# US 7,510,875 B2 · Methods for producing purified adenoviral vectors > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,510,875 B2 - Document ID: US7510875B2 - Status: Granted - Kind: B2 - Application number: 10/033,571 - Filed: 2001-12-27 - Published: 2002-11-28 - Granted: 2009-03-31 - Earliest claimed: 1996-11-20 - Assignees: Introgen Therapuetics, Inc. - Inventors: Shuyuan Zhang; Capucine Thwin; Zheng Wu; Toohyon Cho; Deborah Wilson; Lucetta Caston - Web page: https://patentgrep.com/patent/US7510875B2 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 35/761: Adenovirus - C07K 14/4746: {p53} - C12N 15/86: Viral vectors - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C12N 2710/10045: Special targeting system for viral vectors - C12N 2710/10051: Methods of production or purification of viral material - C12N 2710/10322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2710/10332: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent - C12N 2710/10343: viral genome or elements thereof as genetic vector - C12N 2710/10351: Methods of production or purification of viral material - C12N 2810/6018: Adenoviridae - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production with cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge. ## Claims ### Claim 1 A method for making a purified adenovirus composition comprising: a) growing host cells in a media; b) providing nutrients to said host cells by perfusion or through a fed-batch process; c) infecting said host cells with an adenovirus; d) lysing said host cells to provide a cell lysate comprising adenovirus; and e) purifying adenovirus from said lysate by a process other than the use of cesium chloride density gradient centrifugation to provide a pharmaceutically acceptable purified adenovirus composition having a contaminating nucleic acid content of less than 400 pg per 1010 pfu virus and greater than or equal to about 60 pg per 1010 pfu virus. ### Claim 2 (depends on 1) The method of claim 1, wherein the purified adenovirus composition comprises 70%+/−10% of the starting PFU. ### Claim 3 (depends on 1) The method of claim 1, wherein the purified adenovirus composition comprises a substantially purified adenovirus composition. ### Claim 4 (depends on 1) The method of claim 1, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.2 ng/ml. ### Claim 5 (depends on 1) The method of claim 1, wherein the purified adenovirus composition has an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 6 (depends on 5) The method of claim 5, wherein the purified adenovirus composition has an A260/A280 ratio of 1.27+/−0.03. ### Claim 7 (depends on 1) The method of claim 1, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.8 ng/ml and an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 8 (depends on 1) The method of claim 1, wherein the media is serum-free. ### Claim 9 (depends on 1) The method of claim 1, wherein the host cells are grown in a bioreactor. ### Claim 10 (depends on 1) The method of claim 1, wherein the host cells are grown on microcarriers. ### Claim 11 (depends on 1) The method of claim 1, wherein said purified adenovirus composition comprises an adenoviral vector encoding an exogenous gene construct. ### Claim 12 (depends on 11) The method of claim 11, wherein said exogenous gene construct is operatively linked to a promoter. ### Claim 13 (depends on 12) The method of claim 12, wherein said promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase. ### Claim 14 (depends on 11) The method of claim 11, wherein said exogenous gene construct encodes a therapeutic gene. ### Claim 15 (depends on 14) The method of claim 14, wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisense jun, antisense trk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl , Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-1, MEN II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, G-CSF, thymidine kinase or p53. ### Claim 16 (depends on 15) The method of claim 15, wherein said therapeutic gene encodes p53. ### Claim 17 (depends on 1) The method of claim 1, wherein the adenovirus in said purified adenovirus composition is a replication-incompetent adenovirus. ### Claim 18 (depends on 17) The method of claim 17, wherein the adenovirus in said purified adenovirus composition is lacking at least a portion of the E1-region. ### Claim 19 (depends on 18) The method of claim 18, wherein the adenovirus in said purified adenovirus composition is lacking at least a portion of the E1A and/or E1B region. ### Claim 20 (depends on 17) The method of claim 17, wherein said host cells are capable of complementing replication. ### Claim 21 (depends on 1) The method of claim 1, wherein said host cells are 293 cells. ### Claim 22 (depends on 1) The method of claim 1, wherein said lysate is treated with a nuclease. ### Claim 23 (depends on 1) The method of claim 1, wherein said purified adenovirus composition comprises a pharmaceutically acceptable buffer. ### Claim 24 (depends on 23) The method of claim 23, wherein said purified adenovirus composition provides a unit dose of between 103 and 1015 PFU/dose. ### Claim 25 (depends on 23) The method of claim 23, wherein the purified adenovirus composition provides a unit dose of between 1010 and 1014 PFU/dose. ### Claim 26 (depends on 1) The method of claim 1, wherein said purifying is characterized by a process that includes at least one chromatography step to provide a pharmaceutically acceptable composition. ### Claim 27 (depends on 26) The method of claim 26, wherein said purifying is characterized by a process that includes a single chromatography step to provide a pharmaceutically acceptable composition. ### Claim 28 A method for making a purified adenovirus composition for therapeutic use comprising: a) growing host cells in a media comprising glucose wherein nutrients are provided to said host cells by perfusion or through a fed-batch process; b) infecting said host cells with an adenovirus; c) lysing said host cells by a lysis method other than freeze-thaw to produce a lysate comprising said adenovirus composition; and d) purifying adenovirus from said lysate to provide a purified adenovirus composition for therapeutic use. ### Claim 29 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use comprises 70%+/−10% of the starting PFU. ### Claim 30 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use comprises a substantially purified therapeutic adenovirus composition. ### Claim 31 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has a contaminating nucleic acid concentration of less than 0.8 ng/ml. ### Claim 32 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has a contaminating nucleic acid concentration of less than 0.2 ng/ml. ### Claim 33 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 34 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has an A260/A280 ratio of 1.27+/−0.03. ### Claim 35 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has a contaminating nucleic acid concentration of less than 0.8 ng/ml and an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 36 (depends on 28) The method of claim 28, wherein the purified adenovirus composition for therapeutic use has a BSA content below the detection level of a western blot assay. ### Claim 37 (depends on 28) The method of claim 28, wherein the media is serum-free. ### Claim 38 (depends on 28) The method of claim 28, wherein the host cells are grown in a bioreactor. ### Claim 39 (depends on 28) The method of claim 28, wherein the host cells are grown on microcarriers. ### Claim 40 (depends on 28) The method of claim 28, wherein said purified adenovirus composition for therapeutic use comprises an adenoviral vector encoding an exogenous gene construct. ### Claim 41 (depends on 40) The method of claim 40, wherein said exogenous gene construct is operatively linked to a promoter. ### Claim 42 (depends on 41) The method of claim 41, wherein said promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late, polyoma F9-1, or tyrosmase. ### Claim 43 (depends on 40) The method of claim 40, wherein said exogenous gene construct encodes a therapeutic gene. ### Claim 44 (depends on 43) The method of claim 43, wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisense jun, antisense trk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-1, MEN II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, G-CSF, thymidine kinase or p53. ### Claim 45 (depends on 44) The method of claim 44, wherein said therapeutic gene encodes p53. ### Claim 46 (depends on 28) The method of claim 28, wherein said purified adenovirus composition for therapeutic use is a replication-incompetent adenovirus. ### Claim 47 (depends on 46) The method of claim 46, wherein said purified adenovirus composition for therapeutic use is lacking at least a portion of the E1-region. ### Claim 48 (depends on 47) The method of claim 47, wherein the purified adenovirus composition for therapeutic use is lacking at least a portion of the E1A and/or E1B region. ### Claim 49 (depends on 28) The method of claim 28, wherein said host cells are capable of complementing replication. ### Claim 50 (depends on 28) The method of claim 28, wherein said host cells are 293 cells. ### Claim 51 (depends on 28) The method of claim 28, wherein said lysate is treated with a nuclease. ### Claim 52 (depends on 28) The method of claim 28, wherein said purified adenovirus composition for therapeutic use comprises a pharmaceutically acceptable buffer. ### Claim 53 (depends on 52) The method of claim 52, wherein said purified adenovirus composition for therapeutic use provides a unit dose of between 103 and 1015 PFU/dose. ### Claim 54 (depends on 52) The method of claim 52, wherein the purified adenovirus composition for therapeutic use provides a unit dose of between 1010 and 1014 PFU/dose. ### Claim 55 (depends on 28) The method of claim 28, wherein said purifying is characterized by a process that includes at least one chromatography step capable of providing a purified therapeutic adenovirus composition. ### Claim 56 (depends on 28) The method of claim 28, wherein said purifying is characterized by a process that includes a single chromatography step capable of providing a purified therapeutic adenovirus composition. ### Claim 57 (depends on 28) The method of claim 28, wherein the lysis method other than freeze-thaw is a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication to produce a crude lysate composition comprising adenovirus. ### Claim 58 (depends on 1) The method of claim 1, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.8 ng/ml. ### Claim 59 (depends on 1) The method of claim 1, wherein the purified adenovirus composition has a contaminating nucleic acid content of less than about 140 pg per 1010 pfu virus and greater than or equal to about 60 pg per 1010 pfu virus. ### Claim 60 (depends on 1) The method of claim 1, wherein the particle to pfu ratio is between about 36 and about 38. ### Claim 61 (depends on 1) The method of claim 1, wherein the purified adenovirus composition comprises a level of BSA that is below the detection level of a Western blot assay. ### Claim 62 (depends on 61) The method of claim 61, wherein the purified adenovirus composition comprises 70%+/−10% of the starting PFU. ### Claim 63 (depends on 61) The method of claim 61, wherein the purified adenovirus composition comprises a substantially purified adenovirus composition. ### Claim 64 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.2 ng/ml. ### Claim 65 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 66 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has an A260/A280 ratio of 1.27+/−0.03. ### Claim 67 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.8 ng/ml and an A260/A280 ratio of between about 1.2 and 1.3. ### Claim 68 (depends on 61) The method of claim 61, wherein the media is serum-free. ### Claim 69 (depends on 61) The method of claim 61, wherein the host cells are grown in a bioreactor. ### Claim 70 (depends on 61) The method of claim 61, wherein the host cells are grown on microcarriers. ### Claim 71 (depends on 61) The method of claim 61, wherein said purified adenovirus composition comprises an adenoviral vector encoding an exogenous gene construct. ### Claim 72 (depends on 71) The method of claim 71, wherein said exogenous gene construct is operatively linked to a promoter. ### Claim 73 (depends on 72) The method of claim 72, wherein said promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase. ### Claim 74 (depends on 71) The method of claim 71, wherein said exogenous gene construct encodes a therapeutic gene. ### Claim 75 (depends on 74) The method of claim 74, wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fms, antisense jun, antisense trk, antisense ret, antisense gsp, antisense hst, antisense bcl, antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NE-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, G-CSF, thymidine kinase or p53. ### Claim 76 (depends on 75) The method of claim 75, wherein said therapeutic gene encodes p53. ### Claim 77 (depends on 61) The method of claim 61, wherein the adenovirus in said purified adenovirus composition is a replication-incompetent adenovirus. ### Claim 78 (depends on 77) The method of claim 77, wherein the adenovirus in said purified adenovirus composition is lacking at least a portion of the E1-region. ### Claim 79 (depends on 78) The method of claim 78, wherein the adenovirus in said purified adenovirus composition is lacking at least a portion of the E1A and/or E1B region. ### Claim 80 (depends on 77) The method of claim 77, wherein said host cells are capable of complementing replication. ### Claim 81 (depends on 61) The method of claim 61, wherein said host cells are 293 cells. ### Claim 82 (depends on 61) The method of claim 61, wherein said lysate is treated with a nuclease. ### Claim 83 (depends on 61) The method of claim 61, wherein said purified adenovirus composition comprises a pharmaceutically acceptable buffer. ### Claim 84 (depends on 83) The method of claim 83, wherein said purified adenovirus composition provides a unit dose of between 103 and 1015 PFU/dose. ### Claim 85 (depends on 83) The method of claim 83, wherein the purified adenovirus composition provides a unit dose of between 1010 and 1014 PFU/dose. ### Claim 86 (depends on 61) The method of claim 61, wherein said purifying is characterized by a process that includes at least one chromatography step to provide a pharmaceutically acceptable composition. ### Claim 87 (depends on 61) The method of claim 61, wherein said purifying is characterized by a process that includes a single chromatography step to provide a pharmaceutically acceptable composition. ### Claim 88 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has a contaminating nucleic acid concentration of less than 0.8 ng/ml. ### Claim 89 (depends on 61) The method of claim 61, wherein the purified adenovirus composition has a contaminating nucleic acid content of less than about 140 pg per 1010 pfu virus and greater than or equal to about 60 pg per 1010 pfu virus. ### Claim 90 (depends on 61) The method of claim 61, wherein the particle to pfu ratio is between about 36 and about 38.