MXPA99010045A - Procedure for the purification of recombinant human erythropoyetine from recombinant cell culture supplementers and human erythropoyetin obtained with such procedime - Google Patents

Abstract

The present invention relates to a method for obtaining high purity recombinant human EPO. The claimed process is characterized, furthermore, by avoiding exposing the protein to extreme temperature conditions and to organic solvents that could affect its biological activity or become toxic, its use in humans. The procedure consists of differential precipitation, hydrophobic interaction chromatography, concentration and diafiltration, successive anionic and cationic chromatography, new concentration and diafiltration and gel permeation chromatography. The procedure is distinguished by not using steps of high pressure liquid chromatography and by a high percentage of recovery of EPO. The invention also comprises the EPO obtained according to the procedure described

Description

PROCEDURE FOR ERITROPOYETINE PURIFICATION HUMAN RECOMBINANT FROM SUPPORTING PARTIES CULTIVATION OF CELLULES AND ERYTHROPOYETINAHUMANA RECOMBINANT OBTAINED WITH SUCH PROCEDURE Technical Description of the Invention A method for obtaining erythropoietin (EPO) characterized by a sequence of concatenated separation steps including differential precipitation, hydrophobic interaction liquid chromatography, liquid anion exchange chromatography, liquid cation exchange chromatography and liquid chromatography of molecular exclusion. The EPO obtained by the method described.

TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for the purification of EPO.

STATE OF THE ART EPO is a glycoprotein that stimulates the differentiation of erythroblasts in the bone marrow, thereby increasing the number of erythrocytes in the blood. The average life of erythrocytes in humans is 120 days, for which a human being loses 1/120 of their erythrocytes every day.

This loss must be continuously replenished to keep the number of red blood cells stable.

The existence of the EPO was postulated from the beginning of the century and was definitely demonstrated by Reissman and Erslev in the early 50's. See Carnot, et al., C.R. Acad. Sci., (France), 143, 384-6 (1906); Carnot, et al., C.R. Acad. Sci., (France), 143, 432-5 (1906); Carnot, et al., C.R. Soc. Biol., 111, 344-6 (1906); Carnot, C.R. Soc. Biol., 111, 463-5 (1906); Reissman, Blood, 1950, 5, 372-80 (1950) and Erslev, Blood, 8, 349-57 (1953). The experiments of Reissman and Erslev were quickly confirmed by other researchers. See Hodgson, et al., Blood, 9, 299-309 (1954); Gordon, et al., Proc. Soc. Exp. Biol. Med., 86, 255-8 (1954) and Borsook, et al., Blood, 9, 734-42 (1954). The individualization of the production site sparked a great debate. Successive works led to identify the kidney as the main organ and peritubular interstitial cells as the producer synthesis site. See Jacobson, et al., Nature, 179, 633-4 (1957); Kuratowska, et al., Blood, 18, 527-34 (1961); Fisher, Acta Hematol., 26, 224-32 (1961); Fisher, et al., Nature, 205, 611-2 (1965); Frenkel, et al., Ann. N.Y. Acad. Sci., 149, 1, 292-3 (1968); Busuttil, et al., Proc. Soc. Exp. Biol. Med., 137, 1, 327-30 (1971); Busuttil, Acta Haematol., (Switzerland), 47, 4, 238-42 (1972); Erslev, Blood, 44, 1, 77-85 (1974); Kazal, Ann. Clin. Lab. Sci., 5, 2, 98-109 (1975); Sherwood, et al., Endocrinology, 99, 2, 504-10 (1976); Fisher, Ann. Rev. Pharmacol. Toxicol., 28, 101-22 (1988); Jelkmann, et al., Exp. Hematol., 11, 7, 581-8 (1983); Kurtz, et al., Proc. Natl. Acad. Sci., (USA), 80, 13, 4008-11 (1983); Caro, et al., J. Lab. Clin. Med., 103, 6, 922-31 (1984); Caro, et al., Exp. Hematol., 12, 357 (1984); Schuster, et al., Blood, 70, 1, 316-8 (1986); Bondurant, et al, Mol. Cell. Biol., 6, 7, 2731-3 (1986); Bondurant, et al., Mol. Cell. Biol., 6, 7, 2731-3 (1986); Schuster, et al., Blood, 71, 2, 524-7 (1988); Koury, et al., Blood, 71, 2, 524-7 (1988); Lacombe, et al., J. Clin. Invest., 81, 2, 620-3 (1988); Koury, et al., Blood, 74, 2, 645-51 (1989). A smaller proportion, from 10% to 15% of the total EPO is produced by the liver in adults. See Naughton, et al., J. Surg. Oncol., 12, 3, 227-42 (1979); Liu, et al., J. Surg. Oncol., 15, 2, 121-32 (1980); Dornfest, et al., Ann. Clin. Lab. Sci., 11, 1, 37-46 (1981); Dinkelaar, et al., Exp. Hematol., 9, 7, 796-803 (1981); Caro, et al., Am. J. Physiol., 244, 5 (1983); Dornfest, et al., J. Lab. Clin. Med., 102, 2, 274-85 (1983); Naughton, et al., Ann. Clin. Lab. Sci., 13, 5, 432-8 (1983); Jacobs, et al., Nature, 313, 6005, 806-10 (1985); Erslev, et al., Med. Oncol. Tumor. Pharmacother., 3, 3-4, 159-64 (1986). EPO is produced proportionally to the degree of tissue hypoxia, and its expression grows by increasing the number of producing cells. EPO is a protein that has shown great efficacy for the treatment of anemia caused by different factors, especially anemia of renal origin. However, its therapeutic availability was limited until recently by the lack of a mass production method, since the quantity and quality of the EPO obtained by any of the known extractive systems were insufficient. Recently, the use of recombinant DNA techniques has made it possible to obtain proteins in large quantities. The application of these techniques to eukaryotic cells has allowed the large-scale production of EPO. See US patents 5,688,679 (Powell), 5,547,933 (Lin), 5,756,349 (Lin), 4,703,008 (Lin) and 4,677,195 (Hewick et al.) Once productivity is assured, the next problem is the systems of separation of the EPO, fundamental to guarantee the purity necessary for its application in humans.

Currently there are several techniques for the separation of glycoproteins such as EPO. These techniques include ultrafiltration, electro-focusing on a column, electrophoresis on a flat bed, filtration using gels, electrophoresis, isotachophoresis and several other chromatographic methods. Among the most used chromatography techniques are the ion exchange method and the adsorption chromatography.

The ion exchange method is a separation technique by which the components of the solution are distinguished according to their differential load and isolated by elution, either stepwise or through the application of a continuous gradient, with eluents of varying strength ionic or pH. The method uses a gel or resin matrix, either positive or negative, to induce the sticking or electrostatic adsorption of the components of opposite charge. During desorption or elution the components of the sample are displaced from the resin by the ions present in the solution or buffer used to elute, or by a pH modification that alters the differential charge of the molecule of interest. Reverse phase adsorption chromatography involves the separation of the components of the sample according to their different polarities. The components of the sample are first adsorbed by a resin composed of a silica matrix coated with an organic polymer. Subsequently, when the displacement of the components is verified by non-polar molecules of the eluting solvent, either in stages or through the application of a continuous gradient, selective desorption of the sample is obtained. The separation techniques mentioned above were initially used in the separation of relatively small hydrophobic or hydrophilic molecules. Its application to the separation of larger molecules, such as proteins, and especially to complex proteins such as lipoproteins, nucleoproteins and glycoproteins, is more recent. Several publications illustrate the state of the art regarding the separation of proteins. See Sofer, et al., "Handbook of Process Chromatography", (Academic Press Inc., San Diego, California, 1997); Olson, Ed., "Separation Technology", (Interpharm Press, Inc., Buffalo Grove, Illinois, 1995); Franks, Ed., "Protein Biotechnology", (Humana Press, Totowa, New Jersey, 1993); Deutscher, Ed., "Guide to Protein Purification", Methods of Enzymology, Vol. 182 (Academic Press Inc., San Diego, California, 1991); Seetharam and Sharma, Eds., "Purification and Analysis of Recombinant Proteins", (Marcel Dekker, Inc., New York, New York, 1991); Harris and Angal, Eds., "Protein Purification Applications", (Oxford University Press, Oxford, England, 1990); Brown, et al., Anaytical Biochemistry, 99, 1-21 (1979); Harrison, et al., "VDYAC ™ Comprehensive Guide to Reverse Phase Materials for HPLC," pp. 1-12, (The Sep / A / Ra / Tions Groups, Hesperia, California, 1984). On the other hand, the non-polar solvents recommended or usually used for the separation of proteins and polypeptides by the reverse phase high pressure liquid chromatography technique include reagents, such as acetonitrile, difficult to remove from the protein involved and potentially toxic to the Humans. See Parsons, et al., Endocrinology, 114, 6, 2223-2227 (1984). It should be noted, however, that aqueous solutions of ethanol and formic acid have been used for the elution of proteins. See Takagaki, et al., Journal of Biological Chemistry, 5, 4, 1536-1541 (1980). Previously, hematopoietic factors of interest due to their therapeutic use in humans, such as EPO, thrombopoietin and granulocyte colony stimulating factor, were obtained from urinary sources. See Miyake, et al., Journal of Biological Chemistry, 252, 15, 5558-5564 (1979). The recovery of these proteins from urinary sources was characterized by their low yield, inadequate purity and high cost due to the low level of concentration in which these proteins naturally occur. The application of immunoaffinity chromatographies for the recovery of proteins by the use of monoclonal antibodies specific for the protein of interest is also known. Nevertheless, in the particular case of EPO, by inducing the change of the affinity constant of the monoclonal antibody for the protein to achieve its recovery, the biological activity of EPO is drastically reduced. Recently, several specific methods for the separation of recombinant EPO have been revealed. One of these methods consists in the recovery of the protein by anion exchange chromatography with selective protease removal, followed by reverse phase chromatography and filtration. See US patent 4,667,016 (Lai, et al.) This technique claims a 16% yield of EPO with unknown specific activity and purity. Another proposed method for the separation of recombinant EPO consists of the application of reverse phase high pressure liquid chromatography to a solution of the partially purified protein. See US patent 4,677,195 (Hewick, et al.) This method could not be reproduced in practice.

Description of the Invention Although there is a large amount of information regarding the production of recombinant human EPO, a purification method that results in an EPO suitable for use in humans, with a purity greater than 99% and with absence of contaminants such as: a) aggregate material, b) degraded material, c) spurious proteins and, d) proteases. A purity less than 99% or the presence of any of the mentioned contaminants can be toxic to humans. On the other hand, several of the methods proposed for the purification of EPO are not efficiently applicable on an industrial scale. Some of the known methods incorporate reverse phase high pressure liquid chromatography steps. This method requires, on the one hand, a greater investment in equipment and maintenance compared to other separation methods and, on the other hand, it uses organic solvents that make the purification process more expensive and that are also dangerous, difficult to handle and highly polluting. environment. Other proposed methods are irreproducible in practice or have low recovery of the protein.

The present invention describes an EPO purification system characterized by a high recovery, purity and quality of the protein obtained. An advantage of the method described in this patent is the obtaining of EPO free of proteases and of unwanted molecular variants such as aggregates, degraded and isoelectric point molecules different from the expected one, among others. The EPO obtained by the claimed method has more than 99% purity and can be used to formulate pharmaceutical compounds for use in humans without the need for subsequent purification treatments. The EPO obtained by the claimed process is a heterogeneous protein composed of not less than 5 and not more than 8 isoforms of isoelectric point comprised between 3.0 and 4.5 and possesses a specific biological activity in vivo of more than 100,000 IU / mg of protein measured by the presence of 59Fe in ex-hypoxic polycythaemic mice. Another advantage of the claimed invention is its reproducibility on an industrial scale and its low environmental impact. The claimed process is a clean process that does not use separation steps based on reverse phase high pressure liquid chromatography, nor does it use organic solvents that may be harmful to the environment. A further advantage of the invention is that it avoids subjecting EPO to extreme temperature conditions or exposing it to organic solvents or other solutions that may be aggressive to the protein or toxic for use in humans. The claimed method consists of several separation steps including differential precipitation of supernatants of EPO-containing cell cultures, concentration and diafiltration, and chromatographies by hydrophobic interaction, anion exchange, cation exchange, and molecular exclusion. The following examples illustrate the separation steps followed in the claimed method.

Example 1 Recovery (Clarification) In 30 liters of sterile concentrate (See patent application AR-98-01-05611, of November 6, 1998), from culture supernatants of EPO-producing cells, which contained 10.7 g of EPO, 7,920 grams of ammonium sulphate. The solution is maintained at 4 ° C for 24 h. Several contaminating proteins precipitate while the EPO remains in solution. The product is centrifuged at 5,000 RPM, in a Sorvall brand centrifuge, using an HG4L rotor. This separation step yields 10.7 grams of EPO.

Example 2 Hydrophobic Interaction Chromatography The material from the previous step is chromatographed using a hydrophobic interaction matrix (Phenyl Sepharose 6 Fast Flow low sub.- Pharmacia) according to the following parameters and conditions: 1. Equipment a. Pre-Column 1) Diameter: 10 cm 2) Bed height: 25 cm 3) Matrix a) Q-Sepharose Big Bead. (Pharmacia) b) Volume: 2, 000 ml b. Column 1) Diameter: 20 cm 2) Bed height: 13 cm 3) Matrix: a) Phenyl-Sepharose 6 Fast Flow low sub. (Pharmacia) b) Volume: 4,000 ml Solutions and Buffers a. Buffer A: NaH2PO4 anh. 10 mM, pH 7.2 b. BufFer F: NaH2P04 anh. 10 mM, (NH4) 2S04 1.8 M, pH 7.2 c. Buffer G: NaH2PO4 anh. 150 mM, pH 7.2 d. Isoprol 20% e. 0.5 N NaOH Material a Chromatograph a. Ammonium sulfate supernatant from the previous example b. Sample conditions 1) Volume: 30,000 ml 2) Total protein mass measured by Bradford test: 20-40 grams 3) Approximate concentration measured by Bradford test: 0.6-1.3 mg / ml 4) Conductivity: 190-210 mSi / cm 5) pH: 7.2 4. Sanitization and Balancing of the Pre-column (*) To balance the precolumn, the following solutions or bufFers are sequentially passed through it. the quantities detailed below: 1.0 ve (2 liters) of water; 1.0 ve (2 liters) of 0.5N NaOH; 1.0 ve (2 liters) of BufFer G and finally 1.5 ve (3 liters) of BufFer F. 5. Sanitizing and Balancing the Column (*) In order to balance the column, the following are sequentially passed through it: solutions or buffers in the quantities detailed below: 1.0 ve (4 liters) of water; 1.0 ve (4 liters) of 20% isopropanol; 1.0 ve (4 liters) of water, 1.0 ve (4 liters) of 0.5N NaOH; 1.0 ve (4 liters) of water; 1.0 ve (4 liters) of BufFer G and finally 1.5 ve (6 liters) of BufFer F. 6. Run Conditions Once the precolumn and the column are balanced, the second one is connected after the first one and the seed is sown. material to be chromatographed. Said seeding is carried out at a temperature of 4 ° C at a flow of 100 ml / min. The elution is then carried out at the same flow but at room temperature, passing the following solutions and bufFers in the order and quantities detailed below: 2.5 ve (10 liters) of BufFer F, (after this buffer the precolumn is removed). Once the precolumn is removed, the chromatographic run is continued on the Phenyl Sepharose column on which a gradient of BufFer F-Buffer A is made starting from an 85:15 ratio of said bufFers until reaching a 50:50 ratio of the same in a total volume of 10 ve (40 liters). At the end of the gradient, 1.5 ve (6 liters) of BufFer F-BufFer A are passed in a 30:70 ratio and finally 1.5 ve (6 liters) of water. The selected fractions, containing EPO, are sterilized by filtration through a 0.22 μm porous membrane and kept at 4 ° C. This separation step yields 7.5 grams of EPO. (*) see means column volume Example 3 Concentration and Diafiltration The fractions from the previous example are concentrated and diafiltered according to the following parameters and conditions: 1. Equipment a. Peristaltic pump: Watson Marlow - Cat. No. 302S b. Tubing: Masterflex - Cat. No. 06402-18 c. Concentrator: Prep Scale Millipore CDU F006LC 2. Solutions and BufFers a. Sodium dodecyl sulfate (SDS) 10 mM b. Triton X-100 lmM c. NaOH 0, lN d. Water e. BufFer A: NaH2PO4 anh. 10 mM, pH 7.2 3. Material to Process a. Selected fractions from the previous example b. Sample conditions 1) Volume: 7,000-10,000 ml 2) Conductivity: 170-130 mSi / cm 3) pH: 7.2 4. Procedure First, the cleaning, sanitization and equilibration of the equipment is carried out by passing through the same next sequence of solutions and bufFers: 10 liters of 10 mM SDS; 40 liters of water; 10 liters of Triton X-100 1 mM, 40 liters of water; 10 liters of NaOH 0. ÍN; 40 liters of water and finally 5 liters of BufFer A. In this way the equipment is able to be used to carry out the concentration and diafiltration process against BufFer A of the selected fractions, according to the current methodology. 5. Final Condition of Concentration a. Final volume of concentrate: 2,000 ml b. Diafiltered against: BufFer A c. Conductivity: 1,100-1,550 μSi / cm d. pH: 7.2 This separation step yields 7.2 grams of EPO.

Example 4 Anion Exchange Chromatography The material from the previous example is chromatographed using an anion exchange matrix according to the following parameters and conditions: 1. Equipment a. Column 1) Diameter: 10 cm 2) Bed height: 25 cm 3) Matrix a) Q-Sepharose Fast Flow (Pharmacia) b) Volume: 2,000 ml Solutions and BufFers a. BufFer A: NaH2PO4 anh. 10 mM, pH 7.2 b. BufFer G: NaH2PO4 anh. 150 mM, pH 7.2 c. BufFer N: Ac. 50 mM acetic acid, 500 mM NaCl, pH 4.0 d. BufFer S: Ac. 50 mM acetic acid, pH 4.0 e. NaOH 0.5 N Material to Chromatograph a. Selected fractions of hydrophobic interaction, concentrated and diafiltered. b. Sample conditions 1) Volume: 2,000 ml 2) Total protein mass measured by Bradford test: 8.0-10 grams 3) Approximate concentration measured by Bradford test: 4.0-5.0 mg / ml 4) Conductivity: 1,100-1,550 μSi / cm 5) pH: 7.2 Sanitization and Balancing of the Column (*) In order to balance the column, the following solutions or bufFers are passed sequentially in the quantities They are detailed below: 1.0 ve (2 liters) of water; 1.0 ve (2 liters) of 0.5N NaOH; 1.0 ve (2 liters) of BufFer N; 2.0 ve (4 liters) of BufFer S; 3.0 ve (6 liters) of BufFer G; and finally 2.0 ve (4 liters) of BufFer A. 5. Corrida conditions (*) Once the column is balanced, the material is chromatographed. Said seeding is carried out at room temperature at a flow of 100 ml / min. The elution is then carried out at the same flow and temperature, passing the following solutions and bufFers in the order and quantities detailed below: 1.0 ve (2 liters) of BufFer A and 4.0 ve (8 liters) of BufFer S. Next a gradient of BufFer S-BufFer N is made starting from a 100: 0 ratio until reaching a 50:50 ratio of the same in a total volume of 1.5 ve (3 liters). At the end of the gradient, 1.5 ve (3 liters) of BufFer N are passed. The selected fractions, containing EPO, are sterilized by filtration through a 0.22 μm porous membrane and stored at 4 ° C. This separation step yields 5.9 grams of EPO. (*) see means column volume Example 5 Cationic Exchange Chromatography The material from the previous example is chromatographed using a cation exchange matrix according to the following parameters and conditions: 1. Equipment a. Column 1) Diameter: 10 cm 2) Bed height: 25 cm 3) Matrix a) SP-Sepharose Fast Flow. (Pharmacia) b) Volume: 2,000 ml Solutions and BufFers a. BufFer D: Na2HPO4.12H2O 12.5 mM, Ac. Cítrico.H2O 4 mM, pH 6.0 b. BufFer E: Na2HPO4.12H2O 12.5 mM, Ac. Cítrico.H2O 4 mM, NaCl 0.5M, pH 6.0 c. NaOH 0.5 N Material a Chromatograph a. Fraction selected in the previous example adjusted to pH 6.0 with Ce NaOH. and diluted to a conductivity of 4,800 μS / cm (conductivity equal to BufFer D-BufFerE 93.5: 6.5) b. Sample conditions 1) Volume: 5,000 ml 2) Total protein mass measured by Bradford test: 4.5-6.5 grams 3) Approximate concentration measured by Bradford test: 0.9-1.3 mg / ml 4) Conductivity: 4,800 μS / cm (Same as BufFer D-BufFer E in a ratio of 93.5: 6.5) 5) pH: 6.0 Sanitization and Balancing of the Column (*) To balance the column, proceed to pass through it, in sequentially, the following solutions or bufFers in the quantities detailed below: 1.0 ve (2 liters) of water; 1.0 ve (2 liters) of 0.5N NaOH; 1.0 ve (2 liters) of BufFer E and finally 1.5 ve (3 liters) of BufFer D-BufFer E in a ratio 93.5: 6.5 5. Run Conditions (*) Once the column is balanced, the material is chromatographed . Said seeding is carried out at room temperature at a flow of 100 ml / min. The elution is then carried out at the same flow and temperature, passing the following solutions and bufFers in the order and quantities detailed below: 1.5 ve (3 liters) of BufFer D-BufFer E in a 93.5: 6.5 ratio. Then a gradient of BufFer D-BufFer E is made starting from a 93.5: 6.5 ratio of said bufFer until reaching a 50:50 ratio of the same in a total volume of 2.0 ve (4 liters). At the end of the gradient 1.5 ve (3 liters) of BufFer E are passed. The selected fractions, containing EPO, are sterilized by filtration through a 0.22 mm porous membrane and stored at 4 ° C. This separation step yields 4.2 grams of EPO. (*) see means column volume Example 6 Concentration and Diafiltration The fractions from the previous example are concentrated and diafiltered according to the following parameters and conditions: 1. Equipment a. Peristaltic pump: Watson Marlow - Cat. No. 302S b. Tubing: Masterflex - Cat. No. 06402-18 c. Concentrator: Prep Scale Millipore CDU F002LC 2. Solutions and BufFers a. Sodium dodecyl sulfate (SDS) 10 mM b. Triton X-100 lmM c. NaOH 0, lN d. Water e. BufFer B: NaH2PO4 anh. 10 mM, 0.5 M NaCl, Lactose 0.05 mg ml, pH 7.2 Material to Process a. Fractions selected in the previous example b. Sample conditions 1) Volume: 8,000 mi 2) Total protein mass measured by Bradford test: 3.5-4.5 grams 3) Approximate concentration measured by Bradford test: 0.4-0.6 mg / ml 4) Conductivity: 5,000-8,000 μSi / cm 5) pH: 6.0 Procedure First, the equipment is cleaned, sanitized and balanced by passing the following sequence of solutions and buffeers: 10 liters of 10 mM SDS; 40 liters of water; 10 liters of Triton X-100 1 mM, 40 liters of water; 10 liters of 0.1N NaOH; 40 liters of water and finally 5 liters of BufFer B. In this way the equipment is able to be used to perform the concentration and diafiltration process against BufFer B of the selected fractions, according to the current methodology. Final Condition of Concentration a. Final volume of concentrate: 400 ml b. Diafiltered against: BufFer B c. Conductivity: 15,500-19,000 μSi / cm d. pH: 7.2 e. Conservation: at 4 ° C This separation step yields 4.0 grams of EPO.

Example 7 Molecular Exclusion Chromatography The material from the previous step is chromatographed using a molecular exclusion matrix according to the following parameters and conditions: 1. Equipment a. Column 1) Diameter: 10 cm 2) Bed height: 76 cm 3) Matrix a) Sephacryl S-200 HP (Pharmacia) b) Volume: 6,000 ml 2. Solutions and BufFers a. Buffer B: NaH2PO4 anh. 10 mM, NaCl 0.15 M, Lactose 0.05 mg / ml, pH 7.2 b. NaOH 0.5 N 3. Material to Chromatograph a. Fractions selected in the previous example concentrated b. Sample conditions 1) Volume: 400 ml 2) Total protein mass measured by Bradford test: 3.5-4.5 grams 3) Approximate concentration measured by Bradford test: 8.5-11 mg / ml 4) Conductivity: 15.500-19.000 μSi / cm 5) pH: 7.2 4. Sanitization and Balancing of the Column (*) To balance the column, the following solutions or bufFers are passed sequentially in the quantities detailed below: 1.0 ve ( 6 liters) of water; 1.5 ve (9 liters) of 0.5N NaOH and finally 3.0 ve (18 liters) of Buffer B.

. Copying Conditions (*) Once the column is balanced, 100 ml of the material to be chromatographed is sown. Said seeding is carried out at room temperature at a flow of 35 ml / min. The elution is then carried out at the same flow and temperature, passing 0.75 ve (4.5 liters) of BufFer B. This procedure is repeated 4 times, that is, until the material to be chromatographed is finished. The selected fractions, containing EPO, are sterilized by filtration through a 0.22 μm porous membrane and stored at 4 ° C. This separation step yields 3.2 grams of EPO with 99% purity. (*) ve means column volume The previous step concludes the purification process obtaining recombinant human EPO with a degree of purity higher than 99% and with an overall process yield of approximately 30% following the sequence claimed in claim 2. A sample of the EPO obtained using the process described was submitted to the following analyzes to demonstrate its identity and purity: In a denaturing polyacrylamide gel (SDS-PAGE) the purified EPO observed a broad band of more than 30 kDa molecular weight. See Fig. 1. The purified EPO was recognized by a monoclonal antibody as well as by a polyclonal antibody against human EPO in a "Western Blot" assay. See Fig. 2. The glycanase treatment of the purified EPO proved the existence of the glycosidic chains in quantity and molecular weight according to what was expected for EPO. See Fig. 3. The purified EPO showed to be composed of a series of isoelectric point species or isoforms between 3.0 and 4.5. See Fig. 4. The complete amino acid sequencing of purified EPO showed complete homology with natural human EPO having the following sequence of 165 amino acids, SEQ ID No. 1.

NH2 - -Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Wing Lys Glu Wing Glu Asn lie Thr Thr Gly Cys Wing Glu Hys Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Wing Trp Lys Arg Met Glu Val Gly Gln Gln Wing Val Glu Val Trp Gln Gly Leu Wing Leu Leu Ser Glu Wing Val Leu Arg Gly Gln Wing Leu Leu Val Asn Being Ser Gln Pro Trp Glu Pro Leu Gln Leu Hys Val Asp Lys Wing Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu jArg Wing Ala Leu Gly Wing Gln Lys Glu Ala lie Ser Pro Pro Asp Wing Wing Wing Wing Pro Leu Arg Thr He Thr Wing Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Wing Cys Arg Thr Gly Asp- COOH X glycosylation sites The presence of the four glycosylation sites on the 165 amino acid chain, as well as the complex structure of carbohydrates, and fundamentally the terminal sialic acid residues, were demonstrated together with their correct biological activity in vivo, in the test model of the ex-hypoxic polycythemic mouse, exhibiting total parallelism with the international standard co-subject. A sample of the EPO obtained by the described procedure was subjected to a reverse phase high-pressure liquid chromatography and molecular exclusion analysis. In both cases, a purity greater than 99% was demonstrated. See Figs. 5 and 6.

The following table illustrates the recovery of each separation step of the claimed process.

The following table illustrates the cumulative recovery of the procedure claimed in claim 2.

Description of Diagrams Fig. 1 illustrates an analysis by polyacrylamide gel electrophoresis (SDS-PAGE) of a sample of pure EPO, obtained according to the method described. Molecular weight markers are shown on lanes 1, 4 and 7. In streets 2, 3, 5 and 6, different masses of pure EPO obtained according to the claimed process were run. The purity of the obtained product and its apparent molecular weight of more than 30 kDa can be appreciated, which coincides with that of human urine EPO. Fig. 2 illustrates a "Western Blot" analysis of an EPO sample obtained according to the method described. The identity of the purified EPO is verified, since it is recognized by a human anti-EPO antibody. In Street 1, a human EPO standard was run, in the street 2 molecular weight markers and in the streets 3 to 5 EPO samples obtained according to the claimed method. Fig. 3 illustrates an SDS-PAGE analysis of a pure EPO sample obtained according to the described method, treated with glycanase. Molecular weight markers were run on streets 1, 4 and 8. On streets 2 and 7, untreated EPO is seen. In street 3, EPO treated with O-glycanase was run; the presence of an O-glycosylation is verified. In lane 5, partially degraded EPO was run with N-glycanase; the presence of 3 N-glycosylations with the molecular weights corresponding to those expected for EPO is verified. In lane 6, degraded EPO was run with O-glycanase and N-glycanase, obtaining the expected molecular weight for the fully deglycosylated protein. Fig. 4 illustrates a study of the isoelectric points of pure EPO samples produced according to the described method. The EPO samples were run in streets 2, 3 and 4, the isoelectric point markers in lanes 1 and 5. The presence of the forms corresponding to EPO, with isoelectric points between 3.0 and 4.5 is verified .

Fig. 5 reveals the purity of an EPO sample produced according to the method described using reverse phase high pressure liquid chromatography. Fig. 6 discloses the purity of an EPO sample produced according to the method described using high-pressure liquid chromatography for molecular exclusion.

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Claims (16)

1. Claims
2. Having described and exemplified the nature and main object of the present invention, as well as the manner in which it can be carried out, it is claimed to claim as property and exclusive rights: 1. A procedure for the purification of erythropoietin. recombinant human from cell culture supernatants characterized by including the following steps: a) differential precipitation; b) hydrophobic interaction chromatography; c) concentration and diafiltration; d) anion exchange chromatography; e) cation exchange chromatography; f) concentration and diafiltration; g) gel permeation chromatography. 2. A method, as characterized in claim 1, wherein steps a) through g) are performed in the following sequence: a), b), c), d), e), f) and g).
3. 3. A method, as characterized in claim 1, wherein steps a) through g) are performed in the following sequence: a), c), d), e), b), f) and g).
4. 4. A process, as characterized in claim 1, wherein step a) comprises adding ammonium sulfate to the culture supernatant, followed by centrifugation.
5. 5. A method, as characterized in claim 1, wherein step b) comprises using a hydrophobic interaction matrix.
6. 6. A process, as characterized in claim 5, wherein the hydrophobic interaction matrix used is Phenyl Sepharose 6 Fast Flow.
7. 7. A method, as characterized in claim 1, wherein step d) comprises using an anion exchange matrix.
8. 8. A process, as characterized in claim 7, wherein the anion exchange matrix used is Q-Sepharose Fast Flow.
9. 9. A method, as characterized in claim 1, wherein step e) comprises using a cation exchange matrix.
10. 10. A process, as characterized in claim 9, wherein the cation exchange matrix used is SP-Sepharose Fast Flow.
11. 11. A method, as characterized in claim 1, wherein step g) comprises using a molecular exclusion matrix.
12. 12. A process, as characterized in claim 11, wherein the molecular exclusion matrix used is Sephacryl S-200 HP.
13. 13. An erythropoietin, substantially pure, obtained according to the method characterized in claim 1.
14. 14. An erythropoietin, as characterized in claim 13, wherein said substance has a purity greater than 99% as determined by a gel electrophoresis analysis of polyacrylamide (SDS-PAGE) and reversible phase high-pressure liquid chromatography and molecular exclusion chromatography.
15. 15. An erythropoietin, as characterized in claim 13, wherein said substance is composed of a series of isoforms of isoelectric point comprised between 3.0 and 4.5.
16. 16. An erythropoietin, as characterized in claim 13, wherein said substance shows total homology to the natural human erythropoietin having the following amino acid sequence SEQ ID NO: 1.