Stabilizer for hyaluronidase and liquid formulation comprising hyaluronidase

Abstract

This invention relates to a stable liquid formulation comprising hyaluronidase and a stabilizer for hyaluronidase and more particularly, to a stable liquid formulation by the addition of the stabilizer to the hyaluronidase.

1 . A method of stabilizing a hyaluronidase in liquid formulation, comprising contacting a stabilizer consisting of (i) a buffering agent to provide pH 4.0 to 6.0, (ii) 0.01 to 0.5 v/v % of a non-ionic surfactant and (iii) 0.1 to 5 mM of a chelating agent or MgCl 2 . 
     
     
         2 . The method of  claim 1 , wherein the hyaluronidase has a purity of 95% or higher. 
     
     
         3 . The method of  claim 1 , wherein the hyaluronidase has a specific activity of 70,000 IU/mg or higher. 
     
     
         4 . The method of  claim 1 , wherein the liquid formulation is an injectable formulation. 
     
     
         5 . The method of  claim 1 , wherein the hyaluronidase has a stability of maintaining the activity up to 90% or more with regard to its initial enzymatic activity 100 at a temperature condition of 2 to 8° C. 
     
     
         6 . The method of  claim 1 , wherein the hyaluronidase is an active component of the formulation. 
     
     
         7 . The method of  claim 1 , wherein the chelating agent is EDTA. 
     
     
         8 . The method of  claim 1 , wherein the non-ionic surfactant is selected from the group consisting of polyoxyethylene-sorbitan fatty acid ester and Triton X-100. 
     
     
         9 . The method of  claim 8 , wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and Triton X-100. 
     
     
         10 . The method of  claim 1 , wherein the buffering agent is selected from the group consisting of succinate buffer, acetate buffer, phosphate buffer, citrate buffer, malonate buffer, 2-(N-Morpholino)ethanesulphonic acid(MES) buffer, Tris buffer and glycine buffer. 
     
     
         11 . The method of  claim 1 , wherein the hyaluronidase is derived from sheep, cows, pigs, or humans. 
     
     
         12 . The method of  claim 1 , wherein the hyaluronidase is a recombinant hyaluronidase produced by transducing a mammalian hyaluronidase gene into microbes, animal cells or plant cells, or is an extract derived from sheep, cows, pigs, or humans. 
     
     
         13 . The method of  claim 1 , further comprising purifying the hyaluronidase from a hyaluronidase-containing material. 
     
     
         14 . The method of  claim 13 , further comprising purifying the hyaluronidase from a hyaluronidase-containing material by one or more methods selected from the group consisting of affinity chromatography, ion exchange chromatography, and gel filtration. 
     
     
         15 . The method of  claim 14 , wherein the affinity chromatography is an affinity chromatography in which a matrix is composed of cross-linked agarose beads modified with a modified triazine dye, or an affinity chromatography in which a matrix is composed of ross-linked agarose beads modified with heparin. 
     
     
         16 . The method of  claim 15 , wherein the purifying hyaluronidase from a hyaluronidase-containing material is performed sequentially by using an affinity chromatography in which a matrix is composed of cross-linked agarose beads modified with a modified triazine dye,
 a cation exchange chromatography,   an anion exchange chromatography,   an affinity chromatography in which a matrix is composed of ross-linked agarose beads modified with heparin, and   a gel filtration.