UOX-ALBUMIN CONJUGATE WITH CERTAIN NUMBERS OF ALBUMIN CONJUGATED THERETO, AND MANUFACTURING METHOD THEREOF

Abstract

The present application relates to a method of preparing urate oxidase (Uox) including a non-nature amino acid (NNAA) and Uox prepared thereby. The present application showed that the method of preparing Uox including an NNAA may be effectively used to prolong the half-life of a protein which is difficult to be linked to a carrier.

In addition, the Uox produced by the method may be effectively used for various biopharmaceuticals since its efficacy is maintained and drug persistency increases due to site-specific conjugation of a carrier, a risk of an immune response is reduced, and it is easily separated due to formation of uniform conjugate.

Claims

1. A Uox-carrier conjugate of formula 1: ##STR00010## wherein UoX is a uricase from Aspergillus Flavus formed from 4 uricase monomers (p′); L is a crosslinker; and Car is a carrier, wherein the p′ has an amino acid sequence of SEQ ID NO:1 substituted at at least one amino acid residue selected from the group of tyrosine 8, tyrosine 16, tyrosine 30, tyrosine 46, tyrosine 65, phenylalanine 79, phenylalanine 87, tyrosine 91, tryptophan 106, phenylalanine 120, phenylalanine 159, tryptophan 160, phenylalanine 162, tyrosine 167, tryptophan 174, tryptophan 186, tryptophan 188, phenylalanine 191, phenylalanine 204, tryptophan 208, phenylalanine 219, tyrosine 233, tyrosine 251, tyrosine 258, phenylalanine 259, tryptophan 265, and phenylalanine 279 by a non-natural amino acid selected from p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), L-Homopropargylglycine (HPG), O-propargyl-L-tyrosine (oPa) and p-propargyloxyphenylalanine (pPa), the Uox and the L is linked through the substituted non-natural amino acid, and n=2.

2. The Uox-carrier conjugate according to claim 1, wherein the Car is an albumin.

3. The Uox-carrier conjugate according to claim 1, wherein the p′ has an amino acid sequence of SEQ ID NO:1 substituted at tryptophan 160, or tryptophan 174 by a non-natural amino acid selected from p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), L-Homopropargylglycine (HPG), O-propargyl-L-tyrosine (oPa) and p-propargyloxyphenylalanine (pPa).

4. The Uox-carrier conjugate according to claim 1, wherein the substituted non-natural amino acid is p-Azido-L-phenylalanine (AzF).

5. The Uox-carrier conjugate according to claim 1, wherein the L comprises alkylene, alkenylene, alkynylene, or (CH2O)n.

6. The Uox-carrier conjugate according to claim 5, wherein the L comprises (CH2O).sub.10.

7. The Uox-carrier conjugate according to claim 2, wherein the albumin is a human serum albumin.

8. The Uox-carrier conjugate according to claim 1, wherein the linked structure of the Uox and the L is a bonding structure formed by a click-chemistry reaction.

9. The Uox-carrier conjugate according to claim 8, wherein the linked structure of the Uox and the L is a bonding structure formed by a reaction between dibenzocyclooctyne (DBCO) and azide.

10. The Uox-carrier conjugate according to claim 2, wherein the linked structure of the L and the albumin is a bonding structure formed by a reaction between maleimide (MAL) and a cysteine residue of the albumin.

11. The Uox-carrier conjugate according to claim 1, wherein the Uox is formed from two p′ each of which is not linked to the carrier and two p′ each of which is linked to the carrier.

12. The method for manufacturing the Uox-carrier conjugate according to claim 1, comprising producing a uricase comprising a non-natural amino acid by culturing bacteria by fed-batch culture; separating the uricase comprising a non-natural amino acid; conjugating the uricase comprising a non-natural amino acid with a carrier; and separating the uricase conjugated to the carrier, wherein the separating is performing a hydrophobic interaction chromatography, an anion exchange chromatography, and a size exclusion chromatography.

13. The method for manufacturing the Uox-carrier conjugate according to claim 12, wherein the separating the uricase comprising a non-natural amino acid; is performing a hydrophobic interaction chromatography, an anion exchange chromatography, and a size exclusion chromatography in order.

14. The method for manufacturing the Uox-carrier conjugate according to claim 12, wherein the separating the uricase comprising a non-natural amino acid; is performing an anion exchange chromatography, a hydrophobic interaction chromatography, and a size exclusion chromatography in order.

15. A composition for preventing or treating at least one disease selected from the group of hyperuricemia, gout, deposition of urate crystals in joints, acute gouty arthritis by deposition of urate crystals in joints, urolithiasis, nephrolithiasis, and gouty nephropathy.

16. A food composition for preventing or ameliorating at least one disease selected from the group of hyperuricemia, gout, deposition of urate crystals in joints, acute gouty arthritis by deposition of urate crystals in joints, urolithiasis, nephrolithiasis, and gouty nephropathy.

Description

DESCRIPTION OF DRAWINGS

[0181] FIG. 1 is a diagram showing a result of comparing conventional Uox-W160, 174AzF (6xHis)(template) and a His-tag-removed sequence (deletion) of an Uox expression vector.

[0182] FIG. 2 shows a fed-batch culture profile of an Uox-producing strain.

[0183] FIG. 3 shows a hydrophobic chromatography result for Uox separation.

[0184] FIG. 4 shows an anion exchange chromatography for Uox separation.

[0185] FIGS. 5 and 6 show a size exclusion chromatography result for Uox separation, an SDS-PAGE gel image, and an SEC-HPLC result. Specifically, FIG. 5 shows a size exclusion chromatography for Uox separation and an SDS-PAGE gel image.

[0186] FIG. 6 shows a size exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) result for analyzing Uox purity.

[0187] FIG. 7 shows a result of confirming AzF introduction of Uox through SDS-PAGE analysis.

[0188] FIG. 8 shows a cation exchange chromatography result for separation of an Uox-carrier conjugate.

[0189] FIG. 9 shows an anion exchange chromatography result for separation of an Uox-carrier conjugate.

[0190] FIGS. 10 and 11 shows a size exclusion chromatography result and an SDS-PAGE gel image for separating an Uox-carrier conjugate. Specifically, FIG. 10 shows size exclusion chromatography results of mono-HSA-Uox and di-HSA-Uox according to a reaction molar ratio of Uox:HSA-DBCO=1:1. FIG. 11 shows size exclusion chromatography results of mono-HSA-Uox and di-HSA-Uox according to a reaction molar ratio of Uox:HSA-DBCO=1:3.

[0191] FIG. 12 shows a result of separating mono-HSA-Uox and di-HSA-Uox, analyzed through SEC-HPLC.

[0192] FIG. 13 shows an effect of reducing a blood uric acid level of an Uox-carrier conjugate in a hyperuricemia mouse animal model.

[0193] FIG. 14 shows an effect of reducing a blood uric acid level of a Uox-carrier conjugate in a hyperuricemia rat animal model.

[0194] FIGS. 15 to 17 show kidney autopsy results of a hyperuricemia rat animal model. Specifically, FIG. 15 shows the appearances of the kidneys of animal models.

[0195] FIGS. 16 and 17 show histomorphological changes in animal models.

[0196] FIG. 18 shows an anion exchange chromatography result for Uox separation in a second Uox separation and purification process.

[0197] FIG. 19 shows a hydrophobic interaction chromatography result for Uox separation in a second Uox separation and purification process.

[0198] FIGS. 20 and 21 show a size exclusion chromatography result, an SDS-PAGE gel image and a SEC-HPLC result for Uox separation in a second Uox separation and purification process. Specifically, FIG. 20 shows the size exclusion chromatography result and SDS-PAGE gel image for Uox separation. FIG. 21 shows the SEC-HPLC result for analyzing Uox purity.

[0199] FIG. 22 shows the result of measuring in vitro enzyme activity of Uox-HSA.

[0200] FIG. 23 shows the comparison of the effect of reducing a blood uric acid level between Uox-HSA and competing drugs in hyperuricemia rat animal models.

[0201] FIG. 24 shows the comparison of the pharmacodynamic effect between Uox-HSA and competing drugs in animal models.

[0202] FIG. 25 shows an immunogenic analysis result in animal models.

[0203] FIGS. 26 and 27 show the molecular weight analysis results of Di-HSA-Uox.

[0204] FIGS. 28 and 29 show the results of analyzing the isoelectric point (pI) characteristic of Uox-HSA using cIEF.

[0205] FIG. 30 shows the comparison of a pharmacodynamic effect between mono-HSA-Uox and di-HSA-Uox in SD-Rats.

MODES OF THE INVENTION

[0206] 1. Urate Oxidase (Uox)

[0207] A Urate oxidase (hereinafter, Uox) is an enzyme having a function of degrading uric acid. Since the human body does not produce the Uox, if uric acid is not smoothly degraded, gout may occur. The Uox may be used as a material for treating a disease caused by uric acid, representatively, gout. The Uox is a tetramer having a form in which four monomers with the same structure are linked.

[0208] In one embodiment, Uox may be Aspergillus Flavus-derived Uox. Here, the structure of an Aspergillus Flavus-derived Uox monomer present in nature is SEQ ID NO: 1. In another embodiment, Uox may be microorganism-derived Uox. In still another embodiment, Uox may be mammal-derived Uox.

[0209] In the present application, urate oxidase is indicated as an Uox.

[0210] The Uox has a form in which four Uox monomers (p′) are linked together. In “p” according to the present application, one or more residues of the amino acid sequence of SEQ ID NO: 1 are substituted with NNAAs.

[0211] In one embodiment, the “p′” may have a modified structure in which one or more amino acid residues selected from the group consisting of tyrosine at position 8, tyrosine at position 16, tyrosine at position 30, tyrosine at position 46, tyrosine at position 65, phenylalanine at position 79, phenylalanine at position 87, tyrosine at position 91, tryptophan at position 106, phenylalanine at position 120, phenylalanine at position 159, tryptophan at position 160, phenylalanine at position 162, tyrosine at position 167, tryptophan at position 174, tryptophan at position 186, tryptophan at position 188, phenylalanine at position 191, phenylalanine at position 204, tryptophan at position 208, phenylalanine at position 219, tyrosine at position 233, tyrosine at position 251, tyrosine at position 258, phenylalanine at position 259, tryptophan at position 265 and phenylalanine at position 279 of the amino acid sequence of SEQ ID NO: 1 are substituted with NNAAs. Further, the “p′” may have a structure in which tryptophan at position 160 or phenylalanine at position 174 of the amino acid sequence of SEQ ID NO: 1 are substituted with an NNAA.

[0212] In one embodiment, the substituted NNAA may be p-azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), L-homopropargylglycine (HPG), 0-propargyl-L-tyrosine (oPa) or p-propargyloxyphenylalanine (pPa). Further, the substituted NNAA may be p-azido-L-phenylalanine (AzF).

[0213] 2. Crosslinker

[0214] The scope of the present application includes a crosslinker for conjugating Uox with albumin. The term “crosslinker” used herein is an agent for connecting two proteins, as well as a structure of connecting two proteins in the structure of a conjugate formed by a reaction.

[0215] In the structural formula of the present application, the crosslinker is indicated as L.

[0216] The crosslinker as an agent consists of a functional group for forming a bond with Uox, a functional group for forming a bond with albumin and a structure of connecting them. After forming a conjugate, the functional groups correspond to a connecting structure between Uox and the crosslinker and a connecting structure between the crosslinker and albumin of the conjugate structure.

[0217] In one embodiment, the crosslinker may include a functional group reactive to Uox to be connected with Uox. Further, the functional group reactive to Uox may be a click-chemistry functional group. Furthermore, the click-chemistry functional group may be dibenzocyclooctyne (DBCO), azide, tetrazine, or transcyclooctene. Furthermore, the click-chemistry functional group may be dibenzocyclooctyne.

[0218] In one embodiment, the crosslinker may include a functional group reactive to albumin to be connected with albumin. In one example, the reactive functional group may have reactivity to a thiol (—SH) of a cysteine residue. Here, the reactive functional group includes maleimide (MAL), 3-arylpropiolonitrile, haloacetal, pyridyl disulfide, and conventionally used ones. In another example, the reactive functional group may have reactivity to (—NH.sub.2) of a lysine residue. Here, the reactive functional group includes N-hydroxysuccinimide ester (NHS), imidoester, and conventionally used ones. In still another example, the reactive functional group may be a click-chemistry functional group.

[0219] A structure in which two functional groups of a crosslinker or a structure of connecting structures including the same will be described. In one embodiment, the structure may include an alkylene, alkenylene, alkynylene, aralkylene, arylalkylene or (CH.sub.2O).sub.n. The terms “alkylene,” “alkenylene,” “alkynylene,” “aralkylene,” and “arylalkylene” refer to conventionally understood structures including a hydrocarbon chain and/or an aromatic ring, and are intended to also include all structures including allowable substitutions or hetero atoms. Further, the structure may include (CH.sub.2O).sub.n. Furthermore, the structure may include (CH.sub.2O).sub.10.

[0220] 3. Carrier

[0221] A carrier connected to the conjugate of the present application refers to a material such as a protein or polymer with a longer half-life in the body. In one embodiment, the carrier may be a serum protein. The serum protein is keyhole limpet hemocyanin (KLH), globulin or albumin, which is found in serum and has a longer half-life. In one embodiment, the carrier may be a polymer. In one example, the carrier may be polyethylene glycol (PEG).

[0222] In one embodiment, the carrier may be albumin. Here, the albumin refers to commonly referred to albumin. In one embodiment, the albumin may be mammal-derived albumin. Further, albumin may be human serum albumin (HSA). In one embodiment, the albumin may be wild-type. In another embodiment, the albumin may be genetically manipulated. Further, the genetic manipulation may be manipulation to include NNAA. The “wild type” or “genetically manipulated” used herein is a term indicating a protein state determined on the basis of whether a naturally-found protein and an amino acid sequence are the same.

[0223] In the structural formula of the present application, the carrier is represented as Car, and the albumin is represented as Alb.

[0224] 4. Uox-Carrier Conjugate

[0225] An Uox-carrier conjugate having the structure of following structural formula 1 according to the present invention is provided:

##STR00002##

[0226] In Structural Formula 1, the description of components Uox, L and Car may be applied mutatis mutandis to the above description of 1., 2. and 3.

[0227] Here, n may be 1 to 8. Further, n may be 1 to 4. Preferably, n is 1 or 2. Furthermore, n may be 1. In another example, n may be 2.

[0228] In one example, when n=1, Uox may consist of three p′ monomers to which a carrier is not connected and one p′ monomer to which one carrier is connected. In one example, when n=2, Uox may consist of three p′ monomers to which a carrier is not connected and one p′ monomer to which two carriers are connected. In another example, when n=2, Uox may consist of two p′ monomers to which a carrier is not connected and two p′ monomers to which a carrier is connected.

[0229] In one embodiment, Car may be albumin. Here, a Uox-albumin conjugate having the following Structural Formula 2 is provided:

##STR00003##

[0230] The description of components Uox, L and Alb may be applied mutatis mutandis to the above description of 1., 2. and 3., and also applied to the description of Structural Formula 1.

[0231] 4.1. Click-Chemistry

[0232] The term “click-chemistry” is a chemical concept introduced by K. Barry Sharpless of the Scripps Research Institute to explain a complementary chemical functional group and a chemical reaction, which are designed for two molecules to rapidly and stably make a covalent bond. The click-chemistry refers to a specific reaction, as well as the concept of a rapid and stable reaction. In certain embodiments, the click-chemistry is modularized, has a wide range and high yield, does not have a significant by-product, is stereospecific and physiologically stable, has a great thermodynamic driving force (e.g., >84 kJ/mol), and/or has to have high atom economy. Several reactions are known to satisfy the above conditions:

[0233] (1) Huisgen 1,3-dipolar cycloaddition (e.g., including a Cu(I)-catalytic cycloaddition reaction, and also frequently referred to generically as a “click reaction”; see Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064): copper or ruthenium is generally used as a catalyst;

[0234] [Schematic Diagram of Huisgen 1,3-Dipolar Cycloaddition]

##STR00004##

[0235] (2) Diels-Alder reaction, which is a cycloaddition reaction including, for example, a normal electron-demand Diels-Alder reaction and an inverse electron-demand Diels-Alder reaction, but not limited thereto (i.e., strain-promoted cycloaddition (SPAAC));

[0236] [Schematic Diagram of Diels-Alder Reaction]

##STR00005##

[0237] [Example of Diels-Alder Reaction; TCO and Tetrazine]

##STR00006##

[0238] [Schematic Diagram of Strain-Promoted Cycloaddition Reaction]

##STR00007##

[0239] [Example of Strain-Promoted Cycloaddition Reaction; Azide and DBCO]

##STR00008##

[0240] (3) Nucleophilic addition for small stained rings such as epoxide and aziridine;

[0241] (4) Nucleophilic addition for activated carbonyl group;

[0242] (5) Addition reaction for carbon-carbon double bond or triple bond.

[0243] [Addition Reaction of Thiol and Alkene]

##STR00009##

[0244] The term “click-chemistry functional group” used herein refers to a functional group involved in the click-chemistry. For example, a strained alkyne, for example, cyclooctyne, corresponds to a click-chemistry functional group. Generally, the click-chemistry needs at least two molecules including complementary click-chemistry functional groups, respectively. A pair of click-chemistry functional groups having reactivity is referred to as a “partner click-chemistry functional group” in the present application. For example, in the strain-promoted cycloaddition reaction between azide and cyclooctyne, the azide is a partner click-chemistry functional group of cyclooctyne and other alkynes. Exemplary click-chemistry functional groups used in the present application include a terminal alkyne, an azide, a strained alkyne, a diene, a dienophile, a trans-cyclooctene, an alkene, a thiol and tetrazine, but the present invention is not limited thereto. Other click-chemistry functional groups are known to those of ordinary skill in the art.

[0245] 4.2. Linkage of Uox and Crosslinker

[0246] In Structural Formula 1, the connecting relationship between Uox and L will be described in further detail. The Uox and L are linked together by a functional group reactive to Uox included in L.

[0247] In one embodiment, the Uox and the crosslinker may be linked by a modified NNAA of the p′ as described in 1.

[0248] In one embodiment, the connecting structure of Uox and the crosslinker may be a binding structure formed by click-chemistry. Since an NNAA including a click-chemistry group can be inserted into Uox according to the preparation process of the present application, a conjugate can be prepared using click-chemistry. Since the click-chemistry and its structure is bio-orthogonally formed, the reaction is stably performed and the structure is not easily broken. In one example, the connecting structure of Uox and the crosslinker may be a binding structure formed by a reaction between dibenzocyclooctyne (DBCO) and azide (see 4.1.). Since the NNAA AzF includes azide, as an example, it can be used to form the binding structure.

[0249] 4.3. Linkage of Crosslinker and Albumin

[0250] In Structural Formula 1, the connecting relationship between L and Alb will be explained in further detail. L and Alb are linked together by a functional group of L, reactive to albumin.

[0251] In one embodiment, the crosslinker and the albumin may be connected by a cysteine residue of the albumin. Since the cysteine residue includes a thiol (—SH) having reactivity, it is generally used to prepare a bioconjugate. Here, the connecting structure of the crosslinker and the albumin may be a binding structure formed by the reaction between the functional group and the cysteine residue of the albumin as described in 2. In one example, the connecting structure may be a binding structure formed by a reaction between maleimide (MAL) and a cysteine residue of the albumin.

[0252] In one embodiment, the crosslinker and the albumin may be connected by a lysine residue of the albumin. Since the lysine residue includes a free amine group (—NH.sub.2) having reactivity, it is generally utilized to prepare a bioconjugate. Here, the connecting structure between the crosslinker and the albumin may be a binding structure formed by a reaction between the functional group and a cysteine residue of the albumin as described in 2.

[0253] However, since the present application is characterized by a binding site that does not disturb the activity of Uox and a click-chemistry reaction used for the corresponding binding, the binding between the albumin and the crosslinker corresponds to a part with a low need for problem solving. Therefore, the connecting structure may use any connecting method that can be generally used. The present application is described with the intention not to be greatly limited by the binding structure between the crosslinker and the albumin.

[0254] 5. Efficacy of Uox-Albumin Conjugate

[0255] This section is intended to explain the improved efficacy when a Uox-albumin conjugate having the structure that is able to be characterized by the above-described 1. and 4. is compared with wild-type Uox. Further, this section is for explaining the improved efficacy when being compared with other persistent Uox drugs.

[0256] 5.1. Maintenance and Improvement of Urea Degrading Activity

[0257] Generally, a persistent drug prepared by connecting a macromolecule and a protein is greatly decreased in activity. This is because there is a problem in which the connected macromolecule blocks the active site of a target protein. This problem can be analyzed in two aspects. (1) In the case of a technique of connecting a macromolecule through random substitution such as PEGylation in the conventional art, a connection site of the macromolecule is not specified. For this reason, it is impossible to connect a macromolecule by avoiding the active site. (2) Although specific binding is possible, much research is required to find a connection site which does not suppress the activity of a desired protein. Substitution should occur at a site away from the active site of the desired protein, at the time, a site at which the formation of the tertiary structure of the protein is not inhibited has to be substituted, as well as a site with high solvent accessibility for a high yield. At the same time, since the mode of action of a desired protein in the human body may not be completely defined, it is necessary to find a binding site that does not inhibit additional interactions such as various coenzymes. To this end, efforts are required to select candidate substitution sites according to certain criteria, and to actually experiment with them to select sites with high activity.

[0258] As confirmed in the following Examples 6, 11 and 12, the Uox-albumin conjugate according to the present application (hereinafter, Uox-HSA) has the same or improved activity compared with wild-type Uox. As shown in Table 6, Uox-HSA has similar activity to a wild-type protein (Fasturtec) compared at the same dose, and exhibits activity two-fold or higher than a persistent drug such as KRYSTEXXA. In consideration that the conventional persistent drug does not have as much efficacy compared with the wild-type protein, this effect is very significant. As described above, by blocking an active site, the activity of KRYSTEXXA prepared through random PEGylation was greatly lowered. In addition, having similar activity to the wild-type protein means that the tertiary structure of the protein is completely maintained without blocking the active site, and other additional actions, in addition to a direct reaction with a substrate, are not inhibited.

[0259] 5.2. Enhancement of Persistency

[0260] Since the Uox-HSA of the present application includes albumin participating in an FcRn cycle, a half-life is greatly improved compared with a wild-type protein. As shown in Example 13, Uox-HSA has a half-life 8.7-fold or longer than the wild-type protein. In addition, the effect of enhancing the persistency of Uox-HSA shows a similar value compared with a conventional drug, such as KRYSTEXXA. Therefore, it can be confirmed that the Uox-HSA of the present application has greatly increased persistence, as intended.

[0261] Since the molecular weight of KRYSTEXXA is much greater than Uox-HSA (KRYSTEXXA: 497 kDa, Uox-HSA: 270 kDa), and Uox-HSA uses HSA and thus is expected to further prolong a half-life when being used for a human, efficacy may be considered more positively.

[0262] 5.3. Decrease in Immunogenicity

[0263] Uric acid oxidases mainly used for medical purposes are microorganism-derived proteins. Due to the characteristics of a foreign protein, these uric acid oxidases have high immunogenicity. Therefore, when they are administered to the human body, there is a problem in that they can be degraded by an immune response or symptoms of the immune response may occur. In addition, there is also a problem in that a dosage has to be adjusted to avoid these immune diseases.

[0264] Albumin accounts for the majority of serum proteins. For this reason, albumin is a protein which is very stable and exhibits almost no immunogenicity. As a foreign protein Uox is conjugated with albumin, an effect of reducing the immunogenicity of Uox may be expected.

[0265] According to Example 15, it was possible to confirm that Uox-HSA is actually decreased in immunogenicity compared to a wild-type protein. Further, in consideration that Uox-HSA uses HSA, when being applied to the human body, it can be expected to show a more improved effect.

[0266] 5.4. Di-HSA-Uox has Improved Efficacy Compared with Mono-HSA-Uox

[0267] Di-HSA-Uox of Uox-HSAs according to the present application has improved efficacy compared with mono-HSA-Uox. Since the present application has a half-life prolonging effect and an immunogenicity improving effect by the albumin conjugation, it was expected that as the number of albumins increases, the half-life and immunogenicity would be further improved. In addition, considering preparation difficulty and steric hindrance, it was thought that it would be most appropriate to conjugate two albumins. As a result of verifying this, as confirmed in Examples 14 and 15, it was confirmed that a conjugate to which two albumins are conjugated has more improved physical properties. The di-HSA-Uox according to the present application is a novel material which has not been previously revealed, and has excellent efficacy as described above.

[0268] Hereinafter, the present application will be described in further detail with reference to examples. However, these examples are provided to exemplify the present application, and the scope of the present application is not limited to these examples.

[0269] Hereinafter, experimental materials used in the present application are as follows: p-azido-L-phenylalanine (AzF) was purchased from Chem-Impex International (Wood Dale, Ill.), human serum-derived albumin was purchased from Albumedix (Nottingham, UK), a Vivaspin centrifuge concentrator was purchased from Sartorius Corporation (Bohemia, N.Y.), DBCO-PEG.sub.3-FITC was purchased from Conju-Probe, LLC (San Diego, Calif.), DBCO-PEG.sub.4-MAL was purchased from Click-Chemistry Tools (Scottsdale, Ariz.), and a PD-10 desalting column, a Hitrap phenyl FF hydrophobic interaction column, a HiTrap Q HP cation exchange column, a HiTrap SP HP anion exchange column, a Superdex 200 10/300 GL size exclusion column and AKTA pure 25 L were purchased from GE Health Care (Piscataway, N.J.). As all other chemical reagents, products from Sigma-Aldrich Corporation (St. Louis, Mo., USA) were used.

Example 1. Construction of Expression Vector from which Six his were Removed for Producing Urate Oxidase

[0270] An expression cell line (developed in Prior Patent No. KR 10-1637010) for constructing conventional Uox is inappropriate as a commercial strain because of immunogenicity and the possibility of having an unpredictable side effect in future non-clinical or clinical trials due to six histidines (His) attached to the C-terminus. Accordingly, a commercial strain from which six His are removed was constructed.

[0271] As a vector into which a non-natural amino acid can be inserted, a pEVOL-pAzF plasmid (Plasmid ID: 31186) including an AzF-specific engineered pair consisting of a tyrosyl-tRNA synthetase originating from Methanococcus jannaschii and amber suppressor tRNA was purchased from Addgene (Cambridge, Mass.) and used without additional modification.

[0272] To construct an expression vector from which six His are removed from the C-terminus of the conventional pQE80-Uox plasmid, the following primers were used:

TABLE-US-00002 F: (SEQ ID NO: 6) 5-CTCTCTGAAGAGCAAGCTGTAAGCTTAATTAGCTGAGC-3 R: (SEQ ID NO: 7) 5-GCTCAGCTAATTAAGCTTACAGCTTGCTCTTCAGAGAG-3

[0273] To substitute tryptophansat positions 160 and 174 of Uox with an amber codon (UAG), site-directed mutagenic PCR was performed using the pQE80-Uox as a template. To introduce the amber codon at position 160, 5-CAATTCACAGTTTTAGGGGTTTCTGAG-3 and 5-CTCAGAAACCCCTAAAACTGTGAATTG-3 primers were used, and to introduce the amber codon at position 174, 5-CACTGAAGGAGACTTAGGATAGAATCCTG-3 and 5-CAGGATTCTATCCTAAGTCTCCTTCAGTG-3 primers were used.

[0274] A result of sequencing for the used strain is shown in FIG. 1.

[0275] As confirmed from FIG. 1, it was confirmed that a His-tag was removed from the strain.

Example 2. Fed-Batch Culture Process of UoX Producing Strain

[0276] A fed-batch process that provides a carbon source and a nitrogen source for mass production of a non-natural amino acid-introduced Uox was performed. The strain was E. Coli C321.ΔA.exp[(pEVOL-AzF)(pQE80-Uox.W160,174amb)]. The E. Coli C321.ΔA.exp[(pEVOL-AzF)(pQE80-Uox.W160,174amb)] was prepared by simultaneously transforming E. coli C321.ΔA.exp(Addgene, ID: 49018) with pEVOL-pAzF and the pQE80-UoxW160.174amb of Example 1, and is hereinafter referred to as E. Coli C321.ΔA.exp[(pEVOL-AzO(PQE80-Uox.W160,174amb)].

[0277] The bioreactor was a 75 L bioreactor (Sartorius Corporation, Bohemia, N.Y.). Seed culture was accomplished twice, and started with adjustment of a medium volume of the main culture to 30 L. The culture was performed under conditions of 30° C., pH 7.0, 600 rpm, 1 vvm and 100% DO, and during culture, oxygen was continuously provided. For primary seed culture, a medium (12 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.3 g/L KH.sub.2PO.sub.4, 12.53 g/L K.sub.2HPO.sub.4, 100 μg/mL ampicillin, 35 μg/mL chloramphenicol) was prepared and inoculated (0.75%), and culture was carried out at 37° C. and 200 rpm for 15 hours. For secondary seed culture, 5% of the primary seed culture liquid was inoculated, and then culture was carried out at 30° C., 500 rpm, 1 vvm and 100% DO to an O.D. of 5 or more. For the main culture, a medium (12 g/L tryptone, 12 g/L yeast extract, 3.2 g/L KH.sub.2PO.sub.4, 17.4 g/L K.sub.2HPO.sub.4, 100 μg/mL ampicillin, 35 μg/mL chloramphenicol and 0.1 g/L thiamine) was prepared and inoculated with 13.3% of the secondary seed culture liquid, and culture was carried out at 30° C., pH 7.0, 600 rpm, 1 vvm and DO 100%, and when O.D. reached 140 or more, 2 mM AzF, 1 mM IPTG and 0.2% arabinose were added to induce protein expression. In addition, cell growth was induced while an additional carbon source medium (600 g/L glucose, 1.2 g/L MgSO.sub.4) and an additional nitrogen source medium (240 g/L yeast extract, 1.5 g/L ammonium sulfate) were provided at a constant rate at the time point of the concentration of a carbon source decreasing. After the culture, a pellet was obtained by centrifugation at 4° C. and 6,000 rpm for 10 minutes, and stored at −80° C. for a subsequent experiment.

[0278] The result is shown in FIG. 2.

[0279] As confirmed in FIG. 2, when E. coli cells proliferate to a certain level, growth inhibition occurs due to an increasing pH or depletion of a carbon source, which is a nutrient in a medium, and if it is left unattended, cell disruption is caused. To solve this, the cells were cultured using fed-batch culture providing an additional medium at a constant rate. An initial main culture medium was adjusted to 30 L for culture, and when O.D. reached 140 or more, for AzF and overexpression induction, IPTG and arabinose were added. After ten hours of culture, which is the time when almost all of a carbon source, glucose, is consumed, fed-batch culture was carried out while the additional medium was provided at a constant rate. As a result, after 46 hours, O.D. reached 195.9, and the final yield was 240.6 g/L.

Example 3. Separation and Purification Process for AzF-Introduced Uox-1

[0280] The present application introduces two types of processes for separating and purifying AzF-introduced Uox. A first process is described in detail in Example 3, and the other process is described in detail in Example 8.

[0281] To separate and purify Uox from the collected pellet, three-step chromatography was performed. The pellet was suspended in a suspension buffer (20 mM Tris-HCl pH 8.5, 1 M (NH.sub.4).sub.2SO.sub.4, and 1 mg/mL of lysozyme was added to be dissolved for 30 minutes. A supernatant was obtained by centrifugation at 10,000 rpm for 20 minutes, followed by purification through chromatography. In the first step, a 1 M (NH.sub.4).sub.2SO.sub.4-containing solvent was equilibrated using a hydrophobic column (Hitrap phenyl FF) by hydrophobic interaction chromatography, a sample was injected thereinto, followed by elution with a (NH.sub.4).sub.2SO.sub.4-free solvent. After concentration, the buffer was exchanged with 20 mM Tris-HCl pH 8.5, and then two-step anion exchange chromatography was performed. Elution was performed by an NaCl gradient method using an anion change column (Hitrap Q HP). Subsequently, to increase purity, three-step size exclusion chromatography was performed, separation was performed using a size exclusion column (Superdex 200 10/300 GL), and then analysis was performed using SDS-PAGE and SEC-HPLC columns (Shodex LW803, 8.0×300 mm, 3 μm) to confirm purity. For a comparative experiment, culture and separation were performed by the same method as described above, except an AzF injection method using E. Coli Top10 (pQE80-Uox-WT), to produce Uox-WT.

[0282] The results are shown in FIGS. 3 to 6.

[0283] As confirmed in FIGS. 3 to 5, the total yield of Uox was 1 to 10 mg/g.

[0284] As confirmed in FIG. 6, as analyzed by SEC-HPLC, Uox with 95% or more purity was separated.

[0285] To detect an endotoxin and impurities in the Uox separated and purified with high purity, the presence of a bacterial toxin was determined by an Endotoxin Kinetics method, and for host-derived protein analysis, the impurities were confirmed and quantified using a host cell protein kit.

[0286] As a result, the endotoxin was detected at 5 EU/mg or less, and HCP was detected at 10 ppm or less in the Uox separated and purified with high purity.

Example 4. Confirmation of Whether AzF of UoX was Introduced Using Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC

[0287] Each of Uox-WT and Uox-AzF, each of which was adjusted to a concentration of 10 μM, and 80 μM DBCO-PEG.sub.3-FITC were reacted for 2 hours, and then unreacted DBCO-PEG.sub.3-FITC was removed using a PD-10 column, followed by loading on an SDS-PAGE gel. The reaction with DBCO-PEG.sub.3-FITC was analyzed through SDS-PAGE, and visualized using a Blue/white transilluminator (Bioneer, Daejoen). A triazole was formed by bonding of an azide group and DBCO, and AzF introduction to Uox was verified using the principle that fluorescein isothiocyanate (FITC) is excited in a blue light (470 nm) range to exhibit green.

[0288] The result is shown in FIG. 7.

[0289] As confirmed in FIG. 7, Uox-WT did not exhibit fluorescence, and Uox-AzF exhibited strong fluorescence. This indicates that AzF is introduced at specific positions (W160 and W174) of Uox.

Example 5. Preparation and Separation Processes of Conjugate of HSA and Uox

[0290] As a carrier, human serum albumin (HSA) was used, and only a buffer (PBS pH 7.4) was exchanged without a separate separation process. Cysteine at position 34 of HSA was connected with a bifunctional linker DBCO-PEG.sub.4-MAL through Michael addition, thereby preparing HSA-PEG.sub.4-DBCO. A reaction molar ratio of the albumin and the bifunctional linker DBCO-PEG.sub.4-MAL was 1:1, 1:2, 1:4 or 1:8, and these materials were reacted at room temperature for 2 hours. After the reaction, for removal of a remaining linker and exchange of the buffer, the buffer was exchanged with 20 mM sodium phosphate pH 6.0 using a PD-10 column.

[0291] As a result of confirming the conjugation yield of HSA-PEG.sub.4-DBCO, in a 1:4 reaction, a yield of 93.2% was able to be obtained, and in a 1:8 reaction, a yield of 103.9% was able to be obtained. Considering the high price of the bifunctional linker DBCO-PEG.sub.4-MAL, a 1:4 molar ratio of the albumin and the bifunctional linker was used for an experiment.

[0292] To connect HSA site-specifically to Uox-AzF, a conjugate of Uox and HSA was prepared using strain-promoted azide-alkyne cycloaddition (SPAAC). A click-chemistry reaction for forming a stable triazole by bonding an azide group and DBCO under a Cu-free condition was used. The azide group-introduced Uox-AzF was reacted with HSA-PEG.sub.4-DBCO in a molar ratio of 1:1, 1:1.5, 1:2 or 1:3 at room temperature and then a conjugation yield was calculated by three-step chromatography. In the first step, to remove unreacted albumin, a cation exchange column (Hitrap SP HP) was used. After equilibration with 20 mM sodium phosphate pH 6.0, a sample was injected, and eluted with a 0 to 100% NaCl gradient. Fractions were collected and concentrated, and then the buffer was replaced with 20 mM Bis-Tris pH 6.5. In the second step, to remove remaining Uox, an anion exchange column (Hitrap Q HP) was used. After equilibration with 20 mM Bis-Tris pH 6.5, a sample was injected, and then eluted with a 0 to 100% NaCl gradient. Fractions were collected and concentrated, and then in the third step, mono-HSA-Uox and di-HSA-Uox were purely separated using a size exclusion column (Superdex 200 10/300 GL), and purity was confirmed by SEC-HPLC and SDS-PAGE.

[0293] The results are shown in Table 1 and FIGS. 8 to 12.

TABLE-US-00003 TABLE 1 Conjugation yield according to molar ratio of HSA-DBCO and Uox HSA-DBCO:Uox Conjugation (molar ratio) yield (%)  1:1 5.3 1.5:1  9.2  2:1 11.0  3:1 9.3

[0294] As confirmed in Table 1, the conjugation yield according to the molar ratio of HSA-DBCO and Uox was highest when reacting in a molar ratio of 2:1.

[0295] As confirmed in FIG. 10, a new band having a size of approximately 101 kDa was able to be detected by SDS-PAGE.

[0296] As confirmed in FIG. 11, as the molar ration increases, the proportion of di-HSA-Uox is higher than that of mono-HSA-Uox.

[0297] As confirmed in FIG. 12, the separation of mono-HSA-Uox and di-HSA-Uox was confirmed by SEC-HPLC.

Example 6. Measurement of Enzyme Activity of UoX-HSA

[0298] To analyze the enzyme activity of Uox-HSA, spectroscopy was used. A decreasing concentration of uric acid over time when 100 μM uric acid was reacted with 60 nM Uox-HSA was analyzed using a Ultrospec 2100 pro UV/Visible spectrophotometer (Biochrom, Cambridge, UK) at 293 nm, which is the maximum absorbance wavelength of uric acid. The unit (U/mL) of enzyme activity was obtained by multiplying an absorbance change (Δ.sub.ABS293nm/min) by the total reaction volume, dividing by the molar absorption coefficient of uric acid (12.3 mM.sup.−1 cm.sup.−1), and then dividing by the volume of an enzyme. The unit (U/mL) of enzyme activity was defined as the amount of an enzyme capable of converting 1 μM of uric acid into allantoin per minute at room temperature. An activity per unit mass (specific activity, U/mg) of the enzyme was obtained by dividing by an amount of the enzyme used in the reaction.

[0299] The result is shown in Table 2.

TABLE-US-00004 TABLE 2 Uox-HSA enzyme activity M.W. (kDA) U/mL U/mg Uox-WT 136 0.38 ± 0.002 46.14 ± 0.194 Mono-HSA-Uox 203 0.35 ± 0.008 28.61 ± 0.663 Di-HSA-Uox 270 0.37 ± 0.043 22.63 ± 2.65

[0300] As confirmed in Table 2, the enzyme activities of Uox-WT, mono-HSA-Uox and di-HSA-Uox were measured to be 0.38 U/mL, 0.35 U/mL and 0.37 U/mL, respectively. This confirmed that the albumin conjugation does not affect the enzyme activity of Uox, and the Uox-HSA conjugate maintains activity even with one or two albumin bindings. A difference in the enzyme activity (U/mg) per unit mass is caused by an increase in molecular weight of the albumin-conjugated Uox-HSA conjugate.

[0301] It was confirmed that di-HSA-Uox in which two albumins are conjugated has greater enzyme activity than mono-HSA-Uox in which one albumin is conjugated.

Example 7. Confirmation of Uric Acid Level Reducing Effect of UoX-HSA Using Gout-Induced Animal Model

[0302] An experiment for the uric acid level reducing effect of Uox-HSA using a gout-induced animal model was carried out with mice and rats.

[0303] As experimental animals, 8 to 10-week-old male C57BL mice (20 to 25 g, Samtako) bred in an environment maintained at a humidity of 50±5% and a temperature of 24 to 26° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water. The induction of hyperuricemia was performed by dissolving hypoxanthine, which is a uric acid precursor, in 1 mL of a 3% starch solution and then orally administering it at a concentration of 500 mg/kg, and 10 minutes after administration, dissolving potassium oxonate, which is an Uox inhibitor, in 1 mL of 0.5% sodium carboxymethylcellulose and then intraperitoneally injecting it at a concentration of 250 mg/kg. Allopurinol used as a control and a uric acid-forming inhibitor was orally administered at 50 mg/kg 10 minutes after hypoxanthine and potassium oxonate were intraperitoneally injected. Uox-WT (Rasburicase) and Uox-HSA were intravenously administered at 3.4 mg/kg and 5.0 mg/kg, respectively, to evaluate whether a uric acid level in blood decreases over time. An experimental group was administered Uox-HSA according to an administration route and an administration dose as shown in Table 3 below, and blood was sampled from a caudal vein over time. The uric acid level in blood was quantified by an FRAP method. 30 uL of blood and 300 uL of a working solution (10 mM TPTZ, 20 mM FeCl.sub.3, 300 mM Acetate buffer) were mixed, and absorbance was measured at 593 nm.

[0304] As experimental animals, 7-week-old male Sprague-Dawley rats (250 to 280 g, Samtako) bred under an environment maintained at a humidity of 50±5% and a temperature of 24 to 26° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water. The induction of hyperuricemia was performed by dissolving hypoxanthine, which is a uric acid precursor, in 1 mL of 3% starch solution, and one hour after administration, dissolving potassium oxonate, which is an Uox inhibitor, in 1 mL of 0.5% sodium carboxymethylcellulose and then orally administering it at a concentration of 250 mg/kg for 7 days. Allopurinol used as a control and a uric acid-forming inhibitor was administered for 7 days with the hyperuricemia induction to inhibit the formation of uric acid. Uox-WT (Rasburicase) and Uox-HSA were intravenously administered at 3.4 mg/kg and 5.0 mg/kg, respectively, to evaluate whether a uric acid level in blood decreases over time. An experimental group was administered with Uox-HSA according to an administration route and an administration dose as shown in Table 4 below, and blood was collected from a caudal vein over time, centrifuged at 3,000 rpm for 10 minutes to separate plasma, followed by quantification using a uric acid assay kit (Abnova, Taipei, Taiwan).

TABLE-US-00005 TABLE 3 Group classification and dose of mouse animal models Dose No. of Group Drug Route (mg/kg) animals 1 Control — — 3 2 Hyperurecemia + PBS I.V. — 3 3 Hyperurecemia + Uox-WT I.V. 3.4 3 4 Hyperurecemia + Uox-HSA I.V. 5.0 3 5 Hyperurecemia + Allopurinol Oral 50 3

TABLE-US-00006 TABLE 4 Group classification and dose of rat animal models Dose No. of Group Drug Route (mg/kg) animals 1 Control — — 3 2 Hyperurecemia I.V. — 6 3 Hyperurecemia + Uox-WT I.V. 3.4 6 4 Hyperurecemia + Uox-HSA I.V. 5.0 6 5 Hyperurecemia + Allopurinol Oral 50 4

[0305] The results are shown in FIGS. 13 and 14.

[0306] As confirmed in FIG. 13, the uric acid level in blood began to rapidly decrease 30 minutes to 2 hours after administration of Uox-HSA in a mouse animal model and was maintained for 12 hours. Uox-WT had a decreased uric acid level in blood until 5 hours, but a uric acid level began to be increase after 5 hours.

[0307] As confirmed in FIG. 14, the uric acid in blood began to decrease from 30 minutes and then was maintained until 16 hours after administration of Uox-HSA in rat animal models. This means that the activity of Uox-HSA tends to be maintained and its persistency also increases, compared with Uox-WT (Rasburicase), which is a conventional drug. On the other hand, after administration of the uric acid-forming inhibitor, allopurinol, used as a control, a uric acid level began to decrease until 30 minutes, and began to increase 1 hour after administration.

[0308] In addition, as confirmed in FIGS. 15 to 17, as a result of kidney autopsy, a severe renal damage was observed by allopurinol administration, compared with the other groups.

Example 8: Separation and Purification Process for AzF-Introduced UoX-2

[0309] To separate and purify Uox from the pellet harvested from the process described up to Example 2, 3-step chromatography was performed. The pellet was suspended in a suspension buffer (20 mM Tris-HCl pH 9.0), 1 mg/mL of lysozyme was added, followed by dissolution for 30 minutes. Only a supernatant was obtained by centrifugation at 10,000 rpm for 20 minutes, and then purified by 3-step chromatography. In the first step, elution was performed with a 0.5M NaCl gradient method using an anion exchange column (Hitrap DEAE FF, GE Healthcare). In the second step, following equilibration with a 0.5 M (NH.sub.4).sub.2SO.sub.4-containing solvent through hydrophobic interaction chromatography using a hydrophobic column (Hitrap phenyl(High sub), GE Healthcare), a sample was injected, and then eluted with a 20 mM Tris-HCl pH 9.0 solvent. Subsequently, to increase purity, 3-step size exclusion chromatography was carried out, separation was performed using a size exclusion column (Superdex 200 10/300 GL), and then the purity was confirmed by analysis with SDS-PAGE and SEC-HPLC (AdvanceBio SEC 300A, 7.8×300 mm, 2.7 μm). For a comparative experiment, E. Coli Top10 (pQE80-Uox-WT) was cultured and separated by the same method except a process of injecting AzF to produce Uox-WT.

[0310] The results are shown in FIGS. 18 to 21.

[0311] As confirmed in FIGS. 18 to 20, the total yield of Uox was 1 to 5 mg/g.

[0312] As confirmed in FIG. 21, as a result of SEC-HPLC, Uox with a purity of 95% was separated.

Example 9: Analysis of Molecular Weight of Di-HSA-UoX Through SEC-LC/MS

[0313] The results measured through an experiment of measuring the molecular weight of Di-HSA-Uox are shown in Table 5 and FIGS. 26 and 27 below.

TABLE-US-00007 TABLE 5 Result of confirmation of molecular weight of Uox0di-HSA conjugate Uox-di-rUSA conjugate (PS-P23-2020-0002) Experimental Theoretical mass mass mass Sample (average) (average) Possible difference File name [Da] [Da] compounds [Da] HM01420 34112.7 34111.1 Urate oxidase 1.6 (2× AzF: without N-terminal methionine) 270578.7 270668.6 Tetramer −89.9 (4× Uox + 2× Linker + 2× rHSA) 66437.3 66437.1 Human Serum 0 . . . 2 Albumin (rHSA)

[0314] In FIGS. 26 and 27, A is a total ion chromatogram (TIC) result, B is a separated mass spectrum of Uox (2×AZF, without N-terminal methionine), C is a separated mass spectrum of Uox-HSA (4× Uox+2× Linker+2× rHSA), and D is a separated mass spectrum of HSA.

[0315] As confirmed in FIGS. 26 and 27, the molecular weight of Uox-HSA was measured to be 270578.7 Da, similar to the theoretical value of di-HSA-Uox (270668.6 Da).

Example 10: Analysis of Isoelectric Point (pI) Characteristic of UoX-HSA Using cIEF

[0316] cIEF analysis was carried out for pI analysis of separated and purified Uox-HSA. As analysis equipment, ProteinSimple Maurice was used. The pI of Uox-WT, mono-HSA-Uox and di-HSA-Uox was assessed using a pH 3 to 10 separation range (pI marker). The analysis results are shown in FIGS. 28 and 29.

[0317] As confirmed in FIGS. 28 and 29, Di-HSA-Uox was analyzed as a main peak at pI 6.72, and Uox-WT was detected with various peaks in a PI range from 7.04 to 9.09. Mono-HSA-Uox was detected with various peaks in a pI range from 6.58 to 7.51.

Example 11: Measurement of In Vitro Enzyme Activity of UoX-HSA

[0318] Unless described otherwise, Uox-HSA described in the following example refers to di-HSA-Uox. To analyze the enzyme activity of Uox-HSA, spectroscopy was used. A decreasing concentration of uric acid over time when 100 μM uric acid was reacted with 60 nM Uox-HSA was analyzed using a Hidex Microplate reader (Hidex, Finland) at 293 nm, which is the maximum absorbance wavelength of uric acid. The unit (U/mL) of enzyme activity was obtained by multiplying an absorbance change (Δ.sub.ABS293nm/min) by the total reaction volume, dividing by the molar absorption coefficient of uric acid (12.3 mM.sup.−1 cm.sup.−1), and then dividing by the volume of an enzyme. The unit (U/mL) of enzyme activity was defined as the amount of an enzyme capable of converting 1 μM of uric acid into allantoin per minute at room temperature. An activity per unit mass (specific activity, U/mg) of the enzyme was obtained by dividing by an amount of the enzyme used in the reaction.

[0319] The result is shown in Table 6 and FIG. 22.

TABLE-US-00008 TABLE 6 UoX-HSA enzyme activity U/mL U/mg Uox-HSA 0.13 8.1 FASTURTEC 0.14 17.6 KRYSTEXXA 0.06 7.9

[0320] As confirmed in Table 6, the enzyme activities per unit dose of Uox-HSA, FASTURTEC and KYRSTEXXA were measured to be 0.13 U/mL, 0.14 U/mL and 0.06 U/mL, respectively. Particularly, the activities of Uox-HSA and KRYSTEXXA showed a 2-fold or more difference. The difference between an enzyme activity per unit mass (U/mg) and an enzyme activity per unit dose of the Uox-HSA conjugate is due to an increase in molecular weight of the albumin-conjugated Uox-HSA conjugate. It was possible to confirm that, compared with KRYSTEXXA, the same amount of Uox-HSA has significantly improved activity.

Example 12: Uric Acid Level Reducing Effect of UoX-HSA Using Animal Model in which Gout was Repeatedly Induced

[0321] As experimental animals, 6-week-old male Sparague-Dawley rats (190 to 210 g, Samtako) bred under an environment maintained at a humidity of 50±5% and a temperature of 22±3° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water. The repeated induction of hyperuricemia was carried out by dissolving a uric acid precursor, hypoxanthine, in 1 mL of a 3% starch solution and then orally administering it at a concentration of 500 mg/kg, and 10 minutes after administration, dissolving an Uox inhibitor, potassium oxonate, in 1 mL of 0.5% sodium carboxymethylcellulose and then intraperitoneally administering it at a concentration of 250 mg/kg. The repeated induction was performed a total of 4 times: two days before administration of a test material, one day before administration of a test material, on the day of administration and one day after administration. This experiment was performed to confirm the persistency of the test drug by reinducing hyperuricemia one day after administration of the test drug unlike Example 7.

[0322] As an administration dose of the test drug, Uox-HSA was intravenously administered at 2.0 mg/kg to evaluate whether a uric acid level in blood was reduced over time. As a control drug, FASTURTEC (Sanofi-Aventis) and the only persistent gout therapeutic, KRYSTEXXA (Horizon Pharma), were intravenously administered at 2.0 mg/kg to evaluate whether a uric acid level in blood was reduced over time (Table 7). The uric acid level in blood was quantified using a uric acid assay kit (Abnova, Taipei, Taiwan) after plasma was separated by centrifugation of the blood collected from a caudal vein.

TABLE-US-00009 TABLE 7 Conditions for administration of test drug Dose Dose No. of Group Drug Route (mg/kg) (nmol/kg) animals 1 Hyperurecemia I.V. — — 4 2 Hyperurecemia + I.V. 2.0 7.4 4 Uox-HSA 3 Hyperurecemia + I.V. 2.0 14.6 4 FASTURTEC 4 Hyperurecemia + I.V. 2.0 14.6 4 KRYSTEXXA

[0323] As confirmed in FIG. 23, in all of the Uox-HSA, FASTURTEC and KRYSTEXXA-administered groups of the repeated induction animal models, the uric acid level in blood began to decrease after 30 minutes and was maintained until 12 hours. Afterward, as a result of reinduction at 24 hours, FASTURTEC did not show an effect of reducing uric acid, whereas Uox-HSA and KRYSTEXXA showed an effect of reducing uric acid to a normal level or less. In addition, Uox-HSA showed an equal or higher effect of reducing uric acid to that of KRYSTEXXA with only half the amount. From this, it was possible to confirm that Uox-HSA is a drug having high persistency and excellent activity compared with the conventional Uox drugs.

Example 13: Evaluation of Pharmacodynamic Characteristic in SD-Rt Model

[0324] As experimental animals, 5-week-old male Sparague-Dawley rats (190 to 210 g, Orient Bio) bred in an environment maintained at a humidity of 50±5% and a temperature of 22±3° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water.

[0325] As an administration dose of the test drug, Uox-HSA was intravenously administered at 4.0 mg/kg to evaluate whether a uric acid level in blood was reduced over time. As a control drug, FASTURTEC (Sanofi-Aventis) and the only persistent gout therapeutic, KRYSTEXXA (Horizon Pharma), were intravenously administered at 2.0 mg/kg to evaluate whether a uric acid level in blood was reduced over time. Blood was collected form a caudal vein, centrifuged to separate plasma, followed by analysis by a Uricase activity assay method.

TABLE-US-00010 TABLE 8 Conditions for administration of test drug Dose No. of Group Drug Route Dose (mg/kg) (nmol/kg) animals 1 UoX-HSA I.V. 4.0 14.6 5 2 FASTURTEC I.V. 2.0 14.6 5 3 KRYSTEXXA I.V. 2.0 14.6 5

[0326] As confirmed in FIG. 24 and Table 9 below, FASTURTEC showed a short half-life of 2.1 hours, whereas Uox-HSA showed a half-life of 27 hours, which was approximately 8.7-fold higher than that of FASTURTEC. The persistent gout therapeutic, KRYSTEXXA, showed a half-life of 35.2 hours. The size of KRYSTEXXA is approximately 500 kDa or more so that it is not filtered in the kidney and thus keeps maintaining its activity. Uox-HSA includes two human-derived albumins bound thereto, and the half-life is expected to be more prolonged due to recycle of FcRn recycling by albumin in clinical trials.

TABLE-US-00011 TABLE 9 Result of measurement of half-life of test drug t½ (h) AUC (mU/mL × h) Uox-HSA 27.0 3,739.5 From 0.5 to 168 h FASTURTEC 3.1 788.2 From 0.5 to 24 h KRYSTEXXA 35.2 5,456.0 From 0.5 to 168 h

Example 14: Comparison of Pharmacodynamic Characteristics of Di-HSA-UoX and Mono-HSA-UoX

[0327] As experimental animals, 7-week-old male Sparague-Dawley rats (250 to −280 g, Samtako) bred in an environment maintained at a humidity of 50±5% and a temperature of 24 to 26° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water. Mono-HSA-Uox and di-HSA-Uox were intravenously administered at 5.0 mg/kg and 6.6 mg/kg, respectively, and then blood samples were collected 0, 0.5, 1, 2, 4, 8, 24, 48 and 96 hours after administration to analyze enzyme activity.

[0328] The result of comparing the characteristic of di-HSA-Uox measured in Example 13 and the characteristic of mono-HSA-Uox measured in this example are shown in Table 10 and FIG. 30.

TABLE-US-00012 TABLE 10 Comparison of pharmcodynamic characteristics of di-HAS-Uox and mono-HSA-Uox t½ (h) AUC (mU/mL × h) Di-HSA-Uox 27.0 3739.5 Mono-HSA-Uox 21.8 1243.7

[0329] As confirmed in Table 10, it was able to be confirmed that di-HSA-Uox has a half-life approximately 24% longer than mono-HSA-Uox. This shows that, as expected in the section 5.4., when an albumin binding number increases, an effect of improving half-life is exhibited.

Example 15: Immunogenic Analysis in CD-1 Mouse by 4-Week Repetitive Administration

[0330] As experimental animals, 5-week-old female Hsd:ICR (CD-1) mice (20 to 25 g, Orient Bio) bred in an environment maintained at a humidity of 50±5% and a temperature of 22±3° C. were used for a subsequent experiment after acclimating in a laboratory environment for 1 week with sufficient supply of feed and water.

[0331] As an administration dose of the test drug, Uox-HSA was injected into a thigh muscle at 4.0 mg/kg, and administered once a week for five weeks. As a control drug, FASTURTEC was injected into a thigh muscle at 2.0 mg/kg, and administered once a day for 7 days (Table 11). Blood collected every week for 6 weeks was centrifuged to separate plasma, followed by analysis using an anti-uricase antibody ELISA method.

TABLE-US-00013 TABLE 11 Conditions for administration of test drug for immunogenicity test Group Drug Route Dose (mg/kg) No. of animals 1 Uox-HSA I.M. 4.0 4 2 FASTURTEC I.M. 2.0 4

[0332] As confirmed in FIG. 25 and Table 11, it was possible to confirm that the Uox-HSA-administered group shows slightly less formation of anti-uricase antibodies compared with the FASTURTEC-administered group. It was possible to confirm that the immunogenicity of the foreign material such as Uox is reduced by the conjugation of HSA. Further, since the HSA is human albumin, when being actually administered to a human, it is expected that a more improved immunogenic reducing effect will be exhibited.

[0333] Regarding the above description, it should be understood by those of ordinary skill in the art that the above descriptions of the present application are exemplary, and the exemplary embodiments disclosed herein can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be interpreted that the exemplary embodiments described above are exemplary in all aspects, and are not limitative. The scope of the present application is defined by the appended claims and encompasses all modifications and alterations derived from meanings, the scope and equivalents of the appended claims.