Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof

Abstract

A hyperglycosylated recombinant human coagulation factor IX (FIX) fusion protein, a preparation method therefor, and use thereof. The fusion protein sequentially comprises, from N- to C-terminus, a human FIX, a flexible peptide linker, at least one human chorionic gonadotropin β subunit carboxy-terminal peptide rigid unit, and a half-life extending moiety. The fusion protein has a biological activity similar to that of the recombinant FIX, an extended in vivo activity half-life, and reduced immunogenicity, so as to improve pharmacokinetics and pharmacodynamics.

Claims

1. A fusion protein, wherein the fusion protein has the amino acid sequence as shown in SEQ ID NO: 8.

2. The fusion protein of claim 1, wherein the fusion protein has an activity of >200 IU/mg.

3. A DNA molecule encoding the fusion protein of claim 1, which comprises a sequence as shown in SEQ ID NO:9.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the fusion protein of claim 1.

5. A method for preparing a fusion protein of claim 1, comprising: (a) introducing the DNA sequence encoding a fusion protein of claim 1 into a CHO cell to generate a CHO-derived cell line; (b) screening the high-yielding cell line in step (a) which expresses more than 1 mg/10.sup.6 (million) cells per 24 hours in its growth medium; (c) growing the cell line obtained in step (b) to express the fusion protein; (d) harvesting the fermentation broth obtained in step (c) and isolating and purifying the fusion protein.

6. The method of claim 5, wherein the CHO-derived cell line in step (a) is DXB-11.

7. The method of claim 5, wherein the fusion protein purification in step (d) comprises affinity chromatography and anion exchange chromatography.

8. A method for treating a hemorrhagic disease, comprising administrating a therapeutically effective amount of a fusion protein of claim 1 to a patient, wherein the patient has congenital or acquired FIX deficiency, or the patient has hemophilia B and suffers from spontaneous or surgical bleeding.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the nucleotide sequence (SEQ ID NO: 9) and deduced amino acid sequence (SEQ ID NO: 8) of the fusion protein inserted into the Spe I-EcoR I fragment in expression vector pF9-5B. The mature fusion protein contains hFIX, a flexible peptide linker (underlined with __), a CTP rigid unit (underlined with __) and a vFcγ.sub.2-3 variant.

(2) FIG. 2 shows the SEC-HPLC chromatogram of purified fusion protein F9-5B.

(3) FIG. 3 shows the SDS-PAGE electrophoretogram of purified fusion protein F9-5B.

REFERENCE TO SEQUENCE LISTING

(4) The Sequence Listing associated with the instant disclosure has been submitted electronically herewith as an 27 kilobyte file with File Name (Substitute Sequence Listing.txt), Creation Date (Aug. 17, 2021), Computer System (IBM-PC/MS-DOS/MS-Windows). The Sequence Listing submitted electronically herewith is hereby incorporated by reference into the instant disclosure.

DETAILED DESCRIPTION

(5) hCG-β Carboxyl Terminal Peptide (CTP)

(6) CTP is a short peptide from the carboxyl terminus of the human chorionic gonadotropin (hCG) beta subunit. Four kinds of reproduction-related polypeptide hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), and human chorionic gonadotropin (hCG) contain the same alpha subunit and their respective specific beta subunits. Compared with the other three hormones, hCG has a significantly prolonged in vivo half-life, which is mainly due to the specific carboxyl terminal peptide (CTP) on the hCG β-subunit (Fares F A et al., Proc Natl Acad Sci USA, 1992, 89(10): 4304-4308). The native CTP contains 37 amino acid residues, including four O-glycosylation sites, and sialic acid residues at the terminus. The highly sialylated, negatively charged CTP can resist the clearance by the kidney, thereby prolonging the in vivo half-life of the protein (Fares F A et. al., Proc Natl Acad Sci USA, 1992, 89(10): 4304-4308). The present inventors creatively connect at least one CTP peptide with a flexible peptide linker having an appropriate length to form a new peptide linker, for linking FIX to a half-life extending moiety e.g., an immunoglobulin Fc fragment.

(7) The present inventors have found that the addition of a CTP peptide between FIX and an Fc variant is equivalent to the addition of a rigid peptide linker. On one hand, the addition of the CTP peptide ensures that the N-terminally fused FIX does not affect the binding site in Fc variant for FcRn, thus having no effect on the half-life. In addition, the protein A binding site in Fc is important for purification steps. The addition of CTP ensures that the N-terminally fused FIX will not “cover” its binding site for protein A. Thus the fusion protein can be purified with a cheaper and more suitable filler, which reduces the cost of purification. On the other hand, the addition of a CTP rigid unit prevents the Fc fragment having a size of about 25 kD from interfering with the correct folding of the N-terminally fused FIX, thus leading to no loss or decline of the biological activity/function of the FIX. The rigid CTP peptide containing multiple glycosyl side chains can form a stable steric conformation compared to the random coil of flexible linkers such as (GGGGS)n (SEQ ID NO: 21). This “block” effect causes the FIX and Fc fragment to fold independently into correct three-dimensional conformations without affecting the biological activities of each other. Moreover, the protective effect of the glycosyl side chains of CTP reduces the sensitivity of the peptide linker to proteases, such that the fusion protein is less susceptible to degradation in the linking region.

(8) IgG Fc Variants

(9) Non-Lytic Fc Variants

(10) The Fc element is derived from the constant region (Fc fragment) of immunoglobulin IgG, and plays an important role in eradicating pathogens in immune defense. The Fc-mediated effector functions of IgG function through two mechanisms as follows. (1) After binding to Fc receptors (FcγRs) on the cell surface, pathogens are broken down by phagocytosis or lysis or by killer cells through the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway. (2) Alternatively, after binding to C1q of the first complement component C1, the complement-dependent cytotoxicity (CDC) pathway is triggered and thus pathogen are lysed. Among the four subtypes of human IgG, IgG.sub.1 and IgG.sub.3 are able to bind to FcγRs effectively, and IgG.sub.4 has lower binding affinity for FcγRs. The binding of IgG.sub.2 to FcγRs is too low to be measured, so human IgG.sub.2 has little ADCC effects. In addition, human IgG.sub.1 and IgG.sub.3 can also effectively bind to C1q to activate the complement cascade. Human IgG.sub.2 binds weakly to C1q and IgG.sub.4 does not bind to C1q (Jefferis R et al., Immunol Rev, 1998, 163: 59-76), so the CDC effect of human IgG.sub.2 is also weak. Obviously, none of the native IgG subtypes is well suitable for constructing FIX-Fc fusion proteins. In order to obtain non-lytic Fc variants without effector functions, the most effective method is to mutate the complement- and receptor-binding regions of the Fc segment and adjust the binding affinity of Fc for related receptors to reduce or eliminate ADCC and CDC effects but retain only the biological activity of the functional protein and the long in vivo half-life of the Fc segment without the generation of cytotoxicity. More mutation sites contained in non-lytic Fc variants can be found in Shields R L et al., J Biol Chem, 2001,276(9):6591-604 or China Patent No. CN 201280031137.2.

(11) Fc Variants with Enhanced Affinity to the Neonatal Receptor FcRn

(12) The plasma half-life of IgG depends on its binding to FcRn. Typically, IgG binds to FcRn at pH 6.0 and dissociates from FcRn at pH 7.4 (plasma pH). Through the study on the binding sites of the two, the sites on IgG that bind to FcRn are modified to increase the binding affinity at pH 6.0. It has been proven that mutations of some residues in the human Fcγ domain, which are important for the binding to FcRn, can increase the serum half-life. Mutations in residues T250, M252, S254, T256, V308, E380, M428 and N434 have been reported to increase or decrease the FcRn-binding affinity (Roopenian et al., Nat. Review Immunology 7:715-725, 2007). Trastuzumab (Herceptin, Genentech) variants, disclosed in Korean Patent No. KR 10-1027427, show increased FcRn-binding affinity, and these variants contain one or more amino acid modifications selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, 279Y, 308F and 308Y. Bevacizumab (Avastin, Genentech) variants, provided in Korean Patent No. KR 2010-0099179, show prolonged in vivo half-life and these variants contain amino acid modifications N434S, M252Y/M428L, M252Y/N434S and M428L/N434S. In addition, Hinton et al. also found that two variants T250Q and M428L increased the binding affinity for FcRn by 3 and 7 times, respectively. When the two sites were mutated simultaneously, the binding affinity was increased by 28 times. In rhesus macaque, the M428L or T250Q/M428L variant shows a 2-fold increase in plasma half-life (Paul R. Hinton et al., J Immunol, 2006, 176:346-356). More mutation sites contained in Fc variants with increased binding affinity for FcRn can be found in China Patent No. CN201280066663.2. In addition, studies show that the T250Q/M428L mutations in the Fc regions of five humanized antibodies improve the interaction between the Fc domain and FcRn. Moreover, in subsequent in vivo pharmacokinetic tests, compared to wild-type antibodies, the Fc mutated antibodies show improved pharmacokinetic parameters, such as increased in vivo exposure, reduced clearance, and increased subcutaneous bioavailability, when administered via subcutaneous injection (Datta-Mannan A et al., MAbs. Taylor & Francis, 2012, 4(2):267-273.).

(13) Fusion Protein and Preparation Method Thereof

(14) The fusion protein gene of the present invention is artificially synthesized after codon optimization. Based on the nucleotide sequence of the present invention, one skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods, for example, but not limited to, artificial synthesis or traditional subcloning. For specific methods, see J. Sambrook, Molecular Cloning: A Laboratory Manual. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by segmentally synthesizing nucleotide sequences followed by subcloning.

(15) The present invention also provides an expression vector for mammalian cells comprising a sequence encoding a fusion protein of the present invention and an expression regulatory sequence operably linked thereto. By “operably link” or “operably linked to” is meant a condition in which some portions of a linear DNA sequence are capable of regulating or controlling the activity of other portions of the same linear DNA sequence. For instance, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the sequence.

(16) The expression vector for mammalian cells may be a commercially available vector such as, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT and the like which can be used in a eukaryotic cell expression system. One skilled in the art can select a suitable expression vector based on the host cell.

(17) The coding sequence of the fusion protein of the present invention may be introduced into suitable restriction sites by one skilled in the art by restriction enzyme cleavage and splicing according to a conventional method based on the restriction enzyme map of the known empty expression vector, to produce the recombinant expression vector of the present invention.

(18) The present invention also provides a host cell expressing a fusion protein of the present invention comprising a coding sequence of a fusion protein of the present invention. The host cell is preferably a eukaryotic cell such as, but not limited to, CHO cells, COS cells, 293 cells, RSF cells and the like. In a preferred embodiment of the present invention, the cell is a CHO cell which can better express the fusion protein of the present invention to obtain a fusion protein having good activity and good stability.

(19) The present invention also provides a method for producing a fusion protein of the present invention by using recombinant DNA technology, including the steps of:

(20) 1) providing a nucleic acid sequence encoding a fusion protein;

(21) 2) inserting the nucleic acid sequence of 1) into a suitable expression vector to obtain a recombinant expression vector;

(22) 3) introducing the recombinant expression vector of 2) into a suitable host cell; 4) growing the transfected host cell under conditions suitable for expression;

(23) 5) collecting the supernatant and purifying the fusion protein product.

(24) The coding sequence can be introduced into a host cell by various techniques known in the art such as, but not limited to, calcium phosphate precipitation, lipofection, electroporation, microinjection, viral infection and method using alkali metal ions.

(25) For the culture and expression of host cells, see Olander R M et. al., Dev Biol Stand, 1996, 86:338. The cells and debris in the suspension can be removed by centrifugation and the supernatant is collected.

(26) The fusion protein obtained as described above can be purified to a substantially uniform nature, for example, showing a single band or specific bands on SDS-PAGE electrophoresis. The supernatant is firstly to be concentrated. The concentrated supernatant may be further purified by gel chromatography or by ion exchange chromatography, such as anion exchange chromatography or cation exchange chromatography. The gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like. The Q- or SP-group is a preferred ion exchange group. Finally, the purified product may be further finely purified by methods such as hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography, and the like. All of the above purification steps can be used in different combinations to ultimately obtain proteins with a substantially uniform purity. The expressed fusion protein can be purified by using an affinity chromatography column containing an antibody, receptor or ligand specific for the fusion protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column can be eluted by using conventional methods such as high salt buffer, pH change, and the like.

(27) Pharmaceutical Composition

(28) The present invention also provides a pharmaceutical composition comprising an effective dose of a fusion protein of the present invention and a pharmaceutically acceptable carrier. In general, an effective amount of the fusion protein of the present invention may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8. The term “effective amount” or “effective dose” refers to an amount that yields functional or active effects on humans and/or animals and is acceptable by humans and/or animals. “Pharmaceutically acceptable” ingredients are those that are suitable for use in humans and/or mammals without excessive adverse side effects (e.g., toxicity, irritation and allergies), i.e., substances with a reasonable benefit/risk ratio. The term “pharmaceutically acceptable carrier” refers to a carrier for delivering a therapeutic agent, and the carrier includes various excipients and diluents.

(29) Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the present invention may be prepared in the form of injections, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions described above are preferably manufactured under aseptic conditions. The amount of the active ingredient administered is the therapeutically effective amount. The pharmaceutical formulation of the present invention can also be prepared in a sustained release form.

(30) The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount may be determined by one of ordinary skill in the art based on various factors for example by clinical trials. The factors include, but are not limited to, the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc., the severity of the disease to be treated in a patient, the patient's weight, the patient's immune status, the route of administration, etc.

EXAMPLES

Example 1. Construction of an Expression Plasmid Encoding the FIX Fusion Protein

(31) The gene sequence encoding the full-length FIX and gene sequences encoding flexible peptide linkers with different lengths, CTP rigid peptides with different lengths and different IgG Fc variants were artificially-optimized, CHO cell-biased codons and can be obtained by chemical synthesis. A restriction site, SpeI or EcoRI respectively, were present at each of the 5′-end and 3′-end of the synthesized fragment to facilitate insertion of the target fragment into a specific site of the expression vector. The verified fusion gene was digested with SpeI and EcoRI, and then inserted between corresponding restriction sites in expression plasmid PXY1A1, which was obtained by modifying PCDNA3.1 as a template, to obtain an expression plasmid pF9-5 of the fusion gene. The plasmid PXY1A1 contains, but not limited to, the following important expression elements: 1) a human cytomegalovirus early promoter and an enhancer required for high exogenous expression in mammalian cells; 2) a double selection marker which may confer kanamycin resistance to bacteria and G418 resistance to mammalian cells; 3) an expression cassette of mouse dihydrofolate reductase (DHFR) gene, which allows the fusion gene and DHFR gene to be co-amplified in the presence of methotrexate (MTX) in DHFR gene-deficient host cells (See U.S. Pat. No. 4,399,216). The expression plasmid of the fusion protein was then transfected into a mammalian host cell line. DHFR enzyme-deficient CHO cells are preferred host cell line for stable expression at high levels (See U.S. Pat. No. 4,818,679).

(32) As shown in Table 1, the present invention constructed a series of hFIX fusion proteins comprising flexible peptide linkers of different lengths, CTP rigid units of different compositions, and several different subtypes of IgG Fc (vFc) variant elements. The nucleotide sequence of F9-5B and the translated amino acid sequence thereof are shown in FIG. 1.

(33) TABLE-US-00003 TABLE 1 Composition of several FIX fusion proteins constructed Elemental composition of the fusion Name protein (from N-terminus to C-terminus) F9-5A FIX-L3-CTP.sup.1-vFcγ.sub.1 F9-5B FIX-L2-CTP.sup.2-vFcγ.sub.2-3 F9-5C FIX-L5-CTP.sup.4-vFcγ.sub.4 F9-5D FIX-L1-CTP.sup.3-CTP.sup.3-vFcγ.sub.2-2 F9-5E FIX-L4-CTP.sup.3-vFcγ.sub.2-1 F9-5F FIX-L2-vFcγ.sub.2-3-CTP.sup.2 F9-5G FIX-L4-vFcγ.sub.2-3

Example 2. Transient Expression of Various Fusion Proteins and Determination of in Vitro Activity of the Same

(34) The series of expression plasmids as obtained in Example 1 were respectively transfected into 3×10.sup.7 CHO-K1 cells in a 30 mL shake flask by using DNAFect LT Reagent™ (ATGCell), and the transfected cells were cultured in serum-free growth medium containing 1000 ng/mL of vitamin K1 for 5 days. The concentration of the fusion protein in the supernatant was measured and the activity thereof was determined by the method as described in Example 6. The ELISA results showed that the transient expression levels of these plasmids were similar under these conditions, but the coagulation activities of these fusion proteins showed large differences. The activities of F9-5B, F9-5C, F9-5D and F9-5E were 119.5%, 104.2%, 83.9% and 94.7%, respectively, of that of F9-5A, whose molar specific activity was defined as 100%. The activity of F9-5F was only about 30% of that of F9-5B, probably because that the CTP rigid unit which was placed at the N-terminus of Fc formed a fixed spatial conformation to effectively separate different functional regions of the fusion protein, which facilitated FIX and the Fc part to fold independently into correct three-dimensional conformations, thereby maintaining a high activity. The fusion protein in the supernatant of F9-5G cell culture mostly existed in the form of inactive polymers. This may be because that an over-length peptide linker can not increase the activity of the fusion protein, but instead will cause the protein to fold incorrectly and exist as inactive polymers.

Example 3 Expression of Fusion Proteins in Transfected Cell Lines

(35) The expression plasm ids of the fusion proteins as described above were transfected into mammalian host cell lines to express FIX fusion proteins. DHFR-deficient CHO cells are preferred host cell line for stable expression at high levels (See U.S. Pat. No. 4,818,679). A preferred method of transfection was electroporation, and other methods including calcium phosphate co-deposition, liposome transfection and microinjection might also be used. In electroporation, Gene Pulser Electroporator (Bio-Rad Laboratories) was used at a voltage of 300 V and a capacitance of 1050 μFd, and 50 μg of Pvul-linearized expression plasmid was added to 3×10.sup.7 cells placed in the cuvette. The electroporated cells were transferred to a shake flask containing 30 ml of growth medium. Two days after the transfection, the medium was replaced with a growth medium containing 0.6 mg/mL of G418. The cells were seeded in 96-well plates at a certain concentration and cultured for 12-15 days until large discrete cell clones appeared. Transfectants resistant to the selected drug were screened by an ELISA assay against human IgG Fc. Quantification of the expression of the fusion protein can also be performed by using an ELISA assay against FIX. The wells producing high levels of Fc fusion protein were subcloned by limiting dilutions.

(36) To achieve higher levels of fusion protein expression, co-amplification utilizing the DHFR gene that can be inhibited by an MTX drug is preferred. The transfected gene of the fusion protein was co-amplified with the DHFR gene in growth media containing increasing concentrations of MTX. Subclones with positive DHFR expression were subjected to limiting dilution, and transfectants capable of growing in media containing up to 6 μM of MTX were screened by increasing the selection pressure gradually. The transfectants were measured for secretion rates and cell lines yielding high levels of exogenous protein were screened. Cell lines with a secretion rate of more than about 1, preferably about 2 mg/10.sup.6 [i.e. million] cells/24 h, were adapted to suspension culture by using serum-free growth media. Conditioned media was then used to purify the fusion protein.

Example 4. Production of Fusion Proteins

(37) First, the high-yielding cell lines obtained in Example 3 were subjected to serum-free adaptation culturing in a petri dish and then transferred to a shake flask for suspension adaptation culturing. After these cells were adapted to these culture conditions, they were fed-batch cultured in a 300 mL shake flask, or a perfusion culture was simulated by replacing the medium daily. The CHO-derived cell line expressing the fusion protein F9-5B obtained in Example 3 was fed-batch cultured in a 300 mL shake flask for 14 days. The cumulative yield of the expressed recombinant fusion protein reached 200 mg/L, and the viable cell density reached up to 18×10.sup.6 cells/m L. 1000 mL shake flasks could be used for producing more fusion proteins. In another culture method, the CHO-derived cell line as described above was cultured in a 100 mL shake flask with the medium changed daily. The expressed recombinant fusion protein reached a cumulative yield of about 30 mg/L per day. The viable cell density in the shake flask reached up to 35×10.sup.6 cells/mL. The biological activities of the recombinant fusion proteins produced by the above two methods were equivalent.

Example 5. Purification and Characterization of Fusion Proteins

(38) Affinity chromatography was mainly used in the present invention to purify FIX fusion protein F9-5B. The instrument used for protein purification in this example was AKTA Explorer 100 (GE Healthcare, USA). The reagents used in this example were all analytical-grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

(39) Step 1: affinity chromatography. Sample capture, concentration, and removal of part of contaminants were performed by using Mabselect Sure available from GE or other commercially available recombinant protein A affinity chromatography media, such as Mabselect, Mabselect Sure LX available from GE, anti-alkali Protein A Diamond available from Bestchrom, Toyopearl AF-rProteinA-650F available from TOSOH, rProtein A Bead available from Smart-Lifesciences, MabPurix available from Sepax Technologies, KANEKA KanCapA available from Pall and Eshumono A available from Merck. First, the column was equilibrated with 3-5 column volumes of equilibration buffer (20 mM PB, 140 mM NaCl, pH 6.8-7.4) at a linear flow rate of 50-100 cm/h; the clarified fermentation broth was loaded at a linear flow rate of 50-100 cm/h; after loading, the column was equilibrated with 3-5 column volumes of equilibration buffer (20 mM PB, 140 mM NaCl, pH 6.8-7.4) at a linear flow rate of 50-100 cm/h to rinse unbound components; the column was rinsed with 3-5 column volumes of decontamination buffer 1 (20 mM Citric-Citrate, 0.5 M NaCl, pH 4.8-5.2) at a linear flow rate of 50-100 cm/h to remove part of contaminants; the column was equilibrated with 3-5 column volumes of decontamination buffer 2 (20 mM Citric-Citrate, pH 4.8-5.2) at a linear flow rate of 50-100 cm/h; then the target product was eluted with elution buffer (50 mM NaAc-HAc, 1.0 M Urea, pH 3.0-4.0) at a linear flow rate of no more than 60 cm/h. Products corresponding to the target peak were collected and neutralized to neutral to acidic (pH 4.8-5.2) with 1M Tris, pH 9.0.

(40) Step 2: anion exchange chromatography. Intermediate purification was carried out with Q Sepharase FF available from GE or other commercially available anion exchange chromatography media, such as DEAE Sepharose FF, Q Sepharose HP, Capto Q, Capto DEAE available from GE, Toyopearl GigaCap Q-650 available from TOSOH, DEAE Beads 6FF available from Smart-Lifesciences, Generik MC-Q available from Sepax Technologies, Fractogel EMD TMAE available from Merck, and Q Ceramic HyperD F available from Pall, to decrease the amount of HCP, residual DNA, and shed protein A. The eluent obtained in step 1 still contained a certain proportion of HCP, residual DNA, endotoxin and other contaminants, so it is necessary to remove these contaminants. First, the column was equilibrated with 3-5 column volumes (CVs) of equilibration buffer (40 mM Na.sub.2PO.sub.4-Citric, 0.1 M NaCl, pH 4.8-5.2) at a linear flow rate of 50-100 cm/h; the sample captured by the affinity chromatography was diluted 1 fold with the equilibration buffer and then loaded. The target protein flowed through under this condition. The flow-through samples were collected once the A.sub.280 was raised to 100 mAU. After loading, the column was rinsed equilibration buffer (40 mM Na.sub.2PO.sub.4-Citric, 0.1 M NaCl, pH 4.8-5.2) at a linear flow rate of 50-100 cm/h, and flow-through samples were collected until the A.sub.280 decreased to 100 mAU, at which point the collection was stopped; then the column was rinsed with 3-5 column volumes of regeneration buffer (1M NaCl, 1M NaOH) at a linear flow rate of 50-100 cm/h to regenerate the column. Samples collected were detected for HCP, DNA, Protein A, and SEC-HPLC.

(41) Step 3, affinity chromatography. The final purification was carried out by using Cellufine Sulfate available from JNC or other commercially available affinity chromatography media such as Heparin FF and Heparin HP available from GE to remove aggregates and further remove contaminants such as HCP and DNA. First, the column was rinsed with 3-5 column volumes of equilibration buffer (20 mM PB, 100 mM NaCl, pH 7.0-7.4) at a linear flow rate of 50-100 cm/h; the target protein obtained after the anion chromatography in step 2 was diluted 1 fold with the equilibration buffer to decrease the concentration of organic matters and then loaded; after loading, the column was rinsed with 3-5 column volumes of equilibration buffer (20 mM PB, 100 mM NaCl, pH 7.0-7.4) at a linear flow rate of 50-100 cm/h; the column was then eluted with a linear gradient of salt concentrations, elution buffer: 20 mM PB, 1 M NaCl, pH 7.0-7.4, with elution buffer from 0-100%, 15 column volumes, linear flow rate of no more than 50 cm/h. The eluted fractions were collected in stages, and the collected samples were detected for protein content, SEC-HPLC, activity and HCP content respectively. The specific activity of the protein was calculated to be about 200 IU/mg as determined by protein concentration and protein activity.

(42) Results of the SEC-HPLC chromatography and SDS-PAGE electrophoresis of the sample are shown in FIGS. 2 and 3, respectively. The results of SEC-HPLC showed that the purity of the main peak of the purified fusion protein was more than 90%, and the band pattern in the SDS-PAGE electrophoresis was in line with expectations. The non-reduction electrophoresis contained the fusion protein, and a clear single-strand band was obtained after reduction.

Example 6. Determination of the In Vitro Activity of the Fusion Protein by a Chromogenic Substrate Assay

(43) The activity of the FIX-Fc fusion protein can be determined by a chromogenic substrate assay. In this example the BIOPHEN Factor IX kit (HYPHEN BioMed, Ref. A221802) was used for determination based on the principle as follows. Factor XIa supplied in the kit activates Factor IX presented in the tested sample into FIXa, which forms a thrombin complex with thrombin-activated FVIII:C, phospholipids (PLPs) and calcium ions (Ca.sup.2+) in the presence of thrombin, PLPs and Ca.sup.2+. The enzyme complex activates Factor X in the determination system into an activated form, Xa. The activation activity of the thrombin complex to Factor X is positively correlated with the content of Factor IX in the tested sample. The activity of the activated Factor Xa can be measured by its specific cleavage on a chromogenic substrate (SXa-11), that is, by measuring the absorbance of its cleavage product, pNA, at 405 nm. The absorbance of pNA is directly proportional to the activity of FIXa.

(44) The purified FIX fusion protein F9-5B reached a specific activity of more than 200 IU/mg as determined by the present method.

Example 7 Pharmacokinetic Determination of the Fusion Protein

(45) Male SD rats (SPF grade, purchased from Bikai Experimental Animal Co., Ltd., Shanghai) were pre-fed for 1 week and then randomly divided into 2 groups (2 rats in each group). Rats were intravenously injected with a single dose of 4.5 mg/kg (high-dose group) and 1.5 mg/kg (low-dose group) of fusion protein F9-5B respectively, and investigated for the relationship between drug concentration in blood and time. 0.3 ml of blood was collected from orbits at 0, 1, 3, 6, 24, 48, 72, 96, 120, 144 and 168 hours after administration in the control group and administration group. The blood was allowed to stand at room temperature for 30 min, and centrifuged at 5000 rpm for 10 min to isolate the serum which was then stored at −20° C. The amount of fusion protein in the serum at each time point was determined by an ELISA assay specific for FIX. The main pharmacokinetic parameters were calculated for each group by the software PKSOLVER. The results are shown in Table 2.

(46) TABLE-US-00004 TABLE 2 Pharmacokinetic parameters of FIX fusion protein in SD rats Dose T.sub.1/2 (h) AUC 0-inf_obs Lambda_z(1/h) Vz_obs CI_obs 1.5 mg/kg 29.89 25333.27 0.024 0.60 0.01 4.5 mg/kg 31.57 80620.02 0.021 0.56 0.01

(47) According to the pharmacokinetic data, the in vivo half-life of the high- and low-dose fusion protein F9-5B was 31 and 30 hours, respectively, which was increased by 8 times than the T.sub.1/2 β value of rhFIX (Chinese Patent NO. CN104427994). The fusion protein F9-5B showed an improved half-life compared to rhFIX, demonstrating that the addition of a linker peptide and an Fc variant at the C-terminus of FIX did not interfere with the activity of the fusion protein, but instead produced an unexpected effect on the activity and half-life of the FIX fusion protein. It is speculated that the CTP rigid peptide, which links the FIX to a Fc variant together with a flexible peptide linker, can not only further prolong the in vivo half-life of FIX, but also increase the spatial distance between molecules in the fusion protein by means of the blocking effect resulted from multiple glycosylated side chains, which promotes FIX and the Fc segment to fold independently into correct three-dimensional conformations without affecting biological activities of each other. It can be seen that F9-5B exhibits superior performance in terms of bioavailability and pharmacokinetics compared to rhFIX.

(48) Although preferred embodiments of the present invention have been illustrated and described, it will be understood that various changes may be made by those skilled in the art in light of the teachings herein, without departing from the scope of the invention.

(49) All documents mentioned in the present invention are hereby incorporated by reference to the same extent as if each of the documents is individually recited for reference. It is to be understood that various modifications and changes may be made by those skilled in the art upon reading the above teachings of the present invention, which also fall within the scope of the claims appended hereto.