Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21022—Coagulation factor IXa (3.4.21.22)

Definitions

• the present invention relates generally to novel formulations comprising factor IX, including both highly concentrated, lyophilized, and liquid formulations comprising factor IX suitable for administration via various routes including for example routes such as intravenous, subcutaneous, intramuscular and intradermal.
• factor IX a plasma glycoprotein.
• a deficiency of factor IX characterizes a type of hemophilia (type B).
• Treatment of this disease has traditionally involved intra venous infusion of human piasma-derived protein concentrates of factor IX. Infusion of blood concentrates involves the risk of transmission of various infectious agents, such as viral hepatitis and HIV, or thromboembolic factors.
• An alternative method of producing factor IX, by recombinant DNA techniques, has been described in USPN 4,770,999, Kaufman et al., September 13, 1988.
• the cDNA coding for human factor IX has been isolated, characterized, and cloned into expression vectors. See.
• factor IX protein For example, Choo et al., Nature 299: 178-180 (1982); Fair et al., Blood 64: 194-204 (1984); and Kurachi et ai, Proc. Nat. Acad. Sci. , U.S.A. 79:6461-6464 ( 1982).
• a purification process for a protein results in concentrating the protein.
• This concentrated protein also known as bulk protein, may be in a formulation buffer.
• Bulk protein typically at a concentration of about 2 to at least 20 mg/mL, can then be shipped frozen to a fill/finish facility where it is adjusted to an appropriate dosage concentration and placed into dosage vials or some device suitable for administration, e.g. a pre-fillable syringe.
• the drug product is left in the liquid state and stored and administered as a liquid.
• the drug product is lyophilized, i.e., freeze-dried.
• lyophilized drug product has sufficient stability to be kept in long-term storage, i.e., greater than six months; lyophilized drug product is reconstituted at a later time by adding a suitable administration diluent just prior to patient use.
• Protein stability can be affected inter alia by such factors as ionic strength, pH, temperature, repeated cycles of freeze/ thaw, and exposures to shear forces Active protein may be lost as a result of physical instabilities, including denaturation and aggregation (both soluble and insoluble aggregate formation), as well as chemical instabilities, including, for example, hydrolysis, deamidation, and oxidation, to name just a few
• physical instabilities including denaturation and aggregation (both soluble and insoluble aggregate formation)
• chemical instabilities including, for example, hydrolysis, deamidation, and oxidation
• liquid stability When developing a liquid formulation, many factors are taken into consideration Short-term, i e , less than six months, liquid stability generally depends on avoiding gross structural changes, such as denaturation and aggregation These processes are described in the literature for a number of proteins, and many examples of stabilizing agents exist ("Strategies to Suppress Aggregation of Recombinant Keratinocyte Growth Factor during Liquid Formulation Development", B L Chen et al , i Pharm Sci 83(12) 1657-1661 , (1994), ' Formulation Design of Acidic Fibroblast Growth Factor", P K Tsai et al , Pharm Res 10(5) 649-659 (1993), "The Stabilization of Beta-Lactoglobuhn by Glycine and NaCl",
• More specific types of degradation may include, for example, disulfide bond scrambling, oxidation of oligosaccharides and/or certain residues, deamidation, cyclization, and the like.
• assays are developed to monitor subtle changes so as to monitor the ability of specific excipients to uniquely stabilize the protein of interest.
• the formulation be approximately isotonic and that the pH of the formulation be in a physiologically suitable range upon injection/infusion, otherwise pain and discomfort for the patient may result.
• the choice and amount of buffer used is important to achieve the desired pH range.
• the choice and amount of agents used to modify tonicity is important to assure ease of administration.
• labile proteins such as factor IX
• intravenous the protein is directly available in the blood stream.
• side effects including occlusion and/or fibrin formation, especially in the elderly.
• the patient's veins are particularly small, e.g., in small children, it can be difficult to achieve the requisite therapeutic dose.
• factor IX formulations there are no highly concentrated factor IX formulations commercially available.
• carrier-protein-free, plasma- derived factor IX formulations are freeze-dried products which are reconstituted for use, and are limited to low factor IX concentrations, e.g., about 100 U/mL or less than 1 mg/mL.
• low concentrations are primarily indicated for intravenous administration and not intended for subcutaneous, intramuscular, or intradermal use.
• Alpha Therapeutic Corporation provides lyophilized AlphaNine ® SD, comprising heparin, dextrose, polysorbate 80, and tri(n-butyl) phosphate. This preparation is meant to be stored at temperatures between 2° and 8°C.
• Heparin is to be avoided as it is an anti-coagulant and tri(n-butyl) phosphate is irritating to mucous membranes; thus, this formulation is less than ideal.
• Armour Pharmaceutical Company provides lyophilized Mononine ® , comprising histidine, sodium chloride and mannitol, is similarly meant to be stored at 2° to 8°C. The package insert recommends not storing this formulation for greater than one month at room temperature. There are no liquid nor any highly concentrated factor IX products currently commercially available.
• Easier to handle for the patient are administration forms such as subcutaneous, intramuscular, or intradermal.
• Subcutaneous administration of factor IX is described in Berrettini, Am. J. Hematol. 47:61 (1994) and in WO 93/07890, BTG, Brownlee (published 29 April 1993).
• an Immuno product was used: ImmunineTM; factor IX, heparin, sodium citrate, sodium chloride, and antithrombin III at a concentration of 118 U/mg.
• the product was reportedly poorly and slowly transported into the circulation and the authors concluded that subcutaneous administration was not reliable for treating or preventing bleeding in hemophilia B patients and that even more concentrated forms would be unacceptable in terms of clinical efficacy.
• plasma derived factor IX (pFIX) was administered subcutaneously (at doses of 15-47 U/kg in dogs and at a dose of 30 U/kg in the patient). This resulted in plasma factor IX in dogs which was dose dependent and ranged from 0.8 to 7.6% , with intramuscular giving higher levels.
• plasma factor IX activity reached only 1 % within six hours; this level of activity persisted for 36 hours; the low concentration of plasma-derived factor IX required high volume injections at multiple (10) sites.
• formulations should provide for factor IX stability for greater than one year and for compatibility over a wide range of protein concentration (0.1 mg/mL to greater than
• compositions and methods for providing highly concentrated, lyophilized, and liquid preparations of factor IX useful as bulk protein or useful for administration are stable for at least six months, and preferably up to 36 and 60 months; and can be stored at temperatures ranging from -100°C to 40°C, from -80°C to 0°C, and from -20°C to 10°C.
• the compositions comprise factor IX, tonicity modifiers, cryoprotectants, and, optionally, a buffering agent and/or other excipients which further stabilize factor IX.
• the factor IX concentration ranges from about 0.1 to about 160 mg/mL (equivalent to about 20 to at least 56,000 U/mL), with 1 to 160 mg/mL (250 to 56,000 U/mL) and 0.1 to 10 mg/mL (25 to 2500 U/mL) preferred, the most preferred range depending upon the route of administration.
• Tonicity modifiers include, but are not limited to, salts, sugars, polyols, and amino acids. Suitable amino acids include arginine, glycine and histidine at a concentration of about 10 to 500 mM, with about 10 to 300 mM and about 10 to 200 mM preferred.
• Suitable cryoprotectants include polyols, e.g.
• compositions may also contain a surfactant or detergent, such as polysorbate (e.g. Tween) or polyethyleneglycol (PEG), which may also serve as a cryoprotectant during freezing.
• a surfactant or detergent such as polysorbate (e.g. Tween) or polyethyleneglycol (PEG), which may also serve as a cryoprotectant during freezing.
• the surfactant ranges from about 0.005 to 1 % , with about 0.005 to 0.1 % and about 0.005 to 0.02% preferred.
• the composition may contain an appropriate buffering agent to maintain a physiologically suitable pH, e.g., in the range of about 5.8 to 8.0 with about 6.2 to 7.2 and about 6.5 to 7.0 being preferred.
• Buffering agents preferably include histidine, sodium citrate, potassium citrate, maleic acid, ammonium acetate, Tris, sodium phosphate, potassium phosphate, and diethanolamine, with sodium/potassium citrate preferred, with a preferred pH of about 6.5 to 7.5, and a concentration range of about 1-100 mM, with 5 to 50 mM and 10 to 25 mM preferred.
• small amounts of a chelator such as EDTA are included, at a concentration of 0.05 to 50 mM. or 0.05 to 10 mM, or 0.1 to 5 mM, with about 1 to 5 mM preferred.
• Another aspect of the present invention provides formulations of factor IX suitable for administration in a final dosage form, for example, via intravenous, subcutaneous, intradermal, or intramuscular routes of administration.
• a final dosage form for example, via intravenous, subcutaneous, intradermal, or intramuscular routes of administration.
• large quantities of bulk drug are frozen and can be shipped, if necessary, to a manufacturing site where the bulk drug is filled into small vials; if desired, the final dosage form is a diluted, pH-adjusted form; bulk drug typically comprises a higher protein concentration than finished drug and does not need to be isotonic.
• the finished drug compositions comprise factor IX, tonicity modifiers, cryoprotectants and optionally a buffering agent and/or other excipients which further stabilize factor IX, as described, supra.
• the finished drug formulations are stable for at least six months and preferably up to 36 and 60 months; and can be stored at temperatures ranging from -100 C C to 40°C, from -20°C to 37°C and from 2°C to 8°C.
• the concentrations of the excipients provide a combined osmolality of about 250 to 420 milliosmolal.
• Preferred formulations include factor IX concentrations ranging from about 0.1 to greater than 160 mg/mL (20 U/mL to greater than 56,000 U/mL); with sodium citrate as a buffering agent; some combination of mannitol, sucrose, arginine, and glycine as cryoprotectants and tonicity modifiers; and optionally small amounts of a chelator, such as EDTA (ca. 1 to 5 mM) and/or small amounts of polysorbate
• factor IX 0.1 to greater than 160 mg/mL
• glycine a surfactant and/or buffer (e.g. , histidine) and/or a cryoprotectant (e.g. polysorbate).
• a surfactant and/or buffer e.g. , histidine
• a cryoprotectant e.g. polysorbate
• novel methods of administration of highly concentrated factor IX using both intravenous and subcutaneous routes e.g. , an intravenous dose followed by a subcutaneous dose.
• factor IX includes both plasma derived and recombinantly or synthetically produced Factor IX concentration is conveniently expressed as mg/mL or as U/mL, with 1 mg usually representing > 150 U ⁇ 100 U or more One Unit of activity is defined as the amount of factor IX clotting activity in one milliliter of normal human plasma
• the specific activity is the ratio of clotting activity concentration to protein concentration, expressed as U/mg of protein Patients with hemophilia generally have from ⁇ 1 to 25 % of the factor IX clotting factor as is found in normal human plasma
• amounts specified are understood to be ⁇ about 10% , e g , about 50 mM includes 50 mM ⁇ 5 mM, e g , 4% includes 4% ⁇ 0 4% , etc
• tonicity modifier includes agents which contribute to the osmolality of the solution
• tonicity modifiers include, but are not limited to, ammo acids such as argimne, histidine, and glycine, salts such as sodium chloride, potassium chloride, and sodium citrate, and saccha ⁇ des such as sucrose, glucose, and mannitol. and the like
• cryoprotectant generally includes agents which provide stability to the protein from freezing-induced stresses, however, cryoprotectants may also provide general stability, for example for bulk drug formulations during storage from non-freezing-induced stresses
• cryoprotectants include polyols, and saccha ⁇ des such as mannitol and sucrose, as well as surfactants such as polysorbate, or polyethyleneglycol, and the like While preferred concentrations of cryoprotectant range from about 0 2 to 4% (weight/volume), relatively high concentrations, for example greater than 5 % , are also suitable, the levels used are limited oniy by those customarily used in clinical practice
• the upper concentration limits for bulk drug may be higher than for finished dosage, e g , greater than 5 %
• “Surfactants” generally include those agents which protect the protein from air/solution interface induced stresses and solution/surface induced stresses (e g , resulting m protein aggregation), and may include detergents such as polysorbate-80 (Tween), for example, about 0
• buffering agent encompasses those agents which maintain the solution pH in an acceptable range and may include histidine, phosphate (sodium or potassium), citrate (sodium or potassium), maleic acid, ammonium acetate, tris (tris (hydroxymethyl) ammomethane), diethanolamme, and the like
• the upper concentration limits may be higher for bulk protein than for finished dosage protein forms as is readily appreciated by one skilled in the art
• buffer concentrations can range from several milhmolar up to the upper limit of their solubility, e g., citrate, could be as high as 200 mM
• one skilled in the art would also take into consideration both achieving and maintaining a physiologically appropriate concentration
• Percentages are weight/volume when referring to solids dissolved in solution and volume/volume when referring to liquids mixed into solutions For example, for sucrose, it is dry weight sucrose/volume of solution and for Tween, it is the volume of
• excipients includes pharmaceutically acceptable reagents to provide appropriate tonicity, cryoprotection of the protein, maintenance of pH, and proper conformation of the protein during storage so that substantial retention of biological activity and protein stability is maintained
• Example 1 describes the effect of calcium addition and the effect of pH on clotting activity
• Example 2 describes the effects of specific buffering agents on the formation of high molecular weight aggregates (HMW)
• Example 3 illustrates the use of the invention for higher concentrations of factor IX
• Example 4 illustrates the complexity of excipient interactions in stabilizing factor IX
• Example 5 describes factor IX in various formulations relating to freeze/thaw stability
• Example 6 describes the effects of long term storage, and Examples 7 and 8 illustrate highly concentrated forms and their use
• a well characterized property of factor IX is its ability to bind Ca 2+ ions. Structural studies indicate that Ca :+ binding may confer a more stable structure, reducing the probability of molecular motion ("Structure of the Metal-free ⁇ -Carboxyglutamic Acid-rich Membrane
• Samples are prepared in the formulations set forth in Table I below, at a recombinant factor IX protein concentration of ⁇ 0.5 mg/ml (100 U/ml) and an osmolality of 300 ⁇ 50 milliosmolal. All samples contain a recombinant form of factor IX.
• a set of samples was prepared in the formulations listed in Table 1.
• the formulation of sample A is the formulation used for commercially available plasma-derived lyophilized factor IX (MononineTM). All samples contain a recombinant form of factor IX.
• Factor IX activity is determined according to the method of Pittman, D., et al.. Blood 79:389-397 (1992) utilizing factor IX- deficient blood.
• the ratio of clotting activity to protein concentration, the specific activity, expressed as Units/mg of protein, is given in Table 2.
• An acceptable specific activity is one that is generally no more than 20% higher than the starting specific activity because an unusually high specific activity may indicate an activation-like event, which may have thrombotic implications.
• Activated Factor IX is Factor IX that has been cleaved at residues R 145 - A 146 and R 180 - V 181 and is then able to catalyze clotting. Normally, Factor IX circulates as intact protein and is not converted to its activated form unless there is initiation of the clotting cascade. Injecting someone with activated rhFIX could have thrombotic implications. Therefore inclusion of Ca 2+ at a concentration of 5 mM is destabilizing and is to be avoided.
• Example 2 Effects of Buffer Choice on HMW formation
• Factor IX is prepared in a set of isotonic experimental formulations as summarized in Table 3, including several different excipient combinations for each buffering agent and some including less than 5 mM EDTA. Factor IX concentrations are approximately 1 mg/mL (average 161 U/mL). Samples are assayed for the amount of high molecular weight material (HMW) present and for clotting activity. The formation of significant ( > 3%) amounts of HMW is undesirable and as indicative of physical degradation of factor IX with possible impact on product safety and efficacy. HMW is especially undesirable for subcutaneous administration as aggregated proteins are more immunogenic when given subcutaneously. Morein, B. and K. Simons, Vaccine 3:83. Subunit vaccines against enveloped viruses: virosomes, micelles, and other protein complexes (1985); and Antibodies: A laboratory manual, (page 100), E. Harlow and D. Lane, Cold Spring Harbor Laboratory, 1988.
• HMW high molecular weight material
• Table 4 shows the effects of the different buffering agents on HMW generation as measured by size exclusion chromatography (SEC-HPLC). Samples were stored at 30°C for six weeks. Table 4 gives the average increase expressed as (HMW/total protein x 100%) at six weeks minus that at time zero.
• citrate buffered samples had, on average, the smallest amount of HMW generated, regardless of the other excipients included.
• An appropriate buffer does not allow greater than a 2% increase.
• Aggregates are commonly known to be more immunogenic than monomeric proteins and are generally not acceptable for an intravenous formulation. However, aggregates are even more undesirable for a subcutaneous, intradermal or intramuscular formulation, because these routes of administration are more likely to generate an immune response.
• Another set of formulations is prepared comprising higher concentrations of factor IX; samples are prepared in the formulations listed in Table 5 at a concentration of 8 mg/mL (2000 U/mL). All contain 15 mM sodium citrate and are buffered at pH 6.8, without surfactant. BG4 is slightly hypertonic, the rest are isotonic. Table 5 Sample Formulations
• BG1 2 % sucrose, 2 % arginine-HCl, 1 mM EDTA
• BG2 4% sucrose, 1 % glycine, 1 mM EDTA
• BG4 5 % arginine-HCl, 1 mM EDTA
• BG5 4 % sucrose, 1 % glycine
• factor IX formulations all containing citrate, is prepared as summarized in Table 7 All formulations are isotonic, contain factor IX at concentrations of 1 to 2 mg/mL (average 208 to 481 U/mL), use 10 mM to 15 mM sodium citrate as the pH buffering agent, and are adjusted to pH 6.8.
• Samples are stored at 4°C and assayed at several points in time. After eight months of 4°C storage, nine samples maintain - 100% of the clotting activity of the starting material.
• the formulations of these nine are shown in Table 8 (all include 15 mM sodium citrate, are pH 6.8, and isotonic).
• two more preferred formulations include as follows: (both are buffered at pH 6.8 with 15 mM citrate and are isotonic), 3% mannitol, 1.5 % arginine-HCl; and 3.3% arginine-HCl.
• Example 5 Effects of Freeze/Thaw Cycle
• Samples are prepared in the formulations set forth in Table 10 below, at a protein concentration of - 2 mg/mL (500 U/mL) and an osmolality of 300 ⁇ 50 milliosmolal. All include 10 mM sodium citrate, pH 6.8, and all are prepared both with and without 0.005% Tween-80 (polysorbate).
• Samples of factor IX in each formulation were subjected to five freeze-thaw cycles to determine susceptibility to freezing-induced denaturation, which can result in formation of protein aggregates.
• a series of freeze-thaw cycles is a useful indication of a protein's susceptibility to increased aggregate formation as may be observed during freezing and long- term storage. Samples are assayed for the amount of HMW present. Samples with and without Tween-80 (0.005%) have minimal aggregation (less than 0.15% HMW increase).
• Formulation 1 sodium citrate 0.0075 M to 0.04 M 0.19% to 1 % w/v arginine (-HC1) 0.13 M to 0.16 M 2.8% to 3.3 % w/v sucrose 0 to 0.06 M O to 2% w/v polysorbate-80 0 to 0.0000382 M 0 to 0.005% w/v factor IX 600 to 56,000 Units/mL 0.1 to 160 mg/mL
• Formulation 2 sodium citrate 0.0075 M to 0.04 M 0.19% to 1 % w/v arginine (-HC1) 0.06 M to 0.07 M 1.3 to 1.5% j sucrose 0 to 0.02 M 0 to 0.7% mannitol 0.165 M 3 % polysorbate-80 0 to 0.0000382 M O to 0.005% w/v factor IX 600 to 56,000 Units/mL 0.1 to 160 mg/mL
• Example 6 Effect of Long Term Storage at 4°C
• Factor IX is formulated at 2 mg/mL (500 U/mL) in 15 mM sodium citrate (0.38%), 0.16 M arginine (3.3 %), pH 6.8 and stored for one year at 4'C. The recovery of activity is 95 % and the % HMW is 0.32% . Factor IX is formulated at 2 mg/mL in 15 mM sodium citrate, 3 % mannitol, 1.5% arginine, pH 6.8 and stored for one year at 4"C. The recovery of activity is 76% and the % HMW was 0.36% . The loss of activity is attributed to deamidation.
• Factor IX is formulated at 4000 U/mL, 8000 U/mL, 16,000 U/mL and greater than 30,000 U/mL (i.e. , 16 to greater than 120 mg/mL) in 10 mM histidine. 260 mM glycine, 1 % sucrose, 0.005 % Tween-80, pH 7.0. Factor IX is concentrated by centrifugal concentration in a Centricon-10 and by stir-cell concentration in an Amicon stir cell using a YM-10 membrane. Other methods used for concentrating proteins, especially those using membranes which retain and exclude species based on molecular weight, such as tangential flow filtration, can also be used. In addition, spray-drying can be used with no untoward effects.
• Highly concentrated factor IX is effective when administered subcutaneously, intradermally or intramuscularly. Utilizing a highly concentrated formulation of factor IX, . e. , 4,000 U/mL to greater than 56,000 U/mL, a single site, low volume, subcutaneous injection is possible as is described below.
• Subcutaneous factor IX produces therapeutic levels of factor IX activity in less than three hours after administration.
• the combination dose of an intravenous with a subcutaneous dose provides immediate coagulant protection and improves the efficacy of the subcutaneous dose.
• highly concentrated forms of factor IX can be formulated in the formulations described, supra, in Examples 1-5, and effectively used for administration.

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