MXPA06005967A - Nanofiltration of factor vii solutions to remove virus - Google Patents

Abstract

The present invention relates to a novel method for improving the viral safety of liquid Factor VII compositions, in particular those comprising active Factor VII polypeptides (a Factor VIIa polypeptide).

Description

FILTRATION OF VIRUSES OF LIQUID COMPOSITIONS OF FACTOR VII FIELD OF THE INVENTION The present invention relates to a new method for improving the viral safety of the liquid compositions of factor VII, in particular those comprising the active polypeptides of factor VII (a factor Vlla polypeptide).

ANTECEDENTS OF. THE INVENTION A variety of factors involved in the blood coagulation process have been identified including factor VII (FVII), a plasma glycoprotein. Hemostasis is initiated by the formation of a complex between tissue factor (TF) that is exposed to circulating blood after damage to the spleen wall, and the factor Vlla that is present in the circulation in an amount corresponding to approximately 1% of the total protein mass of factor VII. Factor VII exists in the plasma mainly as a single chain zymogen which is cleaved by FXa to its activated form of two chains, Factor Vlla. The recombinant activated factor Vlla (rFVIIa) has been developed as a prohemostatic agent. The administration of rFVIIa offers a fast and highly effective prohemostatic response in hemophiliacs with Ref.:172710 hemorrhages, who can not be treated with other coagulation factors due to the formation of antibodies. Hemorrhage also in subjects with factor VII deficiency or subjects who have a normal coagulation system but experience excessive bleeding can be successfully treated with factor Vlla. The purification and handling of factor VII must be careful, due to the possibility of degradation of the molecule. Factor VII and factor Vlla, being large molecules (molecular weight approximately 50 kD), are susceptible to mechanical degradation by shearing forces, during purification and filtration. In addition, factor Vlla is an active proteolytic enzyme that degrades other proteins, including factor Vlla. The degradation of factor Vlla mainly involves the cleavage in the heavy chain of the Vlla factor, particularly in the non-amino acids. 290 and 315 in the molecule. Finally, the methionine residues of factor VII and factor Vlla can be oxidized. An object of the present invention is to provide a method for the elimination or inactivation of virus from the liquid compositions of factor VII by means of which method is substantially to preserve the integrity of the substituents of factor VII. WO 96/00237 describes a method of virus filtration of a solution containing a macromolecular protein, for example a protein such as the factor IX plasma protein. WO 98/37086 describes the elimination of viruses from solutions of proteins derived from plasma, by means of nanofiltration using the membrane having an average pore size of 15 nm. Tomokiyo et al., Vox Sanguinis, 2003, 84, 54-64, describes the large-scale production of activated factor VII concentrate, derived from human plasma. The production method involves the step of filtering the viruses from a solution comprising the inactive factor VII.

BRIEF DESCRIPTION OF THE INVENTION In a broad aspect, the invention relates to methods for the elimination and / or inactivation of viruses from the composition of factor VII. The term "virus" as used herein means any ultramicroscopic infectious agent that replicates itself, only within the cells of living hosts, or non-infectious particles derived therefrom. In one embodiment, the virus is infectious. In one embodiment, the virus is a non-infectious viral particle. A first aspect of the present invention relates to a method for removing virus from a liquid composition of factor VII, said method comprising subjecting the solution to nanofiltration using a nanofilter with a pore size of at most 80 nm. A second aspect of the present invention relates to a method for removing viruses from a liquid composition of factor VII, said composition comprises one or more polypeptides of factor VII, at least 5% of one or more factor VII polypeptides are in the activated form, the method comprises subjecting the solution to nanofiltration using a nanofiltration having a pore size of at more than 80 nm. A third aspect of the invention relates to a method for removing viruses from a liquid composition of factor VII, the composition comprises one or more factor VII polypeptides, the liquid composition is substantially free of serum, the method comprises subjecting the solution to nanofiltration using a nanofilter that has a pore size of at most 80 nm. A further aspect of the invention relates to a method for removing viruses from a liquid composition of factor VII, the composition comprising one or more factor VII polypeptides, the method comprising subjecting the solution to nanofiltration using a nanofilter having a size of At the most 80 nm pore, the nanofilter has a membrane made of one or more materials selected from cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF compound, surface modified PVDF, and polyethersulfone. A further aspect of the invention relates to a method for inactivating viruses in a liquid composition of factor VII, the composition comprising one or more factor VII polypeptides, the method comprising the step of combining the composition with a detergent. A further aspect of the invention relates to a method for the high level elimination of the presence of active viruses in a liquid composition of factor VII, the method comprises the steps of: (i) inactivating viruses and (ii) eliminating viruses .

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic illustration of a system suitable for the methods of the invention. The system includes a pressure tank (1) with a supply of compressed air, a pre-filter (2) to remove particles that could otherwise clog the virus filter, and a pressure gauge (P), a filter (3) of virus and a storage tank (4).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for removing or inactivating viruses, including non-enveloped viruses, from a liquid factor VII composition that typically comprises a significant proportion of activated factor VII polypeptides, and thus proteolytically active. The method includes the step of subjecting the liquid composition to factor VII to nanofiltration using a nanofilter having a pore size of at most 80 nm. The method is particularly useful for the removal of enveloped viruses, as well as non-enveloped viruses such as murine leukemia virus (enveloped) that can be eliminated by filters with a pore size of around 50 nm, and porcine parvovirus. . { not wrapped) that can be removed by filters with a pore size of around 20 nm. Liquid compositions of factor VII, for example, those comprising a significant proportion of the activated factor VII polypeptides, can in principle be prepared from the following dehydrated constituents of factor VII, but are more typically obtained from processes of large-scale production, for example processes that involve recombinant techniques. In such processes, a cell culture supernatant is typically harvested subsequently subjected to one or more processing steps to obtain the desired protein, including, without limitation, centrifugation or filtration to remove cells that were not immobilized on the carriers; affinity chromatography, hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic methods (e.g., in preparative isoelectric focusing (IEF), differential solubility (e.g., precipitation with ammonium sulfate), or extraction and the like.See in general, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, JC Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. The purification of factor VII polypeptides may also involve, for example, affinity chromatography on an anti-factor VII antibody column (see example, Wakabayashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem. 27: 7785, 1988) and activation by proteolytic cleavage, using factor Xlla or other proteases having specificity as trypsin, such as, for example, factor IXa, kallikrein, factor IXa, kallikrein, factor Xa, and thrombin See, for example, Osterud et al., Biochem 11: 2853 (1972); Thomas, United States Patent No 4,456,591, and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, a factor VII polypeptide can be activated by passing it through an ion exchange chromatography column, such as Mono Q® (Pharmacia) or the like. The methods of the present invention are particularly useful for large-scale production processes. By the term "large scale" is meant typically methods wherein the volume of the liquid compositions of the factor VII polypeptide is at least 100 liters, such as at least 500 liters, for example, at least 1000 liters, or at least 5000 liters. This is not limiting in any way, since the present invention will work for liquid compositions of the polypeptide to factor VII of at least 100 liters. It has now been found that nanofiltration can be applied even after the volume of the factor VII polypeptide has been partially or fully activated. Thus the methods of the invention are applicable as one of the purification steps of the factor VII polypeptide, typically one of the final steps of the purification process. More specifically, a typical purification process starting from the harvested material of a fermentation broth (or human (or mammalian) plasma can be described as follows: Purification step Possible stages for virus filtration Harvest Capture Intermediate Purification Refining i 4 Pharmacological substance The content of the factor VII polypeptide in the activated form is initially (for example, from the harvest step), typically around 2%, and increases in the course of the purification process to 90% or more or before the polypeptide is obtained as a pharmacological substance. The liquid composition of factor VII subjected to nanofiltration, comprises one or more factor polypeptides VII in a suitable solvent. The solvent is typically water or an aqueous mixture / solution, such as pure water, an aqueous buffer, a water / ethanol mixture, a water / DMSO mixture, or an aqueous saline solution, eg, saline, a solution of urea or a guanidine solution. A suitable aqueous liquid must also comprise a detergent (surfactant). In interesting embodiments, the liquid composition of factor VII is obtained, or originates, from a cell culture supernatant e.g., a cell culture supernatant obtained as described in WO 02/29084. In one embodiment, the liquid composition of factor VII is free of serum, for example, free of animal-derived components. In this way, cell cultures can be cultured in a medium that lacks animal-derived components. An attractive variant thereof is one in which the factor VII polypeptides are produced by cell culture in CHO cells, for example, in CHO cells in medium free of any components of native origin or a medium lacking the animal-derived components, and that they lack proteins ("protein free"). The means for CHO cells can be any commercially available protein-free CHO medium that lacks the animal-derived components or a home-produced medium for CHO cells. In some embodiments, the cells used in the practice of the present invention are adapted for the growth of the suspension in the medium lacking the animal-derived components, such as, for example, in the medium lacking serum. Such adaptation methods are described, for example, in Scharfenberg, et al. , Animal Cell Technology Developments Towards the 21st Century, E.C. Beuvery et al. (Eds.), Kluwer Academic Publishers, pp. 619-623, 1995 (BHK and CHO cells); Cruz, Biotechnol .. Tech. 11: 117-120, 1997 (insect cells); Keen, Cytotechnol, 17: 203-211, 1995 (myeloma cells); Berg et al., Biotechniques 14: 972-978, 1993 (human kidney cells 293). In a particular embodiment, the host cells are BHK 21 or CHO cells that have been engineered to express human factor VII or a factor VII polypeptide, and which have been adapted to develop in the absence of serum or derived components of animals . In an alternative embodiment, the factor VII polypeptide (s) is produced by cell culture in the presence of fetal calf or bovine serum. According to one aspect of the invention, a feature is that a significant proportion, for example, at least 5%, such as at least 7%, for example at least 10%, of one or more factor VII polypeptides are in the activated form (e.g., the cleaved, bioactive form of the factor VII polypeptide (e.g., a factor VII polypeptide)). In additional embodiments, the Vlla factor polypeptide represents 5-70%, such as 7-40%, for example 10-30%, of the mass of one or more factor VII polypeptides. In other embodiments, the Vlla factor polypeptide represents 50-100%, such as 70-100%, for example 80-100%, of the mass of one or more factor VII polypeptides. In other additional embodiments, the Vlla factor polypeptide represents 20-80%, such as 30-70%, for example 30-60% of the mass of one or more factor VII polypeptides. In most modalities, the solution comprises a factor VII polypeptide in inactivated form, as well as a polypeptide of the bioactive factor Vlla, for example, the Vlla factor polypeptide represents less than 100% of the mass of one or more factor VII polypeptides. In the most typical embodiment, the composition comprises an activated Vlla factor polypeptide corresponding to a factor VII polypeptide (inactive), eg, the Vlla factor polypeptide is the factor VII polypeptide in the activated form. In another embodiment, the Vlla factor polypeptide is somewhat different from the activated form of the inactivated factor VII polypeptide. It should be understood of course that the composition in the applied modalities may comprise more than one factor VII polypeptide and more than one Vlla factor polypeptide. The term "one or more factor VII polypeptides" encompasses wild-type factor VII (e.g., a polypeptide having the amino acid sequence described in U.S. Patent No. 4,784,950), as well as variants of factor VII that show substantially the same or an improved biological activity relative to wild-type factor VII. The term "factor VII" is intended to encompass factor VII polypeptides in their non-cleaved form (zymogen), as well as those that have been proteolytically processed to produce their respective bioactive forms, which may be designated Factor Vlla. Typically, factor VII is cleaved between residues 152 and 153 to produce factor Vlla. The term "factor Vlla" specifically means an activated factor VII polypeptide (eg, bioactive, cleaved). In this way, "factor Vlla" is a subgroup in relation to "factor VII". The term "inactive factor VII" specifically means factor VII which is not the factor Vlla. The term "factor VII polypeptide" also encompasses polypeptides, including variants, in which the biological activity of factor Vlla has been substantially modified or reduced to some extent in relation to the activity of wild type factor Vlla, as well as the Factor VII derivatives and factor VII conjugates. These polypeptides include, without limitation, factor VII or factor Vlla within which specific alterations have been introduced into the amino acid sequences, which modify or disrupt the bioactivity of the polypeptide. The term "factor VII derivative" as used herein, is intended to designate wild-type factor VII, factor VII variants that show substantially the same or improved biological activity, relative to wild-type factor VII, and polypeptides related to factor VII, in which one or more of the amino acids of the parent peptide has been chemically and / or enzymatically modified, for example, by alkylation, glycosylation, PEGylation, acylation, ester formation or amide formation or the like . This includes, but is not limited to, the PEGylated human factor, the cysteine-PEGylated human factor Vlla and variants thereof. Non-limiting examples of factor VII derivatives include glucocoglylated FVII derivatives as described in WO 03/31464 and in U.S. Patent Applications Nos. 20040043446, 20040064911, 20040142856, 20040137557 and 20040132640 (Neose Technology, Inc. ); FVII conjugates as described in WO 01/04287, U.S. Patent Application No. 20030165996, and WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, Patent Application. of the United States 20030211094 (University of Minnesota). The term "PEGylated human factor" means the human factor Vlla, which has a PEG molecule conjugated to the human Factor Vlla polypeptide. It should be understood that the PEG molecule can be linked to any part of the Vlla factor polypeptide including any amino acid residue or carbohydrate portion of the Vlla factor polypeptide. The term "cysteine-PEGylated human factor factor" means the factor Vlla having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced into the human factor Vlla. The biological activity of factor Vlla in blood coagulation is derived from its ability to (i) bind to tissue factor (TF) and (ii) to catalyze the proteolytic cleavage of factor XI or factor X to produce activated factor IX or X (factor IXa or Xa, respectively). For the purposes of the invention, the biological activity of factor VII polypeptides ("factor VII biological activity") can be quantified by measuring the ability of a preparation to promote blood coagulation using factor VII deficient plasma and thromboplastin , as described, for example, in U.S. Patent No. 5,997,864 or WO 92/15686. In this assay, biological activity is expressed as the reduction in coagulation time relative to a control sample, and is converted to factor units to "factor VII units" by comparison with a combined human serum standard containing 1 unit / ml of factor VII activity. Alternatively, the biological activity of factor Vlla can be quantified (i) by measuring the ability of factor Vlla (or the factor VII polypeptide) to produce an activated factor X (factor Xa) in a system comprising TF embedded in a lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997); (ii) by measuring the hydrolysis of factor X in an aqueous system ("in vitro proteolysis assay", see below); (iii) by measuring the physical link of factor Vlla (or factor VII polypeptide) to TF using an instrument based on surface plasmon resonance (Person, FEBS Letts, 413: 359-363, 1997); (iv) by measuring the hydrolysis of a synthetic substrate of factor Vlla (or a factor VII polypeptide) ("in vitro hydrolysis assay", see below); or (v) by measuring the generation of thrombin in an in vitro system independent of TF. Factor VII variants that have substantially the same or improved biological activity relative to the wild-type factor Vlla, encompass those that show at least about 25%, such as at least about 50%, such as at least about 75%, such as at least about 90% of the specific activity of factor Vlla that has been produced in the same cell type, when tested in one or more of a coagulation assay, proteolysis assay, or TF binding assay as described previously . In one embodiment, the biological activity is greater than 80% of the biological activity of recombinant wild type human Vlla factor. In yet another embodiment, the biological activity is greater than 90% of the biological activity of the recombinant wild-type human Vlla factor. In a further embodiment, the biological activity is greater than 100% of the biological activity of recombinant wild type human Vlla factor. In a further embodiment, the biological activity is greater than 120% of the biological activity of recombinant wild type human Vlla factor. In a further embodiment, the biological activity is greater than 200% of the biological activity of recombinant wild type human Vlla factor. In an additional mode, the biological activity is greater than 400% of the biological activity of recombinant wild-type human factor Vlla. Factor VII variants that have substantially reduced biological activity relative to wild type factor Vlla, are those that show less than about 25%, such as less than about 10%, such as less than about 5%, such as less than about 1% of the specific activity of wild type factor Vlla that has been produced in the same cell type, when tested in one or more of a coagulation assay, proteolysis assay, or TF binding assay, as described previously . Factor VII variants that have a substantially modified biological activity relative to wild-type factor VII include, without limitation, factor VII variants that show TF-independent proteolytic factor VII activity, and those that bind to TF but do not rupture factor X. Factor VII variants, whether they show substantially the same or a better bioactivity than wild-type factor VII or, alternatively, show substantially modified or reduced bioactivity relative to wild-type factor VII, include, without limitation, polypeptides having an amino acid sequence that differs from the wild type factor VII sequence by insertion, deletion, or substitution of one or more amino acids. Non-limiting examples of factor VII variants that have substantially the same biological activity and wild-type factor VII include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem. Biophys, 352: 182-192, 1998 ); Vlla factor variants that show stability i? increased proteolytic as described in U.S. Patent No. 5,580,560; the Vlla factor that has been proteolytically excised between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol., Bioeng 48: 501-505, 1995); the oxidized forms of the Vlla factor (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999); Factor VII variants as described in PCT / DK02 / 00189; and Factor VII variants that show enhanced proteolytic stability as described in WO 02/38162 (Scripps Research Institute); Factor VII variants that have a modified Gla domain and that show an improved membrane bond as described in WO 99/20767, U.S. Patent Nos. 6017882 and 6747003, U.S. Patent Application No 20030100506 (University of Minnesota), and WO 00/66753, the U.S. Patent Applications Nos. 20010018414, 2004220106, and 200131005, the Patents of the United States Nos. 6762286 and 6693075 (University of Minnesota); and Factor VII variants as described in WO 01/58935, U.S. Patent No. 6806063, the United States Patent Application No. 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO 04/029091 (Maxygen ApS). Non-limiting examples of the factor VII variants having an increased biological activity compared to the wild-type factor Vlla include the factor VII variants as described in WO 01/83725, WO 02/22776, WO 02/077218 , WO 03/27147, WO 03/37932; WO 02/38162 (Scripps Research Institute); and Vlla factor variants with enhanced activity as described in Japanese Patent 2001061479 (Chemo-Sero-Therapeutic Res. Inst.). Non-limiting examples of the factor VII variants having substantially reduced biological activity modified relative to wild-type factor VII include R152E-FIIa (Wildgoose et al., Biochem 29: 3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270: 66-72, 1995), FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15: 515-520, 1998), and the factor Vlla that lacks Gla domain (Nicolaisen et al., FEBS Letts 317: 245-249, 1993). Explicit examples of factor VII polypeptides include, without limitation, wild type factor VII, L305V-FVII, L305V / M306D / D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVH, V158T / M298Q-FVII, V158D / E296V / M298Q-FVII, K337A-FVII, M298Q-FVII, V158D / M298Q-FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / K337A-FVII, V158D / E296V / M298Q / L305V / K337A-FVII, K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII, L305V / K337A-FVII, L305V / V158D -FVII, L305V / E296V-FVII, L305V / M298Q-FVII, L305V / V158T-FVII, L305V / K337A / V158T-FVII, L305V / K337A / M298Q-FVII, L305V / K337A / E296V-FVII, L305V / K337A / V158D-FVII, L305V / V158D / M298Q-FVII, L305V / V158D / E296V-FVII, L305V / V158T / M298Q-FVII, L305V / V158T / E296V-FVII, L305V / E296V / M298Q-FVII, L305V / V158D / E296V / M298Q-FVII, L305V / V158T / E296V / M298Q-FVII, L305V / V158T / K337A / M298Q-FVII, L305V / V158T / E296V / K337A-FVII, L305V / V158D / K337A / M298Q-FVII, L305V / V158D / E296V / K337A-FVII, L305V / V158D / E296V / M298Q / K337A-FVII, L305V / V158T / E296V / M298Q / K337A-FVII, S314E / K316H-FVII, S314E / K316Q-FVII, S314E / L305V-FVII, S314E / K337A-FVII, S314E / V158D-FVII, S314E / E296V-FVII, S314E / M298Q-FVII, S314E / V158T-FVII, K316H / L305V-FVH, K316H / K337A-FVII, K316H / V158D-FVII, K316H / E296V-FVII, K316H / M298Q-FVII, K316H / V158T-FVII, K316Q / L305V-FVII, K316Q / K337A-FVII, K316Q / V158D-FVII, K316Q / E296V-FVII, K316Q / M298Q-FVII, K316Q / V158T-FVII, S314E / L305V / K337A-FVII, S314E / L305V / V158D-FVII, S314E / L305V / E296V-FVII, S314E / L305V / M298Q-FVII, S314E / L305V / V158T-FVII, S314E / L305V / K337A / V158T-FVII, S314E / L305V / K337A / M298Q-FVII, S314E / L305V / K337A / E296V-FVII, S314E / L305V / K337A / V158D-FVII, S314E / L305V / V158D / M298Q-FVII, S314E / L305V / V158D / E296V-FVII, S314E / L305V / V158T / M298Q-FVII, S314E / L305V / V158T / E296V-FVII, S314E / L305V / E296V / M298Q-FVII , S314E / L305V / V158D / E296V / M298Q-FVII, S314E / L305V / V158T / E296V / M298Q-FVII, S314E / L305V / V158T / K337A / M298Q-FVII, S314E / L305V / V158T / E296V / K337A-FVII, S314E / L305V / V158D / K337A / M298Q-FVII, S314E / L305V / V158D / E296V / K337A-FVII, S314E / L305V / V158D / E296V / M298Q / K337A-FVII, S314E / L305V / V158T / E296V / M298Q / K337A-FVII , K316H / L305V / K337A-FVII, K316H / L305V / V158D-FVII, K316H / L305V / E296V-FVII, K316H / L305V / M298Q-FVII, K316H / L305V / V158T-FVII, K316H / L305V / K337A / V158T-FVII , K316H / L305V / K337A / M298Q-FVII, K316H / L305V / K337A / E296V-FVII, K316H / L305V / K337A / V158D-FVII, K316H / L305V / V158D / M298Q-FVII, K316H / L305V / V158D / E296V-FVII , K316H / L305V / V158T / M298Q-FVII, K316H / L305V / V158T / E296V-FVII, K316H / L305V / E296V / M298Q-FVII, K316H / L305V / V158D /? 296V / M298Q-FVII, K316H / L305V / V158T / E296V / M298Q-FVII, K316H / L305V / V158T / K337A / M298Q-FVII, K316H / L305V / V158T / E296V / K337A-FVII, K316H / L305V / V158D / K337A / M298Q-FVII, K316H / L305V / V158D / E296V / K337A-FVII, K316H / L305V / V158D / E296V / M298Q / K337A-FVII, K316H / L305V / V158T / E296V / M298Q / K337A-FVII, K316Q / L305V / K337A-FVII, K316Q / L305V / V158D-FVII, K316Q / L305V / E296V-FVII, K316Q / L305V / M298Q-FVII, K316Q / L305V / V158T-FVII, K316Q / L305V / K337A / V158T-FVII, K316Q / L305V / K337A / M298Q-FVII, K316Q / L305V / K337A / E296V-FVII, K316Q / L305V / K337A / V158D-FVII, K316Q / L305V / V158D / M298Q-FVII, K316Q / L305V / V158D / E296V-FVII, K316Q / L305V / V158T / M298Q-FVII, K316Q / L305V / V158T / E296V-FVII, K316Q / L305V / E296V / M298Q-FVII, K316Q / L305V / V158D / E296V / M298Q- FVII, K316Q / L305V / V158T / E296V / M298Q-FVII, K316Q / L305V / V158T / K337A / M298Q-FVII, K316Q / L305V / V158T / E296V / K337A-FVII, K316Q / L305V / V158D / K337A / M298Q-FVII, K316Q / L305V / V158D / E296V / K337A-FVII, K316Q / L305V / V158D / E296V / M298Q / K337A-FVII, K316Q / L305V / V158T / E296V / M298Q / K337A-FVII, F374Y / K337A-FVII, F374Y / V158D- FVII, F374Y / E296V-FVII, F374Y / M298Q-FVII, F374Y / V158T-FVII, F374Y / S314E-FVII, F374Y / L305V-FVII, F374Y / L305V / K337A-FVII, F374Y / L305V / V158D-FVII, F374Y / L305V / E296V-FVII, F374Y / L305V / M298Q-FVII, F374Y / L305V / V158T-FVII, F374Y / L305V / S314E-FVII, F374Y / K337A / S314E-FVII, F374Y / K337A / V158T-FVII, F374Y / K337A / M298Q-FVII, F374Y / K337A / E296V-FVII, F374Y / K337A / V158D-FVII, F374Y / V158D / S314E-FVII, F374Y / V158 D / M298Q-FVII, F374Y / V158D / E296V-FVII, F374Y / V158T / S314E-FVII, F374Y / V158T / M298Q-FVII, F374Y / V158T / E296V-FVII, F374Y / E296V / S314E-FVII, F374Y / S314E / M298Q-FVII, F374Y / E296V / M298Q-FVII, F374Y / L305V / K337A / V158D-FVII, F374Y / L305V / K337A / E296V-FVII, F374Y / L305V / K337A / M298Q-FVII, F374Y / L305V / K337A / V158T- FVII, F374Y / L305V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V-FVII, F374Y / L305V / V158D / M298Q-FVII, F374Y / L305V / V158D / S314E- • FVII, F374Y / L305V / E296V / M298Q -FVII, F374Y / L305V / E296V / V158T- FVII, F374Y / L305V / E296V / S314E-FVII, F374Y / L305V / M298Q / V158T- FVII, F374Y / L305V / M298Q / S314E-FVII, F374Y / L305V / V158T / S314E - FVII, F374Y / K337A / S314E / V158T-FVII, F374Y / K337A / S314E / M298Q-FVII, F374Y / K337A / S314E / E296V-FVII, F374Y / K337A / S314E / V158D-FVII, F374Y / K337A / V158T / M298Q -FVII, F374Y / K337A / V158T / E296V-FVII, F374Y / K337A / M298Q / E296V-FVII, F374Y / K337A / M298Q / V158D-FVII, F374Y / K337A / E296V / V158D-FVII, F374Y / V158D / S314E / M298Q - FVII, F374Y / V158D / S314E / E296V-FVII, F374Y / V158D / M298Q / E296V-FVII, F374Y / V158T / S314E / E296V- FVII, F374Y / V158T / S314E / M298Q-FVII, F374Y / V158T / M298Q / E296V-FVII, F374Y / E296V / S314E / M298Q-FVII, F374Y / L305V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / K337A / S314E-FVII, F374Y / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A-FVII, F374Y / L305V / E296V / M298Q / S314E-FVII, F374Y / V158D / E296V / M298Q / K33'7A-FVII, F374Y / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / V158D / K337A / S314E-FVII, F374Y / V158D / M298Q / K337A / S314E-FVII, F374Y / V158D / E296V / K337A / S314E- FVII, F374Y / L305V / V158D / E296V / M298Q- FVII, F374Y / L305V / V158D / M298Q / K337A- FVII, F374Y / L305V / V158D / E296V / K337A- FVII, F374Y / L305V / V158D / M298Q / S314E- FVII, F374Y / L305V / V158D / E296V / S314E-FVII, F374Y / V158T / E296V / M298Q / K337A-FVII, F374Y / V158T / E296V / M298Q / S314E-FVII, F374Y / L305V / V158T / K337A / S314E-FVII, F374Y / V158T / M298Q / K337A / S314E-FVII, F374Y / V158T / E296V / K337A / S314E-FVII, F374Y / L305V / V158T / E296V / M298Q-FVII, F374Y / L305V / V158T / M298Q / K337A-FVII, F374Y / L305V / V158T / E296V / K337A-FVII, F374Y / L305V / V158T / M298Q / S314E-FVII, F374Y / L305V / V1 58T / E296V / S314E-FVII, F374Y / E296V / M298Q / K337A / V158T / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / E296V / M298Q / V158T / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158T-FVII, F374Y / L305V / E296V / K337A / V158T / S314E-FVII, F374Y / L305V / M298Q / K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A-FVII, F374Y / L305V / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A / S314E-FVII, S52A-Factor VII, S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor Vlla that lacks Gla domain; and P11Q / K33E-FVII, T106N-FVII, K143N / N145T-FVII, V253IM-FVII, R290IM / A292T-FVII, G291N-FVII, R315N / V317T-FVII, K143N / N145T / R315N / V317T-FVII; and factor VII having substitutions, additions or deletions in the amino acid sequence 233Thr to 240Asn, factor VII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys. In some embodiments, the Vlla factor polypeptide is human factor Vlla (hFVIIa), such as the recombinant human Factor Vlla elaborated (rhFVIIa). In other embodiments, one or more of the factor VII polypeptides comprises a variant of the factor VII sequence. In some embodiments, one or more factor VII polypeptides have a different glycosylation of wild type factor VII.

Nanof i 1 tra ci on The liquid composition of factor VII is subjected to nanofiltration using a nanofilter having a pore size of at most 80 nm. The pore size of the nanofilter is more particularly at more than 50 nm, for example at more than 30 nm, in the range of 10 to 30 nm.

The term "pore size" typically means the size of the smallest viruses that are retained by the filter. Examples of suitable commercially available nanofilters are Asahi Planove 15 N, Asahi Planove 20 N, Asahi Planova 35 N, and Asahi Planova 75 N, all from Asahi Chemical, Tokyo, Japan; Millipore NFR, Millipore NFP, Millipore Viresolve 70, and Millipore Viresolve 180, all from Millipore; and Pall DV20, Pall DV 50, Pall Omega VR 100 K; and 15 nm Bemberg microporous membrane (BMM-15). The nanofilter membrane can be, for example, made from one or more materials selected from cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF composite, surface modified PVDF, polyethersulfone and similar materials. In one embodiment, the material is selected from materials based on polyvinylidene chloride and polyethersulfone-based materials. The nanofiltration can be conducted through the tangential filtration mode or in the dead end filtration mode, as will be understood by the person skilled in the art. In one embodiment, the nanofiltration is conducted in the dead end filtration mode. The pH value of the liquid composition of factor VII after nanofiltration is not considered particularly critical. In this way, the pH value is normally given in view of the conditions applied in the steps of the process, directly from the nanofiltration step. In some embodiments, the pH value is adjusted to that of the liquid composition having a pH in the range of 5.5-10, such as in the range of 7.0-9.5, for example, in the range of 7.6-9.4. , such as in the range of 7.7-9.3, for example in the range of 8.0-9.0 or in the range of 8.3-8.7. In one embodiment, the pH is in the range of 5-7. In one embodiment, the pH is greater than 9.5, such as in the range of 9.5-10. In addition, the concentration of the factor VII polypeptide and the liquid composition is typically also given by the steps of the preceding process, but will normally fall in the range of 0.01-5 mg / ml, such as in the range of 0.05-2.0 mg / ml . In the nanofiltration process it can be conducted using a filtration system as illustrated in figure 1. The process can be conducted as in the following illustrative example: The pressure tank (1) is filled with water for injection (WFI), and the pressure in the tank is raised to 3.5 bar before the virus filter (3), and the filter is washed for 10 minutes. The pressure is reduced to 2 bars and the virus filter (3) is flushed for another 10 minutes. The pressure tank is not emptied of WFI and the process is optionally repeated with a buffer, before the liquid composition of the factor VII is filled into the pressure tank (1). The pressure is raised to 2 bars and is maintained sequentially constant during filtration. The virus filter (3) can be subsequently tested for integrity by standard procedures. The filtrate was collected in a storage tank and can further be processed in order to obtain a pharmaceutical composition comprising a Factor Vlla polypeptide, as a pharmaceutical substance. That said, it is typically advantageous to apply a pre-filtration step before the nanofiltration step in order to remove the larger particles, aggregates, etc., which could otherwise cause the nanofilter to become clogged. Such a pre-filter typically has a pore size in a range of 0.05-0.5 μm. In one embodiment, the pre-filter is a Millipore NFR filter. Alternatively, when using air pressure, a liquid pump placed after the pressure tank can provide atmospheric pressure for filtration. If the liquid composition of the nanofiltered factor VII comprises the inactive factor VII polypeptides, the composition may be subsequently subjected to an activation step, for example, as described in Bj0rn, S. and Thi, L. Res. Disclosures (1986). 269, 564-565, Pedersen, AH & al., Bioche istry (1989), 28, 9331-9336, and Tomokiyo, K. and et al., Vox Sang, 84, 54-64 (2003). Further processing of the composition and the final formulation as a pharmaceutical product can be conducted as described in Jurlander, B. et al., Seminars in Thrombosis and Hemostasis 27, 373-383 (2001).

Nanofiltration of fluid-free, serum-free factor VII polypeptide compositions A separate aspect of the invention, which may include some of all of the foregoing characteristics, relates to a method for removing virus from a liquid composition of factor VII, the composition comprising or more factor VII polypeptides, the liquid composition is substantially free of serum, the method comprises subjecting the solution to nanofiltration using a nanofilter having a pore size of at most 80 nm. An attractive variant of this is one in which factor VII polypeptides are produced by cell culture in CHO cells, for example, in free CHO cells of any naturally occurring components. This aspect of the invention is particularly limited to liquid factor VII compositions in which a certain portion of or of factor VII polypeptides are in the activated form. However, the aforementioned conditions for the first aspect of the invention also apply for this second aspect of the invention, mutatis mutandis.

Nanofiltration of liquid compositions of polypeptide VII via particular filters Another separate aspect of the invention, which may include some of the above characteristics, relates to the method for removing viruses from a liquid composition of factor VII, the composition comprising one or more polypeptides of the Factor VII, the method comprises subjecting the solution to nanofiltration using a nanofilter having a pore size of at most 80 nm, wherein the filter has a membrane made from one or more materials selected from regenerated cellulose such as cuprammonium, fluoride of polyvinylidene hydrophilic (PVDF), PVDF compound, PDVF multiplied surface, and polyethersulfone. In one embodiment, the material is selected from materials based on polyvinylidene fluoride and polyethersulfone-based materials. This aspect of the invention is not particularly limited to liquid factor VII compositions in which a certain proportion of or of the factor VII polypeptides are in activated form. However, the conditions mentioned above, for the first aspect of the invention also applies to this third aspect of the invention, mutatis mutandis.

Inactivation of the virus by the addition of a detergent In yet another aspect, the present invention also relates to a method for inactivating viruses in a liquid composition of factor VII, the composition comprising one or more factor VII polypeptides, the method comprising the step of combine the composition with a detergent. In some embodiments, the detergent is selected from nonionic detergents such as those selected from octylphenoxy polyethoxyethanol, polysorbates, poloxamers, polyoxyethylene alkyl ethers, polyethylene / polypropylene block copolymers, polyethylene glycol (PEG), polyoxyethylene stearates, castor oils polyoxyethylenates Illustrative examples thereof are nonionic detergents such as Triton X-100, Tween®, polysorbate 20, polysorbate 60, polysorbate 80, Brij-35 (polyoxyethylene dodecyl ether), poloxamer 188, poloxamer 407, PEG8000, Pluronic polyols ®, polyoxy-23-lauryl ether, Myrj 49, and Cremophor A.

A particularly useful detergent is an octylphenoxy-polyethoxyethanol of the formula p- ((CH3) 3CH2C (CH2) 2) -C6H4-0- (CH2CH2?) NH wherein n is in the range of 5-15, in particular one where n is 9-10, such as the Triton X-100 detergent. In one embodiment, the detergent is combined with the liquid composition of factor VII to obtain a detergent concentration "in the composition in the range of 0.01-0.5% by weight, such as in the range of 0.05-0.4% by weight, such as in the range of 0.05-0.3% by weight, such as in the range of 0.05-0.2% by weight, such as in the range of 0.05-0.1% by weight., the detergent is combined with the composition at a temperature in the range of 2-12 ° C, such as in the range of 2-9 ° C. For most purposes, it was found undesirable to include a trialkyl phosphate detergent, in this way, the detergent may be substantially free of trialkyl phosphate solvent such as tri (n-butyl) phosphate. In a particular embodiment, the method comprises the steps of combining the factor VII polypeptide composition with Triton X-100 at a concentration of 0.05-0.2% by weight, at a temperature in the range of 2-9 ° C, with the The condition of the detergent is substantially free of trialkylsulfate solvents such as tri (n-butyl) phosphate.

This aspect of the invention is not particularly limited to liquid factor VII compositions in which a certain portion of or of the factor VII polypeptides are in activated form. However, the aforementioned conditions for the first aspect of the invention also apply for this fourth aspect of the invention, mutatis mutandis.

Combination of virus inactivation steps In a further aspect, the present invention relates to a method for the high level elimination of the presence of active viruses in a liquid composition of factor VII, the method comprises the steps of (1) inactivating viruses by the method defined under "virus inactivation by addition of a detergent" and (ii) eliminate the virus by any of the methods defined herein under "nanofiltration" in any order. In one embodiment, the step of inactivating viruses precedes the step of virus removal. Even if it is believed that the individual steps are sufficient for the purpose of eliminating the presence of active viruses, the two methods can be considered, at least partially "orthogonal" in the sense that certain viruses can be more difficult to eliminate by one of the methods, while certain viruses can be more easily eliminated by the other method, and vice versa. In this way, the combination of the two methods will provide an even higher level of safety for the patient for whom the Factor VII polypeptide is intended, and. not least by the doctor who prescribes the factor VII polypeptide drug, and by the regulatory authorities that approve the drug. In this way, the combination of the two methods can have a high commercial value. As described above, the present invention relates to the removal or inactivation of viral particles. The reduction of the amount of viral particles in a particular process step is usually described in logarithmic units (logarithm log 10, or log? 0), where the reduction factor is calculated as the amount of viral particles after the step relative to the amount of viral particles before the process step. For example, if 106 viral particles are found before a step and 102 are found after the step, the reduction is 104, or 4 log? 0. The total reduction of the viral particles from the whole process is described in the same way, and can be calculated by the addition of the virus clearing from each step in the process, the word "clearing" means the elimination of the virus and inactivation of the virus. For a specific viral clearance step to be effective, a virus reduction of at least 4 log10 is preferred first. In one embodiment of the present invention, a filtration step reduces the amount of viral particles with at least 4 log10. In one embodiment of the present invention, a filtration step reduces the amount of viral particles with at least about 5 log10. In one embodiment of the present invention, a step of combining the FVII composition with a detergent inactivates at least about 4 logio of virus. In one embodiment of the present invention, a step of combining the FVII composition with a detergent inactivates at least about 5 log10 of virus. The determination of the amount of the viral particles is known to the person skilled in the art and can be measured in standard TCID50 assays (50% endpoint of the infectious dose of the tissue culture, by me), plate tests or assays. of PCR. TCID50 and plaque assays can be used to measure the concentration of infectious particles, while PCR assays can be used to measure infectious and noninfectious inactivated viral particles.

Modalities of the present invention 1. A method for removing viruses from a factor VII composition, the composition comprises one or more factor VII polypeptides, at least 5% of one or more factor VII polypeptides that are in the activated form, the The method comprises subjecting the solution to nanofiltration using a nanofilter having a pore size of at most 80 nm. 2. The method according to mode 1, wherein at least 7%, for example, at least 10%, of one or more factor VII polypeptides are in the activated form. 3. The method according to mode 1, wherein the activated form of the factor VII polypeptide represents 5-70%, such as 7-40%, for example, 10-30% of the mass of one or more of the polypeptides of the Factor VII. 4. The method according to the embodiment 1, wherein the activated form of the factor VII polypeptide represents 50-100%, such as 70-100%, for example, 80-100% of the mass of one or more of the polypeptides of the Factor VII.

. The method according to mode 1, wherein the activated form of the factor VII polypeptide represents 20-80%, such as 30-70%, for example, 30-60% of the mass of one or more of the polypeptides of the Factor VII. 6. The method according to any of the preceding embodiments, wherein the liquid composition has a pH in the range of 7.0-9.5, for example in the range of 7.6-9.4, such as in the range of 7.7-9.3, by example, in the range of 8.0-9.0 or in the range of 8.3-8.7. 7. The method according to any of the preceding embodiments, wherein the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the range of 0.05-2.0 mg / ml. 8. The method according to any of the preceding embodiments, wherein the pore size of the nanofilter is at most 50 nm, for example at most 30 nm, such as in the range of 10-30 nm. 9. The method according to any of the preceding embodiments, wherein the nanofilter membrane is manufactured from one or more materials selected from cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), compound PDVF, surface modified PDVF, and polyethersulfone .

. The method according to any of the preceding embodiments, wherein the liquid composition of factor VII is obtained, or originates from a cell culture supernatant. 11. The method according to any of the preceding embodiments, wherein the liquid composition is substantially free of serum. 12. The method according to any of embodiments 1-10, wherein the factor VII polypeptide (s) is produced by cell culture in the presence of fetal, bovine or calf sera. 13. The method according to any of the preceding embodiments, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells. 14. The method according to mode 13, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells, in a medium free of any components of natural origin.

. A method for removing virus from a liquid composition of factor VII, the composition comprises one or more factor VII polypeptides, the liquid composition is substantially free of serum, the method comprises attaching the solution to nanofiltration using a nanofilter having a serum size from at most 80 nm. 16. The method according to mode 15, wherein the liquid composition of factor VII is obtained, or originates, from a cell culture supernatant. 17. The method according to any of embodiments 15-16, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells. 18. The method according to mode 17, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells, in a medium free of any components of animal origin. 19. The method according to any of the embodiments 15-18, wherein at least 5% of one or more factor VII polypeptides are in activated form.

. The method according to any of the embodiments 15-19, wherein the liquid composition has a pH in the range of 7.0-9.5, for example in the range of 7.6-9.4, such as in the range of 7.7-9.3, by example, in the range of 8.0-9.0, or in the range of 8.3-8.7. 21. The method according to any of the 15-20 modalities, wherein the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the range of 0.05-2.0. mg / ml. 22. The method according to any of embodiments 15-21, wherein the pore size of the nanofilter is at most 50 nm, for example at more than 30 nm, such as in the range of 10-30 nm. 23. The method according to any of the embodiments 15-22, wherein the nanofilter membrane is manufactured from one or more of the selected materials of cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF compound, modified PVDF superficially, and polyethersulfone. 24. A method for removing virus from a liquid composition to factor VII, the composition comprises one or more factor VII polypeptides, the method comprising subjecting the solution to nanofiltration using a nanofilter having a pore size of at most 80 nm, the nanofilter it has a membrane made from one or more materials selected from cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF compound, surface modified PVDF, and polyethersulfone.

. The method according to the modality 24, where the material is selected from materials based on polyvinylidene chloride and materials based on polyethersulfone. 26. A method according to any of the 24-25 modalities, wherein at least 5% of one or more factor VII polypeptides are in the activated form. 27. The method according to any of the embodiments 24-26, wherein the liquid composition has a pH in the range of 7.0-9.5, for example in the range of 7.6-9.4, such as in the range of 7.7-9.3, by example, in the range of 8.0-9.0, or in the range of 8.3-8.7. 28. The method according to any of the modalities • 24-27, wherein the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the range of 0.05- 2.0 mg / ml. 29. The method according to any of embodiments 24-28, wherein the pore size of the nanofilter is at most 50 nm, for example at more than 30 nm, such as in the range of 10-30 nm.

. The method according to any of the embodiments 24-29, wherein the liquid composition of factor VII is obtained, or originates, from a cell culture supernatant. 31. The method according to any of the embodiments 24-30, wherein the liquid composition is substantially free of serum. 32. The method according to any of the embodiments 24-31, wherein the factor VII polypeptide (s) is produced by cell culture in the presence of fetal, bovine or calf serum. 33. The method according to any of the embodiments 24-32, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells. 34. The method according to embodiment 33, wherein the factor VII polypeptide (s) is produced by cell culture in CHO cells, in a medium free of any components of animal origin.

. A method for inactivating virus from a liquid composition to factor VII, the composition comprising one or more factor VII polypeptides, the method comprising the step of combining the composition with a detergent. 36. The method according to embodiment 35, wherein the detergent is an octylphenoxy-polyethoxyethanol of the formula p- ((CH 3) 3 CH 2 C (CH 2 (2 (-C 6 H 4-0- (CH 2 CH 2)) nH wherein n is in the range from 5-15. 37. The method according to the embodiment 36, wherein the detergent is one where n is 9-10, such as Triton X-100. 38. The method according to embodiment 35, wherein the detergent is selected from the list of Tween®, polysorbate 20, polysorbate 60, and polysorbate 80. 39. The method according to any of embodiments 35-38, wherein the detergent is combined with the liquid composition of factor VII to obtain a detergent concentration in the composition in the range of 0.01-0.3% by weight, such as in the 0.05-0.2% by weight interval. 40. The method according to any of embodiments 35-39, wherein the detergent is combined with the composition at a temperature in the range of 2-12 ° C, such as in the range of 2-9 ° C. 41. The method according to any of embodiments 35-40, wherein the detergent is substantially free of trialkyl phosphate solvents such as tris (n-butyl) phosphate. 42. A method for the high level removal of the presence of active virus in a liquid composition of factor VII, the method comprises the steps of (i) inactivating viruses by the method defined in any of the modes 35-41, and (ii) ) eliminate viruses by any of the methods defined in any of the modes 1-35, in any order. 43. The method according to the modality 42, wherein the step of inactivating the viruses precedes the step of eliminating the viruses.

EXAMPLES Example 1: serum free production of factor VII The following experiment was carried out to produce factor VII in a large pilot scale culture. A CHO Kl cell line transformed with a plasmid encoding factor VII adapted for growth in suspension culture in a medium free of animal-derived components. A bank of the adapted cells was frozen. Cells from the bank were prepared in revolving bottles in suspension culture in free medium of animal-derived components. As the number of cells increased, the volume was gradually increased by the addition of the new medium. When the volume had reached 4 L, and the number of cells had reached> 0.8 x 10s / ml, the contents of the rotating bottles were transferred to a stirred tank reactor of 50 liters (sowing reactor). As the number of cells in the 50 liter reactor increased, the volume was gradually increased by the addition of the new medium. When the volume had reached 50 liters, and the number of cells had reached "1 x 10s / ml, the contents of the 50 liter reactor were transferred to a stirred tank reactor of 500 liters (production reactor). The 500 liter reactor contained macroporous Cytopore 1 carriers (Amersham Biosciences) within which the cells became immobilized within 24 hours after inoculation. The volume in the 500 liter reactor was gradually increased by the addition of the new medium as the number of cells increased. When the volume had reached 450 liters, and the number of cells had reached "2 x 106 / ml, the production phase was started, and a medium change was made every 24 hours: the agitation was stopped to allow the sedimentation of the carriers containing cells, and 80% of the culture supernatant was then harvested and replaced with a new medium. The harvested culture supernatant was filtered to remove untreated cells and cellular debris, and was then transferred for further processing. The 50-liter bioreactor as well as the 500-liter bioreactor was instrumented for temperature control, dissolved oxygen (purging oxygen through the micropurger), stirring speed, top space aeration rate and pH (downstream controls by addition of C02 gas to the upper space). In addition, the 500 liter bioreactor was instrumented for the control of dissolved C02. The measurement of the C02 in line was made by means of a YSI 8500 C02 instrument. The C02 level was controlled by purging the atmospheric air within the liquid through a tube according to the C02 signal. The curve speed was adjusted to 0 liters / per liter of liquid in culture when the C02 concentration was at or below the set point, 0.01-0.05 liter / minute per liter of liquid culture when the C02 concentration was at above the set point. The set point for the dissolved C02 was 160 mmHg.

As mentioned, no base was added to the bioreactor to control the upstream pH. During the production phase, the cell density reached 1-2 x 107 cells / ml, and the concentration of factor VII in the daily harvest was 10-20 mg / 1. PC02 was maintained within the range of 150-170 mmHg. The pH was maintained above 6.70, even when no base was added.

Example 2: Filtration of the eluate from the capture step Solution of the protein to be filtered: 25 liters of FVII solution from the capture step, with the following characteristics. Concentration of FVIl / FVIIa: 630 mg / liters 1.7% of oxidized forms of FVII Degree of activation (for example, percentage of FVIIa): not analyzed Degradation: < 2.2% The filtration was conducted essentially as described herein with reference in Figure 1: Filter: Millipore NFR, 0.08 m2 Pressure: 2 bar Properties of the filtrate: Concentration of FVIl / FVIIa: 610 mg / liters; for example: yield of FVII: 96.8% 1.5% of oxidized forms of FVII Degree of activation (for example percentage of FVIIa): not analyzed Degradation: < 2.2% Example 3: Filtration of the eluate from the capture step. Solution of the protein to be filtered: 185 ml of FVII solution from the capture step, with the following characteristics. Concentration of FVII / FVIIa: 82 mg / liters 3.4% of oxidized forms of FVII Degree of activation (for example, percentage of FVIIa): 19% Degradation: < 3% The filtration was conducted essentially as described herein with reference in Figure 1: Filter: Pall DV 50, 0.0017 m2 Pressure: 2 bar Properties of the filtrate: Concentration of FVIl / FVIIa: 77.1 mg / liters; for example: FVII yield: 94% 4.1% of oxidized forms of FVII Degree of activation (eg percentage of FVIIa): 20% Degradation: < 3% Example 4: Filtration of the eluate from the capture step Solution of the protein to be filtered: 108 ml of the eluate from step 1 with the following characteristics: Concentration of FVIl / FVIIa: 320 mg / liters 3.7% of forms oxidized FVII Degree of activation (eg percentage of FVIIa): 3.3% Degradation: < 0.5% The filtration was conducted essentially as described herein with reference to Figure 1: Filter: Asahi Planova 20 N, 0.001 m2 Pressure: 0.8 bar Properties of the filtrate: FVII / FVIIa concentration: 310 mg / liters; for example: yield of FVII: 100% 3.7% of oxidized forms of FVII Degree of activation (eg percentage of FVIIa): not analyzed Degradation: < 0.5% Example 5: Filtration of the pharmaceutical substance in bulk FVII Solution of the protein to be filtered: 98 ml of the bulk substance FVIIa, with the following characteristics: Concentration of FVII / FVIIa: 1460 mg / liters 2.1% of oxidized forms of FVII Degree of activation (for example percentage of FVIIa): > 90% Degradation: 11.9% The filtration was conducted essentially as described herein with reference to Figure 1: Filter: Millipore NFR, 0.0017 m2 Pressure: 2 bar Properties of the filtrate: FVII / FVIIa concentration: 1320 mg / liters; for example: yield of FVII: 90.4% 2.3% of oxidized forms of FVII Degree of activation (for example, percentage of FVIIa): not analyzed, since the degree of activation in the solution to be filtered is 98% Degradation: 12.3% Example 6: 50 ml virus removal of a solution of the FVII factor polypeptide (see example 1) from the capture step comprising a murine leukemia virus, YY titer of plaque forming units (pfu). The filtration is conducted essentially as described herein with reference to Figure 1: Filter: Millipore NFR, yy cm2 Pressure: YY bar Virus title in the filtrate: xx ufp Calculation factor calculated: xx. It is noted that in relation to this date, the best known method for carrying out the aforementioned invention is that which is clear from the present description of the invention.

Claims (43)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for removing viruses from a factor VII composition, the composition comprises one or more factor VII polypeptides, at least 5% of one or more factor VII polypeptides that are in the activated form, the method characterized in that it comprises subjecting the solution to nanofiltration using a nanofilter that has a pore size of at most 80 nm.

2. The method according to claim 1, characterized in that at least 7%, for example, at least 10%, of one or more factor VII polypeptides are in the activated form.

3. The method according to claim 1, characterized in that the activated form of the factor VII polypeptide represents 5-70%, such as 7-40%, for example, 10-30% of the mass of one or more of the Factor VII polypeptides.

4. The method according to claim 1, characterized in that the activated form of the factor VII polypeptide represents 50-100%, such as 70-100%, for example, 80-100% of the mass of one or more of the Factor VII polypeptides. The method according to claim 1, characterized in that the activated form of the factor VII polypeptide represents 20-80%, such as 30-70%, for example, 30-60% of the mass of one or more of the Factor VII polypeptides. The method according to any of the preceding claims, characterized in that the liquid composition has a pH in the range of 7.0-9.

5, for example in the range of 7.

6-9.4, such as in the range of 7.7-9.3, by example, in the range of 8.0-9.0 or in the range of 8.3-8.

7. The method according to any of the preceding claims, characterized in that the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the range of 0.05-2.0. mg / ml.

The method according to any of the preceding claims, characterized in that the pore size of the nanofilter is at most 50 nm, for example at more than 30 n, such as in the range of 10-30 nm.

The method according to any of the preceding claims, characterized in that the membrane of the nanofilter is manufactured from one or more materials selected from cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), compound PDVF, surface modified PDVF, and polyethersulfone.

10. The method according to any of the preceding claims, characterized in that the liquid composition of factor VII is obtained, or originates from a cell culture supernatant.

11. The method according to any of the preceding claims, characterized in that the liquid composition is substantially free of serum.

The method according to any of claims 1-10, characterized in that the factor VII polypeptide (s) is produced by cell culture in the presence of fetal, bovine or calf sera.

The method according to any of the preceding claims, characterized in that the factor VII polypeptide (s) is produced by cell culture in CHO cells.

14. The method according to the claim 13, characterized in that the factor VII polypeptide (s) are produced by cell culture in CHO cells, in a medium free of any components of natural origin.

15. A method for removing virus from a liquid composition of factor VII, characterized in that it comprises one or more factor VII polypeptides, the liquid composition is substantially free of serum, the method comprises subjecting the solution to nanofiltration using a nanofilter having a size of serum of at most 80 nm.

16. The method according to claim 15, characterized in that the liquid composition of factor VII is obtained, or originates, from a cell culture supernatant.

17. The method according to any of claims 15-16, characterized in that the factor VII polypeptide (s) is produced by cell culture in CHO cells.

18. The method according to claim 17, characterized in that the factor VII polypeptide (s) are produced by cell culture in CHO cells, in a medium free of any components of animal origin.

The method according to any of claims 15-18, characterized in that at least 5% of one or more factor VII polypeptides are in activated form.

The method according to any of claims 15-19, characterized in that the liquid composition has a pH in the range of 7.0-9.5, for example in the range of 7.6-9.4, such as in the range of 7.7-9.3 , for example, in the range of 8.0-9.0, or in the range of 8.3-8.7.

21. The method according to any of claims 15-20, characterized in that the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the 0.05-2.0 mg / ml.

22. The method according to any of claims 15-21, characterized in that the pore size of the nanofilter is at most 50 nm, for example at more than 30 nm, such as in the range of 10-30 nm .

23. The method according to any of claims 15-22, characterized in that the membrane of the nanofilter is manufactured from one or more of the selected materials of cellulose regenerated with cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF compound, Surface modified PVDF, and polyethersulfone.

24. A method for removing virus from a liquid composition to factor VII, characterized in that it comprises one or more factor VII polypeptides, the method comprising subjecting the solution to nanofiltration using a nanofilter having a pore size of at most 80 nm, The nanofilter has a membrane made from one or more materials selected from cellulose regenerated with. cuprammonium, hydrophilic polyvinylidene fluoride (PVDF), PVDF compound, surface modified PVDF, and polyethersulfone.

25. The method of compliance with mode 24, characterized in that the material is selected from materials based on polyvinylidene chloride and polyethersulfone-based materials.

26. A method according to any of claims 24-25, characterized in that at least 5% of one or more VTI factor polypeptides are in the activated form.

27. The method according to any of claims 24-26, characterized in that the liquid composition has a pH in the range of 7.0-9.5, for example in the range of 7.6-9.4, such as in the range of 7.7-9.3 , for example, in the range of 8.0-9.0, or in the range of 8.3-8.7.

28. The method according to any of claims 24-27, characterized in that the concentration of the factor VII polypeptide (s) in the liquid composition is in the range of 0.01-5 mg / ml, such as in the range of 0.05. -2.0 mg / ml.

29. The method according to any of the embodiments 24-28, characterized in that the pore size of the nanofilter is at most 50 nm, for example at more than 30 nm, such as in the range of 10-30 nm .

30. The method according to any of claims 24-29, characterized in that the liquid composition of factor VII is obtained, or is originated, from a cell culture supernatant.

31. The method according to any of claims 24-30, characterized in that the liquid composition is substantially free of serum.

32. The method according to any of claims 24-31, characterized in that the factor VII polypeptide (s) are produced by cell culture in the presence of fetal, bovine or calf serum.

33. The method according to any of claims 24-32, characterized in that the factor VII polypeptide (s) are produced by cell culture in CHO cells.

34. The method according to claim 33, characterized in that the factor VII polypeptide (s) are produced by cell culture in CHO cells, in a medium free of any components of animal origin.

35. A method for inactivating virus from a liquid composition to factor VII, characterized in that it comprises one or more factor VII polypeptides, the method comprising the step of combining the composition with a detergent.

36. The method according to claim 35, characterized in that the detergent is an octylphenoxy-polyethoxyethanol of the formula p- ((CH3) 3CH2C (CH2 (a (-C6H4-0- (CH2CH2?) NH where n is in the range of 5-15,

37. The method according to claim 36, characterized in that the detergent is one wherein n is 9-10, such as Triton X-100.

38. The method according to claim 35, characterized because the detergent is selected from the list of Tween®, polysorbate 20, polysorbate 60, and polysorbate 80.

39. The method according to any of claims 35-38, characterized in that the detergent is combined with the liquid composition of factor VII. to obtain a concentration of the detergent in the composition in the range of 0.01-0.3% by weight, such as in the range of 0.05-0.2% by weight

40. The method according to any of the 5 claims 35-39, characterized because the detergent is combined or with the composition at a temperature in the range of 2-12 ° C, such as in the range of 2-9 ° C.

41. The method according to any of claims 35-40, characterized in that the detergent is Substantially free of trialkylphosphorylate solvents such as tris (n-butyl) phosphate.

42 A method for the high level elimination of the presence of active virus in a liquid composition of factor VII, characterized the method because it comprises the 15 steps of (i) inactivating the viruses by the method defined in any of the modes 35-41, and (ii) eliminating viruses by any of the methods defined in any of the modes 1-35, in any order.

43 The method according to claim 20, characterized in that the step of inactivating the viruses precedes the step of eliminating the viruses.