PEG-Urate Oxidase Conjugates and Use Thereof
A naturally occurring or recombinant urate oxidase (uricase) covalently coupled to poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), wherein an average of 2 to 10 strands of PEG are conjugated to each uricase subunit and the PEG has an average molecular weight between about 5 kDa and 100 kDa. The resulting PEG-uricase conjugates are substantially non-immunogenic and retain at least 75% of the uricolytic activity of the unmodified enzyme.
1 - 41 . (canceled)
42 . An isolated tetrameric uricase produced by a method comprising
applying a solution of uricase to at least one separation column at a pH of 9 to 10.5; and
recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein said one or more fractions are substantially free of uricase aggregates.
43 . The isolated tetrameric uricase of claim 42 , wherein the uricase is a mammalian uricase or a recombinant form thereof.
44 . The isolated tetrameric uricase of claim 42 , wherein the uricase is a fungal or microbial uricase or a recombinant form thereof.
45 . The isolated tetrameric uricase of claim 44 , wherein the fungal or microbial uricase is isolated from Aspergillus flavus, Arthrobacter globiformis , or Candida utilis , or is a recombinant enzyme having substantially the sequence of one of those uricases.
46 . The isolated tetrameric uricase of claim 42 , wherein the uricase is an invertebrate uricase or a recombinant form thereof.
47 . The isolated tetrameric uricase of claim 46 , wherein the invertebrate uricase is isolated from Drosophila melanogaster or Drosophila pseudoobscura , or is a recombinant enzyme having substantially the sequence of one of those uricases.
48 . The isolated tetrameric uricase of claim 42 , wherein the uricase is a plant uricase or a recombinant form thereof.
49 . The isolated tetrameric uricase of claim 48 , wherein the plant uricase is isolated from root nodules of Glycine max or is a recombinant enzyme having substantially the sequence of that uricase.
50 . The isolated tetrameric uricase of claim 42 , each subunit of the uricase being subsequently covalently linked to an average of 2 to 12 strands of PEG to form a PEG-uricase conjugate, wherein each molecule of PEG has a molecular weight of 5 kDa to 100 kDa.
51 . The isolated tetrameric uricase of claim 50 , wherein the PEG has an average molecular weight of 10 kDa to 60 kDa.
52 . The isolated tetrameric uricase of claim 51 , wherein the PEG has an average molecular weight of 20 kDa to 40 kDa.
53 . The isolated tetrameric unease of claim 50 , wherein the average number of covalently coupled strands of PEG is 2 to 10 strands per uricase subunit.
54 . The isolated tetrameric uricase of claim 53 , wherein the average number of covalently coupled strands of PEG is 3 to 8 strands per uricase subunit.
55 . The isolated tetrameric uricase of claim 54 , wherein the average number of covalently coupled strands of PEG is 4 to 6 strands per uricase subunit.
56 . The isolated tetrameric uricase of claim 50 , wherein the strands of PEG are covalently coupled to uricase via linkages selected from the group consisting of urethane linkages, secondary amine linkages, and amide linkages.
57 . The isolated tetrameric uricase of claim 50 , wherein the PEG is linear.
58 . The isolated tetrameric uricase of claim 50 , wherein the PEG is branched.