PEG-urate oxidase conjugates and use thereof
View Patent ↗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. A method for isolating a tetrameric form of urate oxidase (uricase) from a solution of purified uricase comprising tetrameric uricase and uricase aggregates, said method comprising:
(a) separating the solution into fractions on at least one separation column at a pH between about 9 and about 10.5, wherein said column is selected from the group consisting of an ion-exchange column and a size-exclusion column; and
(b) recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein greater than 90% of said isolated uricase in said one or more fractions is in a tetrameric form.
2. The method of claim 1 , wherein said separating is performed at a pH of 10.2.
3. The method of claim 1 , wherein said separating is performed at a pH of 10 to 10.5.
4. The method of claim 1 , further comprising analyzing said one or more fractions to determine at least one property thereof selected from the group consisting of the presence of said tetrameric unease and the absence of said unease aggregates.
5. The method of claim 4 , wherein said analyzing comprises at least one analytical method selected from the group consisting of chromatography, centrifugation, light scattering and electrophoresis.
6. The method of claim 5 , wherein said analytical method is high performance liquid chromatography.
7. The method of claim 1 , wherein the uricase in said solution is selected from the group consisting of recombinant mammalian uricase, uricase from Candida utilis , recombinant soybean uricase, uricase from Arthrobacter globiformis , porcine uricase, and baboon uricase.
8. The method of claim 7 , wherein said uricase is one or more of chimeric, amino-terminally truncated, carboxyl-terminally truncated, amino-terminally and carboxyl-terminally truncated, and mutated.