Claims for Patent: 7,419,808

Summary for Patent: 7,419,808

Title:	Methods and compositions for the production of adenoviral vectors
Abstract:	The present invention addresses the need to improve the yield of adenovirus when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of infection temperatures lower than 37.degree. C. in a cell culture system results in improved yields of adenovirus. In addition, it has been demonstrated that when host cells are grow in a bioreactor, initiating adenovirus infection by diluting the host cells with fresh media and adenovirus results in improved yield of adenovirus. Methods of adenoviral production and purification using infection temperatures less than 37.degree. C. are disclosed. Methods of adenoviral production and purification wherein the host cells are grown in a bioreactor and adenovirus infection is initiated by diluting the host cells with fresh media and adenovirus are also disclosed.
Inventor(s):	Zhang; Shuyuan (Sugar Land, TX), Pham; Hai (Houston, TX)
Assignee:	Introgen Therapeutics, Inc. (Austin, TX)
Application Number:	11/079,986
Patent Claims:	1. A method for producing an adenovirus, comprising: a) producing an adenovirus preparation in a bioreactor, comprising the steps of: (i) preparing a cell culture in the bioreactor by seeding the bioreactor with host cells and media; (ii) growing the host cells in the cell culture, comprising perfusing fresh media through the cell culture without the removal of cells from the bioreactor; (iii) initiating virus infection in the cell culture by diluting the cell culture with fresh media and adenovirus without the removal of cells from the bioreactor; and b) isolating adenovirus from the adenovirus preparation. 2. The method of claim 1, wherein the media is serum-free media. 3. The method of claim 1, wherein the media is protein-free media. 4. The method of claim 1, wherein the media is CD293. 5. The method of claim 1, wherein the bioreactor comprises a bioreactor that uses axial rocking of a planar platform to induce wave motions inside of the bioreactor. 6. The method of claim 5, wherein the wave motions are induced inside of a sterilized bag made of layers of polyethylene vinyl acetate and ethyl vinyl alcohol. 7. The method of claim 1, wherein the bioreactor is a disposable bioreactor. 8. The method of claim 1, wherein the bioreactor is a 10 L bioreactor. 9. The method of claim 1, wherein the bioreactor is a commercially-available bioreactor. 10. The method of claim 1, wherein preparing an adenovirus preparation further comprises monitoring pH of the media. 11. The method of claim 1, wherein preparing an adenovirus preparation further comprises monitoring dissolved oxygen tension in the media. 12. The method of claim 1, wherein preparing an adenovirus preparation further comprises monitoring temperature in the media. 13. The method of claim 1, wherein the media is perfused through a filter. 14. The method of claim 13, wherein the filter is internal to the bioreactor. 15. The method of claim 13, wherein the filter is external to the bioreactor. 16. The method of claim 13, wherein the filter is a floating flat filter. 17. The method of claim 13, wherein spent media is removed from the bioreactor through the filter. 18. The method of claim 1, wherein culture volume is maintained by a load cell used to trigger fresh medium addition. 19. The method of claim 1, wherein preparing an adenovirus preparation further comprises perfusing media beginning on day 3 of host cell growth. 20. The method of claim 1, wherein initiating virus infection is accomplished by diluting the host cells 2-fold to 50-fold with fresh media and adenovirus. 21. The method of claim 20, wherein the host cells are diluted 10-fold with fresh media and adenovirus. 22. The method of claim 1, wherein initiating virus infection comprises adding 20-100 vp/host cell. 23. The method of claim 22, wherein initiating virus infection comprises adding about 50 vp/host cell. 24. The method of claim 1, wherein preparing an adenovirus preparation further comprises allowing virus infection to proceed for about 4 days. 25. The method of claim 1, wherein isolating the adenovirus from the adenovirus preparation occurs at about 4 days after viral infection is completed. 26. The method of claim 1, wherein the adenovirus is a replication-deficient adenovirus. 27. The method of claim 26, wherein the host cells complement the growth of the replication-deficient adenovirus. 28. The method of claim 26, wherein the adenovirus lacks at least a portion of the E1-region. 29. The method of claim 26, wherein the adenovirus is lacking at least a portion of the E1A and/or E1B region. 30. The method of claim 1, wherein the host cells are selected from the group consisting of 293, PER.C6, 911, and IT293SF cells. 31. The method of claim 30, wherein the host cells are 293 cells. 32. The method of claim 1, wherein the adenovirus is a recombinant adenovirus. 33. The method of claim 32, wherein the recombinant adenovirus encodes a recombinant gene that is operatively linked to a promoter. 34. The method of claim 33, wherein the promoter is a tissue-specific promoter or an inducible promoter. 35. The method of claim 33, wherein the promoter is an SV40 EI, RSV LTR, beta-actin, CMV-IE, adenovirus major late, polyoma F9-l, tyrosinase promoter, alpha-fetal protein promoter, or egr-1. 36. The method of claim 33, wherein the recombinant gene is antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fins, 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-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, mda7, fus, interferon .alpha., interferon .beta., interferon .gamma., ADP, p53, ABLI, BLC1, BLC6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1, TNF, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rap1A, cytosine deaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1, DPC-4, FHIT, PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, Rb, zac1, DBCCR-1, rks-3, COX-1, TFPI, PGS, Dp, E2F, ras, myc, neu, raferb, fins, trk, ret, gsp, hst, abl, E1A, p300, VEGF, FGF, thrombospondin, BAI-1, GDAIF, or MCC. 37. The method of claim 33, wherein the recombinant gene is a gene encoding an ACP desaturase, an ACP hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNA polymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, a glucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, a hyaluronidase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lyase, a lysozyme, a pectinesterase, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase, a peptidease, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, a reporter gene, an interleukin, or a cytokine. 38. The method of claim 33, where the recombinant gene is a gene encoding carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione .beta.-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, .beta.-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease copper-transporting ATPase, Wilson's disease copper-transporting ATPase, cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, .alpha.-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase, or human thymidine kinase. 39. The method of claim 33, wherein the recombinant gene encodes growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin, angiotensin I, angiotensin II, .beta.-endorphin, .beta.-melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, .beta.-calcitonin gene related peptide, hypercalcemia of malignancy factor, parathyroid hormone-related protein, parathyroid hormone-related protein, glucagon-like peptide, pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin, vasopressin, vasotocin, enkephalinamide, metorphinamide, alpha melanocyte stimulating hormone, atrial natriuretic factor, amylin, amyloid P component, corticotropin releasing hormone, growth hormone releasing factor, luteinizing hormone-releasing hormone, neuropeptide Y, substance K, substance P, or thyrotropin releasing hormone. 40. The method of claim 33, wherein the recombinant gene is a p53 gene. 41. The method of claim 33, wherein the recombinant gene is a mda7 gene. 42. The method of claim 1, wherein isolating the adenovirus comprises lysing the host cells. 43. The method of claim 42, wherein lysing the host cells is by freeze-thaw, autolysis, or detergent lysis. 44. The method of claim 1, wherein isolating the adenovirus further comprises reducing the concentration of contaminating nucleic acids in the adenovirus preparation. 45. The method of claim 1, wherein isolating the adenovirus further comprises placing the adenovirus into a pharmaceutically acceptable composition. 46. The method of claim 1, wherein isolating the adenovirus further comprises purifying the adenovirus. 47. The method of claim 46, wherein purifying the adenovirus comprises a chromatography step. 48. The method of claim 47, wherein the chromatography step comprises subjecting the adenovirus to more than one chromatographic separation. 49. The method of claim 47, wherein the chromatography step involves subjecting the adenovirus to only one chromatographic separation. 50. The method of claim 49, wherein the chromatographic separation includes ion exchange chromatography. 51. The method of claim 1, further comprising analyzing virus production. 52. The method of claim 51, wherein virus production is analyzed using HPLC. 53. The method of claim 1, wherein isolating the adenovirus further comprises obtaining a purified adenovirus composition having one or more of the following properties: (a) a virus titer of between 1.times.10.sup.9 and about 1.times.10.sup.13 pfu/ml; (b) a virus particle concentration between about 1.times.10.sup.10 and about 2.times.10.sup.13 particles/ml; (c) a particle:pfu ratio between about 10 and about 60; (d) having less than 50 ng BSA per 1.times.10.sup.12 viral particles; (e) between about 50 pg and 1 ng of contaminating human DNA per 1.times.10.sup.12 viral particles; (f) a single HPLC elution peak consisting essentially of 97% to 99% of the area under the peak. 54. An adenovirus composition comprising between 5.times.10.sup.14 and 1.times.10.sup.18 viral particles, prepared by a process in accordance with claim 1. 55. The adenovirus composition of claim 54, wherein the composition is a pharmaceutically-acceptable composition. 56. The method of claim 1, wherein isolating the adenovirus from the adenovirus preparation comprises the steps of: (a) subjecting the adenovirus preparation to chromatography on a first chromatographic medium, whereby adenovirus particles are retained on the first chromatographic medium; (b) eluting adenovirus particles from the first chromatographic medium to produce an eluate of adenovirus particles; (c) subjecting adenovirus particles from the eluate to chromatography on a second chromatographic medium, wherein the second chromatographic medium retains one or more contaminants from the eluate and wherein the second chromotographic medium is not solely a size exclusion medium; and (d) collecting adenovirus particles from the eluate. 57. The method of claim 56, wherein the first chromatographic medium is selected from the group consisting of an anion exchange medium, cation exchange medium, immobilized metal affinity medium, sulfated affinity media, immunoaffinity medium, heparin affinity medium, hydroxyapatite medium, and hydrophobic interaction medium. 58. The method of claim 56, wherein the second chromatographic medium is selected from the group consisting of cation exchange media, anion exchange media, immobilized metal affinity media, sulfated affinity media, dye affinity media, hydroxyapatite media, immunoaffinity media, heparin affinity media, and hydrophobic interaction media. 59. The method of claim 1, wherein isolating the adenovirus from the adenovirus preparation comprises the steps of: (a) subjecting the adenovirus preparation to chromatography on a first chromatographic medium, whereby contaminants from the adenovirus preparation are retained on the first chromatographic medium; (b) subjecting adenovirus particles remaining in the eluant to chromatography on a second chromatographic medium whereby adenovirus particles from the eluant are retained on the second chromatographic medium, wherein when the second chromatographic medium is an anion exchange medium, then the first chromatographic medium is a medium other than a sulfonated polysaccharide affinity medium, and (c) eluting adenovirus particles from the second chromatographic medium. 60. The method of claim 59, wherein the first chromatographic medium is selected from the group consisting of an anion exchange medium, cation exchange medium, immobilized metal affinity medium, sulfated affinity media, immunoaffinity medium, heparin affinity medium, hydroxyapatite medium, and hydrophobic interaction medium. 61. The method of claim 59, wherein the second chromatographic medium is selected from the group consisting of cation exchange media, anion exchange media, immobilized metal affinity media, sulfated affinity media, dye affinity media, hydroxyapatite media, immunoaffinity media, heparin affinity media, and hydrophobic interaction media. 62. The method of claim 20, wherein initiating virus infection is accomplished by diluting the host cells 10-fold to 50-fold with fresh media and adenovirus.

Details for Patent 7,419,808

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	01/15/1974		2023-05-15
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	12/27/1984		2023-05-15
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/15/1985		2023-05-15
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/16/1990		2023-05-15