A method for modifying uricase with polyethylene glycol

Abstract

The present invention belongs to the field of biotechnology and pharmaceuticals, and specifically relates to a method for polyethylene glycol-modified uricase, comprising the following steps: dissolving a uricase solution in a carbonate buffer, adding a PEG modifier and lysine, stirring and reacting at 4 to 30°C for 60 to 240 minutes, and after the reaction is completed, ultrafiltration concentration and molecular sieve purification can be performed to obtain high-purity PEGylated uricase. Lysine is added during the modification process of the present invention, which effectively improves the uniformity of the PEG-modified uricase, reduces the amount of the PEG modifier, and effectively reduces the degradation of PEG. The preparation method of the PEGylated uricase of the present invention is simple, has high raw material utilization, high enzyme activity recovery rate, large processing capacity, and is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and pharmaceuticals, and specifically relates to a method for modifying uricase with polyethylene glycol. Background Art

[0002] Uricase, also known as urate oxidase, can catalyze the oxidation of uric acid into allantoin, carbon dioxide and water. Functional uricase exists in most organisms, but with the evolution of primates, gene mutations in the body lead to the loss of the function of uricase synthesis. Therefore, any increase in purine catabolism or decrease in uric acid excretion in the human body will lead to an increase in uric acid concentration. When the concentration of uric acid in the blood is greater than 7mg / dl (6mg / dl for women), it will cause hyperuricemia. Further development of hyperuricemia will lead to the accumulation of uric acid in the joints of the body, and then form gout. In the treatment of hyperuricemia and gout, uricase recombined by different methods can quickly and effectively reduce uric acid in the patient's blood, and has a good therapeutic effect on patients with severe uricemia, refractory gout and tumor lysis syndrome.

[0003] However, due to the large immunogenicity of uricase and the short half-life in vivo, it can be chemically modified in order to reduce immunogenicity and prolong half-life. Commonly used protein modifiers are polyethylene glycol (PEG), and PEG modification is to covalently link PEG to protein drugs in vitro to achieve the effect of reducing immunogenicity and increasing half-life. Polyethylene glycol is a non-toxic, non-immunogenic water-soluble macromolecular substance that can modify proteins by covalent bond binding. After PEG modification, the immunogenicity of protein drugs is reduced, and the solubility and stability of proteins can be improved, and the clearance of kidneys can be reduced to prolong the half-life of drugs. At present, FDA has approved the listing of multiple PEG-modified recombinant protein drugs. For example, a PEG-modified PEG-modified recombinant pig and baboon uricase disclosed in CN1322141A.

[0004] For PEG saturated modified uricase, in order to achieve saturated modification, an excessive amount of PEG needs to be added. This results in a large amount of PEG used. At the same time, due to the problem of reaction conditions, PEG is easily degraded seriously. In the document International Journal of Pharmaceutics 387 (2010) 215-222, the concentration of uricase is 1 mg / ml, and the molar ratio of PEG to uricase is 370:1. According to the results of its electrophoresis experiment, under this modification condition, some uricases are not modified, and the modification uniformity is low, and the modification degree is also low. In patent CN 102634492A, the protein concentration of uricase is 4 mg / ml, and the molar ratio of PEG to uricase is 40:1-160:1. According to its experimental results, within the test range, as the amount of PEG used increases, the modification degree increases, and the modification uniformity also increases. However, when the amount of PEG added continues to increase, although the modification degree increases, it cannot reach saturated modification. It is not that as the amount of PEG used increases, the uniformity of PEG modification can be further effectively improved. Therefore, it is necessary to provide a method for saturated modification of uricase with PEG with a small amount of PEG and high modification uniformity. Summary of the invention

[0005] In order to solve the defects of poor uniformity and large amount of PEG used in the process of PEG saturated modification of uricase in the prior art, the present invention provides a method for modifying uricase with polyethylene glycol.

[0006] The technical solution of the present invention is as follows:

[0007] A method for modifying uricase with polyethylene glycol comprises the following steps: dissolving a uricase solution in a carbonate buffer, adding a PEG modifier and lysine, stirring and reacting at 4 to 30° C. for 60 to 240 minutes, and after the reaction is completed, ultrafiltration concentration and molecular sieve purification are performed to obtain high-purity PEGylated uricase.

[0008] Preferably, the concentration of the uricase solution is 3 to 8 mg / ml, more preferably 4 to 6 mg / ml.

[0009] Preferably, the carbonate buffer is a sodium carbonate-sodium bicarbonate buffer solution, wherein the salt ion concentration is 0.04-0.5 mol / L, more preferably 0.1-0.2 mol / L.

[0010] Preferably, the carbonate buffer has a pH of 8.0 to 10.5, more preferably 8.5 to 9.0.

[0011] Preferably, the PEG modifier is a monomethoxy polyethylene glycol with a succinimide group at the end, which reacts with uricase to be linked through an amide bond, preferably monomethoxy polyethylene glycol mPEG-SPA of succinimidyl propionate, monomethoxy polyethylene glycol mPEG-SS of succinimidyl succinate, monomethoxy polyethylene glycol mPEG-SSA of succinimidyl succinamide or monomethoxy polyethylene glycol mPEG-NHS of N-hydroxy-succinimide; monomethoxy polyethylene glycol mPEG-SPA of succinimidyl propionate is further preferred.

[0012] Preferably, the molecular weight of the PEG modifier is 5K to 20K, and more preferably the molecular weight is 5K.

[0013] Preferably, the molar ratio of the PEG modifier to uricase is 250-500:1, more preferably 320-410:1.

[0014] Preferably, the molar ratio of lysine in the reaction system to the lysine residue in the uricase protein monomer molecule is 0.5 to 4:1, more preferably 1:1.

[0015] Preferably, the reaction temperature is 4-8°C.

[0016] The starting uricase of the present invention is not particularly limited, and the uricase gene can be derived, cloned or produced from any source, including, for example, from a microorganism or by biotechnology or any combination thereof. For example, uricase can be cloned from a microorganism, including but not limited to Aspergillus flavus, Candida albicans, Arthrobacter protozoa, Streptomyces and Bacillus bacteria, fungi, yeast, Schizosaccharomyces, Aspergillus, Aspergillus and Neurospora; mammals, including pigs (Sus scrofu), squirrel monkeys (Samiri sciureus), baboons (Papio), macaques (Mcaca mulatta), plants including chickpeas (Cicer arietinum), beans (Phaseolus vulgaris, potatoes (Solnum tuberosum) and peas (Pisum sativum); insects including fruit flies (Drosophila).

[0017] For example, the uricase described in the present invention can be prepared by the uricase prepared in CN1561390A, the method of Bioconjugate Chem. 1999, 10, 638-646 or other methods in the prior art.

[0018] Preferably, after ultrafiltration and concentration, the concentration of PEG-modified uricase is 8-12 mg / ml.

[0019] Preferably, the molecular sieve purification includes column packing, equilibration, sample loading and elution processes, and the target product is collected according to the changes in the absorption values ​​of the ultraviolet and differential detectors.

[0020] Further preferably, the molecular sieve filler is Sephacryl S300 filler.

[0021] Further preferably, the mobile phase buffer used in the molecular sieve equilibration and elution process is a phosphate buffer solution, and the phosphate buffer is sodium dihydrogen phosphate or potassium salt / disodium hydrogen phosphate or potassium salt. The concentration is 0.1-0.3 mol / L and the pH is 8.0-10.0.

[0022] The present invention adds lysine during the modification process, effectively improves the uniformity of PEG-modified uricase, reduces the amount of PEG modifier, and effectively reduces the degradation of PEG. The method of the present invention can achieve saturation modification with less PEG modifier, and has good uniformity. After saturation modification of uricase with PEG, high-purity target product can be directly obtained by ultrafiltration concentration and molecular sieve purification. The method of polyethylene glycol-modified uricase of the present invention is simple, has high raw material utilization rate, high enzyme activity recovery rate, large processing capacity, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 1; M: high molecular weight marker; 1: PEG-modified uricase; 2: uricase.

[0024] Figure 2 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 2; M: high molecular weight marker; 1: uricase; 2: PEG-modified uricase.

[0025] Figure 3 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 3; M: high molecular weight marker; 1: PEG-modified uricase; 2: uricase.

[0026] Figure 4 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 4; M: high molecular weight marker; 1: uricase; 2: PEG-modified uricase.

[0027] Figure 5 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 5; M: high molecular weight marker; 1: PEG-modified uricase; 2: uricase.

[0028] Figure 6This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 6; M: high molecular weight marker; 1: uricase; 2: PEG-modified uricase.

[0029] Figure 7 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 7; M: high molecular weight marker; 1: PEG-modified uricase; 2: uricase.

[0030] Figure 8 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Example 8; M: high molecular weight marker; 1: PEG-modified uricase; 2: uricase.

[0031] Fig. 9 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Comparative Example 1; M: high molecular weight marker; 1: uricase; 2: PEG-modified uricase.

[0032] Fig.10 This is the SDS-PAGE electrophoresis diagram of PEG-modified uricase in Comparative Example 2; M: high molecular weight marker; 1: uricase; 2: PEG-modified uricase. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention is described below in conjunction with some specific embodiments. It should be understood that the following examples are only for illustrative purposes and are not intended to limit the present invention. The test methods in the following examples without specifying specific conditions are usually based on conventional conditions or methods in the art.

[0034] The present invention does not limit the source of the raw material uricase, which can be prepared by itself according to the method of the prior art. Other reagents and raw materials can be obtained through commercial channels unless otherwise specified. The modifier mPEG-SPA-5K is a monomethoxy polyethylene glycol with succinimidyl propionate, with a molecular weight of 5K; the modifier mPEG-SPA-20K is a monomethoxy polyethylene glycol with succinimidyl propionate, with a molecular weight of 20K; the modifier mPEG-SS-5K is a monomethoxy polyethylene glycol with succinimidyl succinate, with a molecular weight of 5K; the modifier mPEG-SS-10K is a monomethoxy polyethylene glycol with succinimidyl succinate, with a molecular weight of 10K; the modifier mPEG-SSA-5K is a monomethoxy polyethylene glycol of succinimidyl succinamide, and the modifier mPEG-NHS-5K is a monomethoxy polyethylene glycol of N-hydroxy-succinimide. Pegylated uricase is polyethylene glycol-modified uricase.

[0035] Example 1

[0036] 90 mg uricase (0.66 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (20 ml) at pH 8.5, lysine (11.19 mg, 1:1) was added, and then mPEG-SPA-5K (1.35 g, 410:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of modified uricase activity was 91.4%. The above reaction solution was concentrated by ultrafiltration to a concentration of 9 mg / ml of polyethylene glycol modified uricase for standby use, and 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.15 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase concentrate was loaded onto a molecular sieve chromatography column. After loading, the column was eluted with a 0.15 mol / L sodium dihydrogen phosphate / sodium hydrogen phosphate solution (pH 9.0). The polyethylene glycol modified uricase sample was collected and the enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 95.7%. The sample was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 1 As shown, the SEC-HPLC purity was 99.2%.

[0037] Example 2

[0038] 150 mg uricase (1.09 μmol) was dissolved in 0.04 mol / L sodium carbonate-sodium bicarbonate buffer solution (50 ml) at pH 8.0, lysine (44.76 mg, 1:2.4) was added, and then mPEG-SPA-20K (6.59 g, 302:1) was added, and the reaction was stirred at 10-15°C for 240 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of modified uricase activity was 76.1%. The above reaction solution was concentrated by ultrafiltration to a concentration of 12 mg / ml of polyethylene glycol modified uricase for standby use, and 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.1 mol / L sodium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 10.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.10 mol / L sodium dihydrogen phosphate / potassium hydrogen phosphate solution (pH 10.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 82.7%. The sample was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 2 As shown, the SEC-HPLC purity was 98.7%.

[0039] Example 3

[0040] 60 mg uricase (0.44 μmol) was dissolved in 0.1 mol / L sodium carbonate-sodium bicarbonate buffer solution (10 ml) at pH 10.5, lysine (3.73 mg, 1:0.5) was added, and then mPEG-SS-10K (1.64 g, 373:1) was added, and the reaction was stirred at 20-30°C for 60 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 79.5%. 400 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.3 mol / L potassium dihydrogen phosphate / sodium dihydrogen phosphate solution (pH 8.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.30 mol / L potassium dihydrogen phosphate / sodium dihydrogen phosphate solution (pH 8.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 85.9%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 3 As shown, the SEC-HPLC purity was 99.4%.

[0041] Example 4

[0042] 120 mg uricase (0.88 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (15 ml) at pH 9.0, lysine (14.92 mg, 1:1) was added, and then mPEG-SSA-5K (1.63 g, 370:1) was added, and the reaction was stirred at 4-8°C for 80 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 90.2%. 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.2 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.20 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 91.3%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 4 As shown, the SEC-HPLC purity was 99.1%.

[0043] Example 5

[0044] 100 mg uricase (0.73 μmol) was dissolved in 0.5 mol / L sodium carbonate-sodium bicarbonate buffer solution (10 ml) at pH 9.0, lysine (12.38 mg, 1:1) was added, and then mPEG-NHS-5K (1.36 g, 370:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 92.4%. 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.2 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.20 mol / L sodium dihydrogen phosphate / sodium hydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 92.5%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 5 As shown, the SEC-HPLC purity was 98.7%.

[0045] Example 6

[0046] 80 mg of uricase (0.58 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (10 ml) at pH 9.0, lysine (19.67 mg, 1:2) was added, and then mPEG-SPA-5K (0.94 g, 320:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 90.1%. 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.2 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 10.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto the molecular sieve chromatography column. After loading, it was eluted with 0.20 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 10.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometer. The enzyme activity recovery rate after purification was 88.7%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 5 As shown, the SEC-HPLC purity was 98.8%.

[0047] Example 7

[0048] 80 mg of uricase (0.58 μmol) was dissolved in 0.1 mol / L sodium carbonate-sodium bicarbonate buffer solution (10 ml) at pH 9.0, lysine (59.0 mg, 1:6) was added, and then mPEG-SPA-5K (1.20 g, 410:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 80.8%. 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.3 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.30 mol / L potassium dihydrogen phosphate / dipotassium hydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 79.6%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 7 As shown, the SEC-HPLC purity was 99.8%.

[0049] Example 8

[0050] 90 mg uricase (0.66 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (10 ml) at pH 8.5, lysine (11.19 mg, 1:1) and mPEG-SS-5K (1.22 g, 370:1) were added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of uricase activity after modification was 90.7%. 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.1 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase solution was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.10 mol / L sodium dihydrogen phosphate / sodium hydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 92.4%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Figure 8 As shown, the SEC-HPLC purity was 99.2%.

[0051] Comparative Example 1

[0052] 90 mg uricase (0.66 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (20 ml) at pH 8.5, and then mPEG-SPA-5K (1.35 g, 410:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of modified uricase activity was 74.7%. The above reaction solution was concentrated by ultrafiltration to a concentration of 9 mg / ml of polyethylene glycol modified uricase for standby use, and 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.15 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase concentrate was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.15 mol / L sodium dihydrogen phosphate / sodium dihydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 83.2%. The concentrate was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Fig. 9 As shown, the SEC-HPLC purity was 99.1%.

[0053] Comparative Example 2

[0054] 90 mg uricase (0.66 μmol) was dissolved in 0.2 mol / L sodium carbonate-sodium bicarbonate buffer solution (20 ml) at pH 8.5, cysteine ​​(9.25 mg, 1:1) was added, and then mPEG-SPA-5K (1.35 g, 410:1) was added, and the reaction was stirred at 4-8°C for 120 min. After the reaction was completed, the enzyme activity was determined by uric acid absorption spectrophotometry, and the recovery rate of modified uricase activity was 67.6%. The above reaction solution was concentrated by ultrafiltration to a concentration of 9 mg / ml of polyethylene glycol modified uricase for standby use, and 1600 ml of Sephacryl S300 filler was loaded into the column, and equilibrated with 0.15 mol / L sodium dihydrogen phosphate / disodium hydrogen phosphate solution (pH 9.0) until the column bed volume was constant, and then equilibrated for 2 hours. The polyethylene glycol modified uricase concentrate was loaded onto a molecular sieve chromatography column. After loading, it was eluted with a 0.15 mol / L sodium dihydrogen phosphate / sodium hydrogen phosphate solution (pH 9.0) to collect the polyethylene glycol modified uricase sample. The enzyme activity was determined by uric acid absorption spectrophotometry. The enzyme activity recovery rate after purification was 85.5%. The sample was tested by SDS-PAGE and SEC-HPLC. The SDS-PAGE electrophoresis diagram is shown in the figure below. Fig.10 As shown, the SEC-HPLC purity was 99.2%.

Claims

1. A method for modifying uricase with polyethylene glycol, characterized in that: The method comprises the following steps: dissolving a uricase solution in a carbonate buffer, adding a PEG modifier and lysine, stirring and reacting at 4-30°C for 60-240 minutes, and after the reaction is completed, ultrafiltration concentration and molecular sieve purification are performed to obtain pegylated uricase; the PEG modifier is monomethoxypolyethylene glycol mPEG-SPA with succinimidyl propionate, monomethoxypolyethylene glycol mPEG-SS with succinimidyl succinate, monomethoxypolyethylene glycol mPEG-SSA with succinimidyl succinamide or monomethoxypolyethylene glycol mPEG-NHS with N-hydroxy-succinimide; the molecular weight is 5K-20K; the molar ratio of the PEG modifier to the uricase is 250-500:1, and the molar ratio of lysine in the reaction system to the lysine residue in the uricase protein monomer molecule is 0.5-4:

1.

2. The method according to claim 1, characterized in that The carbonate buffer solution is a sodium carbonate-sodium bicarbonate buffer solution, wherein the salt ion concentration is 0.04-0.5 mol / L.

3. The method according to claim 1, characterized in that The pH of the carbonate buffer solution is 8.0-10.

5.

4. The method according to claim 1, characterized in that The PEG modifier is monomethoxypolyethylene glycol mPEG-SPA of succinimidyl propionate;.

5. The method according to claim 1, characterized in that The molar ratio of PEG modifier to uricase is 320-410:

1.

6. The method according to claim 1, characterized in that After ultrafiltration and concentration, the concentration of PEG-modified uricase was 8-12 mg / ml.

7. The method according to claim 1, characterized in that The molecular sieve filler is Sephacryl S300 filler.

8. The method according to claim 1, characterized in that The mobile phase buffer used in the molecular sieve balancing and elution process is a phosphate buffer solution, and the phosphate buffer is sodium dihydrogen phosphate or potassium salt / disodium hydrogen phosphate or potassium salt.

9. The method according to claim 8, characterized in that The phosphate buffer has a concentration of 0.1-0.3 mol / L and a pH of 8.0-10.0.

Images