MXPA97003097A - Procedure for the production of inhibited forms of active blood factors - Google Patents

Abstract

A process for producing a highly purified preparation of an inhibited form of an activated blood factor is described, which includes the provision of a partially purified preparation containing the blood factor of interest, the treatment of the partially purified preparation to convert the factor blood to an activated form, inhibited, in a simple step, and then the purification of the activated blood factor, inhibited, result

Description

PROCEDURE FOR THE PRODUCTION OF INHIBITED FORMS OF ACTIVATED BLOOD FACTORS FIELD OF THE INVENTION This invention relates to the production of blood factors, and particularly this invention relates to the large-scale production of purified inhibited forms of activated blood factors.

BACKGROUND OF THE INVENTION After the beginning of the coagulation process, blood coagulation proceeds through the sequential activation of certain plasma proenzymes to their enzyme forms. These plasma glycoproteins, including Factor XII, Factor IX, Factor XI, Factor X, Factor VII, and prothrombin, are zymogens of serine proteases. Most of these blood coagulation enzymes are effective on a physiological scale, only when they are assembled in complexes on the membrane surfaces with protein cofactors such as Factor VIII and Factor V. Other blood factors modulate and localize the formation of the REF : 24667 clot, or dissolve blood clots. Activated protein C is a specific enzyme that inactivates the procoagulant components. Calcium ions are involved in many of the component reactions. Blood coagulation follows either the intrinsic pathway, where all the protein components are present in the blood, or the extrinsic pathway, where the tissue factor of the cell membrane protein plays a critical role. Clot formation occurs when fibrinogen is cleaved by thrombin to form fibrin. Blood clots are composed of activated platelets and fibrin. Thrombin is a multifunctional protease that regulates several key biological processes. For example, thrombin is among the most potent of the known platelet activators. In addition, as noted above, thrombin is essential for the cleavage of fibrinogen to fibrin to initiate clot formation. These two elements are involved in normal hemostasis, but in the atherosclerotic arteries can initiate the formation of a thrombus, which is a major factor in the pathogenesis of vaso-occlusive conditions such as myocardial infarction, unstable angina, non-hemorrhagic attack and reocclusion of the coronary arteries after angioplasty or trolitic therapy. Thrombin is also a potent inducer of smooth cell proliferation, and may therefore be involved in a variety of proliferative responses such as restenosis after angioplasty and graft-induced atherosclerosis. In addition, thrombin is chemotactic for leukocytes and may therefore play a role in inflammation. Hoover. R.J. et al., Cell 14: 423 (1978): Etingin, O.R. and collaborators Cell 61: 657 (1990). These observations indicate that the inhibition of thrombin formation or the inhibition of thrombin itself may be effective in the prevention or treatment of thrombosis, limiting restenosis and controlling inflammation. Thrombin formation is the result of proteolytic cleavage of its prothrombin precursor at the Arg-Thr bond at positions 271-272, and the Arg-Ile bond at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is assembled on the membrane surfaces of platelets, monocytes and endothelial cells. The complex consists of Factor Xa (a serine protease), Factor Va (a cofactor), calcium ions and the surface of acid phospholipids. Factor Xa is the activated form of its precursor, Factor X, which is secreted by the liver as a precursor of 58 kDa and is converted to the active form, Factor Xa, in the extrinsic and intrinsic blood clotting pathways . It is known that the circulating levels of Factor X, and of the precursor of the Factor Va, Factor V, are of the order of 10"7 M. There has been no determination of the levels of the active Va and Xa Factors, corresponding. The amino acid sequences and genes of most plasma proteins involved in blood hemostasis are commonly known, such as Factor lia, Factor Va, Factor Vlla, Factor IXa, Factor Xa, Factor Xla, Factor Xlla, Activated Protein C, Activated Protein S, fibrinogen and thrombin. Also commonly known are the amino acid sequences and genes of the precursor forms of these blood factors, and the common methods for their activation or conversion to mature forms. Factor X (Stuart Factor) is an essential component of the blood coagulation cascade (see Figures 1 and 2). Factor X is a member of the vitamin K-dependent family of blood coagulation glycoproteins, which contain gam-a-carboxyglutamyl ("da"), which bind calcium ions, which also includes Factors VII and IX, prothrombin, protein C and protein S. Fuñe B. et al., Cell 53: 505 (1988). Factor X is the zymogen for Factor Xa serine protease. Factor Xa is combined with a cofactor, activated Factor V, calcium, and phospholipids on a membrane surface to form the prothrombinase complex. This enzyme complex converts prothrombin to thrombin, which then converts fibrinogen to fibrin, one of the pathways that result in thrombosis (Colman, RW et al., "Overvie of Hemostasis" in Colman, RW et al. s and Thrombosi s, Basi c Principi es and Clini cal Practi ce, Second Edition (1987), Part I, Section A, Plasma Coagulation Factors, pp. 3-17). Factor X can be purified from natural, synthetic or recombinant sources, by any of a number of different extractive and chromatographic techniques, such as: a combination of ion exchange, affinity chromatography of heparin and hydroxyapatite (Kosow, DP Thromb. Res., 9 (6): 565-573 (1976): sulfated dextran (Miletich, JP et al., Biotechnology Bi ochemi stry 105: 304-310 (1980)): a combination of adsorption with barium citrate , precipitation with ammonium sulfate, ion exchange and affinity chromatography of heparin (Bajaj, SP et al Prep. Bi ochem. 11: 397-412 (1981)); fractionation by Cohn (Monohan, J.B. and collaborators Thromb, Res. 19 (6): 743-755 (1980)); non-sulfated carbohydrate matrices (U.S. Patent No. 4,721,572 and U.S. Patent No. 4,725,673): immunoaffinity chromatography (European Patent Application 0,286,323); hydrophobic interaction chromatography (Freidberg, R.C. et al, Prep Biochem.18 (3): 303-320 (1988)); metal chelate chromatography (PCT / GB88 / 01150); a combination of immunoaffinity and ion exchange (Ahmad, S. S. et al., Thromb Res. 55 (1): 121-133 (1989)); and as a by-product in the purification of other blood coagulation factors (Hrinda, ME et al., "Preclinical Studies of a Monoclonal Antibody-Purified Factor IX, Mononine®" in Seminars in Hema tology 28 (3) Suppl. 6.6-14 ( 1991) and North American Patent No. 5,071,961). Typically, the activation, inactivation and purification of Factor X are achieved separately.

Factor X must be activated to Factor Xa before the protease is incorporated into the prothrombinase complex (Steinberg M. et al, "Activation of Factor X" in Colman, RW et al., Supra, Part I, Section A, Chapter 7, pp. 112-119). Factor Xa is a two-chain molecule linked by a disulfide bond between the two chains. The heavy chain contains the serine protease, the active site similar to trypsin and the N-terminal activation peptide which is glycosylated. The heavy chain has at least three forms, α, β and β, which differ due to the cleavage of a C-terminal peptide in the heavy chain (Aronson, DL et al., Proc. Soc. Exp. Biol. Med. 137 (4): 1262-1266 (1971); Mertens, K. et al., Biochem. J. 185: 647-658 (1980). It is thought that this C-terminal peptide is glycosylated through an O-linked glycosylation. The form a is the full length form of the heavy chain, and the ß and γ forms are cut in. The light chain contains a domain similar to the growth factor, and a number of post-translationally modified amino acid residues, unique, called gamma-carboxy-glutamic acid residues ("GLA's"), which are involved in the delivery of activity through the interactions of the calcium bond, required in the prothrombinase complex (Davie, EW, "The Blood Coagulation Factors : Their cDNAs, Genes and Expression "in Colman, RW et al., Supra. te 1, Section A, pp. 242-268). Factor X can be activated to Factor Xa by any of several methods. Factor X is activated naturally through the extrinsic pathway (Factor VIIa / Tissue Factor complex) or the intrinsic pathway (Villa Factor Factor / Factor IXa-phospholipid-calcium-enzyme complex) (Mertens, K. et al., Biochem J 165: 647-658 (1980); Jesty J., J. Biol. Chem. 261 (19): 8695-8702 (1986): Steinberg, M. et al., Supra.Bauer, K. et al., Bl ood 74 (6): 2007-2015 (1989): Chattopadhyay, A. et al., J. Biol. Chem. 2: 735-739 (1989)). Factbr X can also be activated to Factor Xa by the protease such as the activation enzyme of Russell's Snake Venom Factor X ("RW-X") (Furie, BC et al., Methods in Enzymology 45: 191-205 (1976): DiScipio, RG et al, Biochemistry 16 (24): 5253-5260 (1977), trypsin (Steinberg, M. et al., Supra), or cancer procoagulant (Gordon, SG et al., Bl ood Coagulation and Fibrinolysi s 2: 735-739 (1991)).

It is known that numerous activities of snake venom affect the mechanism of intrinsic coagulation by activating, inhibiting or converting various factors in the cascade of blood coagulation. It is known that snake venoms activate protein C, prothrombin, thrombin-like enzymes, fibrinogenases, and the activities of Factors V and X (NA Marsh, Bl ood Coagulation and Fibrinolysis 5: 399-410 (1994)). Synthetic peptides and peptidomimetics are also known as substrates and inhibitors of serine proteases (Claeson, G., Bl ood Coagulation and Fibrinolysis 5: 411-436 (1994).) A number of protease inhibitors are also known. General and specific serine The various activators and inhibitors are commonly known for many of the blood factors.For example, it is known that Factor I (fibrinogen) is activated by thrombin, Factor II (prothrombin) is known to be activated by Factor Xa and thrombin, Factor V is known to be activated by papain, a Factor V activation protease from Russell's snake venom, plasmin, Factor Xa, chymotrypsin, and trope. mbocitin, and is inactivated by activated protein C; it is known that Factor VII is activated by minor proteolysis, with a signal peptidase and a processing protease; Factor IX is known to be activated by Factor Xla with calcium ions, tissue factor, Factor VII, and Russell-Factor X snake venom, and is known to be inactivated by hirudin and antithrombin III; Factor X is activated by Factors IXa and VII with phospholipid and calcium ions, and by Russell's snake venom; It is known that Factor XI is activated by Factor Xlla and trypsin; It is known that Factor XII is activated by contact with negatively charged surfaces, by sulfatides, trypsin, plasmin, and kallikrein; Protein C is activated by thrombin, etc. See Colman et al., Supra, for the text describing the known activators and inactivators of blood factors. In some circumstances, it is desirable to interfere with the functioning of Factor Xa in order to prevent excessive coagulation. In other circumstances, such as hemophilia, it is desirable to provide a source of Factor Xa independent of the activation process that occurs in normal individuals. The two common forms of hemophilia (hemophilia A and B) involve deficiencies only in the intrinsic pathway of activation, but the operation of the intrinsic pathway does not appear to be successful in stopping the hemorrhage. Similarly, other patients are currently treated for deficiencies of other blood factors (such as VII, X, XI, XIII), or von Willebrand's disease. Factor VII deficiency is not clinically as well defined as hemophilia A or hemophilia B, however, it has been reported that patients with Factor VII deficiency have severe bleeding. Deficiency of protein C is associated with thrombotic risk. Factor Xa, and various other activated blood factors, have not typically been useful as pharmaceuticals due to their extremely short serum half life, which for example is typically only about 30 seconds for Factor Xa. The use of acylation to prolong the half-life of certain blood factors has also been described. For example, Cassels, R. and collaborators, Biochem. Jour. 247: 359-400 (1987), reports that various acylation agents remained attached to urokinase, tPA and the plasminogen-streptokinase-activating complex for periods of time ranging from a half-life of 40 minutes to a longer half-life. 1000 minutes, depending on the nature of the acylating group and the nature of the factor. U.S. Patent No. 4,337,244 describes acylation of tPA or streptokinase. The use of an amidinophenyl group that functions as an arginine analog to temporarily introduce a substituted benzoyl group into the active site for purposes of improving serum stability was discussed by Fears R. et al., Seminars in Thrombosis and Homeostasis 15 : 129-39 (1980) (see also: Fears R. et al, Drugs 33 Suppl 3: 57-63 (1987), Sturzebecher J. et al., Thrombosis Res. 47: 699-703 (1987)), which describes the stabilized acyl derivatives of tPA. The use of the plasminogen activator-streptokinase activator acylated complex ("APSAC") is described in Crabbe S.J. and collaborators, Pharmacotherapy 10: 115-26 (1990). Acylated forms of thrombin have also been described. In general, the methods for the activation, inhibition and recovery of the target blood factor have been multi-step and complex processes. The chemically inactivated forms of Factor Xa can be used in a number of therapeutic indications (U.S. Patent No. 4,285,932; U.S. Patent No. 5,120,537; Benedict, C.R. and collaborators Blood 81 (8): 2059-2066 (1993); American Series No. 08 / 268,003, filed on June 26, 1994; Sinha, U. et al., "Procoagulation Activities of Reversibly Acylated Forms of Factor Xa", presented at the 35th Annual Meeting of the North American Cardiac Association, Saint Louis MO, December 3-7, 1993). Factor Xa can be irreversibly inactivated using chloromethyl ketone derivatives, such as glutamylglycyl-arginyl ("EGR") chloromethyl-ketone, or dansyl-glutamyl-glycyl-arginyl ("DEGR") chloromethyl-ketone (see example Nesheim, H: E: and collaborators Jour. Biol. Chem. 254: 10952 (1979): U.S. Patent No. 5,120,537; Kettner, C. et al., Biochem 17 (22): 4778-4783 (1978); Kettner, C. et al., Biochim, Biophys, Acta 569: 31-40 (1979), Kettner, C. et al., Arch. Biochem. Biophys., 202: 420-430 (1980), Kettner, C. et al. in Enzymology 80 Part C: 826-842 (1981); Kettner, C. et al., Thromb Res. 22: 645-652 (1981); Nesheim, ME et al. "Biol. Chem. 256 (13): 6537 -6540 (1981); Patent North American No. 4,318,904; Lijnen, H.R. and collaborators, Thromb. Res. 34: 431-437 (1984); Williams, B. et al., J. Biol. Chem. 264 (13): 7536-7545 (1989); U.S. Patent No. 5,153,175). This inactivated irreversible X Factor can be used to inhibit the generation of thrombin in vi, and thus be used as an anticoagulant (US Patent No. 5,120,537, and Benedict, C.R. et al., Supra). Factor Xa can be reversibly inactivated using various derivatives of the acylation compounds of 4-amidinophenyl benzoate (or p-amidinophenyl ester hydrochloride) which impart reversibility to various proportions. This reversibly inactivated Factor Xa can be used to promote the formation of thrombin in vi, and thus can be used in prc-coagulant indications (US Pat. No. 4,285,932, North American Serial No. 08 / 268,003, filed on June 26, 1994, and Sinha et al., Supra).

BRIEF DESCRIPTION OF THE INVENTION The invention features a process for the production of a highly purified preparation of an inhibited (ie, permanently or transiently inactivated) form of an activated blood factor, by providing a partially purified preparation containing the blood factor, the treatment of the partially purified preparation for converting the blood factor to an activated, inhibited form, in a single step (and / or in a simple reaction vessel), and then purifying the resulting, inhibited, activated blood factor. The invention provides the production of blood factors activated in a permanently or transiently inhibited manner, at a high purity and at a high yield. In certain embodiments, the methods of the invention can be used to prepare the activated Factor II, inhibited (Factor Ia inhibited), Factor Va activated, inhibited, (Factor Va inhibited), Factor VII activated, inhibited (Factor Vlla inhibited), Protein. Activated C, inhibited, Protein S activated, inhibited, Factor IX activated, inhibited (Factor IXa inhibited), Factor X activated inhibited (Factor Xa inhibited), Factor XI activated inhibited (Factor Xla inhibited), Factor XII activated, inhibited (Factor Xlla) inhibited), and activated fibrinogen, inhibited (Factor I inhibited). The inhibition treatment can immediately follow the activation treatment, with or without a step of the intervening process, or, the activation and inhibition treatments can be carried out concurrently. The partially purified preparation, which contains the blood factor, can be derived from natural, synthetic materials or from recombinant sources. In some embodiments, the inhibition treatment includes the use of a peptidyl-chloromethyl ketone derivative, which is preferably tri-peptidyl or larger, such as EGR-ck or DEGR-ck. In some embodiments, the inhibition treatment includes causing an acyl group to bind at the active site of a blood factor (an activated or zymogen form), where it inhibits clearance and is liable to retard hydrolysis to generate the active form of the blood factor, resulting in an activated blood factor, reversibly inhibited. Other features and advantages will be apparent from the specification and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing a human X Factor, indicating the regions of the molecule.

Figures 2A and 2B are diagrams showing, respectively, the a and ß forms of a human Factor Xa.

Figure 3 is a block diagram, schematic, showing the steps of the process according to an exemplary embodiment of the invention, to produce an inhibited, activated blood factor.

DESCRIPTION OF THE SPECIFIC MODALITIES Definitions of terms As used herein, the terms "blood coagulation factor" and "blood factor" mean and refer to blood factors in general, and particularly to any number of peptides, factors and cofactors, which comprise the cascade of intrinsic or extrinsic blood coagulation in humans , or are involved in the modulation, localization or dissolution of blood clots. Suitable blood factors for use in this invention include, but are not limited to, Factors II, V, VII, IX, X, XI and XII, Proteins C and S, thrombin, fibrinogen, etc., in its forms of zymogen, not activated, activated or inhibited-activated. The term "blood factor" refers to the respective native, synthetic or recombinantly produced polypeptide sequence, as is commonly known. As used herein, the term "impure initial protein fraction" refers to any protein fraction from either natural, synthetic or recombinant sources, which contains the blood factor of interest in combination with other proteins, or in combination with other materials present in the environment where the protein fraction was produced or derived. The term "partially purified preparation" means a preparation that contains a blood factor of interest, and that is substantially or completely free of inhibitors of the blood factor of interest. In certain embodiments, the partially purified preparation is substantially free of chelating agents or contains free calcium in molar excess of any chelating agents that may be present. A partially purified preparation can be at a high level of purity. "X Factor" refers to the sequence of the Single chain or two chain X factor, native, synthetic or recombinantly produced, essentially as shown in Figure 1 or Figure 2, containing at least the heavy chain to which the activation peptide is attached, in its N end, and the light chain. These may or may not be linked through a cleavage sequence as indicated in the figures. "Factor IX", "Factor VII" and "Protein C" refer to the respective protein sequence, either native or recombinantly produced, as is commonly known. The terms "chemical inhibitor" and "chemical inactivator", as used herein, mean and refer to any of a number of reactive peptide or organic molecules, which have the ability to covalently link to the site. active of the activated blood coagulation factor, and to inactivate the activated blood coagulation factor, that is, to inhibit the activity of the activated blood factor. Known reagent compounds include the tri- (or major-) -peptidyl-chloromethyl-ketone derivatives or the tri- (or major-) -peptidyl-arginyl-chloromethyl-ketones to produce irreversibly inhibited compounds, or any of a group of acylating agents that can produce transiently inhibited blood factors. A blood factor that is "activated," as that term is used herein, is one that has been catalytically formed from an inactive zymogen precursor. An activated blood factor that is "inhibited", as that term is used in the present, is one that substantially lacks the expected enzymatic activity for the blood factor, when activated. "Factor Xa" refers to the enzymatically active X Factor, native, synthetic or recombinantly produced, containing only light and heavy chain. The activation peptide is not present in this complex. "Inhibited Factor Xa" means and refers to a modified form of Factor Xa, which is activated in the sense that it combines to form the prothrombinase complex, but which has no serine protease activity by virtue of the modification of your active site. "Acylated Factor Xa" or "AcXa", unless otherwise specified, refers to Factor Xa, whether recombinantly produced or not, wherein the catalytic domain of serine has been blocked with a substituent that provides Acyl-Factor Xa with a serum half-life of at least 5-10 minutes, preferably more than 15 minutes, and which releases Factor Xa actively, over this period of time. The serum half-life can be measured directly in vivo using an appropriately labeled form. However, it is preferable to evaluate the ability of extended AcXa to generate the active Factor Xa, within the required time frame in vi tro, using as a criterion the in vi tro assays for which Factor Xa is a catalyst. Under these conditions, the appropriate forms of Acylated Factor Xa for the invention, include those having a constant rate for hydrolysis in aqueous isotonic media at pH 7.4 and 37 ° C, such that a half-life of about 5 minutes is achieved at several hours. The half-life can be determined directly in vi tro by measuring the hydrolysis rate of acylated Factor Xa, if desired, using its ability to activate coagulation, or the prothrombinase reaction as criteria for Xa formation. The blood factors described in this invention are defined herein as any isolated polypeptide sequence, which possesses a biological property of the naturally occurring blood factor polypeptide, comprising a commonly known polypeptide sequence, variants and homologs thereof, and the like of mammal or other animals. "Biological property" for purposes herein, means an effector in vi or antigenic function or activity that is directly or indirectly performed by a blood factor (either in its native or denatured conformation), or by any subsequence thereof. Effector functions include binding to the receptor, any enzymatic activity, or enzyme-modulating activity, any binding activity to carriers, any hormonal activity, any activity in the promotion or inhibition of adhesion of cells to an extracellular matrix or molecules of the cell surface, or any structural paper. However, effector functions do not include antigenic functions, for example, possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring blood factor polypeptide. Ordinarily, the blood factors claimed herein will have an amino acid sequence having at least 75% sequential amino acid identity with a commonly known sequence, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% The identity or homology with respect to a sequence of the commonly known blood factor is defined herein as the percentage of amino acid residues in the candidate sequence, which are identical with the amino acid residues of the known blood factor, after the alignment of the sequences and the introduction of empty spaces, if necessary, to achieve maximum percentage homology, and not considering any conservative substitutions as part of the sequential identity. None of the N-terminal, C-terminal or internal extensions, deletions or insertions within the blood factor sequence, will be built to affect homology.

Thus, polypeptides of permanently or transiently inactivated blood factors, and blood factors with prolonged plasma half-lives that can be elaborated according to this invention, each include the blood factor sequence; the fragments thereof having a consecutive sequence of at least 5, 10, 15, 20, 25, 30 or 40 amino acid residues from a commonly known sequence of the blood factor; variants of the amino acid sequences of a commonly known sequence of the blood factor, wherein an amino acid residue has been inserted at the N-terminus or at the C-terminus a, or within, the sequence of the blood factor or its fragment as defined above; variants of the amino acid sequence of the commonly known sequence of the blood factor or its fragment, as defined above, have been replaced by other residues. Blood factor polypeptides include those that contain predetermined mutations by, for example, site-directed mutagenesis or PCR mutagenesis, and other animal species of the blood factor polypeptides such as the blood factors of rabbit, rat, pig, non-human primates , equines, murines and sheep, and alleles or other variants of natural origin of the aforementioned sequences and human sequences, derived from the blood factor commonly known by their fragments as defined above, wherein the blood factor or its fragments have been covalently modified by substitution, chemical, enzymatic or other appropriate means, with a different portion of an amino acid of natural origin (eg, a detectable portion such as an enzyme or radioisotope): glycosylation variants of the blood factor (insertion of a site of glycosylation or suppression of any if glycosylation thio by suppression, insertion or substitution of the appropriate amino acids): and soluble forms of the blood factor.

Modes for carrying out the invention general As summarized above, this invention provides a process for the production of large-scale quantities of active, chemically inactivated (for example chemically-inhibited) blood factors from an initial, impure protein fraction. In general, the process includes one or more steps to obtain a partially purified preparation containing the blood factor of interest; the treatment step of the partially purified fraction to inactivate and inhibit the blood factor; and the steps to complete the purification of the activated, inhibited, resulting blood factor. The inhibition treatment can immediately follow the activation treatment, without the passage of the intervention process; or the activation and inhibition treatments can be carried out concurrently. Figure 3 shows a block diagram that underlines a preferred embodiment of the process of this invention for the production of reversibly or irreversibly inhibited forms of blood coagulation factors, with specific reference to Factor X inactivated with EGR-ck. With reference to the preferred embodiment exemplified in Figure 3, the initial material is a plasma fraction, preferably virally inactivated, which contains the blood factor of interest. The starting material may alternatively be a product of recombinant expression of the blood factor. The initial material can be initially processed, for example through an affinity purification chromatography column (e.g., an immunoaffinity column), to produce the partially purified preparation containing the blood factor of interest. As shown in Figure 3, a highly specific affinity purification step is used so that the resulting elution pool contains the desired blood coagulation factor at a high level of purity. The partially purified preparation can then be concentrated and / or diafiltered in an appropriate buffer, to carry out the activation and inactivation (inhibition) treatments. In Figure 3, the blood factor of interest is Factor X, which can be activated using RW-X, purified from Vípera russelli venom, to produce Factor Xa; and Factor Xa can be inactivated using a peptidyl-chloromethyl ketone or an acylating agent. Here, the preparation is treated concurrently with RW-X and EGR-ck, to produce EGR-Factor Xa. After this, a series of final purification steps are carried out to bring the activated, inhibited, blood factor of interest to a desired level of purity. Particularly, as in the example of Figure 3, the treated preparation can be subjected to an additional viral clearance step, an ion exchange step to remove various contaminants and, optionally, an additional inactivation step (here using EGR-ck) to clean substantially all remaining activated factor. The product can then be concentrated and diafiltered in a storage buffer.

Partial purification of the source material that contains the blood factor Any of a variety of techniques and combinations of techniques, known in the art, can be used to partially purify the preparation, to make it ready for the activation and inhibition treatment. Preferably, the partially purified preparation contains substantially no inhibitors of the blood factor of interest, and preferably does not contain blood coagulation factors other than the factor of interest, although other zymogen factors may be present. For example, where Factor X is the blood factor of interest, and Russell's snake venom (RW-X) is used as an activating agent, the partially purified preparation must be substantially free of Factors V and IX, since Factors V and IX are also subject to activation by RW-X. Where the blood factor is dependent on calcium, the use of chelators should be avoided, unless free calcium is present in a molar excess of the chelant. For this reason, EDTA and EGTA buffers are less preferred. Preferably (for improved yield), but not necessarily, the blood factor of interest is present in the partially purified preparation at about 50% purity, more preferably at about 80% purity, and still more preferably at a purity of approximately 90%. Preferred techniques for partial purification include, for example, column chromatographic techniques using immunoaffinity, heparin-affinity and hydroxylapatite chromatography, sulphated dextrans, ion exchange, metal-chelate chromatography, non-sulfated carbohydrate matrices, fractionation of Cohn, hydrophobic interaction chromatography ("HIC"), and precipitation with ammonium sulfate, DEAE resins are appropriate, and preferably (but not necessarily) anion exchange chromatography can be used, also preferred are any of the various Quaternary amine columns, for example the "Q" columns. In certain preferred embodiments, an immunoaffinity resin is prepared and used according to methods generally accepted in the art. Preferred resins include tresyl activated agarose, under the registered trademark Affinica® by Schlercher and Scheull, as well as other tresyl activated resins, aldehyde-activated resins, triazine-activated resins, hydrazide-activated resins, azlactone-activated resins and others Typically, using standard techniques, a line of hybridoma cells that produce a monoclonal antibody with specificity for the target blood factor is obtained. The cell line is then injected into mice in order to conveniently produce amounts of the monoclonal antibody in ascites fluid, however, recombinant production or other antibody production techniques / antibody fragments can be advantageously used. The monoclonal antibody can then be chromatographically purified using standard techniques such as protein A chromatography and ion exchange for a purity greater than 98%. The required amount of resin can be prepared according to the manufacturer's instructions and the resin and antibody can be exchanged with buffer inside the coupling damper. In a preferred embodiment, the coupling buffer contained 0.1 M sodium carbonate at pH 8.5, and the antibody solution was incubated overnight with the tresyl activated resin at 2-8 ° C, to allow efficient coupling of the antibody. The antibody can be coupled to the resin according to methods known in the art, commonly at proportions of between 1-10 mg of antibody per milliliter of resin. After the coupling step, the bound resin is washed and blocked, typically according to the manufacturer's instructions, and packed in an appropriate chromatography column (either radial or axial flow geometries) for use in the purification of the blood factor of interest. In a particularly preferred embodiment, anti-Factor X immunoaffinity chromatography is established using an immunoaffinity resin made as described above, and used to partially purify a plasma fraction containing Factor X as the blood factor of interest. A frozen Factor X containing the plasma fraction (for example, a column washing fraction of Factor IX affinity chromatography, or a DEAE eluate or calcium phosphate from a plasma fractionation process, Cohn fractionation, is obtained. etc.); the plasma fraction may have been treated with heat or with solvent-detergent to reduce the potential viral load and the pH is adjusted to neutral pH ± 0.5 units. The plasma fraction is thawed at 2 to 8 ° C, filtered on a 0.2 μm filter and applied to an anti-Factor X immunoaffinity column, equilibrated with phosphate buffered saline, at 2-8 ° C. The residence time of the load through the column is adjusted to be greater than or equal to 5 minutes. Once an appropriate load has been applied for the bonding capacity of the column (generally 0.1-1.0 mg of antigen / ml of resin), the column is washed with a volume of phosphate buffered saline, at least equal to 10 volumes of the column. After sufficient washing of the column, the purified antigen, Factor X, is eluted using 0.1 M CAPS buffer containing 25 mM sodium chloride, pH 6.5-11.3. The elution pool that is more than 90% of Factor X is immediately titrated to neutral pH ± 0.5 units with concentrated HEPES buffer (2-3 M).

The step of activation and inhibition In the activation and inhibition step, an inhibition (inactivation) treatment is carried out concurrently with an activation treatment: or an inactivation treatment follows an activation treatment with or without the intervention of processing steps. The techniques for activating and inactivating any of the various blood factors are known in the art and are discussed above by way of background.

Irreversible inactivation In general, irreversible inactivation can be achieved by any of a variety of methods discussed above, including irreversible inactivation by chloromethyl ketone derivatives or by the use of small molecules that covalently and irreversibly bind to the active site of the blood factor. In the particularly preferred embodiments, a preparation containing Factor X, purified as described above, can be treated to activate and inhibit Factor X as follows. Purified X Factor is concentrated to approximately 1 mg / ml using a Filtron ultrafiltration system with 8 kDa MWCO Omega type membranes, or other equivalent system. Concentrated X Factor can be diafiltered in 50 mM Tris buffer, 25 mM sodium chloride, pH 7.5 or can be directly activated, without buffer exchange, using the Russell Snake Venom Factor X activation enzyme (RW-X) ) at a mass: mass ratio of Factor X: RW-X of between 1000: 1 to 20: 1 at 18-37 ° C in the presence of 5 mM calcium chloride for at least 5-10 minutes. Purified RW-Factor X can be prepared through a number of previously published processes (Williams, WJ et al., Biochem. J. 84: 52-62 (1962); Kisiel, W. et al., Biochem. 15 (22 ): 4901-4906 (1976) and Takeya, H. et al., J. Biol. Chem. 267 (20): 14109-14117 (1992)) from crude RW. The reaction can be stopped after one hour with the addition of EDTA at 10 mM. Activated Factor X (Factor Xa) can be either simultaneously or sequentially reacted with a covalent inhibitor, either a tripeptide-chloromethyl ketone or acylating agents (e.g., 4-amidinophenyl benzoate variants or others as discussed further below), at a mole ratio of 20: 1 of inhibitor: Factor X for at least 30 minutes at room temperature in order to inactivate (ie, inhibit) Factor Xa.

Reversible inactivation Techniques for reversibly and irreversibly inactivating activated blood factors are described in co-pending US Patent Application Serial No. 08 / 268,003, filed on June 29, 1994, the pertinent parts of which are incorporated by reference herein. In general, reversible (i.e., transient) inactivation can be carried out by any of a variety of methods, including the binding of an antibody / antibody fragment to the active region, the binding of the portion that sterically blocks the domain proteolytic or other active domain, or the incorporation of a chemical portion which blocks the domain of the active blood factor, and is gradually released from the blood factor. In the particularly preferred embodiments of this invention, the blood factor is transiently inactivated upon acylation. Reversible inactivation can be achieved using benzamidines, which are good reversible inhibitors of trypsin-like enzymes. The amidino cationic group of the inhibitor interacts with an enzyme carboxylate located at the bottom of the SI subsite. A wide variety of substituted benzamidines, such as thrombin and plasmin inhibitors, have been investigated and are suitable for the practice of this invention (see, for example, Andrews, JM et al., Jour. Med. Chem. 21: 1202- 07 (1978)). Extensive studies have been reported on compounds containing two benzamidine portions, which are also desirable for the practice of this invention (see, for example, Tidwell, R.R. and collaborators, Thrombosi s Research 19: 339-49 (1980)). 1,2-bis (5-amidino-2-benzofuranyl) -ethane is also useful for transient inhibition, and is known to inhibit Factor Xa with a Ki of 570 Nm. Also suitable for transient inactivation in the activation / inhibition step according to the invention, are Kunitz inhibitors (a class of protease inhibitors widely studied). The inhibitor of bovine pancreatic trypsin (aprotinin) and the inhibitor of the tissue factor pathway (also known as LAC1) belong to this class. The dissociation constants (T) can be in the range of 17 weeks to 11 seconds (Gebhard, W. et al., Proteinase Inhibitors, (1986) Elsevier). Aprotinin competitively inhibits Factor Vlla with a Ki of 30 μM (Chabbat, J. et al, Thrombosi s Research, 71: 205-15 (1993)). The treatment for inhibiting the activated blood factors by acylation according to the invention proceeds by the standard acylation reaction of the corresponding blood factor, either recombinantly produced or isolated from the plasma, according to procedures analogous to those described, by example, or referred to in Cassels, R. and collaborators, Biochem. Jour. 247: 395-400 (1987), or US Patent No. 4,337,244. In certain embodiments in the activation / inhibition step according to the invention, the partially purified preparation containing the blood factor is treated with a three to thirty-fold molar excess of an acylating agent in a buffer of neutral pH at temperature ambient. The catalytic activity is followed over a time course of about one to sixty, and preferably ten to thirty minutes to ensure the desired level of inactivation of the protein. The reagent is preferably prepared as a 0.1 M solution in DMSO or water, and added to the protein at pH 7.5. The blocked protein is subjected to chromatography (preferably on a gel filtration column or ion exchange) at pH 5.0 to remove excess reagent. The protein can be stored at pH 5.0 from -70 ° C to -80 ° C before further use. Active site acyl groups, suitable for use in this invention, include benzoyl, p- or o-methyl (toluoyl), p- or o-methoxy (p is a more preferred anisoyl), p- or o-fluoro -benzoyl-dimethyl-acryloyl (3,3 or 3,4). Compounds difluoro, CH3CO-benzene (acetyl group), CH3CONH-benzene (acetanilide), p- or o-ethoxy (or other alkyl groups), and guanidino-benzoyl. Suitable esters for use in this invention include the 4-toluoyl ester, the 3, 3-dimethylacrityl ester, the cyclohexylidenacetyl ester, the cyclohex-1-carbonyl ester, the 1-methylcyclohexylidenacetyl ester, the 4-methyl ester, -aminobenzoyl, the p-amidinophenyl ester of p-anisic acid, the p-amidinophenyl ester of o-anisic acid, the p-amidinophenyl ester of 3,4-di ethylbenzoic acid, the p-amidinophenyl ester of benzoic acid, the p-amidinophenyl ester of 3,3-dimethylacrylic acid, and PDAEB (4-N- (2-N '- (3- (2-pyridyldithio) -propenyl) amino-ethyl) -amino-benzoyl ester. Generally, the acylating agent will be the activated form of a non-toxic acid which provides a saturated, unsaturated or aromatic ring of 5 or 6 carbon atoms, to which a carboxyl is substituted.The ring may contain additional substitutions, such as amino , alkoxy, alkyl, addition ring systems, or any other substitute non-toxic hearer, that does not interfere. For Factor X and other blood factors that have a catalytically active serine domain, any compound capable of acylating the hydroxyl group of serine or otherwise blocking the catalytic domain of serine in a reversible manner is appropriate for the synthesis of an acylated blood factor. As described in U.S. Patent No. 4,337,244, in general, acylation agents can be used either direct or reverse. For direct acylating agents, the acylation portion is itself attracted to the catalytic site of Factor Xa or another blood factor, in the reverse acylation process, the leaving group is thus attracted. The acylated form of the blood factor is then purified from the reaction mixture using standard purification techniques, including dialysis, chromatography, selective extraction, and the like. Potent acylating agents such as the 3-alkoxy-4-chloroisocoumarines have been reported for a variety of serine proteases (Harper, JW et al., Jour Am. Chem. Soc. 106: 7618-19 (1984): Harper, J: W. and collaborators Biochemistry 24: 7200-13 (1985)), and are suitable for use in the activation / inhibition step according to the invention. The stability of the acylated enzymes is dependent on the alkoxy groups, small groups that give transiently stable acylated enzymes (T less than 2 hours). The compounds produced according to the processes of this invention, which serve as diagnostic and / or pharmaceutical products of the acylated blood factor, must have an appropriate rate of deacylation which ensures an appropriate clearance time, in vi ve. Acylated proteins react in a time, temperature and pH dependent manner. Typically, deacylation is faster at 37 ° C than at room temperature, and is faster at pH 8.0 than at pH 7.5. The speed of deacylation can be measured by having a half-life of at least 5 minutes in the buffer, using the proto-binase and / or coagulation tests. Deacylation can be measured directly as described in R.A.G. Smith et al., Progress in Fibrinolysi, Vol. VII, pp. 227-31 (1985. Churchill Livingstone). Prothrombinase and coagulation assays are described in D.L. Wolf et al., Jour. Biol. Chem. 266: 13726 (1991). In certain preferred embodiments, the deacylation of acylated Factor Xa is carried out by incubation in an appropriate pH solution and testing aliquots in an amidolytic or coagulation assay. Relative activity is calculated as a percentage of the equivalent amount of active Factor Xa, carried through the same incubations. The preferred assay for the acylated Vlla Factor involves multiple steps. The acylated enzyme is incubated in an appropriate buffer at a protein concentration of 160 nM. At each time point, an aliquot is diluted to 0.16 nM and incubated with lipidated tissue factor (0.25 nM) for 1 minute at room temperature. The Factor VIIa / Tissue Factor mixture is then used for the activation of Factor X and results in Factor Xa tested in an amidolytic assay.

Purification after the activation and inhibition step After the treatment step of the partially purified preparation, to activate and inhibit the blood factor, any of a variety of subsequent purification techniques and combinations of techniques, known in the art, such as those discussed above, can be used to carry the activated blood factor, inhibited, to a degree of acceptable, final purity. In a particularly preferred embodiment, the product resulting from the activation and inhibition step described above, namely inhibited Factor Xa, is ultrafiltered at room temperature using a Millipore Viresolve® unit, or equivalent process to further remove potential contaminants (e.g. , IgG, RW-X, X Factor, etc.). For a standard Millipore Viresolve® membrane unit of 92.90 dm2 (one square foot), for example, typically the cross flow rate is maintained at 1.0-1.7 liter / minute, while the permeation rate is controlled at between 5 and 60 milliliters / minute. A Virésolve® unit of 92.90 dm2 (one square foot) has sufficient membrane capacity to filter at least one gram of inhibited Factor Xa, at a concentration of approximately 0.7 ± 0.3 mg / ml, however other parameters are appropriate for the practice of this invention. In order to achieve enhanced recovery of the product, after the filtration is complete, the system should be rinsed more than two, and preferably at least five times with at least 75 ml each time, using the appropriate buffer used in the reaction , pH 6.0-7.5 (although it must be recognized that other buffers, volumes and time may be desired for a particular application). The resulting permeate can then be directly charged at room temperature or another convenient temperature onto an anion exchange chromatography column or other type of chromatographies. In this particularly preferred embodiment, the DEAE Fractogel resin with a loading capacity of at least 8 mg of product per ml of resin is used. When working with permanently inactivated compounds, the column is pre-equilibrated with phosphate buffer at pH 6.5 (or pH 5.0-5.5 for reversibly inactivated compounds) containing less than 0.2 M sodium chloride. The sample mass, appropriate for the load capacity, of the column, is applied and once the application is completed, the column is washed with at least 5 volumes of phosphate buffer column at pH 6.5 (or pH 5.0 -5.5) that does not contain sodium chloride. A step change is made to wash the column with at least 5 volumes of phosphate buffer column at pH 6.5 (or pH 5.0-5.5), containing 0.2 M sodium chloride. The product is then eluted with a step change phosphate buffer pH 6.5 (or pH 5.0-5.5) containing 0.3 M sodium chloride. If desired, the elution pool from the Fractogel DEAE column (or other purification step) can be incubated with the inhibitory agent a second time, in order to reduce the level of residual Factor Xa (or other blood factor) that can be copurified with the inhibited Factor Xa (or other blood factor). The elution combination from the Fractogel DEAE column (or the second inactivation step) can then be concentrated and diafiltered if desired in the final storage buffer, of choice, using for example a Filtron ultrafiltration system with MWCO type Omega membranes. of 8 kDa.

Therapeutic Uses and other uses of Blood Factors When used in vivo for therapy, the blood factors of the present invention are administered to the patient in therapeutically effective amounts (e.g., amounts having desired therapeutic effect). These will normally be administered parenterally. The dosage and dosage regimen will depend on the degree of coagulation disorder, of the characteristics of the activated or inhibited particular blood factor, used, for example, of its therapeutic index, of the patient and of the patient's clinical history. Advantageously, the blood factor is administered in a form of intensive care, or continuously over a period of 1 to 2 weeks, or over a period of years intravenously to treat disorders in vascular function. Optionally, administration is made during the course of adjunctive therapy such as angiography, angioplasty, thrombolysis, stenting, surgery or cardiac / valve / artery / vein transplantation, combined cycles of therapies with or anticoagulants, including inhibitors of platelet aggregation, or as part of the therapeutic administration of another cardiovascular modulator agent. For parenteral administration the blood factors will be formulated in a unit dose injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can be used. Liposomes can be used as carriers. The vehicle may contain minor amounts of additives such as substances that increase isotonicity and chemical stability, for example, buffers and preservatives. Blood factors will typically be formulated in such vehicles at concentrations of about 0.1 mg / ml to 100 mg / ml. The blood factor compositions used in therapy are formulated, and the doses established in a manner consistent with good medical practice, taking into account the disorder to be treated, the condition of the individual patient, the site of distribution of the composition, the method of administration and other factors known to practitioners. The blood factor compositions are prepared for administration according to the description of blood factor preparation, described below.

EXAMPLES The following examples illustrate the invention by specific reference to the production of inhibited Factor X. The examples are intended to be illustrative only, and not to limit the scope of the invention.

Example 1: Preparation of immunoaffinity re? In an example of the practice of this invention for the preparation of an anti-Factor X immunoaffinity column, the anti-Factor X monoclonal antibody, derived from crude mouse ascites, was purified by means of protein chromatography, Fast Flow in S-sepharose and Fast Flow in DEAE, in sequential steps and then exchanged with buffer in 0.1 M sodium carbonate buffer, pH 8.5, in the preparation for ligating the tresyl activated agarose (Schleicher and Schuell) . 5 liters of the antibody solution at 2 mg / ml were ligated for not less than 12 hours at 4 ° C, to an equal volume of the prepared resin activated with tresyl. The mixture was continuously stirred in order to maintain optimal contact between the resin and the antibody solution. After coupling, the supernatant was recovered from the resin suspension and assayed by an absorbance measurement for protein concentration. The resin was then incubated for at least 12 hours at 4 ° C, with 0.1 M Tris buffer, pH 8.5, in order to block the remaining unreacted tresyl sites on the activated resin. After blocking, the supernatant was collected again and assayed by an absorbance measurement. The resin was then washed with 10 volumes of 20 mM potassium phosphate buffer at pH 7.0, which contained 1.0 M sodium chloride. The high salt wash was also collected and tested for absorbance. The binding efficiency of the antibody from the process was determined to be greater than 98% when calculated by taking the difference between the known amount of the antibody in the initial mixture and the mass of the antibody recovered in the sum of the supernatant samples, and then dividing between the initial mass of the antibody. The dynamic binding capacity of the antigen was determined by packing a one milliliter column of the coupled resin, and applying Factor X to the column, after it had equilibrated with phosphate buffered saline. Factor X was then eluted with 0.1 M CAPS buffer, pH 10.5, and neutralized with 2 M HEPES at pH 7.5, for the measurement of absorbance. The binding capacity of Factor X, dynamic, was determined as at least 0.1 ± 0.2 mg of Factor X per milliliter of resin.

Example 2: Partial purification of Factor X from a source of natural plasma.

In one example of the practice of this invention for the purification of X Factor from a natural plasma source, using an anti-Factor X immunoaffinity column, a 50 ml sample from the washing fraction of a purification step by Factor IX affinity that contains approximately 2.2 mg of total protein per ml (based on the total dye binding protein assay) of which 50% of the total protein was Factor X, was applied directly to a radial flow column of 100 ml containing anti-X Factor bound to tresyl activated agarose, pre-equilibrated with phosphate-buffered saline. The sample charge was applied at a flow rate of 20 ml per minute, to provide a residence time of at least 5 minutes. After the flow to the peak returned to the baseline by washing with phosphate buffered saline, the column was then washed with phosphate buffered saline containing 0.5 M sodium chloride for at least 3 column volumes. Factor X was then eluted with 0.1 M CAPS buffer containing 0.025 M sodium chloride at pH 10.5 in approximately 2.5 column volumes. The elution pool was assayed by absorbance measurement, total protein assay and SDS-PAGE. The analyzes showed that the elution pool is predominantly a single band, with no detectable major contaminant bands, and that less than 15% of the loaded X Factor flowed through the column when the column was loaded to at least 67% capacity .

Example 3: Activation and inhibition of Factor X and anion exchange chromatography of the reaction mixture.

In an example of practice of this invention for the conversion of Factor X to the inhibited form of Factor Xa and the subsequent purification, 11 mg of Factor X purified by immunoaffinity were reacted at room temperature for one hour with EGR-ck (have worked similarly Peptisyntha, Belgium, EGR-ck obtained from Calbrochem San Diego, CA: from Bachem, Torrance, CA: or from Bachem AG, Switzerland), and RW-X (they have operated similarly Haemtech, Burlington, VT; RW -X obtained from ERL., Indianapolis Indiana) at a molar ratio of 20: 1 EGR-ck: X Factor and a mass ratio of 1: 250 RW-X: X Factor, respectively, in the presence of 5 mM calcium chloride . The RW-X reaction was stopped by the addition of concentrated EDTA at 10 mM. The reaction mixture was analyzed by Size Exclusion Liquid Chromatography (HPLC) and a specific chromogenic assay for Factor Xa. The results showed that the reaction that converts Factor X to Factor Xa had proceeded to more than 80% of the conversion, and that the residual Factor Xa was less than 400 nanograms per milliliter of the solution. The mixture of the solution was then directly applied to a 1.2 ml DEAE column (Fractogel 650 ME Merck, Darmstadt, Federal Republic of Germany), other anion exchange resins, for example, a Fast Flow and Fast Flow DEAE obtained from Pharmacia, Uppsala, Sweden, Poros O, PEI Poros, Poros IIQ obtained from PerSeptive Biosystems, Boston MA, etc., which have worked in a similar manner) pre-equilibrated with 20 mM sodium phosphate buffer, pH 6.5 containing sodium chloride 0.2 M. The cross-flow was washed to the baseline with 20 mM sodium phosphate buffer, pH 6.5, and then the column was washed with at least 5 column volumes each time with 20 mM sodium phosphate buffer, pH 6.5, containing 0.15 M, 0.2 M and 0.25 M sodium chloride. The inhibited Factor Xa was eluted with one step for 0.3 M sodium chloride in 20 mM sodium phosphate buffer, pH 6.5, in a total of 8 volumes of column. The column was then washed with a high saline solution, 1.0 M sodium chloride and then treated with 0.5 N sodium hydroxide. The total protein and SDS-PAGE assays were performed on all eluted fractions together with HPLC of Size exclusion, reverse phase HPLC and ELISA 's of contaminant, on the combined elution. The HPLC results indicated that the elution pool had a purity greater than 89% by both HPLC methods. The total recovery of protein from the step resulted in a mass balance of 97%, indicating good recovery of all the protein loaded on the column. Pollutant assays indicated that the passage of DEAE was able to clear contaminants such as anti-Factor X and RW-X IgG at levels of at least 500 times. In addition, the SDS-PAGE gels indicated that many contaminant bands had been removed from the loading sample during side-to-side flow and washing steps prior to elution. This result indicates that the binding capacity of DEAE for Factor Xa inhibited was at least 6 milligrams of Factor Xa inhibited per milliliter of resin.

Example 4: Ultrafiltration of the reaction mixture using Millipore Viresolve *.

In one example of the practice of this invention for the ultrafiltration of the reaction mixture, a small area module NMWCO of 70 kDa (Millipore, Viresolve® 70, containing 9.3 cm2 (0.01 square foot) of membrane area) was used) . Factor X (14.5 ml at 0.5 milligrams per milliliter or 29 mg) was reacted with RW-X and EGR-ck as described in Example 3 above. The reaction mixture was then recirculated over the small area module at a transverse flow rate of 12 ml per minute with a peristaltic pump for 30 minutes to balance the system. Several permeated at different flow rates, in the range of 0.15 to 1.0 ml per minute, were collected and tested by absorbance measurement at 280 nm. All permeate flow rates showed more than 90% passage of the protein when a representative permeate sample was collected. Thus, for large-scale purposes, a volume reduction experiment was conducted to examine the recovery of total protein after passage of a fixed amount of protein. A total protein recovery greater than 80% was achieved without incorporated washing steps. Additional washing steps can be included if desired, to increase recovery. This experiment showed that for every 92.9 dm2 (1 square foot) of membrane area, at least one gram of total protein (at a concentration of 0.5 mg per ml) was processed from the reaction mixture, with more than 80% of recovery in a time between half an hour and an hour.

Example 5: Large scale production of EGR-Xa actor, highly purified: Preparation of immunoaffinity resin.

Examples 5 to 10 illustrate a process according to this invention for the large-scale production of highly purified EGR-Factor Xa. Although each of these examples remains independently, it can also be understood that these are processes that were orchestrated in sequence, with the product from each of Examples 5 to 9, which is used for the steps described in the following example. To prepare an immunoaffinity resin specific for Factor X, ten grams of an anti-Factor X murine monoclonal antibody derived from highly purified ascites was coupled to 2.2 liters of low substitution activated monoaldehyde resin, Actigel® ALD (Sterogene, ARcadia, CA). The coupling reaction was carried out on a 180 mm Moduline® column (Amicon, Beverly, MA), in 0.1 M sodium phosphate buffer, 0.1 M sodium cyanoborohydride, pH 7.0 for 20 ± 4 hours at 2-8 ° C. The mixture was maintained as a homogeneous suspension through the use of continuous agitation with a top mixer. After coupling, the resin was allowed to stand and the effluent from the column was collected and tested for the presence of antibody. Less than 5% of the original antibody was detected in the supernatant by an absorbance measurement at 280 nm. The resin was then washed with 20 liters of 0.1 M sodium phosphate buffer, 0.5 M sodium chloride sequentially, with the product from each of Examples 5 to 9 used for the steps described in the following example. To prepare a specific immunoaffinity resin for Factor X, ten grams of an anti-Factor X monoclonal antibody, murine, highly purified ascites derivative, was ligated to 2.2 liters of low substituted monoaldehyde activated resin, Actigel® ALD ( Sterogene, Arcadia, CA). The coupling reaction was carried out on a Moduline * 180 mm column (Amicon, Beverly, MA), in 0.1 M sodium phosphate buffer, 0.1 M sodium cyanoborohydride, pH 7.0 for 20 ± 4 hours at 2-8 ° C. The mixture was maintained as a homogeneous suspension through the use of continuous agitation with a top mixer. After coupling, the resin was allowed to settle and the effluent from the column was collected and tested for the presence of antibody. Less than 5% of the original antibody was detected in the supernatant by an absorbance measurement at 280 nm. The resin was then washed with 20 liters of 0.1 M sodium phosphate buffer, 0.5 M sodium chloride, pH 7.0, to remove the non-specifically bound protein. Unbound, unbound monoaldehyde binding sites were blocked by recirculating 0.1M ethanolamine, 0.1M sodium cyanoborohydride, pH 7.0 through the resin for approximately 6 ± 2 hours at 2-8 ° C. The resin was then intensively washed and equilibrated (greater than 40 liters) with 20 mM Tris buffer, 150 mM sodium chloride, pH 7.5, to remove all traces of sodium cyanoborohydride. After final washing and equilibration, the effluent from the column showed no detectable levels of sodium cyanoborohydride. The binding capacity of the resin for Factor X was determined to be at least 100 μg / ml.

Example 6: Large scale production of highly purified EGR-Factor Xa: purification by immunoaffinity chromatography.

A partially purified plasma fraction, treated with solvent-detergent, containing Factor X, was obtained from an authorized manufacturer of plasma products (Alpha Therapeutic Corporation City of Industry, California). This material was supplied preconcentrated and frozen in a sodium citrate / sodium chloride buffer system, pH 6.8. The total protein concentration was measured at between 1.4 and 1.5 mg / ml, using a total Bradford dye binding protein assay (BioRad, Hercules, California). The concentration of Factor X was measured at approximately 1.0 ± 0.1 mg / ml based on the results of a reverse phase HPLC assay (RP-HPLC) and a deficient coagulation assay on Factor X (1 unit = 10 μg of X Factor). 1.3 liters of the plasma fraction containing Factor X was thawed for 16-24 hours at 2-8 ° C and then filtered through a 0.2 μm filter (Millipak® 20, Millipore, Bedford, MA) to remove any particulate material. Based on the bonding capacity of the resin, four cycles of the immunoaffinity column were required to process the 1.3 liters of the plasma fraction containing Factor X. In this way, the plasma fraction containing the X Factor, filtered, it was divided into four almost equal volumes (330 ml ± 20 ml) for application to the anti-Factor X immunoaffinity column (prepared as described above). For each cycle, the immunoaffinity column (at 2-8 ° C) was equilibrated with 3-5 column volumes of 20 mM Tris buffer, 150 mM sodium chloride, pH 7.5. After equilibration, the plasma fraction containing the filtered Factor X was then charged to a residence time of 5 minutes per column volume and the flow to the peak was washed to the baseline with at least 7 additional column volumes of buffer 20 mM Tris, 150 mM sodium chloride, pH 7.5. Factor X was eluted with a pH step using 0.1 M CAPS buffer, 25 mM sodium chloride, pH 10.5. Each pool of the immunoaffinity elution (in approximately 2-4 volumes of columan) was immediately titrated to pH 7.5 ± 0.2 with 2.0 M HEPES added at 110 ± 10 mM. Absorbance and RP-HPLC measurements confirmed that a total of 1.03 ± 0.03 grams of highly purified X Factor, recovered from four cycles (236.5 mg, 249.4 mg, 291 mg and 250.5 mg for cycles one through four) was recovered. respectively). Thus, the cumulative yield of Factor X in the combined elution for the immunoaffinity step was 78.0 ± 5.0%, and the total recovery of Factor X, including Factor X, which flowed through the column and was not captured. , was 88.0 ± 5.0%. The four elution pools were then stored at 2-8 ° C for one or two days before further processing.

Example 7: Large scale production of highly purified EGR-Factor Xa: Filtration and concentration of the combination by immunoaffinity elution.

Because the pools were stored at 2-8 ° C, and to add another viral reduction step, the neutral pH immunoaffinity elution pools prepared as described in Example 6 were filtered through a 0.04 filter. μm (Sealkleen®, 464.5 cm2 (0.5 square feet), Pall Corporation, East Hills, NY) at 2-8 ° C. The filtered immunoaffinity elution pools were then concentrated at 2-8 ° C to 1.0 ± 0.1 mg / ml, using a Minisette® ultrafiltration system (Filtron, Northborough, MA), maintaining four Omega-type ultrafiltration cassettes of 8 kDa, of 696.8 cm2 (0.75 square feet). Transmembrane pressure was maintained at 1.05 ± 0.07 kg / cm2 (15 ± 1 psig), while the transverse flow velocity and filtration rates were 2.56 ± 0.2 liters / minute and 0.28 ± 0.02 liters / minute throughout of the course of the concentration step. The final concentration of Factor X was verified by a measurement of absorbance at 280 nm. No protein was detected in any of the filtered, tested samples, and the recovery of Factor X for these two steps was thus greater than 99.0%.

Example 8: Large scale production of highly purified EGR-Factor Xa: Activation / inactivation of Factor X.

Concentrated X Factor, prepared as described in Example 7, was activated simultaneously with Russell's Snake Venom Factor X activation enzyme (RW-X, Haematologic Technologies, Inc. Essex Junction, VT) by the addition of RW-X at a mass ratio of 250: 1 (Factor X: RW-X), and inactivated with Glu-Gly-Arg-chloromethyl-ketone (EGR-ck, Peptisyntha, Brussels, Belgium), added at a molar ratio of 20: 1 (EGR-ck: Factor X). The activation reaction was initiated by the addition of 1.0 M calcium chloride to a final concentration of 5 mM, and was carried out at room temperature of 21 ± 3 ° C. The activation reaction was stopped after one hour by the addition of 0.5 M EDTA, pH 8.0 to a final concentration of 10 mM. The percentage conversion by mass of Factor X was greater than 95.0%, as determined by Size Exclusion HPLC (SE-HPLC). The final concentrations of Factor X and EGR-Xa after the reaction by RP-HPLC were 0.07 mg / ml and 0.9 mg / ml, respectively. The total mass of EGR-Xa recovered after the reaction step was approximately 882.0 ± 45.0 mg. The post-reaction mixture was kept at 2-8 ° C overnight for additional processing the next day.

Example 9: Large scale production of highly purified EGR-Factor Xa: Viral reduction step.

In order to remove impurities and contaminants (eg, virus, RW-X, and IgG), the reaction mixture prepared as described in Example 8 was ultrafiltered through a cut ultrafiltration module, of nominal molecular weight of 70 kDa, of 92.9 dm2 (1 square foot) (Viresolve®, Millipore, Bedford, MA). Prior to ultrafiltration, the post-reaction mixture was filtered through a 0.45 μm microfilter (Corning, Corning, NY). The filtered post-reaction mixture was then ultrafiltered at an initial cross-flow rate of 0.76 ± 0.1 liters / minute and eventually increased to a final cross-flow rate of approximately 1.2 ± 0.1 liters / minute. The flow rate of the permeate was initially controlled at 16.0 ± 1.0 ml / minute and then decreased to 8.0 ± 1.0 ml / minute in an attempt to increase recovery. The permeate was continuously tested for the passage of the protein using absorbance measurements and showed a relatively constant 50% step over the course of the two hours of filtration. After the total retained volume reached approximately a retention volume or 50 ± 20 ml, the retentate was washed with 50 ± 20 ml using 0.1 M CAPS, 25 mM sodium chloride, 110 mM HEPES, pH 7.5, the wash was repeated six times more for a total of seven washes using a total of 425 ml of wash buffer. The recovery of EGR-Xa from this step was greater than 90% and the total balance of protein mass was 100.0 ± 5.0%.

Example 10: Large scale production of highly purified EGR-Factor Xa: anion exchange chromatography.

In a final purification step to minimize contaminants and impurities, the ultrafiltered reaction mixture of Example 9 was then directly loaded onto a 5 x 20 cm XK chromatography column, (Pharmacia, Piscataway, NJ), containing 125 ± 10 Mi from anion exchange resin at 650 M DEAE Fractogel® (EM Science, Gibbstown, NJ). The column was pre-equilibrated with 5-10 column volumes of 20 mM potassium phosphate buffer, 0.2 M sodium chloride, 10 mM EDTA, pH 6.5. After completion of the sample load, the column was first washed with 20 mM potassium phosphate buffer, 10 mM EDTA, pH 6.5, until the peak of side-to-side flow had returned to between 10% of the line base. The column was then washed with 20 mM potassium phosphate buffer, 0.2 M sodium chloride, 10 mM EDTA, pH 6.5 for 5-10 column volumes. The EGR-Xa was then eluted using 20 mM potassium phosphate buffer, 0.3 M sodium chloride, 10 mM EDTA, pH 6.5. The elution peak was collected in 8 ± 1 column volumes. The elution pool was tested for total protein concentration (Bradford and absorbance measurements), purity (RP-HPLC), residual X and Xa (RP-HPLC and chromogenic, respectively), and residual RW-X and anti IgG -Factor X (ELISA). Approximately 750 ± 50 mg of total protein were recovered, containing more than 95% of EGR-Xa (forms a and ß in a ratio of 30 to 1), less than 4% of Factor X and less than 150 parts per million of Factor Xa residual, less than 10 parts per million RW-X and undetectable levels of anti-Factor X IgG. Table 1 summarizes the individual step yields of Factor X / EGR-Xa and the total yield of the process for the production of EGR -Xa from a plasma fraction containing X Factor, partially purified, as described in Examples 10-15. The resulting EGR-Xa product showed more than 100% clot inhibition activity in an in vitro coagulation assay (aPTT) when compared to a commercially available EGR-Xa standard (Haematologic Technologies, Inc. Essex Junction VT) .

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (16)

1. A method for the preparation of an inhibited form of an activated blood factor, characterized in the process because it comprises the steps of the provision of a partially purified preparation containing the blood factor, the treatment of the partially purified preparation to convert the blood factor to a activated blood factor, and to convert the activated blood factor to an inhibited form in a simple step, and then the recovery of the activated, inhibited, resulting blood factor.

2. The process or process according to claim 1, characterized in that the treatment step of the partially purified preparation comprises the reaction of the preparation with a blood factor activating agent, and the reaction of the preparation with a blood factor inhibiting agent. , activated.

3. The process according to claim 2, characterized in that the reaction with the blood factor activating agent and the reaction of the preparation with an activated blood factor inhibiting agent are carried out concurrently.

4. The process according to claim 2, characterized in that the reaction of the preparation with an activated blood factor inhibiting agent is carried out before the reaction is carried out with a blood factor activating agent.

5. The process according to claim 2, characterized in that the reaction of the preparation with an activated blood factor inhibiting agent is carried out after the reaction is carried out with a blood factor activating agent.

6. The process according to claim 1, characterized in that the blood factor is one selected from the group consisting of Factors II, V, VII, IX, X, XI, XII, Protein C, Protein S, and fibrinogen.

7. A process for the production of a highly purified preparation of an inhibited form of an activated blood factor, characterized in the process because it comprises "the steps of the provision of a partially purified preparation containing the blood factor, the treatment of the partially purified preparation for converting the blood factor to an activated blood factor, and to convert the activated blood factor to an inhibited form in a simple reaction vessel, and then recovering the activated, inhibited, resulting blood factor.

8. The process according to claim 7, characterized in that the conversion to an activated blood factor and said conversion to an inhibited form are carried out without intervening process steps.

9. The process according to claim 7, characterized in that the inhibited activated blood factor is recovered to a level of purity appropriate for pharmaceutical administration.

10. The process according to claim 7, characterized in that the inhibited activated blood factor is recovered using immunoaffinity chromatography using an antigen-specific monoclonal antibody, coupled to an activated resin selected from the group consisting of: agarose, cross-linked agarose, dextran , crosslinked polysaccharide, polymethyl methacrylate, and resin based on synthetic polymer.

11. The process according to claim 7, characterized in that the inhibited activated blood factor is recovered using immunoaffinity chromatography using an antigen-specific monoclonal antibody, coupled to an activated resin, and wherein the activated resin utilizes a selected activation chemistry. from the group consisting of: tresyl, azlactone, aldehyde, hydrazide, N-hydroxy-succinimide and triazine.

12. The process according to claim 7, characterized in that the inhibited activated blood factor is recovered using an anion exchange column having an anion exchange group bound to a naturally derived polysaccharide or to a synthetically derived polymer matrix.

13. The process according to claim 7, characterized in that the partially purified blood factor is treated with an activation enzyme in solution.

14. The process according to claim 7, characterized in that the partially purified blood factor is treated with an immobilized activation enzyme.

15. A process for the production of a highly purified preparation of an inhibited form of activated Factor X, characterized the process because it comprises the steps of providing a partially purified preparation containing an X Factor, treating the partially purified preparation concurrently with a activator of Factor X, and with an activated Factor X inhibitor, and then the recovery of activated Factor X, inhibited, resulting.

16. The process according to claim 15, characterized in that the activator of Factor X is selected from the group consisting of RW-X, trypsin, Factor Vlla and Factor IXa.