Concentrated protein preparations of bone morphogenetic proteins and methods of use thereof

Abstract

Disclosed herein are heretofore undescribed preparations of highly concentrated, solubilized proteins, such as but not limited to, Bone Morphogenetic Proteins. Such protein preparations can be formulated in an aqueous carrier at protein concentrations in excess of 10 mg/ml when using the methods of manufacture taught herein. Such methods yield stable protein preparations in either solubilized or lyophilized form. The protein preparations of the present invention are particularly beneficial when administered either locally or systemically, in part, because low administration volumes can be accomplished. This is especially important for local treatment of certain anatomic locations such as, for example, the synovial fluid of a joint when treating osteoarthritis with BMP-7 or the intradiscal space when treating degenerative disc disease with BMP-7.

We claim: 
     
         1 . A preparation of bone morphogenetic protein comprising:
 an aqueous carrier; and,   bone morphogenetic protein solubilized in said carrier at a concentration of at least about 10 mg/ml.   
     
     
         2 . The protein preparation of  claim 1  wherein said protein concentration is greater than about 20 mg/ml. 
     
     
         3 . The protein preparation of  claim 1  wherein said aqueous carrier has an ionic strength of at least about 10 mM. 
     
     
         4 . The protein preparation of  claim 3  wherein said aqueous carrier has an ionic strength of no more than about 100 mM. 
     
     
         5 . The protein preparation of  claim 3  wherein said aqueous carrier has an ionic strength of no more than about 50 mM. 
     
     
         6 . The protein preparation of  claim 3  wherein said aqueous carrier has an ionic strength of no more than about 20 mM. 
     
     
         7 . The protein preparation of  claim 1  wherein said aqueous carrier has an ionic strength of no more than about 10 mM. 
     
     
         8 . The protein preparation of  claim 1  wherein said aqueous carrier has a pH of at least about 2. 
     
     
         9 . The protein preparation of  claim 4  wherein said aqueous carrier has a pH of no more than about 5. 
     
     
         10 . The protein preparation of  claim 8  wherein said aqueous carrier has a pH of about 3. 
     
     
         11 . The protein preparation of  claim 4  or  9  wherein the protein concentration is about 20-60 mg/ml; the ionic strength is about 0-50 mM; and the pH is about 2.5-4. 
     
     
         12 . The protein preparation of  claim 11  wherein the protein is BMP-7 in a concentration of more than 20 mg/ml; the ionic strength is about 10 mM; and the pH is about 3.5. 
     
     
         13 . The protein preparation of  claim 11  wherein the preparation is solubilized at a temperature of about 4° C. to about 25° C. 
     
     
         14 . The protein preparation of  claim 1  further comprising a stabilizing excipient. 
     
     
         15 . The protein preparation of  claim 13  wherein the stabilizing excipient is selected from the group consisting of: sugars, polyols, surfactants, and any combination thereof. 
     
     
         16 . The protein preparation of  claim 1  wherein the aqueous carrier comprises a buffer of the single acidic group type selected from the group consisting of: potassium phosphate, proprionic acid, lactic acid, trifloroacetic acid and acetic acid; or the two acidic group type selected from the group consisting of: sodium glutamate and sodium succinate. 
     
     
         17 . The protein preparation of  claim 1  wherein the preparation is lyophilized or a reconstituted lyophilate. 
     
     
         18 . The protein preparation of  claim 1  wherein the protein is a cysteine knot protein. 
     
     
         19 . The protein preparation of  claim 18  wherein the protein is not a member of the TGF-beta superfamily of proteins. 
     
     
         20 . The protein preparation of  claim 18  wherein the protein is mono- or dimeric. 
     
     
         21 . The protein preparation of  claim 18  wherein the molecular weight of the protein is 25-50 kd. 
     
     
         22 . The protein preparation of  claim 18  wherein the protein is a basic protein having a pI range of about 5-10. 
     
     
         23 . The protein preparation of  claim 18  wherein the protein is selected from the group of cysteine knot proteins consisting of: PDGF, VEGF and NGF. 
     
     
         24 . The protein preparation of  claim 1  wherein the protein is a member of the TGF-beta superfamily. 
     
     
         25 . The protein preparation of  claim 1  wherein the protein is a member of the BMP subfamily of the TGF-beta superfamily of proteins. 
     
     
         26 . The protein preparation of  claim 25  wherein the protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6 GDF-7, and sequence variants of any one of the foregoing. 
     
     
         27 . The protein preparation of  claim 25  wherein the protein is selected from the group consisting of GDF-5, GDF-6 and GDF-7. 
     
     
         28 . The protein preparation of  claim 25  wherein the protein is selected from the group consisting of BMP-7. 
     
     
         29 . The protein preparation of  claim 1  wherein the protein is a protein having at least about 50% amino acid sequence identity with a member of the BMP subfamily within the conserved C-terminal cysteine-rich domain. 
     
     
         30 . A method of treating a skeletal tissue disorder, injury or disease, the method comprising the step of:
 administering to a subject in need thereof the protein preparation of  claim 1 , wherein said protein preparation is in a dose effective to treat said skeletal tissue disorder, injury or disease.   
     
     
         31 . The method of  claim 16  wherein the skeletal tissue is mineralized or non-mineralized skeletal tissue. 
     
     
         32 . The method of  claim 31  wherein the protein preparation is in an amount effective to treat a skeletal tissue disorder, injury or disease selected from the group consisting of metabolic bone disease, osteoarthritis, osteochondral disease, rheumatoid arthritis, osteoporosis, bone fractures, Paget's disease, periodontitis, and dentinogenesis. 
     
     
         33 . The method of  claim 31  wherein the protein preparation is in an amount effective to treat a non-mineralized skeletal tissue disorder, injury or disease selected from the group consisting of osteoarthritis, osteochondral disease or defect, chondral disease or defect, rheumatoid arthritis, trauma-induced and inflammation-induced cartilage degeneration, age-related cartilage degeneration, articular cartilage injuries and diseases, full thickness cartilage defects, superficial cartilage defects, sequelae of systemic lupus erythematosis, sequelae of scleroderma, periodontal tissue regeneration, hierniation and rupture of intervertebral discs, degenerative diseases of the intervertebral disc, osteocondrosis, and injuries and diseases of ligament, tendon, synovial capsule, synovial membrane and meniscal tissues. 
     
     
         34 . The method of  claim 30  wherein the protein preparation is in an amount effective to ameliorate tissue injury selected from the group consisting of: trauma-induced and inflammation-induced cartilage degeneration, articular cartilage injuries, full thickness cartilage defects, superficial cartilage defects, hierniation and rupture of intervertebral discs, degeneration of intervertebral discs due to an injury(s), and injuries of ligament, tendon, synovial capsule, synovial membrane and meniscal tissues. 
     
     
         35 . The method of  claim 30  wherein the disease is osteoarthritis. 
     
     
         36 . The method of  claim 30  wherein the effective dose is administered to the synovial space of a knee, hip or articulating joint. 
     
     
         37 . The method of  claim 30  wherein the disease is degenerative disc disease. 
     
     
         38 . The method of  claim 37  wherein the effective dose is administered to the intradiscal space, the nucleus pulposus or the annulus fibrosus. 
     
     
         39 . The method of  claim 30  wherein the administering step is local or systemic. 
     
     
         40 . The method of  claim 30  wherein the effective dose is about 1 mg to 30 mg in about 100 microliters to 3 ml. 
     
     
         41 . The method of  claim 30  wherein the method further comprises the step of admixing said protein preparation with a suitable matrix material prior to administration. 
     
     
         42 . The method of  claim 30  wherein the protein is selected from the group consisting of: BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6 GDF-7, and sequence variants of any one of the foregoing 
     
     
         43 . The method of  claim 30  wherein the protein is BMP-7. 
     
     
         44 . The method of  claim 30  wherein the administering step is performed pre- or post-surgery. 
     
     
         45 . The method of  claim 30  wherein the administering step is performed more than once. 
     
     
         46 . A method of treating a non-skeletal tissue, the method comprising the step of; administering to a subject in need thereof the protein preparation of  claim 1 , wherein said protein preparation is in a dose effective to treat said non-skeletal tissue disorder, injury or disease. 
     
     
         47 . The method of  claim 46  wherein the protein preparation is in an amount effective to treat the disorder, injury or disease of a non-skeletal tissue selected from the group consisting of: liver disease, liver resection, hepatectomy, renal disease, chronic renal failure, central nervous system ischemia or trauma, neuropathy, motor neuron injury, dendritic cell deficiencies and abnormalities, Parkinson's disease, ophthalmic disease, ocular scarring, retinal scarring, and ulcerative diseases of the gastrointestinal tract, fibrosis, fibrotic disorders, scleroderma, and pulmonary fibrosis. 
     
     
         48 . A kit comprising a lyophilized bone morphogenetic protein and a reconstitution diluent, wherein the protein and the diluent are in separate containers, and wherein the amount of diluent is sufficient only for preparation of solubilized protein at a concentration of at least about 10 mg/ml. 
     
     
         49 . The kit of  claim 48  further comprising a matrix. 
     
     
         50 . The kit of  claim 48  further comprising an implantable device suitable for coating with the solubilized protein. 
     
     
         51 . A method for manufacturing a concentrated form of lyophilized recombinant bone morphogenetic protein, the method comprising the steps of:
 (1) providing a processing solution of recombinant bone morphogenetic protein, the weight per volume of protein in said processing solution being less than the weight per volume specified for lyophilization;   (2) adjusting the weight per volume of protein in said processing solution to produce a lyophilization solution, the weight per volume of protein in said lyophilization solution being greater than that of the processing solution;   (3) filling a vial suitable for lyophilization with a specified volume of said lyophilization solution; and,   (4) lyophilizing said lyophilization solution to manufacture a concentrated form of lyophilized recombinant bone morphogenetic protein.   
     
     
         52 . The method of  claim 51  further comprising the step of reconstituting said lyophilized recombinant bone morphogenetic protein in a reconstitution diluent wherein the volume of diluent is the same as the volume in step 3. 
     
     
         53 . The method of  claim 51  further comprising the step of reconstituting said lyophilized recombinant bone morphogenetic protein in a reconstitution diluent wherein the volume of diluent is less than the volume in step 3. 
     
     
         54 . The method of  claim 51  wherein the concentrated form is achieved post-processing and contains solubilized bone morphogenetic protein in a concentration of at least 10 mg/ml. 
     
     
         55 . A concentrated form of recombinant bone morphogenetic protein according to  claim 51 . 
     
     
         56 . A kit for the treatment of a disorder, injury or disease comprising a lyophilized bone morphogenetic protein and a reconstitution diluent, wherein the protein and the diluent are in separate containers, and further wherein the kit comprises a plurality of separate containers each containing an amount of diluent, such that the amounts of diluent are sufficient to prepare solubilized protein preparations ranging in concentration from at least about 2 mg/ml to about 60 mg/ml for use in the treatment of a disorder, injury or disease. 
     
     
         57 . The kit of  claim 56  wherein the concentration and volume of solubilized protein preparations are customized for treatment of the disorder, injury or disease. 
     
     
         58 . A method for preparing a concentrated form of bone morphogenetic protein, the method comprising the steps of:
 (1) providing a processing solution of bone morphogenetic protein, the weight per volume of protein in said processing solution being the same as the weight per volume specified for lyophilization;   (2) filling a vial suitable for lyophilization with a specified volume of said processing solution thereby producing a lyophilization solution;   (3) lyophilizing said lyophilization solution; and,   (4) packaging the lyophilized lyophilization solution together with a specified volume of reconstitution diluent, the volume of said diluent being less than the specified fill volume of step 3.   
     
     
         59 . The method of  claim 58  further comprising the step of adjusting the weight per volume of protein in said processing solution to produce an adjusted lyophilization solution, the weight per volume of protein in said adjusted lyophilization solution being greater than that of the processing solution. 
     
     
         60 . A method for preparing a concentrated form of protein in lyophilized or other reconstitutible non-liquid form, the method comprising the steps of:
 (1) providing a processing solution of protein, the weight per volume of protein in said processing solution being the same as the weight per volume specified for lyophilization or other reconsitutible non-liquid form;   (2) providing a vial suitable for lyophilization (or other form) for containing a specified volume of said processing solution thereby producing a lyophilization (or other form) solution;   wherein the weight per volume of protein in each of the processing and lyophilization (or other form) solution is at least 2 mg/ml.   
     
     
         61 . The method of  claim 60  further comprising the step of
 providing a lyophilized form of the lyophilization solution or a reconstitutible form of the other non-liquid form. 
 
     
     
         62 . The method of  claim 60  or  61  further comprising the step of providing a specified volume of diluent for rehydrating the lyophilized form or reconstituting the other reconstitutible non-liquid form, the volume of said diluent being sufficient to permit preparation of an aqueous protein preparation having at least 2 mg/ml; or further comprising the step of providing instructions for rehydrating the lyophilized form or reconstituting the other reconstitutible non-liquid form so as to product an aqueous protein preparation having at least 2 mg/ml.