KR101006070B1 - Liquid composition of factor vii polypeptides - Google Patents

Abstract

(Iv) Factor Ⅶ Polypeptide

(Ii) a buffer suitable for maintaining a pH in the range of about 4 to about 8

(Iii) a buffer selected from the list of calcium salts, magnesium salts or mixtures thereof, wherein the concentration of (iii) is at least 15 mM.

Factor VIII Polypeptide

Description

LIQUID COMPOSITION OF FACTOR VII POLYPEPTIDES

The present invention relates to liquid aqueous compositions containing Factor VII polypeptides and methods of making and using such compositions. More specifically, the present invention relates to liquid compositions stabilized against chemical and / or physical degradation.

 Many factors related to the blood coagulation process have been identified, including factor VII and plasma glycoproteins. Hemostasis begins with the formation of a complex between tissue factor (TF) exposed to circulating blood and F 혈관 a present in the blood in an amount corresponding to about 1% of the total protein level after damage to the blood vessel wall. FVIIa is predominantly present in the plasma as a single chain simogen, which is cleaved by FXa into a two-chain activated form of FVIIa. Recombinant activated factor VIIa (rFVIIa) has developed into a prostatic hemostatic agent. Administration of rFVIIa provides a fast and highly effective pro-hemostatic response in bleeding subjects who cannot be treated with coagulation factor products due to antibody formation. Bleeding subjects with factor VII deficiency or those with normal coagulation but with excessive bleeding can also be successfully treated with FVIIa.

It is desirable to have a dosage form of factor VIIa suitable for storage and delivery. Ideally, the drug product is stored and administered as a liquid. Alternatively, the drug product is lyophilized, ie freeze-dried and then reconstituted by addition of an appropriate diluent just prior to use in the patient. Ideally, the drug product has sufficient stability to maintain for long periods of storage, i.e., 6 months or more.

The decision of whether to keep the terminated drug product in liquid or lyophilized is usually based on the stability of the protein drug in that form. Protein stability can be affected by factors such as ionic strength, pH, temperature, repeated cycles of freeze / thaw and exposure to shear stress. Active proteins are lost as a result of physical instability, including denaturation and aggregation (soluble and insoluble aggregate forms) and chemical instability, which can name minorities, for example, hydrolysis, deaminoation and oxidation. For a general review of the stability of protein drugs, see, eg, Manning, et al., Pharmaceutical Research 6: 903-918 (1989).

While the possible occurrence of protein instability is widely accepted, it is not possible to predict specific instability problems of specific proteins. Such instability results in protein by-products or derivatives having low activity, increased toxicity and / or increased immunogenicity. In essence, protein precipitation leads to thrombosis, a syringe that is disproportionate in dosage form and amount.

In addition, post-translational modifications such as, for example, gammacarboxylation of certain glutamic acid residues at the N-terminus and addition of carbohydrate side branches, provide a potential site that is sensitive to changes in storage. In addition, specifically for factor VIIa, with serine proteases, fragmentation due to autocatalysis occurs (enzymatic degradation). Thus, the stability and efficacy of the protein composition are directly related to the stability. Maintaining stability in liquid form is generally different from lyophilized form because of the greatly increased likelihood of molecular motion and thus the increased likelihood of molecular interaction. Maintaining stability in concentrated form is also different due to the tendency to aggregate form at increased protein concentrations.

Many factors are taken into account when developing liquid compositions. For short periods of time, up to six months, liquid stability generally depends on avoiding overall structural changes such as denaturation and aggregation. This process is described in the literature for many proteins and there are many examples of stabilizers. Buffers that are effective in stabilizing one protein are known to actually act to destabilize another. Once a protein is stable to overall structural changes, developing a liquid composition with long term stability (eg, 6 months or longer) is more dependent on stabilizing the protein from the type of degradation specific for that protein. More specific types of degradation include, for example, disulfide bond mixing, oxidation of certain residues, deaminoation, cyclization. It is not always possible to point out each type of degradation, but assays have been developed to adjust for minor changes in order to adjust the ability of specific excipients to stabilize only that protein.

In addition to considering safety, excipients are generally selected that have been approved by various global medical coordination agencies. The pH of the composition is preferably in a physiologically appropriate range upon injection / infusion, otherwise pain and anxiety of the patient results.

For a general review of protein compositions, see, for example, Cleland et al .: The development of stable protein compositions: A closer look at protein aggretation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10 (4): 307-377; and Wang et al., Parenteral compositions of proteins and peptides: Stability and stabilizers, Journal of Parenteral Science and Technology 1988 (Supplement), 42 (2S).

The other literature on stabilization of proteins follows.

U. S. 20010031721 A1 (American Home Productss) relates to Factor IX compositions that are highly concentrated, lyophilized and liquid.

U. S. 5,770, 700 (Genetics Institute) is associated with the Liquid Factor IX composition.

WO 97/19687 (American Red Cross) relates to liquid compositions of plasma proteins, in particular Factor VII and Factor IX.

US Pat. No. 4,297,344 discloses coagulation factors II and VIII, antithrombin III and plasminogen against heat by adding selected amino acids such as glycine, alanine, hydroxyproline, glutamine and aminobutyric acid and carbohydrates such as monosaccharides, oligosaccharides or sugar alcohols. Stabilization of is started.

Factor VIIa undergoes several degradation pathways, in particular aggregation (dimerization), oxidation and autolysis cleavage (cutting the peptide backbone). In addition, precipitation occurs. Many of these reactions can be significantly slowed by removing water from the protein. However, the development of aqueous compositions for factor VIIa has the advantage of eliminating restoration errors, thus increasing administration accuracy and clinically simplifying the use of the product to increase patient compliance. Ideally, the composition of factor Xa should be stable for at least 6 months for a wide range of protein concentrations. This makes the method of administration flexible. In general, more highly concentrated forms allow for the administration of smaller volumes, which is highly desirable from the point of view of the patient. Liquid compositions have an advantage over lyophilized products in terms of ease of administration and use.

Today, the only commercially available, recombinantly produced FVII polypeptide composition is a lyophilized Factor FVIIa product reconstituted prior to use; It contains a relatively low factor VIIa concentration, for example about 0.6 mg / ml. A small bottle of NovoSeven (Novo Nordisk A / S, Denmark) (1.2 mg) contains 1.2 mg recombinant human factor VIIa, 5.84 mg NaCl, 2.94 mg CaCl ₂ , 2 H ₂ 0, 2.64 mg GlyGly, 0.14 mg polysorbate 80 And 60.0 mg mannitol; It is reconstituted to pH 5.5 with 2.0 ml water for injection (WFI). Once reconstituted, the protein solution is stable for use for 24 hours. Thus, liquid or concentrated Factor VII products for use are not currently available.

Therefore, there is a need in the art for a method for improving the stability, increasing concentrations, maintaining activity levels and providing liquid compositions suitable for storage, including human factor VIIa (chemical and / or physical stability). . Accordingly, it is an object of the present invention to provide an aqueous Factor VIII polypeptide composition that provides an acceptable adjustment of chemical and / or physical degradation products, such as enzymatic degradation or autocatalytic products.

Summary of the invention

The inventors have found that factor VII or an analog thereof (“factor VII polypeptide”) and calcium or magnesium salts or mixtures thereof are in a pH range of about 4 to about 8 when formulated in aqueous solution with buffer at a concentration of at least 15 mM. It was found to be stable.

In one aspect, the present invention provides a pharmaceutical composition comprising (i) a Factor VII polypeptide, (ii) a buffer suitable for maintaining a pH in the range of about 4.0 to about 8.0; (iii) providing a liquid aqueous composition comprising a buffer selected from the list of calcium salts, magnesium salts or mixtures thereof, and the concentration of (iii) is at least 15 mM.

In another embodiment, the buffer (iii) is present at a concentration of at least about 25 mM, such as 50 mM, 100 mM, 200 mM, 400 mM, 800 mM, 900 mM or at least 1000 mM.

In another embodiment, the composition further comprises (iv) an ionic strength modifier.

In another embodiment, the ionic strength modifiers are: neutral salts such as sodium chloride; amino acid ; Or a small peptide or a mixture of at least two of the above modifiers. In a preferred embodiment, the ionic strength modifier is sodium chloride.

In other embodiments, the buffer (iv) is such as 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 400 mM, 800 mM, 1000 mM, 1200 mM, 1500 mM, 1800 mM, 2000 mM or at least 2200 mM. Present at a concentration of at least about 5 mM.

In a series of embodiments, the buffer (iii) (calcium and / or magnesium salt) is about 25 mM to about 1000 mM, about 50 mM to about 1000 mM, about 100 mM to about 1000 mM, about 200 mM to about 1000 mM , About 300 mM to about 1000 mM, about 400 mM to about 1000 mM, about 500 mM to about 1000 mM, about 600 mM to about 1000 mM, about 700 mM to about 1000 mM; About 15 mM to about 800 mM, about 25 mM to about 800 mM, about 50 mM to about 800 mM, about 100 mM to about 800 mM, about 200 mM to about 800 mM, about 300 mM to about 800 mM, from about 400 mM to about 800 mM, about 500 mM to about 800 mM; About 15 mM to about 600 mM, about 25 mM to about 600 mM, about 50 mM to about 600 mM, about 100 mM to about 600 mM, about 200 mM to about 600 mM, about 300 mM to about 600 mM; And from about 15 mM to about 1000 mM, such as about 15 mM to about 400 mM, about 25 mM to about 400 mM, about 50 mM to about 400 mM, or about 100 mM to about 400 mM.

In a series of embodiments, the buffer (iv) (ion intensity modifier) is about 25 mM to about 2200 mM, about 50 mM to about 2200 mM, about 100 mM to about 2200 mM, about 200 mM to about 2200 mM, about 400 mM to about 2200 mM, about 600 mM to about 2200 mM, about 800 mM to about 2200 mM, about 1000 mM to about 2200 mM, about 1200 mM to about 2200 mM, about 1400 mM to about 2200 mM, about 1600 mM To about 2200 mM, about 1800 mM to about 2200 mM or about 2000 mM to about 2200 mM; About 5 mM to about 1800 mM, about 25 mM to about 1800 mM, about 50 mM to about 1800 mM, about 100 mM to about 1800 mM, about 200 mM to about 1800 mM, about 400 mM to about 1800 mM, about 600 mM to about 1800 mM, about 800 mM to about 1800 mM, about 1000 mM to about 1800 mM, about 1200 mM to about 1800 mM, about 1400 mM to about 1800 mM, about 1600 mM to about 1800 mM; About 5 mM to about 1500 mM, about 25 mM to about 1400 mM, about 50 mM to about 1500 mM, about 100 mM to about 1500 mM, about 200 mM to about 1500 mM, about 400 mM to about 1500 mM, about 600 mM to about 1500 mM, about 800 mM to about 1500 mM, about 1000 mM to about 1500 mM, about 1200 mM to about 1500 mM; About 5 mM to about 1200 mM, about 25 mM to about 1200 mM, about 50 mM to about 1200 mM, about 100 mM to about 1200 mM, about 200 mM to about 1200 mM, about 400 mM to about 1200 mM, about 600 and from about 5 mM to about 2200 mM, such as from about mM to about 1200 mM or from about 800 mM to about 1200 mM.

In one preferred embodiment, the total concentration of buffers (iii) and (iv) is about 100 mM to about 2500 mM, about 200 mM to about 2500 mM, about 400 mM to about 2500 mM, about 600 mM to about 2500 mM, About 800 mM to about 2500 mM, about 1000 mM to about 2500 mM, about 1200 mM to about 2500 mM, about 1400 mM to about 2500 mM, about 1600 mM to about 2500 mM, about 1800 mM to about 2500 mM or about 2000 mM to about 2500 mM; About 50 mM to about 2000 mM, about 100 mM to about 2000 mM, about 200 mM to about 2000 mM, about 400 mM to about 2000 mM, about 600 mM to about 2000 mM, about 800 mM to about 2000 mM, about 1000 mM to about 2000 mM, about 1200 mM to about 2000 mM, about 1400 mM to about 2000 mM or about 1600 mM to about 2000 mM; About 50 mM to about 1600 mM, about 100 mM to about 1600 mM, about 200 mM to about 1600 mM, about 400 mM to about 1600 mM, about 600 mM to about 1600 mM, about 800 mM to about 1600 mM, about 1000 and from about 50 mM to about 2500 mM, such as from about mM to about 1600 mM or from about 1200 mM to about 1600 mM.

In one embodiment, the buffers (iii) and (iv) are present at a concentration of about 600 to about 800 mM of (iii) and 0 to about 5 mM of (iv); In another embodiment, buffers (iii) and (iv) are present at a concentration of about 300 to about 500 mM of (iii) and about 1100 to 1300 mM of (iv); In another embodiment, buffers (iii) and (iv) are present at a concentration of about 100 to about 300 mM of (iii) and at a concentration of about 1500 to about 1900 mM of (iv); In another embodiment, the buffers (iii) and (iv) are present at a concentration of about 50 to about 150 mM of (iii) and at a concentration of about 1800 to about 2300 mM of (iv).

In other embodiments, the ionic strength of the composition is at least 50, such as at least 75, 100, 150, 200, 250, 400, 500, 650, 800, 1000, 1200, 1600, 2000, 2400, 2800 or at least 3200.

In another embodiment, the calcium salt is selected from the list of calcium chloride, calcium acetate, calcium gluconate and calcium laurate. In another embodiment, the magnesium salt is selected from the list of salts of magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, magnesium laurate and strong acid.

In a preferred embodiment, the buffer (iii) is selected from the list of calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate or mixtures thereof; The ionic strength modifier (iv) is sodium chloride.

In further embodiments, the composition further comprises (v) a tonicity modifier.

In another embodiment, the enteric modifier (v) is a neutral salt; Monosaccharides, disaccharides are polysaccharides; Sugar alcohols; amino acid; Or a small peptide or a list of at least two mixtures of said modifiers.

In one embodiment, the enteric modifier (v) is about 1 to about 500 mM; About 1 to about 300 mM; About 10 to about 200 mM; Or at a concentration of about 20 to about 150 mM.

In further embodiments, the composition further comprises (vi) a nonionic surfactant.

In one embodiment, the nonionic surfactant is present in an amount from about 0.005 to about 2.0% by weight.

In other embodiments, the nonionic surfactants include polysorbates or poloxamers or polyoxyethylene alkyl ethers; Preferably poloxamer 188 or poloxamer 407 or polysorbate 20 or polysorbate 80 or polyoxy 23 lauryl ether.

In further embodiments, the composition further comprises (iii) an antioxidant. In another embodiment, the antioxidant is D- or L-methionine; Methionine analogs; Methionine-containing peptides; Ascorbic acid; Cysteine; Methionine-homologs such as homocysteine; Glutathione. In a preferred embodiment, the antioxidant is L-methionine. In one embodiment, the antioxidant is present at a concentration of about 0.1 to about 5.0 mg / ml.

In one embodiment, the pH of the composition is about 4.5 to about 7.0; About 5.0 and about 7.0; About 5.5 to about 7.0; Or from about 4.0 to about 7.0, such as from about 6.0 to about 7.0.

In one embodiment, suitable buffers for maintaining pH in the range of about 4.0 to about 8.0 are citrate, acetate, histidine, malate, phosphate, tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lactate, article Lysyl-glycine, PIPES, acid and salt of glycine or a buffer selected from the list of mixtures of at least two of the above buffers.

In one embodiment, the concentration of buffer is about 1 mM to 100 mM; 1 mM to about 50 mM; About 1 mM to about 25 mM; About 2 mM to about 20 mM; Or about 10 mM.

In a further embodiment, the composition further comprises (iii) a preservative. In one embodiment, the preservative is selected from the list of phenol, benzyl alcohol ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride and benzatonium chloride

In one embodiment, the composition is isotonic; In others, it is faulty. In one embodiment, the composition is formulated for pharmaceutical administration. In one embodiment, the composition is stable and / or stabilized at 2-8 ° C. for at least 6 months.

In other embodiments, the Factor V polypeptide is human factor VIIa; Recombinant human factor VIIa; Factor VII-associated polypeptides; Factor VII sequence variants; Or the factor VII polypeptide and the activity of the factor VII polypeptide and the activity of the natural human factor VIIa (wild-type FVIIa) are at least about 1.25, preferably at least about 2.0, or when tested in an "in vitro proteolytic assay" as described herein; 4.0, most preferably at least about 8.0. In one embodiment, the Factor V polypeptide has a different glycosylation than wild type human factor VII.

In other embodiments, the factor VII polypeptide is from about 0.1 mg / ml to about 10 mg / ml; About 0.5 mg / ml to about 5.0 mg / ml; About 0.6 mg / ml to about 4.0 mg / ml; about 1.0 mg / ml to about 4.0 mg / ml; about 0.1 mg / ml to about 5 mg / ml; About 0.1 mg / ml to about 4.0 mg / ml; About 0.1 mg / ml to about 2 mg / ml; Or from about 0.1 mg / ml to about 1.5 mg / ml.

In a further aspect, the present invention also provides a composition comprising (i) a factor VII polypeptide; (ii) a buffer suitable for maintaining pH in the range of about 4.0 to about 8.0; (iii) providing a liquid, aqueous composition of Factor VII polypeptide, comprising providing Factor VII polypeptide in a solution comprising a buffer selected from the list of calcium salts, magnesium salts, or mixtures thereof (iii) ) Concentration is at least 15 mM.

In a further aspect, the present invention relates to the use of the composition to prepare a medicament for treating Factor VII-response syndrome.

In a further aspect, the present invention provides a method for treating Factor VII-responsive syndrome, i.e., (i) Factor VII polypeptides, (ii) in a range of about 4.0 to about 8.0 buffers suitable for maintaining pH; (iii) a method comprising administering to a subject in need thereof an effective amount of an aqueous liquid composition comprising a buffer selected from the list of calcium salts, magnesium salts or mixtures thereof, the concentration of (iii) being at least 15 mM.

In another embodiment, the syndrome is selected from the group consisting of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, hypoplatelets, von Willebrand's disease, the presence of a coagulation factor inhibitor, surgery, intracranial bleeding, trauma and anticoagulation therapy do.

The composition according to the invention is a composition which is useful, stable and preferably prepared for use of the factor VII polypeptide. The composition is stable up to at least 6 months and preferably 36 months when stored at a temperature in the range of 2-8 ° C. The composition is chemically and / or physically stable and especially chemically when stored at 2-8 ° C. for at least 6 months.

“Stable” refers to at least 50% of the initial biological activity after storage for 6 months at 2 to 8 ° C., after which the composition is measured in particular in one step coagulation assay (Example II) as described in WO 92/15686. It means to keep. In summary, the sample to be tested is diluted in 50 mM Tris (pH 7.5) and 100 μl of 0.1% BSA is incubated with 100 μl of Factor VIII deficient plasma and 200 μl of thromboplastin C containing 10 mM Ca ²⁺ . . Clotting time is measured and compared to a standard curve using a mixture of normal human plasma that is citrated at the reference standard or serial dilutions.

Preferably the stable composition retains at least 80% of its initial activity after storage at 2-8 ° C. for 6 months.

The term “stabilized” is used interchangeably with “relatively stable” and after storage at 2-8 ° C. for at least 6 months, the composition is subjected to the following degradation products: (i) enzymatic degradation products, (ii) aggregates ( Dimers, oligomers, polymers), (iii) oxidized forms, and (iv) deaminoated forms relative to the amount of corresponding degradation products contained in the solution of reconstituted NovoSeven® product stored for similar time under similar conditions. It means containing a small amount of.

The term "physically stable" designates a composition that remains visually clear. Physical stability of the composition is assessed by means of visual inspection and turbidity after storage of the composition at different temperatures for various times.

Visual inspection of the composition is performed in light that is precisely focused on a dark background. The composition is classified as physically unstable when showing visual turbidity.

The term 'physical stability' of a Factor VIII polypeptide relates to the formation of insoluble and / or soluble aggregates in the form of dimers, oligomers and polymers of the Factor VIII polypeptide and in the form of structural modifications and denaturation of the molecule.

The term “chemically stable” refers to a composition that maintains at least 50% of its initial biological activity after storage for 6 months at 2-8 ° C. as measured in a substantially one step coagulation assay as described in WO 92/15686. Specifies

The term “chemical stability” relates to the formation of chemical changes in the Factor VII polypeptide upon storage in solution at accelerated conditions. Examples are enzymatic degradations that result in hydrolysis, deaminoation and oxidation and formation of factor VII polypeptide fragments. In particular, sulfur containing amino acids are susceptible to oxidation with the formation of the corresponding sulfoxides.

The composition includes factor VII polypeptides, calcium and / or magnesium ions, buffers, and, optionally, other excipients that further stabilize the Factor VII polypeptide, including ionic strength modifiers and enteric modifiers. Factor VII polypeptide concentrations range from about 0.1 to about 10 mg / mL.

As used herein, “ion strength modifier” includes a buffer that contributes to the ionic strength of the solution. Buffers are not limited and include, for example, neutral salts such as sodium chloride or potassium chloride; amino acid ; Small peptides (eg, having 2 to 5 amino acid residues such as glycylglycine) or a mixture of at least two of these modifiers. Preferred buffer is sodium chloride. The ionic strength modifier is at least about 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 400 mM, 800 mM, 1000 mM, 1200 mM, 1500 mM, 1800 mM, 2000 mM or at least 2200 mM Present in concentration.

As used herein, the term "field modifier" includes buffers that contribute to the osmotic pressure of a solution. Intestinal modifiers include, but are not limited to, amino acids; Small peptides (eg having 2 to 5 amino acid residues); Neutral salts; Monosaccharides or disaccharides; Polysaccharides; Sugar alcohols or mixtures of at least two of the above modifiers. Examples of enteric modifiers are not limited and include sodium chloride, potassium chloride, sodium citrate, sucrose, glucose, glycylglycine and mannitol. Normally, the modifying agent is from about 1 to about 500 mM, depending on the other ingredients present; About 1 to about 300 mM; About 10 to about 200 mM; Or at a concentration of about 20 to about 150 mM. For example, neutral salts such as sodium chloride or potassium chloride are used.

"Neutral salt" means a salt that is not an acid or a base when dissolved in an aqueous solution.

The term “suitable buffer for maintaining pH in the range of about 4.0 to about 8.0” includes about 4.0 to about 7.0, about 4.5 to about 7.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 7.0, about 5.5 Maintaining a solution pH in an acceptable range of about 4.0 to about 8.0, such as from about 6.5, about 6.0 to about 7.0, about 5.0 to about 6.0, about 6.4 to about 6.6, or about 6.5, about 5.2 to about 5.7, or about 5.5. Buffers. The term is used interchangeably with "buffer". This is not limited and includes citrate (sodium or potassium), acetate (ammonium, sodium or calcium), histidine (L-histidine), maleate, phosphate (sodium or potassium), tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lactate, glutamic acid, glycylglycine, PIPES, acids and salts of glycine or mixtures of at least two of these buffers. The buffer concentration range is selected to maintain the desired pH of the solution. The buffer is a mixture of at least two buffers, and the mixture may provide a pH value in a specific range. In alternative embodiments, the buffer concentration is about 1 mM to 100 mM; 1 mM to about 50 mM; About 1 mM to about 25 mM; About 2 mM to about 20 mM; Or about 10 mM.

Optionally, the composition also contains a surfactant or surfactant. "Surfactants" or "surfactants" generally include buffers that protect proteins from atmospheric / solution interface induced stresses and solution / surface induced stresses (eg, forming protein aggregates). Surfactants are not preferably limited and include polysorbates such as polysorbate 20 or 80 (eg Tween®); Nonionic surfactants including polyoxy ethylene alkyl ethers such as poloxamer 188 or 407 or poloxamers (eg pluronic® polyols) and other ethylene / polypropylene block polymers or polyethylene glycols (PEG) such as PEG8000. The amount of surfactant present is in the range of about 0.005 to about 2.0%.

Optionally, the composition comprises an antioxidant. Antioxidants are not limited and include ascorbic acid, cysteine, homocysteine, cysteine, cystathion, methionine, glutathione and other peptides containing cysteine or methionine, in particular peptides having 2 to 5 amino acid residues (at least one residue being methionine or Cysteine residues, preferred being methionine, especially L-methionine. Antioxidants are included at concentrations of 0.1 to 5 mg / ml, such as 0.1 to 4, 0.1 to 3, 0.1 to 2 or 0.5 to 2 mg / ml.

Preservatives are also included in the composition to prevent microbial growth and to allow for "multiple use" packaging of the FVIII polypeptide. Preservatives include phenol, benzyl alcohol ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride and benzethium chloride. Preservatives are usually included at concentrations of 0.1 to 20 mg / ml, depending on the pH range and type of preservative. Optionally, the composition also includes a buffer that can inhibit deaminoation.

As used herein, it is understood that the specified amount is ± about 10%. For example, about 50 mM comprises 50 mM ± 5 mM; For example, 4% is 4% ± 0.4% and the like.

The percentage is (weight / weight) when referring to a solid dissolved in a solution and a liquid mixed into the solution. In Tween, for example, this is the weight of 100% stock / weight of the solution.

The term "ion intensity" is the ionic strength of the solution (μ) defined by the formula: u = 1 / 2∑ ([i] (Zi ² )), N is the ionic strength, [i] is the molar concentration of the ions and Zi Is the charge of the ion (James Fritz and George Schenk: Quantitative Analytical Chemistry, 1979). In other embodiments of the invention, the ionic strength of the composition is at least 50, such as at least 75, 100, 150, 200, 250, 400, 500, 650, 800, 1000, 1200, 1600, 2000, 2400, 2800 or at least 3200.

The term "isotropic" means "isotonic with serum", ie, at about 300 ± 50 milliosmol / kg. Intestinal tract refers to measuring the osmotic pressure of a solution before administration. The term "failure" means to designate an osmotic level above the physiological level of serum, such as at least 300 ± 50 milliosmol / kg.

The term "pharmaceutically effective amount" or "effective amount" is an effective dosage as determined by one of skill in the art to titrate the dosage to achieve the desired response. Factors needed when considering dosage include efficacy, bioavailability, desired pharmacokinetic / pharmacodynamic profile, treatment conditions, patient-related factors (e.g. weight, health, age, etc.), co-administration of drugs (e.g. anticoagulants). Presence, time of administration, or other factors known to the medical practitioner.

The term “treatment” is defined as the management and care of a test subject, eg, a mammal, in particular a human, for the purpose of treating a disease, condition or condition and the administration of a factor Ⅶ polypeptide to prevent the onset of symptoms or complications, or symptoms or complications. Includes the alleviation or elimination of diseases, conditions or diseases. A pharmaceutical composition according to the invention containing a Factor VII polypeptide is administered parenterally to a subject in need of such treatment. Parenteral administration is performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a syringe such as a pen. Alternatively, parenteral administration can be performed by means of an infusion pump.

Factor VIIa concentrations are conveniently expressed in mg / mL or IU / mL, with 1 mg usually representing 43000-56000 IU or more.

How to use:

Preparations of the invention include, for example and without limitation, coagulation factor deficiency (eg, deficiency of hemophilia A and B or coagulation factor XI or shock); It is used to treat factor VIII-responsive syndromes such as thrombocytopenia or von Willebrand's disease or bleeding disorders, including those caused by excessive bleeding from a coagulation factor inhibitor or cause. The preparation is also administered to a patient involved in surgery or other trauma or to patients undergoing anticoagulation therapy.

Factor VII Polypeptides Prepared According to the Invention :

The term “human factor VII” or “FVII” designates human factor VII produced by methods including natural resource extraction and purification and recombinant cell culture systems. Its sequence and features are described, for example, in US Pat. 4,784, 950. The term includes, for example, biologically active human factor Ⅶ equivalents in which one or more amino acids in the entire sequence are different. In addition, the terminology used herein includes substitution, deletion and insertion or post-translational modification of factor VII amino acid variants. As used herein, “factor VII polypeptide” includes without limitation Factor VII and Factor VII-related polypeptides. A "factor VII related polypeptide" includes, without limitation, a Factor VII polypeptide (eg, Factor VII variants) that is chemically altered with respect to Human Factor VII and / or comprises one or more amino acid sequence alterations with respect to Human Factor VII and / or Or an amino acid sequence truncated with respect to human factor VIII (eg, factor VIII fragment). Such factor VII related polypeptides exhibit different properties with respect to human factor VII, including stability, phospholipid binding, altered specific activity, and the like.

The term “factor-associated polypeptide” includes those that, without limitation, proteolytically process a non-cleaved (simogen) form of the polypeptide to produce each bioactive form designated by factor VIIa. Typically, factor VII is cleaved between residues 152 and 153 to produce factor VIIa. The term “factor VII” refers to polypeptides having amino acid sequences 1-406 (as disclosed in US Pat. No. 4,784, 950) of wild type human factor VII and other species, such as cattle, pigs, cats, murine animals, salmon factor VII, without limitation Derived wild type factor VII, said factor VII derived from blood or plasma or produced by recombinant means. It further includes natural allelic variants of Factor VII that exist and occur in individuals. In addition, the degree and location of glycosylation or other post-translational modifications will depend on the nature of the host cell and host cell environment selected.

As used herein, a “factor VII related polypeptide” is a polypeptide that exhibits, without limitation, a factor VIIa biological activity that is altered or reduced with respect to the activity of wild-type human factor VIIa and a biological activity that is substantially the same or improved with respect to wild-type human factor VIIa. It includes. Such polypeptides include, without limitation, chemically altered Factor VII or Factor VII a and Factor VII variants incorporating specific amino acid sequence alterations that alter or disrupt the bioactivity of the polypeptide.

This further includes polypeptides with slightly altered amino acid sequences, eg, polypeptides with altered N-terminus including substituted or added N-terminal amino acids and / or chemically altered polypeptides with respect to human factor VIIa.

Factor VII related polypeptides, including variants of Factor VII, exhibiting substantially the same or better bioactivity than wild type Factor VII, or alternatively exhibiting substantially altered or reduced bioactivity with respect to wild type Factor VII, without limitation , Polypeptides having one or more amino acids inserted, deleted or substituted with an amino acid sequence different from that of the wild type factor VII.

Factor VII related polypeptides, including variants that exhibit substantially the same or improved biological activity with respect to wild type Factor VIIa, are the same cells as tested in one or more coagulation assays, protein lysis assays, or TF binding assays as described herein. 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 preferred of the specific activity of wild type factor VIIa produced in the type Preferably at least about 120% and most preferably at least about 130%.

In some embodiments the factor VII polypeptide is a Factor VII related polypeptide and, in certain variants, is tested for the activity of the Factor VII polypeptide and the original human factor VIIa (wild-type FVIIa) when tested in an "in vitro hydrolysis assay" (see "Test" below). In a variant, wherein the ratio between activities is at least about 1.25; in another embodiment, the ratio is at least about 2.0; In other embodiments, the ratio is at least about 4.0. In some embodiments of the invention, the factor VII polypeptide is a Factor VII related polypeptide and in certain variants the activity of the Factor VII polypeptide and the original human Factor VIIa when tested in an "in vitro hydrolysis assay" (see "assay" below) The ratio between the activities of (wild type FVIIa) is at least about 1.25: in another embodiment, the ratio is at least about 4.0; In other embodiments, the ratio is at least about 8. 0.

In some embodiments, the factor VII polypeptide is US Pat. Human factor VII as disclosed in 4,784, 950 (wild-type factor VII). In some embodiments, the factor VII polypeptide is human factor VIIa. In a series of embodiments, the factor VII polypeptide is at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about the term 0%, most preferably of the specific biological activity of human factor VIIa Factor VII related polypeptides representing at least about 160%.

In some embodiments, the factor VII polypeptide has an amino acid sequence that is different from that of wild type factor VII by inserting, deleting, or replacing one or more amino acids.

In a series of embodiments, the Factor V polypeptides are described in U. S. Patent No. Polypeptides that exhibit at least about 70%, preferably at least about 80%, more preferably at least about 90% and most preferably at least about 95% identity with the sequence of wild type factor VII as disclosed in 4,784, 950. Amino acid sequence homology / identity can be determined by using a suitable computer program for sequence alignment such as, for example, the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic acid Re-search, 22: 4673-4680). Is determined from a conveniently aligned sequence

Non-limiting examples of Factor VII variants having substantially the same or improved homology with wild type Factor VII include S52A-FVIII, S60A-FVIII (lino et al., Arch. Biochem. Biophys. 352: 182-192,1998); L305V-FⅦ, L305V / M306D / D309S-FⅦ, L305I-FⅦ, L305T-FⅦ, F374P-FⅦ, V158T / M298Q-FⅦ, V158D / E296V / M298Q-FⅦ, K337A-FⅦ, M298Q-FⅦ, V158D / M298Q FⅦ, L305V / K337A-FⅦ, V158D / E296V / M298Q / L305V-FⅦ, V158D / E296V / M298Q / K337A-FⅦ, V158D / E296V / M298Q / L305V / K337A-FⅦ, K157A-FⅦ, E296V96 M298Q-FVIII, V158D / E296V-FVIII, V158D / M298K-FVIII and S336G-FVIII; FVIIa variants exhibiting increased TF independent activity as disclosed in WO 01/83725 and WO 02/22776; U. S. Patent No. FVIIa variants exhibiting increased proteolytic stability as disclosed in 5,580, 560; Factor VIIa proteolytically cleaved between residues 290 and 291 or residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); Oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54,1999).

Biological Activity of Factor VII Polypeptides:

The biological activity of factor VIIa in coagulation may be due to (i) proteolytic cleavage of factor IX or factor X to bind to tissue factor (TF) and (ii) to produce activated factor IX or X (factor IXa or Xa, respectively). It comes from the ability to catalyze.

For the purposes of the present invention, the biological activity of Factor VII polypeptides ("Factor VII biological activity") is described in US Pat. Factor VII deficient plasma and thromboplastin as described in 5,997, 864 or WO 92/15686 are used to measure and quantify the ability of an agent to promote coagulation. In this assay, biological activity is expressed as a decrease in clotting time with respect to the control sample and converted to “factor Ⅶ units” compared to a mixed human serum standard comprising 1 unit / ml factor Ⅶ activity. Alternatively, factor VIIa biological activity is

Measuring the ability of Factor VIIa or Factor VII-related polypeptides to produce activated Factor X (Factor Xa) in a system comprising TF embedded in a lipid membrane and Factor X (Persson et al., J. Biol. Chem. 272: 19919 -19924,1997);

Determining factor X hydrolysis in an aqueous solution system (“in vitro protein dissolution assay”, see below);

Measuring physical binding of VIIa or Factor VII-associated polypeptides to TF using a tool based on surface plasmon resonance (Persson, FEBS Lettes. 413: 359-363,1997);

Measuring the hydrolysis of the synthetic substrate by the Factor VIIa and / or Factor VII-related polypeptides (“In Vitro Hydrolysis Assay”, see below);

-Measure and quantify thrombin generation in TF-independent in vitro systems.

Assays Suitable to Determine Biological Activity of Factor VII Polypeptides :

Factor VII polypeptides useful in accordance with the present invention are selected by appropriate assays that can be performed as simple preliminary in vitro tests. Accordingly, the present specification discloses a simple test (called an "in vitro hydrolysis assay") for the activity of a factor VII polypeptide.

In Vitro Hydrolysis Assay (Test 1)

Native (wild-type) Factor VIIa and Factor VIIa variants (referred to as "factor VIIa") are assayed for specific activity. Simultaneous assays can be made for direct comparison with specific activity. Assays are performed on microtiter plates (MaxiSorp, Nunc, Denmark). Chromic Substrate D-lle-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM contains 0.1 M NaCl, 5 mM CaCl ₂ and 1 mg / ml bovine serum albumin at pH 7.4 Is added to Factor VIIa (final concentration 100 nM) in 50 mM Hepes. Absorbance at 405 nm is measured continuously with a Spectra Max 340 plate reader (Molecular Devices, USA). Absorbance during 20 min culture after subtracting the absorbance from the enzyme-free blank well is used to calculate the ratio between the activity of the variant and wild type factor VIIa:

Ratio = (A _{405 nm} Factor VIIa variant) / (A _{405 nm} Factor VIIa wild type).

Based on this, factor VIIa variants having a activity comparable to or higher than the original factor VIIa, eg, the ratio of the activity of the variant to that of the original factor VII (wild-type FVII) is about 1.0. The variants above can be identified.

The activity of the Factor VII polypeptide is achieved by using a physiological substrate, such as Factor X, at an appropriate concentration of 100-1000 nM to measure Factor Xa produced after addition of the appropriate chromogenic substrate (e.g. S-2765) ("In vitro Hydrolysis assay "). In addition, the activity assay is performed at physiological temperature.

In vitro protein lysis assay (assay 2)

The original (wild-type) Factor VIIa and Factor VIIa variants (referred to as “factor VIIa”) are simultaneously assayed to directly compare specific activity. Assays are performed on microtiter plates (MaxiSoScience, Nunc, Denmark). Factor VIIa (10 nM) and Factor X (0.8 microM) were incubated for 15 minutes at pH 7.4 in 100 ml 50 mM Hepes containing 0.1 M NaCl, 5 mM CaCl ₂ and 1 mg / ml bovine serum albumin. Factor X cleavage is stopped by adding 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg / ml bovine serum albumin. The amount of factor Xa produced is measured by addition of the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Absorbance at 405 nm is measured continuously with SpectraMax ^™ 340 plate reader (Molecular Devices, USA). Absorbance for 10 minutes after subtracting the absorbance from the blank wells without FVIIa is used to calculate the ratio of the proteolytic activity of the variant to wild type factor VIIa:

Ratio = (A405 nm Factor VIIa variant) / (A405 nm Factor VIIa wild type).

Based on this, it is possible to identify factor VIIa variants with comparable or higher activity than native factor VIIa, such as variants where the ratio between the activity of the variant and that of the native factor VII (wild type FVIII) is about 1.0 or greater.

The ability to generate thrombin of factor VII or factor VII related polypeptides can be measured in assays (assay 4) containing appropriate coagulation factors and inhibitors (minus factor VII when mimicking hemophilia A conditions) and activated platelets at physiological concentrations. (As described in p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99,542-547, incorporated herein by reference).

The activity of the factor VII polypeptide is also measured essentially using a one-step coagulation assay (assay 4) as described in WO 92/15686 or US Pat. No. 5,997,864. In summary, the sample to be tested is diluted in 50 mM Tris (pH 7.5) and 100 μl of 0.1% BSA is incubated with 100 μl of Factor VIII deficient plasma and 200 μl of thromboplastin C containing 10 mM Ca ²⁺ . . Coagulation times are measured and compared to standard curves using a mixture of normal human plasma that is citrated at the reference standard or serial dilutions.

Preparation and Purification of Factor VII Polypeptides:

Human purified factor VIIa suitable for use in the present invention is preferably Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or European Patent No. Prepared by DNA recombination technique as described in 200.421 (ZymoGenetics, Inc.). Factor VIII is also described in Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. lnvest. 71: 1836-1841,1983. In this way, a factor VII is produced which has no detectable amount of other coagulation factors. As a final purification step, more purified Factor VII formulations are obtained by methods including additional gel filtration. Factor VII is converted to Factor VIIa activated by known means, namely, various other plasma proteins such as Factor XIIa, IXa or Xa. Alternatively, as described in Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), factor VII is passed through an ion-exchange chromatography column such as Mono Q® (Pharmacia fine Chemicals) or the like. Activate it.

Factor VII related polypeptides are prepared by wild type Factor VII or recombinant techniques. Factor VII related polypeptides having an amino acid sequence altered in comparison to wild type factor VII can be modified by changing the amino acid codon or by removing some of the amino acid codons from the nucleic acid encoding the natural factor VII by known means such as, for example, site specific variants. It is prepared by altering the nucleic acid sequence encoding VIII.

It is evident to those skilled in the art that they can be substituted outside the region critical to the function of factor VIIa and form an active polypeptide. Amino acid residues that are essential and unsubstituted for the activity of Factor VII polypeptides are known to those skilled in the art, such as site specific variants or alanine scanning variants (see, eg, Cunningham and Wells, 1989, Science 244: 1081-1085). Check accordingly. In later techniques, variants are introduced and formed at all positively charged residues of the molecule and tested for coagulation crosslinking activity to identify amino acid residues that are critical for the activity of the molecule. Sites of substrate enzyme interactions can be determined by analysis of three-dimensional structures such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (de Vos et al., 1992, Science 255: 306-312; Smith et al. , 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64.

Introduction of the variant into the nucleic acid sequence to exchange one nucleotide for another nucleotide is carried out by site specific mutagenesis using methods known in the art. Particularly useful is the process of using a highly twisted double stranded DNA vector with two synthetic primers containing the insert and the desired variant. Oligonucleotide primers complementary to opposite strands of the vector are amplified during the temperature cycle by means of Pfu DNA polymerase. As soon as the primers are bound, an altered plasmid containing staggered nicks is produced. After the temperature cycle, the product is digested with the parent DNA template and treated with Dpnl specific for methylated and hemimethylated DNA to select synthesized DNA containing variants. Other methods known in the art for generating, identifying and isolating variants may be used, such as gene shuffling or phage display techniques.

Isolating the polypeptide from the cell comprises removing cell culture medium comprising the desired product from the adherent cell culture; Centrifugation or filtration to remove unattached cells; others and the like.

Optionally, factor VII polypeptides can be further purified. Purification includes, without limitation, affinity chromatography such as anti-Factor® antibody columns (see, eg, Wakabayashi et al., J. Biol. Chem. 261: 11097,1986; and Thim et al., Biochem. 27: 7785,1988); Hydrophobic interaction chromatography; Ion exchange chromatography; Size exclusion chromatography; Electrophoresis is performed by methods known in the art, including preliminary isoelectric point separation (IEF), differential solubility (eg ammonium sulfate precipitation), or extraction, etc. Generally, Scopes, Protein Purification, See Springer-Verlag, New York, 1982; and Protein Purification, JC Janson and Lars Ryden, editor s, VCH Publishers, New York, 1989. Subsequent purification, preparation preferably comprises a nonfactor VII or factor derived from the host cell. (Iii) less than about 10%, more preferably less than about 5%, most preferably less than about 1% of the weight of the related polypeptide.

Factor VII polypeptides are activated by proteolytic cleavage using trypsin-like XIIa or other proteases such as factor IXa, kallikrein, factor Xa and thrombin. Osterud et al., Biochem. 11: 2853 (1972); Thomas, US Patent No. 4,456, 591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, the Factor VII polypeptide is activated by passing through an ion exchange chromatography column such as Mono Q® (Pharmacia) or the like. The activated Factor VII or Factor VII related polypeptides formed are formulated and administered as described below.

Figure 1 shows the amount of FVIII aggregates and FVIII fragments after 3 months storage at 2-8 ° C.

The following examples illustrate the practice of the present invention. This embodiment is for illustrative purposes only and does not limit the claimed scope of the invention.

Example 1

Test method

The amount of aggregation is determined by non-denatured size exclusion HPLC. The amount of oxidized form is determined by RP-HPLC. The amount of enzymatic degradation form is determined by RP-HPLC. Unmodified size exclusion chromatography is run on a Waters Protein Pak 300 SW column, 7.5 × 300 mm, using 0.2 M ammonium sulfate, 5% 2-propanol pH 7.0 as the mobile phase. Flow rate: 0.5 ml / min. Detection: 215 nm. Load: 25 μg FVIIa.

Reverse phase HPLC is performed on a proprietary 4.5 × 250 mm butyl bonded silica column with a particle size of 5 μm and a pore size of 300 mm 3. Column temperature: 70 ° C. A-buffer: 0.1% v / v trifluoroacetic acid. B-buffer: 0.09% v / v trifluoroacetic acid, 80% v / v acetonitrile. The column eluted with a linear gradient of X to (X + 13)% B within 30 minutes. X was adjusted to elute FⅦa with a retention time of about 26 minutes. Flow rate: 1.0 ml / min. Detection: 214 nm. Load: 25 μg FVIIa.

Example 2

Composition preparation

Generally, aqueous FVIIa composition samples for analysis in these experimental examples are prepared from bulk solutions purified by buffer exchange in a gel filtration column. The composition additive is contained in the elution buffer in the final ratio or added to the eluate. The resulting solution is sterile filtered using a sterile membrane filter (0.2 micron pore size or equivalent) and filled with sterile glass vials and plugged and sealed with butyl rubber stoppers and aluminum flip-off type caps.                 

Example 3

Effect of pH on Chemical / Physical Stability

pH 3,3. 5,4. 0,4. 5,5. 0,5. RFVa aqueous containing 1.4 mg rFVa / mL, 50 mM sodium chloride, 10 mM calcium chloride and 10 mM glycylglycine, a mixture of acetate and histidine, adjusted to 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 Small bottles of the composition are incubated at elevated temperatures of 2-8 ° C. or 30 ° C., removed at various time points, assayed for pH changes, and chemical stability is determined by RP-HPLC and GP-HPLC.

 After storage for 3 months at 2-8 ° C., the aqueous composition showed no significant change in pH. Non-denatured size exclusion HPLC performed on samples stored at 2-8 ° C. for 3 months showed no significant aggregation of drug product above pH value 5.5 (FIG. 1). RP-HPLC performed on these samples showed no significant increase in protein fragmentation or oxidation in the pH range 4.5-5.5.

1 shows data three months after storage at 2-8 ° C. FIG. The initial amount of coagulation amount is about 0.5% and the initial amount of fragments is about 9%.

Example 4

Buffer capacity of other buffers

A small bottle of rFⅦa aqueous composition containing one of the following buffer materials: malic acid, acetic acid, histidine, glutamic acid and citric acid at a concentration of 1.0 mg rFⅦa / mL, 50 mM sodium chloride, 10 mM calcium chloride and 10 mM glycylglycine Incubate at elevated storage temperatures of 2-8 ° C. or 30 ° C. by months. The pH was adjusted to 5.5 at the start because it produced the least amount of degradation product (FIG. 1). The measurement of pH in the composition containing glycylglycine showed an increase up to 6.2 during the storage period. In the same period, the other composition showed a stable value of 5.5 +/- 0.1.

Example 5

Physical Stability of Aqueous Compositions Containing Various Surfactants

12 different compositions were prepared. The composition is:

rFⅦa 0.75mg / ml

NaCl 2.92 mg / ml

CaCl ₂ , 2H ₂ 0 1.47mg / ml

Glycylglycine 1.32mg / ml

Surfactant / Solvent x mg / ml

pH 5.5

The concentrations of surfactants / solvents tested are described in the table below. The composition is prepared from a liquid bulk solution of rFVIIa. A stock solution of surfactant / solvent is prepared in a buffer containing NaCl, CaCl ₂ , 2H ₂ O and glycylglycine at the concentrations described above. The rFⅦa bulk and surfactant solution were mixed and the pH of the solution was adjusted to 5.5. The composition was filtered (0.2 μm) and filled into small bottles (1 ml solution per small bottle).

The appearance of the composition was determined by visual inspection and the absorbance of the composition was determined at 400 nm. Subsequently, the small bottle was shaken (800 / min) at room temperature for 19 hours. After shaking, the appearance and absorbance were determined at 400 nm. The results are reported in the table below.

Surfactants
type Conc.
(mg / ml) Exterior Absorbance (400nm) I'm after I'm after increase None (criteria) - Handful of particles Very cloudy Tween®80 0.1 Very few
particle Sunny, a few particles 0.0044 0.0036 0.0008 Tween®20  0.1 Very few
particle Sunny, a few particles 0.0039 0.0101 0.0062 Poloxamer 188 1.0 Very few
particle Sunny, a few particles  0.0063 0.0027 0.0036 Pluronic®
F127 1.0 Very few
particle Sunny, a few particles 0.0000 0.0048 0.0048 Polyethylene Glycol 400 0.1 Very few
particle muddiness 0.0076 1.5708 1.5632 Polyethylene Glycol 4000 0.5 Handful of particles Very cloudy 0.0108 1.6624 1.6516 Brij® 0.1 Very few
particle Sunny, a few particles 0.0028 0.0015 0.0013 Myrj®59 0.1 Very few
particle Sunny, a few particles 0.0002 0.1110 0.1108 Myrj®52 0.1 Very few
particle Sunny, a few particles 0.0009 0.9390 0.9381 LPCM 0.1 Very few
particle Sunny, a few particles 0.0026 0.0012 0.0014 Glycerol 1.0 Very few
particle muddiness 0.0040 1.4064 1.4024

"Part." = "Particle"

The results suggest that the criteria (without the addition of surfactants / solvents) are visually cloudy upon shaking and a significant increase in absorbance is observed at 400 nm. Tween ® 20 (= Polysorbate 20), Tween ® 80 (= Polysorbate 80), Poloxamer 188, Pluronic ® F127 (= Poloxamer 407), Brij® 35 (= Polyoxyl 23 lauryl ether) And the addition of LPCM (= α-lysophosphatidylchlorine myristoyl) completely prevented the increase in turbidity and uptake, with a slight increase in turbidity (compared to the reference) Myrj®59 (= polyjade Yarn 100 stearate) and Myrj® 52 (= polyoxyl 40 stearate). Glycerol, polyethylene glycol 400 or polyethylene glycol 4000 could not prevent an increase in turbidity at the concentrations used in this experiment.

Example 6

Chemical Stability of Aqueous Compositions Containing Methionine as Antioxidant

Three different compositions were prepared. The composition is:

rFⅦa 0.75mg / ml

NaCl 2.92mg / ml

CaCl ₂ , 2H ₂ 0 1.47 mg / ml

Glycylglycine 1.32 mg / ml

Methionine 0 or 0. 25 or 1. 0 mg / ml

pH 6.5

The composition was prepared from a liquid bulk solution of rFVIIa. Methionine was dissolved in a buffer containing NaCl, CaCl ₂ , 2H ₂ O and glycylglycine at the concentrations cited above. The rFⅦa bulk and methionine solutions were mixed and the pH was adjusted to 6.5 in the solution. The composition is filtered (0.2 μm) and placed in small bottles (1 ml solution per small bottle). Small bottles were stored at 5 ° C, 25 ° C, and 40 ° C. The sample was taken out and analyzed for the amount of oxidized form at the time described in the table below (RP-HPLC). The table shows the amount of oxidized form (%).

Methionine
start  25 ℃ 40 ℃ 25 ℃ 40 ℃ 5 ℃ 0 (standard) 2.4 4.4 7.5 4.4 12.8 3.1 0.25 1.7 2.4 5.3 2.8 9.9 1.9 1.0 1.6 2.3 5.0 2.6 9.6 1.3

The results suggest that the addition of methionine lowers the rate of oxidation in the composition.

Example 7

Chemical Stability of Aqueous Compositions Containing Calcium Chloride

Four different compositions were prepared. The composition is:

rFⅦa 1.0 mg / ml

NaCl 2.92 mg / ml

CaCl ₂ , 2 H ₂ 0 1.47 mg / ml (10mM), 29.4 mg / mL (200mM), 58.8 mg / mL (400mM)

               And 117.6 mg / mL (800 mM), respectively

Glycylglycine 1.32 mg / ml

 pH 7.0

The composition was prepared from a liquid bulk solution of rFVIIa. Calcium chloride is dissolved in a buffer containing NaCl and glycylglycine to mix with rFⅦa bulk to produce the concentrations recited above. After mixing the pH of the solution was adjusted to 7.0. The composition is filtered (0.2 μm) and placed in a small bottle (1 ml solution per small bottle). Small bottles were stored at 5 ° C.

The activity of factor VII (IU / ml) is determined by a coagulation assay.

Composition
Amount of calcium chloride 0 mdr
IU / ml 3 mdr
IU / ml  5 ℃ 1 (10 mM) 54444 33623  2 (200 mM) 59917 46528 3 (400 mM) 54680 59370 4 (800 mM) 51773 52801

Claims (34)

(Iii) a factor VII polypeptide, (Ii) a buffer suitable for maintaining a pH in the range of 4.0 to 8.0, (Iii) a buffer selected from calcium salts, magnesium salts or mixtures thereof, and (vii) comprises an antioxidant present at a concentration of 0.1-5.0 mg / ml, Wherein the concentration of (iii) is at least 200 mM, the liquid aqueous composition for treatment of bleeding disease. The composition of claim 1, wherein the composition further comprises (iv) an ionic strength modifier. 3. The method of claim 2, wherein the ionic strength modifier (iv) comprises a neutral salt comprising sodium chloride; amino acid; Small peptides; Or a mixture of at least two of said modifiers. 4. The composition of claim 3 wherein the ionic strength modifier (iv) is sodium chloride. The composition of any one of claims 1 to 4, wherein the buffer (iii) is present at a concentration of 200 mM to 1000 mM. 5. The composition of claim 2, wherein the ionic strength modifier (iv) is present at a concentration of at least 5 mM. 6. The composition according to any one of claims 1 to 4, wherein the calcium salt is selected from the list of calcium chloride, calcium acetate, calcium gluconate and calcium rabbitrate. The composition according to any one of claims 1 to 4, wherein the magnesium salt is selected from the list of magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, magnesium rabbitate. 7. A composition according to claim 6, wherein the buffer (iii) is selected from the list of calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate or mixtures thereof and the ionic strength modifier (iv) is sodium chloride. . The composition according to any one of claims 1 to 4, further comprising (v) an enteric modifier. 11. The method of claim 10, wherein the enteric modifier (v) comprises a neutral salt; Monosaccharides, disaccharides or polysaccharides; Sugar alcohols; amino acid; Or a small peptide or a mixture of at least two of said modifiers. The composition of claim 10, wherein the enteric modifier (v) is present at a concentration of 1 to 500 mM. 13. The composition of claim 12, wherein the concentration is 10-250 mM. The composition of any of claims 1 to 4, further comprising (vi) a nonionic surfactant. 15. The nonionic surfactant according to claim 14, wherein the nonionic surfactant is a polysorbate or poloxamer or polyoxyethylene alkyl ether such as poloxamer 188 or poloxamer 407 or polysorbate 20 or polysorbate 80 or polyoxy 23 lauryl ether. A composition, characterized in that. The method of claim 1, wherein the antioxidant is selected from the group consisting of D- or L-methionine; Methionine analogs; Methionine-containing peptides, methionine homologs, ascorbic acid; Cysteine, homocysteine; A composition, characterized in that it is selected from glutathione and cystathionine. The composition of claim 16, wherein the antioxidant is L-methionine. The composition of claim 1, wherein the antioxidant is present at a concentration of 0.1 mg / ml to 4 mg / ml. 5. The composition of claim 1, wherein the pH is maintained at 4.0 to 7.0. 6. The buffer according to any one of claims 1 to 4, wherein the appropriate buffer for maintaining a pH in the range of 4.0 to 8.0 is citrate, acetate, histidine, malate, phosphate, tartaric acid, succinic acid, MES, HEPES, imidazole. , TRIS, lactate, glycylglycine, PIPES, acid and salt of glycine or a buffer selected from the list of mixtures of at least two of said buffers. The composition of claim 20, wherein the concentration of buffer is between 1 mM and 50 mM. The composition of claim 21, wherein the concentration of buffer is 10 mM. The process according to any one of claims 1 to 4, comprising phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride and benzatonium chloride. A composition comprising the same (iv) preservative. The composition according to any one of claims 1 to 4, which is isotonic. The composition according to any one of claims 1 to 4, which is formulated for pharmaceutical administration. 5. The composition of claim 1, which is stable at 2-8 ° C. for at least 6 months. 6. 5. The composition of claim 1, wherein the factor VII polypeptide is human factor VIIa comprising recombinantly produced human factor VIIa. 6. 5. The composition of claim 1, wherein the Factor VII polypeptide is Factor VII sequence variant. 6. The composition of claim 28, wherein the ratio between the activity of the Factor VII polypeptide and the activity of the native human factor VIIa (wild type FVIIa) is at least 1.25 when tested in an in vitro proteolytic assay. 5. The composition of claim 1, wherein the Factor VIII polypeptide is present at a concentration of 0.1 mg / ml to 10 mg / ml. 6. A method for preparing a liquid aqueous composition for treating a bleeding disease of a Factor VII polypeptide, (Ii) a buffer suitable for maintaining a pH in the range of 4.0 to 8.0, (Iii) a buffer selected from calcium salts, magnesium salts or mixtures thereof, and (vii) comprises an antioxidant present at a concentration of 0.1-5.0 mg / ml, Wherein the concentration of (iii) comprises providing the factor VII polypeptide in a solution of at least 200 mM. delete delete delete

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