The improved timeline 1400 is shown in FIG. 14A, while the traditional timeline 1450 is presented in FIG.14B

[0128] The improved timeline 1400 demonstrates a more efficient process, with shorter durations for multiple stages of production compared to the traditional timeline 1450.

[0129] In some aspects, the improved timeline 1400 may be achieved by optimizing various factors in the bioreactor system 100, such as the linear speed of circulation of media / buffer, the height of the reactor bed, even cell growth, pH during transfection, and the flow rate of recirculation. These optimizations may result in a more efficient process for producing gene therapy products, as represented by the improved timeline 1400.

[0130] In some cases, the improved timeline 1400 may also be achieved by using specific transfection reagents, such as calcium phosphate, which may increase transfection efficacy and potentially reduce costs associated with the use of expensive transfection reagents. The use of these specific transfection reagents may contribute to the shorter durations for each stage of production in the improved timeline 1400.

[0131] In some embodiments, the improved timeline 1400 may also be achieved by using a recirculation system that allows for the continuous exchange of media and nutrients between the reactor vessel 102 and the recirculation mixing vessel 104. This continuous exchange may enhance the efficacy of transfection, potentially contributing to the shorter durations for each stage of production in the improved timeline 1400.

[0132] In contrast, the traditional timeline 1450 may represent a process for producing gene therapy products using traditional methods, such as roller bottle culture. These traditional methods may be less efficient than the improved process represented by the improved timeline 1400, resultingin longer durations for each stage of production. Traditional methods may also be limited in a capability to scale up.

[0133] In some aspects, the traditional timeline 1450 may be characterized by inefficiencies in cell transfection and difficulties in purification due to cellular debris. These challenges may contribute to the longer durations for each stage of production in the traditional timeline 1450

[0134] In some cases, the traditional timeline 1450 may also be characterized by the use of expensive transfection reagents, which may increase the cost of producing gene therapy products. These costs may also contribute to the longer durations for each stage of production in the traditional timeline 1450.WSGR Docket No. 69389-701.601

[0135] Overall, the comparison between the improved timeline 1400 and the traditional timeline 1450 may highlightthe efficiency gains in the improved process for producing gene therapy products.

[0136] Referring to FIG. 15A and FIG. 15B, the bioreactor system 100 may be scalable, allowing for increased production of gene therapy products. The scalability of the bioreactor system 100 may be achieved by adjusting the dimensions of the reactor vessel 102. In some aspects, the reactor vessel 102 may be configured to maintain its performance even when scaled up to larger sizes. This scalability may allow for the production of gene therapy products at a larger scale, potentially enhancing the productivity and cost-effectiveness of the bioreactor system 100.

[0137] FIG. 15A shows a reactor 1500 with a capacity of 200 m2. FIG. 15B depicts a large scale reactor 1550 with a capacity of 600 m2.

[0138] In some cases, the reactor vessel 102 may be scaled up by increasing its height rather than its diameter. This may allow for the maintenance of a consistent linear speed of circulation of media / buffer throughout the reactor vessel 102, even when the reactor vessel 102 is scaled up. Maintaining a consistent linear speed of circulation may be critical for ensuring even cell growth and efficient transfection within the reactor vessel 102.

[0139] Additional means of scaling an automation are also considered. For example, as illustrated in FIG. 16, a bioreactor system 1600 may include a plurality of reactor vessels 102 interfaced to a single recirculation mixing vessel 104. The recirculation mixing vessel 104 may be scaled to incorporate sufficient media for the plurality of reactors vessels 102. The recirculation mixing vessel 104 may be interfaced to the reactor vessels 102 via a manifold 1602. In some aspects, the manifold 1602 may include automated valving allowing for a controllable flow of media to each of the plurality of reactor vessels 102. In some embodiments, gas flow maybe similarly manifolded 1604 to the plurality of reactor vessels 102

[0140] In some aspects, robotics may be employed to automate various stages of the gene therapy production process within the bioreactor system. Robotic systems may be used for precise and consistent cell seeding, media exchange, transfection mixture introduction, and harvesting of gene therapy products. These automated systems may control the circulation of media, adjust pH levels, regulate gas supply, and perform rinse or wash procedures with minimal human intervention. Robotics may also be utilized for sample collection and analysis, allowing for real-time monitoring and adjustment of culture conditions. By automating these processes, the use of robotics may potentially increase reproducibility,WSGR Docket No. 69389-701.601reduce human error, and improve overall efficiency and scalability of gene therapy product production.

[0141] In some aspects, as illustrated in FIG. 18, the recirculation mixing vessel 104 may be configured to expel used media during the production. The used media may be separate into waste 1704 and product 1702. Fresh media 1706 may be received by the recirculation mixing vessel 104 for continued production.

[0142] In some embodiments, the replacement of media in the recirculation mixing vessel 104 may be automatic. For example, after a first time period (e.g., 48 hours post transduction), an automatic replacement of media may occur. The system may automatically consider this output media waste 1704 (e.g., assumethe majority of the collected capsids are empty). After a second time period (e.g., 72 hours post transduction), a second replacement of media may occur with the output further processed a product 1702, as described in reference to FIG. 10. In some embodiments, other monitored information (e.g., pH, temperature, metabolites, etc.) may be considered in triggering media replacement.

[0143] In some aspects, a plurality of additional media replacements may occur, extending the production lifecycle of the bioreactor system 100. For example, the production and harvest illustrated in FIG. 14A may be repeated multiple times based on a single inoculation and growth. The extended production lifecycle may greatly increase the production yield efficiency over time.

[0144] In alternative embodiments, the replacement of media may be manual. The bioreactor system 100 may include a means of signaling (e.g., a speaker, a light, a digital transmission, or a combination thereof) for a media replacement under the conditions described above in reference to an automatic media replacement.

[0145] In some aspects, the bioreactor system 100 may include a process for introducing a transfection mixture 502 into the system. The transfection mixture 502 may be introduced via the recirculation mixing vessel 104. The transfection mixture 502 may include a combination of DNA and a transfection reagent. In some cases, the transfection reagent may be calcium phosphate, which may increase transfection efficacy. The DNA may contain the genetic material to be transferred into the cells, while the reagent may facilitate the uptake of the DNA by the cells.

[0146] In some embodiments, the transfection mixture 502 may be prepared separately and then added to the recirculation mixing vessel 104. The transfection mixture 502 may be introduced into the recirculation mixing vessel 104 in a controlled manner, such as by using a pump or a valve. The transfection mixture 502 may then be circulated through theWSGR Docket No. 69389-701.601recirculation path 106 and into the reactor vessel 102, where it may come into contact with the cells growing on the fixed-bed substrate.

[0147] In some aspects, the circulation of the transfection mixture 502 through the bioreactor system 100 may be controlled to ensure efficient transfection of the cells. The recirculation rate between the recirculation mixing vessel 104 and the reactor vessel 102 may be adjusted to optimize the exposure of the cells to the transfection mixture 502. For example, the recirculation rate may be increased to ensure that the transfection mixture 502 is evenly distributed throughout the reactor vessel 102, thereby maximizing the contact between the cells and the transfection mixture 502.

[0148] In some cases, the transfection mixture 502 may be introduced into the recirculation mixing vessel 104 at a specific stage of the cell culture process. For example, the transfection mixture 502 may be introduced after the cells have been seeded and grown to a sufficient density on the fixed-bed substrate. The introduction of the transfection mixture 502 may be timed to coincide with a specific phase of the cell growth cycle, such as the exponential growth phase, to maximize the efficiency of transfection.

[0149] In some embodiments, the concentration of the plasmid in the transfection mixture 502 may be optimized for efficient transfection. For instance, the concentration of the plasmid(s) may be approximately 0.3 pg / cm2. This concentration may be optimal for promoting efficient transfection within the bioreactor system 100. In some cases, the concentration of the plasmid(s) may be adjusted based on the specific requirements of the cell culture process, such as the type of cells being cultured, the desired product yield, or the stage of the production process.

[0150] In some aspects, alternative transfection reagents may be used in the bioreactor system 100. For example, polymer-based or liposome-based reagents may be used as transfection reagents. These alternative transfection reagents may provide additional options for optimizing the transfection process within the bioreactor system 100.

[0151] In some cases, the bioreactor system 100 may include a process for incubating the cells with the transfection mixture 502. The incubation process may allow for sufficient time for the cells to uptake the DNA from the transfection mixture 502. The duration of the incubation process may be optimized based on the specific requirements of the cell culture process, such as the type of cells being cultured, the desired product yield, or the stage of the production process.

[0152] In some embodiments, the pH during the incubation process may be controlled to optimize the efficiency of transfection. For example, the pH may be maintained between 7.0WSGR Docket No. 69389-701.601and 7.8 during the incubation process. This pH range may be optimal for promoting efficient transfection within the bioreactor system 100. In some cases, the pH may be adjusted based on the specific requirements of the cell culture process, such as the type of cells being cultured, the desired product yield, or the stage of the production process.

[0153] In some aspects, the bioreactor system 100 may include a pH control system that maintains different pH levels during different stages of the cell culture process. For instance, during a pre-transfection phase, the pH control system may maintain a pH of approximately 7.6. This pH level may be optimal for the pre-transfection phase, potentially enhancing the efficiency of the sub sequent transfection process. In some cases, the pH control system may include abase pump connected to the recirculation path 106 for adjusting the pH of media circulating through the system. The base pump may be configured to add a base to the media in the recirculation path 106, thereby adjusting the pH of the media.

[0154] During a transfection phase, the pH control system may maintain a pH of approximately 7.4. This pH level may be optimal for the transfection phase, potentially enhancing the efficiency of transfection. The pH control system may adjust the pH of the media in the recirculation path 106 as soon as the system limit is reached. This rapid adjustment of pH may prevent the creation of local microenvironments within the reactor vessel 102 that could be damaging to cell growth.

[0155] In some embodiments, the bioreactor system 100 may be configured to produce lentiviral vectors. The production of lentiviral vectors may involve a similar process to that described above, with cells being seeded on the fixed -bed substrate, media and nutrients being circulated between the reactor vessel 102 and the recirculation mixing vessel 104, and a transfection mixture being introduced into the system. However, the production of lentiviral vectors may not require the lysis of cells to harvest the product. Instead, the lentiviral vectors may be continuously harvested from the supernatant of the bioreactor system 100. This continuous harvesting capability may enhance the productivity and efficiency of the bioreactor system 100 for lentiviral vector production.

[0156] Further provided herein are methods for transfecting cells. In some cases, the methods may be implemented using the bioreactor systems described herein.

[0157] In one aspect, a method of transfecting cells in a fixed-bed bioreactor is provided. In some cases, the method comprises providing or obtaining a fixed -bed bioreactor comprising a plurality of cells attached to a substrate; and contacting the plurality of cells with a cell culture media comprising a nucleic acid payload and a transfection reagent for at least 7 hours, such that the plurality of cells are transfected with the nucleic acid payload.WSGR Docket No. 69389-701.601

[0158] In another aspect, provided herein is a method for culturing cells in a fixed -bed bioreactor. In some cases, the method comprises the method providing or obtaining a fixed -bed bioreactor comprising a plurality of cells attached to a substrate and a volume of cell culture media in contact with the plurality of cells; and replacing the volume of cell culture media at least about 0.5 times per hour.

[0159] In some cases, the fixed-bed bioreactor comprises a volume of cell culture media (e.g., a bioreactor vessel volume), and the volume of cell culture media is replaced at least about 0.5 times per hour during the contacting. In some cases, the volume of cell culture media is replaced from about 0.5 to about 5 times per hour during the contacting. The rate of volume replacement can be adjusted, e.g., using the bioreactor systems, as described herein.

[0160] In some cases, the method further comprises adjusting the pH of the cell culture media to maintain the pH of the cell culture media within about 5% of a target pH during the contacting. In some cases, the adjusting comprises monitoring the pH of the cell culture media, and adjusting a perfusion rate to alter the pH of the cell culture media. The pH can be monitored, e.g., using the pH sensors described herein. The pH levels can be adjusted, e.g., based on feedback from a pH sensors, e.g., using the bioreactor systems described herein.

[0161] In some cases, the method comprises monitoring or detecting a level of at least one cell waste product in the cell culture media, and adjusting a perfusion rate to alter the amount of the at least one cell waste product, such as lactate, in the cell culture media. In some cases, the method comprises monitoring or detecting a level of glucose in the cell culture media, and adjusting a perfusion rate to alter the amount of glucose in the cell culture media. Waste products, cell metabolites, etc. in the media can be monitored, e.g., by using the sensors described herein, and the levels of waste products, cell metabolites, etc. can be adjusted, e.g., by altering the perfusion rate of the system, e.g., using the bioreactor systems described herein.

[0162] In some cases, the method further comprises adjusting a perfusion rate to maintain a concentration of the transfection reagent within about 25% of a target transfection concentration. In some cases, the transfection reagent comprises calcium phosphate. In some cases, the nucleic acid payload comprises a viral plasmid.

[0163] In some cases, the method may involve transfecting the cells with a viral plasmid(s) for producing viral particles. In some cases, the viral particles are adeno -associated viral (AAV) particles. In some cases, the plurality of cells comprise HEK293 cells. In some embodiments, the plurality of cells comprise Vero cells. In some cases, the method results in at least about 2E13 vg / L AAV particles. In other cases, the viral particles are lentiviralWSGR Docket No. 69389-701.601particles. In some cases, the method further comprises harvesting a plurality of viral particles from the plurality of cells.

[0164] In some cases, the method further comprises replacing the cell culture media after the contacting with fresh cell culture media for a period of time. In some cases, the harvesting occurs at least 48 hours after the contacting. In some cases, the method further comprises removing empty capsids prior to the harvesting. In some cases, the contacting of comprises continuously contacting the plurality of cells with the cell culture media during the contacting.

[0165] In another aspect, provided herein is a method of producing a biologic. The method may comprise providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate; continuously contacting, for a first time period, the plurality of cells with a first volume of cell culture media comprising a transfection reagent and a plasmid, such that at least one cell is transfected with the plasmid and produces the biologic; replacing the first volume of cell culture media with a second volume of cell culture media that does not contain the transfection reagent and the plasmid by continuously contacting, for a second time period, the plurality of cells with the second volume of cell culture medial; replacing the second volume of cell culture media with a third volume of cell culture media by continuously contacting, for a third time period, the plurality of cells with the third volume of cell culture media; and collecting the biologic during a fourth time period.

[0166] In some cases, the biologic is a plurality of viral particles. In some cases, the plasmid is at least one viral plasmid. In some cases, the plurality of viral particles comprise adeno-associated viral (AAV) particles. In some cases, the plasmid is selected from the group consisting of: a helper plasmid, a plasmid containing Rep and / or Cap genes, a transfer plasmid, and any combination thereof. In some cases, the first time period is at least 7 hours. In some cases, the second time period is at least 12 hours. In some cases, the third time period is from about 24 hours to about 48 hours after the second time period. In some cases, the fourth time period is from about 48 hours to about 72 hours after the second time period. In some cases, the method further comprises contacting the plurality of cells with a lysis buffer to lyse the plurality of cells and release the AAV particles into the lysis buffer. In some cases, the method further comprises collecting the lysis buffer containing the AAV particles. In some cases, the method further comprises discarding the third volume of cell culture media. In some cases, at least about 70% of the AAV particles in the third volume of cell culture media are empty AAV particles. In some cases, at least about 35% of the AAV particles collected in (e) are full AAV particles. In some cases, the plurality of viral particlesWSGR Docket No. 69389-701.601are lentiviral particles, retroviral particles, or adenoviral particles. The methods provided herein are not limited to viral particles. In some cases, the biologic is a protein or polypeptide. In some cases, the biologic is an antibody or fragment thereof.

[0167] In some cases, any one or any combination of the steps comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour. In some cases, any one or any combination of the steps comprises completely replacing the full bioreactor volume of cell culture media at a rate of from about 0.5 times to about 4 times per hour.

[0168] In some cases, the method further comprises replacing the third volume of cell culture media with a fourth volume of cell culture media by continuously contacting, for a fifth time period, the plurality of cells with the fourth volume of cell culture media. In some cases, the fifth time period is from about 72 hours to about 96 hours after the second time period. In some cases, the method further comprises, replacing the fourth volume of cell culture media with a fifth volume of cell culture media by continuously contacting, for a sixth time period, the plurality of cells with the fifth volume of cell culture media. In some cases, the sixth time period is greater than about 96 hours after the second time period. In some cases, the collecting comprises continuously perfusing the bioreactor with a volume of cell culture media or lysis buffer during the course of the fourth time period.

[0169] In some cases, the fixed-bed bioreactor is coupled to a recirculation system. In some cases, the recirculation system is coupled to a plurality of fixed-bed bioreactors.

[0170] In another aspect, provided herein are compositions, e.g., that can be produced using the systems and methods provided herein. In one aspect, provided herein is a composition comprising at least 2E13 vg / L viral particles, and no amount or an undetectable amount of a lipid-based or a polymer-based transfection reagent. In some cases, the undetectable amount is an amount of the lipid-based or polymer-based transfection reagent that is undetectable by high-performance liquid chromatography (HPLC). In some cases, the polymer -based transfection reagent is a polyethylenimine (PEI) transfection reagent. In some cases, the composition further comprises an amount of calcium detectable by HPLC. In some cases, the viral particles are AAV particles.

[0171] Described herein, in some aspects, is a method utilizing a system described herein to grow cells to an increased density, where the increase in cell density is superior compared to other comparable system for cell culturing (e.g., CARBO system). FIG. 23 illustrates a nonlimiting example of a procedure for culturing and grow the cells (e.g., Vero cells). By seed trainingthe Vero cells in roller bottles, Vero cells can be growing robustly (e.g., about 7.5E7WSGR Docket No. 69389-701.601cells per roller bottle at harvest). The reactor can be seeded at a density of about 1X102-5X106cells / cm2. In some embodiments, the cells can be seeded at a density of about 1X103-5X105cells / cm2on the fixed-bed substrate. In some embodiments, the cells can be seeded at about IxlO3, 5xl03, 7.5xl03, or IxlO4cells / cm2on the fixed-bed substrate. In some embodiments, the cells can grow for at least about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours. In some embodiments, cell growth can be terminated after about24 hours, 48 hours, 72 hours, 78 hours, 84hours, 90 hours, 96 hours, or 120 hours. In some embodiments, cell growth can be terminated at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours for distribution study. In some embodiments, the cells, during growth, can be monitored for metabolite or waste presence or concentration. For example, FIG.24 illustrates glucose or lactate trends of the Vero cells at numerous hours after inoculation. In some embodiments, an equilibrium can be maintained between the reactor and the recirculation vessel. In some embodiments, the recirculation vessel contains majority of growth nutrients. FIG.25 illustrates cell density of the Vero cells cultured by the system and the method described herein. 7.5E3 cells / cmA2 of the Vero cells were seeded and grew for 90 hours. About 100 samples were cut from various part of the reactor, positions of the samples shown FIG. 25. In some embodiments, the cells, after seeding or growth, do not exhibit deceased cell growth rate compared to the cells from before seeding. In some embodiments, the cells, after seeding or growth, do not exhibit increased cell death compared to the cells from before seeding. For example, FIG. 26 illustrates Vero cell viability (4X to show global view) of robust cell growth and minimum cell death when cultured by the system and the method described herein.

[0172] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0173] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.WSGR Docket No. 69389-701.601

[0174] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0175] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof ’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0176] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are describedin the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0177] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, forexamplean increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2 -fold, atleast 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0178] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by atleast 10% as compared to a reference level, for example a decreaseWSGR Docket No. 69389-701.601by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non -detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0179] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0180] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0181] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22,23, 24,25, 26,27, 28,29, 30,31, 32,33, 34,35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47,48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub -range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0182] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided byWSGR Docket No. 69389-701.601way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLES

[0183] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.Example 1. Kinetic study of AAV viral vector production

[0184] This example illustrates utilization of a specialized clone of HEK293 cells, suited for fixed-bed bioreactors, followed by a simple, chemically defined transfection reagent. Further enhanced by a perfusion process via recirculation, the result illustrated a unique, scalable, and cost-effective vector manufacturing process, indicating that this was a suitable and potentially better method for commercial manufacturing of AAV viral vectors (FIG. 18A). It has been suggested that the standard transient transfection method used for vector production, in either suspension or adherent cell culture, over-saturates the host cell’s ability to process the ingested plasmid material; the transfected host cells never recover from the initial transfection shock before lysis and harvest at the typical 72 hours harvest. This standard process leads to overwhelming empty capsid generation, and possible other impurities. However, no solution exists to solve the problem. The process illustrated in Example 1, the clear physical separation of the host cell and circulating media, allowed examination of extended kinetics of vector production post transfection. This was achieved by utilizing fractional harvests that allowed for the exploration and evaluation of both host cell health and capsid / vector formation kinetics over an extended period.WSGR Docket No. 69389-701.601

[0185] Three kinetic experiments were conducted using the same materials and methods but varying durations. (FIG. 18B): Experiment 1: Three-day / 72 hour run; Experiment 2: Five-day / 114 hour run; and Experiment 3 : Seven-day / 162 hour run. In all three experiments, the specialized HEK293 cells were inoculated into the fixed-bed bioreactor and cultured to achieve a target transfection cell density, at which time perfusion via recirculation transfection reagent was allowed to occur for ~18 hours. Starting one day post-transfection (day 2), the supernatant was harvested daily and replaced with fresh media. In Experiment 3, before lysis, samples strips embedded in the reactor were retrieved. Both the specialized HEK293 cells’ viability and their continued ability to divide and support viral vector production through re-culturing were examined. FIG. 19 illustrates cumulative viral genome yield in supernatant. In all three experiments, significant viral genome levels were detected in the supernatant on each day of harvest via dPCR. By extending harvest to 7 days posttransfection, the cumulative viral genome yield in the supernatant increased by 181% compared to the 3 -Day harvest. Without lysing cells, 7.33E14 vgin the supernatant alone was achieved. FIG. 20A illustrates viral genome yields from supernatant vs. lysate. With the perfusion -based, extended harvest process, vectors were allowed to mature and secrete into the supernatant without the need to lyse the host cells. The percentage of viral genome yield in the supernatant increased from 41% after 3 days to 88% after 7 days, while the lysate percentage decreased from 59% to 12%, respectively. This significantly decreased the residual host cell protein burden and allowed further utilization of these host progeny cells (FIGs21A-D) FIG.20B illustrates significant reduction in HCP impurity. A unique feature of the process in this example was the physical separation of the host specialized HEK293 cells and capsids / vectors in circulating media. By not lysing the host cells, the residual host cell protein burden was significantly decreased from what was already a lower baseline comparing to other production platforms. Data shown here are from the 7 day experiment. Even greater reduction was observed in 3 day and 5 day experiment. FIGs. 21 A-C illustrate sustained cell viability. Two sample strips from different locations were removed from the fixed-bed bioreactor 7 days post-transfection and stained with a Live / Dead cell imaging kit. Three representative images were chosen - green represents viable cells and red represents dead cells. Both sample strips showed high cell viability at 7-days post-transfection, while significant intracellular viral genome levels were still being detected. FIG. 21D illustrates longterm host cell viability and productivity post transfection. Cells were harvested from the bioreactor 7 days post-transfection and re-seeded onto a T-flask. After 3 days (or 10 days post-transfection), cellsWSGR Docket No. 69389-701.601maintained good health and reached high confluency, as shown in FIG. 2 ID, with viral genome levels still being detected in the supernatant at this point. After 12 days posttransfection, no intracellular vector was detected after lysing of the cells. This indicated that meaningful vector production in cells extended to around 10 days after transfection. FIGs.22A-F illustrate changing AUC profile of daily harvest media over the course of 7 days. Daily harvested material were sent to a commercial testing lab for direct AUC examination. The material was not processed at all. The clear product peak profiles is a testament to the level of purity and titer of our harvest material. The more immediate harvest (day 2) showed consistently high levels of empty capsid formation with the lack of clear full capsid peak. This is similar to what has beenreported. More interestingly, as time progresses, the daily harvest started to gradually skew towards full capsid generation, reaching -70% at later time points. FIG. 22G illustrates improved full capsid content. Daily full capsid yields were graphed with the total capsid yields, calculated with the measured ratios from AUC (FIGs. 22A-F), along with the cumulative % full of the supernatant harvests starting on Day 3. By discarding the Day 2 supernatant, which contained a large majority of empty capsids, a greater than 52% full capsids at harvest in the supernatant material was achieved. The 3-Day and 5-Day harvest experiments showed similar trends, in thatthe large majority of empty capsids were produced 2 days post-transfection, with much greater full capsid production thereafter.

[0186] As shown in this example, the perfusion-based, fixed-bed bioreactor production system and process was optimized for adherent cells. This production process can be further coupled with Fractional Timed Harvests to improve production efficiency. This allows extended study of the kinetics of AAV viral vector production in a commercial-scale adherent system past historical kinetic observations of 72 hours, allowing study of host cell behavior post-transfection in relation to vector formation over an extended duration. These results demonstrated that the process described in this example was gentle and effective in maintaining the health of host cells to realize their full production potential of AAV viral vectors, unlike current transfection methods that seekto maximize fed -batch productivity via additives, harsh reagents and supplements in a suspension or adherent environment. As shown in this example, host cells can thrive in an adherent, perfusion-based, hyperintensified bioreactor environment. During transfection, a perfusion via recirculation method can be utilized and the host cells can recover from the initial transfection shock quickly and continue to generate vectors for an extended period of time. Furthermore, the host cells such as the specialized HEK293 cells described herein can continue to divide, generate viralWSGR Docket No. 69389-701.601vectors 10 days post initial transfection, and behave more like transient producer cells. During the extended vector production kinetic study, the perfusion based process coupled with Fractional Timed Harvest showed improved full capsidcontent overtime. The early time point results are consistent with previous reports and findings that there is a significant wave of empty particles and likely other impurities more immediately after transfection. As such, the process described herein allows user to easily discard any portion of the harvest (especially the early time points) and greatly improve harvest quality with minimal reagents, less toxic procedurally to host cells, and maximum of host cells’ capability in vector generation, transforming the cells into more producer cell like.

[0187] With the process illustrated in this example, it was shown that the perfusion via recirculation process allowed for ultra-low density (1E3 & 5E2 cells / cmA2) seeding with the same even cell distribution and reaching a confluency / density readily usable for transfection based vector production. The robustness of cell growth at 5E2 cells / cmA2 showed supporting evidence that further reduction of seeding density to 250 cells / cmA2 was feasible. A vial of frozen host cells (clone of HEK293) at lE7cell could be thawed (-75% survival rate) and seeded into a reactor (<500 cells / cmA2) and grown into confluency, which bypassed the burdensome seed-train and paved way for upstream simplification and further automation.Example 2. Vero cell culturing

[0188] Vero cells were cultured, expanded, or monitored by the system and method described herein. FIG.23 illustrates a non-limiting example of a procedure for culturing Vero cells. By seed trainingthe Vero cells in roller bottles, Vero cells were growing robustly (about 7.5E7 cells per roller bottle at harvest). The reactor was seeded at 7.5E3 cells / cmA2. Cell growth was terminated after 90 hours for distribution study. FIG. 24 illustrates glucose or lactate trends of the Vero cells at numerous hours after inoculation. Equilibrium was maintained between the reactor and the recirculation vessel, which contained majority of the growth nutrients. FIG.25 illustrates cell density of the Vero cells. 7.5E3 cells / cmA2 of the Vero cells were seeded and grew for 90 hours. About 100 samples were cut from various part of the reactor, positions of the samples shown FIG.25. Cells were spread evenly at a cell density of 1 -2E5 / cmA2 throughout the reactor bed. Cell density was relatively low in the mid-section of the bed, indicating more growth potential. Red dots on the graph indicated samples cut from the second layer of nonwoven mesh. The samples showed consistent growth between first and second layer. Overall average cell density after 90 hours of growth was 1.28E5 cells / cmA2. FIG. 26 illustrates Vero cell viability (4X to show global view) of robust cell growth and minimum cell death.WSGR Docket No. 69389-701.601

[0189] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually and separately indicated to be incorporated by reference for all purposes.

Claims

WSGR Docket No. 69389-701.601CLAIMSWhat is claimed is:

1. A method of transfecting cells in a fixed-bed bioreactor, the method comprising:(a) providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate; and(b) contacting the plurality of cells with a cell culture media comprising a nucleic acid payload and a transfection reagent for at least 7 hours, such that the plurality of cells are transfected with the nucleic acid payload.

2. The method of claim 1, wherein the fixed-bed bioreactor comprises a volume of cell culture media, and the volume of cell culture media is replaced at least about 0.5 times per hour during the contacting.

3. The method of claim 2, wherein the volume of cell culture media is replaced from about 0.5 to about 5 times per hour during the contacting.

4. The method of any one of claims 1-3, further comprising adjusting the pH of the cell culture media to maintain the pH of the cell culture media within about 5% of a target pH during the contacting.

5. The method of claim 4, wherein the adjusting comprises monitoring the pH of the cell culture media, and adjusting a perfusion rate to alter the pH of the cell culture media.

6. The method of claim 4, wherein the adjusting comprises monitoring or detecting a level of at least one cell waste product in the cell culture media, and adjusting a perfusion rate to alter the amount of the at least one cell waste product in the cell culture media.

7. The method of claim 6, wherein the at least one cell waste product is or comprises lactate.

8. The method of claim 4, wherein the adjusting comprises monitoring or detecting a level of glucose in the cell culture media, and adjusting a perfusion rate to alter the amount of glucose in the cell culture media.

9. The method of any one of claims 1-8, further comprising adjusting a perfusion rate to maintain a concentration of the transfection reagent within about 25% of a target transfection concentration.

10. The method of any one of claims 1-9, wherein the fixed-bed bioreactor is coupled to a recirculation system.

11. The method of claim 10, wherein the fixed -bed bioreactor is coupled to the recirculation system via a closed loop.

12. The method of claim 10 or 11, wherein the recirculation system comprises at least one pump for adjusting a perfusion rate.WSGR Docket No. 69389-701.60113. The method of any one of claims 1-12, wherein the transfection reagent comprises calcium phosphate.

14. The method of any one of claims 1-13, wherein the fixed-bed bioreactor comprises at least one sensor.

15. The method of claim 14, wherein the at least one sensor comprises a pH sensor.

16. The method of claim 14 or 15, wherein the at least one sensor comprises a glucose sensor.

17. The method of any one of claims 14-16, wherein the at least one sensor comprises a lactate sensor.

18. The method of any one of claims 14-17, wherein the at least one sensor comprises a temperature sensor.

19. The method of any one of claims 14-18, wherein the at least one sensor comprises a dissolved oxygen sensor.

20. The method of any one of claims 14-19, wherein the at least one sensor comprises a pressure sensor.

21. The method of any one of claims 1-20, wherein the nucleic acid pay load comprises a viral plasmid.

22. The method of any one of claims 1 -21, wherein the plurality of cells comprises HEK293 cells.

23. The method of any one of claims 1-21, wherein the plurality of cells comprises Vero cells.

24. The method of any one of claims 1 -23, further comprising harvesting a plurality of viral particles from the plurality of cells.

25. The method of claim 24, wherein the viral particles are or comprise adeno-associated viral (AAV) particles or lentiviral particles.

26. The method of claims 24, wherein the viral particles express an antigen.

27. The method of any one of claims 24-26, wherein the viral particles comprise AAV particles and the method results in at least 2E13 vg / L AAV particles.

28. The method of any one of claims 24-27, further comprising replacing the cell culture media after the contacting with fresh cell culture media for a period of time.

29. The method of claim 28, wherein the harvesting occurs at least 24 hours, 48 hours, or 72 hours after the contacting of (b).

30. The method of any one of claims 24-29, further comprising removing empty capsids prior to the harvesting.WSGR Docket No. 69389-701.60131. The method of any one of claims 1 -30, wherein the contacting of (b) comprises continuously contacting the plurality of cells with the cell culture media during the contacting.

32. A method of culturing cells in a fixed-bed bioreactor, the method comprising:(a) providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate and a volume of cell culture media in contact with the plurality of cells; and(b) replacing the volume of cell culture media at least about 0.5 times per hour.

33. The method of claim 32, wherein the volume of cell culture media is completely replaced from about 0.5 times to about 4 times per hour.

34. The method of claim 32 or 33, further comprising monitoring the pH of the cell culture media.

35. The method of claim 34, further comprising adjusting a perfusion rate of the cell culture media to maintain a pH within about 5% of a target pH.

36. The method of any one of claims 32-35, further comprising monitoring or detecting one or more cell waste products in the cell culture media.

37. The method of claim 36, further comprising adjusting a perfusion rate to control a level of the one or more cell waste products in the cell culture media.

38. The method of claim 37, wherein the cell waste product is or comprises lactate, glucose, or both.

39. A method of producing a biologic, the method comprising:(a) providing or obtaining a fixed-bed bioreactor comprising a plurality of cells attached to a substrate;(b) continuously contacting, for a first time period, the plurality of cells with a first volume of cell culture media comprising a transfection reagent and a plasmid, such that at least one cell is transfected with the plasmid and produces a biologic;(c) replacing the first volume of cell culture media with a second volume of cell culture media that does not contain the transfection reagent and the plasmid by continuously contacting, for a second time period, the plurality of cells with the second volume of cell culture media;(d) replacing the second volume of cell culture media with a third volume of cell culture media by continuously contacting, for a third time period, the plurality of cells with the third volume of cell culture media; and(e) collecting the biologic during a fourth time period.WSGR Docket No. 69389-701.60140. The method of claim 39, wherein the biologic is or comprises a plurality of viral particles.

41. The method of claim 39, wherein the plasmid is or comprises at least one viral plasmid.

42. The method of claim 39 or 40, wherein the plurality of viral particles comprise adeno-associated viral (AAV) particles.

43. The method of any one of claims 40-42, wherein the plurality of viral particles expresses an antigen.

44. The method of claim 41, wherein the plasmid is selected from the group consisting of: a helper plasmid, a plasmid containing Rep and / or Cap genes, a transgene plasmid, and any combination thereof.

45. The method of any one of claims 39-44, wherein the first time period is at least 7 hours.

46. The method of any one of claims 39-45, wherein the second time period is at least 12 hours.

47. The method of any one of claims 39-46, wherein the third time period is from about 24 hours to about 48 hours after (b).

48. The method of any one of claims 39-47, wherein the fourth time period is from about 48 hours to about 72 hours after (b).

49. The method of any one of claims 39-48, further comprising contacting the plurality of cells with a lysis buffer to lyse the plurality of cells and release the AAV particles into the lysis buffer.

50. The method of claim 49, further comprising collecting the lysis buffer containing the AAV particles.

51. The method of any one of claims 39-50, comprising discarding the third volume of cell culture media.

52. The method of claim 51, wherein at least about 70% of the AAV particles in the third volume of cell culture media are empty AAV particles.

53. The method of any one of claims 42-52, wherein at least about 35% of the AAV particles collected in (e) are full AAV particles.

54. The method of claim 40, wherein the plurality of viral particles are or comprises lentiviral particles, retroviral particles, or adenoviral particles.

55. The method of claim 39, wherein the biologic is or comprises a protein or polypeptide.

56. The method of claim 39, wherein the biologic is or comprises an antibody or fragment thereof.

57. The method of claim 39, wherein the biologic is or comprises a vaccine.

58. The method of claim 39, wherein the biologic is or comprises an antigen.WSGR Docket No. 69389-701.60159. The method of any one of claims 39-58, wherein any one or any combination of (b), (c), (d), or (e) comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour.

60. The method of claim 59, wherein any one or any combination of (b), (c), (d), or (e) comprises completely replacing the full bioreactor volume of cell culture media at a rate of from about 0.5 times to about 4 times per hour.

61. The method of any one of claims 39-60, further comprising, prior to (e), (f) replacing the third volume of cell culture media with a fourth volume of cell culture media by continuously contacting, for a fifth time period, the plurality of cells with the fourth volume of cell culture media.

62. The method of claim 61, wherein the fifth time period is from about 72 hours to about 96 hours after (b).

63. The method of claim 61 or 62, further comprising, prior to (e) and after (f), (g) replacing the fourth volume of cell culture media with a fifth volume of cell culture media by continuously contacting, for a sixth time period, the plurality of cells with the fifth volume of cell culture media.

64. The method of claim 63, wherein the sixth time period is greater than about 96 hours after (b).

65. The method of any one of claims 61-64, wherein (f), (g), or both comprises completely replacing a full bioreactor volume of cell culture media at a rate of at least about 0.5 times per hour.

66. The method of any one of claims 39-65, wherein the collecting of (e) comprises continuously perfusing the bioreactor with a volume of cell culture media or lysis buffer during the course of the fourth time period.

67. The method of any one of claims 39-66, wherein the fixed-bed bioreactor is coupled to a recirculation system.

68. The method of claim 67, wherein the recirculation system is coupled to a plurality of fixed-bed bioreactors.

69. A composition comprising at least 2E13 vg / L viral particles, and no amount or an undetectable amount of a lipid-based or a polymer-based transfection reagent.

70. The composition of claim 69, wherein the undetectable amount is an amount of the lipid-based or polymer-based transfection reagent that is undetectable by high-performance liquid chromatography (HPLC).WSGR Docket No. 69389-701.60171. The composition of claim 69 or 70, wherein the polymer-based transfection reagent is a polyethylenimine (PEI) transfection reagent.

72. The composition of any one of claims 69-71, further comprising an amount of calcium detectable by HPLC.

73. The composition of any one of claims 69-72, wherein the viral particles comprise AAV particles.

74. A bioreactor system for producing gene therapy products, comprising:(a) a reactor vessel containing a fixed-bed substrate for cell growth;(b) a recirculation mixing vessel fluidically connected to the reactor vessel;(c) a recirculation path forming a closed loop between the reactor vessel and the recirculation mixing vessel;(d) a pump configured to control a recirculation rate between the recirculation mixing vessel and the reactor vessel; and(e) a gas supply connected to the system to provide gases for cell growth and metabolism.

75. The bioreactor system of claim 74, wherein the fixed-bed substrate comprises a plurality of layers of material.

76. The bioreactor system of claim 75, wherein the plurality of layers of material comprise at least one cell immobilization layer and at least one flow distribution layer.

77. The bioreactor system of claim 76, wherein the at least one cell immobilization layer comprises a fibrous structure for cell attachment.

78. The bioreactor system of any one of claims 74-77, wherein the pump is configured to control the recirculation rate to achieve an exchange rate of between 0.5 and 5 reactor volumes per hour.

79. The bioreactor system of any one of claims 74-78, further comprising a pH control system configured to maintain pH within a predetermined range during cell growth, transfection, and production.

80. The bioreactor system of claim 79, wherein the pH control system comprises pH sensor, wherein in response to detecting an out-of-range pH, the pH control system is configured to release a solution into the recirculation mixing vessel.

81. The bioreactor system of claim 79, wherein the pH control system comprises a base pump connected to the recirculation path for adjusting pH of media circulating through the system.

82. A method for producing a biologic in a bioreactor system, the method comprising:(a) seeding cells on a fixed-bed substrate in a reactor vessel;WSGR Docket No. 69389-701.601(b) circulating media between the reactor vessel and a recirculation mixing vessel through a closed loop recirculation path or a uni-directional perfusion;(c) adjusting pH in the reactor vessel using the recirculation path;(d) introducing a transfection mixture into the system;(e) incubating the cells with the transfection mixture; and(f) harvesting the biologic from the system.

83. The method of claim 82, wherein seeding the cells comprises seeding at a density of about 1X102-5X106cells / cm2on the fixed-bed substrate.

84. The method of claim 83, wherein seeding the cells comprises seeding at the density of about 1X103-5X105cells / cm2on the fixed-bed substrate.

85. The method of claim 84, wherein seeding the cells comprises seeding at the density at about IxlO3, 5xl03, 7.5xl03, or IxlO4cells / cm2on the fixed-bed substrate.

86. The method of any one of claims 82-85, wherein adjustingthe pH comprises maintaining a pH between 7.0 and 7.8 during cell growth and transfection.

87. The method of claim 86, wherein adjustingthe pH comprises maintaining a pH of approximately 7.6 during a pre-transfection phase and a pH of approximately 7.4 during a transfection phase.

88. The method of any one of claims 82-87, wherein circulating the media comprises controlling a recirculation rate, via the pump, to achieve an exchange rate of between 0.5 and 4 reactor volumes per hour.

89. The method of any one of claims 82-88, wherein the transfection mixture comprises 8 -20% of an overall working volume of the bioreactor system.

90. The method of any one of claims 82-89, wherein harvesting the biologic comprises:(a) obtaining a supernatant from the reactor vessel;(b) performing at least one rinse or wash of the fixed-bed substrate; and(c) lysing cells remaining on the fixed-bed substrate.

91. The method of any one of claims 82-90, wherein the transfection mix containing media is exchanged with serum-free media between 12 - 36 hours after transfection, and the serum-free media is recirculated through the recirculation path utilizing an exchange rate of 0.5 and 5 reactor vessel volumes per hour via pump.

92. The method of any one of claims 82-91, wherein the recirculation mixing vessel monitors and controls temperature, pH, glucose, and lactate.WSGR Docket No. 69389-701.60193. The method of claim 92, further comprising modulating recirculation speed using the recirculation mixing vessel to control at least one of pH, temperature, glucose, and lactate in the reactor vessel.

94. The method of any one of claims 82-93, wherein the biologic comprises a gene therapy product.

95. The method of any one of claims 82-93, wherein the biologic comprises a virus.

96. The method of claim 95, wherein the virus expresses antigen.

97. The method of claim 95 or 96, wherein the biologic comprises a vaccine.

98. The method of any one of claims 84-97, wherein the cells grow from the density of about lxl03-5xl05cells / cm2to a second density of about IxlOMxlO6cells / cm2.

99. The method of claim 98, wherein the cells grow to the second density of about l.lxlO5, 1.2xl05, 1.3xl05, 1.4xl05, or 1.2xl05cells / cm2.

100. The method of any one of claims 83-97, wherein the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density.

101. The method of claim 100, wherein the cells grow to a second density that is at least two, four, six, eight, 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times of the density.

102. The method of any one of claims 98-101, wherein the cells grow to the second density at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding.

103. The method of any one of claims 82-102, wherein the cells do not exhibit decreased cell growth at about 24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding.

104. The method of any one of claims 82-103, wherein the cells do not exhibit increased cell death at about24 hours, 48 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, or 120 hours after seeding.

105. A method of monitoring cells cultured on the fixed-bed substrate of any one of claims 1 -104, comprising monitoring the cells for a plurality of days.

106. The method of claim 105, wherein the cells are transfected before or after of said monitoring.

107. The method of claim 105 or 106, wherein the cells are cultured on the fixed-bed substrate for at least one, two, three, four, five, six, seven, eight, nine, 10, 11, or 12 days.

108. The method of any one of claims 105-107, wherein the cells are monitored without lysis.WSGR Docket No. 69389-701.601109. The method of any one of claims 105-108, wherein the monitoring results in decreased toxicity on the cells.

110. The method of any one of claims 105-109, wherein the method allows the cells to generate progeny cells.

111. The method of claim 110, wherein the cells and the progeny cells produce a biologic.

112. The method of claim 110, wherein the cells or the progeny cells can be seeded on a second fixed-bed substrate.

113. The method of claim 112, wherein the cells or the progeny ells produce the biologic when seeded on the second fixed-bed progeny.

114. The method of any one of claims 105-113, wherein the monitoring comprises monitoring the cells or the biologic.

115. The method of claim 114, wherein the monitoring comprises monitoring a viability of the cells or the progeny cells.

116. The method of claim 114, wherein the monitoring comprising monitoring a quality of the biologic produced by the cells or the progeny cells.

117. The method of any one of claims 111-116, wherein the biologic comprises antibody, antigen, viral particle, viral capsid, vector, or a combination thereof.

118. The method of claim 114, wherein the monitoring results in increased quality of the biologic produced by the cells or the progeny cells.

119. The method of any one of claims 105-118, wherein the cells or the progeny cells can be transfected or re -transfected to produce the biologic.

120. The method of claim 119, wherein the cells or the progeny cells can be reseeded or transfected more than one time.

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