A method for reducing host cell dna contamination in recombinant adeno-associated virus

Abstract

The application belongs to the technical field of biotechnology, and discloses a method for reducing the residual host cell DNA (HCD) in a recombinant adeno-associated virus (rAAV). The method can significantly reduce the residual HCD in the inner and outer capsids of rAAV particles by adding Emricasan reagent to the cell culture solution during rAAV production. Compared with other rAAV production processes, the method can improve the purity of rAAV products, thereby improving the safety and drugability of rAAV related gene therapy products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for reducing the residual host cell DNA in recombinant adeno-associated virus. BACKGROUND

[0002] Gene therapy is to introduce, modify or remove specific genes into patient cells to repair or compensate for defective genes, so as to achieve the purpose of treating or preventing genetic diseases. In gene therapy, nucleic acid delivery mediated by viral vectors is the main way of gene introduction into cells. Recombinant adeno-associated virus (rAAV) vectors have many advantages such as long-term stable expression of exogenous genes, no integration into host genome, infection of dividing / non-dividing cells, wide range of host cells, strong specificity, low immunogenicity, etc., making them safe and effective gene therapy tools, and thus being considered as one of the most promising gene therapy vectors, which has been applied to gene therapy and clinical research of various diseases.

[0003] At present, multiple rAAV production platforms based on different host cell lines have been established, mainly including plasmid transfection production system based on HEK293 / Hela cells, production system based on insect cells and baculovirus, packaging cell or inducible packaging cell method, etc. In host cells, after the expression of capsid proteins and replication proteins, the viral genome is replicated and packaged into newly assembled capsids, thereby producing rAAV particles. In this process, host cell genome DNA, plasmid DNA used, etc. are also randomly and incorrectly packaged into the capsid. Outside the capsid, these non-target genome contaminant DNAs also attach to the outside of the viral particles or the capsid. The produced rAAV is subjected to further purification and concentration steps to remove various impurities produced during rAAV packaging, thereby improving the concentration and purity of rAAV. During the purification process, the addition of Benzonase can effectively remove most of the contaminant DNA outside the capsid, but the contaminant DNA inside the capsid cannot be effectively removed. These residual contaminant DNAs in rAAV preparations may trigger unnecessary immune responses of the host, cause unpredictable biological effects of gene expression, risk of integration into the host cell genome, etc., which adversely affect the efficacy and safety of gene therapy. Therefore, it is urgent to further study how to reduce the residual contaminant DNA in rAAV production, so as to improve the safety and effectiveness of rAAV, and thus accelerate the process of rAAV-mediated gene therapy. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art and provide a method for reducing the residual host cell DNA in recombinant adeno-associated virus, so as to effectively reduce the residual level of host cell DNA (HCD) inside and outside the capsid in rAAV preparations.

[0005] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a kit for reducing host cell DNA residual in recombinant adeno-associated virus, comprising 1.0 μM-5.0 μM of Emricasan, or a salt thereof, or a stereochemical isomer thereof.

[0007] Emricasan is shown as formula (I):

[0008]

[0009] As a preferred embodiment of the kit of the present application, it further comprises 30 U / mL-40 U / mL of Benzonase.

[0010] In a second aspect, the present application provides a composition for reducing host cell DNA residual in recombinant adeno-associated virus, comprising 1.0 μM-5.0 μM of Emricasan and 30 U / mL-40 U / mL of Benzonase.

[0011] In a third aspect, the present application provides application of Emricasan in preparation of a preparation for reducing host cell DNA residual in recombinant adeno-associated virus.

[0012] As a preferred embodiment of the application of the present application, the concentration of Emricasan is greater than 10 nM.

[0013] In a fourth aspect, the present application provides a method for reducing host cell DNA residual in recombinant adeno-associated virus, wherein Emricasan is added to cell culture solution when cells are cultured in rAAV.

[0014] As a preferred embodiment of the method of the present application, the concentration of Emricasan is greater than 10 nM.

[0015] As a further preferred embodiment of the method of the present application, the concentration of Emricasan is 1.0 μM-5.0 μM.

[0016] As a preferred embodiment of the method of the present application, the cells are at least one of HEK293, HEK-derived cells, CHO, CHO-derived cells, Hela cells, Vero cells, and SF-9 cells.

[0017] As a preferred embodiment of the method of the present application, the cells are cultured by suspension culture or adherent culture.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application can significantly reduce the residual HCD in the rAAV particle capsid by adding Emricasan reagent to the cell culture solution during rAAV production. Compared with other rAAV production processes, this method improves the purity of rAAV products, thereby improving the safety and drugability of rAAV related gene therapy products. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Statistical chart of genome titer and HCD residual of Emricasan added, no Benzonase treated lysate sample.

[0021] Figure 2 Statistical chart of genome titer and HCD residual of Emricasan added, no Benzonase treated primary purification sample.

[0022] Figure 3 Statistical chart of genome titer and HCD residual of Emricasan added, Benzonase treated primary purification sample.

[0023] Figure 4 Statistical chart of genome titer and HCD residual of Emricasan added, Benzonase treated final purification concentrated product. DETAILED DESCRIPTION

[0024] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described below in conjunction with specific examples. Those skilled in the art should understand that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0025] The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0026] Example 1: Genome titer and HCD residual of Emricasan added, no Benzonase treated lysate sample

[0027] (1) Cell recovery and subculture

[0028] Remove the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank and immediately place them in a 37°C water bath with gentle shaking to rapidly thaw the cells. Inside a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL of Dynamis (Gibco, Cat#A2661501) medium. Then, place the culture flask in a 37°C, 5% CO2 incubator with shaking at 100rpm for 2–3 days.

[0029] Inside the biosafety cabinet, take 0.5 mL of suspension cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, passage the cells (passage every 2-3 days). At a final cell concentration of 3.0E+05-5.0E+5 cells / mL, take an appropriate volume of suspension cell culture and place it in a 250 mL culture flask, adding Dynamis medium to a total volume of 120 mL. Incubate the flask at 37°C with 5% CO2 at 100 rpm for 2-3 days.

[0030] (2) Transfect cells with the rAAV triple plasmid

[0031] On the day of transfection, 0.5 mL of suspended cells were collected for cell counting and viability testing. The cells were then diluted to 2.6E+06 cells / mL using Dynamis medium. The cell dilution was aliquoted into 125 mL culture flasks at 20 mL / flask, and Emricasan was added to final concentrations of 0 μM, 3.0 μM, and 5.0 μM, respectively. The culture flasks were then incubated at 37°C with a 5% CO2 incubator and shaken at 100 rpm.

[0032] The plasmids pGOI-eGFP (Addgene: 68550), pRC-AAV9 (GenBank: KF032296), and pHelper (GenBank: AF369965) were mixed at a ratio of 28 μg:33 μg:7.0 μg per bottle, and added to 1.0 mL of DMEM medium. The mixture was then pipetted and thoroughly mixed. 68 μL of the mixture was added... (Polyplus, Cat#101000044) Transfection reagent, mix well by pipetting, and let stand at room temperature for 30 minutes. Add DNA / The transfection mixture was added to a bottle containing suspension cells and placed in a 37°C, 5% CO2 incubator with shaking at 100 rpm.

[0033] (3) Genomic titer and HCD residue determination

[0034] On day 3 post-transfection, 10% cell lysis buffer was added to the culture flask, and the flask was incubated at 37°C with 5% CO2 and shaken at 100 rpm for 2 h. 1.0 mL of the lysis buffer was transferred to a 1.5 mL centrifuge tube and centrifuged at 4000 rpm for 3 min. 100 μL of the supernatant was used to determine genomic titer and HCD residual levels.

[0035] The genomic titer of rAAV samples was determined by qPCR using the AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03). First, the rAAV lysate supernatant was digested with DNase I (TaKaRa, Cat#2270A) and diluted 4000-fold as a qPCR template. ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') and ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers. Serially diluted plasmid standards were used as quantitative references. A standard curve was constructed using the serially diluted plasmid references, and the genomic titer of the rAAV samples was calculated based on the standard curve. The genomic titer test results showed no significant difference in genomic titer between the 3.0 μM and 5.0 μM M micasan groups compared to the control group. Figure 1 ).

[0036] HCD residues were determined by qPCR using the HEK293 Residual DNA Fragment Analysis Kit (Hubei Shenke, Cat#1103176): rAAV lysis supernatant was diluted 200 μL and used as the qPCR template; the reaction system was prepared according to the kit instructions, and serially diluted HEK293 DNA quantitative reference standards were set up. A standard curve was constructed using the serially diluted HEK293 DNA quantitative reference standards. The HCD residue amount in the rAAV samples was calculated based on the standard curve, and the HCD residue level was calculated based on the corresponding genomic titer.

[0037] The HCD residue test results showed that the HCD residue level in the rAAV lysis buffer samples without Benzonase treatment was relatively high; compared with the Control group, the HCD residue levels in the groups with added 3.0 μM and 5.0 μM Emricasan were significantly reduced to 0.06 and 0.05 times, respectively. Figure 1 ).

[0038] Example 2: Genomic titer and HCD residue in pre-purified samples with added Emricasan and without Benzonase treatment

[0039] (1) Cell resuscitation and passage culture

[0040] Remove the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank and immediately place them in a 37°C water bath with gentle shaking to rapidly thaw the cells. Inside a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL of Dynamis (Gibco, Cat#A2661501) medium. Then, place the culture flask in a 37°C, 5% CO2 incubator with shaking at 100rpm for approximately 2–3 days.

[0041] Inside the biosafety cabinet, take 0.5 mL of suspension cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, passage the cells (passage every 2-3 days). At a final cell concentration of 3.0E+05-5.0E+05 cells / mL, take an appropriate volume of suspension cell culture and place it in a 250 mL culture flask, adding Dynamis medium to a total volume of 120 mL. Incubate the flask at 37°C with 5% CO2 at 100 rpm for approximately 2-3 days.

[0042] (2) Transfect cells with the rAAV triple plasmid

[0043] On the day of transfection, 0.5 mL of suspended cells were collected for cell counting and viability testing. The cells were then diluted to 2.6E+06 cells / mL using Dynamis medium. The cell dilution was aliquoted into 125 mL culture flasks at 20 mL / flask, and Emricasan was added to final concentrations of 0 μM, 1.0 μM, and 2.0 μM, respectively. The culture flasks were then incubated at 37°C with a 5% CO2 incubator and shaken at 100 rpm.

[0044] Mix pGOI-eGFP, pRC-AAV1 (Addgene: 112862), and pHelper plasmid at a ratio of 28 μg:33 μg:7.0 μg per bottle, add to 1.0 mL of DMEM medium, and mix thoroughly by pipetting. Add 68 μL of [the mixture / medium]... (Polyplus, Cat#101000044) Transfection reagent, mix well by pipetting, and let stand at room temperature for 30 minutes. The transfection mixture was added to a bottle containing suspension cells and placed in a 37°C, 5% CO2 incubator with shaking at 100 rpm.

[0045] (3) Genomic titer and HCD residue determination

[0046] On day 3 post-transfection, 10% cell lysis buffer was added to the vial, and the cells were incubated at 37°C with 5% CO2 and shaken at 100 rpm for 2 hours for lysis. The samples were then loaded into POROS. TM GoPure TM In an AAV9 pre-packed column (ThermoFisher, Cat#A36650), impurities were washed with 0.1M PBS buffer (pH=7.4), followed by elution with 3 mL glycine-hydrochloric acid buffer (pH2.7), and the sample was collected and neutralized in 1M Tris-HCl buffer (pH=8.0) containing 8% of the purified sample. 100 μL of this initially purified sample was used to determine genomic titer and HCD residual levels.

[0047] The genomic titer of rAAV samples was determined by qPCR using the AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03). First, the pre-purified rAAV samples were digested with DNase I (TaKaRa, Cat#2270A) and diluted 40,000 times as a qPCR template. ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers. Serially diluted plasmid standards were used as quantitative references. A standard curve was constructed using the serially diluted plasmid references, and the genomic titer of the rAAV samples was calculated based on the standard curve. The genomic titer test results showed that the genomic titer level in the pre-purified rAAV samples without Benzonase treatment was low; compared to the Control group, the genomic titers of the 1.0 μM and 2.0 μM Emricasan groups were significantly different. Figure 2 ).

[0048] HCD residues were determined by qPCR using the HEK293 Residual DNA Fragment Analysis Kit (Hubei Shenke, Cat#1103176): The pre-purified rAAV sample was diluted 100 times and used as a qPCR template. The reaction system was prepared according to the kit instructions, and serially diluted HEK293 DNA quantitative reference standards were set up. A standard curve was constructed using the serially diluted HEK293 DNA quantitative reference standards. The HCD residue amount in the rAAV sample was calculated based on the standard curve, and the HCD residue level was calculated based on the corresponding genomic titer.

[0049] The HCD residue test results showed that the HCD residue level was high in the untreated rAAV pre-purified samples; compared with the control group, the HCD residue levels in the groups with added 1.0 μM and 2.0 μM Emricasan were significantly reduced to 0.04 and 0.02 times, respectively.Figure 2 ).

[0050] Example 3: Genomic titer and HCD residue in pre-purified samples treated with Emricasan and Benzosenase

[0051] (1) Cell resuscitation and passage culture

[0052] Remove the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank and immediately place them in a 37°C water bath with gentle shaking to rapidly thaw the cells. Inside a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL of Dynamis (Gibco, Cat#A2661501) medium. Then, place the culture flask in a 37°C, 5% CO2 incubator with shaking at 100rpm for approximately 2–3 days.

[0053] Inside the biosafety cabinet, take 0.5 mL of suspension cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, passage the cells (passage every 2-3 days). At a final cell concentration of 3.0E+05-5.0E+5 cells / mL, take an appropriate volume of suspension cell culture and place it in a 250 mL culture flask, adding Dynamis medium to a total volume of 120 mL. Incubate the flask at 37°C in a 5% CO2 incubator with shaking at 100 rpm for approximately 2-3 days.

[0054] (2) Transfect cells with the rAAV triple plasmid

[0055] On the day of transfection, 0.5 mL of suspended cells were collected for cell counting and viability testing. The cells were then diluted to 2.6E+06 cells / mL using Dynamis medium. The cell dilution was aliquoted into 125 mL culture flasks at 20 mL / flask, and Emricasan was added to final concentrations of 0 μM, 1.0 μM, and 2.0 μM, respectively. The culture flasks were then incubated at 37°C with a 5% CO2 incubator and shaken at 100 rpm.

[0056] Mix pGOI-eGFP, pRC-AAV1, and pHelper plasmids at a ratio of 28 μg:33 μg:7.0 μg per vial, add to 1.0 mL of DMEM medium, and mix thoroughly by pipetting. Add 68 μL of [the mixture / medium]... (Polyplus, Cat#101000044) Transfection reagent, mix well by pipetting, and let stand at room temperature for 30 minutes. Add DNA / The transfection mixture was added to a bottle containing suspension cells and placed in a 37°C, 5% CO2 incubator with shaking at 100 rpm.

[0057] (3) Genomic titer and HCD residue determination

[0058] On day 3 post-transfection, 10% cell lysis buffer and 40 U / mL Benzonase were added to the vial, and the cells were incubated at 37°C with 5% CO2 and shaken at 100 rpm for 2 hours for lysis. The samples were then loaded into POROS. TM GoPure TM In an AAV9 pre-packed column (ThermoFisher, Cat#A36650), impurities were washed with 0.1M PBS buffer (pH=7.4), followed by elution with 3 mL glycine-hydrochloric acid buffer (pH2.7), and the sample was collected and neutralized in 1M Tris-HCl buffer (pH=8.0) containing 8% of the purified sample. 100 μL of this initially purified sample was used to determine genomic titer and HCD residual levels.

[0059] The genomic titer of rAAV samples was determined by qPCR using the AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03). First, the initially purified rAAV samples were digested with DNase I (TaKaRa, Cat#2270A) and diluted 40,000-fold as qPCR templates. ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers. Serially diluted plasmid standards were set as quantitative references. A standard curve was constructed using the serially diluted plasmid references, and the genomic titer of the rAAV samples was calculated based on the standard curve. The genomic titer test results showed a slight increase in genomic titer compared to the control group with the addition of 1.0 μM and 2.0 μM methylasan. Figure 3 ).

[0060] HCD residues were determined by qPCR using the HEK293 Residual DNA Fragment Analysis Kit (Hubei Shenke, Cat#1103176): The pre-purified rAAV sample was diluted 100 times and used as a qPCR template. The reaction system was prepared according to the kit instructions, and serially diluted HEK293 DNA quantitative reference standards were set up. A standard curve was constructed using the serially diluted HEK293 DNA quantitative reference standards. The HCD residue amount in the rAAV sample was calculated based on the standard curve, and the HCD residue level was calculated based on the corresponding genomic titer.

[0061] The HCD residue test results showed that the HCD residue level in the rAAV pre-purified samples treated with Benzonase was very low; compared with the Control group, the HCD residue level in the groups with added 1.0 μM and 2.0 μM Emricasan was significantly reduced to 0.08 and 0.07 times, respectively. Figure 3 ).

[0062] Example 4: Genomic titer and HCD residue of the final purified and concentrated product treated with Emricasan and Benzosenase

[0063] (1) Cell resuscitation and passage culture

[0064] Remove the frozen Viral Production Cells 2.0 (Gibco, Cat#A49784) suspension cells from the liquid nitrogen tank and immediately place them in a 37°C water bath with gentle shaking to rapidly thaw the cells. Inside a biosafety cabinet, transfer the thawed cells to a 125mL culture flask containing 20mL of Dynamis (Gibco, Cat#A2661501) medium. Then, place the culture flask in a 37°C, 5% CO2 incubator with shaking at 100rpm for approximately 2–3 days.

[0065] Inside the biosafety cabinet, take 0.5 mL of suspension cells for cell counting and viability testing. When the cell density is greater than 3.0E+06 cells / mL and the cell viability is greater than 90%, passage the cells (passage every 2-3 days). At a final cell concentration of 3.0E+05-5.0E+05 cells / mL, take an appropriate volume of suspension cell culture and place it in a 250 mL culture flask, adding Dynamis medium to a total volume of 120 mL. Incubate the flask at 37°C with 5% CO2 at 100 rpm for approximately 2-3 days.

[0066] (2) Transfect cells with the rAAV triple plasmid

[0067] On the day of transfection, 0.5 mL of suspended cells were collected for cell counting and viability testing. The cells were then diluted to 2.6E+06 cells / mL using Dynamis medium. The cell dilution was aliquoted into 125 mL culture flasks at 20 mL / flask, and Emricasan was added to final concentrations of 0 μM, 3.0 μM, and 5.0 μM, respectively. The culture flasks were then incubated at 37°C with a 5% CO2 incubator and shaken at 100 rpm.

[0068] Mix pGOI-eGFP, pRC-AAV9, and pHelper plasmid at a ratio of 28 μg:33 μg:7.0 μg per flask, add to 1.0 mL of DMEM medium, and mix thoroughly by pipetting. Add 68 μL of [the mixture / medium]... (Polyplus, Cat#101000044) Transfection reagent, mix well by pipetting, and let stand at room temperature for 30 minutes. Add DNA / The transfection mixture was added to a bottle containing suspension cells and placed in a 37°C, 5% CO2 incubator with shaking at 100 rpm.

[0069] (3) Genomic titer and HCD residue determination

[0070] On day 3 post-transfection, cell lysis buffer to a final concentration of 10% was added to the culture flask, and the mixture was shaken for approximately 30 minutes to lyse. The lysis buffer was initially filtered through sterile filter paper, dissolved in 20 mL of virus diluent, and digested with Benzonase to a final concentration of 40 U / mL. Large particles were removed by centrifugation. Then, density gradient centrifugation was performed using iodixanol as the medium, followed by ultrafiltration to further purify and concentrate the rAAV virus particles, bringing the volume to 0.2 mL. 50 μL of this final purified and concentrated sample was used to determine genomic titer and HCD residual levels.

[0071] The genomic titer of rAAV samples was determined by qPCR using the AceQ Universal SYBR qPCR master kit (Vazyme, Cat#Q511-03). First, the rAAV samples were digested with DNase I (TaKaRa, Cat#2270A) to achieve final purification and concentration, and then diluted 80,000 times as a qPCR template. ITR-qF (5'-GGAACCCCTAGTGATGGAGTT-3') / ITR-qR (5'-CGGCCTCAGTGAGCGA-3') were used as primers. Serially diluted plasmid standards were set as quantitative references. A standard curve was constructed using the serially diluted plasmid references, and the genomic titer of the rAAV samples was calculated based on the standard curve. The genomic titer test results showed no significant difference in genomic titer between the 3.0 μM and 5.0 μM M micasan groups compared to the control group. Figure 4 ).

[0072] HCD residues were determined by qPCR using the HEK293 Residual DNA Fragment Analysis Kit (Hubei Shenke, Cat#1103176): The final purified and concentrated rAAV sample was diluted 200 μL and used as the qPCR template. The reaction system was prepared according to the kit instructions, and serially diluted HEK293 DNA quantitative reference standards were set up. A standard curve was constructed using the serially diluted HEK293 DNA quantitative reference standards. The HCD residue amount in the rAAV sample was calculated based on the standard curve, and the HCD residue level was calculated based on the corresponding genomic titer.

[0073] The HCD residue test results showed that the HCD residue level in the final purified and concentrated rAAV sample treated with Benzonase was very low; compared with the Control group, the HCD residue levels in the groups with the addition of 3.0 μM and 5.0 μM Emricasan were significantly reduced to 0.02 and 0.03 times, respectively. Figure 4 ).

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A kit for reducing host cell DNA residue in recombinant adeno-associated virus, characterized in that, It includes Emricasan, or its salts, or its stereochemical isomers, at concentrations of 1.0 μM to 5.0 μM; it also includes Benzonase at concentrations of 30 U / mL to 40 U / mL.

2. A composition for reducing host cell DNA residue in recombinant adeno-associated virus, characterized in that, This includes Emricasan at concentrations of 1.0 μM to 5.0 μM and Benzosenase at concentrations of 30 U / mL to 40 U / mL.

3. The application of Emricasan in the preparation of formulations that reduce host cell DNA residue in recombinant adeno-associated virus, characterized in that, The concentration of Emricasan was 1.0 μM-5.0 μM.

4. A method for reducing host cell DNA residue in recombinant adeno-associated virus, characterized in that, In rAAV production, Emricasan is added to the cell culture medium during cell culture; the concentration of Emricasan is 1.0 μM-5.0 μM.

5. The method according to claim 4, characterized in that, The cells are at least one of HEK293, HEK-derived cells, CHO, CHO-derived cells, HeLa cells, Vero cells, and SF-9 cells.

6. The method according to claim 4, characterized in that, The cultured cells are cultured using suspension culture or adherent culture.

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