AU2014250714B2 - Stabilised compositions of factor VII polypeptides - Google Patents

Abstract

The invention relates to chemically as well as physically stable kits and compositions comprising polypeptides, in particular Factor VII or Factor VII-related polypeptides, such that these compositions can be stored, handled and used at room temperature.

Description

Stabilised Compositions of Factor VII polypeptides

The present application is a divisional application of Australian Patent Application No. 2011203354, which is incorporated in its entirety herein by reference.

FIELD OF INVENTION

The present invention relates to kits comprising chemically as well as physically stable compositions comprising Factor VII or a Factor VIl-related polypeptide such that these compositions can be stored, handled and used at room temperature.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Medicaments containing polypeptides are complex compositions. When developing such a medicament several parameters need to be considered. By example, the medicament needs to be effective, safe and lead to good patient compliance. Moreover, the medicament may be formulated for parenteral administration using pharmaceutically acceptable excipients, which will have to meet with the approval of various world-wide medical regulatory agencies. For the purpose of parenteral administration, it is highly desirable that the formulation is approximately isotonic and that the pH of the formulation is in a physiologically suitable range upon injection/infusion, otherwise it may result in pain and discomfort for the patient. For a general review of polypeptide formulations, see, for example, Cleland et al.: The development of stable protein formulations: A closer look at protein aggregation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10(4): 307-377; and Wang etal., Parenteral formulations of polypeptides and peptides: Stability and stabilizers, Journal of Parenteral Science and Technology 1988 (Supplement), 42 (2S).

However, for medicaments comprising polypeptides the safety may directly be related to the physical and chemical stability of the polypeptide. Polypeptides are susceptible to physical degradation, including denaturation and aggregation such as the formation of soluble or insoluble aggregates in the form of dimers, ligomers and polymers, or to chemical degradation, including for example, hydrolysis, deamidation and oxidation. Consequently, the said physical and chemical instability may lead to loss of activity of the polypeptide, formation of toxic and immunogenic degradation products, in case of coagulation factor polypeptides there is serious risk of introducing thrombosis upon injection of the degraded polypeptides, clogging of needles used for injections and risk of non-homogeneity, to name just a few.

Thus, compositions comprising polypeptides need to be stabilised so as allowing storage and handling at ambient temperatures. One approach of stabilising a polypeptide relates to removal of water from the polypeptide, e.g. such as providing the polypeptide in the form of a lyophilised cake, the final matter obtained in a freeze-drying process. However, the freeze-drying process itself is also harmful to polypeptides; during freeze-drying, the polypeptide solution is first cooled until adequately frozen and bulk water in the polypeptide solution will form ice at this stage. The polypeptide is hereby prone to freeze-induced stress resulting in deformation and precipitation. In the next step, the so-called primary drying stage, the ice sublimes and in the secondary drying - stage, adsorbed or bound water is removed under elevated temperatures. During this water removal, the polypeptides may loose their proper conformation that is provided mainfy through hydrogen Ponding,

Therefore, to preserve polypeptide conformation, activity and stability during freeze-drying, the polypeptide solution needs to be supplemented with sufficient amounts of proper excipients with cryoprotectant and/or iyoprotectant properties so as to protect the polypeptide from freeze-induced stress and/or stress during removal of water, respectively, U.S. 20010031721 A1 (American Home Products) concerns highly concentrated, iyophilised, and liquid Factor DC formulations. WO 97/26909 (Genetics Institute) concerns iyophilised preparations of Factor IX suitable for storage and administration. The preparations may comprise sucrose or mannitol as a cryoprotectant. WO 95/28954 (Genetics Institute) concerns preparations of Factor IX suitable for storage and administration. The preparations may comprise sucrose as a cryoprotectant.

Additionally, when providing a Iyophilised product, an essential feature relates to the properties of the Iyophilised cake. It needs to have good properties as to its form and structure, l.e. it should not coitapse in that such collapsed cakes can be hard or even impossible to dissolve (reconstitute) before use. Conversely, the physical structure of the Iyophilised cake may not be too loosen and soft. Therefore, one or more so-called bulking agents are added to the polypeptide solution before freeze-drying.

Apart from choosing the right bulking agents it is also essential to avoid excipients which destabilises the physical properties of the cake. The concentration of these substances should be as low as possible. Furthermore, it is important that the reconstituted solution is not too hypotonic or hypertonic as this will cause injection Inconvenience or even pain for the patient when administered. Therefore, it is normally necessary to add tonicity to the composition. Another excipient could be a buffer substance in order to keep the pH of the reconstituted solution stable during storage.

Vitamin K-dependent polypeptides are a group of polypeptides involved in the blood clotting process; the group include factor VII, factor DC, factor X, factor II, Protein C, Protein S, gas6, and bone matrix Gfa polypeptide or can be a protease selected from the group consisting of factor Vila, factor IXa, factor Xa, factor Ila, and activated protein C. Factors Vila, IXa, and Xa are particularly useful proteases. Factor VEI Is a polypeptides involved in the biood clotting process. It can be made by recombinant techniques or prepared from plasma and is widely used in treatment of bleeding episodes in haemophilia patients.

Factor VII is a polypeptide involved in the blood clotting process. Today, Factor Vila can be made by recombinant techniques (rFVIIa) and is widely used as a pro-haemostatic agent. Factor VII (human wild-type) has been described in U.S. Patent No. 4,784,950. rFVIIa offers today a rapid and highly effective pro-haemostatic response in haemophilic individuals experiencing bleeding. Advantageously, rFVIIa can be used for treating haemophilic individuals that cannot be treated with other coagulation factor products due to antibody formation. Also individuals suffering from Factor VII deficiency or individuals having a normal coagulation system but still experiencing excessive bleeding can be treated successfully with rFVIIa.

Today, recombinantly-made FVII polypeptide is provided as freeze-dried product that is meant to be stored at temperatures between about 2 and about 8 °C. The requirement of cooled conditions causes a burden to and is inconvenient for the manufacturer or provider as well as the end user (the patient).

The actual recombinantly-made FVII product is NovoSeven® (Novo Nordisk A/S, Denmark) that consists of 1.2 mg recombinant human Factor Vila, 5.84 mg NaCI, 2.94 mg CaCI2, 2 H20, 2.64 mg Glycylglycine, 0.14 mg polysorbate 80 and 60.0 mg mannitol. When reconstituted by 2.0 ml of water for injection (WFI), the pH is 5.5 and the thus prepared FVII-containing solution is sufficiently stable for 24 hours at room temperature.

The present investigators have found that upon storage of the lyophilised NovoSeven® product for 6 months at 25 °C about 6 to 7 % w/w of the initial content of the rFVIIa is present in the form of aggregates.

Thus, compositions comprising Factor VII polypeptides need to be stabilised so as allowing storage and handling at ambient temperatures.

In some preferred embodiments, the present invention provides improved compositions, kits, and methods for producing these, wherein the dry compositions comprising the polypeptides are stabilized against chemical and physical degradation (such as, e.g., forming less dimer/oligomer degradation forms); with good properties of the lyophilised cake as to its form and structure, i.e. it should not collapse; with good and stable physical structure of the lyophilised cake; where the dry composition is devoid of excipients which destabilise the physical properties of the cake, e.g., by decreasing the eutectic melting point and thus increasing the risk of collapse of the cake; wherein the reconstituted composition prepared by dissolving the dry polypeptide-containing composition in the administration vehicle is isotonic, or closely isotonic, and has a well-defined pH (pH-stable). Particularly, the present invention relates to improved compositions comprising Factor VII polypeptides, substantially without the presence of degradation products and without decreased activity of the Factor VII polypeptides, preferable after prolonged storage at ambient conditions, e.g. for at least 6 months. Furthermore, some embodiments of the invention relate to stable compositions suitable for parenteral administration so as not to cause any inconvenience for the patient.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a kit comprising: a) in a first unit form, a composition comprising human Factor Vila and a combination of a saccharide, mannitol, CaCI2 and a surfactant wherein the composition has a moisture content of not more than 3%, and container means for containing said first unit form; and b) in a second unit form, an administration vehicle comprising a solvent for reconstitution (solution) of said composition comprising: (i) histidine suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent, wherein the histidine is present in an amount of from 0.1 mM to 100 mM; and, optionally, (ii) a tonicity-modifying agent in an amount sufficient to make essentially isotonic the reconstituted solution resulting from dissolving the composition of the first unit form in the administration vehicle of the second unit form, and container means for containing said second unit form, when used to produce a reconstituted human Factor Vila solution.

According to a second aspect, the present invention provides a method for preparing a liquid formulation of human Factor Vila, the method comprising the steps of: a) providing a first and a second unit form as defined in the first aspect; and b) mixing said first and second unit forms so as to provide a dissolved liquid solution of the composition in the administration vehicle.

According to a third aspect, the present invention provides a method of treating a Factor VII factor-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a liquid formulation of human Factor Vila prepared by the method of the second aspect, wherein said syndrome is selected from the group consisting of: acquired or congenital haemophilia A; acquired or congenital haemophilia B; Factor XI deficiency; Factor VII deficiency; Glanzmann thrombastenia; thrombocytopenia; von Willebrand’s disease; presence of a clotting factor inhibitor, such as inhibitors to factors VIII or IX; surgery; trauma; dilutional coagulophathy; and anticoagulant therapy.

According to a fourth aspect, the present invention provides use of a liquid formulation of human Factor Vila prepared by the method of the second aspect in the manufacture of a medicament for treating a Factor Vll-responsive syndrome, wherein said syndrome is selected from the group consisting of acquired or congenital haemophilia A; acquired or congenital haemophilia B; Factor XI deficiency; Factor VII deficiency; Glanzmann thrombastenia; thrombocytopenia; von Willebrand’s disease; presence of a clotting factor inhibitor, such as inhibitors to factors VIII or IX; surgery; trauma; dilutional coagulophathy; and anticoagulant therapy.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

It has been found by the present investigators that polypeptide-containing medicaments can be provided as a kit of parts comprising a first unit form consisting of a dry (e.g., a freeze-dried) composition comprising a polypeptide and at least one stabilizing agent wherein the composition has a moisture content of not more than about 3%, and container means for containing said first unit form; and, in container means for containing such a unit, a second unit form consisting of an administration vehicle comprising a solvent for solution (reconstitution) of said composition and at least one of the components selected from the list of: (i) an agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent in an amount of from about 0.1 mM to 100 mM; and (ii) a tonicity-modifying agent in an amount sufficient to make essentially isotonic the reconstituted solution resulting from dissolving the composition of the first unit form in the administration vehicle of the second unit form.

Substances which usually are present in the formulation like buffer substances and tonicity modifiers will very often decrease the eutectic melting point and will increase the risk of collapse of the cake. If these substances are present during freeze drying the temperature of the ice during the primary drying must be lowered to avoid collapse and consequently the time for freeze drying is prolonged. The concentration of these substances in the freeze-dried cake should be as low as possible or they should be completely avoided. Instead they may beneficially be added to the reconstitution liquid.

By lowering the concentration of these excipients or completely removing them, the reconstituted solution will in some instances become hypotonic and it is necessary the add tonicity modifiers to the solvent so as to obtain a solution with the needed tonicity, such as isotonicity, or closely so (“essentially isotonic”). Another necessary excipient in the solvent could be a buffer substance in order to keep the pH of the reconstituted solution stable during storage.

The kit of parts is sufficiently stable so as to allow for storage at room temperature for about at least 8 months.

Furthermore, it has been found that Factor VII polypeptides can be provided in a composition that is sufficient stable so as to allow for storage at room temperature for about at least 8 months. The investigators have found that the stabilisation relates to the proper combining of some pharmaceutically acceptable excipients.

Accordingly, the present invention relates to a kit (containing a pharmaceutical medicament/treatment), said kit comprising a) a composition comprising a polypeptide and at least one stabilizing agent, wherein the composition has a moisture content of not more than about 3%, in a first unit form, and container means for containing said first unit form; and b) an administration vehicle comprising a solvent for reconstitution (solution) of said composition and at least one of the components selected from the list of: (i) an agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent, wherein the agent is present in an amount of from about 0.1 mM to 100 mM, and (ii) a tonicity-modifying agent in an amount sufficient to make the reconstituted solution resulting from dissolving the composition of the first unit form in the administration vehicle of the second unit form essentially isotonic; in a second unit form, and container means for containing said second unit form.

In a further aspect, the invention relates to a method for preparing a liquid formulation of a polypeptide, the method comprising the steps of: a) providing a first and a second unit form as described above, and b) mixing said first and second unit forms so as to provide a dissolved liquid solution of the composition in the administration vehicle.

In other aspects, the invention relates to a method for treating a coagulation factor-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a liquid formulation of a coagulation factor prepared by the method described above, and to the use of said formulation for the preparation of a medicament in the form of a kit as defined above for treatment of a Factor Vll-responsive syndrome.

In another aspect, the invention relates to a composition comprising a Factor VII polypeptide, and at least one stabilizing agent selected from the group consisting of a) a combination of an antioxidant and mannitol; b) a combination of methionine and a polyol; c) a combination of a saccharide and mannitol; d) a combination of sucrose and a polyol; and e) methionine; and a polysorbate surfactant in an amount of from about 0.06 to 0.08 mg/mL; said composition having a moisture content of not more than about 3%.

In yet a further aspect, the invention relates to a method of preparing the above defined compositions, comprising the steps of: i) providing a Factor VII polypeptide in a solution comprising at least one stabilizing agent selected from the group consisting of a) a combination of an antioxidant and mannitol; b) a combination of methionine and a polyol; c) a combination of a saccharide and mannitol; d) a combination of sucrose and a polyol; and e) methionine; and a polysorbate surfactant in an amount of from about 0.06 to 0.08 mg/mL; and ii) processing said solution so as to obtain a solid composition with a moisture content not more than about 3 % w/w.

In other aspects, the invention relates to a method for treating a FVII-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a composition as defined above, and to the use of Factor VII polypeptide for the preparation of a medicament for treating a Factor Vll-responsive syndrome, said medicament comprising a composition as defined above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to storage-stable kits and compositions comprising polypeptides, including FVIII polypeptides, Vitamin K-dependent polypeptides, and FVII polypeptides. The compositions can be stored at room temperature for an extended period of time without causing substantial degradation of the polypeptide. By room temperature is meant the ambient temperature inside a room; it normally ranges from about 5eC to about 40eC, such as from about 10eC to 30eC, or 15eC to 25eC.

By proper predetermined combination of particular pharmaceutically acceptable excipients, the present investigators have provided stabilised compositions comprising polypeptides, particularly Factor VII polypeptides, thus allowing the compositions to be stored at room temperature for an extended period of time such as at least about 8 months. Advantageously, the stabilised compositions need not to be stored at cooled conditions, such as between 2 and 8 °C.

The present invention also concerns storage-stable compositions that are stable for at least about 8 months upon storage at about 30 °C. The composition is preferably stored in the dark. Thus, the present invention makes it possible to store such compositions at room temperature without increasing the risk of adverse events to the patient administering such compositions. Advantageously, the improved storage-stability will also result in reduced cost in that no special cooled conditions are required upon storage, further resulting in more convenient handling of the composition by the user.

Polypeptides to be formulated in accordance with the present invention includes, without limitation, blood coagulation factors including vitamin K-dependent polypeptides, such as, e.g., without limitation, factor VIII, factor V, factor XI, factor VII, factor IX, factor X, factor II, Protein C, Protein S, gas6, and bone matrix Gla polypeptide; activated FVIII, factor Va, factor Xla, factor Vila, factor IXa, factor Xa, factor I la, and activated Protein C.

The term “Vitamin K-dependent polypeptide” includes polypeptides selected from the group consisting of factor VII, factor IX, factor X, factor II, Protein C, Protein S, gas6, and bone matrix Gla polypeptide or can be a protease selected from the group consisting of factor Vila, factor IXa, - factor Xa, factor Ha, and activated Protein C. Factors Vila, IXa, and Xa are particularly useful proteases.

The term "Factor VII polypeptide" is denoted to mean any Factor VU polypeptide that is effective in preventing or treating Weeding. This includes Factor VII polypeptides derived from blood or plasma, or produced by recombinant means.

As used herein, "Factor VII polypeptide" encompasses, without limitation, Factor VII, including variants thereof, as well as Factor Vll-retated polypeptides, Factor VII derivatives and Factor VII conjugates. The term "Factor VII" is intended to encompass, without imitation, polypeptides having the amino acid sequence 1-406 of wild-type human Factor VII (as disclosed in U.S, Patent No. 4,784,950), as well as wiid-type Factor VII derived from other species, such as, e.g., bovine, porcine, canine, murine, and saimon, said Factor VII derived from biood or piasma, or produced by recombinant means, it further encompasses natural afieiic variations of Factor VII that may exist and occur from one individual to another, Aiso, the degree and location of glycosyiation or other post-translation modifications may vary depending on the chosen host cells ahd the nature of the host cellular environment. The term "Factor VII" is aiso intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteoiytically processed to yield their respective bloactive forms, which may be designated Factor Vila.

Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor Vila,

The term "Factor VII derivative" as used herein, is intended to designate a FVIX polypeptide exhibiting substantially the same or improved biological activity relative to wiid-type Factor VII, in which one or more of the amino acids of the parent peptide have been genetically and/or chemically and/or enzymatically modified, e.g. by alkylation, glycosyiation, PEGyiation, acylation, ester formation or amide formation or tire Sike, This includes but is not iimited to PEGylated human Factor Vila, cystelne-PEGyiated human Factor Vila and variants thereof. Non-limiting examples of Factor VII derivatives inetudes GiycoPegyiated FVH derivatives as disclosed in WO 03/31464 and US Patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, and US 20040132640 (Neose Technologies, Inc,); FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 {Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota),

The term "improved biological activity" refers to FVII polypeptides with 1) substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila or is) to FVII polypeptides with substantially the same or increased TF binding activity compared to recombinant wild type human Factor Vila or lit) to FVII polypeptides with substantially the same or increased half life in biood piasma compared to recombinant wild type human Factor VBa.The term "PEGylated human Factor Vila" means human Factor Vila, having a PEG molecule conjugated to a human Factor Vila polypeptide. It is to be understood, that the PEG motecuie may be attached to any part of the Factor Vila poiypeptide including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide. The term "cysteine-PEGytated human Factor Vila" means Factor Vila having a PEG molecule conjugated to a suifhydryl group of a cysteine introduced in human Factor Vila.

As mentioned, the term "Factor VII polypeptides" is also denoted to mean "Factor VH~refated polypeptides" The term "Factor Vll-related polypeptides" are intended to encompass such polypeptides in their uncieaved (zymogen) form, as well as those that have been proteoiytically processed to yield their respective bioactive forms. As used herein, "Factor VII-related polypeptides" encompass, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wiid-type human Factor VII and polypeptides wherein the biological activity has been substantially reduced relative to the activity of wiid-type human factor Vila (as disclosed in US Patent No, 4,784,950), These polypeptides include, without limitation, Factor VII or Factor Vila that has been chemically modified, such as, e.g,, by reacting factor VII with an irreversible inhibitor such as an organophosphor compound, a suifdnyf fluoride, a peptide halomethyi ketone or an azapeptide, or by acylation, by non-iimiting example, and Factor VII variants into which specific amino add seguence alterations have been introduced that slightly modify or Improve the biological activity of the polypeptide, such as, e.g., polypeptides wherein the catalytic activity of factor Vila is inhibited by chemicaS derivatization of the catalytic site, or triad.

The term "catalytic site" or "active site”, when used herein with reference to FVIIa, refer to the catalytic and zymogen substrate binding site, including the "Si” site of FVIIa as that term is defined by Schecter, L and Berger, A., (1967) Siochem, Siophys, Res. Common, 7:157-162. The catalytic site of human and bovine Factor VII proteins comprises the amino acids Ser344, Asp242, and Hisl93 (subscript numbering Indicating position in the sequence) that are forming a so-called catalytic "triad”. The catalytic sites in Factor VII from other mammalian species may be determined using presently available techniques including, among others, protein Isolation and amino acid sequence analysis. Catalytic sites may also be determined by aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined (Sigler et at, 3, Mol. Biot, 35:143-164 (1968)) and therefrom determining from said alignment the analogous active site residues.

Factor VTI-related polypeptides, including variants, having substantially the same or improved biological activity relative to wiid-type Factor Vila encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific activity of wiid-type Factor Vila that has been produced in the same celt type, when tested In one or more of a dotting assay, proteolysis assay, or IF binding assay as described in the present specification.

Factor Vll-related polypeptides, including variants, wherein the biologicai activity has been substantially reduced relative to the activity of wiid-type human factor Vila encompass those polypeptides that exhibit less than about 25%, more preferably iess than about 10%, or 5%, or 3%, or 2%, and most preferably less than about 1% of the specific activity of wiid-type factor Vila, when tested in one or more of a clotting assay, FIXa or FXa generation assay, amidoiysis or proteolysis assay as described within the present specification

In some embodiments the Factor Vll polypeptides are Factor VII-related polypeptides, in particular variants, wherein the ratio between the activity of said Factor VII polypeptide and the activity of native human Factor Vila (wild-type FVIIa} is at least about 1.25 when tested in the “In Vitro Hydrolysis Assay” (see Examples, General Methods, below); in other embodiments, the ratio is at least about 2.0; in further embodiments, toe ratio is at least about 4.0. In some embodiments of toe invention, the Factor VII polypeptides are Factor Vll-reiated polypeptides, in particular variants, wherein the ratio between toe activity of said Factor VII polypeptide and the activity of native human Factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Proteolysis Assay" {see Examples, General Methods, below); in other embodiments, toe ratio is at ieast about 2.0; in further embodiments, the ratio is at ieast about 4.0; in further embodiments, the ratio Is at least about 8,0.

Non-limiting examples of Factor VII variants having substantially toe same or improved bloiogicai activity as wild-type Factor VII include S52A-FVII, S6QA-FVII (Lino et al., Arch. Blochem. Biophys. 352: 182-192, 1998); L3Q5V-FVÏI, 130SV/M3Ö6D/D309S-FVII, L305I-FVII, L3Ö5T-FVII, F374P-FVII, V15ST/M298Q-FVII, V158D/E296V/M298Q-FVÏÏ, K337A-FVÏI, M298Q-FVII, V158D/M298Q-FVÏI, L385V/K337A-FVÏI, V158O/E296V/H298Q/L305V-FVII, V1.58D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVU, E296V-FVII, E296V/M298Q-FVH, V1S8D/E296V-FVH, V158P/M298K-FVII, and 5336G-FVII; FVIIa variants exhibiting increased proteolytic stability as disclosed in U S. Patent No, 5,580,560; Factor Vila that has been proteolyticatly cleaved between residues 290 and 291 or between residues 3lS and 316 (Molierup et a!., Biotechnol. Bioeng. 48:501-505, 1995); oxidized forms of Factor Vila (Kornfeit et a!., Arch, Biochem, Biophys. 363:43-54, 1999); FVII variants as disclosed in PCT/DK02/ÖÖ189 (corresponding to WO 02/077218); and FVII variants exhibiting increased proteolytic stability as disclosed in WO 02/38162 {Scripps Research Institute); FVII variants having a modified Gta-domain and exhibiting an enhanced membrane binding as disclosed in WO 99/20767, US patents US 6017882 and US 6747003, US patent application 20030100506 (University of Minnesota) and WO 00/66753, US patent applications US 20010018414, US 2004220106, and US 200131005, US patents US 6762286 and US 6693075 (University of Minnesota); and FVII variants as disclosed in WO 01/58935, US patent US 6806063, US patent application 2ÖÖ3ÖÖ9633S (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO 04/029091 (Maxygen ApS); FVII variants having increased bioiogica! activity compared to wild-type FVIIa as disclosed in WÖ 01/83725, WO 02/22776, WO 02/077218, PCT/OKÖ2/ÖÖ635, Danish patent application PA 2002 01423, Danish patent application PA 200101627; WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in IP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).

Non-limiting examples of factor VII polypeptides having substantially reduced bloiogicai activity relative to wiid-fcype factor VII indude Rl52E-FVIIa (Wiidgoose et a!., Biochem 29:3413-3420, 1990), S344A-FVIÏa {Kazama et at., 1, Biot. Chem. 270:66-72, 1995), FFR-FVIIa (Hoist et at., Eur, X Vase, Endovasc. Surg, 15:515-520,1998),, and factor Vila lacking the Gia domain, (Nicolaisen et af,, FEBS Letts, 317:245-249, 1993). Non-limiting examples also include human FVIIa, which has the lysine residue In position 341 replaced by another amino acid residue; human FVIIa, which has the serine residue in position 344 replaced by another amino acid residue ; human FVIIa, which has the aspartic acid residue in position 242 replaced by another amino acid residue; human FVIIa, which has the histidine residue in position 193 replaced by another amino acid residue; FVII-(K341A); FVIHS344A); FVII-(D242A); fVÏÏ-(H193A}; Phe-Phe-Arg-FVEt (FFR-FVII), D-Phe-Phe-Arg-FVII (D-FFR-FVII), Phe-Pro-Arg-FVII (FPR-FVII), D-Phe-Pro-Arg-FVII (O-FPR-FVII), L-Giu-GSy-Arg-FVII (EGR-FVII) and D-Giu-Gly-Arg-FVII (D-E6R-FVII), Oansyi-Phe-Phe-Arg-FVII, Dansyi-D-Phe-Phe-Arg-FVII, Dansyi-Phe-Pre-Arg-FVII, Dansyi-D-Phe-Pro-Arg-FVH, Dansyi-L-Giu-Gly-Arg-PVII and Dansyi-D-Giu-Giy-Arg-FViL Non-limiting examples of chemically modified factor VII polypeptides and sequence variants are described, e.g., in u.S. Patent No. 5,997,854,

Exampfes of factor VII or factor Vll-related polypeptides include, without limitation, wild-type Factor VII, 1305V-FVII, L305V/M3060/D309S-FVn, L30SI-FVII, L305T~FVII, F374P-FVÜ, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q~FVII, Vi58D/M298Q'FVII, L305V/K33?A~FVII, V158Ü/E296V/M298Q/L305V-FVH, V158D/E296V/M298Q/K337A-FVH, V158D/E295V/M29SQ/L305V/K337A-FVii, K157A-FVH, E295V-FVII, E295V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVH, and S336G-FVÏI, L305V/K337A-FVII, L30SV/VI58D-FVII, L3Ö5V/E296V-FVH, L305V/M298Q-FVII, L305V/V1S8T~FVII, L305V/K337A/VI58T-FVII, L305V/K337A/H298Q-FVII, 13Ö5V/K337A/E296V-FVII, L305V/I037A/V1S80-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVH, L305V/V158T/H298Q-FVII, L305V/V158T/E296V-FVXI, L305V/E296V/M298Q-FVII, L30SV/V158D/E296V/M298Q-FVU, L305V/V1S8T/E296V/M298Q-FVI1, l3g5WVt58T/K337A/M298Q~FVn, L305V/V15ST/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVn, L3Q5V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FV1I, S314E/K316Q-FVII, S314E/L305V-FVI3, S314E/K337A-FVII, S314E/V158D-FVIÏ, S314E/E296V-FVII, S3I4EW298Q-FVÏI, S3i4E/VlS8T-FVn, K316B/L30SV-FV!I, K316H/K33 7A-FVI1, K316H/V158D-FVII, S016H/E296V-FVII, K316H/M298Q-FVÏI, K316H/V158T-FVII, K316Q/1305V-FVH, K31$Q/K337A-PVII, K315Q/V158D-FVII, K316Q/E296V-FVH, K3I5Q/H298Q-FVU, K316Q/V1S8T~FVÏI, S314E/L395V/K337A-FVII, S314E/L305V/V1S8O-FVII, S314E/L305V/E295V-FVII, S314E/L305V/M298Q-PVII, S314E/O05V/Vl58T~FVII, S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVn, S3i4E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVU, S314E/L3Ö5V/V158D/M298Q-FVII, S314E/L305V/V158D/E295V-FVII, S314E/L305V/V158T/f4298Q-FVlI, S314E/L385V/V158T/E295V~ FVII, S314E/1305V/E296V/M298Q-FVH, S314E/L305V/V158D/E296V/M298Q-FVII, S314E/L305V/V158T/E296V/M298Q-FV1I, S314E/L305V/V158T/K337A/M298Q-FVII, S314E/O05V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVO, S314E/I.3ÖSV/VI58D/E296V/K337AFVIÏ, S3i4E/L305V/Vi58O/E296V/M298Q/K337A~FVn, S314E/L3Ö5V/V158T/E296V/M298Q/K337A-FVII, K3l6H/L305V/iO37A-FVII, K316H/130SV/V1580-FVII, K316H/1305V/E298V-FVII, K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T~FVII, K316B/L305V/K337A/M298Q-FVII, K3l6H/t305V/K33?A/E296V-- FVII, K316H/L3Ö5V/K337A/V158D-FVII, K316H/P05V/VIS80/M298Q-FVII, K316H/f305V/V158D/E296V~FVII, K316H/L305V./V158T/M298Q-FVn, K316H/L305V/V158T/E296V-' FVII, K316H/L30SV/E296V/M298O-FVII, K3l6H/L3Ö5V/ViSSO/E296WM298Q-FVn, K316H/L305V/Vi58T/E296V/M298Q-FVn, K316H/L3G5V/V158T/037A/M298Q~FVÏI, K31&amp;H/L305V/V158T/E296V/K337A-FVII, K316H/L305V/V1S8D/K337A/H298Q-FVII, K316H/1305V/V158D/E296V/K337A -FVII, K316H/L305V/Vi58D/E296V/M298Q/K337A-FVn, K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/E305V/K337A-FVI1, K316Q/L3Ö5V/VI58D-FVII, K3i6Q/L3ÖSV/£296V-FVIif K316Q/L305V/M298Q-FVÏI, K316Q/L305V/V158T"FVn, K316Q/L305V/K337A/V158T-FVII, K3i6Q/L3Ö5V/K337A/M298Q-FVÏI, K316Q/L305V/K337A/E296V-FVH, K3i6Q/L305V/K337A/V158D~FVII, K31 &amp;Q/L3Ö5V/V1S8D/H298Q-FVII, K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/Vl58T/M298Q-FVn, K3I6Q/L305V/V158T/E296V-FVII, K316Q/L305V/E29&amp;V/H298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305 V/V1S8T/E296V/M298Q*FVII, O16Q/E305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V1S8T/Ë296V/K337A-FVII, K3l6Q/l30SV/VlS8D/K337A/«298Q-FVn, K316Q/L305V/V158D/E296V/K337A -FVII, K316Q/L305V/V158D/E296V/M298Q/K337A-FVII, K316Q/L305V/V158T/E29&amp;V/M298Q/K33?A~FVII, F374Y/K337A-FVII, F374Y/V158D-FVO, F374Y/E296V-FVII, F374Y/H298Q-FVII, F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L3ÖW/K337A-FVII, F374Y/L30SV/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L30SV/M298Q-FVII, F374Y/i-3Ö5V/Vi58T~FVH, F374Y/L3Q5V/S314E-FVH, F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVB, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII, F374Y/K337A/V158D-FVII, F374Y/V158D/S314£~FVÏI, F374Y/V15SD/M298Q-FVII, F374Y/V158D/E296V~FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII, F374Y/V158T/E296V-FVH, F374Y/E29<SV/S314E-FVIÏ, F374Y/S314t/M298Q”FVH, F374Y/E296V/H298Q-FVII, F3 74Y/L3ö5V/K33?A/V158D-FVn, F374Y/130SV/K337A/E296V-FVII:, F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVIÏ, F374Y/L305V/K337A/S314E-FVIÏ, F374Y/L3ÜSV/V158D/E29&amp;V-FVIT, F374Y/L30SV/V158D/M298Q-FVII, F374Y/l30SV/VlS8D/S3i4E-FVU, F374Y/130SV/E296V/M298Q-FVH, F374Y/l3ÖSV/E296V/V158T-FVn, F374¥/U85V/E296V/S3i4E-FVII, F374Y/L30SV/M298Q/V158T-FVH, F374Y/L305V/M298Q/S314E-FVn, F374Y/L305V/V158T/S314E-FVH, F374Y/K337A/S3l4E/V158T-FVn, F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M29SQ-FVH, F374Y/K337A/Vi58T/£296V~FVn, F374Y/K337A/M298Q/E29SV-FVII, F374Y/K337A/M298Q/V1SSO-FVII, F374Y/K337A/E296V/V1580-FVII, F374Y/V158D/S314E/M298Q-FVH, F374Y/V158D/S314E/E296V-FVH, F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVH, F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-fVH, F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S3.14E-FV!1, F374Y/L3Ö 5V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/fG37A/S314E- FVII, F374Y/L305V/E296V/M298Q/K337A -FVII, F374Y/E305V/E296V/M298Q/S314£-FVII, F374Y/V158D/E296V/M298Q/K337A-FVIX, F374Y/Vi580/E296V/M298Q/S314E-FVII( F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVH, F374Y/V158D/E296V/K337A/S314E-FVIX, F374Y/L305V/Vl58D/E296V/M298Q-FVn, F374Y/L3Ö5V/V158D/M298Q/K337A-FVII, F3?4Y/1305V/V158D/E29&amp;WK337A~FVII, P3?4Y/t3Ö5V/V158D/M298Q/S314E-FVÏI, F374Y/L3Ö5V/V158D/E296V/S314E-FVII, F374Y/VÏ58T/E296V/M298Q/K337A-FVÏÏ, F374Y/Vi58T/E296V/M298Q/S314E-FVlï, F3?4Y/L3Ö5V/V158T/K33?A/S314E~FVII, F374Y/V158T/M298Q/K337A/S314E-FVII, F374Y/V158T/E296V/K337A/S314E-FVU, F374Y/L305V/V158T/E296V/M298Q-FVII, F374Y/L305V/V158T/M298Q/K337A-FV1I, F374Y/L3G5V/V158T/E296V/K337A-FVII, F374Y/ L3ö5V/Vi53T/M298Q/S31 4 E-FVII, F374Y/1305V/V1S8T/E296V/S3 14E-FVÏÏ, F374Y/E296V/M298Q/037A/V15ST/S314E-FVÏI, F374Y/V15SD/E296V/M298Q/K337A/S314E-FVXI, F374Y/L3Ö5V/Vl58D/E296V/M298Q/S314E-FVn, F374Y/L305V/£296V/M298Q/Vi58T/S314E~FVH, F3?4Y/1305V/E296V/M298Q/K337A/V15ST-FVÏÏ, F374Y/L30SV/E296¥/K337AA/158T/S3l4E-FVn, F374Y/L305V/M298Q/K337A/V158T/S314E-FVII, F374Y/L30SV/V158D/E296V/iM298Q/K337A-FVIIi F374Y/OQ5V/Vl58D/E296V/K33?A/S314E~FVII, F374Y/L305V/Vi58D/M298Q/K337A/S3i4E-FVII, F374Y/L30SV/ E296V/H298Q/ K337A/V1S 8T/S314E-FVII, F374Y/l30SVyViS8D/E296V/M29SQ/K337A/S3l4E-FVII, S52A-Factor VII, S6GA~Factor VO; and PllQ/K33E-FVn, T106N-FVH, K143N/N145T-FVII, V2S3N-FVII, R290N/A292T-FVÏÏ, G291N-FVH, R315N/V317T~FVII, K143 N/N145T/R31SN/V317T-FV1I; FVH having substitutions, additions Or deletions in the amino acid sequence from 233Thr to 240Asn, PVII having substitutions, additions or deletions in the amino acid sequence from 394Arg to 329Cys, and EVIÏ having substitutions, deletions, or additions in the amino acid sequence ISei53-Arg223.

For purposes of the invention, biological activity of Factor VII polypeptides {"Factor VH bioiogicai activity") may be quantified by measuring the abifity of a preparation to promote Wood dotting using Factor VH-defident plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864. In this assay, bioiogicai activity is expressed as the reduction in dotting time relative to a control sample and is converted to "Factor VII units" by comparison with a pooSed human serum standard containing 1 unit/m! Factor VII activity, Alternatively, Factor Vila biological activity may be quantified by 0} Measuring the ability of Factor Vila or a Factor Vila -related poiypepüde to produce activated Factor X {Factor Xa) in a system comprising TF embedded in a iipid membrane and Factor X, {Persson et a!,, 3. Biot Chem. 272:19919-19924, 1997); 01) Measuring Factor X hydrolysis in an aqueous system (In Vitro Proteolysis Assay”, see Examples, General Methods, below); (ili) Measuring the physical binding of Factor Vila or a Factor Vila -related polypeptide to TF using an instrument based on surface piasmon resonance (Persson, FEBS Letts, 413:359-363, 1997); and (tv) Measuring hydroiysis of a synthetic substrate by Factor Vila and/or a Factor Vila -related polypeptide ("In Vitro Hydroiysis Assay” see Examples, General Methods, beiow); and (v) Measuring generation of thrombin in a TF-independent in vitro system.

The term "Factor VII bioiogicai activity" or "Factor VII activity” is intended to indude the ability to generate thrombin; the term also includes the ability to generate thrombin on the surface of activated platelets in the absence of tissue Factor. “Examples, General Methods" of the present specification describes in detail assays useful for assaying FVII biological activity.

Moreover, throughout the present specification, the terms below have the following meaning;

The term “kit" or “kit of parts" is intended to mean a combination of a dry product, in a first unit, containing a polypeptide and one or more stabilizing agents; and an administration vehicle consisting of a solvent suitable for dissolving the dry product of the first unit, in combination with at least one buffering agent in an amount of from about from about 0.1 mM to 100 mM, and/or at least one tonicity modifying agent, in a second unit. The kit contains a pharmaceutical treatment, parttouiariy of bleeding episodes. Before use, the dry composition of the first unit is mixed with and dissolved in the administration vehicle contained in the second unit, thereby providing a medicament ready for use.

The medicament ready for use is essentially isotonic.

The term “administration vehicle" is intended to encompass pharmaoeuticaiiy acceptable, preferably sterile liquids suitable for administration by injectable means, such as infusion or injection, e.g., by intravenous, subcutaneous, or intramuscular injection. The administration vehicie is preferably aqueous. The administration vehicle comprises a solvent, or a mixture of solvents, suitable for reconstitution (solution) of the polypeptide composition (e.g., Water for Injecöon/WFI), and one or more agents suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent in an amount of from about 0,1 mM to 100 mM; and/or one or more tonicity modifying agents in an amount sufficient to make essentially Isotonic the reconstituted solution resuiting from dissolving the composition of the first unit form in the administration vehicle of the second unit form.

The administration vehicle may contain further substances, such as metaf salts, e.g., calcium and/or magnesium salts, amino adds, e.g., giycyigiydne.

The reconstituted compositions are intended for parenteral administration for prophylactic and/or therapeutic treatment.

An "effective amount" of a polypeptide refers to tire amount of polypeptide which, when administered in accordance with the invention, produces a measurable improvement in at least one clinical parameter of haemostasis known to those of ordinary skill in the art.

It will be understood that an effective amount of a polypeptide may vary according to the subject's haemostatic status, which, in turn, may be reflected in one or more dinical parameters, including, e.g., relative levels of drculating coagulation factors; amount of blood test; rate of bleeding; hematocrit, and the like. It wii! be further understood that the singte-dose-effective amount may be determined by those of ordinary skill in the art by routine experimentation, by constructing a matrix of values and testing different points in the matrix.

The term "stabilizing" is intended to encompass minimising the formation of aggregates {insoluble and/or soluble) and/or chemica! degradation as well as providing maintenance of pH and proper conformation of the polypeptide during storage or production of the compositions so that substantial retention of biological activity and polypeptide stability is maintained, Moreover, stabilising is aiso denoted to mean iyoprotection and cryoprotection of the poiypeptide during production of the compositions at freeze-drying conditions.

The term "stabilizing agent" is intended to encompass substances, or a mixture of substances, that are able to stabilize a poiypeptide during storage or production of a composition comprising the polypeptide.

The term "structural stabilisation" or "structure! stability" is intended to encompass the ability to form a lyophilised plug or cake with good properties and looks, e.g. such that it does not coiiapse and is readily dissolved before use.

The term "storage-stable" is intended to define a product that is stabilised upon storage at temperatures between S°C - 40°C and remains within pre-selected product specifications for a suitable time period - often several months.

The term "physical stability” of Factor VO polypeptides relates to the formation of insoluble and/or soluble aggregates in the form of dimeric, oligomeric and polymeric forms of Factor VII polypeptides as well as any structural deformation and denaturation of the molecule.

The term "chemical stability" is intended to relate to the formation of any chemical change in the Factor VII polypeptides upon storage in dissolved or solid state at accelerated conditions. By example are hydrolysis, deamidation and oxidation. In particular, the sulphur-containing amino acids are prone to oxidation with the formation of the corresponding sulphoxides.

The term "cryoprotectants” as used herein generally include agents, which provide stability to the poiypeptide from freezing-induced stresses. Examples of cryoprotectants include poiyois such as, for example, mannitol, and indude saccharides such as, for example, sucrose, as well as including surfactants such as, for example, pofysorbate, poloxamer or polyethylene glycol, and the tike. Cryoprotectants also contribute to the tonldty of the formulations.

The term "lyoprotectant" as used herein includes agents that provide stability to the poiypeptide during water removal upon the drying process of the lyophilisation process. For example by maintaining the proper conformation of the polypeptide, Examples of lyoprotectants include saccharides, in particular di- or trisaccharides. Cryoprotectants may aiso have lyoprotectant effects,

Tim term "agent suitabie for keeping the pH in the range of 3 to 9", or "buffering agent", encompasses those agents that maintain the solution pH in an acceptable range between 3,0 and 9.0. Typical examples of agents capable of Keeping the pH within a range of 3 to 9 are the acid form or salts of citric acid, acetic add, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, PIPES, imidazole, IRIS, ladle acid, giutaric acid and glycySgfycine, It Is to be understood that a combination of agents, wherein the combination of agents Is suitable for maintaining the pH in the above-described range, may aiso be used in the present invention.

The term “tyophiiised cake" as used herein is denoted to encompass the sofid composition obtained upon processing a dissolved or at ieast a partly dissolved composition under conditions involving at least one step of cooling said dissoived/partiy dissolved composition to ice followed by at least one step of vacuum drying.

The term “iyophiiization” and "freeze-drying" encompasses a process during which liquid is removed from a dissolved or at ieast partly dissolved composition under conditions invoiving at least one step of cooling the dissolved or partly dissolved solution to ice followed by vacuum drying. Lyophiiization, or freeze-drying, is the most common process for making solid polypeptide pharmaceuticals. The process consists of two major steps: freezing of a polypeptide solution, and drying of the frozen solid under vacuum. The drying step is further divided into two phases: primary and secondary drying. The primary drying removes the frozen water (sublimation of ice) and the secondary drying removes the non-frozen "bound" water (desorption of water). More detailed analysis of each Iyophiiization step is provided in, e.g., Wang et al, Internationa! Joumai of Pharmaceutics 203 (2000): 1-60 (see section 4, page 16 ff.j.

Typically, a composition is freeze-dried by filling into vials, freezing on the shelves of the freeze-dryer, after which a vacuum is established and the shelves heated to implement primary drying (or sublimation of ice). Thereafter, secondary drying (or desorption of sorbed water) takes place at a higher temperature until the completion of the process, i.e., where the composition contains a sufficiently tow content of moisture (water). Methods for freeze-drying are generally known in the art, see, for example, Wang et al. International Journal of Pharmaceutics 203 (2000): 1-60,

It is within the ordinary skill of the practitioner to optimize toe freeze-drying conditions in regard of temperature($}, time(s) at each temperature, and also pressure that is to be used during the process for a specific composition.

The term "moisture content" is meant to encompass water associated with the product, including, without limitation, water in adsorbed form, such as unfrozen water entrapped in or adsorbed to the frozen solute phase and/or associated with the amorphous phase or adsorbed to toe crystalline solid. The term "water content" is used interchangeably with "moisture content". The desired residua! moisture ievef (moisture content) is a function of toe duration and the temperature of the secondary drying step. Several methods for determining the residua! moisture content during iyophiiization are known in the art; for example, an electronic hygrometer or a residua! gas analyser may be used. Moisture contents of freeze-dried formulations can be determined by several methods known in the art, such as, for example, foss-on-drying, Kari Fischer titration, thermal gravimetric analysis ( IGA), gas chromatography (GC), or near IR (see, e.g. Wang et al. international Journal of Pharmaceutics 203 (2000): i-60). Methods for determining water contents (moisture contents) are also described in both the European and U,S. Pharmacopoeias. For example, determination of water content can be performed by Kart Fischer couiometric titration as described in the U.S. Pharmacopoeia (USP <921, Ic>) or the European Phamacopoeia (EP <2.5.32>).

In brief, the method is as follows:

Determination of water content by couiometric titration: The Kari Fischer reaction is used in the couiometric determination of water based upon the quantitative reaction of water with suiphur dioxide and iodine in an anhydrous medium. Iodine is produced eieetrochemicaliy in the reaction cel! by oxidation of iodide. The iodine produced at the anode reacts immediately with the water and the suiphur dioxide contained in the reaction ceil. The amount of water in the substance is directly proportional to the quantity of electricity up until the titration end-point. When aii of the water in the ceii has been consumed, the end-point is reached and thus an excess of iodine appears which is detected eiectrometricatiy thus indicating the end-point. The percentage water content present in the substance is then calculated.

Moisture content may be defined in terms of the weight of the sample in the via! at the time of analysis (i,e, solids plus the water present- tailed wet weight basis) or it may be defined in terms where it is corrected for the measured water in the sample (l.e, dry weight basis), In case of freeze-dried products with low moisture contents the two measurements (wet weight basis vs. dry weight basis) yield very similar resuits. As used herein, moisture contents are defined in terms of the solids plus the water present (Le,, wet weight basis).

The term "bulking agent"' generally includes agents, which provide good tyophilised cake properties, which form a pharmaceutically elegant product, which help the polypeptide overcome various stresses, shear/freeztng for example, associated with iyophitisation processes, and which help to maintain polypeptide activity levels during the (feeze-drying process and subsequent storage. Non-limiting examples of bulking agents include mannitol, glycine, sucrose, factose.

These agents may also contribute to the tonicity of the formulations.

Isotonic solutions have a tonicity within the physiological range of the blood, peritonea! fluid or other relevant body fluids. By isotonicity is meant a solution with an osmotic pressure corresponding to the osmotic pressure of a 0.9% Nad solution (> 286 mOsM), The term "essentially isotonic" is denoted to mean a tonicity corresponding to the osmotic pressure of a saline solution containing from about 0,7 to about i.5% NaCi, such as, e.g., from about 0.8 to about 1.3%, about 0,8 to about 1,1%, about 0,8 to about 1.0%, or about 0,9% NaCi.

The term "tonicity modifier" or "tonicity modifying agent" is denoted to mean any agent, or mixture of agents, capable of adjusting the tonicity of the composition such that upon dissolving the composition at the time of use, the dissolved (or reconstituted) composition Is essentially isotonic. Obviously, the tonicity of the reconstituted solution may depend on both tbe contents of tonieity-modifying agents in the dry composition and in the reconstitution solution.

Tonicity modifying agents include, without limitation, components selected from the Sist of: sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, mannitol, glycerol, propylene giycoi, or mixtures of two or more of these.

Amounts of the above tonicity modifying agents suitable for providing a composition having a tonicity as defined above are, for example, from 0 to about 9 mg/m! of sodium chloride, from 0 to about 17 mg/mi of calcium chloride dihydrate, from 0 to about 51 mg/mi of mannitoi, from 0 to about 26 mg/mi of glycerol, from 0 to about 21 mg/m! of propylene giycoi, depending on whether the individual tonicity modifier is used alone or in combination with one or more tonicity modifiers.

The term "surfactants" generally include those agents, which protect the polypeptide from air/solution interface-induced stresses and solution/surface induced-stresses. For exampie surfactants may protect the polypeptide from aggregation. Suitable surfactants may include e.g. polysorbafces, polyoxyethylene alky! ethers such as Brij 35®, or poioxamer such as Tween 20, Tween 80, or poioxamer 188. Preferred surfactants are poioxamers, e.g, Poioxamer 188, Poioxamer 407; polyoxyethylene alkyl ethers, e.g. Brij 35®, Cremophor A25, Sympatens AlM/230; and polysorbates/Tweens, e.g, Poiysorbate 20, Poiysorbate 80. More preferred are Poioxamers, e.g, Poioxamer 188, and Tweens, e.g. Tween 20 and Tween 80, Typically, the surfactants are added in an amount of from 0.005 to 5 mg/mi. Preferred amounts are from 0,01 to 3 mg/mi, more preferred from 0.01 to 0,3 mg/mi for Tween 20 and/or Tween 80 and from 0.05 to 3.0 mg/mi for Poioxamer 188.

The term "initial content" relates to the amount of Factor VH polypeptides added to a composition at the time of preparation. The concentration given herein (mg/mi) refer to either the concentration in the solution of Factor VII polypeptide before removing the moisture (e.g, before freeze-drying) or in the reconstituted composition, or is referred as % w/w, which then relates to the concentration in the solid composition, e.g. the iyophiiised cake.

As used herein, amounts specified are understood to be * about 10%; thus about 50 mH includes 50 mM ± 5mM, 4% indudes 4% A 0.4%, etc.

As stated above, the present investigators have contributed essentially to the art by stabilising Factor VII polypeptides thereby allowing long-term storage without causing Increased risk and Inconvenience to the user.

The present investigators have found that a number of crucia! parameters need to be adjusted in stabilising Factor VII polypeptides. One important parameter relates, at least in part, to the moisture content, e.g. water. The moisture content should be limited. As a further essentia! parameter, the composition should at least include one stabilizing agent.

Stabilizing agents indude, without limitation, antioxidants, saccharides, polyols, surfactants, and agents suitable for maintaining pH in a predetermined range.

In one embodiment of the present invention, a proper stabilizing agent includes the combination of at least two groups of pharmaceutically acceptable excipients seiected from the group consisting of antioxidants, saccharides and polyols. The saccharides and polyois have lyoprotectant and/or cryoprotectant properties that may be important, at Seast In part, in the event where the composition is freeze-dried, In genera!, improved stability may be achieved, in part, by the proper combination of at least two of these groups of excipients. However, more specifically it was found that when said combination comprises a saccharide (sucrose) or an antioxidant (methionine), the stabilising effect may be even more significant. Moreover, it was also surprisingly found that methionine prevents oxidative degradation of the Factor VII polypeptides.

As stated, in one embodiment of the invention the stabilising agent indudes combining at least two groups of pharmaceutically acceptable excipients.

According to the invention, the Factor VII polypeptide is meant to encompass the polypeptides as described above. In suitable embodiments of the invention, the Factor VII poiypeptide is seiected from the group consisting of Human Factor Vila, Recombinant Human Factor Vila and a Factor VII Sequence Variant, Preferably, the Factor VII Polypeptide is Human Factor Vila or Recombinant Human Factor Vila or a Factor VII-reiated polypeptide wherein the ratio between the activity of said Factor VII-reiated polypeptide and wild-type Factor VII is at least 1,25 when tested in one or more of the ’"In Vitro Proteolysis Assay" and the "in Vitro Hydrolysis Assay" as described in the present specification.

As stated, the moisture content should be limited. For the purposes of the present invention, the Factor VII polypeptides, when provided In bulk, may be provided in solid or liquid form. However, typicafiy the Factor VII polypeptides, when provided in bulk, are in liquid form. Thus, further processing of the buik poiypeptides for the manufacturing of compositions requires the steps of adding suitable excipients and removing the liquid from the buik, said addition of excipients may be carried out before or after removing the liquid. One such mean for removing liquid from a polypeptide relates to freeze-drying. Therefore, in a preferred embodiment of the present invention, the composition is in the form of a iyophilised cake. However, the present invention does not preclude other processes that are suitable for removing the liquid from the buik polypeptide so as to achieve a solid composition with moisture content of not more than about 3% w/w.

Moreover, according to the invention, the moisture content is preferably not more than about 2.5% w/w, preferably not more than about 2% w/w, most preferably not more than about 1.5% w/w.

As may be understood, the invention relates, in part, to limiting the degradation of Factor VII polypeptides during preparation, e.g. during admixing of excipients and removing of liquid so as to achieve a solid composition with moisture content of the most 3% w/w, and to limiting said degradation from the time of manufacturing the soiid composition until the time of use, e.g. until the time when the composition is to be administered by a patient.

Therefore, as a still further parameter so stabilising kits anti compositions comprising Factor VII polypeptides, the pH should be kept in the pH range within 3 to 9 when dissolved in aqueous solvent, such as, e.g., pure water or aqueous buffer. That is to say that the pH in the polypeptide solution at the time before removing the moisture content, e.g, before freeze-drying, should be kept within a pH of about 3 to about 9, Advantageously, this pH range is aiso within the desired physiological range, thereby causing no harm to the user upon administering the composition by parenteral means. Preferably, the pH of the solution is from about 4,0 to about 9,0, such as 4,0 to 8.0, 4,0 to 7,5, 4.0 to 7.0, 4,5 to 7.0, 4.5 to 6.8, 4.5 to 6.5, 5.0 to 7,0, 5.0 to 6.5, 5.0 to 6.0, 5.5 to 6,0, or about 5.5 to about 6.5 such as about 5.5, 5,6, 5.7, 5.8, 5.9, 6,0, 6,1, 6,2, 6.3, 6,4, or 6.5.

In suitable embodiments of the invention, the agent suitable for keeping the pH in the range of 3 to 9 Is selected from the group consisting of acid or salts of citric acid, acetic acid, histidine, malic acid, phosphoric acid, tartaric add, succinic add, MES, HEPES, imidazole, THIS, factie acid, gfutaric acid, PIPES anti giycyigiydne, or a mixture of at least two such listed agents, wherein the mixture is able to provide a pH value in the specified range.

Furthermore, the suitable agent for keeping the pH in the range of 3 to 9 may also be a mixture of at least two such listed agents, wherein the mixture is able to provide a pH vaiue in the specified range. The concentration of the suitable agents is in the range of from about 0,1 mH to 100 mM; from about O.imH to about 5GmM; such as from about O.imM to about 40mM; from about O.lmM to about 35mM; from about O.lmM to about 30mM; from about O.SrnM to about 25mM; from about ImM to about 20mM; from about lmM to about 15mM; from about SmM to about 20mM; or from about 5mM to about 15mH,

In one embodiment of Sbe invention, the agent suitabie for keeping the pH in the range of 3 to 9 is histidine, preferably L-histidine,

Degradation of the Factor VS polypeptide by the oxidative pathway as weli as by the aggregation pathway are sensitive parameters of stability,

Typically, the compositions are stabilised upon termination of the freeze-drying such that less than 5% w/w, such as iess than 4, 3 or 2% w/w of the initial content of Factor VII polypeptide is converted into its oxidised forms. The initial content of said Factor VII polypeptide being the amount added to toe composition upon preparation of the composition before toe freeze-drying step. Moreover, less than S% w/w, such as less than 4.0%, 3.0%, 2.5%, 2%, 1.5%, or less than 1% w/w of the initial content of Factor VII polypeptide is recovered as aggregate forms, as determined by conventional analytical methods (such as, for example, as described in the Examples of the present application).

The present investigators have found that further degradation (i.e., as calculated from the time of termination of the manufacturing process untii 8 months of storage at 30 °C) of a Factor VII polypeptide is minima! upon storage under ambient conditions. It was found that compositions comprising an antioxidant (methionine) are more stable towards oxidative degradation of the Factor VII polypeptide.

Therefore, suitable compositions have a Simited increase in the content of oxidised forms upon storage for at least 8 months at ambient conditions,

That is to say that in still more interesting embodiments, the composition is stable such that no more than about 6% w/w of the initial content of Factor VII polypeptide is additionally degraded into oxidised forms upon storage of the composition for B months at 30 °C after termination of the manufacturing process, e.g, freeze-drying process. In further suitabie embodiments thereof, not more than about 5, 4, 3, 2, or 1.5 % w/w or of the Factor VII polypeptide is additionaiiy converted into oxidised forms, as calculated from the time of termination of the manufacturing process until 8 months of storage at 30 eC. In these embodiments of the invention the compositions are stable such that not more than about 5% (4, 3, 2, or 1.5 %) w/w of the initial content of Factor VII polypeptide is converted to oxidised forms upon storage of said composition at 30 “C for 8 months. As stated above, the initial content relates to the amount of Factor VII polypeptide added to the composition upon preparation of the composition before the freeze-drying step,

As indicated, the degradation of Factor VII polypeptides by the aggregation pathway may also be regarded as an essential stability indicating parameter.

Thus, interesting embodiments of the invention relate to compositions that are stable such that not more than about 5% w/w of the initial content of Factor VII polypeptide is converted to aggregates upon storage of said composition at 30 X, for 8 months. As stated above the initial content of said Factor VII poiypeptide being the amount added to the composition upon preparation of the composition before the freeze-drying step. By proper optimisation of, at feast in part, the contents of saccharides, polyols and antioxidants, the composition is stabfe such that not more than about 4.0%, 3.035 w/w, such as 2,5, 2,0,1.5, or 1,0% w/w, of the initial content of Factor VII poiypeptide is converted to aggregates upon storage of said composition at 30 ®C for 8 months.

Thus, advantageously, the compositions of the invention have iow contents of oxidised forms and aggregates upon termination of the manufacturing process, i.e. upon termination of the freezedrying process, and thus the compositions according to the invention are characterised by having a iow initial content of oxidised forms and aggregates before being subjected to storage, e,g, not more than about 5% w/w, such as 4%, 3%, or 2% w/w of the initial contents of Factor VII polypeptide is converted into an oxidised form, and iess than 5% w/w, such as not more than about 4,0%, 3.0%, 2,5%, 2%, 1.5%, or not more than about 1% w/w, is converted into a dimeric or higher-order polymeric form upon termination of the manufacturing process

Moreover and advantageously, the kits and compositions of the invention are storage-stable, e.g. iess than 10% w/w, such as 5%, 5%, 4%, 3%, 2%, or 1,5% w/w of the initial contents of Factor VII poiypeptide is converted into an oxidised form, and less than 5% w/w, such as 4%, 3%, 2.5%, 2%, 1,5%, or 1% w/w is converted into a dimeric or higher-order polymeric form upon storage at 30 °C for at least 8 months in the dark.

As mentioned, said improved stability relates to the proper combination of particular excipients. According to the present invention, the stabilising agents may be selected from the group of saccharides, polyols and antioxidants. In suitable embodiments, the saccharides of Interest are di-and tri-saccharides and polysaccharides such that the saccharides may be selected from the group consisting of sucrose, dextrose, lactose, maltose, trehalose, cydodextrins, maltodextrins and dextrans, Moreover, in some embodiments, the poiyoi is selected from the group consisting of mannitol, sorbitol and xylitol. In still interesting embodiments, the antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, giuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giuthatione.

It is understood that the saccharide and poiyoi excipients, respectively, may afso be a mixture of at least two such listed agents. In one series of embodiments of the invention, the saccharide excipient used is a combination of at least two dl-, tri- and/or polysaccharides, such as, for example, sucrose in combination with cyclodextrin, trehalose in combination with cyciodextrin, sucrose in combination with dextrao, or sucrose in combination with lactose. In one series of embodiments of the invention, the poiyoi excipient used is a combination of at (east two polyols, such as, for example, mannitoi In combination with sorbitol, mannitol in combination with xyiitoi, or sorbitoi In combination with xyiitoi. In one series of embodiments of the invention, the antioxidant excipient used is a combination of at least two antioxidants, such as, for example, methionine in combination with one or more of homocysteine, cysteine, cystathionine, giuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giuthatione.

In particular interesting embodiments, the antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, giuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giuthatione. In a preferred embodimènt, the antioxidant is methionine.

In further interesting embodiments of the invention, the poiyols are to be present in an amount ranging from about 5% w/w to about 90% w/w. Preferably, the amount of the poiyoi is to be present in a range from about 18% w/w to about 88% w/w, such as from about 18% w/w to about 83% w/w, 25% to 80%, 30% to 65%, 30% to 80%, 40% to 80%, 50% to 80%, 30% to 75%, 40% to 75%, 50% to 75%, or from about 50% to about 70% w/w.

The poiyoi are to be present in an amount ranging from about 0.5 to 75 mg/ml, such as from about 2 to 60 mg/mt, 5 mg/mi to 55 mg/ml, 8 to 45 mg/mt, 10 to 40 mg/mi, 10 to 30 mg/mi, or from about 2 to 45 mg/mi, 5 mg/ml to 45 mg/mi, 5 to 35 mg/mi, 5 to 25 mg/ml, 5 to 20 mg/ml, 20 to 40 mg/mt, or such as from about 20 to 30 mg/ml,

Moreover, in interesting embodiments thereof as wefi as in some other interesting embodiments of the invention, the saccharide is to be present in the composition in an amount ranging from about 0 to about 85% w/w. In further interesting embodiments thereof, the amount ranges from about 3% w/w to about 80% w/w, such as from about 7% w/w to about 75% w/w, 10% to 70%, 10% to 50%, 20% to 50%, 10% to 40%, or from about 10% w/w to about 35% w/w.

Hie saccharide should be in an amount ranging from about 0.5 to 75 mg/ml, such as from about 2 to 60 mg/mi, from about 5 mg/m! to 55 mg/ml, from about 8 to 45 rng/mi, from about 10 to 4Ö mg/mi, from about 10 to 30 mg/mi, or from about 2 to 45 mg/mi, from about 5 mg/mi to 45 mg/mi, from about 5 to 35 mg/mi, from about 5 to 25 mg/mi, such as from about 5 to 20 mg/ml.

The antioxidant should be provided in an amount ranging from about 0,05 to 10 mg/mi, preferably from about 0,1 to 5 mg/ml, more preferabiy from about 0.1 mg/mi to 2,5 mg/ml, even more preferably from about 0.1 to 2 mg/ml, most preferably from about 0,1 to 1 mg/mi.

The ratio between the polyol and the saccharide needs to be property adjusted. In suitable embodiments of the Invention, said polyo! Is in a weight ratio relative to said saccharide ranging from about 100:1 to 1:50. In even more suitable embodiments thereof, said weight ratio is from about 50:1 to 1:10, more preferably from about 20:1 to 1*5. In other suitable embodiments, the weight ratio relates to ranges from about 10:1 to 1:2, and from about 6:1 to 1:2. Suitable embodiments relate to those wherein said sugar aicohoi is in a weight ratio relative to said saccharide ranging from about4:1 to 1:1, such as from about4:1 to 3:2 or from about 1:1 to 3:2,

In some embodiments of the invention, the poSyof is mannito! and in still further embodiments the saccharide is sucrose. Moreover, In still further embodiments the antioxidant is methionine.

In stiil preferred embodiments of the invention, the composition further comprises other pharmaceutical excipients acting as bulking agent. That is to say that bulking agents other than mannitol are included in the compositions, In particular, buiking agents are included in compositions prepared by freeze-drying.

Initial contents of Factor VII poiypeptide in the composition is preferably from about 0.6 mg/ml to about 10.0 mg/ml, such as from about 0,6 mg/ml to about 8 mg/ml, from about 0.6 mg/ml to about 6 mg/ml, from about 0,6 mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 4 mg/ml, from about 0.6 mg/ml to about 3 mg/ml, from about 1.0 mg/ml to about 5 mg/ml, from about 1,0 mg/ml to about 4 mg/ml, or from about 1.0 mg/ml to about 3 mg/ml, e.g. , about 1.0 mg/ml, about 2.0 mg/ml, about 3.0 mg/ml, about 4,0 mg/ml, or about 5.0 mg/ml.

In one embodiment, the composition contained in the first unit form of the kit comprises: Factor VII polypeptide, Mannitol, Sucrose, and polysorbate, preferably polysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the list of: CaCI2, NaCi, and Giycyigiycine, In one embodiment, the Factor VII polypeptide is human Factor Vila.

In one embodiment, the composition contained in the first unit form of the kit comprises: Factor VIX polypeptide, Mannitol, Sucrose, methionine, and poiysorbate, preferably poiysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the list of: CaCi2, NaCi, and GSyeylgiycine, In one embodiment, the Factor VII polypeptide Is human Factor Vila,

In another embodiment, the composition contained in the first unit form of the kit comprises Factor VII poiypeptide, Mannitoi, Sucrose, Histidine, and poiysorbate, preferably poiysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the tist of: CaCI2, NaCi, and Glycylgiycine. In one embodiment, the Factor VII polypeptide is human Factor Vila.

In one embodiment, the composition contained in the first unit form of the kit comprises: Factor VII polypeptide, Mannitoi, Sucrose, methionine, Histidine, and poiysorbate, preferably poiysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5,0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the list of: CaC(2, NaCi, and Glycylgiycine. In one embodiment, the Factor VII polypeptide is human Factor Vila,

In another embodiment, the composition contained in the first unit form of the kit comprises Factor VII poiypeptide, Mannitoi, Sucrose, and Poioxamer 188, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the fist of: CaCI2, NaQ, and Glycylgiycine. In one embodiment, the Factor VII poiypeptide is human Factor Vila.

In another embodiment, the composition contained in the first unit form of the kit comprises Factor VII polypeptide, Mannitoi, Sucrose, methionine, and Poioxamer 188, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from the list of: CaC12, NaCi, and Glycylgiycine. In one embodiment, the Factor VII poiypeptide is human Factor Vila.

In another embodiment, the composition contained in the first unit form of the kit comprises Factor VII poiypeptide, Mannitoi, Sucrose, Histidine, and Poioxamer 188, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7,0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components selected from tfrefist of: Ca€i2, NaCi, and Gtycyigiydne. In one embodiment, the Factor VU poiypeptide is human Factor Vila,

In another embodiment, the composition contained in the first unit form of the kit comprises Factor VII polypeptide. Mannitol, Sucrose, Histidine, methionine, and Poioxamer 188, has a moisture content of not mom than about 3%, and has a pH in the range of 5,0 to 7.0 when the composition is dissolved in water. In one embodiment, the composition further comprises one or more components seiected from the list of; CaCI2, NaCi, and Giycyigiycine. in one embodiment, the Factor VII polypeptide is human Factor Vila.

In one embodiment, the administration vehide contained in the second unit form of the kit comprises: Water, and histidine. In a further embodiment, the vehide further comprises one or more components seiected from the iist of; CaC!2, NaCI, and Giycyigiycine. In a further embodiment thereof, the vehicle comprises one or more components seiected from the iist of:

CaC12 in a concentration of about 5-15 mM, NaCI in a concentration of about 30 to 60 mM, such as, e.g., about 40mM or about SOmM.

In a preferred embodiment, the method for preparing a stable Factor VII polypeptide comprises freeze-drying. The freeze-drying relates to a process, wherein the solution comprising said Factor VU polypeptide 1$ Riled into iyophifisation vials or the like. Said Factor VII polypeptide may optionally be subjected to sterile filtration before start of freeze-drying. Cooling is applied to the shelves of the freeze-drier in order to freeze the vials and the solution below critical product temperatures. Water Is removed by introducing vacuum and condensation of water vapour on the ice-condenser of the freeze-drier. When the product is dry, usuaiiy less than 3% residual moisture content: (e.g., measured by Kart Fischer couiometric titration as described above), the viais are dosed and capped. Manufacturing is finalised and the composition is now in a form of a iyophilised cake.

The reconstituted compositions are intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenteralSy, he., intravenously, subcutanousiy, or Intramuscularly, or they are administered by way of continuous or puisative infusion.

Therefore, a stili further aspect of the invention relates to the use of the solid stabilised composition for the preparation of a medicament for treating a coagulation factor-responsive syndrome. In one embodiment, the invention reiates to the use of Factor VH polypeptide for the preparation of a medicament for treating a Factor VII-responsive syndrome. in different embodiments, said Factor VII-responsive syndrome is haemophilia A, haemophilia B, Factor XI deficiency, Factor VII deficiency, thrombocytopenia, von Wiiiebrand's disease, presence of a clotting Factor inhibitor, surgery or trauma. Additionally, Factor VII-responsive syndrome may be associated with anticoaguiant therapy.

As stated the compositions of the invention is in soitd form. Accordingly, in a suitable embodiment the medicament should be suitable for being dissolved, which aiiows for parenterai administration of the medicament. Thus, when administering the compositions to a patient, it comprises the step of dissolving the composition in a suitable liquid prior to the administering step.

Abbreviations used herein: FVXX: Coaguiation Factor VII in its single chain form FVIIa: Coaguiation Factor VII in its cleaved, activated two-chain form rFVII (rFVIIa): Recombinant Factor VII (recombinant Factor Vila)

Embodiments:

In one series of embodiments of the invention, the first unit form of the kit comprises the excipients, and amounts thereof, and has the pH as shown in the list of formuiations I to 48: (Tabie i)

5 In another series of embodiments, the first unit form of the kit comprises the excipients, and , amounts thereof, and has the pH as shown in the fist of formulations 100 to 124; (Table 2)

In one series of embodiments, the concentration of FVII polypeptide In any one of compositions 1 to 48 and IOC to 124 is from about 0.6 mg/mL to about 10,0 mg/ml, such as from about 0-6 mg/ml to about 8 mg/ml, from about 0.6 mg/mL to about 5 mg/mL, from about 0.6 mg/mL to about 3 mg/ml, from about 1.0 mg/ml to about 5 mg/ml, or from about 1,0 mg/ml to about 3 mg/mL , ,,

In another series of embodiments, the concentration of FVII polypeptide in any one of compositions 1 to 48 and 100 to 124 is selected from the fist of: about 0,6 mg/ml, about 0,7 mg/ml, about 0.8 mg/mL, about 0.9 mg/ml, about 1.0 mg/ml, about l.l mg/ml, about 1.2 mg/ml, about 1,3 mg/ml, about 1,4 mg/ml, about 1.5 mg/ml, about 1,6 mg/ml, about 1,7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2,1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2,6 mg/ml, about 2,7 mg/ml, about 2.8 mg/ml, about 2,9 mg/ml, about 3.0 mg/ml, about 3,1 mg/ml, about 3.2 mg/ml, about 3,3 mg/mL, about 3.4 mg/ml, about 3.5 mg/ml, about 3,6 mg/ml, about 3.7 mg/ml, about 3.8 mg/mL, about 3.9 mg/ml, and about 4,0 mg/ml

In one series of embodiments, the concentration of polysorbate in formulations 1 to 48 and 100 to 124 is from about 0.05 to 0,08 mg/ml, such as, from about 0,06 to 0,08 mg/ml, or about 0.07 mg/ml.

In a another series of embodiments, the first unit forms comprise a composition as described in any one of compositions 1 to 48 and 100 to 124, and the second unit form comprises L-histidine in an amount of from about 0.5 mg/ml to 3 mg/ml, such as, from about 1.0 to about 2 .0 mg/ml, or about 1.55 mg/ML,

In yet another series of embodiments, the first unit forms comprise a composition as described in any one of compositions 1 to 48 and 100 to 124, and the second unit form comprises from about 30 to about 60 mM Nad, such as, about 40 mM, about 45 mM, or about 50 mM NaCI.

In yet another series of embodiments, the first unit forms comprise a composition as described in any one of compositions 1 to 48 and 100 to 124, and the second unit form comprises from about 30 to about 60 mM NaCi, such as, about 40 mM, about 45 mM, or about 50 mM NaO; and L-histidine in an amount of from about 0,5 mg/ml to 3 mg/ml, such as, from about 1.0 to about 2.0 mg/ml, or about 1.55 mg/ML.

Another aspect of the invention is the provision of novei compositions comprising a Factor VII polypeptide and at ieast one stabilizing agent selected from the group consisting of a) a combination of an antioxidant and mannitol; b) a combination of methionine and a poiyoi; c) a combination of a saccharide and mannitol; d) a combination of sucrose and a poiyoi; e) methionine; and a poiysorbate surfactant in an amount of from about 0,06 to 0,08 mg/ml; said composition having a moisture content of not more than about 3%;

In one embodiment, the combination of an antioxidant and mannitol (a) further comprises a saccharide. In another embodiment, the combination ©f methionine and a poiyoi {b> further comprises a saccharide. In another embodiment, the combination of a saccharide and mannitol (c) further comprises an antioxidant. In another embodiment, the combination of sucrose and a poiyoi {d> further comprises an antioxidant.

In an interesting embodiment, the antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, giuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giuthatione, or mixtures thereof; preferably methionine, or mixtures containing methionine. In another interesting embodiment, the the saccharide is selected from the group consisting of sucrose, dextrose, lactose, maltose, trehalose, cyciodextrins, maltodextrins and dextrans, or mixtures thereof; preferably sucrose, or mixtures containing sucrose. In yet another interesting impediment, the poiyoi is selected from the group consisting of mannitol, sorbitol and xyiitoi, or mixtures thereof; preferably mannitol, or mixtures containing mannitol

The compositions may further comprise an agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent. Non-limiting examples of such agents as wei! as preferred pH ranges have been described above.

The composition may further comprise a tonicity modifier, Non-iimitingexamples of such tonidty modifiers have been described above.

The polysorbate surfactant is selected from the group consisting of pofysorbate 20 or 80, preferably potysorbate 80,

In one embodiment of the invention, the novel compositions are selected from the list of; {Table 3)

In one embodiment thereof, the compositions are selected from the iist of; (Table. 4}

In one embodiment thereof, the compositions are selected from the list of: (Table 5}

In one series of embodiments, the concentration of poiysorbate in formulations I to XVI is from about 0.05 to 0,08 mg/mt, such as, from about 0.06 to Ö.08 mg/mL, or about 0,07 mg/mL

In one series of embodiments, the poiysorbate in formulations I to XVI is poiysorbate 20, e,g. Tween 28™. In one series of embodiments, the FVII polypeptide in formulations I to XVI is poiysorbate SO, e.g., Tween 80w.

In yet another embodiment, the compositions are selected from the list of: (Table 6) _____ . - -.....

_.......................................... ”

Itable 6> contTI).......................................................... "

' ' ””

In one series of embodiments, the formulations I to XXVIII further comprise one or more components selected from the list of:

Ca2+, preferably in an amount of from about 5 to about IS rnM, such as about 10 mM, and preferably as CaD2 x2H20;

NaCi, preferably in an amount of about SO mM, or about 40 mM, e g., 39 mM;

Histidine, preferably L-Histidine, preferably in an amount of about 10 mM; and Giycyiglycine, e.g,, in an amount of about 10 mM

In another series of embodiments, the compositions contain the excipients, and amounts thereof, as described in any one of Formulations I to XXVIII but has a pH of pH 5.5, or 5.6, or 5.7, or, 5,8, Or S.9, Or 6,1, or 6,2, or 6.3, or 6.4, or 6,5,

In one series of embodiments, the concentration of FVii polypeptide in formulations I to XVI is about 1,0 mg/ml.

In one series of embodiments, the FVII polypeptide in formulations I to XXVm is wild-type human factor Vila.

In one series of embodiments, the FVII polypeptide in formulations I to XXVIII is a FVII variant.

In different series of embodiments, the FVH polypeptide in formulations i to XXVHI is selected from the list of: L305V-FVH, 13Ö5V/M306Ö/D3Q9S-FVIÏ, L305I-FVÏI, L3Ö5T-FVH, F374P-FVH, V158T/M29SQ-FVII, V158D/E296V/M298Q~FVIÏ, K337A-PVII, M298Q-FVH, V1S8D/M298Q-FVII, L3Q5V/K337A-FVI1, V158D/E296V/M298Q/L305V-FVII, V158D/E296V7M298Q/'K337A-FVII, V158D/E296V/M298Q/t305V/K337A~FVII, K157A-FVH, E296V-FVH, E296V/M298Q-FVIÏ, V158D/E296V-FVII, V158D/M298K-FVÏ1, and S336G--FVII;

In different series of embodiments, the Factor VII polypeptide in any one of compositions 1 to 48 and 100 to 124 Is selected from the list of: From about 0.6 mg/ml to about 10.0 mg/ml, such as from about 0.6 mg/ml to about 8 mg/ml, from about 0,6 mg/ml to about 6 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, from about 0,6 mg/ml to about 4 mg/ml, from about 0.6 mg/ml to about 3 mg/ml, from about 1.0 mg/ml to about 5 mg/ml, from about 1.0 mg/ml to about 4 mg/ml, from about 1.0 mg/ml to about 3 mg/ml, about 0.6 mg/ml, about 0,7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1,0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1,8 mg/mL, about 1.9 mg/ml, about 2,0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2,3 mg/ml, about 2,4 mg/ml, about 2,5 mg/ml, about 2,6 mg/ml, about 2.7 mg/ml, about 2,8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about 3,1 mg/ml, about 3.2 mg/ml, about 3,3 mg/ml, about 3.4 mg/ml, about 3.5 mg/ml, about 3.6 mg/ml, about 3.7 mg/ml, about 3,8

mg/ml, about 3,9 mg/ml, and about 4.0 mg/mL

In another aspect, the invention provides a method of preparing the novel compositions defined in Tables 3 to 6, comprising the steps of: i) providing a Factor VII polypeptide irt a soiution comprising at teast one stabilizing agent selected from the group consisting of a) a combination of an antioxidant and mannitoi; b) a combination of methionine and a poiyoi; c) a combination of a saccharide and mannitoi; d) a combination of sucrose and a poiyoi; and e) methionine is) processing said soiution so as to obtain a solid composition with a moisture content not more than about 3 % w/w.

In one embodiment, the poiyoi is present in an amount ranging from about 0,5 to about 75 mg/mi, preferabiy from about 2 to 45 mg/mi, such as from about 5 mg/ml to 45 mg/mi, from about 5 to 35 mg/ml, from about 5 to 25 mg/ml, 5 to 20 mg/mi, 20 to 40 mg/mi, or from about 20 to about 30 mg/ml,

In one embodiment, the saccharide is present in an amount ranging from about 0,5 to 75 mg/mi, preferabiy from about 2 to 45 mg/mi, such as from about 5 mg/mf to 45 mg/mi, from about 5 to 35 mg/mi, from about 5 to 25 mg/ml, or from about 5 to 20 mg/mi,

In one embodiment, the antioxidant is in an amount ranging from about 0,05 to 10 mg/mf, preferably from about 0.1 to 5 mg/ml, more preferabiy from about 0.1 mg/ml to 2.5 mg/mi, even more preferabiy from about 0.1 to 2 mg/mi, most preferabiy from about 0.1 to 1 mg/mi.

In one embodiment, the saccharide is sucrose. In one embodiment, the antioxidant is methionine. In one embodiment, the poiyoi is mannitoi. In one embodiment, the processing comprises freezedrying.

The novel compositions of the present invention are reconstituted using an acceptable, preferabiy sterile, diluent or carrier, preferabiy an aqueous carrier. Non-limiting examples of aqueous carriers include Water for Injection (WFI) as weli as solvents as described in the present specification (above) containing at least one of tbe components selected from the fist of; (i) an agent suitable for keeping tbe pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent in an amount of from about 0.1 mM to 100 mM; and (ii) a tonicity modifying agent in an amount sufficient to make essentially isotonic the reconstituted soiution. In one embodiment, the carrier is WFI; in another embodiment, the solvent comprises histidine.

Embodiments of the invention

Embodiment 1: A kit containing a pharmaceutical medicament, said kit comprising a) a composition comprising a poiypeptide and at teast one stabilizing agent, wherein the composition has a moisture content of not more than about 3%, in a first unit ferm, and container means for containing said first unit form; and. b} in a second unit form, an administration vehicle comprising a solvent for reconstitution {solution} of said composition and at least one of the components selected from the list of: (iii) an agent suitable for keeping the pH of said composition in the range of 3 to § when dissolved In aqueous solvent, wherein the agent is present in an amount of from about 0,1 mH to 100 mM, (iv) a tonicity modifying agent In an amount sufficient to make essentially isotonic the reconstituted solution resulting from dissolving the composition of the first unit form in the administration vehicle of the second unit form; and container means for containing said second unit form.

Embodiment 2: A kit in accordance with Embodiment 1, wherein the first unit form comprises at least one component selected from the group of; surfactants, antioxidants, saccharides, and polyols.

Embodiment 3: A kit in accordance with embodiment 2 or embodiment 2, wherein the second unit form further comprises at feast one component selected from the group of: surfactants, antioxidants, saccharides, and polyols.

Embodiment 4: A kit in accordance with any one of embodiments i to 3, wherein the polypeptide is a blood coagulation factor, such as Factor VIII, Factor ÏX, Factor X, Factor II, Factor V, Factor VIL

Embodiment 5: A kit in accordance with any one of embodiments 1 to 3, wherein the polypeptide Is a vitamin K-dependent polypeptide, such as Factor VII, Factor EX, Factor X, Factor II, Protein C, Protein S, protrombln. ' -/

Embodiment 6: A kit in accordance with embodiments 4 or 5, wherein the coagulation factor polypeptide is selected from the list of; human Factor VIE, human Factor Vila, a Factor VII-reiated polypeptide, human Factor IX, human Factor X, activated human Protein C.

Embodiment ?: A kit In accordance with embodiment 6, wherein the FVII-reiated polypeptide is a factor VII variant sefected from the list of: L3Q5V-FVII, L305V/M3Ö6D/D3Ö9S-FVII, L305I-FVII, DÖ5T-FVH, F374P-FVII, V158T/M298Q-FVIÏ, V1S80/E296V/M298Q-FVÏI, K337A-FVH, M298Q-FVII, V158D/M298Q-FVII, t3Q5V/K33?A-FVn, VlS8D/E296V/M29SQ/L3fiSV-FVH, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K1S7A-FVII, E296V-FVII, É296V/F! 298Q- FVII, V158D/E296V-FVII, V158D/M298ii~FVn, and S3366-FVII.

Embodiment 8; A kit in accordance with embodiment 6, wherein the FVII-reiated polypeptide is a factor VII variant wherein the ratio between the activity of said Factor VII variant and human factor Vila (wild-type Factor VII} is at least 1.25 when tested in one or more of the "In Vitro Proteolysis Assay" and the "in Vitro Hydrolysis Assay" as described in the present specification.

Embodiment 9: A kst in accordance with any one of embodiments 1 to 8, wherein the "agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent" is present in an amount of from about Ö.imM to about SOmM; such as from about O.lmH to about 40mM; horn about O.imM to about 35mM; from about O.imM to about 30mM; from about O.SmM to about 25mM; from about IroM to about 20mM; bom about imM to about l5mM, from about 5mM to about 20mM; or from about 5mM to about 15mM.

Embodiment 10: A kit in accordance with any one of embodiments 1 to 9, wherein the "agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissoived in aqueous solvent " is selected from the list of: citric acid, acetic acid, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, imidazole, This, lactic add, giutaric add, PIPES and gtycyigiycine, or a mixture of at (east two such listed agents, wherein the mixture is able to provide a pH vaiue in the specified range.

Embodiment 11: A kit in accordance with any one of embodiments 1 to 10, wherein the second unit form comprises an agent suitable for keeping the pH of said composition in the range of 4 to 7 when dissolved in aqueous solvent; preferably in the range of 4.5 to 7.5, such as 5 to 7, or 5,5 to 6,5.

Embodiment 12 : A kit in accordance with any one of embodiments 1 to 11, wherein the second unit form comprises histidine

Embodiment 13: A kit in accordance with any one of embodiments 1 to 12, wherein the "tonicity modifying agent" is selected from the fist of: Sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, mannitol, glycerol, propylene glycol, or a mixture of at least two such listed modifying agents; preferably selected from the list of; sodium chloride mannitoi, glycerol, propylene glycol, calcium chloride, or mixtures thereof.

Embodiment 14; A kit in accordance with embodiment 13, wherein the tonicity modifying agent comprises Ca2+ or Mg2+.

Embodiment 15; A kit in accordance with any one of embodiments 1 to 14, wherein one or both of the first and second unit forms further contain a preservative.

Embodiment 16: A kit in accordance with any one of die preceding embodiments; wherein the first unit form comprises at least one stabilizing agent selected from the group consisting of a) a combination of an antioxidant and mannitol; b) a combination of methionine and a polyol; c) a combination of a saccharide and mannitol; d) a combination of sucrose and a poiyol; e) methionine; and f} a surfactant said composition having a moisture content of not mors than about 3%.

Embodiment 17: A kit in accordance with embodiment 16, wherein the combination of an antioxidant and mannitol further comprises a saccharide.

Embodiment 18: A kit in accordance with embodiment 16, wherein the combination of methionine and a poiyoi further comprises a saccharide.

Embodiment 19; A kit in accordance with embodiment 16, wherein the combination of a saccharide and mannitoi further comprises an antioxidant.

Embodiment 20: A kit in accordance with embodiment 16, wherein the combination of sucrose and a poiyoi further comprises an antioxidant.

Embodiment 21: A kit in accordance with any one of embodiments 16 or 17, wherein the combination of an antioxidant and mannitol (a) further comprises a surfactant

Embodiment 22: A kit in accordance with any one of embodiments 16 or 18, wherein the combination of methionine and a poiyoi (b) further comprises a surfactant.

Embodiment 23 : A kit in accordance with any one of embodiments 16 or 19, wherein the combination of a saccharide and mannitoi (c) further comprises a surfactant.

Embodiment 24: A kit in accordance with any one of embodiments 16 or 20, wherein the combination of sucrose and a poiyoi (d) further comprises a surfactant.

Embodiment 25: A kit in accordance with embodiment 16, wherein the stabi!i2ing agent is a combination of a surfactant and methionine (e).

Embodiment 26: A kit in accordance with any one of the preceding embodiments, wherein the antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, gfuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giutbatione.

Embodiment 27: A kit in accordance with any one of the preceding embodiments, wherein the saccharide Is selected from the group consisting of sucrose, dextrose, iactose, maltose, trehaio.se, eyefodextrins, maftodextrins and dextrans.

Embodiment 28: A kit in accordance with any one of the preceding embodiments, wherein the poiyoi is seiected from the group consisting of mannitoi, sorbltoi and xytitol.

Embodiment 29; A kit in accordance with any one of the preceding embodiments, wherein the composition of the first unit form is stable such that not more than about 5% w/w of the initial content of Factor VII polypeptide is converted to aggregates upon storage of said composition at 30 °C for 8 months, preferably not more than about 4,0% w/w, 3.0% w/w, 2.5% w/w, 2.0% w/w, 1.5% w/w, or not more than about 1,0% w/w,

Embodiment 30: A kit in accordance with any one of the preceding embodiments, wherein the composition of the first unit form is stable such that not more than about 6% w/w of the initial content of Factor VII polypeptide is converted to oxidised forms upon storage of said composition at 30 ®C for 8 months, preferably not more than about 5% w/w, 4,0% w/w, 3.0% w/w, 2,5% w/w, 2,0% w/w, or not more than about 1.5% w/w.

Embodiment 31: A kit in accordance with any one of the preceding embodiments, wherein said poiyoi is in an amount ranging from about 5% w/w to 90% w/w, preferably from about 18% w/w to 88% w/w, 18% to 83%, 25% to 80%, 40% to 80%, 50% to 80%, or from about 50% w/w to 70% w/w,

Embodiment 32: A kit in accordance with any one of the preceding embodiments, wherein said saccharide is in an amount ranging from about 0 to 85% w/w, preferably from about 3% w/w to 80% w/w, 7% to 75%, 10% to 70%, 10% to 50%, .10% to 40%, or from about 10% w/w to 35% w/w.

Embodiment 33: A kit in accordance with any one of the preceding embodiments, wherein said poiyoi is in a weight ratio relative to said saccharide ranging from about 100:1 to 1:50, preferably from about 50:1 to 1:10; 20:1 to 1:5; 10:1 to 1:2; 6:1 to 1:2; 4;i to 1:1; or from about 4:1 to 3:2,

Embodiment 34: A kit in accordance with any one of the preceding embodiments, wherein the first unit form further comprises a tonicity modifier.

Embodiment 35; A kit in accordance with any one of the preceding embodiments, wherein the surfactant is selected from toe group consisting of poiysorbates, such as polysorbate 20 or 80; polyoxyethylene aikyl ethers, such as Srij 35®; or poioxamers, such as Pofoxamer 188 or 407; and other ethylene/polypropylene block polymers or poiyethyienegtycol {PEG} such as PEG8Ö00,

Embodiment 36: The kit in accordance with any one of the preceding embodiments, wherein the saccharide is sucrose.

Embodiment 37: The kit in accordance with any one of tbe preceding embodiments, wherein the poiyoi is mannitol.

Embodiment 38: The kit in accordance with any one of the preceding embodiments, wherein Factor VII polypeptide is present in a concentration of from about 0,$ mg/mi to about 10.Ö mg/mi, such as from about 0.6 mg/mi to about 6 mg/mi, from about 0.6 mg/mi to about 5 mg/mi, or from about 0.6 mg/mi to about 4 mg/mi.

Embodiment 39: The kit to accordance with any one of the preceding embodiments, wherein said moisture content is not more than about 2,5% w/w, preferably not more than about 2% w/w, most preferably not more than about 1,5% w/w

Embodiment 40: The kit in accordance with any one of the preceding embodiments, wherein the first unit form is a iyophilised cake.

Embodiment 41: The kit in accordance with any one of the preceding embodiments, wherein the first unit ferm comprises: Factor VII poiypeptide, Mannitol, Sucrose, and a surfactant selected from a poiysorbate or a poioxamer, such as Tween 80® or Poioxamer 188®.

Embodiment 42: The kit in accordance with embodiment 41, which further contains methionine.

Embodiment 43; A kit in accordance with any one of the preceding embodiments 41 or 42, wherein the second unit form contains L-histidine in an amount of from about O.lmM to about 50mM; such as from about O.lmM to about 40mM; from about O.lmM to about 35mM; from about O.lmM to about 30mM; from about 0.5mM to about 25mM; from about ImM to about 20mM; from about ImM to about l5mM; from about 5mM to about 20mM; or from about 5mM to about 15mM.

Embodiment 44: A kit in accordance with any one of the preceding embodiments 41 to 43, wherein the second unit form further contains one or more components selected from the list oh CaC12, NaCi, and Giycyigiycine.

Embodiment 45: A method fbr preparing a liquid formuiation of a poiypeptide, the method comprising the steps of: a) providing a first and a second unit form as described in any one of embodiments 1 to 44; b) mixing said first and second unit forms so as to provide a dissolved liquid solution of the composition in the administration vehicle.

Embodiment 46: A method for treating a coagulation factor-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a liquid formulation of a coagulation factor prepared by the method of embodiment 45.

Embodiment 47: A method in accordance with embodiment 46, wherein the coagulation factor-responsive syndrome is haemophilia, and the coagulation factor is Factor VIII or Factor IX; or the syndrome is sepsis, and the coagulation factor is protein C or activated protein C,

Embodiment 48: A method in accordance with embodiment 46 for treating a FVII-responsive syndrome, comprising administering to a subject in need thereof art effective amount of a liquid formulation of said biological agent prepared by the method of embodiment 45.

Embodiment 49: The method in accordance with embodiment 48, wherein said syndrome is selected from the group consisting of haemophiiia A, haemophliia B, Factor XI deficiency, Factor VII deficiency, thrombocytopenia, von Witiebrand's disease, presence of a clotting factor inhibitor, surgery, trauma, ditutionai coagulopathy, and anticoagulant therapy.

Embodiment 50: Use of a Factor VII polypeptide for the preparation of a medicament in the form of a kit as defined in any one of embodiments I to 44 for treatment of a Factor VII-responsive syndrome.

Embodiment 51: A composition comprising a Factor VII polypeptide, and at least one stabilizing agent selected from the group consisting of a} a combination of an antioxidant and mannitol; b) a combination of methionine and a polyol; c) a combination of a saccharide and mannitol; d) a combination of sucrose and a polyol; e) methionine; and a polysorbate surfactant in an amount of from about 0,06 to 0.08 mg/rnl; said composition having a moisture content of not more than about 3%;

Embodiment 52: The composition in accordance with embodiment 51, wherein the combination of an antioxidant and mannitol further comprises a saccharide.

Embodiment 53: The composition in accordance with embodiment 51, wherein the combination of methionine and a poiyoi further comprises a saccharide.

Embodiment 54: The composition in accordance with embodiment 51, wherein the combination of a saccharide and mannitol further comprises an antioxidant.

Embodiment 55: The composition In accordance with embodiment 51, wherein the combination of sucrose and a poiyoi further comprises an antioxidant.

Embodiment 56: The composition in accordance with any one of the preceding embodiments Si to 55, wherein die antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, gluthatiooe, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and gluthatione.

Embodiment 57: The composition in accordance with any one of the preceding embodiments Si to 56, wherein the saccharide is selected from the group consisting of sucrose, dextrose, lactose, maitose, trehalose, cyclodextrins, maltodextrins and dextrans.

Embodiment 58: The composition in accordance with any one of the preceding embodiments 51 to 57, wherein the poiyol is selected from the group consisting of mannitol, sorbitol and xyftoi.

Embodiment 59: The composition in accordance with any one of the preceding embodiments 51 to 58, wherein the composition is stable such that not more than about 5% w/w of the initial content of Factor VII polypeptide is converted to aggregates upon storage of said composition at 30 *C for 8 months, preferably not more than about 4.0% w/w, 3.0% w/w, 2.5% w/w, 2.0% w/w, 1.5% w/w, or not more than about 1.0% w/w,

Embodiment 60: The composition in accordance with any one of the preceding embodiments 51 to 59, wherein the composition is stable such that not more than about 6% w/w of the initial content of Factor VH polypeptide is converted to oxidised forms upon storage of said composition at 30 *C for S months, preferably not more than about 5% w/w, 4.0% w/w, 3,0% w/w, 2.5% w/w, 2,0% w/w, or not more than about 1.5% w/w.

Embodiment 61: The composition in accordance with any one of the preceding embodiments 51 to 60, wherein said polyol is in an amount ranging from about 5% w/w to 90% w/w, preferably from about 18% w/w to 88% w/w, 18% to 83%, 25% to 80%, 40% to 80%, 50% to 80%, or from about 50% w/w to 70% w/w.

Embodiment 62: The composition in accordance with any one of the preceding embodiments 51 to 61, wherein said saccharide is In an amount ranging from about 0 to 85% w/w, preferably from about 3% w/w to 80% w/w, 7% to 75%, 10% to 70%, 10% to 50%, 10% to 40%, or from about 10% w/w to 35% w/w.

Embodiment 63: The composition in accordance with any one of the preceding embodiments 51 to 62, wherein said polyol is in a weight ratio relative to said saccharide ranging from about 100:1 to 1:50, preferably from about 50:1 to 1:10; 20:1 to 1:5; 10:1 to 1:2; 6:1 to 1:2; 4:1 to 1:1; or from about 4;1 to 3:2,

Embodiment 64: The composition in accordance with any one of the preceding embodiments 51 to 63, further comprising an agent suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent, preferably the pH is in the range of 4 to 7, more preferred in the range of 4.5 to 6,5, even more preferred in the range of 5,5 to 6,5.

Embodiment 65: The composition in accordance with embodiment 64, wherein said agent is selected from the group consisting of: citric add, acetic add, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, imidazol, lactic add, gtutaric acid, PIPES and giycylglycine, or a mixture of at (east two such listed agents, wherein die mixture (s able to provide a pH value in the specified range.

Embodiment 66: The composition in accordance with any one of the preceding embodiments 51 to 65, further comprising a tonicity modifier.

Embodiment 67: The composition in accordance with embodiment 66, wherein the tonicity modifier is selected from the group consisting of: sodium acetate, sodium factate, sodium chloride, potassium chloride, caicium chloride, mannitoi, glycerol, and propylene glycol.

Embodiment 68: The composition in accordance with any one of the preceding embodiments 51 to 67, wherein the surfactant is selected from the group consisting of poiysorbate 20 or 80, preferably polysorbate 80.

Embodiment 69: The composition in accordance with any one of the preceding embodiments SI to 68, wherein the saccharide is sucrose.

Embodiment 70: The composition in accordance with any one of the preceding embodiments 51 to 69, wherein the polyol is mannitol.

Embodiment 71: The composition in accordance with any one of the preceding embodiments 51 to 70, wherein the Factor VII Polypeptide is selected from the group consisting of Human Factor Vila, Recombinant Human Factor Vila and a Factor VII Sequence Variant.

Embodiment 72: The composition in accordance with embodiment 71, wherein the Factor VII Polypeptide is Human Factor Vila or Recombinant Human Factor Vila.

Embodiment 73: The composition In accordance with any one of the preceding embodiments 51 to 72, wherein the Factor VII Polypeptide is a Factor VÏI~reiated polypeptide wherein the ratio between the activity of said Factor Vll-related polypeptide and wild-type Factor VII is at least 1.25 when tested in one or more of the "In Vitro Proteolysis Assay" and the "in Vitro Hydrolysis Assay” as described in the present specification.

Embodiment 74: "me composition in accordance with any one of the preceding embodiments 51 to 73, wherein Factor VII polypeptide is present in a concentration of from about 0.6 mg/mi to about 10.0 mg/mi, such as from about 0,6 mg/mi to about 6 mg/mi, horn about 0.6 mg/mi to about 5 mg/ml, or from about 0,6 mg/mi to about 4 mg/mi.

Embodiment 75: The composition in accordance with any one of the preceding embodiments 51 to 74, wherein said moisture content is not more than about 2.5% w/w, preferably not more than about 2% w/w, most preferably not more than about 1.5% w/w

Embodiment 7δ; The composition in accordance with any one of the preceding embodiments 51 to 75, wherein the composition is a lyophiiised cake.

Embodiment 77: The composition in accordance with any one of the preceding embodiments, wherein the composition comprises: Factor VII polypeptide, Msnnitoi, Sucrose, and poioxamer SO, such as Tween SO®.

Embodiment 78: The composition in accordance with embodiment 77, which further contains methionine.

Embodiment 70: The composition in accordance with any one of the preceding embodiments 77 to 78, which farther contains l-histidine.

Embodiment 80; The composition in accordance with any one of the preceding embodiments 77 to 79, which further contains one or more components seiected from the iist of: Caö2, NaCi, and GiycyJglycine.

Embodiments 81: Compositions in accordance with any one of the preceding embodiments 51 to 80, seiected from the iist of:

Embodiment 82: Compositions in accordance with embodiment 81, selected from the list of;

Embodiment 83: Compositions in accordance with any one of the preceding embodiments 81 or 82, containing poiysorbate 80 in an amount of about Ö.07 mg/mL.

Embodiment 84: Compositions in accordance with any one of the preceding embodiments 81 to 83, containing FVIIa polypeptide in an amount of about 1,0 mg/mL

Embodiment 85: Compositions in accordance with any one of the preceding embodiments 81 to 84, further comprising at ieast one of the components selected from the list of: Ca2+ in an amount of about 10 mM, preferabiy as CaCi2 x2H20; NaC! in an amount of about 50 mN or about 40 mM, e.g., 39 mM; Histidine, preferabiy L-Histidine in an amount of about 10 mM,

Embodiment 86: Compositions in accordance with any one of the preceding embodiments 81 to 85, having a pH of 5.5, or 5,6, or 5.7, or, 5,8, or 5,9, or 6.0, or 6,1, or 6,2, or 6,3, or 6.4, or 6,5,

Embodiment 87: A method of preparing the compositions defined in embodiments 51 to 86, comprising the steps of: i) providing a Factor VII polypeptide in a solution comprising at ieast one stabilizing agent seiected from the group consisting of a) a combination of an antioxidant and mannitoi; b) a combination of methionine and a poiyoi; c} a combination of a saccharide and mannitoi; d) a combination of sucrose and a poiyoi; and e) methionine; and a polysorbate surfactant in an amount of from about 0,06 to 0.08 mg/mL; ii) processing said solution so as to obtain a solid composition with a moisture content not more than about 3 % w/w.

Embodiment 88: A method in accordance with embodiment 87, wherein itse antioxidant is seiected from the group consisting of homocysteine, cysteine, cystathionine, methionine, giuthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and giuthatione.

Embodiment 89: A method in accordance with embodiment 87, wherein the saccharide is seiected from the group consisting of sucrose, dextrose, iactose, maltose, trehalose, cyciodextrins, maitodextrins and dextrans.

Embodiment 90: A method in accordance with embodiment 87, wherein the poiyoi is seiected from the group consisting of mannitoi, sorbitol and xyiitol.

Embodiment 91: A method in accordance with embodiment 87, wherein the poiyoi is present in an amount ranging from about 0.5 to about 75 mg/mi, preferabiy from about 2 to 45 mg/mi, such as from about 5 mg/mt to 45 mg/mi, from about 5 to 35 mg/mi, from about S to 25 mg/ml, 5 to 20 mg/ml, 20 to 40 mg/mi, or from about 20 to about 30 mg/mi,

Embodiment 92: A method in accordance with embodiment 87, wherein the saccharide is present in an amount ranging from about O.S to 75 mg/mi, preferably from about 2 to 45 mg/ml, such as from about 5 mg/mf to 45 mg/mf, from about 5 to 35 mg/mi, from about 5 to 25 mg/ml, or from about 5 to 20 mg/mi.

Embodiment 93: A method in accordance with embodiment 87, wherein the antioxidant is in an amount ranging from about 0.05 to 10 mg/mi, preferably from about 0.1 to 5 mg/ml, more preferably from about 0.1 mg/m! to 2.5 mg/mi, even more preferabiy from about 0.1 to 2 mg/ml, most preferabiy from about 0.1 to 1 mg/mi.

Embodiment 94: A method in accordance with embodiment 87, wherein the saccharide is sucrose.

Embodiment 95: A method in accordance with embodiment 87, wherein the antioxidant is methionine.

Embodiment 96: A method in accordance with embodiment 87, wherein the poiyo) is mannitol.

Embodiment 97: A method in accordance with embodiment 87, wherein the processing comprises freeze-drying.

Embodiment 98: A method for treating a FVII-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a composition as defined in any one of embodiments 51 to 86.

Embodiment 99: The method in accordance with embodiment 98, wherein said syndrome is selected from the group consisting of haemophilia A, haemophilia 8, factor XI deficiency, Factor VII deficiency, thrombocytopenia, von Wiliebrand's disease, presence of a dotting factor inhibitor, surgery, trauma, dilutional coagulopathy, and anticoagulant therapy.

Embodiment 100: Use of Factor VII polypeptide for the preparation of a medicament for treating a Factor VII-responsive syndrome, said medicament comprising a composition as defined in any one of embodiments 51 to 86.

Embodiment 101: The use in accordance with embodiment 100, wherein said syndrome is selected from the group consisting of haemophilia A, haemophilia 8, Factor XT deficiency, Factor VII deficiency, thrombocytopenia, von Wiliebrand's disease, presence of a dotting factor inhibitor, surgery, trauma, and anticoagulant therapy.

Hie following examples are offered by way of illustration, not by way of limitation:

EXAMPLES

General methods Example 1

Analytical methods used in determining stability indicating parameters; A. Determination of Oxidised forms by Reverse Phase HPIC (RP-HPLC): HPLC Column: 4*5x250 mm column packed with butyibonded silica with a particle size of $pm and pore size 3ÖQ&amp;, Column temperature; 70=0. Eluent A; water 99.9% v/v and triffuoracetie acid 0.1 % v/v. Eluent B: acetonitrile 80% v/v. triffuoracetie acid 0,09% v/v and water 19.91 % v/v. The column was eluted with a linear gradient from X% B to (X+l3)% B in 30 minutes. Flow rate: 1.0 ml/min. Detection: 214 run,

The oxidised forms are methionine sulfoxides of Factor VII Polypeptides, For example the two main derivatives of FVII are Met(0)298 FVII and Met(O)306 FVII.

The content of oxidised forms is expressed as the percentage of the initial amount of Factor VII in the composition upon preparation that is recovered as oxidised forms of Factor VII. 8. Determination of aggregates of Factor VII polypeptides by High Performance Gel Permeation Chromatography (GP-HPLC). GP-HPLC was run on a Waters Polypeptide Pak 300 SW column, 7,5x300 mm. using 0.2 M ammoniumsulfat pH 7.0 containing 5% isopropanoi as the mobile phase. Flow rate: 0,5 mi/min and detection: 215 nm.

The content of aggregates is expressed as the percentage of the initial amount of Factor VH in the composition upon preparation that is recovered as dimeric, oligomeric and polymeric forms of Factor VII,

Example 2

Assays for testing biological activity of Factor VII polypeptides; lest. for. factar..ym.ac.ti.v.ii;y.;. A suitable assay for testing for Factor Vila activity and thereby selecting suitable Factor Vila variants can be performed as a simple preliminary in vitro test: (the "In Vitro Hydrolysis Assay").

FnVfrrojiydrojysis Assay

Native (wild-type) Factor Vila and Factor Vila variant (both hereafter referred to as "Factor Vila") may be assayed tor specific activities. They may also be assayed in parallel to directly compare their specific activities. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenlc substrate D-Ile-Pro-Arg-p-nitroanilide ($-2288, Chromogenix, Sweden), final concentration 1 mM, is added to Factor Vila (final concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0,3 M NaCl, 5 mM CaCI2 and 1 mo/ml bovine serum albumin. The absorbance at 405 nm is measured continuously in a SpectraMax™ 340 piate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank weii containing no enzyme, is used to calculate the ratio between the activities of variant and wild-type Factor Vila;

Ratio = (A^os nm Factor Vila variant)/! A40S nm Factor Vila wild-type).

Based thereon, Factor Vila variants with an activity comparable to or higher than native Factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native Factor VII (wild-type FVIÏ) is around, versus above 1.0,

The activity of Factor Vila or Factor Vila variants may aiso be measured using a physiological substrate such as Factor X, suitably at a concentration of 100-3000 nM, where the Factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. $-2765) ("the In Vitro Proteolysis Assay”). In addition, the activity assay may be run at physiological temperature.

In Vitro ProteotvsisAssav

Native (wild-type) Factor Vila and Factor Vila variant (both hereafter referred to as '‘Factor Vila") are assayed in parallel to directly compare their specific activities. The assay is carried out in a microöter plate (MaxiSorp, Nunc, Denmark). Factor Vila (30 nM) and Factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCI2 and 1 mg/mi bovine serum albumin, are Incubated for 15 min. Factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0,1 M Nad, 20 mM EDTA and 1 mg/ml bovine serum albumin. The amount of Factor Xa generated Is measured by addition of the chromogenic substrate Z-O-Arg-Gly-Arg-p-nitroanillde (S-2765, Chromogenix, Sweden), final concentration 0,5 mM, Hie absorbance at 405 nm is measured continuously in a SpectraMax™ 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtraction of tbe absorbance in a biank well containing no FVIIa, is used to calculate the ratio between the proteolytic activities of variant and wild-type Factor Vila;

Ratio = (A405 nm Factor VHa variant)/(A405 nm Factor Vila wild-type).

Based thereon, Factor Vila variants with an activity comparable to or higher than native Factor VHa may be identified, such as, for example, variants where the ratio between tire activity of the variant and the activity of native Factor VII (wild-type FVII) is around, versus above 1,0. I3mrftb.ih..sengr.aho.n..assa.y·

The ability of Factor VII or Factor VII~related polypeptides or Factor VIII or Factor VIII-reiated polypeptides (e.g., variants) to generate thrombin can be measured in an assay comprising ait relevant coagulation Factors and inhibitors at physiological concentrations and activated platelets (as described on p, 543 in Monroe et ai. (1997) Brit, 1. Haematol. 99, 542-547 which is hereby incorporated as reference), ösL35S3yi

The activity of the Factor VII poiypeptides may also be measured using a one-stage dot assay (assay 4) essentially as described in WO 92/15686 or US 5,997,864. Briefly, the sample to be tested is diluted in 50 mM Tris (pH 7.5), 0,1% BSA and 100 pL is incubated with 100 pt of Factor VII deficient plasma and 200 pi of thromboplastin C containing 10 mM Ca:;+. Clotting times are measured and compared to a standard curve using a reference standard or a pool of titrated normal human piasma in serial dilution.

Working examples Example 3

Stability data for Compositions of rFVIIa freeze dried with and without Histidine, respectively,

It will be seen that it is an advantage to freeze-dry without the presence of Histidine as less Oimers/Oligomers will be formed.

Compositions

For reconstitution of compositions Ai,€l, Ei, Gl, II, and Ki Histidine soivens 1,55 mg/ml wiil be used; for composition Bl, 01, Fl, HI, 11, and LI will be used Water for injection (WFI),

Example 4

Stability data for Compositions of rFVHa freeze dried with and without Histidine, and/or GlycyigJycme, and/or NaCi, respectively

It witi be seen that it is an advantage to freeze-dry without the presence of NaQ as this wii! decrease the risk of coiiapse of the cake.

For reconstitution, Histidine soivens 1,55 mg/ml wit! be used for Composition A2,12, and Q2; for composition B2, 12, and R2 wilt be used Water for injection (WFI).

For reconstitution, Histidine soivens 1,55 mg/ml will be used for Composition C2, K2, and S2; for composition D2,12, and T2 wilt be used Water for injection {WFi).

For reconstitution, Histidine soivens 1,55 mg/ml with NaCf 2,92 mg/ml wifi be used for Composition E2, M2, and U2; for composition F2, N2, and V2 will be used Saiine Water 2.92 mg/ml.

For reconstitution, Histidine soivens 1,55 mg/ml with NaCi 2,92 mg/ml wiii be used for Composition G2, 02, and X2; for composition H2, P2, and Y2 wifi be used Saline Water 2.92 mg/ml,

Example 5

Manufacturing of compositions in general the compositions were prepared fern a purified buik solution. Excipients were added, and the solution was diluted to the desired concentration of rFVlIa. The resulting solution was sterile filtered using a sterilised membrane filter (0,2 micron pore size or equivalent) and filled into sterfie glass viais, Tbe viais were freeze-dried, closed with rubber stoppers, and sealed with aluminium Hip-off type caps.

Example €

Comparison of content of soluble aggregates formed in formulations with and without addition of L~histldine before freeze-drying

The following formulations were prepared (all concentrations stated in mg/ml):

The formulations were prepared as described in example 5 using a filling volume of S.3 mi. After freeze-drying the contents of dimer, oligomer, and polymer forms were measured by GP-HPIC as described in example 3. The resuits are stated below:

Hie resuits show that formulations 1 and 2 without addition of L-histidine had Sower contents of dimer and oiigomer forms after freeze-drying as compared to the corresponding formulations 3 and 4, which contained L-hlstidine.

Exampie 7

Stability of a freeze-dried rFVXIa formulation A formulation containing rFVIIa 1.0 mg/mi

NaCi 2.34 mg/mi

CaCt2, 2H20 1,47 mg/ml

Giycylgiycine 1.32 mg/mi

Polysorbate 80 0.07 mg/mi t-Methionine 0.5 mg/mi

Mannitol 25 mg/ml

Sucrose 10 mg/ml pH 6.0 was prepared by as described in example S using filling volumes of 1.1 ml.

The formulation was placed at 25°C and 40°C in darkness. Sampfes were collected according to the tables below. After reconstitution of the freeze-dried product in 10 mH L-histidine solvent, the contents of dlmer/otlgomer/polymer and oxidised forms were measured as described in exampie 3, while the activity was determined by one-stage clot assay as described in exampie 4.

n.d.: not determined Example 8

The influence of sodium chloride on the vtsua! appearance of the freeze dried cake was investigated in a factorial design study. Among the variable parameters was the content of sodium chloride. The result of four formulations included in the study is shown in the table

Ail formulations further contained: rFVIIa 1.0 mg/ml

Calcium chloride 1,47 mg/ml

Gtycylglycine 1,32 mg/ml

Potysorbate 80 O.i mg/ml

Mannitol 40 mg/ml

Sucrose 10 mg/ml

In addition formulations 1 and 2 contained 0,5 mg/ml methionine, pH was adjusted to; 6.0 (formulations 1 and 4} 5.0 (formulations 2 and 3} example 9

Stability data for Compositions of FVII polypeptide

It will be seen that the compositions are stable with regard to formation of dimers/ollgomers after storage at 25"C for 6 months.

Compositions

For reconstitution of compositions A-9 to H-9 water for injection (WFI) wiii be used.

For reconstitution of compositions 8-9, D-9, F~9, and H-9 Histidine soivens 1.55 mg/ml may also be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A-9 to H-9 but having a pH of 4.5 were atso prepared. Furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9 to H-9 but having a pH of 5.5 were also prepared.

For reconstitution of compositions A-9-ί to H-9-1 Water for injection (WFI) wiii be used.

For reconstitution of compositions B-9-1, D-9-1, F-9-1, and H-9-1 Histidine solvens 1.5S mg/mL may aiso be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A 9-1 to H-9-1 but having a pH of 4.5 were also prepared. Furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9-1 to H-9-1 but having a pH of 5.5 were aiso prepared.

For reconstitution of compositions A-9-2 to H-9-2 Water for injection (WFI) wili be used.

For reconstitution of compositions 8-9-2, D-9-2, F-9-2, and H-9-2 Histidine soivens 1.55 mg/ml may aiso be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A-9-2 to H-9-2 but having a pH of 4,5 were aiso prepared, furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9-2 to H-9-2 but having a pH of 5,5 were aiso prepared.

For reconstitution of compositions A-9-3 to H-9-3 Water for injection (WFI) wilt be used.

For reconstitution of compositions 8-9-3, 0-9-3, F-9-3, and H-9-3 Histidine soivens 1.55 mg/mi. may also be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A-9-3 to H-9-3 but having a pH of 4,5 were aiso prepared. Furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9-3 to H-9-3 but having a pH of 5.5 were aiso prepared.

For reconstitution of compositions A-9-4 to H-9-4 Wafer for injection {WFI} wifi be used.

For reconstitution of compositions 8-9-4, 0-9-4, F-9-4, and H-9-4 Histidine soivens l.SS mg/mt may also be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A-9-4 to H-9-4 but having a pH of 4.5 were atso prepared. Furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9-4 to H-9-4 but having a pH of S.S were also prepared.

For reconstitution of compositions A-9-5 to H-9-5 Water for injection (WFI) wiit be used.

For reconstitution of compositions 8-9-5* D-9-5, F-9-5, and H-9-5 Histidine soivens 1,55 mg/ml may also be used.

Compositions containing the same ingredients and amounts of ingredients as compositions A-9-5 to B-9-5 but having a pH of 4.5 were also prepared. Furthermore, compositions containing the same ingredients and amounts of ingredients as compositions A-9-5 to H-9-5 hut having a pH of 5.5 were aiso prepared.

Claims (20)

1. A kit comprising: a) in a first unit form, a composition comprising human Factor Vila and a combination of a saccharide, mannitol, CaCI2 and a surfactant wherein the composition has a moisture content of not more than 3%, and container means for containing said first unit form; and b) in a second unit form, an administration vehicle comprising a solvent for reconstitution (solution) of said composition comprising: (i) histidine suitable for keeping the pH of said composition in the range of 3 to 9 when dissolved in aqueous solvent, wherein the histidine is present in an amount of from 0.1 mM to 100 mM; and, optionally, (ii) a tonicity-modifying agent in an amount sufficient to make essentially isotonic the reconstituted solution resulting from dissolving the composition of the first unit form in the administration vehicle of the second unit form, and container means for containing said second unit form, when used to produce a reconstituted human Factor Vila solution.

2. A kit according to claim 1, wherein the second unit form comprises histidine suitable for keeping the pH of said composition in the range of 4 to 7 when dissolved in aqueous solvent; preferably in the range of 4.5 to 7.5, such as 5 to 7.

3. A kit according to claim 1 or claim 2, wherein the second unit form comprises histidine suitable for keeping the pH of said composition in the range of 5.5 to 6.5.

4. A kit according to any one of the preceding claims, wherein the second unit form comprises histidine in an amount of from about 0.1mM to about 50mM; from 0.1mM to 40mM; from 0.1mM to 35mM; from 0.1mM to 30mM; from 0.5mM to 25mM; from 1mM to 20mM; from 1mM to 15mM; or from 5mM to 20mM.

5. A kit according to any one of the preceding claims, wherein the second unit form comprises histidine in an amount of from about from 5mM to 15mM.

6. A kit according to any one of the preceding claims, wherein the composition contained in the first unit form of the kit further comprises an antioxidant.

7. A kit according to any one of the preceding claims, wherein the surfactant is a poloxamer or a polysorbate.

8. A kit according to claim 6, wherein the antioxidant is selected from the group consisting of homocysteine, cysteine, cystathionine, methionine, gluthatione, and peptides containing any one of homocysteine, cysteine, cystathionine, methionine and gluthatione.

9. A kit according to claim 8, wherein the antioxidant is methionine.

10. A kit according to any one of the preceding claims, wherein the saccharide is selected from the group consisting of sucrose, dextrose, lactose, maltose, trehalose, cyclodextrins, maltodextrins and dextran.

11. A kit according to any one of the preceding claims, wherein the saccharide is sucrose.

12. The kit according to any one of the preceding claims, wherein human Factor Vila is present in a concentration of from about 0.6 mg/ml to about 10.0 mg/ml, such as from about 0.6 mg/ml to about 6 mg/ml, or from about 0.6 mg/ml to about 5 mg/ml in said reconstituted Factor Vila solution.

13. The kit according to any one of the preceding claims, wherein human Factor Vila is present in a concentration of from about 0.6 mg/ml to about 4 mg/ml in said reconstituted Factor Vila solution.

14. A kit according to any one of the preceding claims, wherein the composition contained in the first unit form of the kit comprises: human Factor Vila; Mannitol; Sucrose; and polysorbate, preferably polysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water.

15. A kit according to any one of the preceding claims, wherein the composition contained in the first unit form of the kit comprises: human Factor Vila; Mannitol; Sucrose; methionine; and polysorbate, preferably polysorbate 20 or 80, has a moisture content of not more than about 3%, and has a pH in the range of 5.0 to 7.0 when the composition is dissolved in water.

16. A kit according to claim 14 or claim 15, wherein said composition further comprises one or more components selected from the list of NaCI and Glycylglycine.

17. A kit according to claim 16, wherein in said reconstituted Factor Vila solution: the human Factor Vila is present at a concentration of about 1 mg/ml; NaCI is present at a concentration of about 40mM (2.34 mg/ml); CaCI2 dihydrate is present at a concentration of about 10mM (1.47 mg/ml); Glycylglycine is present at a concentration of about 10mM (1.32 mg/ml); Mannitol is present at a concentration of about 25mg/ml; Sucrose is present at a concentration of about 10mg/ml; L-methionine is present at a concentration of about 0.5 mg/ml; and polysorbate 80 is present at a concentration of about 0.07 mg/ml, and L-histidine is present at a concentration of about 10mM (1.55mg/ml), wherein said solution has a pH of about 6.

18. A method for preparing a liquid formulation of human Factor Vila, the method comprising the steps of: a) providing a first and a second unit form as defined in any one of claims 1 to 17; and b) mixing said first and second unit forms so as to provide a dissolved liquid solution of the composition in the administration vehicle.

19. A method of treating a Factor VII factor-responsive syndrome, comprising administering to a subject in need thereof an effective amount of a liquid formulation of human Factor Vila prepared by the method of claim 18, wherein said syndrome is selected from the group consisting of: acquired or congenital haemophilia A; acquired or congenital haemophilia B; Factor XI deficiency; Factor VII deficiency; Glanzmann thrombastenia; thrombocytopenia; von Willebrand’s disease; presence of a clotting factor inhibitor, such as inhibitors to factors VIII or IX; surgery; trauma; dilutional coagulophathy; and anticoagulant therapy.

20. Use of a liquid formulation of human Factor Vila prepared by the method of claim 18 in the manufacture of a medicament for treating a Factor Vll-responsive syndrome, wherein said syndrome is selected from the group consisting of acquired or congenital haemophilia A; acquired or congenital haemophilia B; Factor XI deficiency; Factor VII deficiency; Glanzmann thrombastenia; thrombocytopenia; von Willebrand’s disease; presence of a clotting factor inhibitor, such as inhibitors to factors VIII or IX; surgery; trauma; dilutional coagulophathy; and anticoagulant therapy.