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US 5,290,763 AGranted
Osteoinductive protein mixtures and purification processes
AssigneeIntermedics Orthopedics/Denver, Inc.
InventorsJames W. Poser, James J. Benedict
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AssigneeIntermedics Orthopedics/Denver, Inc.
InventorsJames W. Poser, James J. Benedict
Estimating the term…
A method for purifying bone-derived osteoinductive factors including an ultrafiltration process, an anion exchange process, a cation exchange process, and a reverse phase HPLC process. The ultrafiltration process preferably includes a first ultrafiltration step using a membrane having a nominal molecular weight cutoff of approximately 100 kilodaltons (kD) and a second ultrafiltration step employing a membrane having a nominal molecular weight cutoff of approximately 10 kD. For the anion exchange process, a strongly cationic resin is used, preferably having quaternary amine functional groups. Typically, the eluant for the anion exchange process has a conductivity from about 10,260 micromhos (.mu.mhos) (1.026.times.10.sup.-2 siemens (S)) to about 11,200 .mu.mhos (1.120.times.10.sup.-2 S). For the cation exchange process, a strongly anionic resin is used, preferably having sulfonic acid functional groups. The eluant for the cation exchange process typically has a conductivity from about 39,100 .mu.mhos (3.91.times.10.sup.-2 S) to about 82,700 .mu.mhos (8.27.times.10.sup.-2 S) or more. The HPLC process typically utilizes a column containing hydrocarbon-modified silica packing material. The osteoinductive proteins can be eluted from the HPLC column with an acetonitrile solution in combination with aqueous trifluoracetic acid. The purification processes yield osteoinductively active protein mixtures.
An osteinductive mixture of proteins that is soluble in water at a concentration of 10 milligrams of said protein mixture per milliliter of water at room temperature comprising an amino acid composition of from about 20.7 to about 26.1 mole percent acidic amino acids, about 11.3 to about 15.7 mole percent hydroxy amino acids, about 37.6 to about 42.4 mole percent aliphatic amino acids, about 5.8 to about 7.9 mole percent aromatic amino acids and about 13.3 to about 19.9 mole percent basic amino acids, wherein said mixture of proteins achieves a histological grade of at least about 3 when subcutaneously implanted for 21 days at a protein:substrate weight ratio of about 1:600.
An osteoinductive mixture of proteins that is soluble in water at a concentration of 10 milligrams of said protein mixture per milliliter of water at room temperature which, when subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, results in a reduced or non-reduced gel profile comprising substantially all of the protein bands shown in FIG. 1, wherein said mixture of proteins achieves a histological grade of at least about 3 when subcutaneously implanted for 21 days at a protein:substrate weight ratio of about 1:600.
An osteoinductive mixture of proteins that is soluble in water at a concentration of 10 milligrams of said protein mixture per milliliter of water at room temperature that achieves a histological grade of at least about 3 when subcutaneously implanted for 21 days at a protein:substrate weight ratio of about 1:600 produced by a process comprising:
An osteoinductive mixture of proteins that is soluble in water at a concentration of 10 milligrams of said protein mixture per milliliter of water at room temperature that achieves a histological grade of at least about 3 when subcutaneously implanted for 21 days at a protein:substrate weight ratio of about 1:600 produced by a process comprising:
Dependencies are inferred from the claim text.