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 uricase from a solution of purified uricase, said purified uricase comprising tetrameric uricase and uricase aggregates, said method comprising:
separating the solution into fractions on at least one separation column at a pH of 9 to 10.5, wherein said column is selected from the group consisting of an ion-exchange column and a size-exclusion column; and
recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein said one or more fractions contain less than 10% of uricase aggregates larger than tetramers.
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 fractions to determine at least one property selected from the group consisting of the presence of said tetrameric uricase and the absence of said uricase aggregates.
5. The method of claim 4 , wherein said analyzing comprises at least one analysis selected from the group consisting of chromatography, centrifugation, light scattering and electrophoresis.
6. The method of claim 5 , wherein said analyzing comprises high performance liquid chromatography.