Title of Invention

"METHOD FOR MANUFACTURING HIGH PURIFIED FACTOR IX"

Abstract

The present invention relates to a method for manufacturing a highly purified human blood coagulation factor IX, and more preferably, to a method for manufacturing a highly purified human blood coagulation factor IX by subjecting a plasma-derived or recombinant material containing a human blood coagulation factor IX, and by using of ion chromatography and affinity chromatography and inactivating or removing viruses, thereby obtaining a highly purified, safe coagulation factor IX having a specific activity of above 150 IU/mg, with substantially inactivation or removal of all impure proteins and viruses.

Full Text

Technical Field The present invention relates to a method for manufacturing a highly purified human blood coagulation factor IX, and more preferably, to a method for manufacturing a highly purified human blood coagulation factor IX by subjecting a plasma-derived or recombinant material containing a human blood coagulation factor IX, and by using of ion chromatography and affinity chromatography and inactivating or removing viruses, thereby obtaining a highly purified, safe coagulation factor IX having a specific activity of above 150 IU/mg, with substantially inactivation or removal of all impure proteins and viruses. Background Art A human blood coagulation factor IX (abbreviated to Factor IX) is a glycoprotein that is indispensable for a coagulation cascade. Hemophilia B is caused by the absence or deficiency of Factor IX due to a hereditary cause or a pathological cause. The hemophilia B is a rare hereditary disease that primarily affects newborn male babies having prevalence of one of 30,000 ~ 50,000. Cure of the hemophilia B is performed by administering Factor IX concentrates. Factor IX is synthesized in the liver and has similar features like as other vitamin K-dependent glycoproteins. Vitamin K-dependent glycoproteins include Factor II, Factor VII, Factor X, and so on. The Factor IX, a kind of serine protease, is a monomeric glycoprotein containing about 17.5% glucose comprising 4.7% hexose, 6.8% N-acetylhexsamine, and 6% sialic acid. Factor IX has a molecular weight of 55 ~ 75 kDa (Daltons) according to the amount of carbohydrate contents. The exact molecular weight of only proteins, which is calculated based on amino acid sequences, is 47,054 Da. 12 gamma(ﻻ )-carboxyglutamic acid domains play important roles in the activation of Factor IX and thus Factor IX is activated in the presence of phospholipid and calcium ion (Ca2+). The diversity of isoelectric point (pi) of Factor IX depends on the kind and amount of carbohydrates present therein (Journal of Chromatography (1999) 844, 119). Factor IX is activated by the release of activation peptide fragments consisting of 35 amino acids due to cleavage of an arginine(Arg)-alanine(Ala) bond by Factor XIa at sites between Arg145and Ala146 and between Argigo and Val181actor IX without the activated peptides is referred to an active form of Factor IX to distinguish the natural form of Factor IX. The activation of Factor IX is also stimulated by a phospholipid complex comprising Factor VIIa Factor III, and calcium ion (Ca ) (Fig. 1). Factor IX is present in the human plasma in a very low concentration of about 0.1 u M (5 µg/ml). To treat a patient of hemophilia B, high doses of whole blood or plasma should be required. Such high dose blood administration may, however, involve severe side effects, which results from excessive administration of other proteins such as fibrinogen in the plasma. To overcome the side effects, Factor IX concentrates were developed for the first time in the late 1950s. To isolate Factor IX concentrates, Factor IX is first collected from the plasma and then adsorbed into barium sulfate, followed by precipitation and washing to remove other protein components from Factor IX adsorbed into barium sulfate. Later, barium sulfates were replaced with tricalcium phosphates which are nontoxic salts. The process of isolating Factor IX concentrates by adsorption and precipitation may also cause concentration of other vitamin K-dependent glycoproteins such as Factor II, Factor VII, Factor X, and so on. These concentrates are termed a prothrombin complex concentrates (PCC). Factor IX complex obtained above has been widely used as the therapeutic agent of the hemophilia B since the 1960s. A cold ethanol fraction process, which has been used to obtain albumin or globulin from plasma, was not suited to the separation of Factor IX complex. Thus, such conventional methods cannot be advantageously employed to purification of Factor IX. To solve the disadvantage, a column process using anion exchange chromatography has been developed. In the developed process, Factor VIII-rich fractions are removed by cryoprecipitation, and Factor IX present in the cryoprecipitate-free plasma is adsorbed into an anion exchange resin and then is purified. However, this process may lead to adsorption of other proteins such as Factor II, Factor VII, or Factor X. In spite of such low purified Factor IX, the Factor IX complex is currently in use for the treatment of patients with the hemophilia B. Unfortunately, there are several findings reporting that administration of Factor IX complex cause incidence of symptoms such as venous thrombosis or disseminated intravascular coagulation (Thrombosis and Haemostasis (1995) 73, 584. Thrombosis and Haemostasis (1998) 79, 778). Such side effects presumably result from hypercoagulation states caused by excessive amounts of coagulant proteins or unnecessary thrombogenic components of the concentrates when administered along with Factor IX. In order to alleviate side effects of Factor IX complex, an approach for removing Factor II, Factor VII, Factor X, and other proteins from the Factor IX complex was attempted, and a highly purified Factor IX had become commercially available from the 1990s. The highly purified Factor IX was obtained by subjecting concentrated Factor IX complex to anion exchange chromatography followed by heparin gel or monoclonal antibody chromatography, thereby removing Factor II, Factor VII, Factor X, and other proteins from the Factor IX complex. Thus Factor IX complex is currently being replaced by the highly purified Factor IX. A recombinant coagulation factor IX using the Chinese hamster ovary (CHO) cell line was developed and manufactured by Genetic Institute in the trade name of Benefix in 1997. The most serious problem in the treatment of blood-derived coagulation factors is the risk of viral infection due to plasma-derived viruses such as HIV, HBV, HCV, and the like. To ensure virus safety of the blood-derived coagulation factors, a virus removal is an indispensable process for ensuring the safety of blood-derived coagulation factors. Virus inactivation is generally performed by solvent/detergent (S/D) treatment. To achieve a more effective method of virus removal, a process of affinity chromatography can be added in the process of virus inactivation. The most effective method of virus removal is a nanofiltration technique using a 20 nm grade nanofilter. However, this technique requires a process of preliminarily purifying a coagulation factor to obtain a highly purified coagulation factor, otherwise aggregation or clogging of proteins make the nanofilter useless. Use of monoclonal antibodies is effective in selectively separating Factor IX from other coagulation factors. However, since animal cells has been used for antibody production, it is impossible to avoid the risks of viral transmission due to viruses derived from the animal cells. Furthermore, side effects can be appeared according to eluting antibodies during chromatography. It is also impossible to overcome the problem associated with viral infection when Factor IX is isolated from a recombinant cell culture media. Accordingly, there is a need a method for manufacturing a highly purified Factor IX, which has a purity of greater than 99% and a safety inactivated and removed of viruses. Disclosure Technical Problem To solve the above problems, it is an object of the present invention to provide a method for manufacturing a highly purified Factor IX for eliminating side effects such as venous thrombosis or disseminated intravascular coagulation (DIC), which resulting from hypercoagulable states caused by excessive amounts of coagulant proteins or unnecessary thrombogenic components of the concentrates when administered along with Factor IX. Technical Solution Hereinafter, a method for manufacturing a highly purified Factor IX according to the present invention will be described in detail. Fig. 2 is a process flow diagram illustrating a method for manufacturing a highly purified Factor IX according to the present invention. A solution containing Factor IX used in the present invention is not particularly restricted in their forms. In the method for manufacturing a highly purified Factor IX according to the present invention, cryoprecipitate-free human plasma is preferably used as a starting material. In addition, examples of the starting substance include plasma fractions, cell culture broth of recombinant Factor IX, and solution containing the Factor EX, and so on. In an embodiment of the present invention, a method for manufacturing a highly purified Factor IX from a material containing the Factor IX as a starting material provided aprimarily eluting a solution containing the Factor IX by anion exchange chromatography, followed inactivating viruses by Solvent/Detergent (S/D) treatment, secondarily eluting the resultant by anion exchange chromatography, concentrating and filtrating the secondarily eluted product, obtaining eluate by heparin affinity chromatography, collecting an unbound solution by cation exchange chromatography for further purification, and removing viruses by subjecting the collected unbound solution to nanofiltration. In detail, in order to remove impure proteins remaining in the plasma protein solution eluted by the anion exchange chromatography, the eluate is purified by heparin affinity chromatography using heparin as a ligand for a blood coagulation factor. Then, the eluate is subjected to cation exchange chromatography to collect an unbound solution that is not adsorbed into a cation exchanger, thereby removing other impurities remaining in the eluate. Since heparin is used as a ligand in the heparin affinity chromatography, a variety of resin materials including Sepharose and agarose and polyacrylamide can be used as a support, but not limited to herein. Particularly, a heparin Sepharose 6FF column is currently preferred. According to the present invention, when eluate is purified by heparin affinity chromatography, an equilibration buffer used prior to application of the solution containing Factor IX and a solution containing Factor IX preferably have ion strengths of not greater than 20 mS/cm and pH range of between 5.0 and 9.0. In addition, a washing buffer used for washing other proteins adsorbed into a resin preferably has ion strengths of 10 ~ 20 mS/cm. Furthermore, an elution buffer used for eluting the solution containing Factor IX preferably has ion strengths of 20 ~ 50 mS/cm. A particularly preferred embodiment relates to the isolation of Factor IX by cation exchange chromatography, for example by using the following materials: weak cations such as carboxymethyl- (CM-), or carboxy- (C-); and strong cations such as sulfo- (S-), sulfomethyl- (SM-), sulfoethyl- (SE-), sulfopropyl- (SP-), or phospho- (P-). In addition, a variety of column resins can be used, including Sepharose, Sephadex, agarose, Sephacel, Polystyrene, Polyacrylate, Cellulose, and Toyopearl. In the present invention, the cation exchange chromatography is preferably performed at a pH level in a range of between 3.0 and 6.0 such that an unbound solution that is not adsorbed into a column is collected for purification. Although an equilibration buffer used in the cation exchange chromatography can be used in various manners, the solution containing the equilibration buffer used prior to application of the solution containing Factor IX and the solution containing Factor IX have ion strength in a range of between about 10 and about 50 mS/cm. Advantageous Effects The present invention provides a method for manufacturing a highly purified Factor IX. More particularly, the method for manufacturing a highly purified Factor IX according to the present invention comprise of subjecting a plasma-derived or recombinant material containing a human blood coagulation factor IX to ion chromatography and affinity chromatography and inactivating or removing viruses, thereby obtaining a highly purified, safe coagulation factor IX having a specific activity of above 150 IU/mg, with substantially inactivation or removal of all impure proteins and viruses. Description of Drawings Fig. 1 shows intrinsic and extrinsic coagulation pathways; Fig. 2 is a process flow diagram illustrating a method for manufacturing a highly purified Factor IX according to the present invention; Fig. 3 shows results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis identifying purity of Factor IX manufactured by the present invention and otiier commercially available Factor IX products in non-reducing (A) and reducing (B) condition. M: Standard marker (myosin; 198kDa, galactosidase; 115kDa, BSA; 93kDa, ovalbumin; 49.8kDa, carbonic anhydrase; 35.8kDa, soybean trypsin inhibitor; 29.2kDa, lysozyme; 21.3kDa, aprotinin; 6.4kDa); Lane 1: highly purified Factor IX according to method mentioned in table 2; Lane 2: highly purified Factor IX according to method mentioned in table 3; Lane 3: highly purified Factor IX according to method mentioned in table 4; Lane 4: Facnyne (Greencross); Lane 5: Mononine (ZLB Behring); Lane 6: Octanyne (Octapharma); Lane 7: Berinin HS (ZLB Behring); and Lane 8: Immunine (Baxter). FIG. 4 is a graph showing the results of tests for evaluating stability of activity expressed as the titer carried out under CM-Sepharose fast flow (FF) purification conditions. Best Mode for Carrying Out the Invention EXAMPLE 1: Purification of Factor IX by anion exchange chromatography (conventional Process) A cryoprecipitate-free plasma or cell culture medium of recombinant Factor IX as a starting material was mixed with DEAE-Sephadex A-50, prewashed with buffer, and stirred for 2 hours at 40C. Subsequently, the gel to which Factor IX was now bound was separated by filtration or centrifugation. After washing, Factor IX was eluted by a buffer with high salt concentration. A pH of the eluate was appropriately adjusted to 7.5 and then the eluate was subjected to dialysis and concentration for storage at -70 0C. The concentrate was aliquoted and measured the activity under the standards and test methods of blood formulations and recombinant formulations as stipulated by the KFDA (Korea Food and Drug Administration). The above fraction containing Factor IX was subjected to perform a virus inactivation process by S/D treatment. A post-inactivation solution was adsorbed into a DEAE-toyopearl 650M column for second purification, followed by collecting an eluate. EXAMPLE 2: Purification of Factor IX by Heparin affinity chromatography 10 ml of the Factor IX complex solution prepared by the purification in Example 1 was conditioned to have an ion strength of not greater than 20 mS/cm, and pH 7.0, and an equilibration buffer (20 mM sodium citrate, pH 7.0) was induced to a Heparin-Sepharose 6FF column (column conditions: 1 cm in diameter, 18 cm in height, 1.0 ml/min in flow rate, and room temperature) for equilibration. Meanwhile, a sample was loaded for adsorption, washed with the equilibration buffer plus 0.1 M NaCl, and then eluted using 0.25 M NaCl. A protein concentration of the eluate was determined and SDS-PAGE was carried out. The activity was measured using a coagulation timer KC10 manufactured by Amelung, Germany. A specific activity of the eluate preparation from Example 1 was compared with that of the eluate preparation from Example 2. As the result, the specific activity of the Heparin-Sepharose 6FF column eluate of Example 2 was about 60-90 IU/mg, which showed an increase of at least 10 times that of the anion exchange column eluate of Example 1. EXAMPLE 3: Purification of Factor IX by cation exchange chromatography 10 ml of the Factor IX complex solution prepared by the purification in Example 2 was conditioned to have an ion strength of not greater than 20 mS/cm, and pH 4.0, and an equilibration buffer (20 mM citrate in 0.30 ~ 0.4 M NaCl, pH 4.0) was induced to an equilibrated CM-Sepharose 6FF column (column conditions: 1 cm in diameter, 5 cm in height, and 1.0 ml/min in flow rate) for collecting an unbound solution. A protein concentration of the unbound solution was determined and SDS-PAGE analysis was carried out. A specific activity of the unbound solution was at least 150 IU/mg. Fig. 3 shows results of SDS-PAGE analysis carried out to determine purity of Factor IX manufactured by the present invention and other commercially available Factor IX products. As shown in Fig. 3, Factor IX manufactured by the present invention had little impure proteins, suggesting that they had higher purity levels than other Factor IX products. EXAMPLES 4 - 6: Large-scale Purification of Factor IX To confirm large-scale feasibility of the highly purified Factor IX according to the present invention, scale-up experiments were carried out several times. In each of the scale-up experiments, various data items, including specific activity, purification fold, yield, and so on, are shown in Tables 1 through 6 below. Table 1 (Table Removed) As shown in Tables 1 through 6, repeated practices of the present invention confirmed that highly purified Factors DC have the specific activity of at least 150 IU/mg in final concentrates. The final concentrates were placed into vials each by an appropriate amount and lyophilized to be used as test samples of prolonged stability and preclinical samples. EXAMPLE 7: Purification of Factor IX from cell culture medium CHO cells cloned with the Factor IX were cultured in serum free media, and a 5L culture media removed the CHO cells was treated as the same methods as mentioned in Examples 1 through 3, except of S/D treatment, and thereby Factor DC was purified. Table 7 shows various data items, including specific activity, purification fold, yield, and so on. Table 7 (Table Removed) (Table Removed) EXAMPLE 8: Virus inacrivation and removal process In virus inactivation and nanofiltration steps, clearance tests were conducted on various viruses, including HIV (Human Immunodeficiency Virus), BHV (Bovine Herpes Virus), BVDV (Bovine Viral Diarrhea Virus), HAV (Hepatitis A Virus), EMCV (Encephalo Mycarditis Virus), PPV (Porcine Parvovirus), and so on. The results are shown in Tables 8 and 9 below. Virus clearance values shown in Table 8 are log factors obtained by subtracting titers of processing from titers of stock viruses used, while virus reduction values shown in Table 9 are log factors of values obtained by subtracting titers of processing from spiked titers of stock viruses after spiking the stock viruses into target viruses. Table 8 Virus clearance on the processing of plasma-derived Factor IX (Table Removed) Table 9 Virus reduction on the processing of plasma-derived Factor IX (Table Removed) Industrial Applicability The present invention is industrially applicable by providing a method for manufacturing a highly purified Factor IX comprise of subjecting a plasma-derived or recombinant material containing a human blood coagulation factor IX to ion chromatography and affinity chromatography and inactivating or removing viruses, thereby obtaining a highly purified, safe coagulation factor IX having a specific activity of above 150 IU/mg, with substantially inactivation or removal of all impure proteins and viruses. We claim: 1. A method for manufacturing a highly purified human blood coagulation factor IX (Factor IX) using a material containing Factor IX as a starting material, the method comprising: collecting a solution containing Factor IX by performing anion exchange chromatography one or more times; eluting the solution containing Factor IX by performing heparin affinity chromatography; collecting an unbound solution by performing cation exchange chromatography and removing viruses by subjecting the collected unbound solution to nanofiltration. 2. The method of claim 1, in the step on heparin affinity chromatography, wherein equilibration buffer and the solution containing the Factor IX have ion strength of not greater than 20 mS/cm at a pH range of 5.0 ~ 9.0, a washing buffer has ion strength of 10 ~ 20 mS/cm, and an elution buffer has ion strength of 20 ~ 50 mS/cm. 3. The method of claim 1, wherein a column functional group for the cation exchange chromatography is selected from the group consisting of carboxymethyl- (CM-), carboxy-(C-), sulfo- (S-), sulfomethyl- (SM-), sulfoethyl- (SE-), sulfopropyl- (SP-), and phospho- (P-), and a column resin is selected from the group consisting of Sepharose, Sephadex, agarose, Sephacel, Polystyrene, Polyacrylate, Cellulose, and Toyopearl. 4. The method of claim 1, wherein the collecting of the unbound solution by the cation exchange chromatography is performed at a pH in a range of between 3.0 and 6.0. 5. The method of claim 1, wherein the collecting of the unbound solution by the cation exchange chromatography is performed using a solution containing an equilibration buffer and Factor IX having ion strength in a range of 10 ~ 50 mS/cm. 6. The method of claim 1, wherein the material containing Factor IX is a human plasma-derived or cell culture medium of recombinant Factor IX. 7. A method for manufacturing a highly purified human blood coagulation factor IX, substantially as hereinbefore described with reference to the foregoing examples and accompanying drawings.

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3915-delnp-2008-1-Correspondence-Others-(19-06-2013).pdf

3915-delnp-2008-1-Form-1-(19-06-2013).pdf

3915-delnp-2008-1-Petition-137-(19-06-2013).pdf

3915-delnp-2008-abstract.pdf

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3915-delnp-2008-Correspondence Others-(20-12-2012).pdf

3915-delnp-2008-Correspondence-Others-(19-06-2013).pdf

3915-delnp-2008-correspondence-others.pdf

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3915-delnp-2008-Form-3-(20-12-2012).pdf

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3915-delnp-2008-pct-101.pdf

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3915-delnp-2008-pct-304.pdf