Methods of purifying chondroitinase and stable formulations thereof
View Patent ↗An aspect of the present invention relates to stable formulations of chondroitinase and to methods of purifying chondroitinase. The methods of purifying chondroitinase includes the steps of extracting the enzyme from a cell, separating the chondroitinase from the crude cell extract using cation-exchange chromatography, removing impurities through gel filtration chromatography, and removing endotoxin through an anion-exchange membrane to produce a purified chondroitinase.
1. A stable formulation comprising a chondroitinase and a sodium phosphate buffer at a concentration of about 750-1000 mM, wherein the chondroitinase retains at least 50% of activity at about 24 hours.
2. The formulation of claim 1 , wherein said chondroitinase is purified.
3. The formulation of claim 1 wherein said chondroitinase is selected from the group consisting of chondroitinase ABCI, chondroitinase ABCII, chondroitinase AC, chondroitinase B mammalian enzymes with chondroitinase-like activity, Hyal1, Hyal2, Hyal3, Hyal4 and PH20.
4. The formulation of claim 1 , wherein said chondroitinase is chondroitinase ABCI.
5. The formulation of claim 1 , wherein said chondroitinase is chondroitinase AC.
6. The formulation of claim 1 further comprising sodium acetate.
7. The formulation of claim 1 , wherein said formulation is at a pH of about 7.4.
8. The formulation of claim 3 , wherein the chondroitinase ABC I is an amino acid sequence of SEQ ID NO: 2.
9. The formulation of claim 1 , wherein the sodium phosphate buffer concentration is about 750 mM.
10. The formulation of claim 1 , wherein the sodium phosphate buffer concentration is about 1000 mM.
11. The formulation of claim 1 , wherein said chondroitinase is chondroitinase ABCII.
12. The formulation of claim 1 , wherein said chondroitinase is chondroitinase B.
13. The formulation of claim 1 , wherein said chondroitinase is chondroitinase Hyal1.
14. The formulation of claim 1 , wherein said chondroitinase is chondroitinase Hyal2.
15. The formulation of claim 1 , wherein said chondroitinase is chondroitinase Hyal3.
16. The formulation of claim 1 , wherein said chondroitinase is chondroitinase Hyal4.
17. The formulation of claim 1 , wherein said chondroitinase is chondroitinase PH20.