Expression, Purification, and Characterization of Recombinant y ...

Vol. 261, No. 21, Issue of July 25, pp. 9622-9628 1986 Printed in C.S.A.

T H EJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Expression, Purification, and Characterization of Recombinant y-Carboxylated Factor IX Synthesized in Chinese Hamster Ovary Cells” (Received for publication, January 21, 1986)

Randal J. Kaufmant, Louise C. Wasley$, Barbara C. FurieQ,Bruce FurieQ, and Charles B. Shoemaker$ From the $Genetics Institute, Cambridge, Massachusetts 02140 and the $Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, Department of Medicine, New England Medical Center and Tufts UniversitySchool of Medicine, Boston, Massachusetts 02111

Factor IX has been expressed to high levels within a recombinant host cell and thebiologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integratedDNA was amplified to a high copy number by selection for increasingly higher expression levels of the markergene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secretingover 100 bg/ ml Factor IX antigen and up to 1.5 pglml native Factor IX antigen havebeen obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culturemedia. The 7-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and y-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 unitslmg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of 7-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results representthe firstpurification and characterization of a biologically active, y-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.

Factor IX is a zymogen of a serine protease that is an important component of the intrinsic pathway of the blood coagulation cascade. This protein (M, 56,000) is synthesized in the liver and undergoes extensive post-translational modification prior to secretion (for review, see Refs. 1 and 2). These modifications involve glycosylation, vitamin K-depend-

* Supported in part by Grants HL21543 and HL18834 from the National Institutes of Health and a grant from Seragen, Inc. (to B. C. F. and B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

ent y-carboxylation of 12 amino-terminal glutamic acid residues (3), and P-hydroxylation of a single aspartic acid residue (4). The y-carboxyglutamic acid residues confer the metal binding properties that characterize Factor IX and the other vitamin K-dependent blood clotting proteins (5-7). In the presence of metal ions, Factor IX undergoes a conformational change (8-10) that is essential for the expression of coagulant activity. The function of the P-hydroxyaspartic acid at residue 64 is unknown. Factor IX is deficient or functionally defective in a form of hemophilia (hemophilia B); an X chromosome-linked bleeding disorder. Factor IX-deficient patients are treated with human plasma or partially purified Factor IX preparations derived from human plasma as a source of the missing Factor IX. Recombinant Factor IX produced by cells in tissue culture offers special therapeutic potentialif biologically active Factor IX can be produced in large quantities and the active Factor IX purified efficiently to homogeneity. The humanFactor IX gene has been previously cloned (11-14) and active Factor IX expressed at a low level (15-17). We report the application of recombinant DNA technology to thedevelopment of Chinese hamster ovary cell lines that synthesize biologically active Factor IX. The active fraction of the recombinant Factor IX has been purified to homogeneity and shown to be nearly equivalent to plasma-derived Factor IX. EXPERIMENTALPROCEDURES

Isolation of Factor ZX cDNA Clones-A unique oligonucleotide (5‘pGTACAGGAGCAAACACC-3’)homologous to Factor IX mRNA approximately 475 base pairs downstream from the initiator codon (11,12) was synthesized and labeled with [Y-~’P]ATPusing polynucleotide kinase. The resultant probe was used to screen a human liver double-stranded cDNA library in XGT10; the library was prepared as previously described (18). Approximately 100,000 recombinant phage plaques were screened on duplicate filters (19). Hybridization was at 42 “cfor 40 h in 5 X ssc (1 X ssc is 0.15 M NaCl, 15 mM sodium citrate), 5 X Denhardt’s reagent (20), 0.5% SDS,’ and 10 mM EDTA. The filters were washed extensively at 42 “C in 2 X SSC. Three duplicate positives were plaque-purified, and phage DNA was extracted, purified from plate stocks (21), and analyzed by restriction enzyme mapping. Complete two-stranded DNA sequences of the largest cDNA were obtained following subcloning into M13 phage vectors (22) by the dideoxy chain termination method (23) using synthetic oligonucleotide primers. The clone contained all of the coding sequence COOH-terminal of the eleventh codon (11)and the entire 3”untranslated sequence. Recombination of Factor ZX cDNA withFactor ZX Genomic Exon Z to Obtain a Full-length Factor ZX cDNA Clone-A genomic library from a human XXXX chromosome cell line was prepared in Charon The abbreviations used are: SDS, sodium dodecyl sulfate; CHO, Chinese hamster ovary; kb, kilobases.

9622

Recombinant y-Carboxylated

Human Factor IX

9623

prepared with RIPA buffer (radioimmunoprecipitation buffer: 150 mM NaCl, 20mM Tris, pH 7.4, 0.1% (w/v) SDS, 1%(w/v) sodium deoxycholate, 1%(w/v) Triton X-100, 5 mM EDTA, 0.05% (w/v) sodium azide with 1 mM phenylmethanesulfonyl fluoride) and stored a t -20 "C as described (25). Protein samples and immunoprecipitates were separated by electrophoresis on SDS 6% polyacrylamide gels (29) and analyzed by autoradiography after treatmentwith the fluor Enhance (New England Nuclear). Genomic DNA Analysis-High molecular weight DNA wasisolated (30) and 10 pg digested to completion and separated by electrophoresis on 1.1% agarose gels for Southern blot transfer (27, 31). Filters were hybridized (28) to either dihydrofolate reductase or Factor IX 32Pradiolabeled probes prepared by nick translation (32) of DNA fragments isolated from agarose gels. Factor I X Coagulant Assay-Factor IX coagulant activity was determined with a two-stage assay using Factor IX-deficient plasma (33). One unit of Factor IX activity represents the amount of Factor IX in 1 ml of normal human pooled plasma. The protein concentration of purified recombinant Factor IX and plasma-derived Factor IX was measured using AI%at 280 nm of 13.3. ImmunochemicalAssay of Factor ZX-For purposes of comparison, Factor IX was purified from plasma by immunoaffinity chromatography as previously described (10). This preparation migrates as a single band upon SDS gel electrophoresis. Factor IX antigen and the antigenic determinant expressed on the metal-stabilized conformer of Factor IX were assayed using anti-Factor IXtotal antibodies and anti-Factor IX conformation-specific antibodies in a solution-phase radioimmunoassay using '2sI-labeled Factor IX (34). The antibodies were prepared as previously described (34). Factor IX was iodinated with NalZ5Iusing the lactoperoxidase method (35). The '%I-labeled Factor IX was repurified by passage over an anti-Factor IX:Ca(II) antibody column (10). The displacement of '2SI-labeledFactor IX from the anti-Factor IXantibodies was studied using a competition 5' CTAGAGGCCTCTGCA- 3' M) or radioimmunoassay. Anti-Factor 1X:total antibodies (1.2 X 3' -GATCTCCGGAG - 5' anti-Factor IX:Mg(II) antibodies (1.3 X lo-' M) were added to a reaction mixture which included varying concentrations of competiThe adapted DNA was cloned into anM13 mpll PstIvector. Recom- tors (plasma-derived Factor IX standard, partially purified and pubinant plaques were screened with an oligonucleotide homologous to rified recombinant Factor IX) and '261-labeledFactor IX (1 X 10"' the first 17 base pairs of Factor IX coding sequence mRNA. One M). All competitors were diluted in Tris-buffered saline in 1 mM subclone was isolated in which thePstIadapter had ligated 19 benzamidine, 0.01% Tween 20,0.1% bovine serum albumin containnucleotides upstream of the initiator codon and contained the entire ing 1mM CaC12.After incubation a t 4 'C overnight, 1ml of goat antiFactor IX coding sequence and 1030 nucleotides of 3"untranslated rabbit immunoglobulin (25 mg/ml in 0.1 M Tris, pH 7.4,0.15 M NaC1, sequence. The resultantplasmid was sequenced by the dideoxy chain 0.1% NaN3, 2.5% PEG-6000) was added to each tube, mixed vigortermination method (23) using synthetic oligonucleotideprimers. The ously, and allowed to standfor 15 min. The precipitate which formed nucleotide sequence of the coding region was in complete agreement was removedby centrifugation and was assayed for 'I in a Beckman with that published by Choo et al. (12). Gamma 8000 spectrometer. A 2.5-kb fragment containing the Factor IX coding region was then Factor IX antigen was also monitored using a solid-phase enzymeisolated and inserted into the PstI siteof the mammalian expression linked imrnunosorbent assay system. Microtiter plates were coated vector pQ2, in place of y-interferon (25). The resultant Factor IX with an anti-human Factor IX murine monoclonal antibody (Hybriexpression plasmid, designated p91023-IX, contained the Factor IX tech). After the plates were washed, conditioned medium containing coding region in the proper orientation with respect to theadenovirus Factor IX or purified human plasma Factor IX was added. Factor IX major late promoter. bound to the solid phase was detected with rabbit anti-Factor IX Cell Culture, DNA Transfection, and Cell Line Selection-Growth (Calbiochem-Behring). An alkaline phosphatase-conjugated goat and maintenance of the dihydrofolate reductase-deficient CHO anti-rabbit IgG (Zymed) was applied and the bound rabbit immunoDUKX-Bll cell line has been described (26, 27). DUKX-B11 cells globulin measured by hydrolysis of nitrophenyl phosphate. were transfected with a mixture of the Factor IX expression plasmid Immunoaffinity Purification of Carboxylated Recombinant Factor (25 pg, ~91023-IX) and a dihydrofolate reductase expression plasmid IX-Carboxylated recombinant Factor IX was purified from the (2.5 pg, pAdD26SVpA#3,28) by CaPO, coprecipitation as described culture medium of Chinese hamster ovary cells by immunoaffinity (27). After transfection, the cells were fed with a-medium containing chromatography using conformation-specific antibodies (10). In this 10% fetal bovine serum and thymidine, adenosine, deoxyadenosine, strategy, antibodies are employed to bind to forms of Factor IX that penicillin, and streptomycin (10 pg/ml each). Two days later the cells can express specific antigenic determinants only in the presence of were subcultured into a-medium with 10% dialyzed fetal bovine metal ions. A-y-carboxy- and des-y-carboxy recombinant Factor IX serum, penicillin, and streptomycin, but lacking nucleosides. Cells can not assume the metal-stabilized conformation and thus do not were fed again with the selective media after 4-5 days, and colonies bind to these antibodies. Culture medium (930 ml) was concentrated appeared 10-12 days after subculturing. Initial transformants were at 4 "C by ultrafiltration to a volume of420 ml using a PM30 pooled (approximately 25 transformants per pool) and grown in membrane (Amicon). The crude Factor IX preparation was dialyzed increasing concentrationsof methotrexate starting from 0.02 p~ and three times against 10 volumes of 3 mM CaCl2,0.05 M Tris-HC1, 0.5 extending up to 200 p~ methotrexate (0.02, 0.2, 0.5, 1.0, 5.0, 20, and M NaCl and applied to an anti-Factor IXCa(I1)-Sepharose column 200 p M ) . The selected pool (5a3) was then cloned in 200 p~ metho- (1.5 X 2 cm) as previously described. The column was washed extrexate. haustively with the equilibration buffer and then eluted with 10 mM Factor I X Synthesis-The synthesis of Factor IX was monitored EDTA, 0.05 M Tris-HC1, 0.15 M NaC1. by labeling cells (2 X lo6 cells/lO-cm dish) with 1 ml of methionineAmino-terminal Sequence Analysis-Automated Edman degradafree medium containing 0.5 mCi of [%]methionine (specific activity tion was performed on an Applied Biosystems Model 470A gas-phase greater than 800 Ci/Gmol; New England Nuclear). Cells were incu- protein sequenator equipped with a Model 120 PTH Analyzer (36). bated for 4 h at 37 "C, and the conditioned medium was assayed by Recombinant Factor IX was dialyzed against water prior to analysis. immunoprecipitation with a murine anti-Factor IX monoclonal any-CarboxyglutamicAcid Analysis-Amino acid analyses were pertibody (Hybritech) and rabbitanti-mouse immunoglobulin as the formed on a Beckman Model 119CL amino acid analyzer equipped immunoadsorbent. Foranalysis of cell extracts, cell lysates were with a Beckman Model 126 data system. The proteins were hydro-

4A (18) and screened with a Factor IX cDNA probe. Hybridizing recombinant phage were isolated, plaque-purified, andthe DNA isolated. Restriction mapping, Southern analysis, and DNA sequencing permitted identification of five recombinant phage-containing inserts which, when overlapped at common sequences, coded the entire 35-kb Factor IX gene (12). A 4.5-kb HindIII fragment containing the Factor IX promoter and exon 1 of Factor IX was subcloned into Charon 21A. In a separate construction, the largest Factor IX cDNA (2.5 kb) was subcloned into PiAN7 plasmid, a derivative of piVX (21). This plasmid therefore contained 57 base pairs of exon I sequence within the Factor IX cDNA. The exon I containing Charon 21A phage were plated onbacteria harboring the recombinant PiAN7 plasmid and subsequently harvested as a plate stock. Phage which had recombined with the supF containing PiAN7 plasmid were selected by plating onto the F- bacterial line W3009 (21). Approximately one in 2 X lo' phage had become sup F+. One isolate was chosen for large scale DNA preparation and shown by restriction mapping and subsequent sequence analysis to have recombined homologously with the PiAN7 plasmid within the 57-base pair region of homology. This phage contained the Factor IX promoter and exon I fused to the entire Factor IX coding sequence, thus creating a "minigene" without introns. Construction of Factor I X Expression Plusmid p91023-IX-To replace the weaker Factor IX promoter with a stronger promoter for efficient expression in mammalian cells, the Factor IX coding region was excised separate from its promoter and inserted into a derivative of the mammalian expression vector p91023(B) (24) by the following procedure. A 3.5-kb XbaI fragment containingthe Factor IX minigene was purified and digested with exonuclease I11 for the time required to degrade the 5' noncoding sequence. The DNA was then treated with S1 endonuclease and Klenow fragment of polymerase 1 and blunt-end ligated to a synthetic PstI adapter

-

9624

Human Factor IX

Recombinant ?-Carboxylated

lyzed in 2 M KOH for 16 h at 110 "C (37). The y-carboxyglutamic acid composition was quantitated after alkaline hydrolysis by automated amino acid analysis using a ninhydrin detection system.

5a3

CHO 0

RESULTS

Expression of Factor IX in Chinese Hamster Ovary CellsThe constructionof the Factor IX expression vector is shown diagramatically in Fig. 1. This vector, p91023-IX, contains the adenovirus major late promoter including a cDNA copy of the adenovirus tripartite leader, the SV40 enhancer element, andorigin of replication upstream from the adenovirus major late promoter, and the adenovirus virus associated (VA) genes (25). Plasmid ~91023-IXwas introduced with a selectable dihydrofolate reductasegene (pAdD26SVpA#3) (28) into dihydrofolate reductase-deficient Chinese hamster ovary cells bycalcium phosphate-mediatedDNAtransfectionasdescribed (38). Cells selected for the dihydrofolate reductaseat a low level, as positive phenotype expressed Factor IX determined by immunoprecipitation of conditioned media after ["S]methionine labeling of the cells. The transformants were then pooled andselectedfor growth insequentially increasing concentrationsof methotrexate starting from0.02 p~ and extending up to200 p ~ Results . from Southern blot analysis indicated that upon selection to high methotrexate resistance, the transfected Factor IX gene was amplified to approximately 500copies per cell comparedto known amounts of ~91023-IXDNA mixed with wild-type CHO DNA (Fig. 2). The DNA restriction pattern of sequences that hybridize to the Factor IX coding region in the 5-a-3 cell line indicated a rearrangementoccurred which resultedinan altered PstIdigestion pattern. Since primer extension analysis molor enhoncer late promoter

f

23

8-

Ir

4 0 .

4

- 9.5

-6.4 - 4.2

-2.2 - 1.8

- 0.53

. .

Adenovlrus

. 0' 0

Tet

-

0.

0

0.

b

-a

\

P'

-0

4 -

FIG. 2. Southern blot analysis of amplified Factor IX genes. DNA was prepared from the original 5-a-3 poolof dihydrofolatereductase-positive transformants and from the same cells selected for growth in 0.1, 0.5, and 20 p~ methotrexate. DNA samples (10 pg) were digested with PstI and the resultant Southern blot was hybridized to a nick-translatedPstI fragment spanning the Factor IX coding region. Also shown is a reconstruction of CHO DNA with increasing amounts of ~91023-IXranging from 20 to 1000 pg.

using reverse transcriptase had demonstrated that the 5' end of the Factor IXmRNA is correctly initiated, the rearrangeEl ment which occurred in the Factor IX expression vector must or1 have altered sequences downstream of the Factor IX coding 3'UT region (Fig. 1).Northern blot analysisof Factor IX mRNAin these cells indicated several species of very high molecular weight Factor IX mRNA (greater than 28 S) (data notshown); this represents furtherevidence that the3' region has become cDNA polyA slgnal FIG. 1. Schematic diagram of the Factor IX expression vec- rearranged, removing the SV40 polyadenylation site from its tor. The Factor IX expression vector ~91023-IXcontains the SV40 position downstreamof the Factor IXcDNA. origin (ori) of replication including the transcriptional enhancer, the Analysis of Factor IX synthesis by immune precipitation adenovirus major late promoter (including the adenovirus tripartite of [3sS]methionine-labeled protein indicated an increase in leader and a 5' splice site, a 3' splice site (3'SS) derived from an immunoglobulin gene), the Factor IX coding region, a noncoding Factor IX antigen in thecell extracts and in the conditioned gene amplification (Fig. 3). In cell dihydrofolate reductase (DHFR) cDNA insert, the SV40 early poly- media corresponding to the of a band migratinga t 56,000 increased adenylation site, the adenovirus virus associated (VA) genes, the extracts, the intensity tetracycline-resistance gene (TetR) and the pBR322 sequences needed as cells were selectedforhigherdegrees of methotrexate for propagation in Escherichia coli. For details of the components of resistance. The M,56,000 species probably represents intrathis vector see Kaufman (25). The Factor IX cDNA is flanked by PstI restriction sites as shown. The protein coding region (W) and 3'- cellular Factor IX prior to extensive post-translational glyuntranslated region (3'UT, 0 )are indicated. CHO transformant 5a3 cosylation. The conditionedmedia exhibited two broad bands likely contains a rearrangement in the 3"untranslated region of the of Factor IX antigen migratingat 67,000 and 72,000 (Fig. 3). Factor IX cDNA. The two bands may result from differences in processing or

Recombinant y-Carboxylated HumanFactor IX

Cell Extracts

9625

Tota I Media

Media Y

FIG. 3. Polyacrylamide gelelectrophoresis of intracellular and secreted Factor IX from CHO cells. The original 5-CY-3 pool of dihydrofolate reductase-positive transformants and the same cells selected for resistance to increasing concentrationsof methotrexate,asindicated ( p methotrexate), ~ werelabeled (500 pCi/ml ["S]methionine). Extracts were prepared and anaa lyzed by immunoprecipitationwith Factor IX monoclonal antibody (Hybritech) and immunoadsorption with rabbit anti-mouse immunoglobulin. Vitamin Kt (1 pg/ml) was added at the time of labeling. The right panel depicts the total secreted protein of the original CHO dihydrofolate reductase- cell lineand a Factor IX producing subclone (lG8)resistant to 200 pM methotrexate monitored after a 4-h incubation with [Y3] methionine.

o w = w v -

"""

-200

*I 6

-92.5

-200

- 92.5

aJ -- 92.5

Y Y

- 46

glycosylation. Results inFig. 3 demonstrate that there appearsditioned medium from any of the 503 CHO cell lines. Since to be no alteration in the electrophoretic mobility of Factor the synthesisof biologically active Factor IX is dependent on vitamin K, the effect of vitamin K addition was monitored. IX produced upon selection for high levels of FactorIX of increasing concentrationsof vitamin expression. Factor IX antigen levels accounted for a signifi- Results after addition cant proportion of the total protein secreted from a highly K, (3-phytylmenadione, Sigma), for 24 h prior toassay, indiof activeFactor IX. Thevitamin K, methotrexate-resistantclone (Fig. 3, clone 1G8). Further catedthepresence analysis of the secreted Factor IX antigenby enzyme-linked requirement for Factor IX activity appeared to be saturated immunosorbent assay indicated a 3,000-fold increase in the a t 5 pg/ml.vitamin K, (Fig. 5). Addition of a water-soluble FactorIXantigenuponmethotrexateresistance selection form of vitamin K, vitamin K3 (menadione), alsoelicited from 0 to 20 p~ methotrexate (Fig. 4). The level of Factor IX similar levels of active Factor IX, except a 500-fold lower antigen in the 5-0-3 pool of cells in 20 p~ methotrexate was concentration of vitamin KI (10 ng/ml) produced the optimal effect (data not shown). The specificity of the vitamin K, 43.4 pg/ml. expression was demonstrated Factor IX coagulant activity was not detected in the con- dependence on active Factor IX by adding warfarin(1pg/ml), a specific antagonist of vitamin 60

CHO Cell Selection Media (503h e ) [Methotrexate] (pM)

FIG. 4. Relationship between Factor IX activity and Factor IX antigen as a function of increasing methotrexate resistance. The 5-CY-3cellswereselectedfor increasingmethotrexate resistance. At each level of methotrexate resistance, samples were taken from media containing 1 pg/ml vitamin KI for determination of Factor IX activity in Factor IX-deficient plasma (0)and Factor IX antigen by enzyme-linked immunosorbent assay(0).

Vttamtn K, (pg/ml)

FIG. 5. Factor IX activity and antigen levels in CHO cells as a function of vitamin K1 concentration. Approximately 4 X

lo6 logarithmically growing cells were rinsed four times with serumfreemedium containing increasing concentrations of vitamin Kt. After 24 h a t 37 "C, aliquots of conditioned media were frozen in a dry ice/ethanol bath and stored at -70 "Cuntil assayed. All results are expressed as rnilliunits/ml/lO6 cells/day. Vitamin K, (0); warfarin (0).

Recombinant y-Carboxylated

9626

K action. Warfarin blocked the appearance of active Factor IX (Fig. 5). The addition of vitamin K had no apparent effect on the levels or mobility of Factor IX antigen (Fig. 3). The selection of cells resistant to methotrexate was associated with a significant increase in the production of Factor IX antigen. Although Factor IX antigen increased corresponding to gene copy number and methotrexate resistance, the level of Factor IX activitydid not show the same corresponding increase (Fig. 4). The maximal Factor IX antigen production was about 40 pg/ml in cells selected in 20 p~ methotrexate. Factor IX activity levels were maximal at approximately 800 milliunits/ml at resistance 0.1 p M methotrexate.This plateau may reflect saturation of the y-carboxylation of Factor IX at high levels of FactorIX expression. However, alternative explanations, such as the availability of vitamin K, cannot be ruled out at this time. Analysis of the Factor IX Protein Expressed in CHO CellsConditioned medium from the 5-a-3 cells selected in 20 p~ methotrexate was harvested following growth in vitamin K and evaluated immunochemically for Factor IX levels with two different rabbitanti-FactorIX antibodies. Thetotal Factor IX antigen, measured using an affinity purified antiFactor IX antibody in a solution-phase radioimmunoassay, was 23 pg/ml. The subpopulation of Factor IX that was sufficiently carboxylated so that it underwent the metalinduced conformational transition was assayed using conformation-specific antibodies to Factor IX that bind to Factor IX only in thepresence of metal ions (10). The expression of specific metal-stabilized antigenicdeterminantscorrelates closely to coagulant activity. In thisanalysis, the native Factor IX antigen concentration was 1.5 pg/ml. These resvlts indicate thatonly a fractionof the Factor IXexpressed is carboxylated and biologically active. The 5a3 cells were cloned by limiting dilution, and17 stable cell lines were obtained. The native Factor IX antigen and the total Factor IX antigen were determined by radioimmunoassay in culture medium obtained from each clone. The degree of y-carboxylation and the total Factor IX antigen secreted varied significantly among these clones (Table I). TABLEI Immunochemical analysis of native Factor IX antigen and total Factor IX antigen in culture media of recombinant Factor IXproducing Chinese hamster ovary cell subclones Using a radioimmunoassay directly analogous to those previously describedfor native prothrombinand total prothrombinantigen, antiFactor IX:Ca(II) and anti-Factor IX antibodies were used to measure the metal-stabilized conformer of Factor IX and total Factor IX, remectivelv (46). Cell line

S23/20D/K3 1B6 1C4 523/1E8 lFlO 1G1 1G8 1H6 lHlO 4A1 523/20D/lE8 4H1 4Hll 4C8 4D3 4F6 52311D11

Native Factor IX

Total Factor IX

dm1

dm1

1.46 0.84 1.17 0.15 1.37 1.38 1.52 0.29 0.98 0.76 0.50 0.65 0.79 0.43 1.20 1.08 0.27

97.5 34.5 188.0 16.2 152.0 84.5 43.0 119.0 146.0 100.5 28.8 70.5 18.4 41.0 63.5 90.0 45.0

Human Factor I X The highest native Factor IX antigen observed was 1.5 pg/ ml, and thehighest total Factor IX antigen observed was 188 pg/ml. The ratio of carboxylated Factor IX to totalFactor IX varied considerably, from 0.2 to 4.4% in the Factor IXamplified cells. The well-carboxylated Factor IX in the conditioned media was isolated by immunoaffinity chromatography using conformation-specific antibodies (10). This method selectively isolates well-carboxylated Factor IX that is able to undergo the y-carboxyglutamic acid-dependent conformational transitions that metal ions induce. After concentrating the tissue culturesupernatant by ultrafiltration, the crude material, containing both active and inactive species of Factor IX, was applied to a column of anti-Factor IX:Ca(II)-Sepharose in the presence of 3 mM CaClZ. The material that failed to bind lacked Factor IX coagulant activity and native Factor IX antigen. The bound Factor IX was eluted with EDTA, as previously described (10). The Factor IX activity (100%) was recovered in the EDTA eluate. A 6250-fold purification of Factor IXfrom culture medium wasobserved, based upon the specific activity of Factor IX in the culture medium and in the purified preparation. The specific activity of the recombinant Factor IX was measured on the basis of direct measurement of the coagulant activity using Factor IX-deficient plasma and the protein concentration using an EzsOnm of 13.3. The specific activity of recombinant Factor IX was 75 units/mg. In multiple preparations, the specific activity varied between 35-75 units/mg, compared to 150 units/mg measured for plasma-derived Factor IX. The purified recombinant Factor IX migrated as a single band on SDS gels in the presence of 8-mercaptoethanol (Fig. 6). Its electrophoretic mobility was identical to that of Factor IX derived from human plasma. Using a radioimmunoassay incorporating anti-Factor IX:Mg(II) antibodies (equivalent to Me1 (34)) in thepresence of calcium, the crossreactivity of antibody with recombinant Factor IXand plasma-derived Factor IX was nearly identical. The NHz-terminal sequence of the first 17 residues of the recombinant Factor IX was established by automated Edman degradation. Approximately 100 pmol of recombinant Factor IX were applied to a gas-phase sequenator. As shown in Table 11, the amino acid sequences of the NHz termini of recombinant Factor IX and plasma-derived Factor IX are identical. As the y-carboxyglutamic acid derivative is not removed from the filter in the standard Edman degradation, it is notable that residues 7, 8, 15, and 17 are blank and specifically show no glutamic acid. This is consistent with the idea that the purified recombinant Factor IX is well-carboxylated at the initial four y-carboxyglutamic acid positions. The y-carboxyglutamate content of the recombinant Factor TX was determined by subjecting the purified protein to alkaline hydrolysis and amino acid analysis. Purified recombinant Factor IX contained, on average, 6.5 k 1.0 mol of ycarboxyglutamic acid residues per mol of protein. DISCUSSION

Hemophilia B is a sex-linked hemorrhagic disease caused by a deficiency of Factor IX activity in the blood. To treat bleeding episodes, patientsare infused with fresh frozen plasma or Factor IX concentrates derived from plasma. The Factor IX concentrates are only partially purified, are enriched for viral contaminants of the plasma, and can activate blood clotting pathologically due to thepresence of contaminating activated clotting enzymes. The use of recombinant DNA technology to express Factor IX in mammalian cells offers an alternative source of Factor IX.

Recombinant y-Carboxylated Human

1 2

Factor IX

9627

IX precursor to y-carboxylate certainglutamic acid residues to generate a biologically active Factor IXmolecule. Recently, several other groupshave demonstrated the expression of active Factor IX in mammalian systems using recombinant DNA technology. The Factor IX gene was introduced into hep G2 cells (15, 16), a humanhepatomaline known to produce active vitamin K-dependent blood clotting proteins (43), mouse fibroblasts (16), and baby hamster kidney cells (17). Active Factor IXwas detected after concentrationof the Factor IX in the conditioned media and partial purification using barium citrate adsorption. Wehaveshown levels of Factor IX antigen in the tissue culture supernatant as high as 180 pg/ml, or about 30-fold higher than the level of Factor IX antigen in plasma. Thib expression level is two orders of magnitude higher than that reported recently (16, 17). The amount of active, carboxylated Factor IX in the conditioned medium was about 1.5 pg/ml. It would appear that thehighly amplified Factor IX expression saturated the available carboxylation activity, limiting the amount of Factor IX precursor that becomes y-carboxylated. We are currently working to increase the vitamin K-dependent carboxylase activity so that thecarboxylation of Factor IX canbe maximized. In spite of expression of both active (carboxylated) and inactive (acarboxy or des-y-carboxy) Factor IX, we were able to isolate the active recombinant Factor IXcompare and it to FIG. 6. SDS polyacrylamide gel analysis of affinity-puriplasma-derived Factor IX. Conformation-specific antibodies, fied recombinant Factor IX. Concentrated tissue culture superdirected at a metal-stabilized conformer of Factor IX, offer a natant (420ml) was dialyzed into 3 mM CaCI?and applied to an anti- facile method for immunoaffinity purification of the carboxFactor IX:Ca(II)-Sepharosecolumn equilibrated in Tris-buffered saline, 3 mMCaC12, as previously described. After the column was ylated Factor IXspecies. Under-carboxylated Factor IX spewashed free of unbound protein, the bound Factor IX was eluted with cies, many of which contain sufficient y-carboxyglutamic acid 10 mM EDTA, Tris-buffered saline. Recombinant FactorIX and to bind to barium salts, do not bind to these conformationplasma-derived Factor IX, both purified by immunoaffinity chroma- specific antibodies. Therefore,only the well-carboxylated Factography using conformation-specific antibodies, were analyzed on tor IX is bound to the affinity column. This bound protein 10% SDS polyacrylamide gels in the presence of 8-mercaptoethanol. may be easily eluted by the introduction of EDTA, which The gels were stained with Coomassie Blue R-250.Lane 1, plasmacauses a change in the conformationof Factor IX anddissoderived Factor I X , lane 2, recombinant Factor IX. ciation of the antibody-antigencomplex. The isolated recombinant Factor IX yielded a single band TABLE I1 upon SDS gel electrophoresis and migratedidentically to Amino-terminal seauence of recombinant Factor ZX plasma-derived Factor IX. Furthermore, the NH2-terminal Recombinant ResiduePlasma sequence of the recombinant Factor IXwas identical to that Factor IX Factor IX of plasma-derived Factor IX. By direct analysis, the recomPWl binant Factor IX was shown to contain y-carboxyglutamic 1 41 ?Lr acid. The y-carboxyglutamic acid content was less for the 2 Asn 47 recombinant Factor IX than that for plasma Factor IX and 3 17 Ser 4 14 GlY likely reflects purification, in part, of partially carboxylated 5 15 LYS forms of Factor IX that have the requisite y-carboxyglutamic 6 Leu 23 acids to undergo the metal-induced conformational transition. 7 - (Gla)* It is notable that the recombinant Factor enriched IX, on the 8 - (Gla) basis of its ability to express metal-dependent conformational 9 15 Phe 10 15 Val determinants,hasabout50% of the specific activity of 11 8 Gln plasma-derived, fully carboxylated Factor IX. These results 12 13 GlY suggest that some of the y-carboxyglutamic acids in Factor 13 10 Asn IX may not playa functional role. By analogy to prothrombin 12 14 Leu (44, 45), some partially carboxylated forms are likely to be - (Gla) 15 16 partially active if nonessential y-carboxyglutamic acids are Arg 17 - (Gla) missing. Yet, the absence of y-carboxyglutamicacid 16 in human prothrombin isassociated with the totalloss of mema -, not identified. Gla, y-carboxyglutamic acid. branebindingandcoagulantactivity (45), indicatingthe presence of essential y-carboxyglutamic acid residues. The We describe the introduction into and amplificationof the lower specific activity of the purified recombinant Factor IX, human Factor IX gene in Chinese hamster ovary cells. This as compared to plasma Factor IX, islikely explained on the of which express system has previously been used to produce several different basis of heterogeneous forms of Factor IX, all proteins at a high level (38-42). Although CHO cells were not partial or full activity. Although there is need to optimizey-carboxylation, we known previously to have the enzymatic machinery for ycarboxylation, we have demonstrated that thesecells, in the have demonstrateda mammalian recombinantsystem capable presence of vitamin K, are capable of processing the Factor of generating high levels of Factor IX. Using the purification

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Recombinant y-Carboxylated Human Factor IX

system described, it will be possible to prepare pure recombinant Factor IX for the treatment of hemophilia B. Furthermore, site-specific mutagenesis of the recombinant Factor IX should allow systematic examination of the substrate requirements for the vitamin K-dependent carboxylase and evaluation of the structure-function relationship of Factor IX.

20. 21. 22.

Acknowledgments-The technical assistance of Margaret Jacobs, Sharon Pagano, and David Schubert is gratefully acknowledged.

23.

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