Methods of production and use of liquid formulations of plasma proteins

Abstract

The present invention relates to the preparation and use of liquid formulations of plasma proteins, particularly blood coagulation factors. More specifically, the present invention relates to stable liquid formulations of Factor VIII and Factor IX that can be administered by injection or infusion to provide a constant level of the coagulation factor in the blood.

Description

This application is a divisional of U.S. application No. 08/758,560, filed Nov. 29, 1996, now U.S. Pat. No. 5,925,738, which claims priority under 35 U.S.C. §119(e) from provisional applicationNo. 60/007,866, filed Dec. 1, 1995.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference.

"Plasma" as used herein refer to the fluid, non-cellular portion of the blood of humans or animals as found prior to coagulation. It is distinguished from serum, which is obtained after coagulation.

"Plasma protein" as used herein refers to the soluble proteins found in the plasma of normal humans or animals. These include but are not limited to coagulation proteins, antibodies, albumin, lipoproteins, complement proteins, and the like.

"Vitamin K-dependent plasma proteins" as used herein refer to those proteins found in the plasma of a normal individual or animal that are members of the coagulation cascade, either as procoagulants or anticoagulants, and whose synthesis requires the presence of vitamin K. These are present in proenzyme form or activated form. Vitamin K-dependent plasma proteins include: Factor II; Factor VII; Factor IX; Factor X; Protein C; Protein S; and Protein Z.

"Non-vitamin K-dependent plasma proteins" as used herein refer to those proteins found in the plasma of a normal individual or animal that are members of the coagulation cascade, either as procoagulants, anticoagulants, or cofactors, and whose synthesis does not require the presence of vitamin K. These are present in proenzyme form, non-activated cofactor form, or activated form. Non-vitamin K-dependent plasma proteins include Factor VIII and von Willebrand's factor.

"Factor IX" (or "coagulation Factor IX") as used herein refers to a plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. A congenital X-linked deficiency of biologically active Factor IX results in Hemophilia B, a potentially life-threatening disorder.

"Factor VIII" (or "coagulation Factor VIII") as used herein refers to a plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. A congenital X-linked deficiency of biologically active Factor IX results in Hemophilia A, a potentially life-threatening disorder.

"Liquid formulation" as used herein refers to a composition of matter that is found as a liquid, characterized by free movement of the constituent molecules among themselves but without the tendency to separate. Liquid formulations include aqueous and non-aqueous liquids.

"Aqueous liquid formulation" as used herein refers to a liquid composition that contains water as a component.

"Non-aqueous liquid formulation" as used herein refers to a liquid composition that does not contain water as a component.

"Hydrophilic non-aqueous liquid formulation" as used herein refers to a liquid composition that does not contain water as a component and that contains liquids that have a strong affinity for water.

"Mixed aqueous/non-aqueous liquid formulation" as used herein refers to a liquid composition that contains a mixture of water and an additional liquid composition.

"Body temperature" as used herein refers to the normal physiological temperature of a human or animal. The average body temperature of a human is 37°C Centigrade (C.).

"Refrigeration temperature" as used herein refers to the temperature for cold storage that does not allow freezing. One refrigeration temperature is 4°C Centigrade (C.).

"Reconstitution" as used herein refers to the dissolution or resuspension of a solid material into a liquid solution or suspension by the addition of a liquid.

"Rehydration" as used herein refers to the reconstitution to a liquid state by the addition of water.

"Prophylaxis" as used herein refers to the administration of treatment to prevent the onset of symptoms.

"Continuous delivery" as used herein refers to the uninterrupted introduction of a material into the body of a human or animal.

"Injection" as used herein refers to the introduction of a liquid by force into the body of a human or animal.

"Hydrogel" as used herein refers to a semisolid composition constituting a substantial proportion of water, and in which polymers or mixtures thereof are dissolved or dispersed.

"Intranasal delivery" as used herein refers to the introduction of material into the body of a human or animal by absorption through the mucous membranes of the nasal passages and sinuses.

"Inhalation delivery" as used herein refers to the introduction of material into the body of a human or animal by absorption through the lungs.

"Oral delivery" as used herein refers to the introduction of a material into the body of a human or animal by ingestion through the mouth.

"Parenteral delivery" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

"Stability" as used herein refers to the retention of those characteristics of a material that determine activity and/or function.

"Pharmaceutically acceptable carrier" as used herein refers to a non-toxic solid, semisolid or liquid filter, diluent, encapsulating material or formulation auxiliary of any type.

"Patient" as used herein refers to human or animal individuals receiving medical care and/or treatment.

"Congenital deficiency" as used herein refers to the condition of an individual that lacks, as a result of heredity, a compound found in normal individuals. Congenital deficiencies are permanent absent transplantation or genetic intervention, which at this time are not guaranteed cures.

"Acquired deficiency" as used herein refers to the condition of an individual that lacks, as a result of a non-congenital influence, a compound found in normal individuals. Acquired deficiencies are frequently the transient result of other conditions or their treatment, but are nonetheless debilitating and life threatening.

Preferred Embodiments

A first preferred embodiment of the present invention is directed to a stable liquid formulation containing one or more plasma proteins. Preferably, the stable liquid formulation of the present invention is stable, i.e., at least 50% of the activity of the plasma protein(s) contained in the formulation is maintained, for at least one day (24 hours), more preferably for at least 13 days, and most preferably for at least 30 days at 37°C C. or for at least 540 days at 4°C C. The activity of plasma proteins may be determined according to any of the methods and techniques known to those skilled in the art.

According to this embodiment of the present invention, any plasma protein may be contained in the stable liquid formulation. These plasma proteins may be in the form of proenzymes or cofactors that have not yet been activated or they may be in the activated form. Suitable plasma proteins may be obtained from human or mammalian plasma by any of the purification methods known and available to those skilled in the art; from supernatants or pastes of recombinant tissue culture, viruses, yeast, bacteria, or the like that contain a gene that expresses a human or mammalian plasma protein which has been introduced according to standard recombinant DNA techniques; or from the fluids (e.g blood, milk, lymph, urine or the like) or transgenic animals that contain a gene that expresses a human plasma protein which has been introduced according to standard transgenic techniques. The inventive liquid formulation may optionally further contain one or more additional proteins as desired, including: other plasma proteins; protease inhibitors, such as thrombin inhibitors; carrier proteins, including but not limited to von Willebrand Factor; peptides; and derivatives thereof.

In a particularly preferred embodiment of the present invention, the plasma protein is a vitamin K-dependent plasma protein. Illustrative examples of such vitamin K-dependent plasma proteins include, but are not limited to, Factor II, Factor VII, Factor IX, Factor X, Protein C, Protein S and Protein Z. Preferably, the vitamin K-dependent plasma protein is Factor IX.

In another particularly preferred embodiment of the present invention, the plasma protein is a non-vitamin K-dependent plasma protein. Non-vitamin K-dependent plasma proteins include, but are not limited to, Factor VIII and von Willebrand's factor. Preferably, the non-vitamin K-dependent plasma protein is Factor VIII.

In yet another particularly preferred embodiment of the present invention, the liquid formulation is an aqueous liquid formulation. Preferably, the aqueous liquid formulation contains one or more pH buffering compound to maintain the pH of the formulation at a predetermined level, such as in the range of 5.8 to 6.8. Preferably, the pH buffering compound used in the aqueous liquid formulation is an amino acid or mixture of amino acids; more preferably, the pH buffering compound is histidine or a mixture of amino acids one of which is histidine.

Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of 5.8 to 6.8, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions.

The pH buffering compound may be present in any amount suitable to maintain the pH of the aqueous liquid formulation at a predetermined level. Preferably, when the pH buffering compound is an amino acid, the concentration of the amino acid is between 0.1 millimole/liter (0.1 mM) and 1000 mM (1 M). More preferably, the concentration of the amino acid is between 5 mM and 100 mM, most preferably between 10 mM and 50 mM.

Preferably, the pH buffering compound maintains the pH of the aqueous formulation at a level of at least 4∅ More preferably, when the plasma protein is Factor IX, the pH buffering compounds maintains the pH at a level between 5.5 and 8.0, even more preferably at a level between 5.5 and 6.8, and most preferably between 5.8 and 6.2.

The inventive aqueous liquid formulation preferably may also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g, tonicity, osmolality and/or oncotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. Preferably, the osmotic modulating agent does not chelate calcium ions.

The osmotic modulating agent may be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents.

The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation. Preferably, the osmotic modulating agent (s) may be present in an amount sufficient to modulate the osmolality of the inventive formulation to between 50 and 1000 milliosmoles/L (mOsm/L), more preferably between 100 and 500 mOsm/L, and most preferably between 150 and 350 mOsm/L.

If a salt or combination of salts is employed as the osmotic modulating agent, the total concentration thereof is preferably between 1 mM and 1 M, more preferably between 25 mM and 500 mM, most preferably between 50 mM and 150 mM.

The aqueous liquid formulation preferably further contains a sucrose of multivalent metal ions, such as calcium ions. Any multivalent metal ion that helps stabilize the inventive liquid formulation and that will not adversely affect recipient individuals may be used. Suitable metal ions may be determined empirically by one skilled in the art based on these two criteria and suitable sources of such metal ions are known, and include inorganic and organic salts.

Preferably, the aqueous liquid formulation contains a source of divalent metal ions, such as calcium ions, magnesium ions and/or manganese ions. More preferably, the aqueous liquid formulation contains a source of calcium ions, such as calcium chloride.

The concentration of divalent metal ions, such as calcium ions, in the aqueous liquid formulations is preferably between 0.1 mM and 1 M, more preferably between 2 mM and 200 mM, and most preferably between 10 mM and 100 mM.

In a preferred embodiment of the inventive aqueous liquid formulation, the plasma protein is Factor IX, the pH buffering compound is the amino acid histidine, the osmotic modulating agent is sodium chloride and the source of calcium ions is calcium chloride. Preferably, the concentration of histidine is about 10 mM, the concentration of NaCl is about 100 mM, and the concentration of calcium ions varies with the pH of the formulation. Preferably, when the pH of the aqueous formulation is about 6.2, the concentration of calcium chloride is about 10 mM; when the pH is about 6.0, the concentration of CaCl₂ is between 10 mM and 30 mM; and when the pH is about 5.8, the concentration of CaCl₂ is between 10 mM and 100 mM.

In another particularly preferred embodiment of the present invention, the liquid formulation is a non-aqueous liquid formulation. Any suitable non-aqueous liquid may be employed in this embodiment of the present invention provided that it provides stability to the plasma protein(s) contained therein. Preferably, the non-aqueous liquid is a hydrophilic liquid. Illustrative examples of suitable non-aqueous liquids include: glycerol; dimethyl sulfoxide (DM SO); polydimethylsiloxane (PM S); ethylene glycols, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol ("PEG") 200, PEG 300, and PEG 400; and propylene glycols, such as dipropylene glycol, tripropylene glycol, polypropylene glycol ("PPG") 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.

Preferably, the plasma protein is freeze-dried prior to incorporation into the non-aqueous liquid formulation. More preferably, the plasma protein is freeze-dried Factor VIII or freeze-dried Factor IX.

In a highly preferred embodiment of the inventive non-aqueous liquid formulation, the plasma protein is Factor IX and the non-aqueous liquid is an ethylene glycol, such as PEG 200, or a propylene glycol, such as tripropylene glycol, PPG 425, PPG 725, PPG 1000 or PPG 2000. In another highly preferred embodiment of the inventive non-aqueous liquid formulation, the plasma protein is Factor VIII and the non-aqueous liquid is a propylene glycol, such as PPG 425, PPG 775 or PPG 1000.

In another particularly preferred embodiment of the present invention, the liquid formulation is a mixed aqueous/non-aqueous liquid formulation. Any suitable non-aqueous liquid formulation, such as those described above, may be employed in this embodiment of the present invention along with any aqueous liquid formulation, such as those described above, provided that the mixed aqueous/non-aqueous liquid formulation provides stability to the plasma protein(s) contained therein. Preferably, the non-aqueous liquid in such a formulation is a hydrophilic liquid. Illustrative examples of suitable non-aqueous liquids include: glycerol; DM SO; PM S; ethylene glycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols, such as PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.

The inventive stable liquid formulation permits storage of one or more plasma proteins in a frozen or an unfrozen liquid state. Preferably, the stable liquid formulation is stored at a temperature of at least -170°C C., more preferably at a temperature of at least 0°C C., even more preferably at a temperature between 0.1°C C. and 42°C C., and most preferably between 4°C C. and 37°C C.

The stable liquid formulation of the present invention maintains at least 50% of the activity of the plasma protein for at least 24 hours in an unfrozen state. Preferably, at least 50% of the activity is maintained for a period of at least 2 days, more preferably at least 30 days, even more preferably at least 180 days, still more preferably at least 540 days, and most preferably at least 3 years.

It will be appreciated that conditions such as hemophilia caused by a decrease in the standard or normal level of activity of one or more plasma proteins in an individual can be treated by administration of one or more of the plasma proteins, such as Factor VIII (for treatment of Hemophilia A) or Factor IX (for treatment of Hemophilia B). Accordingly, a second preferred embodiment of the present invention is directed to a method of treating a patient having a deficiency of one or more plasma proteins which comprises administering to that individual the stable liquid formulation of the present invention.

Continuous administration may be performed according to any of the methods and techniques know to those skilled in the art, including continuous injection or infusion using a surgically implantable pump, such as an Arrow Model 3000 (Arrow International, Walpole, Mass.) or MiniMed 2001 (MiniMed Technologies, Sylmar, Calif.); injection or infusion using an externally worn pump, such as the MiniMed 504S (MiniMed Technologies) or the H-Tron V100 (Disetronic Medical Systems, Minnetonka, Minn.); or diffusion from a biologically-derived bio-resorbable hydrogel, such as a chitosan hydrogel or N,O-carboxymethyl chitosan (NOC-chitosan) hydrogel, alone or in combination with a charged or uncharged polymeric agent to control hydrogel porosity and/or hydrogel stability and/or kinetics of protein release, such as polylysine or polypropylene glycol; or diffusion from a synthetically-derived bio-resorbable hydrogel, such as polypropylene glycol, alone or in combination with a charged or uncharged polymeric agent to control hydrogel porosity and/or hydrogel stability and/or kinetics of protein relaease, such as polylysine.

The plasma protein(s) for continuous administration is preferably formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of plasma protein for purposes herein is thus determined by such considerations.

The total pharmaceutically effective amount of plasma protein administered parenterally per dose will preferably be sufficient to provide the plasma protein in a concentration of between about 1 unit/mL to about 2000 units/mL. More preferably, this dose is sufficient to provide a concentration of between about 50 units/mL to about 1500 units/mL, even more preferably between about 100 units/mL to about 1000 units/mL, and most preferably about 600 units/mL. A factor in selecting an appropriate dose is the result obtained, as measured by increases in the level of plasma protein in circulation. The length of treatment needed to observe changes and the interval following treatment for responses to occur may vary depending on the desired effect and the individual being treated.

As a general proposition, the purity of plasma protein formulated according to the present invention will preferably be an appropriate purity known to one of ordinary skill in the relevant art to lead to the optimal stability of the protein. For example, when the plasma protein is Factor IX, the Factor IX is preferably of ultrahigh purity. Preferably, the plasma protein has been subjected to multiple chromatographic purification steps, such as affinity chromatography and preferably immunoaffinity chromatography, to remove substances which cause fragmentation, activation and/or degradation of the plasma protein during manufacture, storage and/or use. Illustrative examples of such substances that are preferably removed by purification include thrombin and Factor IXa; other protein contaminants, such as inter-alpha trypsin inhibitor and pre-alpha trypsin inhibitor; non-protein contaminants, such as lipids; and mixtures of protein and non-protein contaminants, such as lipoproteins.

The concentration of plasma protein(s) employed in the formulations of the present invention is preferably selected to optimize the stability of the plasma protein as may be determined empirically by one skilled in the relevant art. For example, when the plasma protein is Factor IX, the stability of the Factor IX in the inventive formulations is greatest at lower concentrations and tends to decrease as the concentration of Factor IX increases. At Factor IX concentrations below about 100 units/mL (0.4 mg/mL), for example, in one embodiment of the inventive formulations (0.01 M histidine, 0.10 M sodium chloride, 0.01 M calcium chloride, pH 6.8), immunoaffinity-purified Factor IX prepared by the ARC showed no evidence of activation to Factor IXa when incubated at 37°C C. for 56 days. At higher Factor IX concentrations (200-600 units/mL), however, the same immunoaffinity-purified Factor IX showed evidence of activation when incubated at 37°C C., the extent of activation increasing with increasing protein concentration (FIG. 4). In more acidic embodiments of the inventive formulations, such as pH 5.8-6.2, 600 units/mL (2.4 mg/mL) of the same immunoaffinity-purified Factor IX was incubated with much lower, if any, activation FIGS. 5A-5B and 8-14A-M.

In a preferred embodiment of the present invention, one or more plasma proteins are contained in a pharmaceutically acceptable carrier. The carrier preferably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetate, lactate, tartrate, and other organic acids or their salts; tris-hydroxymethylaminomethane (TRIS), bicarbonate, carbonate, and other organic bases and their salts; antioxidants, such as ascorbic acid; low molecular weight (for example, less than about ten residues) polypeptides, e.g., polyarginine, polylysine, polyglutamate and polyaspartate; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), and polyethylene glycols (PEGs); amino acids, such as glycine, glutamic acid, aspartic acid, histidine, lysine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose, dextrins or sulfated carbohydrate derivatives, such as heparin, chondroitin sulfate or dextran sulfate; polyvalent metal ions, such as divalent metal ions including calcium ions, magnesium ions and manganese ions; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; counterions, such as sodium or ammonium; and/or nonionic surfactants, such as polysorbates or poloxamers.

Such compositions may be administered orally, rectally, parenterally, subcutaneously, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray, into the lungs or as an inhalant. Preferably, the pharmaceutically acceptable carrier is a biologically-derived bioresorbable hydrogel, such as a chitin hydrogel or chitosan hydrogel.

The plasma protein (or proteins) may also suitably be administered by sustained-release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, but are not limited to, polylactides (U.S. Pat. No. 3,773,919; European Patent No. 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) or ethylene vinyl acetate (R. Langer et al., J. Biomed Mater. Res. 15:167-277 (1981); R. Langer, Chem. Tech. 12:98-105 (1982)), or poly-D-(-)-3-hydroxybutyric acid (European Patent No. 133,988). Sustained-release plasma protein compositions also include liposomally entrapped plasma proteins. Liposomes containing one or more plasma proteins may be prepared by any of the methods known to those skilled in the art, for example, as described in DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

Compositions containing one or more plasma proteins to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes), by gamma irradiation, or any other suitable means known to those skilled in the art. Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

These compositions ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous plasma protein solution, such as an aqueous solution of Factor IX or Factor VIII, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized material using sterile Water-for-Injection (WFI).

The present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions of the present invention may be employed in conjunction with other therapeutic compounds.

A preferred embodiment of the present invention provides a plasma protein in a stable liquid as part of a kit. A more preferred embodiment provides a plasma protein in a stable liquid in a kit whereby the kit provides for the delivery of the plasma protein by injection. A yet more preferred embodiment provides a plasma protein in a stable liquid in a kit whereby the kit provides for injection by and includes but is not limited to a syringe and/or needle. A still more preferred embodiment provides a plasma protein in a stable liquid in a kit whereby the kit provides for the injection of the liquid into a reservoir of a continuous injection or infusion system for intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, or intranasal delivery.

Another still more preferred embodiment provides a plasma protein in a stable liquid in a kit whereby the kit provides for the incorporation of the liquid into a hydrogel and the subsequent introduction of the plasma protein-containing hydrogel by injection, such as subcutaneous, intradermal, intramuscular, intraperitoneal, and the like; by topical application for transdermal, intranasal, by buccal absorption through skin surfaces, mucous membranes, and the like; or by inhalation into the nose and/or lungs. Yet another more preferred embodiment provides for the delivery of a plasma protein in a stable liquid in microspheres or hydrogel for injection or implantation, including but not limited to injection, such as subcutaneous, intradermal, intramuscular, intraperitoneal, and the like.

The various embodiments of the present invention have several advantages over previously used compositions and methods.

A first advantage is that the plasma protein formulation of the present invention is ready to use without on-site preparation. This enables delivery of the protein sooner than those lyophilized formulations that require rehydration.

A second advantage is that the present invention enhances stability of the plasma protein in solution. The formulation of the present invention allows storage of the protein in liquid for longer periods at a given level of activity than is possible with rehydrated previously used formulations. This stability is enhanced during storage under refrigeration and at body temperature. Stability at body temperature enables the plasma protein to be held in a reservoir either subcutaneously or on the body surface in a continuous injection or infusion delivery system. It also permits delivery by diffusion from a supplemented hydrogel whereby the composition of the present invention is incorporated into the hydrogel prior to injection or infusion.

A third advantage of the present invention is the ability to store the plasma protein without the additional cost and restriction of refrigeration. While the present invention demonstrates as an example extended storage of Factor IX under refrigeration, 4°C C., for over one year without loss of activity, it also demonstrates storage at body temperature, 37°C C., for over one month. For situations where refrigeration is not possible, the present invention permits the storage of ready-to-inject plasma proteins at room temperature, for use either prophylactically or in emergency situations.

A fourth advantage of the present invention is the ability to prophylactically treat congenital or acquired plasma protein deficiency in a manner that provides a continuous level of the plasma protein that more closely resembles the level found in normal plasma and achieves normal hemostasis. It is the uninterrupted condition of normal hemostasis that prevents the progressive, cumulative, unrepairable tissue damage that permanently debilitates patients. The elimination of the risk of bleeding episodes and their consequent damage also significantly enhances the quality of life for the patients and their families, enabling younger patients to more fully participate in activities normal for young children, and for older patients to hold jobs and contribute to society.

The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

All patents and publications referred to herein are expressly incorporated by reference.

VI. EXAMPLES

Materials

Coagulation Factor IX, Heat Treated (CFIX-HT): Intermediate-purity Factor IX concentrate prepared from pooled human plasma by anion-exchange chromatography, to capture vitamin K-dependent clotting factors, followed by sulfated-dextran chromatography, to separate these factors; freeze dried and dry-heat treated to reduce viral infectivity. Manufactured for ARC by Baxter/Hyland, Glendale, Calif. Product consists of 10-20% Factor IX, very small amounts of Factors II and X. The bulk of the protein (70-80%) is Inter-alpha Trypsin Inhibitor (IaI).

Coagulation Factor IX. Solvent and Detergent Treated (CFIX-SD): Intermediate-purity Factor IX concentrate prepared by the same chromatographic steps as CFIX-HT, but treated with Tri-n-butyl phosphate (TNBP) and Triton X-100 inactivate viruses prior to the final chromatography step. Otherwise similar to CFIX-HT.

Factor IX-M (Hyland): An ultra high-purity concentrate prepared from pooled human plasma in pilot scale manufacturing at the Baxter/Hyland plant in Glendale Calif. Process utilizes anion-exchange chromatography, solvent/detergent treatment, immunoaffinity chromatography on a 7.5 liter column of immobilized metal-ion dependent anti-human Factor IX monoclonal antibody (anti-FIX-Mab), and a final anion-exchange polishing step.

Factor IX-M (JHL): An ultra-high purity concentrate prepared at the ARC Jerome Holland Laboratory in Rockville Md. CFIX-SD was reconstituted, applied to a one liter of anti-FIX Mab resin in the presence of 40 mM MgCl₂, washed with 1 M NaCl/10 mM MgCl₂, and eluted with citrate/NaCl buffer. Preparations designated "w/DEAE" were further purified on an anion-exchange column to diminish levels of mouse IgG and other contaminants. Specific activity and SDS-PAGE indicate the purity of all FIX-M preps were greater than or equal to 95%.

Alphanine (ARC/A9): High-purity Factor IX concentrate prepared for ARC, from a DEAE eluate from pooled human ARC plasma, by Alpha Pharmaceuticals according to their proprietary purification method by subjecting this eluate to barium citrate adsorption, solvent/detergent treatment and two affinity chromatographic steps on dextran sulfate agarose; provided lyophilized in Alpha proprietary formulation. Dialyzed into ARC formulation for stability testing.

Mononine (ARC/M9): High-purity Factor IX concentrate is manufactured by Armour Pharmaceuticals by a method including immunoaffinity chromatography on an immobilized monoclonal antibody to Factor IX, followed by chromatography on hexylamine agarose.

EXAMPLE 1

Effect of Buffer, Divalent Cations, and Other Excipients on the Stability of Coagulation Factor IX

CFIX-M (JHL)/DEAE was dialyzed into 0.01 M histidine, 0.1 M NaCl, pH 6.8 (histidine-saline) or was left in 0.02 M sodium citrate (NaCit), 0.11 NaCl, pH 6.8 (citrate-saline). An aliquot of the solution was mixed with an equal volume of 2X additive, prepared at twice the desired final concentration in the appropriate buffer (histidine-saline or citrate-silane). The formulated solutions were sterile filtered, aseptically dispensed into sterilized tubes, incubated at 37°C C. or 4°C C. for the designated length of time, and frozen until the end of the study, when the samples were thawed and assayed.

Factor IX coagulation assays were performed by a one-stage method using Kontakt brand APTT (Pacific Haemostasis) and congenital Factor IX-deficient plasma (George King or Universal Reagents). The standard was a lyophilized CFIX-SD concentrate. Working dilutions of Factor IX were prepared on 0.05 M imidazole buffer, pH 7.3, containing 0.1 M NaCl, 0. 1% bovine serum albumin (BSA), and 0.01% Tween 20 to protect the protein from loss and denaturation at surfaces. The samples were assayed on an MLA Electra 900 Automatic Coagulation Timer. Two or more standard curves were included for all runs.

In vitro half-lives (T_½), the time in days at which Factor IX clotting activity declined to 50% of the original activity, were determined from straight-line semi-log plots of the log of the % of initial activity remaining versus days of incubation.

Results

The stability of samples is shown in Table 1, below:

TABLE 1
In Vitro Half-life (T½ in Days) of Factor IX Clotting Activity
(100 Units/ml) at pH 6.8
		T½,
NO. FORMULATION	T½, 37°C C.	4°C C.
1 0.01M Hist/0.1M NaCl	12.0	310
2 0.01M Hist/0.1M NaCl + 10 mM CaCl2	41	540
3 0.01M Hist/0.1M NaCl + 10 mM MgCl2	10	305
4 0.02M NaCit/0.11M NaCl	10.0	110
5 0.02M NaCit/0.11M NaCl + 40 mM CaCl2	5	300
6 0.01M Hist/0.1M NaCl + Heparin, 2 u/mL	8	--
7 0.01M Hist/0.1M NaCl + Heparin, 2 u/ml +	36	--
10 mM CaCl2
8 0.01M Hist/0.1M NaCl + 1 mM Choline	9	--
Factor IX-M (JHL) w/ DEAE polishing column

The indications of Example 1 are:

(1) Histidine provides greater stability than citrate, especially at 4°C C. (No. 1 vs No. 4).

(2) Calcium cannot be replaced by magnesium (No. 3 vs. No. 2).

(3) The presence of heparin does not improve Factor IX stability (No. 6, No. 7).

EXAMPLE 2

Effect of Various Excipients on Stability of Coagulation Factor IX in Histidine-saline

CFIX-M (JHL) in citrate-saline was diluted 1:10 in 0.01 M histidine, 0.1 M NaCl, pH 6.8 (histidine-saline) to give a final composition of 0.009 M histidine, 0.002 M citrate, 0.1 M NaCl, pH 6.8 (Buffer A) or was dialyzed against histidine-saline (Buffer B). Thereafter, the samples were treated as in Example 1, above, with the following changes:

(1) Samples were incubated at 37°C C., as indicated on the table below.

(2) Factor IX coagulation assays were performed on a Lancer Coagulyzer II.

Results

The stability of samples is shown in Table 2, below:

TABLE 2
In Vitro Half-life (T½ in Days) of Clotting Activity of Factor IX-M at
37°C C.
NUMBER	FORMULATION	T½ (DAYS)
1	0.009M Histidine, 0.002M citrate, 0.1M NaCl,	2.2
	pH 6.0
2	0.009M Histidine, 0.002M citrate, 0.1M NaCl,	4.2
	pH 8.0
3	0.009M Histidine, 0.002M citrate, 0.1M NaCl,	5.0
	pH 6.8 (Buffer A)
4	0.05M Glycine in Buffer A	2.8
5	0.5M Glycine in Buffer A	3.0
6	0.1% PEG 4000 in Buffer A	4.0
7	1.0% PEG 4000 in Buffer A	5.0
8	0.1% Albumin (BSA) in Buffer A	1.3
9	1.0% Albumin (BSA) in Buffer A	1.8
10	0.01% Tween in Buffer A	3.0
11	0.10% Tween in Buffer A	5.0
12	0.01M Histidine, 0.1M NaCl, pH 6.8	5.0
	(Buffer B)
13	0.1M Glycine, 2.5 mM CaCl2 in Buffer B	28.0
14	5 mM CaCl2 in Buffer B	30.0
15	100 mM CaCl2 in Buffer B	30.0
Factor IX-M (JHL) w/o DEAE polishing column

The initial indications are:

(1) The absence of calcium may inhibit the stability of the Factor IX.

(2) Addition of the other tested excipients--glycine, polyethylene glycol (PEG), albumin or Tween--cannot compensate for an absence of calcium.

(3) Factor IX is destabilized by albumin.

EXAMPLE 3

Effect of pH, Purity and Excipients on Stability of Coagulation Factor IX

Factor IX-M (JHL) with or without DEAE polishing, CFIX-SD, and ine in ARC formulation (ARC/A9) were treated as in Example 2 above.

Results

The stability of samples is shown in Table 3, below:

TABLE 3
In Vitro Half-life (T½ in Days) of Clotting Activity of Factor IX at 37°C C.
				T½
NO.	FORMULATION	pH	FIX	37°C C.
1	0.01M Hist/0.1M NaCl + 10 mM CaCl2	6.8	DEAE	49
2	0.01M Hist/0.1M NaCl	6.8	DEAE	12
3	Hist/NaCl + 20% Sucrose + 10 mM	6.8	DEAE	42
	CaCl2
4	Hist/NaCl + 0.5M Glycine + 20%	6.8	DEAE	37
	Sucrose + 10 mM CaCl2
5	Hist/NaCl + 10 mM CaCl	6.8	CFIX-SD	18*
6	Hist/NaCl	6.9	CFIX-SD	7
7	Hist/NaCl + 10 mM CaCl2	6.8	ARC/A9	17
8	Hist/NaCl	6.9	ARC/A9	2.5
9	Hist/NaCl + 10 mM CaCl2	6.0	DEAE	43
10	Hist/NaCl + 10 mM CaCl2	6.8	DEAE	43
11	Hist/NaCl + 10 mM CaCl2	7.4	DEAE	35
12	Hist/NaCl + 10 mM CaCl2	8.0	DEAE	16
13	Hist/NaCl + 0.5M Lysine + 10 mM	6.8	DEAE	6
	CaCl2 + 20% Sucrose
14	0.01M Hist/0.1M NaCl	6.8	FIX-M	8
FIX-M = Factor IX-M (JHL) w/o DEAE polishing column
DEAE Factor IX-M (JHL) w/DEAE polishing column
ARC/A9 = ARC Alphanine-SD
CFIX-SD = ARC CFIX-SD

The date of Table 3 are graphed in FIG. 2.

The dications of Example 3 are:

(1) Factor IX is destabilized by impurities: CFIX-SD has much lower stability with and without CaCl₂ than FIX-M/DEAE (Nos. 5, 6 vs. Nos. 1, 2); ARC/A9 has much lower stability with and without CaCl₂ than FIX-M/DEAE (Nos. 7, 8 vs. Nos. 1, 2); FIX-M without DEAE polishing has lower stability than with DEAE polishing (No. 14 vs. No. 2).

(2) Factor IX is destabilized by lysine and glycine.

(3) The buffer cited by Octapharma (See No. 13, WO 91-10439) does not provide the stability of the present invention (No. 13).

(4) The range of pH that provides the greatest stability of Factor IX is 6.0 to 6.8.

EXAMPLE 4

Stability of Coagulation Factor IX in Non-aqueous Liquid Formulations

CFIX-M (JHL) is formulated as a lyophilized mixture of the following components:

(1) 38 milligrams (mg) Coagulation Factor IX

(2) 70 mg NaCl

(3) 290 mg glycine

(4) 8.8 mg CaCl₂

(5) 6 mg histidine.

Aliquots (100 mg) of this dry mixture were resuspended and/or dissolved by syringe in 900-1000 microliters of one of the solvents (polypropylene glycol (molecular weight 425)(PPG); poly(dimethyl siloxane)(PM S); glycerol; or water). These were then aliquoted as 100 microliter samples into ten vials, sealed, and incubated at 37°C C. Factor IX coagulation assays were performed as described in Example 1.

Results

The stability of samples is shown in Table 4 below:

TABLE 4
In Vitro Stability of Factor IX in Non-Aqueous Liquid Formulations
		FINAL ACTIVITY
SOLVENT	HALF LIFE (Days)	(% original) AT 14 DAYS
PMS	5.5	23
PPG	14	50
Glycerol	11	36
Water	12	29

The data of Table 4 are graphed in FIG. 6.

The results indicate:

(1) PPG provided the greatest stability for Coagulation Factor IX.

(2) PM S provided the least stability for Coagulation Factor IX.

EXAMPLE 5

Stability of Coagulation Factor VIII in Non-aqueous Liquid Formulations

Coagulation Factor VIII (Baxter AHFM) is provided as a lyophilized mixture that provides a final composition in 7.5 mL of:

(1) 1000-1200 units Coagulation Factor VIII (133-160 units/mL)

(2) Human Albumin, 16.7 mg/mL

(3) Polyethylene glycol, 2.0 mg/mL

(4) 73 millimoles/liter Histidine

(5) 40 millimoles/liter Glycine. A 100 mg aliquot of this powder was resuspended and/or dissolved in 2.5 mL of solvent (polypropylene glycol (molecular weight 425)(PPG); polyethylene glycol (molecular weight 300)(PPG); or water) by syringe-to-syringe mixing and distributed into 100 microliter aliquots. These vials were then incubated at 37°C C.

Factor VIII activity assays were performed according to the method used in Example I for Factor IX, but using Mega I (Office of Biologics Research and Review, Bethesda Md.) as a Factor VIII standard in place of the Factor IX standard, and using Factor VIII-depleted plasma (Universal Reagents) in place of the Factor IX-deficient plasma.

Results

The stability of samples is shown in Table 5, below:

TABLE 5
In Vitro Stability of Factor VIII in Non-Aqueous Liquid Formulations
SOLVENT	HALF LIFE (Days)	FINAL ACTIVITY (% original)
PPG	23	44
PEG	3	0
Water	18	29

The data of Table 5 is graphed in FIG. 7.

The results indicate that both PPG and water demonstrated an activity spike at 7 days, with a marked decrease following. PEG as a solvent resulted in an almost immediate inactivation of Factor VIII, without the activity spike preceding.

This Example, and Example 4 above with Factor IX in non-aqueous liquid formulations, may be considered preliminary experiments to test the concept of using non-aqueous solvents for clotting factor stabilization. The spike in activity at day 7 with Factor VIII in PPG and water, and with Factor IX in PPG, may be due to activation caused by bacterial contamination of the materials during syringe-to-syringe mixing. The experimental technique was modified and improved in subsequent experiments with non-aqueous liquid formulations, described in Example 8 (Table 7, FIGS. 16 and 17) and Example 9 below (FIGS. 18 and 19).

EXAMPLE 6

Optimization of Aqueous Formulations for Factor IX at 37°C C.

For optimization of pH and calcium concentration, highly purified Factor IX-M with DEA polishing was prepared at a relatively high concentration (2.4 mg/mL; 600 units/mL), a condition under which the protein tends to be activated and fragmented during extended incubation at 37°C C. The formulations contained 10 mM histidine, 0.1 M sodium chloride and calcium chloride at one of the following concentrations: 10 mM, 30 mM and 100 mM. Each formulation was adjusted to one of the following pH values: 5.8, 6.0 and 6.2. The formulations were sterile filtered, dispensed aseptically into autoclaved polypropylene tubes and incubated at 37°C C.

Samples of each formulation were removed at weekly intervals and frozen at -80°C C. After 56 days, all samples were thawed and analyzed for Factor IX clotting activity by a one-stage APTT coagulation assay with a substrate of Factor IX-deficient plasma. Percent activity was plotted versus days of incubation and the in vitro half-life (T_½) was determined as the time in days at which the curve crossed the 50% activity line.

Results

Decay curves of % initial clotting activity are shown in FIGS. 8-10. When results at each of the three pH values were plotted for 10 mM CaCl₂ (FIG. 8), the half-life of clotting activity varied from 38 days to 58 days, with a slightly biphasic curve (having an initial steady decrease for several weeks followed by a gently rising curve or change in slope) suggestive of possible activation at pH 6.2. Similarly, at 30 mM CaCl₂, clear evidence of activation appeared in the pH 6.2 samples (FIG. 9). At 100 mM CaCl₂, there is a slight indication of possible activation at pH 6.0 and pH 6.2 after 40 days (FIG. 10). Table 6 below summarizes the half-life values, and indicates where activation appears possible or likely based on the slight biphasic nature of the decay curves. It appears from this summary that pH 5.8 is superior to pH 6.0 or pH 6.2.

In FIGS. 11, 12 and 13, the three CaCl₂ concentrations are compared at each pH value. The shallower decay curves seen at 100 mM CaCl₂ at both pH 6.0 and pH 6.2 may reflect Factor IX activation. This highest CaCl₂ concentration may be detrimental to long-term stability. In addition, there was clear evidence of activation at 30 mM CaCl₂ at pH 6.2, and this condition may also be suboptimal.

SDS-PAGE gels with Coomassie Blue staining for the nine pH/CaCl₂ combinations incubated at 37°C C. are shown in FIGS. 14A-14D for days 0 and 56 and FIGS. 14E-14M (reduced gels) for all time points. Gels were run in reduced and non-reduced condition (with or without 2-mercaptoethanol added to dissolve disulfide bonds holding fragments together). At zero time, Factor IX looked the same in all formulations, except that in 100 mM CaCl₂ there was high molecular weight smearing in the reduced gels (probably due to non-specific salt effects). High molecular weight smearing was not seen when the 100 mM CaCl₂ samples were diluted with water FIGS. 14K-14M. At 56 days, there was fragmentation in all samples, but clear differences between samples were evident in the reduced gel in FIG. 14D. The best conditions with least fragmentation and the greatest amount of intact monomer appeared in lanes 6-7 (30 mM Ca at pH 5.8-6.0) and lanes 9-10 (100 mM Ca at pH 5.8-6.0). The time studies in FIGS. 14E-14M confirm that the least amount of fragmentation was observed at pH 5.8-6.0 at 30 mM Ca or greater.

Based on the clotting activity, the best CaCl₂ concentration appears to be 30-100 mM. At pH 5.8 and 6.0, however, the stability of Factor IX (T_½) increased with increasing CaCl₂ concentration. The shallower curves observed at higher CaCl₂ concentrations probably indicate improved stability rather than Factor IX activation; SDS-PAGE data suggest that CaCl₂ concentrations of ≧30 mM are best at pH 5.8 or 6∅ Previous results (data now shown) indicate that the stability of Factor IX is very short at pH 5.5.

TABLE 6
Factor IX Clotting Activity T½ Values and Possible Activation upon
Incubation of High-Purity Factor IX (600 units/mL) at 37°C C. in Liquid
Formulations Containing 10-100 mM CaCl2 at pH 5.8-6.2
	FORMULATION
	OF
	10 mM
	HISTIDINE.	CLOTTING ACTIVITY HALF-LIFE
	0.1M NaCl PLUS	pH 5.8	pH 6.0	pH 6.2
	10	mM CaCl2	47	38	58
					(activated?)
	30	mM CaCl2	52	56	?
					(activated?)
	100	mM CaCl2	57	>60	>60
				(activated?)	(activated?)

EXAMPLE 7

Evaluation of Aqueous Factor IX Formulation at 4°C C.

Formulation handling and assays were performed as in Example 6. Six formulations containing various concentrations of Factor IX (25 units/mL -600 units/mL) were prepared with 10 mM histidine, 0.1 M NaCl, 10 mM CaCl₂, pH 6.8. Samples of each formulation were incubated in a cold room (4°C C.), frozen and assayed for clotting activity.

Results

FIG. 15 shows the decay curves for samples incubated at 4°C C. Elevation of clotting activity above the initial 100% activity, indicative of Factor IX activation, is evident at 600 units/mL (70 days), 300 units/mL (140 days), 200 units/mL (210 days) and 100 units/mL (410 days). No evidence of activation was detected at 20 units/mL or 50 units/mL up to 410 days. At the 410 day time point, more than 80% of the initial activity remained in these samples.

EXAMPLE 8

Non-aqueous Formulations For Factor VIII

The freeze-dried Factor VIII used in this Example was Antihemophilic Factor, Human, Method M, Solvent and Detergent Treated (AHF-M) prepared from American Red Cross donor plasma by Baxter Healthcare Hyland Division (Glendale, Calif.). For each experiment, a vial of AHF-M (approximately 1000 units) was suspended in 10 mL of a non-aqueous solvents by adding the solvent aseptically with a hypodermic needle to the freeze-dried Factor VIII powder in an unopened vial, and allowing the suspension to liquefy. Among the solvents tested were: glycerol, dimethyl sulfoxide (DM SO), ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (PEG) 200, PEG 300, PEG 400, dipropylene glycol, tripropylene glycol, polypropylene glycol (PPG) 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000. Each suspension was aseptically aliquoted into sterilized polypropylene tubes. The tubes were incubated at 37°C C., removed at various times, diluted and assayed for clotting activity.

Results

In some non-aqueous solvents (glycerol, DM SO, ethylene glycol), Factor VIII activity appeared to be rapidly inactivated based on undectable activity levels even in unincubated zero time controls (Table 7). These solvents were not found to inhibit the clotting assay when diluted to the concentrations present in diluted Factor VIII samples.

The clotting activity of Factor VIII in dipropylene glycol, tripropylene glycol and polyethylene glycol was also lost quickly; half-life values in these solvents were four days or less.

In contrast, in PPG 425, PPG 725 and PPG 1000, Factor VIII activity was maintained as well as, or better than, the activity of Factor VIII dissolved in water (FIG. 16; Table 7). While the half-life of Factor VIII in aqueous solution was 34 days, the half-life was 42 days in PPG 425, ≧60 days in PPG 725, and ≧90 days in PPG 1000.

When plotted against PPG oligomer size (i.e. the number of monomer units found in the PPG polymer), half-life values for Factor VIII clotting activity are nearly linear with oligomer size up to about PPG 1000, an of 17 monomer units (FIG. 17). Larger PPG polymers (PPG 2000 3000) also gave high stability, but the viscosity of these polymers riations due to affinity in obtaining accurate sample volumes. PPG eared to give lower stability.

PEG was ineffective in stabilizing Factor VIII up to PEG 400 size=4 monomer units) (FIG. 16; Table 7). Higher oligomers of PEG were solid at room temperature and so were not further evaluated.

TABLE 7
In Vitro Half-life (T½ in Days) of Freeze-Dried Factor VIII (AHF-M)
Suspended in Non-Aqueous Liquid Formulations at 37°C C.
NUMBER	SOLVENT	T½ (DAYS)
1	Water	34
2	DMSO	0
3	Glycerol	0
4	PPG 134 (Dipropylene Glycol)	3
5	PPG 192 (Tripropylene Glycol)	4
6	PPG 425 (7-mer)	42
7	PPG 725 (12-mer)	≧60
8	PPG 1000 (17-mer)	≧90
9	PEG 150 (Triethylene Glycol)	0
10	PEG 200 (Tetraethylene Glycol)	2
11	PEG 300 (7-mer)	∼3
12	PEG 400 (9-mer)	∼3

EXAMPLE 9

Non-aqueous Formulations for factor IX

Immunoaffinity-purified Factor IX was freeze-dried in 2.5 mL volumes in as Vitis freeze-dryer. For each experiment, 7 mg of dried Factor IX was suspended in 10 mL of non-aqueous solvent. The solvents tested were: PEG 200, PEG 300, PEG 400, tripropylene glycol, PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000. Each suspension was aseptically aliquoted into sterilized polypropylene tubes that were incubated at 37°C C. for various times, removed, diluted and assayed for clotting activity.

In contrast to the results obtained with Factor VIII in Example 8, polyethylene glycols offered some stability to Factor IX, as shown in FIG. 18. While PEG 300 and PEG 400 each gave a half-life value of under 5 days, the half-life with PEG 200 (tetraethylene glycol) was 17 days (which is less than half the T_½in aqueous solution).

Polypropylene glycols stabilized Factor IX to a significant extent. In tripropylene glycol, the half-life was about 56 days, compared with 39 days in aqueous solution. In PPG 425, PPG 725, PPG 1000 and PPG 2000, the half-life was not reached after 56 days (FIG. 19; irregularities were probably due to sampling errors).

EXAMPLE 10

In Vitro Delivery of Clotting Factors with Implantable Pumps

Two Arrow Model 3000 pumps (Arrow International, Walpole, Mass.) were used, which have a reservoir volume of 30 mL and a delivery rate of 2 mL/day. These pumps are licensed in the United States for implantation in the abdominal area, with intrathecal or hepatic artery access.

Aqueous solutions of Factor VIII (AHF-M) and Factor IX were injected into the pump septum/port according to the standard protocol for filling the pump. Each pump was placed in a water bath at 37°C C. to activate the pumping mechanism, and the contents were delivered through an internal sterile filter via an exit catheter into a collection tube. Samples were collected daily for 13 days and the volume and clotting activity were measured.

Results

Effluent volume was maintained accurately at 2 mL/day for both pumps (FIGS. 20 and 21).

Factor VIII activity was 95% lost by day 6 (FIG. 20). This was determined to be caused, however, by bacterial contamination when sterility was compromised during the process of filling the pump with AHF-M solution (bacterial contamination was confirmed by agar plate colony and culture assessment).

Factor IX activity in the effluent from the pump decreased relatively steadily throughout the 13 days of operation (FIG. 21). The half-life was 18-20 days, about half that observed in polypropylene tubes. At day 13, the effluent activity was 60% of the starting activity. The units of Factor IX delivered per day was 185-210 units for the first five days, decreasing to 160-170 units during days 6-11 and decreasing further to 110-140 units for the final two days (FIG. 22). No bacteria were detected in the Factor IX effluent.

EXAMPLE 11

Biocompatibility/Delivery of Factor VIII with Implantable Pump

Two Arrow Model 3000 pumps (Arrow International, Walpole, Mass.) with delivery rates of 1 mL/day were filled aseptically with a sterile aqueous solution of Factor VIII (reconstituted AHF-M, Lot No. 2935501 AA; 80 Factor VIII clotting units/mL). One pump was placed in a 37°C C. water bath to activate the pumping mechanism, and the contents were delivered via a 0.2 micron filter, glass flow constrictor and silicone rubber exit catheter into a collection tube as described in Example 10. Effluent samples were collected every one to four days and clotting activity was measured by one-stage APTT assay.

To determine the biocompatibility of Factor VIII with the titanium pump reservoir, the exit catheter of the second filled pump was tied in a knot to prevent solution outflow and maintain the entire 30 mL of Factor VIII solution in the reservoir. Samples were withdrawn aseptically from the reservoir with a sterile hypodermic syringe needle every one to four days, and Factor VIII clotting activity was measured as described above.

Results

The activity of Factor VIII samples which were allowed to flow from the pump and those taken from the pump reservoir under stasis did not differ significantly (FIG. 23). The dip in the first samples of pump effluent was due to dilution of the effluent with the saline solution used to purge the pump tubing.

When the decay curve for Factor VIII sampled from the titanium pump reservoir under static conditions was compared to the decay curve for Factor VIII incubated in polypropylene tubes in an earlier experiment, the decay was more rapid in the pump than in the polypropylene tubes (FIG. 24). The in vitro half-life in the titanium reservoir was approximately 22 days, compared to a half-life of about 38 days in polypropylene tubes. This faster decay in the pump reservoir may reflect increased binding, denaturation or fragmentation of Factor VIII in the presence of the titanium surface of the reservoir under the conditions used for testing. Alternatively, the polypropylene surface of the tubes may, in fact, stabilize Factor VIII by some unknown mechanism.

The lower observed stability of Factor VIII incubated at 37 °C C. in the titanium reservoir of the Arrow Model 3000 pump compared to the observed stability of Factor VIII incubated at 37°C C. in polypropylene tubes suggests a difference in the biocompatibility of these two materials with Factor VIII. No difference was observed, however, in the stability of Factor VIII incubated statically inside the pump reservoir and Factor VIII pumped out through the glass/silicone rubber exit catheter. This suggests that the glass and rubber surfaces do not alter the activity of the Factor VIII exiting the pump.

EXAMPLE 12

Biocompatibility/Delivery of Factor IX with Implantable Pump

As in Example 11, two Arrow Model 3000 pumps (Arrow International, Walpole, Mass.) with delivery rates of 1 mL/day were filled aseptically with a sterile-filtered aqueous solution of immunity-purified Factor IX (100 Factor IX clotting units/mL) in 10 mM histidine, 0.10 M NaCi, 10 mM CaCl2, pH 6.2. One pump was placed in a 37°C C. water bath to activate the pumping mechanism, and the contents were delivered via a 0.2 micron filter, glass flow constrictor and silicone rubber exit catheter into a collection tube as described above. Effluent samples were collected every one to two and Factor IX clotting activity was measured by one-stage APTT assay.

To determine the biocompatibility of Factor IX with the titanium pump reservoir, the exit catheter of the second filled pump was tied in a knot to prevent solution outflow and maintain the entire 30 mL of Factor IX solution in the reservoir. Samples were withdrawn aseptically from the reservoir with a sterile hypodermic syringe needle every one to two days, and Factor VIII clotting activity was measured as described above.

Results

The activity of Factor IX samples which were allowed to flow from the pump and those taken from the pump reservoir under stasis did not differ significantly (FIG. 25). Both decay curves extrapolate to half-life values of about 25 days.

When the decay curve for Factor IX sampled from the titanium pump reservoir under static conditions was compared to the decay curve for Factor IX incubated in polypropylene tubes in an earlier experiment, the in vitro half-life in the titanium reservoir was approximately 25 days, compared to a half-life of about 35 days in polypropylene tubes (FIG. 26). Although the half-life in the titanium reservoir is shorter, the rate of decay appears to be similar in both the titanium reservoir and polypropylene tubes (i.e. the slopes of the decay curves are roughly equal). The lower half-life of Factor IX in the titanium reservoir appears to be due to an initial drop (about 25%) in Factor IX activity at the time of the first assay (day 2), suggesting that a portion of the Factor IX is bound to the inner surface of the pump reservoir but the remainder is unaffected.

As with Factor VIII, no difference was observed between Factor IX held statically inside the pump reservoir and Factor IX pumped out through the glass/silicone rubber exit catheter, indicating that glass and silicone rubber do not alter the potency of the Factor IX exiting the pump. The half-life of Factor IX incubated at 37°C C. in the titanium pump reservoir was lower than that of Factor IX incubated at 37°C C. in polypropylene tubes, but this appeared to be due to an initial drop in Factor IX content in the pump reservoir, followed by a rate of decay that was about the same for both materials.

Claims

1. A method of delivering a plasma protein to a mammal in need thereof, said method comprising the steps of:

(a) preparing a stable (at least 30 days at 37°C C.) liquid formulation of at least one plasma protein selected from the group consisting of: (i) a stable aqueous liquid formulation of at least one plasma protein, (ii) a stable non-aqueous liquid formulation of at least one plasma protein, and (iii) a stable mixed aqueous and non-aqueous liquid formulation of at least one plasma protein;

(b) incorporating said stable liquid formulation of at least one plasma protein into a continuous administration system; and

(c) introducing said continuous administration system into amammal in need of said plasma protein,

wherein said plasma protein is delivered from said continuous administration system into said mammal, and

wherein said stable aqueous liquid formulation of at least one plasma protein is selected from the group consisting of:

(i) a stable aqueous liquid formulation comprising: (a) a plasma protein; (b) a ph buffering compound; (c) a source of calcium ions; (d) an osmotic modulating agent in a concentration of 1-500 mM; and (e) water;

(ii) a stable aqueous liquid formulation comprising: (a) aplasma protein; (b) a ph buffering compound; (c) calcium chloride in a concentration of 0. 1-40 mM; (d) an osmotic modulating agent; and (e) water; and

(iii) a stable aqueous liquid formulation comprising: (a) a plasma protein; (b) a ph buffering compound in a concentration of 0.1-100 mM; (c) a source of calcium ions; (d) an osmotic modulating agent; and (e) water; and

wherein said stable non-aqueous liquid formulation comprises: (a) a plasma protein; and (b) a non-aqueous liquid, and contains substantially no water, and

wherein said stable mixed aqueous and non-aqueous liquid formulation of a plasma protein comprises: (a) a plasma protein; (b) a non-aqueous liquid selected from the group consisting of: glycerol, dimethyl sulfoxide (DM SO), ethylene glycol diethylene glycol, triethylene glycol, PEG 200, PEG 300, PEG 400, dipropylene glycol, tripropylene glycol, PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000; and (c) water.

2. The method according to claim 1, wherein said continuous administration system is an implantable pump.

3. The method according to claim 1, wherein osmotic modulating agent is sodium chloride (NaCl).

4. The method according to claim 1, wherein said stable liquid formulation of at least one plasma protein is a stable aqueous liquid formulation comprising:

(a) a plasma protein;

(b) a ph buffering compound;

(c) calcium chloride (CaCl₂);

(d) an osmotic modulating agent; and

(e) water.

5. The method according to claim 1, wherein said stable liquid formulation of at least one plasma protein is a stable non-aqueous liquid formulation of at least one plasma protein comprising:

(a) a plasma protein; and

(b) a non-aqueous liquid.

6. The method according to claim 1, wherein said plasma protein is a vitamin K-dependent plasma protein selected from the group consisting of Factor II, Factor VII, Factor IX, Factor X, protein C, protein S and protein Z.

7. The method according to claim 1, wherein said plasma protein is a non-vitamin K-dependent plasma protein selected from the group consisting of Factor VIII and von Willebrand Factor.

8. The method according to claim 1, wherein said continuous delivery system is a microsphere.

9. The method according to claim 8, wherein said microsphere is selected from the group consisting of liposomes and semi-permeable polymeric microcapsules.

10. The method according to claim 1, wherein said ph buffering compound is an amino acid.

11. The method according to claim 10, wherein said amino acid is histidine.

12. The method according to claim 1, wherein said non-aqueous liquid is a hydrophilic non-aqueous liquid.

13. The method according to claim 12, wherein said hydrophilic non-aqueous liquid is a polyethylene glycol or a polypropylene glycol.

14. The method according to claim 1, wherein said continuous administration system is a bio-resorbable hydrogel.

15. The method according to claim 14, wherein said bio-resorbable hydrogel contains a charged or uncharged polymeric agent to control hydrogel porosity.

16. The method according to claim 14, wherein said bio-resorbable hydrogel contains a charged or uncharged polymeric agent to control hydrogel stability.

17. The method according to claim 14, wherein said bio-resorbable hydrogel contains a charged or uncharged polymeric agent to control kinetics of plasma protein delivery.

18. The method according to claim 14, wherein said bio-resorbable hydrogel is a biologically-derived bio-resorbable hydrogel.

19. The method according to claim 18, wherein said biologically-derived bio-resorbable hydrogel is selected from the group consisting of a chitosan hydrogel and a N,O-carboxymethyl chitosan hydrogel.

20. The method according to claim 14, wherein said bio-resorbable hydrogel is a synthetically-derived bio-resorbable hydrogel.

21. The method according to claim 20, wherein said synthetically-derived bio-resorbable hydrogel is a hydrogel derived from propylene glycol or ethylene glycol.

22. A method of delivering a plasma protein to a mammal in need thereof, said method comprising the steps of:

(a) preparing a stable (at least 30 days at 37°C C.) liquid formulation of at least one plasma protein selected from the group consisting of: (i) a stable aqueous liquid formulation of at least one plasma protein, (ii) a stable non-aqueous liquid formulation of at least one plasma protein, and (iii) a stable mixed aqueous and non-aqueous liquid formulation of at least one plasma protein; and

(b) introducing said stable liquid formulation of at least one plasma protein into a mammal in need thereof,

wherein said plasma protein is delivered into said mammal, and

wherein said stable aqueous liquid formulation of at least one plasma protein is selected from the group consisting of:

(i) a stable aqueous liquid formulation comprising: (a) a plasma protein; (b) a ph buffering compound; (c) a source of calcium ions; (d) an osmotic modulating agent in a concentration of 1-500 mM; and (e) water;

(ii) a stable aqueous liquid formulation comprising: (a) a plasma protein; (b) a ph buffering compound; (c) calcium chloride in a concentration of 0. 1-40 mM; (d) an osmotic modulating agent; and (e) water; and

(iii) a stable aqueous liquid formulation comprising: (a) a plasma protein; (b) a ph buffering compound in a concentration of 0.1-100 mM; (c) a source of calcium ions; (d) an osmotic modulating agent; and (e) water, and

wherein said stable non-aqueous liquid formulation comprises: (a) a plasma protein; and (b) a non-aqueous liquid, and contains substantially no water, and

wherein said stable mixed aqueous and non-aqueous liquid formulation of a plasma protein comprises: (a) a plasma protein; (b) a non-aqueous liquid selected from the group consisting of: glycerol, dimethyl sulfoxide (DM SO), ethylene glycol, diethylene glycol, triethylene glycol, PEG 200, PEG 300, PEG 400, diproyplene glycol, tripropylene glycol, PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000; and (c) water.

23. The method according to claim 22, wherein said stable liquid formulation is introduced into said mammal as a subcutaneous bolus.