MXPA97003470A - Factor v purification process - Google Patents

Abstract

The present invention relates to a process for purifying recombinant coagulation factor VIII by loading an aqueous solution containing factor VIII onto a hydrophobic interaction chromatography (HIC) gel, wherein at least one surfactant is present in the solution aqueous and / or a buffer solution used to balance the gel before loading the aqueous solution onto the gel. The presence of a surfactant when the solution containing factor VIII is loaded onto the HIC gel makes it possible to efficiently separate the intact and active Factor VIII molecules from the molecules with structural deviations. With the present invention it is also possible to considerably reduce the DNA content and / or cellular CHO contaminants and increase the activity of the Factor VIII product to a degree hitherto unknown. The invention further relates to an aqueous solution containing recombinant factor VIII which has been purified according to the current process and use of such aqueous solution, for the preparation of a medicament for administration to a patient having the symptoms of hemophilic

Description

FACTOR VIII PURIFICATION PROCESS FIELD OF THE INVENTION The present invention relates to a process for purifying recombinant coagulation factor VIII by loading an aqueous solution containing factor VIII on a hydrophobic interaction chromatography gel.

(HIC), wherein at least one surfactant is presented in the aqueous solution and / or a buffer used to balance the gel before loading the aqueous solution onto the gel. The presence of a surfactant when the solution containing factor VIII is loaded onto the HIC gel makes it possible to efficiently separate intact and active factor VIII molecules from molecules with structural deviations. With the present invention, it is also possible to considerably reduce the content of DNA and / or CHO cellular contaminants and to retain the activity of the factor VIII product to an extent hitherto unknown. The invention further relates to an aqueous solution containing recombinant factor VIII which has been purified according to the process and current use of such aqueous solution, for the preparation of a medicament for administration to a patient having the symptoms of hemophilia. Also, the invention relates to a method for the treatment of hemophilia by administering a therapeutically effective amount of recombinant factor VIII which has been purified according to the present process. BACKGROUND OF THE INVENTION Hemophilia is a hereditary disease that has been known for centuries, but only within the last four decades has it been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10,000 males that are born alive. The disease is caused by a strongly decreasing level or the absence of biologically active coagulation factor VIII (antihemophilic factor), which is a protein normally present in plasma. The clinical manifestation of hemophilia A is a strong tendency to bleed and before the treatment with factor VIII concentrates was introduced, the average age of affected patients was less than 20 years. Factor VIII concentrates obtained from plasma have been available for approximately three decades. This has considerably improved the situation for the treatment of hemophilia patients and has offered them the possibility of living a normal life. Until recently, therapeutic factor VIII concentrates have been prepared by plasma fractionation. However, methods available for the production of factor VIII in a cell culture using recombinant DNA techniques have been available for some years as reported in for example W. Wood et al., Nature, 312, p. 330-37 (1984) and EP-A-0 160 457. Factor VIII concentrates derived from human plasma contain several forms of fully active fragmented factor VIII, as described by Andersson et al., Proc. Nati Acad. Sci. USA, 83, p. 2979-83 (May 1986). The smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by metal ion (s). Reference is made herein to EP-A-0 197 901. Pharmacia AB of Stockholm, Sweden, has developed a recombinant factor VIII product which corresponds to the plasma factor VIII form of 170 kDa in the therapeutic factor VIII concentrates. The truncated recombinant factor VIII molecule is called r-VIII SQ and is produced by Chinese Hamster Ovary (CHO) cells in a cell culture process in a serum-free medium. The structure and biochemistry of recombinant factor VIII products has been described generally by Kaufman in Trends in Biotechnology, 9 (1991) and Hematology (Hematology), 63, p. 155-65 (1991). The structure and biochemistry of r-VIII SQ has been described in WO-A-9109122. High performance hydrophobic interaction chromatography (HIC) is a suitable separation technique for purifying proteins. The general characteristics and conditions suitable for carrying out an HIC stage have been described by K-0 Eriksson in Protein Purification; Principies, High Resolution Methds, and Applications (Protein Purification, Principles, High Resolution Methods, and Applications), VCH Publishers, Inc. New York, p. 207-226 (1989). In this technique, proteins are eluted from relatively weak hydrophobic static phases using a solution with decreasing ionic strength, introducing a surfactant, which changes the polarity of the solvent and / or simply lowers the temperature. Such relatively benign conditions favor the recovery of proteins with essentially retained activity. The factors that affect adsorption and desorption in HCl are thoroughly reviewed by T. Arakawa and L. Owers Narhi in Biotechnol. Appl. Biochem., 13, p. 151-172 (1991). The influence of surfactants on the interaction of various proteins with an HIC gel (resin) has been described by J. J. Buckley and D. B.

Wetlaufer, J. Chrom. 518, p. 99-110 (1990). However, in this reference, the main purpose was to evaluate the influence of surfactants on gradient elution profiles. The various gels (resins) that exhibit hydrophobic or semihydrophobic ligands have been described, for example agaroses derived with aminoalkyl or diaminoalkyl groups. In the prior art, there are examples of purification of factor VIII by the use of such gels. For example, butyl-agarose gel was prepared by derivatizing Sepharose® 4B (sold by Pharmacia AB of Uppsala, Sweden) with butyl-amine, using a CNBr coupling technique. The resulting gel was used for the purification of factor VIII, as reported by Th. Vukovich et al. in Folia Haematol. Leipzig, 107 (1), p. 148-151 (1979) and Th. Vukovich and cois, in Haemostasis and Thrombosis (Hematosis and Thrombosis) (Proc. Serono Symp.), G. G. Neri Serneri and C. R. M. Prentice eds. , 15, p. 407-410 (1979). A mixture of protein was adsorbed on this gel at a very low ionic strength. The desorption was carried out by increasing the concentration of sodium chloride. This clearly indicates that the forces used were predominantly electrostatic, that is, the mechanisms involved were those normally referred to as ion exchange chromatography (IEC). In addition, it has been reported, for example by B.-L. Johansson and I. Drevin in J. Chrom. , 321, p. 335-342 (1985), that the CNBr coupling technique creates a group of iso-urea between the ligand and the matrix, which is positively charged at an acidic and neutral pH. This profoundly influences the total properties of the gel. Morgenthaler has compared a series of agaroses derived with aminoalkyl- or diaminoalkyl groups for the purification of factor VIII, as described in Thromb. Haemostas., 47 (2), p. 124 (1982). It was found that it is difficult to obtain a reversible link of factor VIII with the agarose gels derived with aminoalkyl groups (Sepharose® alkane). In this way, induced salt elution was only obtained to a certain degree and only from gels derived with short aminoalkyl groups using the CNBr coupling technique. The elution of ethylene glycol fails regardless of the length of the alkyl chain. In contrast, it has been found that by adding detergents at a concentration of > 0.1%, the desorption of factor VIII from such columns could be achieved (EP-A-0 209 041). However, in the examples shown, the capacity of the HIC columns and the concentration of the eluted factor were low. EP-A-0 286 323 relates to a two-step process for purifying polypeptides, especially factor VIII. The first stage uses immobilized antibodies and the second stage is based on an affinity region. The affinity region can be an ion exchange gel or an HIC gel. Detergents are basically referred to as active agents during the inactivation of the virus. In the prior art, they are also mentioned to suppress the intermolecular association mediated by ionic forces, but there is no information about the use of detergents during the adsorption phase on the affinity region. US-A-4, 743, 680 also refers to regulatory compositions during the purification of factor VIII (antihemophilic factor or AHF), by column chromatography. It is mentioned that detergents promote the elution of a mixed function affinity chromatography gel, such as aminohexyl Sepharose®. There is no information in US-A-4, 743, 680 about the use of detergents during the adsorption phase on a hydrophobic resin. The difficulties encountered in applying HIC to the purification of factor VIII, can be attributed to the problems encountered when trying to establish an adequate retention window as well as elution conditions. A suitable retention window means that the factor VIII molecules are retained on the surface of the HIC gel by hydrophobic interaction, while other impurities, mainly nucleic acids and proteins are retained to a lesser degree or, preferably, not at all. A suitable retention window for a factor VIII also means that the factor VIII molecules can be eluted without too severe conditions, thus avoiding denaturation. Such a window can be obtained by selecting a suitable gel consisting of a matrix and hydrophobic ligands attached thereto. The type and density of the ligands strongly influence the interaction between the factor VIII molecules and the surface of the HIC gel, and by this the retention window. Additional parameters that influence the retention window are, for example, ionic strength, temperature, pH and column length. It is of particular importance for the selectivity of the process that such non-specific adsorption factors as positive charges are avoided, since nucleic acids, which are negatively charged, are tolerated at an extremely low level in the preparation of therapeutic proteins. Thus, in the purification of therapeutic proteins produced by a recombinant DNA technique, it is well known that considerable problems are encountered when trying to reduce the DNA content to a very low level stipulated by the Food and Drug Administration (FDA) of the EU A recent example is set forth in P. Ng and G. Mitra of Miles Inc., in the U.S., J. Chrom., A 658, p. 459 (1994), where the DNA concentration was reduced but only by about 1 ng per dose of a therapeutic protein as determined by the 2P R3 DNA hybridization method. Pure hydrophobic interaction chromatography, using uncharged gels in combination with light elution conditions, would provide another dimension for purifying recombinant factor VIII, since an additional separation criterion would be used in comparison to those already used in ion exchange and Immunoaffinity chromatography. This would increase the number of process techniques available to optimize the total purification of recombinant factor VIII. In addition, it would be valuable to find conditions for a moderately strong adsorption, allowing desorption by light elution conditions, since too strong an adsorption could change the conformation of the protein. DESCRIPTION OF THE INVENTION An object of the present invention is to provide an efficient purification process, to produce a highly concentrated and very pure recombinant factor VIII solution. Another object of the present invention is to provide an efficient process, where activity is essentially retained. Another object of the present invention is to reduce the residence time in the stage, while still providing a product of high purity. Still another object of the present invention is to provide a process step, making it possible to fractionate the factor VIII molecules intact from the molecules with structural deviations. The above objects are fulfilled by the present invention, which relates to a process for purifying factor VIII of recombinant coagulation of contaminants by charging an aqueous solution containing factor VIII on a hydrophobic interaction chromatography (HIC) gel, where at least one surfactant is present in the aqueous solution and / or a buffer used to balance the gel before loading the aqueous solution onto the gel. The inventor of the present invention has surprisingly found that hydrophobic interaction chromatography can be used to advantage if a surfactant is present and the ionic strength is carefully controlled, when the solution containing recombinant factor VIII is loaded onto the HIC stage. In this way, the forces that adsorb factor VIII molecules on the surface are sufficient but not strong enough to make it difficult or even impossible to desorb the same molecules. Accordingly, the inventor of the present invention has managed to provide the hitherto unexposed retention window for purified recombinant factor VIII in a HIC step. With the help of the present invention it is possible to reduce the DNA content considerably. DNA reduction is typically 102 over the HIC stage. Toward the end of a sequence of chromatography steps the content is typically reduced to below about 10 pg / 1000 Ul VIII: C, which is a level currently established by the US FDA. for our knowledge, such low level has not been previously exposed. In addition, the content of Chinese Hamster Ovary (CHO) cell contaminants can be greatly reduced. The HIC step can be used in various positions of a purification sequence, as will be apparent below. In all the positions examined, it is possible to fractionate the factor VIII molecules to efficiently place the factor VIII molecules with structural deviations. In the present invention, factor VIII is recombinant and can be a full-length factor VIII or preferably an omission derivative of full-length factor VIII having coagulant activity. More preferably, the omission derivative is the recombinant factor VIII SQ (r-VIII SQ). Derivative of omission is understood to be factor VIII, in which all or part of domain B is missing, while the coagulant activity is retained. Hydrophobic interaction chromatography should be carried out on gels with charge-free ligands, suitably aliphatic or aromatic, hydrophobic subject to various commercially available matrices. The ligands can be coupled to the matrix by conventional coupling techniques that give charge-free ligands. The most common suitable example of such a technique is the glycidyl ether coupling process. In this technique, a hydroxyl group containing polymer is reacted with a glycidyl ether containing the desired alkyl or aryl group at one end. In another technique, an agarose matrix is first activated with glycidoxypropyltrimethoxy silane in water. The immobilization of the ligands is then carried out in the alcohol that is about to be coupled to the gel. In yet another suitable technique, an agarose matrix is first activated with a bis-epoxide, such as 1,4-butanediol diglycidyl ether. The obtained epoxy activated gel can be coupled to a wide range of ligands, for example a suitable aminoalkyl or alkyl mercaptan. Additional available techniques are for example activation with 1,1 '-carbonyl-diimidazole and activation with divinyl sulfone. The gels resulting from the techniques described above are free-loading within the entire pH range, that is, they are truly free of charge, thus giving only hydrophobic interactions with the factor VIII molecules. Reference is made here to Jansson and cois, in Phases of Packaging and Static in Chromatographic Techniques, Marcel Dekker Inc., New York, p. 762-766 and K-0 Eriksson in Protein Purification; Principies, High REsolution Methods, and Applications (Protein Purification, Principles, High Resolution Methods, and Applications), VCH Publishers, Inc., New York, p. 214-217 (1989). In the present invention, the aliphatic ligand is suitably selected from a group of alkyls consisting of propyl, butyl, pentyl, hexyl, heptyl or octyl, preferably butyl. It is also suitable to select the oligoethylene glycol ligand, -0- (CH2-CH2-0) n-CH2-CH2-OH, where n < 10. An example of a suitable HIC gel with oligoethylene glycol ligands is Toyopearl®HicPak ™ Sampler Ether-650M sold by TosoHaas of Philadelphia, E.U.A. The alkyl ligand can be straight (normal alkyl) or branched (iso- or neoalkyl). The aromatic group is preferably phenyl. The matrix can be selected from various strongly hydrophilic matrices for example agarose matrices such as a wide variety of Sepharose® matrices sold by Pharmacia Biotech of Uppsala, Sweden, organic polymer matrices such as TSK-GEL: s sold by Tosoh Corp. of Tokyo, Japan, or highly porous organic polymer matrices sold by Per Septive Biosystems of Boston, USA The matrix is preferably an agarose matrix. Suitable agarose matrices in the present invention are, apart from Sepharose®, Minileak® sold by Kem-En-Tec A / S of Copenague, Denmark and Bio-Gel A sold by Bio-Rad, of Brussels, Belgium. Preferably, the matrix is degraded allowing a rapid flow (FF) and by this a high production capacity. More preferably, the hydrophobic interaction chromatography of the present invention is carried out on a 4 FF gel of Butyl Sepharose®. The short alkyl groups containing 4 carbon atoms allow a suitably strong interaction between the surface of HIC and the factor VIII molecules to allow a pronounced separation under light desorption conditions. The surfactant of the present invention is suitably a non-ionic surfactant, or, more precisely, a zero-net charge surfactant. Preferably, the surfactant is selected from the group consisting of block copolymers, polyoxyethylene sorbitan fatty esters and alkyl ethoxylates. Suitable examples of the block copolymers are combinations of polypropylene glycol and polyethylene glycol, for example Pluronic® sold by BASF in Germany. Suitable examples of polyoxyethylene sorbitan fatty esters are polyoxyethylene- (20) -sorbitan monolaurate, for example Tween® 80, and polyoxyethylene- (20) -sorbitone monooleate, for example Tween®, both sold by ICI From great britain. Suitable examples of alkyl ethoxylates are Triton® X-100 sold by Union Carbide in E.U.A. A surfactant is also suitably present in the desorption of factor VIII molecules from the surface of HIC. In this way, production can be improved compared to elution in the absence of a surfactant. The surfactant present in the desorption may be the same or different from that used in the adsorption. The concentration of surfactant in the aqueous solution of factor VIII loaded on the HIC should be in the range of 0.004 to 1.0% (w / w), suitably from 0.007 to 0.5% (w / w) and preferably from 0.01 to 0.09 % (w / w).

If a buffer solution is used to balance the gel before loading, the concentration of surfactant in said regulator may be the same as in the aqueous solution of factor VIII loaded on the HIC. However, it is also possible to use a regulator with a considerably lower surfactant concentration, for example one tenth of the above concentration for the aqueous solution of factor VIII. In this case, the gel is balanced by applying the regulator of a larger volume, for example 10 times, to reach an adequate concentration of surfactant on the gel. Preferably, the gel is equilibrated with a surfactant before being charged with an aqueous solution of Factor VI11 containing the same or another surfactant. The concentration of surfactant when factor VIII of the HIC is eluted should be up to 0.5% (w / w), suitably from 0.004 to 0.2% (w / w) and preferably from 0.01 to 0.09% (w / w). The ionic strength of the solution that is charged on the HIC stage as well as the ionic strength of the elution solution are important for the type of purification obtained as well as the efficiency of the purification. In this way, to make possible an efficient separation basically of Factor VIII and DNA, the ionic strength of the solution being loaded onto the HIC gel must be greater than or equal to the ionic strength of the solution used to elute factor VIII from the HIC gel. Otherwise, the stage will be a conventional ion exchange stage. In addition, to obtain a reversible adsorption of the Factor VIII molecules, the ionic strength of the solution being loaded onto the HIC stage must be in the range of from 0.3 to 4 M, suitably from 0.6 to 2 M, and preferably from 1 to 1.5 M. The ionic strength of the solution used to elute the factor VIII from the HIC stage should be up to 1 M when the elution is started, suitably up to 0.8 M, and preferably from 0.2 to 0.6 M. Ionic resistance can be maintained constant throughout the elution, or reduced in a linear or staggered manner or combinations thereof. Apart from decreasing the ionic resistance, the Factor VIII molecules can be eluted, that is, the hydrophobic interaction can be reduced by changing the polarity of the solvent, adding detergents and / or decreasing the temperature. The polarity of the solvent can change when adding, for example, ethylene glycol or (iso) propanol. The ionic strength of the solution that is being loaded onto the HIC stage as well as the solution used to elute factor VIII is suitably obtained by the presence of an alkali metal chloride, for example sodium chloride or potassium chloride, or acetate of ammonia, or any combination thereof. Preferably, use is made of ammonia acetate. The effect of adsorption can be improved by the presence in the solution loaded on the HIC stage of at least one compound selected from the group consisting of monosaccharides, disaccharides and sugar alcohols, preferably sorbitol. This is especially applicable when the HIC step follows a cation exchange chromatography step of initial concentration. The concentration of the mono- or disaccharide or sugar alcohol in the solution should be at least 5% (w / w), preferably at least 10% (w / w). The solution that is being loaded onto the HIC for the adsorption of factor VIII on the surface of the gel can have a pH in the range of from about 5 to about 8, suitably from 5.8 to 7.3 and preferably from 6.1 to 6.8. When the desorption of the purified factor VIII from the gel surface is about to happen, the pH of the elution solution should be in the range of from 5.8 to 7.3, suitably from 6.1 to 6.8. The adsorption as well as the desorption of factor VIII in the HIC stage are suitably carried out at room temperature, ie at a temperature of 18 to 25 ° C. In this way, complicated and expensive temperature regulating equipment can be discarded. However, it is also possible to adsorb at room temperature and desorb by decreasing the temperature to, for example, about 4 ° C. The HIC step of the present invention can be combined with various other steps and at various positions in a sequence to purify recombinant factor VIII. In this way, the HIC step can be carried out after an initial concentration step, for example a cation exchange chromatography step. This can also be carried out after an immuno-affinity chromatography (IAC) step. However, preferably, the HIC step is carried out after an anion exchange chromatography step. This provides a process where the high ionic strength of the eluate from the anion exchange step is used to benefit the subsequent HIC stage. In the present invention, the HIC step can be repeated to give a total of two, three or even more steps of HIC in a purification sequence. The use of several stages of HIC can reduce the content of impurities in an additional way, and at the same time increase the concentration of factor VIII. Of course, these and other advantages have to be weighed against the increase in equipment costs. If at least two stages of HIC are used, they can be used with or without intermediate process steps. The following examples are proposed to further illustrate the present invention, without limiting the scope of the invention. EXPERIMENTS Preparation of recombinant factor VIII The production of recombinant Factor VIII SQ (r-VIII SQ) was carried out essentially as described in patent WO-A-9109122, example 1-3. A CHO cell line deficient in DHFR (DG44N.Y.) Underwent electrophoresis with an expression vector containing the r-VIII SQ gene and an expression vector containing the dihydrofolate reductase gene. After selection on the selective medium the surviving colonies were amplified through growth in amounts of methotrexate, increasing in a staggered manner. The supernatant of the resulting colonies was selected individually for the activity of factor VIII. A production clone was chosen and this was subsequently adapted to the development of the serum-free suspension in a defined medium and finally a large-scale cell culture process was developed. The supernatant was collected after certain periods of time and subsequently purified as described below. Example 1 The conditioned medium (containing unborn bovine serum) was then clarified and concentrated by tangential flow filtration. After freezing and melting, the solution was adjusted with 20 mmol / 1 of imidazole. Sodium chloride in a concentration of 1.0 mol / 1 and calcium chloride in 5 mmol / 1 was added. The solution was loaded onto an immunoaffinity chromatography gel, where the ligand was a monoclonal antibody (mAb, called 8A4) directed towards the heavy chain of Factor VIII. After rinsing, factor VIII was eluted with a regulator containing 50 mmol / 1 CaCl 2 and 50% ethylene glycol. The mAb eluate was loaded onto an anion exchange column, Q Sepharose® FF sold by Pharmacia AB of Uppsala, Sweden. After rinsing, factor VIII was eluted with a regulator containing 50 mmol / l histidine, 0.6 mol / l NaCl, 1 mmol / l CaCl 2 and 0. 2 g / 1 Tween 80®, pH 6.8. An adsorption experiment was carried out batchwise using Butyl Sepahrose® gel of 4 FF according to the invention. The chromatography gel by hydrophobic interaction was rinsed with water and 0.1 g of dry gel were then distributed each in Eppendorff 8 tubes. The gels were equilibrated at room temperature in 1.2 ml of regulator containing 50 mmol / 1 histidine and 1 mmol / 1 CaCl2 and with varying pH and salt concentration (NaCl) and surfactant (Tween 80®) as given in the Table below. After the addition of 40 μl per tube of Q eluate containing factor VIII with an activity of 1630 Ul VIII: C / ml, the tubes were turned end-over-end at 10 rpm. At the indicated time, the tubes were centrifuged at 2000 rpm for 1 minute, and samples (150 μl) were separated from the supernatant and stored frozen until analysis. Elution was carried out by the addition of 0.5 ml of a solution containing 25 mmol / 1 of histidine, 0.5 mmol / l of CaCl 2 and 0.4 mol / l of NaCl. The pH was 6.8 and no surfactant was present in the elution solution. The tubes were then rotated for 30 minutes and subsequently centrifuged as set forth above. A second elution was carried out by doing the same procedure with the water. The procoagulant activity of Factor VIII was determined by the use of a chromogenic substrate method, Factor Kit VIII Coatest® (Chromogenix AB of Sweden). The relative standard deviation (RSD) of the method is 7%. The activity of factor VIII and its recovery in eluate are evident from the following Table. TABLE I Discontinuous Adsorption of Q-eluate on Butyl Sepharose® 4 FF. Dependence of pH, concentration of NaCl and presence of Tween 80® on adsorption and recovery kinetics in the eluate. Test Sal Surfac Activity of Recovery Factor (NaCl) tante VIII in the eluate 3 ',% mol / 1 (Tween supernatant,, Ul / ml 80®) Time, minutes g / i11 No pH 02) 3 6 12 lo. 2o. 1 6.8 1.0 0.2 55 24 15 10 48 8 2 7.8 1.0 0.2 55 26 15 9 26 6 3 6.8 0.8 0.2 52 27 22 23 27 4 4 7.8 0.8 0.2 53 28 24 26 19 3 6.8 1.0 0 44 2.8 0.9 0.7 0 0 6 7.8 1.0 0 47 1.5 0.8 0.5 12 0 7 6.8 0.8 0 40 1.7 1.5 1.3 0 0 8 7.8 0.8 0 37 1.4 1.2 1.0 0 0 1) By calculation, the concentration before the addition was 0.007 g / 1, due to the regulatory composition of Q eluate. 2) By calculation, the start activity should be 52 IU / ml. 3) In comparison to the loaded. As is evident from the Table, the presence of a surfactant on the HIC gel before loading of Factor VIII dramatically increases the activity as well as the recovery of Factor VIII, compared to the tests where the surfactant was absent. Example 2 The conditioned medium used in Example 1 was treated as in Example 1, up to and including the elution of factor VIII from the Q Sepharose® FF column used in Example 1. Subsequently, an experiment of discontinuous mode adsorption using Butyl gel Sepharose® 4 FF of Example 1, according to the invention. The gel was rinsed with water and 0.1 g of dry gel were then distributed each in Eppendorff 9 tubes. Another set of 9 tubes was prepared with 1.2 ml of regulator containing 50 mmol / 1 histidine and 1 mmol / 1 CaCl 2 and with pH of 6.8, and with varying salt concentration (NaCl) and surfactant (Tween 80®) as given in the Table below. 40 μl of Q-eluate, containing factor VIII with an activity of 1630 Ul VIII: C / ml, were added to each of the tubes. After sampling, 1.0 ml of each solution was distributed to a tube of Butyl gel, which was turned end-over-ext emo at 10 rpm. At the indicated time, the tubes were centrifuged at 2000 rpm for 1 minute, and samples (40 μl) were removed from the supernatant and stored frozen until analysis. Elution was carried out by the addition of 1.0 ml of a solution containing 50 mmol / 1 histidine and 1 mmol / 1 CaCl 2. The pH was 6.8 and no surfactant was present in the elution solution. The tubes were then rotated for 20 minutes and subsequently centrifuged as set forth above. The activity of factor VIII and the recovery in eluate are evident from the following Table. TABLE II Discontinuous Adsorption of Q-Eluate on Butyl Sepharose® 4 FF. Dependence on the concentration of NaCl Tween 80® on the adsorption of Factor VI11 and recovery in the eluate.

Salt (NaCl) Surfactant Recovery Activity (Tween 80®) Factor VIII in eluate 3 'added in the supernatant after 20 min Test No mol / 1 g / i11 Ul / ml% 1 0.3 0 2.4 0 2 0.6 0 5.3 1.4 3 0.9 0 0.67 3.9 4 0.3 0.04 23 1.3 5 0.6 0.04 6.1 6.4 6 0.9 0.04 1.8 14 7 0.3 0.20 41 4.4 8 0.6 0.20 30 17 9 0.9 0.20 9.2 43 1), 2) and 3) See footnotes to Table I. As is evident from the Table, the presence of a surfactant on the HIC gel before loading Factor VIII dramatically increases activity as well as the recovery of Factor VIII, in comparison to the tests where the surfactant was absent. Example 3 Recombinant factor VIII was produced according to the method described under Experiments. In this case the production medium contained human serum albumin but without unborn bovine serum.

The conditioned medium was clarified by filtration, the pH was adjusted, and then the filtrate was loaded onto a column of S Sepharose® FF (column volume 31). After rinsing, factor VIII was eluted with a salt buffer containing 5 mM CaCl 2 and 0.02% Triton® X-100. This step of cation exchange chromatography was carried out at 2-8 ° C. The eluate from the S Sepharose® FF (eluate-S) stage was frozen until further purification. A column of Butyl Sepharose® 4 FF (column volume 77 ml) was equilibrated at room temperature with a regulator containing 1 M sorbitol, 1.2 M NaCl, 0.1 M NH 4 Ac, 5 mM CaCl 2 and 0. 02% Triton® X-100, pH of 6.8. The S-eluate was melted and adjusted to the composition of the equilibrium regulator. It was then charged at room temperature over the HIC column, which was subsequently rinsed with 4 column volumes of equilibrium buffer. The elution was carried out with a regulator containing 0.4 M NaCl, 0.02 M NH4Ac, 5 mM CaCl2 and 0.02% Triton® X-100, pH 6.8. The HIC eluate was further purified by immunoaffinity chromatography, resulting in a DNA content of 11 pg / kUI. Finally, after an anion exchange chromatography step, the DNA content was found to be below 2.2 pg / IU. This low content was not achieved without the HIC step. The activity of Factor VI11 and the ratio of DNA and factor VIII are evident from the following Table. TABLE III Purification of Factor VIII on Butyl Sepharose® 4FF in a position after basic isolation Applied Eluted Volume of DNA Activity, eluate-HIC, main fraction 96 101 x 10"68 x 10 eluate-HIC, tail fraction 55 12 x 10 '117 x 10' 1) The DNA was determined according to a Threshold methodology. The sample was denatured to generate single-strand (ss) DNA. A binding protein and a monoclonal antibody, both specific for ss DNA, are used to form a complex. To continue the reaction both an enzyme link on the antibody and the streptavidin / biotin affinity system are used. This method is more sensitive than the most widely used hybridization technique. 2) A dose of Factor VIII has been defined as 1000 Ul (l kUI). As is apparent from the Table, the use of the HIC step of the present invention enables a considerable reduction in DNA content. Example 4 An S-eluate was prepared according to Example 3.

A column of Butyl Sepharose® 4 FF (column volume 1.21) was equilibrated at room temperature with a regulator containing 1 M sorbitol, 1.1 M NaCl, 0.1 M NH4Ac, 5 mM CaCl2 and 0.02% Triton® X-100, pH 6.8. The S-eluate was melted and then adjusted to the composition of the equilibrium regulator. Then, it was charged at room temperature over the HIC column, which was subsequently rinsed with 4 column volumes of equilibrium regulator. The elution was carried out with a regulator containing 0.75 M of sorbitol, 0.32 M of NaCl, 0.1 M of NH4Ac, 5 mM of CaCl2 and 0.02% of Triton® X-100, pH 6.8. TABLE IV Purification of Factor VIII on Butyl Sepharose® 4FF in a position after basic isolation Fraction Volume of Activity of DNA1 'Protein fraction Cellular Factor VIII CHO3' 1 IU pg / kui2 'ng / kUI eluate-S 2. 1 12 x 10 '67 x 10"14 x 10 ° eluate-HIC 6 .5 6 .9 x 102 60 x 103 12 x 105 1) and 2) See footnotes in Table III. 3) CHO cell protein was determined using an ELISA method using antibodies raised to an S-eluate prepared from a medium conditioned from a blank cell culture (without production of Factor VIII). This assay can also detect other cellular components of CHO. As is apparent from the Table, the use of the HIC step of the present invention enables a reduction in DNA content by a factor of typically 102. In addition, the content of CHO cells can be reduced by a factor of about 101. Example 5 An S-eluate was prepared according to Example 3. 700 ml of the eluate-S melted and the temperature was adjusted to room temperature. Inactivation of the virus was carried out by incubation for 30 min with tri-n-butyl phosphate (TNBP) and Triton® X-100 at a final concentration of 0.3% (v / v) and 1.0% (v / v), respectively. An immunoaffinity column of monoclonal antibody (mAb) with a volume of 260 ml was equilibrated with an S-eluate buffer containing the corresponding amounts of virus inactivation chemicals. The Factor VIII solution was then loaded onto the mAb column, which was subsequently rinsed. The elution was carried out with a regulator containing 50% ethylene glycol. A 49 ml column of Butyl Sepharose® 4 FF was equilibrated with a regulator containing 50 mM histidine, 1.4 M NH4Ac, 10% ethylene glycol, 50 mM CaCl2 and 0.02% Triton® X-100, pH 6.4. . 35 ml of the eluate from the immunoaffinity column (eluate-mAb) were diluted 5 times in a regulator to a final composition according to the equilibrium regulator and subsequently the column was loaded at a linear flow rate of 35 cm / ha. HIC, which was then rinsed with 5 column volumes of the equilibrium regulator. The column was finally eluted at a linear flow rate of 35 cm / h with a regulator containing 50 mM histidine, 0.5 M NH4Ac, 50 mM CaCl2 and 0.02% Triton® X-100, pH 6.4. TABLE V Purification of Factor VIII on Butyl Sepharose® 4FF at a position after the immunoaffinity stage Fraction Volume of DNA Activity "Protein fraction Cellular Factor VIII CHO3 'mi Ul pg / kUI2' ng / kUI eluate-mAb 200 109 x 10"32 x 10" 10 x 10"eluate-HIC 70 87 x 103 < 27 11 X? O2 1), 2) and 3) See footnotes in Table IV. As is apparent from the Table, the use of the HIC step of the present invention towards the end of a purification sequence, makes possible the production of a final product with a very low DNA content. In addition, the content of CHO cells can be reduced by a factor of about 101. EXAMPLE 6 An eluate mAb was prepared according to Example 5.

A Q Sepharose® column was pre-equilibrated to a high concentration of sodium chloride, and then equilibrated with a regulator of the same composition as the immunoaffinity column was eluted. The eluate-mAb was loaded, and the column was then rinsed with an equilibrium regulator followed by a rinsing regulator of physiological ionic strength. The column was eluted by raising the sodium chloride concentration to 0.6 M. No detergent was used for rinsing and elution of the Q column. A Butyl column was equilibrated Sepharose® 4 FF with a regulator containing 50 mM of histidine, 1.4 M of NH4Ac, 50 mM of CaCl2 and 0.02% of Tween 80, pH of 6.8. NH4Ac was added to the Q-eluate in a final concentration of 1.0 M and 0.02% Tween® 80. This solution was loaded onto the column of butyl gel in a linear flow rate of 60 cm / h. The column was then rinsed by 5 column volumes of equilibrium regulator and then eluted in a linear flow rate of 35 cm / h with a regulator containing 50 mM histidine, 0.5 M NH 4 Ac, 50 mM CaCl 2 and 0.02 % of Tween® 80, pH of 6.8. TABLE VI Purification of Factor VIII on Butyl Sepharose® 4FF at a post-stage position of Sepharose® Q Stage VI II: C on VIII: C on DNA1 'Protein chromatography eluate, stage i of Sep. cellular CHO3 'Ul / ml% pg / kUI21 ng / kUI mAb 2. 2 x 10"82 32 x 10" 10 x 10" Q 17. 9 X 103 70 10 x 102 4. 0 x 102 HIC 26 7 x 103 96 4. 0 1. 3 x 102 1), 2) and 3) See footnotes in Table IV. As is evident from the Table, the use of the HIC step of the present invention towards the end of a suitable purification sequence, makes possible the production of an eluate with a very high activity of Factor VIII in combination with very low contents of DNA and CHO cellular proteins.

Example 7 A Q-eluate was prepared as described in Example 6, however with the use of a deposit of several S-eluates. A column of Butyl Sepharose® 4 FF was equilibrated with a regulator containing 50 mM histidine, 1. 3 M NH4Ac, 50 mM CaCl2, and 0.02% Tween® 80, pH 6.8. The Q eluate was diluted with a double-volume salt buffer and a final concentration of 1.0 M NH4Ac and 0.02% Tween® 80. This solution was loaded onto the column of butyl gel in a linear flow rate of 60 cm. / h. Rinsing and elution was carried out as described in Example 6. TABLE VII Purification of Factor VIII on Butyl Sepharose® 4 FF in a position after a step of Sepharose® Q Factor VIII Activity Specific Cellular Protein Fraction Activity Production Ul / mg DNA, 11 CHO3 'Ul / ml% protein pg / kUI2) ng / kUI eluate-Q 36.9 x 10"100 134 153 eluate-HIC 41.7 x 103 77 17 x 103 4) 6.7 19 1), 2) and 3) See footnotes in Table IV. 4) Determined after regulator change by gel permeation chromatography. As is evident from the Table, the use of the HIC step of the present invention towards the end of a suitable purification sequence, makes possible the production of an eluate with an extremely high specific activity of Factor VIII in combination with very high contents. low DNA and CHO cellular proteins.

Claims (20)

1. NOVELTY OF THE INVENTION Having described the present invention is considered as a novelty and therefore it is claimed as property described in the following claims. A process for purifying recombinant coagulation factor VIII by loading an aqueous solution containing factor VIII onto a hydrophobic interaction chromatography (HIC) gel, characterized in that at least one surfactant is present in the aqueous solution and / or a solution regulator used to balance the gel before loading the aqueous solution onto the gel.
2. 2. A process according to claim 1, characterized in that said at least one surfactant is present both in the aqueous solution and in the buffer solution.
3. 3. A process according to claim 1 or 2, characterized in that the surfactant is a non-ionic surfactant.
4. 4. A process according to claim 3, characterized in that the nonionic surfactant is selected from the group consisting of polyoxyethylene sorbitan block co-polymers or fatty acid esters.
5. 5. A process according to any of the previous claims, characterized in that the concentration of surfactant in the aqueous solution is in the range of 0.01 to 0.09% (w / w).
6. 6. A process according to any of the preceding claims, characterized in that the aqueous solution contains at least one salt selected from the group consisting of alkali metal chlorides and ammonia acetate.
7. A process according to any of the previous claims, characterized in that the aqueous solution contains at least one compound selected from the group consisting of monosaccharides, disaccharides and sugar alcohols.
8. A process according to any of the previous claims, characterized in that the aqueous solution loaded on the gel has an ionic strength that is greater than, or equal to, the ionic strength of the solution used to elute factor VIII from the gel.
9. 9. A process according to any of the previous claims, characterized in that the factor VIII adsorbed by the gel is eluted with a solution with ionic strength of up to 1 M.
10. 10. A process according to any of the previous claims, characterized in that the factor VIII adsorbed The gel is eluted with a solution containing 0.01 to 0.09% (w / w) of a surfactant.
11. 11. A process according to any of the previous claims, characterized in that the aqueous solution loaded on the gel and the solution used to elute factor VIII has a pH in the range of from 6.1 to 6.8.
12. 12. A process according to any of the preceding claims, characterized in that the gel is free of charge within the range of the whole pH used for purification.
13. 13. A process according to claim 12, characterized in that the gel comprises an agarose matrix to which the hydrophobic ligands have been subjected.
14. A process according to claim 13, characterized in that the ligands are selected from the group consisting of propyl, butyl, pentyl, hexyl, heptyl, octyl, phenyl and oligoethylene glycols.
15. 15. A process according to any of claims 12 to 14, characterized in that the ligands are butyl ligands subject to the agarose matrix by glycidyl ether couplings.
16. 16. A process according to any of the previous claims, characterized in that the recombinant coagulation factor VIII is a derivative by omission of a total length factor VIII with retained coagulant activity.
17. 17. A process according to claim 16, characterized in that the default derivative of factor VIII is a recombinant factor VIII SQ (r-VIII SQ) derived by default.
18. 18. A process according to any of the previous claims, characterized in that the step of chromatography by hydrophobic interaction is preceded by an anion exchange chromatography step.
19. 19. A process according to any of the preceding claims, characterized in that the step of chromatography by hydrophobic interaction is carried out at least twice.
20. 20. A method for treating hemophilia by administering a therapeutically effective amount of recombinant Factor VIII that has been purified according to any of claims 1-19. SUMMARY The present invention relates to a process for purifying recombinant coagulation factor VI11 by loading an aqueous solution containing factor VIII onto a hydrophobic interaction chromatography (HIC) gel, wherein at least one surfactant is present in the aqueous solution and / or a buffer solution used to balance the gel before loading the aqueous solution onto the gel. The presence of a surfactant when the solution containing factor VIII is loaded onto the HIC gel makes it possible to efficiently separate the intact and active Factor VIII molecules from the molecules with structural deviations. With the present invention it is also possible to considerably reduce the DNA content and / or cellular CHO contaminants and increase the activity of the Factor VIII product to a degree hitherto unknown. The invention further relates to an aqueous solution containing recombinant factor VIII which has been purified according to the process and current use of such aqueous solution, for the preparation of a medicament for administration to a patient having the symptoms of hemophilia. Also, the invention relates to a method for the treatment of hemophilia by administering a therapeutically effective amount of recombinant Factor VIII which has been purified according to the current process.