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° 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° 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.

1 . A method for producing an adenovirus, comprising: 
 a) preparing an adenovirus preparation, comprising the steps of: 
 (i) growing host cells in media; and  
 (ii) infecting the host cells with an adenovirus at a growth-permissive temperature of less than 37° C.; and  
   b) isolating adenovirus from the adenovirus preparation.    
     
     
         2 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature greater than 31° C. but less than 37° C.  
     
     
         3 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 32° C. to 36° C.  
     
     
         4 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 33° C. to 36° C.  
     
     
         5 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 34° C. to 36° C.  
     
     
         6 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 35° C. to 36° C.  
     
     
         7 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 32° C. to less than 37° C.  
     
     
         8 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 33° C. to less than 37° C.  
     
     
         9 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 34° C. to less than 37° C.  
     
     
         10 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 35° C. to less than 37° C.  
     
     
         11 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature within the range of 36° C. to less than 37° C.  
     
     
         12 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature of about 36° C.  
     
     
         13 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature of about 35° C.  
     
     
         14 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature of about 34° C.  
     
     
         15 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature of about 33° C.  
     
     
         16 . The method of  claim 1 , wherein infecting the host cells with an adenovirus is conducted at a temperature of about 32° C.  
     
     
         17 . The method of  claim 1 , wherein the media is DMEM+2% FBS.  
     
     
         18 . The method of  claim 1 , wherein the glucose concentration in said media is maintained between about 0.5 and about 3.0 gm glucose/liter.  
     
     
         19 . A method for producing an adenovirus, comprising: 
 a) preparing an adenovirus preparation, comprising the steps of: 
 (i) growing host cells in media in a bioreactor;  
 (ii) initiating virus infection by diluting the host cells with fresh media and adenovirus; and  
   b) isolating adenovirus from the adenovirus preparation.    
     
     
         20 . The method of  claim 19 , wherein the media is serum-free media.  
     
     
         21 . The method of  claim 19 , wherein the media is protein-free media.  
     
     
         22 . The method of  claim 19 , wherein the protein-free media is CD293.  
     
     
         23 . The method of  claim 19 , wherein the bioreactor comprises a bioreactor that uses axial rocking of a planar platform to induce wave motions inside of the bioreactor.  
     
     
         24 . The method of  claim 23 , wherein the wave motions are induced inside of a sterilized bag made of layers of polyethylene vinyl acetate and ethyl vinyl alcohol.  
     
     
         25 . The method of  claim 19 , wherein the bioreactor is a disposable bioreactor.  
     
     
         26 . The method of  claim 19 , wherein the bioreactor is a 10 L bioreactor.  
     
     
         27 . The method of  claim 19 , wherein the bioreactor is a commercially-available bioreactor.  
     
     
         28 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises monitoring pH of the media.  
     
     
         29 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises monitoring dissolved oxygen tension in the media.  
     
     
         30 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises monitoring temperature in the media.  
     
     
         31 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises perfusing the media through a filter.  
     
     
         32 . The method of  claim 31 , wherein the filter is internal to the bioreactor.  
     
     
         33 . The method of  claim 31 , wherein the filter is external to the bioreactor.  
     
     
         34 . The method of  claim 31 , wherein the filter is a floating flat filter.  
     
     
         35 . The method of  claim 31 , wherein preparing an adenovirus preparation further comprises removing spent media from the bioreactor through the filter.  
     
     
         36 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises maintaining culture volume by a load cell used to trigger fresh medium addition.  
     
     
         37 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises perfusing media beginning on day 3 of host cell growth.  
     
     
         38 . The method of  claim 19 , wherein diluting the host cells with media is accomplished by diluting the host cells 2-fold to 50-fold with fresh media and adenovirus.  
     
     
         39 . The method of  claim 38 , wherein the host cells are diluted 10-fold with fresh media and adenovirus.  
     
     
         40 . The method of  claim 19 , further comprising initiating virus infection in a second bioreactor.  
     
     
         41 . The method of  claim 19 , wherein initiating virus infection comprises adding 20-100 vp/host cell.  
     
     
         42 . The method of  claim 41 , wherein initiating virus infection comprises adding about 50 vp/host cell.  
     
     
         43 . The method of  claim 19 , wherein preparing an adenovirus preparation further comprises allowing virus infection to proceed for about 4 days.  
     
     
         44 . The method of  claim 19 , wherein isolating the adenovirus from the adenovirus preparation occurs at about 4 days after viral infection is completed.  
     
     
         45 . The method of  claim 19 , wherein the host cells complement the growth of the replication-deficient adenovirus.  
     
     
         46 . The method of  claim 19 , wherein the adenovirus is a replication-deficient adenovirus.  
     
     
         47 . The method of  claim 46 , wherein the adenovirus lacks at least a portion of the E1-region.  
     
     
         48 . The method of  claim 46 , wherein the adenovirus is lacking at least a portion of the E1A and/or E1B region.  
     
     
         49 . The method of  claim 19 , wherein the host cells are selected from the group consisting of 293, HEK293, PER.C6, 911, and IT293SF cells.  
     
     
         50 . The method of  claim 49 , wherein the host cells are HEK293 cells.  
     
     
         51 . The method of  claim 19 , wherein the adenovirus is a recombinant adenovirus.  
     
     
         52 . The method of  claim 51 , wherein the recombinant adenovirus encodes a recombinant gene that is operatively linked to a promoter.  
     
     
         53 . The method of  claim 52 , wherein the promoter is a tissue-specific promoter or an inducible promoter.  
     
     
         54 . The method of  claim 52 , wherein the promoter is an SV40 E1, RSV LTR, α-actin, CMV-IE, adenovirus major late, polyoma F9-1, tyrosinase promoter, α-fetal protein promoter, or egr-1.  
     
     
         55 . The method of  claim 52 , wherein the recombinant gene is 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-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 α; interferon β, interferon γ, 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, MYCLI, 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, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, VEGF, FGF, thrombospondin, BAI-1, GDAIF, or MCC.  
     
     
         56 . The method of  claim 52 , 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.  
     
     
         57 . The method of  claim 52 , 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, α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase, or human thymidine kinase.  
     
     
         58 . The method of  claim 52 , 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, β-endorphin, β-melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, β-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.  
     
     
         59 . The method of  claim 52 , wherein the recombinant gene is a p53 gene.  
     
     
         60 . The method of  claim 52 , wherein the recombinant gene is a mda7 gene.  
     
     
         61 . The method of  claim 19 , wherein isolating the adenovirus comprises lysing the host cells.  
     
     
         62 . The method of  claim 61 , wherein lysing the host cells is by freeze-thaw, autolysis, or detergent lysis.  
     
     
         63 . The method of  claim 19 , wherein isolating the adenovirus further comprises reducing the concentration of contaminating nucleic acids in the adenovirus preparation.  
     
     
         64 . The method of  claim 19 , wherein isolating the adenovirus further comprises placing the adenovirus into a pharmaceutically acceptable composition.  
     
     
         65 . The method of  claim 19 , wherein isolating the adenovirus further comprises purifying the adenovirus.  
     
     
         66 . The method of  claim 65 , wherein purifying the adenovirus comprises a chromatography step.  
     
     
         67 . The method of  claim 66 , wherein the chromatography step comprises subjecting the adenovirus to more than one chromatographic separation.  
     
     
         68 . The method of  claim 66 , wherein the chromatography step involves subjecting the adenovirus to only one chromatographic separation.  
     
     
         69 . The method of  claim 68 , wherein the chromatographic separation includes ion exchange chromatography.  
     
     
         70 . The method of  claim 19 , further comprising analyzing virus production.  
     
     
         71 . The method of  claim 70 , wherein virus production is analyzed using HPLC.  
     
     
         72 . The method of  claim 19 , 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 1×10 9  and about 1×1013 pfu/ml;    (b) a virus particle concentration between about 1×10 10  and about 2×10 13  particles/ml;    (c) a particle:pfu ration between about 10 and about 60;    (d) having less than 50 ng BSA per 1×10e12 viral particles;    (e) between about 50 pg and 1 ng of contaminating human DNA per 1×10 viral particles;    (f) a single HPLC elution peak consisting essentially of 97% to 99% of the area under the peak.    
     
     
         73 . An adenovirus composition comprising between 5×10 14  and 1×10 18  viral particles, prepared by a process in accordance with  claim 1  or  19 .  
     
     
         74 . The adenovirus composition of  claim 73 , wherein the composition is a pharmaceutically-acceptable composition.  
     
     
         75 . The method of  claim 19 , 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.    
     
     
         76 . The method of  claim 75 , 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, hydroxyapetite medium, and hydrophobin interaction medium.  
     
     
         77 . The method of  claim 75 , 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, hydroxyapetite media, immunoaffinity media, heparin affinity media, and hydrophobic interaction media.  
     
     
         78 . The method of  claim 19 , 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 wheregy 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.    
     
     
         79 . The method of  claim 78 , 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, hydroxyapetite medium, and hydrophobin interaction medium.  
     
     
         80 . The method of  claim 78 , 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, hydroxyapetite media, immunoaffinity media, heparin affinity media, and hydrophobic interaction media.