Precursor N-acetylgalactosamine-4 sulfatase, methods of treatment using said enzyme and methods for producing and purifying said enzyme

Abstract

The present invention provides a highly purified recombinant, human precursor N-acetylgalactosamine-4-sulfatase and biologically active mutants, fragments and analogs thereof as well as pharmaceutical formulations comprising highly purified recombinant human precursor N-acetylgalactosamine-4-sulfatase. The invention also provides methods for treating diseases caused all or in part by deficiencies in human N-acetylgalactosamine-4-sulfatase including MPS VI and methods for producing and purifying the recombinant precursor enzyme to a highly purified form.

1 - 23 . (canceled)  
     
     
         24 . A method for producing a recombinant precursor N-acetylgalactosamine-4-sulfatase enzyme comprising the steps of: 
 (a) growing a cell transfected with a DNA encoding all or a biologically active fragment or mutant of a human N-acetylgalactosamine-4-sulfatase enzyme,    (b) introducing the transfected cells into a bioreactor,    (c) supplying a growth medium to the bioreactor,    (d) harvesting said medium containing said enzyme; and    (e) substantially removing the transfected cells from the said harvest medium.    
     
     
         25 . The method of  claim 24  wherein said cell is a mammalian cell.  
     
     
         26 . The method of  claim 25  wherein said mammalian cell is a Chinese Hamster Ovary cell.  
     
     
         27 . The method of  claim 24  wherein the transfected cells are grown on a growth medium comprising a JRH Excell 302 medium supplemented with one or more agents selected from the group consisting of L-glutamine, glucose, hypoxanthine/thymidine, serine, asparagine and folic acid.  
     
     
         28 . The method of  claim 24  wherein said growth medium does not contain G418:  
     
     
         29 . The method of  claim 24  wherein the transfected cells are grown in a bioreactor for up to about 45 days.  
     
     
         30 . The method of  claim 24  wherein the transfected cells are grown in a bioreactor for up to about 90 days.  
     
     
         31 . The method of  claim 24  wherein the transfected cells are substantially separated from the media containing the enzyme through successive membranes.  
     
     
         32 . The method of  claim 31  wherein the successive membranes are 4.0-0.75 μm nomimal, 0.45 μm and 0.2 μm.  
     
     
         33 - 61 . (canceled)  
     
     
         62 . A method to purify a N-acetylgalactosamine-4-sulfatase or biologically active fragment, analog or mutant thereof comprising: 
 (a) obtaining a fluid containing said N-acetylgalactosamine-4-sulfatase or biologically active fragment, analog or mutant thereof;    (b) reducing the proteolytic activity of any protease in said fluid able to cleave said N-acetylgalactosamine-4-sulfatase or biologically active fragment, analog or mutant thereof, wherein said reducing does not harm said N-acetylgalactosamine-4-sulfatase or biologically active fragment, analog or mutant thereof;    (c) contacting the fluid with a Cibracon blue dye interaction chromatography resin;    (d) contacting the fluid with a copper chelation chromatography resin;    (e) contacting the fluid with a phenyl hydrophobic interaction chromatography resin;    (f) recovering said N-acetylgalactosamine-4-sulfatase or biologically active fragment, analog or mutant thereof; wherein steps (c), (d) and (e) can be performed in any temporal sequence.    
     
     
         63 . A pharmaceutical composition comprising precursor N-acetylgalactosamine-4-sulfatase and a polyoxyethylenesorbitan at a concentration ranging from about 0.002% to about 0.008% (weight/volume).  
     
     
         64 . The pharmaceutical composition of  claim 63  wherein said polyoxyethylene sorbitan is polyoxyethylene sorbitan 80 at a concentration of 0.005% (weight/volume).  
     
     
         65 . A method for treating a disease caused all or in part by a deficiency in N-acetylgalactosamine-4-sulfatase activity comprising the step of administering to a human subject in need of such treatment an effective amount of a composition comprising recombinant human N-acetylgalactosamine-4-sulfatase (rhASB).  
     
     
         66 . The method of  claim 65  wherein the amount of rhASB is effective to provide one or more beneficial effects selected from the group consisting of joint mobility, joint pain, joint stiffness, exercise tolerance, exercise endurance, pulmonary function, visual acuity, and activities of daily living.  
     
     
         67 . A method for treating mucopolysaccharidosis VI (MPS VI) comprising the step of administering to a human subject in need of such treatment an effective amount of a composition comprising recombinant human N-acetylgalactosamine-4-sulfatase (rhASB).  
     
     
         68 . The method of  claim 67  wherein the amount of rhASB is effective to provide one or more beneficial effects selected from the group consisting of joint mobility, joint pain, joint stiffness, exercise tolerance, exercise endurance, pulmonary function, visual acuity, and activities of daily living.  
     
     
         69 .- 74 . (canceled)