HIGH YIElD PRODUCTION OF ACTIVE FACTOR IX

Abstract: High yields of active Factor IX are produced by culturing a CHO cell line transfected with chromosomally-integrated Factor IX cDNA in medium to which vitamin K is added.

Description: HIGH YIELD PRODUCTION OF ACTIVE FACTOR IX This " invention relates generally to the cloning and expression in high yield of Factor IX, and more particularly, to the production of biologically-active Factor IX by means of culturing mammalian cells into media containing vitamin K which Factor IX cDNA has been chromosomally-integrated. The plasma glyσoprotein. Factor IX, plays a critical role in the blood-clotting process. Normally synthesized in the liver. Factor IX requires vitamin K activity for the γ-σarboxylation of its 12 amino-terminal glutamic acid residues. A deficiency of Factor IX in the body characterizes a type of hemophilia (type B) . Treatment for this disease is presently limited to intravenous tranfusion of human plasma protein concentrates of Factor IX. However, in addition to the practical disadvantages of time and expense, transfusion of blood concentrates involves the risk of transmission of viral hepatitis, acquired immune deficiency syndrome or thromboemboliσ diseases to the recipient. An alternative method of producing Factor IX, other than extraction from human plasma, is therefore highly desirable. The application of recombinant DNA techniques to the production of Factor IX has elicited considerable information about the protein. The cDNA coding for human Factor IX has been isolated, characterized, and cloned into expression vectors. See, e.g., K. H. Choo et al, "Molecular Cloning of the Gene for Human Anti-hemophilic Factor IX', Nature, Vol. 299: 178-180 (Sept. 1982) and K. Kurachi et al, "Iso- lation and Characterization of a cDNA Coding for Human Factor IX," Proσ. Natl. Acad. Sci. U.S.A.. Vol. 79: 6461-65 (November 1982) . PCT patent application WO 84/00360 published February 16, 1984 describes the identification and cloning of a human Factor IX nucleotide sequence or fragments thereof for use primarily as diagnostic probes. This application 5 only prophetically refers to the production of a human Factor IX polypeptide through growth in mammalian tissue culture cells, preferably a hepatoma cell line. The inven¬ tors* subsequent article, D. S. Anson et al, "Expression of Active Human Clotting Factor IX from Recombinant DNA Clones o in Mammalian Cells", Nature, Vol. 315, pp 683-685 (June 20, 1985) , describes the production of very low levels of a pur¬ portedly active human Factor IXpolypeptide from a transformed rat hepatoma cell line. (See page 685, column 1) European patent application 162,782, publishedNovember s li, 1985, refers to construction of a recombinant viral vector containing a sequence coding for human Factor IX. Biologically active human Factor IX in low yields is assert- edly obtained from bacterial cells containing the episomally integrated recombinant Factor IX sequence. The influence 20 of viral components on growth of the host cells and glyσo- protein of the protein however can result in unreliable and varied batches of protein. [See, also H. de la Salle et al, "Active γ-σarboxylated human Factor IX expressed using recombinant DNA techniques," Nature, 316: 268-270 (July 18, 25 1985)3 Another recent report, S. Busby et al. "Expression of active human Factor IX in transfected cells," Nature, 316: 271-273 (July 18, 1985) also refers to expression of low 30 levels of recombinant Factor IX in BHK cells co-transfected with a neo gene marker. 35 Even these recent studies therefore demonstrate the continued difficulty in obtaining a stable system for producing high yields of Factor IX in biologically active and reliably consistent form, In accordance with the present invention it is sur¬ prisingly discovered that high yields of biologically active Factor IX protein can be produced by culturing a CHO cell line containing chromosomally integrated Factor IX cDNA and adding heterologous vitamin K to the culture medium for a predetermined time prior to harvesting the polypeptide. Even cells that have not previously been demonstrated to provide γ- carboxylation, can be shown to provide active Factor IX by this method. Vitamin K is added to the culture medium in the form of Kl or K3. Where K3 is the vitamin of choice, a concentrate of from about 0.1 ng to 10 ug of vitamin can be added per ml of cell culture, with the preferred concentration range of between 5 ng to 10 ug. Alternatively, vitamin Kl may be added in a desired concentration range of between about 10 ng to 50 ug of vitamin per ml of media or preferably 100 ng to 100 ug of vitamin per ml of media. The length of time required to produce the maximum quantity of active Factor IX can be easily determined by simpleexperimentation, i.e. analyzingaliquots ofconditioned medium at various time intervals for the amount of active Factor IX present until the maximum concentration is deter¬ mined. In another embodiment of the present invention, other biologically active blood coagulation proteins and plasma proteins containing gamma-carboxyglutamic acid are produced from cells transformed with σhromoso ally-integrated cDNA coding for such proteins by adding vitamin K to the cell culture medium. Examples of such blood coagulation proteins and plasma proteins produced in biologically active form from cDNA in accordance with the present invention include prothrombi ' n. Factor X, Factor VII, Protein C, 'Protein S, and the like. The biologically active Factor IX produced by the eucaryotiσ expression of the cloned Factor IX gene in accordance with the present invention can be used for the in vivo treatment of humans by physicians. The amount of active ingredient will, of course, depend upon the severity of the condition being treated, the route of administration chosen, and the specific ' activity of the active Factor IX. The active Factor IX produced according to the present invention may be administered by any route and in any pharmaceutical formulation and dosage which are typical for the natural serum-derived Factor IX. Desirably, the route, formulation and dosage regimens will take into account differences in activity, if any, between the natural Factor IX and the protein" produced as described herein. The following examples refer to the initial isolation and cloning of the Factor IX cDNA, sequencing thereof and the preparation of an expression vector system capable of expressing active Factor IX. Fig. 1 illustrates the structure of expression plasmids p91023-IX and pAaD26SVpA3. EXAMPLE 1 Isolation of human Factor IX cDNA clones A unique oligonucleotide with the sequence 5'pGTACAGGAGCAAACACC-3 ■ OHwas synthesizedwhichwashomologous to Factor IX RNA approximately 475 bp downstream from the initiator codon (Kurachi et al., 1982 supra; Choo et al., 1982, supra) . This oligonucleotide was labeled at the 5' end using polynucleotide kinase (New England Biolabs) and Y- 32 P-ATP (New England Nuclear) . Human liver double-stranded cDNA was prepared (Maniatis et al.. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, 1982) and inserted into a GT10 vector as described by Toole et al. , Nature, 313: 342-347, (1984) . Approximately 100,000 recombinant phage plaques were screened on duplicate filters with the labeled oligonucletide probe by the method of Woo et al. , Proσ. Nat'l. Acad. Sci USA. 75:3688-3692, (1978) . Hybridi¬ zation was at 42°C for 40 hours in 5X SSC, 5X Denhardt's, 0.5% by weight SDS and lOmM EDTA and washed extensively at 42° in 2X SSC. Duplicate positives were plaque purified and DNA was prepared from plate stocks [Maniatis et al., (1982) , supra] and analyzed by restriction digestion. EXAMPLE 2 Isolation of human Factor IX σenomic clones Three human genomiσ libraries were screened using human Factor IX cDNA nick-translated probes by the method of Benton, et al.. Science, 19^:180-182 (1977). The first library screened was an amplified Haelll/Alul partial library in Charon 4A [Lawn et al., Cell, 35: 1157-1174, (1978) ]. The second library was prepared by the method described by Maniatis et al., (1982) supra using a Sau3A partial digest cloned into Charon 28. A third library was prepared from the human lymphoblastoid line GM1202A (NIGMS mutant cell repository) which contains four X chromosomes. In this case Sau3A partial DNA was cloned as above into Jl (kindly supplied by J. Mullins) a derivative of L47.1 [Loenen, et al.. Gene .10:249 (1980)] in which polylinkers have been inserted replacing the two small EcoRl-BamHl fragments. Positive hybridizing plaques were purified and the phage DNA prepared following C s Cl2 step gradient purifi- cation of a one liter liquid lysate [Maniatis et al., (1982) , supra] . EXAMPLE 3 DNA sequence analysis DNA sequences were obtained following subσloning into M13 phage vectors [Norrander et al.. Gene, 26:101-106 (1983)] by the dideoxy chain termination method [Sanger et al., Proσ. Nat'l. Acad. Sci USA. 74 5463-5467 (1977)] using synthetic oligonucleotide primers. The Factor IX cDNA was subcloned into M13 vectors following an ExoIII generated series of deletions according to the following procedures. A plasmid containing the region to be sequenced is digested with a restriction enzyme which cuts at a unique site to one side of the region to be sequenced. The DNA (approximately 20ug) is ethanol precipitated and dissolved in lOOul of a solution containing 50mM Tris, pH 8.0, lmM MgCl 2 , lmM 2-mercaptoethanol (ExoIII buffer). The tube is warmed to 30°C and 200 units of_ExoIII (Pharmacia P-L Bio- chemicals) are added. Based on the estimated degradation rate under these conditions of approximately 200 base pairs/min/end, aliquots are quenched at 30 second intervals thereby generating ends throughout the region to be sequenc¬ ed. The aliquots are quenched by pooling into a tube containing 300ul of a solution containing 500mM NaCl and 20mM EDTA, pH 8.0 (Exo quench). The deleted DNA is ethanol precipitated and dissolved in 80ul of H 2 0. Mild SI digestion is initiated by the addition of 80ul of a solution containing 60mM sodium acetate, 2mM ZnSθ4, 500mM NaCl, 10% by weight glycerol, pH 4.6 and 50 units SI nuσlease (Sigma). After 15 minutes at 20°C the reaction is quenched with 40ul of 500mM Tris-HCl, 1M NaCl, pH 8.0 and ethanol precipitated. Blunt ends are created by subsequent treatment with 5 units of Klenow fragment of DNA polymerase (BRL) in a lOOul solution containing lOmM Tris-HCl, lOmM MgCl 2 lOmM 2- mercaptoethanol, lOOuM dXTP's (containing dATP, dCTP, DGTP, s dTTP) , pH 7.5 at 37°C for 15 minutes. The reaction is phenol extracted and the solution centrifuged through a 1ml G50 spin column (prepared in a 1ml syringe in lOmM Tris, pH 8.0) to recover lOOul. The DNA is ethanol precipitated and then digested by o a restriction enzyme (that leaves "sticky" ends) which cuts on the opposite end of the region to be sequenced than the original restriction cut site prior to ExoIII. Following digestion, the DNA fragments are resolved on a Tris-acetate agarose gel. Gel slices which correspond to approximately s 200 bp size classes throughout the size range desired are removed. One fragment end would be from an ExoIII generated blunt end within the region to be sequenced and the other end from the re-cut site. The DNA fragments are purified by glass powder affinity [Volgelstein and Gillespie, Proc.- 0 Nat'l. A'σad. Sci. USA. 7_6:615-619 (1979)] following dis¬ solution in Nal. These DNA fragments are then ligated into M13 cloning vectors in an assymetriσ manner wherein the blunt end is nearest the universal primer binding site. M13 recombinant 5 plaques which hybridize to a probe made specifically for the region over which sequence is desired are selected for subsequent preparation of single-stranded template DNA [Sanger et al., (1977), supra] . Generally it is necessary to sequence with universal primer only one or two isolates 30 from each size class to obtain overlapping sequence covering the entire region to be sequenced up to 4 kb. Second-strand sequence can be obtained by repeating the procedure, deleting 35 from the opposite side of the region to be sequenced. EXAMPLE 4 Recombination of Factor IX cDNA with Factor IX Exon 1 A 4.5 kb Hindi11 fragment containing the Factor IX 5 promoter and 5' coding region was cloned into Charon 21A. A 2.5 kb fragment of Factor IX cDNA containing the entire Factor IX coding sequence from the eleventh codon of the signal sequence coding region to the Xbal site 66 bp from the polyA tail was subcloned into AN7 plasmid (a derivative o of VX) . [See Maniatis et al., (1982), supra] . There were 57 bp of exon 1 sequence within the Factor IX cDNA. The exon 1 containing Charon 21A phage were plated on bacteria harboring the recombinant AN7 plasmid and subsequently harvested as a plate stock. Phage which had recombined s with the supF containing AN7 plasmid could now be selected by plating onto the supF" bacterial line W3110 [Maniatis et al., (1982), supra] . Approximately one in 2 x 10 4 phage were supF + . One isolate was chosen for large scale DNA preparation and shown by restriction mapping and subsequent sequence 0 analysis to have recombined correctly with the AN7 plasmid within the 57 bp region of homology to obtain a Factor IX "mini gene". EXAMPLE 5 Exonuσleolvtic removal of 5' non-coding sequence 5 The 2.3kb Xba fragment containing the Factor IX "mini gene" was purified from AN7 by glass powder affinity. Approximately 5 ug of the fragment was digested with 50 units of ExoIII in 40ul of ExoIII buffer. After 1 minute, 8ul aliquots were removed to 120ul of Exo quench at 15 0 second intervals The DNA was treated with exonuσlease SI nuσlease and Klenow fragment of polymeyase 1 as above and then ligated to Pst adapters 35 5'p-CTAGAGGCCTCTGCA GATCTCCGGAG-OH 5' The adapted DNA was then put onto a Tris-acetate agarose gel and DNA of approximately 2.3kb was purified by glass powder affinity and ligated into a M13mpll Pst vector. Recombinant plaques were screened with an oligonucleotide homologous to the first 17bp of Factor IX coding sequence RNA. Positive hybridizing plaques were sequenced with the same oligonucleotide to find the deletion end point at the 5' end of the gene. Universal primer sequencing determined the 3' deletion end point. The entire sequence of the biologically active recombinant Factor IX cDNA is shown in Table I below. TABLE I AAT T T ' * •3 AAA3G . r* ■ .-V..-.(_'_QUO TGAACATGAV -.AT C-CAGAA TCACCAGGCC TC-ATCΛ CΛT TGCCT i7A GGA7A7C7AC ΓCAGTGCTGA ATGTACAGTT TTTCTTGATC ATGAAAACGC C.-iACπΛΛATT Z75-\ \ τ ZZZC C.'V.AGA3 T TAATTCAGGT AAATTGGAAG AGTTTGTTCA AGGGAACCTT GAGAGA AAT GTATGGπAGA AAΛG7G7AG7 TTTGAAGAAG CACGAGAAGT TTTTGAAAAC ACTGAAAGAA CAAC7GAATT TTGGAAGCAG TA7G77GA7G GAGATCAGTG TGAGTCCAAT CCATGTTTAA ATGGCGGCAG TTGCAAGGAT GACAT7AA77 CC7A7GAA7G TTGGTGTCCC TTTGGATTTG AAGGAAAGAA CTGTGAATTA GATGTAACAT G7AACAT7AA GAA7GGCAGA TGCGAGCAGT TTTGTAAΛAA TAGTGCTGAT AACAAGG7GG 777GCTCCT6 7AC7GAGGGA TATCGAC77G CAGAAAACCA GAAGTCCTGT GπACCAGCAG 7GCCA7TTCC ATGTGGAAGA GTTTCTGTTT CACAAACTTC TAAGC7CACC CGTGCTGAGA CTGTTTTTCC TGATGTGGAC TATGTAAATT CTACTGAAGC TGAAACCA77 TTGGA7AACΛ TCACTCAAAG CACCCAATCA TTTAATGACT TCAC7CGGG7 7G77GG7GGA GA GA7GCCA AACCAGG7CA ATTCCCTTGG CAGGTTGT7T TGAATGGTAA AG.7TGATGCA 7TCTGTGGAG GC7C7ATCGT 7AA7GAAAAA TGGATTGTAA C7GCTGCCCA CTGTGTTGAA AC7GG7G77A AAA77ACAG7 TGTCGCAGGT GAACA7AA7A TTGAGGAGAC AGAACATACA GAGCAAAAGC GAΛA7GTGAT TCGAATTA77 CC7CACCACA ACTACΛATGC AGCTATTAAT AAGTACAACC AT6ACATTGC CCT7C7GGAA C7GGACGAAC CC77AG7GCT AAACAGCTAC GTTACACCTA TTTGCATTGC TGACAAGGAA TACACGAACA 7C77CC7CAA A777GGATCT GGCTATGTAA GTGGCTGGGG AAGAGTCTTC CACAAAGGGA GATCAGCT7T AGTTCT7CAG 7ACC77AGAG TTCCAC7TGT 7GACCGAGCC ACATGTCTTC GATCTACAAA G7TCACCATC 7A7AACAACA 7G77C7G7GC TGGCTTCCAT GAAGGAGGTA GAGATTCATG TCAAGGAGAT AGTGGGGGAC CCCA7G7TAC TG AGTGGAA GGGACCAGTT •TCTTAACTGG AATTATTAGC TGGGGTGAAG AG7G7GCAAT GAAAGΞCAAA TATGGAήTAT ATACCAAGGT ATCCCGGTAT GTCAACTGGA TTAAGGAAAA AACAAAGCTC ACTTAA T — - -. - - r - rr or. •* .. • *.&ft i oo ATTTCCAAGG TTAATTCATT GGAATTGAAA ATTAACΛGGG CCTCTCACTA ACTAATCACT TTCCCATCTT TTGTTAGATT TGAATATATA CAT-TCTATGA TCA7TGCT7T TTCTCTTTAC AGGGGAGAAT TTCATATT77 ACCTGAGCAA ATTGAT7AGA AAATG3AACC ACTAGAGGAA TA7AA7G7G7 TAGGAAATTA CAG7CA7TTC TGAAGCCCAG C CTTGACAA AATTG7GAAG T7AAA77C7C CAC7C7G7CC ATCAGATAC7 π i oo i lulli. ACT TGGCAA CTAACTCAC7 CAA7777CCC rCC7TAGCAG CA7TCCA7C7 TCCCSATCTT CTT7GCT7C7 CCAACCAAAA CATCATGTTT A77AG77C7G 7A7ACAGTAC AGGA7C7TTG i i π' i uTA 7CACGAGAAG GCTCAGTACC ACACTCATGA AGAAAGAACή CAGGAGTAGC TGAGπGGCTA AAACTCATCA AAAACAC7AC TCCTTTTCCT CTACCCTATT CC7CAATCTT TTACCTTTTC CAAATTCCCA ATTCCCCAAA 7CAGT7TTTC TCTTTCTTAC TCCCTCTCTC CCTTTTACCC TCCATGGTCG TTrAAGGAGA GATGGGGAGC ATCATTCTGT TATACTTCTG TACACAGTTA TACATGTCTA TCAAACCCA5 ACTTGCT7CC ATAGTGGAGA CTTGCTTTTC AGAACATAGG GATGAAGTAA GGTGCCTGAA AAGTTTGGGG GAAAAG777C TTTCAGAGAG T7AAGTTA77 TTATATATAT AATATATATA TAAAA7A7A7 AήTATACAAT A7AAA7A7A7 AGTGTGTGTG 7G7ATGCGTG TGTGTAGACA CACACGCATA CACACA7A7A ATGGAAGCAA 7AAGCCA77C TTAAGAGC7T G7A7GGTTAT GGAGGTCTGA CTAGGCA7GA 7TTCACGAAG GCAAGATTGG CA7A7CAG77 GTAACTAAAA AAGC7GACA7 TGACCCAGAC A7A7TGTACT CTTTCTAAArt ATAATAATAA 7AATGCTAAC AGAAAGAAGA GAACCGTTCG TTTGCAATCT ACAGCTAGTA GAGACTTGAG GAAGAATTCA ACAGTGTGTC TTCAGCAGTG TTCAGAGCCA AGCAAGAAGT TGAAG7TGCC TήGACCAGAG GACATAAGTA TCATG7C7CC TTTAACTAGC ATACCCCGAA GTGGAGAAGG ATGCAGCAGG CTCAAAGGCΛ Tή.AGTCATTC ^rtπ I ι_rt i_ι_π ACTήAGTTGT CC7TTTCTGG TTTCGTGTTC ACCATGGAAC ATTTTGATTA TAGTTAATCC 77C7A7C77G AATCTTCTAG AGAGTTGCTG ACCAACTGAC GTATGTTTCC CTTTGTGAAT TAATAAACTG GTG77C7GGT TCAAAAAAAA AA [Start and stop codons are underlined. The coding region is separated from the 3 • and 5' untranslated regions by a space. ] EXAMPLE 6 Monitoring Factor IX Expression To prepare samples for the following Factor IX assay, approximately 4x10 6 logarithmically growing cells were rinsed four times (5 ml each) with serum-free media containing additions as indicated. After 24 hours at 37°C samples were taken, frozen in a dry ice/ethanol bath, and stored at 70°C until assayed. A. Factor IX Elisa Microtitre plates were coated with human Factor IX murine monoclonal antibody (Hybritech) . The plates were washed, and sample or standard Factor IX preparations were added after dilution into alpha media. . The Factor IX standard was purified human Factor IX diluted from 4.0ug to l.Ong. The secondary antibody (rabbit anti Factor IX, Calbiochem) was applied and washed, and alkaline phosphatase conjugated goat anti-rabbit IgG (Zymea) was applied. The substrate was alkaline phosphate table (Sigma-#104) diluted in diethanolamine and results were read at 410nm. B. Factor IX Clotting Assays The one stage activated partial thromboplastin time assay as described by R. Biggs, Human Blood Coagulation Haemostasis and Thrombosis (Ed. 1) , Oxford, Blackwell, Scientific, pg. 614 (1972) was carried out with equal volumes of: 1) activated partial thromboplastin reagent (general diagnostics) 2) Factor IX deficient plasma (George B. King Biomedical) and 3) normal pooled plasma as standard (George B. King Biomedical) or the sample. One unit of activity is defined as that amount present in 1ml of normal pooled plasma. ' EXAMPLE 7 Expression of Factor IX in Mammalian Cells The Factor IX coding sequence was inserted into the Pst 1 site of plasmid p91023(A) which is described in Example 3 of Japanese patent publication No. 12288/86, published January 20, 1986 which corresponds to United States patent application Ser. No. 677,813, filed December 4, 1983. The resulting clones are screened for proper orientation of the Factor IX gene with those having proper orientation selected as p91023-IX. The Factor IX expression vector, p91023-IX, contains the SV40 enhancer upstream from the AdMLP, a cDNA copy of the adenovirus tripartite leader (TPL) , the adenovirus VA genes, [Kaufman, PNAS. 82:689-693 (1985)], and the inserted Factor IX cDNA coding region upstream from the DHFR coding region. EcoRl(Rl), BamHl(bam) and Xhol(X) restriction sites are indicated. The DHFR expression vector, pAdD26SVp (3) [Kaufman, et al., Mol. Cell. Bio. , 2.:1304-1309 (1982)], contains the adenovirus major late promoter including the first late leader and 5" splice site. The leader exon from the RNA transcript is spliced properly to an introduced 3 1 splice site. The vector contains the SV40 early polyadenylation site, 2.7 KB derived from pSVOa [Mellon et al.. Cell. 22:279-288 (1981)] which contains the ColEl origin of replication, a pBR322 derivative lacking sequences detrimental to replication in mammalian cells, tetracycline resistance, and the SV40 origin of replication. The Factor IX expression vectorp91023-IXwas introduced with pAdD26SVpA3, into DHFR deficient Chinese hamster ovary cells by calcium phosphate mediated DNA transfection. (See Figure 1.) Cells selected for the DHFR positive phenotype expressed Factor IX at a low level. Then the transformants were pooled and selected for growth in the following sequen¬ tially increasing concentrations of methotrexate (MTX) , i.e., 0.02, 0.1, 0.5, 1.0, 5.0 and 20uM. Cells selected in this manner contain amplified copies of the introduced DHFR genes as well as amplified Factor IX genes. The amplified genes were generally localized to one or two specific chromosomes [Kaufman et al., Mol. Cell. Bio. 2, 699-711 (1983)]. DUKX Bll DHFR deficient CHO cells (Urlaub, et al, Proc. Nat'l Acad. Sci. 77:4210-4220, 1980; Kaufman, et al, J. Mol. Biol. 159: 601-621, 1982) were transfected with a mixture of 25ug of p91023-IX and 2.5 ug of pAdD26SVpA3 (Kaufman & Sharp, op. cit) by calcium-phosphate. Trans- formants were selected in media lacking nucleosides as described (Kaufman & Sharp, op. cit) . A similar transfection which gave similar results included the addition of 2.5ug of pSV2 Neo [Southern, et al, J. Mol. APPI. Genet...1:327-341 (1982)]. For the latter transfection, the DHFR + transfor ants • were initially selected in media lacking nucleosides with the addition of the antibiotic G418 (lmg/ml) in order to select for the pSV2 neo marker. The addition of a G418 resistance marker can be useful to facilitate transfer of chromosomes containing transfected DNA into other cells where selection directly for the amplified DHFR gene copies is not possible. Initial transformants were pooled (approximately 25 transformants/pool) and subjected to growth in increasing concentrations of MTX. These pools were analyzed for Factor IX expression by 35 S-methionine labeling and immune precipitation of the conditioned media and cell extracts with a monoclonal antibody which recognizes human Factor IX. A band of approximately 55k daltons was observed in the cell extracts and its level increased as cells were selected for higher degrees of MTX resistance. When the conditoned medium was similarly analyzed, a heterogenous smear was observed which ranged from 72k daltons down to 55k daltons. The smear heterogeneity is likely due to the heterogeneity of glycosylation of the secreted material. In addition, the level of the secreted Factor IX antigen determined by Elisa increased approximately 3000-fold upon selection to) 20 um MTX. (See Table II) The level of Factor IX antigen (i.e. not biologically active) in the 5α3 pool of cells was determined to be 43.4ug/ml. TABLE II Factor IX antigen in conditioned medium of cells selected for growth in increasing concentrations of MTX. TMTX] UG/ML POOL 5 3 0 0 . 015 5 or 3 0 . 02 0. 15 5 α 3 0 . 1 6. 9 5 α 3 0 . 5 29 . 4 5 α 3 5 . 0 36. 0 5 α 3 20 . 0 43 . 4 EXAMPLE 8 Expression of Factor IX activity in CHO cells When the conditioned media from the Factor IX producing o CHO cells (5α3 in various concentrations of MTX) was analyzed for Factor IX activity, there was no activity detected above background. Vitamin K was added to the medium con¬ taining the CHO cells which produce Factor IX antigen. 5 Table III shows the results of monitoring Factor IX activity after adding increasing concentrations of. vitamin Kl (3-phytylmenadione, Sigma) to the CHO Factor IX producing cells (5 α 3, 20 urn MTX) for 24 hours. Significantly, as * the concentration of vitamin Kl increased to 5ug/ml, so did the Factor IX activity in the conditioned medium. Vitamin Kl had no effect on activity when added back to conditioned media immediately before the assay. Thus, in the presence of vitamin Kl, the CHO cells produced up to .275 units of active Factor IX/ml/day/10 6 cells. To determine the validity of a concern that very little of the fat soluble vitamin Kl was actually being delivered to the cell, a similar effect has been demonstrated for the water soluble derivative vitamin K3 (menadione, Sigma) . Lower levels of K3 are required for maximal activity (0.005ug/ml) to be obtained with vitamin K. The specificity of the vitamin K3 dependence on Factor IX activity was demonstrated by adding warfarin (at lug/ml) , a specific antagonist of vitamin K, which blocked the appearance of o active Factor IX. TABLE III Factor IX activitv v. vitamin Kl • 5 Cells Vitamin Kl m Units/ml/day 5 α 3 (20um) lOOng/ml 42.5 5 α 3 (20um) 500ng/ml 105.0 5 3 (20um) lug/ml 192.0 5 α 3 (20um) 5ug/ml 275.0 0 5 α 3 (20um) lOug/ml 268.0 CHO 5ug/ml 17.5 Thus the specific activity of factor from the CHO cells is 5 16.5 mU/ug of Factor IX. Since the level of Factor IX in normal plasma is 1 unit/ml/5ug of Factor IX protein, the specific activity (units of Factor IX activity/mg of Factor IX protein from CHO cells is 9% that derived from normal plasma. The present invention has been described in detail including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art may make modifications and Improvements within the spirit and scope of this invention which is defined by the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for producing biologically active Factor IX in high yield comprising culturing a CHO cell line transfected with chroraosomally-integrated Factor IX cDNA in medium containing vitamin K. 2. The method according to claim 1, wherein the cDNA comprises the cDNA sequence in Table 1. 3. The method according to claim 1, wherein vitamin K is selected from the group consisting of vitamin Kl and vitamin K3. 4. The method according to claim 3, wherein said medium comprises a concentration of about O.lng to 50ug vitamin K3/ml medium. 5. The method according to claim 4, wherein said medium comprises a concentration of about 5ng to about lOug vitamin K3/ml medium. 6. The method according to claim 3, wherein said medium comprises a concentration of about lOng to about "50ug vitamin Kl/ml medium. 7. The method according to claim 6, wherein said medium comprises a concentration of about lOOng to about lOOug vitamin Kl/ml medium. 8. A method for producing a biologically active γ-carboxylated protein in high yield comprising culturing a cell line transfected with chromosomally-integrated cDNA coding for said protein in medium containing vitamin K. 9. The method according to claim 8 further comprising employing as said protein a protein selected from the group consisting of Factor IX, Factor X, Factor VII, Protein C and Protein S. 10. The method according to claim 8 further comprising selecting said vitamin K from the group consisting of vitamin Kl, vitamin K3, and a water soluble vitamin K.

Inventors: KAUFMAN RANDAL J, SHOEMAKER CHARLES B, WASLEY LOUISE C, KAUFMAN, RANDAL, J., SHOEMAKER, CHARLES, B., WASLEY, LOUISE, C.
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1986
Main classification: C12P21
IPC codes: C12N5, C12N9, C12N15, C12P21, C12R1