Purified urate oxidase preparation, composition comprising the same, and purifying method thereof

Purified uric acid oxidase preparation, composition containing same, and purification method thereof

<p>Part of the research described in this application was made with the support of the US-Israel Binational Industrial Research and Development Foundation. Accordingly, the United States Government may have certain rights in this invention.</p><p>The present invention relates to the purification and chemical modification of proteins to extend their circulating life and reduce their immunogenicity. More specifically, the present invention is a poly (ethylene glycol) (poly (ethylene glycol)) or poly (ethylene oxide) (poly (ethylene oxide)) before the conjugation (conjugation) of urate oxidase (Urate Oxidase) (Uricase) uricase)) to remove aggregates larger than octamers. This substantially eliminates its immunogenicity without impairing the uricase activity of uricase.</p>

<p>Statements included in this background are not admissions of prior art, but rather reflect the inventors' own subjective statements and interpretations of the state of the art at the time this invention was made. These interpretations are private and may include the insights of the inventors not heretofore disclosed, which in themselves are not part of the prior art.</p><p>Uric acid oxidases (uricases; EC1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, i.e., allantoin, a more readily secreted purine metabolite. Humans fail to produce enzymatically active uricases as a result of several mutations in the gene for uricases acquired during the course of evolution to higher primates. Wu, All X, (1992),<i>J. Mol. Evol</i> 34: 78-84. Thus, in sensitive people, the presence of an excess concentration of uric acid in the blood (hyperuricemia) and the presence of an excess concentration of uric acid in the urine (hyperuricosuria) causes painful arthritis (gout), imbalance It can cause urate deposition (gout nodules) and nephritis. In some infected patients, over-the-counter medications, such as allopurinol (an inhibitor of uric acid synthesis), produce treatment-limiting side effects or do not adequately treat these symptoms. Hande, K.R., Ildong., (1984)<i>Am J. Med. </i>76:47-56; Fam, AG, (1990)<i>Bailliere's Clin Rheumatol</i> 4:177-192. Injecting uricase can provide at least temporary relief of hyperuricemia and hypouric acidosis. However, since uricase is a foreign protein to humans,<i>Aspergillus flavus</i>(<i>Aspergillus flavus</i>), even the first injection of unmodified protein from <i>Leukemia</i> 11:1813-1816), and also immunological responses limit its utility for long-term or intermittent treatment. Donadio, D. et al., (1981)<i>Nouv Press Med</i> 10:711-712; Leaustic, M. et al., (1983)<i>Rev. Rhum Mal Osteoartic</i> 50:553-554. </p><p>U.S. Patent Application Serial No. 09/370,084 and published International Application PCT/US99/17514 disclose poly(ethylene) having substantially reduced immunogenicity while retaining at least about 75% of the uricase activity of unconjugated uricase. glycol)-uric acid oxidase (PEG-uricase) is disclosed. In one such purified uricase, each subunit is covalently bound to an average of 2 to 10 strands of PEG, wherein each molecule of PEG has a molecular weight between about 5 kDa and 100 kDa. can have</p><p>Aggregation of proteins is known to increase their immunogenicity. This understanding includes methods for intentionally aggregating proteins by exposure to glutaraldehyde prior to use in the manufacture of vaccines to form cross-linking or by treatment such as thermal denaturation, or antisera. contributed to the development of a method for immunizing animals to produce</p><p>Unintentional aggregation of proteins is also possible with therapeutic proteins, such as human gamma globulin (Henney et al. (1968)). <i>N. Engl. J. Med.</i>278: 2244-2246) and human growth hormone (Moore et al. (1980)) <i>J. Clin. Endocrinol. Metab</i>. 51: 691-697) is recognized to contribute to immunization or sensitization during clinical use. The contribution of aggregates to the immunogenicity of human interferon alpha was found in BALB/c mice (Braun et al. (1997)).<i>Pharm. Res.</i> 14: 1472-1478), and an enzyme-linked immunosorbent assay (ELISA) was developed for their determination (Braun et al. (1997)). <i>Pharm. Res.</i> 14: 1394-1400). </p>

<solutionproblem><p>In contrast to the known effects of aggregation on the immunogenicity of proteins, there are no reports of the effects of aggregation on the immunogenicity of proteins conjugated to poly(alkylene glycol) such as PEG. There is a need for poly(alkylene glycol)-uricase conjugates that substantially eliminate the immunogenicity of uricase without reducing uric acid activity. The present invention provides such a composition.</p></solutionproblem><meansproblemsolution><p>According to one aspect of the present invention, as a purified urate oxidase (uricase) preparation, 0% to 2% of aggregates of the uricase preparation are larger than that of the octamer, Case preparations are provided with uricase preparations including tetramer and octamer forms of uricase. </p><p>According to another aspect of the present invention, there is provided a composition comprising a uricase fraction, wherein 0% to 2% of the aggregates of the uricase fraction are larger than the octamers in the fraction, and the fraction is amino-terminal. In , it is a recombinant urcase that is truncated at the carboxy terminus, or at both the amino and carboxy terminus, and the truncated uricase is enzymatically active in tetramer form and octamer form. There is provided a composition comprising a uricase fraction, characterized in that it is in the form.</p><p>According to another aspect of the present invention, there is provided a method for purifying a uricase preparation having reduced immunogenicity, the method comprising the steps of providing a uricase mixture comprising an octameric uricase and a uricase aggregate larger than octamer And, separating part or all of the uricase aggregate larger than the octamer from the uricase mixture, and recovering the remainder including the octamer, wherein the uricase preparation present in the remainder 0% to 2% of aggregates are larger than octamers, and the uricase preparation includes tetrameric and octameric forms of uricase, and the separating step is ion-exchange chromatography, size- There is provided a method for purifying a uricase preparation, characterized in that it is selected from the group consisting of exclusion chromatography and ultrafiltration.</p><p>Preferably, the separating comprises detecting an aggregate larger than the octamer in the uricase fraction and excluding the fraction containing the aggregate, </p><p>More preferably, the detecting comprises a light scattering measurement method.</p><p>According to another aspect of the present invention, it includes a urate oxidase (uricase) preparation, wherein the uricase preparation is tetramer and octamer form of uricase A pharmaceutical composition for lowering the level of uric acid in a body fluid or tissue, characterized in that 0% to 2% of the aggregates of the uricase preparation are larger than the octamer, or</p><p>and a urate oxidase (uricase) preparation, wherein the uricase preparation includes tetramer and octamer forms of uricase, the uricase preparation is provided a pharmaceutical composition for lowering the level of uric acid in a body fluid or tissue, characterized in that there are few uricase aggregates larger than the octameric form.</p><p>Moreover, conjugation of proteins with poly(alkylene glycols), particularly PEG, results in conjugates with reduced immunogenicity and increased persistence in the bloodstream. In the intention to prepare conjugates of uricase that are substantially non-immunogenic while retaining almost all of the uricase activity of the unmodified uricase preparation, the presence of trace amounts of large aggregates of uricase in the starting material is PEG conjugates prepared from uricases containing such aggregates were found to be surprisingly effective in causing both the formation and accelerated clearance of antibodies from circulation. It occurs after repeated scans of the gate, which is detrimental. Surprisingly, the present inventors show that increased immunogenicity and accelerated clearance are greater than native tetramers, i.e. aggregates of well-defined medium-sized uricase subunits (eg aggregates containing eight subunits (8). was not due to the presence of mers)). The octameric form of uricase is at sufficiently high concentrations in most uricase preparations that it can be detected by its UV light absorption (eg at 214 nm or 276 nm) or other measurements on protein concentration or its contribution to refractive index. exists as Nevertheless, in contrast to the much smaller amount of much larger aggregates that cannot be detected by UV absorption under the conditions tested but are soon detected by static (Raleigh) or dynamic light scattering. Thus, it was found that the octamers themselves contribute minimally to the immunogenicity and accelerated clearance of the PEG-uricase conjugate. Therefore, it has been found that removing such traces of very large aggregates prior to conjugation with PEG surprisingly reduces the immunogenicity and accelerated clearance of the resulting PEG-uricase conjugates. </p><p>One embodiment of the present invention is a purified uric acid oxidase (uricase) with few aggregates larger than octamers. Preferably, the uricase is a mammalian uricase. More preferably, the uricase is porcine liver, bovine liver or sheep liver uricase. In one aspect of this preferred embodiment, the uricase is recombinant. In another aspect of this preferred embodiment, the uricase substantially has the sequence of a porcine, bovine, sheep or baboon liver uricase. Advantageously, the uricase is chimeric. Preferably, the uricase is a PKS uricase. In another aspect of this preferred embodiment, the uricase substantially has the sequence of baboon liver uricase in which tyrosine at position 97 is substituted with histidine. Preferably, the uricase comprises an amino terminus and a carboxy terminus, wherein the uricase is truncated at one or both termini. Advantageously, the uricase is a fungal or microbial uricase. Preferably, the fungal or microbial uricase is<i>Aspergillus flavus (Aspergillus flavus), Arthrobacter globiformis (Arthrobacter globiformis), Bacillus sp. (Bacillus sp.)</i> or <i>Candida utilis</i>or a recombinant enzyme substantially having the sequence of one of the uricases. Alternatively, the uricase is an invertebrate uricase. Preferably, the invertebrate uricase is<i>Drosophila melanogaster</i> or <i>Drosophila pseudoobscura</i>or a recombinant enzyme substantially having the sequence of one of the uricases. In another aspect of this preferred embodiment, the uricase is a plant uricase. Preferably, the plant uricase is<i>Glycine max</i>It is a recombinant enzyme isolated from the root nodule of, or having substantially the sequence of its uricase. </p><p>In one aspect of this preferred embodiment, the uricase described above is conjugated to poly(ethylene glycol) or poly(ethylene oxide) under conditions in which the uricase in the conjugate is substantially free of aggregates larger than octamers. . Preferably, the uricase is conjugated to poly(ethylene glycol) or poly(ethylene oxide) via a urethane (carbamate), secondary amine or amide linkage. In one aspect of this preferred embodiment, the poly(ethylene glycol) is monomethoxy poly(ethylene glycol). In another aspect of this preferred embodiment, the poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 5 kDa and 30 kDa. Preferably, the poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 10 kDa and 20 kDa. Advantageously, the average number of strands of the poly(ethylene glycol) or poly(ethylene oxide) is between about 2 and 12 strands per uricase subunit. More advantageously, the average number of strands of the poly(ethylene glycol) or poly(ethylene oxide) is between about 6 and 10 per uricase subunit. Most advantageously, the average number of strands of the poly(ethylene glycol) or poly(ethylene oxide) is between about 7 and 9 per uricase subunit. Preferably, the poly(ethylene glycol) or poly(ethylene oxide) is linear. Alternatively, poly(ethylene glycol) or poly(ethylene oxide) is branched.</p><p>The present invention also provides a pharmaceutical composition for lowering the level of uric acid in a body fluid or tissue, said composition comprising the above-described uricase and a pharmaceutically acceptable carrier. Preferably, the composition is stabilized by lyophilization and dissolved by reconstitution to provide a suitable solution for parenteral administration.</p><p>Another embodiment of the present invention is a method for purifying uricase with reduced immunogenicity, separating uricase aggregates larger than octamers from uricase fractions, and separating such aggregates from purified uricase including the step of excluding. Preferably, the separation step comprises detecting aggregates larger than octamers from at least some of the uricase fractions and excluding the fraction containing the aggregates. Advantageously, said detecting step comprises a measurement of light scattering.</p><p>The present invention also provides an isolated uricase prepared by the method described above.</p></meansproblemsolution><effectiveness><p>According to the present invention, all of the objects of the present invention described above can be achieved.</p></effectiveness>

<p>Previous studies have shown that when a significant decrease in the immunogenicity and/or antigenicity of uricase is achieved by conjugation with PEG (PEGylation), it is necessarily accompanied by a significant decrease in uric acid activity. The present invention includes the observation that trace amounts of uric oxidase aggregates larger than octamers contribute substantially to the induction of immunogenicity and accelerated clearance of PEG-uricase conjugates. These findings seem to apply mostly to other proteins besides uricase, including interferons and growth factors.</p><p>The safety, convenience and cost-effectiveness of biopharmaceuticals are all adversely affected by a decrease in their efficacy and consequently the need to increase the administered dosage. Therefore, there is a need for safe and effective alternatives to reduce elevated levels of uric acid in body fluids including blood and urine. The present invention provides a method for preparing uricase that excludes uricase aggregates larger than octamers for use in the synthesis of PEG-uricase. This PEG-uricase retains all or almost all of the uric acid activity of the unmodified enzyme. The present invention also provides purified uricases that are substantially free of aggregates larger than octamers. The phrase "substantially free" indicates that the purified uricase contains no more than about 2%, preferably no more than about 1% aggregates larger than octamers.</p><p>The present invention provides a method for purifying uricase that allows aggregates larger than octamers to be excluded from the purified preparation. Because these larger aggregates are highly immunogenic, their presence in purified uricase preparations is undesirable. Since aggregates may be too dilute to be detected by UV absorption, the method involves monitoring the column fractions in addition to UV absorption at 280 nm, or by light scattering rather than UV absorption. The purified uricase is then conjugated to water-soluble polymers, preferably poly(ethylene glycol) or poly(ethylene oxide), as described in co-pending U.S. Patent Application Serial No. 09/370,084.</p><p>Removal of aggregated uricase from a preparation consisting predominantly of tetrameric uricase can be accomplished by size-exclusion chromatography, ion-exchange chromatography, ultrafiltration through microporous membranes, and centrifugation, including ultracentrifugation. Including, it can be achieved using methods known to those of ordinary skill in the art. Separation methods may include separation and analysis of fractions, and rejection, ie exclusion, of those fractions containing an excess of large aggregates. Results The uricase preparations are more suitable for the synthesis of substantially non-immunogenic uricase conjugates than unfractionated uricases. For long-term administration, it is important that PEG conjugates of proteins such as eg PEG-uricase have low immunogenicity and do not result in progressively faster clearance from the bloodstream even after repeated administration.</p><p>The present invention also provides pharmaceutical compositions of polymer-uricase conjugates. These conjugates are substantially non-immunogenic and retain at least 75%, preferably 85%, more preferably 95% or more of the uric acid activity of the unmodified enzyme. Uricases suitable for conjugation to water-soluble polymers include bacteria, fungi (fungi), and naturally occurring urate oxidase isolated from tissues of plants and vertebrates and invertebrates, as well as mutated, hybridized, and/or or recombinants of uricase comprising a truncated enzymatically active variant of uricase. Water-soluble polymers suitable for use in the present invention include linear and branched poly(ethylene glycol) or poly(ethylene oxide), both commonly known as PEG. Examples of branched PEGs are the subject of US Pat. No. 5,643,575. A preferred example of a linear PEG is of the general formula CH<sb>3</sb>O-(CH<sb>2</sb>CH<sb>2</sb>O)<sb>n</sb>monomethoxy PEG of H, where n varies between about 100 and about 2,300. </p><p>One embodiment of the present invention is a conjugate of uric acid oxidase (uricase) that retains at least about 75% of the uricase activity of unconjugated uricase and has substantially reduced immunogenicity. The uricase according to this aspect of the invention may be recombinant. The uricase, whether recombinant or not, may be of mammalian origin. In one aspect of this embodiment, the uricase may be porcine, bovine or sheep liver uricase. In another aspect of this embodiment, the uricase may be chimeric. The chimeric uricase may contain portions of porcine liver and/or baboon liver uricase. For example, the chimeric uricase may be a porcine uricase comprising the mutations R291K and T301S (PKS uricase). Alternatively, the uricase may be a baboon uricase in which tyrosine 97 is substituted with histidine, so that the specific activity of uricase can be increased by at least about 60%. The uricase of the present invention, of whatever origin, may be truncated at the amino terminus, at the carboxyl terminus, or at both termini. Likewise, the uricase may be a fungal or microbial uricase. In one aspect of this embodiment, the fungal or microbial uricase is<i>Aspergillus flavus (Aspergillus flavus), Arthrobacter globiformis (Arthrobacter globiformis), Bacillus sp. (Bacillus sp.)</i> or <i>Candida utilis</i>It may be a naturally occurring form or a recombinant form of a uricase from Alternatively, the uricase is, for example,<i>Drosophila melanogaster</i> or <i>Drosophila pseudoobscura</i> It may be an invertebrate uricase, such as a naturally occurring form or a recombinant form of the uricase. The uricase of the present invention can also be used, for example, in soybean root tuber (<i>Glycine max</i>) or a plant uricase, such as a naturally occurring or recombinant form of uricase from PEG may have an average molecular weight between about 5 kDa and 100 kDa; Preferably, the PEG may have an average molecular weight between about 8 kDa and 60 kDa; More preferably, the PEG may have an average molecular weight between about 10 kDa and about 40 kDa, such as between 10 and 20 kDa. The average number of covalently linked strands of PEG can be from 2 to 12 strands per uricase subunit; Preferably, the average number of covalently linked strands may be 6 to 10 per subunit; More preferably, the average number of strands of PEG may be 7 to 9 per subunit. In one aspect of this embodiment, the uricase may be a tetramer. Strands of PEG can be covalently linked to uricase via urethane (carbamate) linkages, secondary amine linkages and/or amide linkages. When the uricase is a recombinant form of any of the uricases mentioned herein, the recombinant form may have a substantially naturally-occurring form of the sequence.</p><p>One preferred mammalian uricase is a recombinant porcine-baby chimeric uricase, which consists of portions of the sequence of porcine liver and baboon liver uricase, all of which were first described by Wu et al. (1989). It was decided. One example of such a chimeric uricase contains the first 288 amino acids from the porcine sequence (SEQ ID NO: 1) and the last 16 amino acids from the non-sequence (SEQ ID NO: 2). Hershfield, et al., International Publication WO 00/08196, Urate Oxidase, published February 17, 2000. Since the latter sequence differs from the pig sequence only in two positions (lysine (K) instead of arginine at residue 291, 301 has a serine (S) instead of a threonine at position B), this mutation is called pig-KS, or PKS urcase (SEQ ID NO: 3). Since PKS uricase has one more lysine residue, it has one more potential site for PEGylation than the porcine or baboon sequence. </p><p>cDNAs for various mammalian uricases, including PKS uricase, were subcloned, and optimal conditions for expression in E. coli were determined using standard methods. Erlich, H.A., (Ed.) (1989)<i>PCR Technology, Principles and Applications for DNA Amplification.</i> New York: Stockton Press; Sambrook, J, et al., (1989)<i> Molecular Cloning. A Laboratory Manual, Second Edition.</i>Cold Spring Harbor, NY: See Cold Spring Harbor Laboratory Press. Recombinant uricases were extracted and purified, and their stability and activity were assessed using modifications of standard assays. Fridovich, I (1965)<i>J. Biol Chem</i> 240: 2491-2494; Nishimura, et al. (1979), and Examples 1-5.</p><p>In one embodiment of the present invention, uricase can be conjugated to a relatively small number of PEG strands via a biologically stable and non-toxic covalent bond. Such linkages may include urethane (carbamate) linkages, secondary amine linkages, and amide linkages. A variety of activated PEGs suitable for such conjugation are described in Huntsville, AL. material from Shearwater Polymers.</p><p>For example, a urethane bond to uricase can be formed by incubating uricase in the presence of a p-nitrophenyl carbonate (NPC) or succinimidyl carbonate (SC) derivative of PEG. SC-PEG can be synthesized using the procedure described in U.S. Patent No. 5,612,460. NPC-PEG was described in Veronese, FM, et al., (1985)<i>Appl Biochem Biotechnol</i> 11: It can be synthesized by reacting PEG with p-nitrophenyl chloroformate according to the methods described in 141-152 and U.S. Patent No. 5,286,637. The method described in the '637 patent is applied to PEGs with higher molecular weights by adjusting the concentrations of the reactants to maintain similar stoichiometry. Another method of NPC-PEG synthesis is described in Buttner W. et al., West German Patent Specification DD 279 486 A1.</p><p>Amide linkages to uricase can be obtained using N-hydroxysuccinimide esters of carboxylic acid derivatives of PEG (Shearwater Polymers). Secondary amine linkages are 2,2,2-trifluoroethanesulfonyl PEG (tresyl PEG; Shearwater Polymers), or PEG aldehyde (Shearwater Polymers) and sodium cyanoborohydride for reductive alkylation using cyanoborohydride. can be formed by</p><p>For conjugates containing PEG with a molecular weight of 10 kDa, the maximum number of strands of PEG bound per subunit while retaining at least 75% of the uric acid activity of the unmodified enzyme is determined by a mammalian uricase (e.g. PKS cage). about 12 strands for casein, a mutein of porcine uricase (see assay conditions in Example 5). The latter degree of PEGylation corresponds to about 40% of the total amino groups. In one embodiment of the invention, the average number of strands of PEG bound per uricase subunit is between about 2 and 12. In a preferred embodiment, the average number of strands of PEG bound per uricase subunit is between about 6 and 10. In a more preferred embodiment, the average number of covalently linked strands of PEG per uricase subunit is between about 7 and 9. In another embodiment, the molecular weight of the PEG used for the binding reaction is between about 5 kDa and 30 kDa, preferably between about 10 kDa and 20 kDa. </p><p>There are several factors that can influence the choice of the appropriate molecular weight and strand number of PEG to bind to a given form of uricase. In general, reducing or eliminating immunogenicity without significant loss of uric acid activity may require binding of a relatively large number of strands of a lower molecular weight PEG compared to a relatively small number of strands of a higher molecular weight PEG. . Likewise, each different form of uricase may have different titration conditions for both size and strand number. The appropriate number of strands of PEG and the molecular weight of the PEG can be readily determined using the methods described herein.</p><p>When PEG conjugates of mammalian uricases were prepared from purified tetrameric and octameric forms of the enzyme (comprising 4 or 8 subunits of about 35 kDa), they were found to contain large aggregates. In contrast to the moderate immunogenicity of the PEG conjugates of the case preparations (see Figure 6) and the very high immunogenicity of the unmodified enzyme, markedly reduced immunogenicity in mice. </p><p>Purified preparations of naturally occurring and recombinant uricases generally contain a mixture of very large enzyme aggregates along with tetrameric (140-kDa) and octameric (280-kDa) forms. The percentage of each uricase preparation in tetrameric or octameric form generally varies from about 20% to 95% (see Figures 2-4). Despite evidence that unpegylated aggregates of several other proteins are highly immunogenic (eg, Moore, WV, et al., (1980))<i>J. Clin. Endocrinol Metab</i> 51: 691-697), previous studies with PEG-uricase did not describe any efforts to limit the content of aggregates, indicating that the potential immunogenicity of PEG-modified aggregates was not taken into account. . On the basis of our observations, it is likely that such aggregates were present in enzyme preparations used for the previous synthesis of PEG-uricase. Their presence seems to have made the task of making non-immunogenic conjugates more difficult. In addition, it is likely that the significant loss of uric acid activity observed in previous efforts to pegylate uricases was associated with a higher number of strands of bound low molecular weight PEG. On the other hand, the PEGylation and uricase purification methods described herein are, at least, with certain uricases, such as PKS uricase (a mutein of porcine uricase) and thermophilic.<i>Bacillus sp. (Bacillus sp.)</i>For enzymes from </p><p>In another preferred embodiment, substantially all large aggregates of the enzyme are subjected to ion-exchange chromatography at a pH of about 9 to 10.5, preferably 10.2, prior to conjugation of the resulting substantially aggregate-free uricase preparation to PEG. It can be removed by chromatography (FIGS. 1-3) or size-exclusion chromatography. The molecular weight of the uricase for each fraction from the preparative column can be determined using, for example, HPLC, conventional size-exclusion chromatography, centrifugation, light scattering, gel electrophoresis in non-denaturing buffer or capillary electrophoresis. It can be monitored by any size-dependent analysis technique. For aggregate-free uricases isolated using size-exclusion chromatography, fractions containing only enzymes between 140-kDa and 280-kDa can be pooled and used for conjugation to PEG. For tetrameric and octameric uricases separated using ion-exchange chromatography, fractions from the ion-exchange column showed significant amounts of tetrameric and octameric forms without large aggregates which fractions were detected by light scattering. can be analyzed for size to determine whether it contains Thus, undesirable large aggregates in the purified product may constitute as small as about 1% of the total urcase, or less.</p><p>The results presented here indicate that, even when heavily PEGylated, PKS uricase forms larger than octamers resulted in accelerated clearance (FIG. 5) and were somewhat immunogenic in mice (FIG. 6). Conversely, conjugates prepared from uricase, essentially free of large aggregates (which can be detected by light scattering), could be re-injected at least six times at weekly intervals with much lower evidence of accelerated clearance (Figure 5). ), there was no formation of detectable antibodies, as measured by a sensitive enzyme-linked immunoassay ( FIG. 6 ). The use of highly purified tetrameric or octameric uricases allows the improved conjugates of the present invention to be further distinguished from previously described PEG-uricase preparations. Conversely, the presence of a significant content of large aggregates in the uricase preparations used by some previous investigators has led them to bind to high strand counts of PEG in an effort to suppress immunogenicity. As a result, the enzymatic activity of the resulting conjugate was significantly reduced. </p><p>The PEG-uricase conjugates of the present invention are useful for lowering uric acid levels in tissues and body fluids of mammals, preferably humans, and are therefore useful in treating gout, tophi and renal insufficiency, organ transplantation ( organ transplantaion) and for the treatment of elevated uric acid levels accompanying symptoms including malignancy. The PEG-uricase conjugate can be injected into a mammal having excess uric acid levels by one of many routes, including intravenous, subcutaneous, intradermal, intramuscular, and intraperitoneal routes. have. Alternatively, they can be nebulized and inhaled. Patton JS. (1996)<i>Adv. Drug Delivery Rev.</i> 19: See 3-36 and U.S. Patent No. 5,458,135. The effective dosage of the PEG-uricase of the present invention will depend on the level of uric acid and the size of the individual. In one embodiment of this aspect according to the invention, the PEG-uricase may be administered in an amount ranging from about 10 μg to about 1 g in a pharmaceutically acceptable excipient or diluent. In a preferred embodiment, the amount administered is between about 100 μg and 500 mg. More preferably, the conjugated uricase is administered in an amount between 1 mg and 100 mg, such as 5 mg, 20 mg, or 50 mg. Mass given for a dosage in embodiments refers to the amount of protein in the conjugate.</p><p>Pharmaceutical formulations containing PEG-uricase are described, for example, in Gennaro, AR. (Ed.) (1990). <i>Remington's Pharmaceutical Sciences</i>, 18th Edition Easton, PA: Mack Publishing Co., may be prepared by conventional techniques. Suitable excipients for the preparation of injectable solutions include, for example, phosphate buffered saline, lactated Ringer's solution, water, polyols and glycerol. Pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions, or emulsions and sterile powders for reconstitution immediately prior to use into sterile injectable solutions or dispersions. is composed by Such formulations may contain additional ingredients such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, buffers, antioxidants and diluents.</p><p>The PEG-uricase may also be provided as a controlled release composition for implantation into a subject for sustained suppression of elevated uric acid levels in a body fluid. For example, polylactic acid, polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate, gellan gum, chitosan, agarose, multilamellar liposomes and many other conventional depots. (depot) agents include bioerodible or biodegradable substances that can be formulated with a biologically active composition. These substances, when implanted or injected, slowly disintegrate, releasing the active substance into the surrounding tissue. For example, one method of encapsulating PEG-uricase includes the method disclosed in U.S. Patent No. 5,653,974. The use of bioerodible, biodegradable and other depot agents is broadly contemplated in the present invention. The use of matrix entrapment systems and infusion pumps for delivery of PEG-uricase is also within the scope of the present invention. PEG-uricase may also be usefully enclosed in micelles or liposomes. Liposome encapsulation techniques are well known in the art. Lasic D. et al., (Eds.) (1995)<i>Stealth Liposomes.</i> See Boca Raton, FL: CRC Press. </p><p>The PEG-uricase pharmaceutical compositions of the present invention can be used, for example, in organ transplant recipients (Venkataseshan, VS, et al., (1990)). <i>Nephron</i> 56:317-321) and patients with some malignancies, which would reduce the need for hemodialysis in patients at high risk for urate-induced kidney disease. In patients with high levels of crystalline urate (gout nodules), such pharmaceutical compositions will improve quality of life faster than currently available therapies.</p><p>The following examples, which are not intended to limit the invention in any sense, illustrate these various aspects. These examples describe PEG-uricases prepared by binding activated PEG (eg, a p-nitrophenyl carbonate derivative) to a mutein of porcine uricase. These examples provide those skilled in the art with substantially non-immunogenic uricase conjugates that retain at least about 75% of the uric acid activity of the unmodified enzyme and are well suited for long-term administration. Guidance for manufacturing is provided.</p><p>Example 1</p><p><u>Preparative Ion-Exchange Chromatography of Uricase</u></p><p>Preparative ion-exchange chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) apparatus (Amersham Pharmacia, Piscataway, NJ). A mono Q column (1×10 cm, Amersham Pharmacia) was run with a gradient from 50 mM sodium carbonate, pH 10.3, 0.1 M NaCl (buffer A) to 50 mM sodium carbonate, pH 10.3, 0.6 M NaCl (buffer B). Eluted at a flow rate of 0.5 ml/min and the sample was loaded at a lower flow rate. This technique was used to fractionate 25 mL of PKS uricase (pH 10.3) solution. PKS uricase was obtained from Bio-Technology General limited (Rehovot, Israel). The latter is a recombinant porcine uricase in which one lysine (K) residue and one serine (S) residue are substituted with one arginine and one threonine residue, respectively, in the parental pig sequence (Lee et al. (Lee et al. (Lee et al. (Lee et al. (Lee et al. 1988) Science 239: 1288-1291; Wu et al. (1989) Proc. Natil. Acad. Sci. USA 86: 9412-9416). After the sample was loaded, the column was washed with 100 mL of Buffer A. The uricase peak started eluting at the end of a 31 mL linear gradient of 0-26% Buffer B. Most of the uricase was eluted isocratically with 7 mL of buffer containing 26% Buffer B. The remainder of the recovered uricase was eluted by a linear 89-mL gradient of 26% to 100% buffer B. Fractions of 4 mL or 6 mL were combined. Fractions #4-11 were assayed for uricase and total protein (FIG. 1), and analyzed by size-exclusion high performance liquid chromatography (HPLC) as described in Example 2 ( 2 and 3). The remaining fractions #5-10 were bound to PEG as described in Example 3. Based on the analysis results in Example 2, as shown in Fig. 1, the PEG conjugates of fractions #5 and #6 were combined as a "low-salt pool", and fractions #7-10. Their PEG-conjugates were combined as a "high-salt pool".</p><p>Example 2</p><p><u>Size-Exclusion Chroma of Urcasease Monitored by Light Scattering and UV Absorption</u>topography</p><p>Size-exclusion HPLC was performed at room temperature, on a Superdex 200 column (1×30 cm, Amersham Pharmacia Biotech), on selected fractions from preparative mono Q chromatography of unfractionated PKS uricase and PKS uricase of Example 1. was performed for Eluates from the Absorption Monitor (UV 2000) of Thermo Separations HPLC (Sunnyvale, CA) were analyzed by light scattering at 90° to the incident light, using a MiniDawn detector from Wyatt Technologies (Santa barbara, CA). .</p><p>The results presented in Figures 2-4 show the resolution between tetramers, octamers and larger aggregates of the uricase subunit and the different ratios of signals detected from these uricase forms in various samples. Unlike the absorption signal, which is directly proportional to the concentration, the light scattering signal is proportional to the product of the size and concentration of the light scattering unit. The resulting sensitivity of the light scattering detector to very small amounts of highly aggregated uricase revealed the presence of the largest aggregates, which eluted at or near the void volume (approximately 7 mL).</p><p>Example 3</p><p><u>Synthesis of PEG-Uricase Conjugates</u></p><p>Unfractionated PKS uricase (from Bio-Technology General Limited) and uricase in fractions from the mono Q column of Example 1 were p-nitrophenyl of PEG obtained from Shearwater Polymers (Huntsville, AL.). A carbonate derivative (NPC-PEG) was used to bind to 10-kDa PEG. The preparation of NPC-PEG from PEG using phenylchloroformate has been described in several reports (e.g., Veronese, FM, et al., (1985)).<i>Appl. biochem. Biotechnol.</i> 11: 141-152; Kito, M. et al., (1996)<i>J. Clin. Biochem Nutr.</i> 21: 101-111) NPC-PEG has also been used in the synthesis of PEG-protein conjugates by previous researchers including the present inventors (eg, Veronese et al,<i> supra</i>; Sherman, MR, et al., in JM Harris, et al., (Eds.)<i>Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680</i>(pp. 155-176) Washington, DC: American Chemical Society. The number of strands of 10-kDa PEG bound to each subunit of uricase is determined by Kunitani, M. et al., (1991).<i>J. Chromatogr.</i> 588: determined to be six (6) by the method described in 125-137. </p><p>Example 4</p><p><u>In vivo serum persistence and immunogenicity of uricase and PEG-uricase</u></p><p>The PEG conjugate of recombinant mammalian uricase prepared according to the method of Example 3 was adjusted to 1 mg protein/mL in phosphate-buffered saline (PBS) at pH 7.4 for injection. . Samples were frozen and stored until analysis or injection. Samples were heated to 37° C. within 1 hour prior to injection into groups of 8 BALB/c female mice. The group of mice had a mean body weight in the range of 18-22 g at the beginning of the study.</p><p>Body weights of all mice were monitored and evidence of adverse reactions to injections or other evidence of poor health was recorded. Twenty-four hours after six weekly injections, animals were anesthetized with ketamine, and 100-200 μL of blood was instilled into the eyeballs, except for sacrifice (drawing of blood) when larger volumes of blood were collected. pulled from the back Serum was prepared from clotted blood at 2-8°C for 4 to 32 hours. Serum was stored at -20°C. Serum was assayed for uric acid activity as described in Example 5 and for antibodies to uricase as described in Example 6.</p><p>Example 5</p><p><u>Uric acid degradation of PEG-uricase in serum from mice injected with PEG-uricase </u>assay of activity</p><p>An activity assay based on ultraviolet light absorption (UV assay) was performed against I. Fridovich (<i>J. Biol Chem.</i> (1965) 240: 2491-2494) within a microplate adaptation of the method. Absorbance at 292 nm, using a SpectraMAX 250 microplate reader from Molecular Devices (Sunnyvale, CA), in 96-well plates with UV-transparent bottom, at room temperature for 15 min. was monitored for a decrease in This data was analyzed by finding the maximum slope (as milli-absorption units per minute) of absorbance measurements made during the interval during which the substrate was oxidized between 10 and 40%. The results obtained by this assay are shown in FIGS. 1 and 5 .</p><p>The mean half-life in the sera of mice first injected with PKS uricase (6×10-kDa PEG PKS) bound to six strands of 10-kDa PEG per subunit was determined from sera obtained at 24 and 72 hours post-injection. based on the data of 29±4 hours. </p><p>In separate experiments, detectable uric acid activity in sera of mice injected with PEG-uricase decreased during storage at -20°C, and the maximum recovery of this activity was 4 h at 37°C, prior to assay. It has been established that it is obtained by culturing. Figure 5 shows the recovery of uric acid-degrading activity after weekly repeated injections with 6×10-kDa PEG-PKS uricase, as in Example 1, before PEGylation according to the method of Example 3; It shows that the maximum when purified by column chromatography. The recovery was the highest after injection of the conjugate prepared from the high salt elution pool of Example 1 (see Figure 1), which had a minimal content of very large aggregates (light scattering profiles of fractions 7-10 in Figure 3). purple). Moderate recovery was obtained with the conjugate prepared from the low salt elution pool from the Mono Q column of Example 1, and the lowest recovery was obtained with the conjugate prepared from unfractionated PKS uricase, which was very It has the highest content of large aggregates (see Fig. 2). Whether using the UV assay described above, P. Fossati et al. (<i>J. Clin. Chem</i> (1980) 26: 227-231), and whether or not the serum was incubated at 37° C. prior to assay, in serum after repeated injections. The same sequence of recovered relative activity (high salt pool &gt; low salt pool &gt; unfractionated uricase) was observed. </p><p>Example 6</p><p><u>Enzyme-Linked Immunosorbent Gum of Serum from Mice Injected with PEG-Uricase</u>ELISA: enzyme-linked immunosorbent assay </p><p>A non-competitive ELISA assay was performed on porcine uricase bound to 96-well Immulon 2 plates (Dynex Technologies, from VWR Scientific, San Francisco, CA). The primary antisera were from mice injected with 6×10-kDa PEG conjugate or uricase prepared according to the method of Example 3. The secondary antibody was goat anti-mouse IgG bound to horseradish peroxidase (Calbiochem-Novabiochem #401 253, La Jolla, CA) and the substrate was B. Porstmann et al. (J. Clin. Chem. Clin. Biochem. (1981) 19: 435-440) o-phenylenediamine dihydrochloride (Sigma P-9187, St. Louis, MO).</p><p>6 shows the results of a non-competitive ELISA assay. The results showed that 6×10-kDa PEG-PKS uricase synthesized according to the method of Example 3 from the high salt eluate from the Mono Q column of Example 1 (shown in FIG. 1 ) was injected weekly for 6 weeks. This indicates that none of the 8 mice produced a detectable immune response. Several mice injected with a conjugate prepared from unfractionated PKS uricase according to the method of Example 3 showed a low but detectable immune response. The highest immune response occurred in mice injected with the conjugate prepared according to the method of Example 3 from the low-salt elution pool from the Mono Q column of Example 1.</p><p>As described in Example 2, without the benefit of a light scattering detector for size-exclusion HPLC analysis, the presence of the largest aggregates, not in the octameric form of uricase, was observed in Example 5 (Figure 5). ), accompanied by progressively reduced recovery of the PEG-uricase conjugate after repeated injections and an increase in immunogenicity in BALB/c mice, as observed in Example 6 (Figure 6). It would not have been clear that These results have important implications for the specification of uricases to be used as starting materials for the preparation of PEG-uricases for clinical use.</p><p>While the foregoing invention has been described in some detail by way of drawings and examples for clarity of understanding, it will be appreciated by those of ordinary skill in the art that, in light of the teachings of the present invention, without departing from the spirit and scope of what is described and claimed, It will be clear that some changes or modifications may be made there.</p>