WO2022214086A1 - Uricase, pharmaceutical composition thereof and use thereof - Google Patents
Uricase, pharmaceutical composition thereof and use thereof Download PDFInfo
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- WO2022214086A1 WO2022214086A1 PCT/CN2022/085916 CN2022085916W WO2022214086A1 WO 2022214086 A1 WO2022214086 A1 WO 2022214086A1 CN 2022085916 W CN2022085916 W CN 2022085916W WO 2022214086 A1 WO2022214086 A1 WO 2022214086A1
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- WIPO (PCT)
- Prior art keywords
- uricase
- seq
- buffer
- sodium chloride
- pharmaceutical composition
- Prior art date
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Definitions
- Gout and hyperuricemia are common diseases that endanger human health. According to statistics, the incidence of hyperuricemia has reached 10% of the population. Gout is an acute arthritis caused by the deposition of urate crystals in the joints due to high blood uric acid levels. On the one hand, with the changes in people's diet and living habits, the intake of high-protein and high-purine foods has increased, and the number of gout patients has increased year by year; on the other hand, some malignant tumors, especially leukemia and lymphoma patients after radiotherapy and chemotherapy , In a short period of time, there will be a large amount of uric acid deposited in the kidneys and the dosage form will cause renal failure.
- One aspect of the present invention provides a uricase comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof that retains enzymatic activity, wherein the mutation of the variant relative to SEQ ID NO: 9 comprises: Replacement of amino acid residues within 17 N-terminal and/or within 10 C-terminal amino acid residues.
- the mutation of the variant relative to SEQ ID NO:9 further comprises: a substitution mutation at position 205 and/or position 208, wherein the position numbering is relative to the position numbering of SEQ ID NO:3.
- the substitution mutation is that S at position 205 is replaced by D or E, and G at position 208 is replaced by R, K or S.
- the substitution mutation is S205D or G208R or a combination thereof.
- the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to that of SEQ ID NO: 3, and the PEG is covalently attached through cysteine residues at these positions.
- the uricase conjugate is the PEG-modified uricase of the present invention.
- the uricase of the above pharmaceutical composition comprises the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:17, respectively.
- the concentration of uricase or uricase conjugate in the pharmaceutical composition is about 1-300 mg/mL, preferably about 1-250 mg/mL, preferably about 1-200 mg/mL, preferably about 5-80mg/mL, more preferably 5-40mg/mL; more preferably 5-20mg/mL; more preferably, the above-mentioned uricase concentration is about 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL mL, 10mg/mL, 12mg/mL, 14mg/mL, 16mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/m
- the above buffer is Tris hydrochloric acid (Tris-HCl) buffer.
- the Tris hydrochloride buffer is made up of 1-30 mM HCl and 1-30 mM Tris (tris.
- the Tris HCl buffer is made up of 11.8 mM Tris-HCl and 8.2 mM Tris.
- the Tris HCl buffer is made up of 16.5 mM Tris-HCl and 3.5 mM Tris.
- Tris hydrochloric acid buffer is made from 20 mM Tris, with the addition of concentrated hydrochloric acid to adjust the pH to 7.5 or 8.0.
- the above-mentioned stabilizer is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or The stabilizer is trehalose with a concentration of about 100-300 mM; or the stabilizer is arginine hydrochloride with a concentration of about 30-200 mM.
- the aforementioned stabilizer is trehalose.
- the above-mentioned stabilizer is trehalose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-300 mM, and a non-limiting example of the above-mentioned trehalose concentration is about 180 mM , 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220mM.
- the above stabilizer is a combination of sodium chloride and trehalose. In some aspects, the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM trehalose, preferably about 30-150 mM sodium chloride in combination with about 40-200 mM trehalose, preferably about 30-200 mM trehalose A combination of 30-100 mM sodium chloride and about 100-180 mM trehalose, non-limiting examples of the above stabilizers are about 50 mM sodium chloride and about 140 mM trehalose.
- the above pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188.
- the above-mentioned surfactant is selected from polysorbate 80.
- Figure 1 Comparison of specific enzyme activities of various uricases.
- a “phosphate buffer” is a buffer that includes phosphate ions.
- phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, dipotassium hydrogen phosphate-potassium dihydrogen phosphate, and the like.
- the preferred phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
- a “citrate buffer” is a buffer that includes citrate ions.
- citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
- a preferred citrate buffer is a citric acid-sodium citrate buffer.
- stabilizer means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
- Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine, or their metabolites, as defined below.
- the temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
- the antibody exhibits substantially no evidence of, for example, aggregation, precipitation and/or denaturation upon visual inspection of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography, the antibody is in the drug combination "maintain its physical stability". Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
- Tumor lysis syndrome refers to a group of clinical manifestations mainly manifested by hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia and acute renal failure due to the massive lysis of tumor cells and the release of intracellular metabolites in a short period of time. syndrome.
- amino acid sequences of the uricase variants respectively comprise the sequences shown in any one of SEQ ID NOs: 10-20, wherein the amino acid sequences of SEQ ID NOs: 12-20 are shown below :
- the polymer is PEG
- the uricase conjugate of the present invention is a PEG-modified uricase, also referred to herein as PEG-uricase.
- PEG can be covalently attached through cysteine residues present on the uricase.
- the uricase has 1, 2, 3, 4 or 5 surface accessible uricases in 1, 2, 3, 4 or 5 positions selected from positions 35, 82, 142, 194, 216, 287 and 288 Cysteine residues, where the position numbers are relative to those of SEQ ID NO:3, through which the PEG is covalently attached.
- the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to that of SEQ ID NO: 3, and the PEG is covalently attached through cysteine residues at these positions.
- the buffer in the pharmaceutical composition of the present invention can be selected from phosphate buffer and Tris hydrochloric acid buffer to provide the pharmaceutical composition of the present invention with 6.0-8.5, preferably about 6.5-8.5, preferably about 7.0-8.5, preferably A pH of about 7.5-8.5, preferably about 7.5-8.1, more preferably about 7.8.
- the pH of the buffer used in the pharmaceutical composition of the present invention may be 6.0-8.5, preferably about 6.5-8.5, preferably about 7.0-8.5, preferably about 7.5-8.5, preferably about 7.5-8.1 , more preferably about 7.8.
- the buffer in the pharmaceutical composition of the present invention can be Tris hydrochloric acid buffer.
- the Tris hydrochloride buffer is made up of 1-30 mM HCl and 1-30 mM Tris (tris.
- the Tris HCl buffer is made up of 11.8 mM Tris-HCl and 8.2 mM Tris.
- the Tris HCl buffer is made up of 16.5 mM Tris-HCl and 3.5 mM Tris.
- Tris hydrochloric acid buffer is made from 20 mM Tris, with the addition of concentrated hydrochloric acid to adjust the pH to 7.5 or 8.0.
- the buffer in the pharmaceutical composition of the present invention, can be a phosphate buffer with a pH of 7.5-8.1, and its concentration in the pharmaceutical composition is 10-30 mM; and the pharmaceutical composition contains 5-40 mg/mL
- the pharmaceutical composition of the present invention can be a liquid preparation or a lyophilized preparation.
- nucleotide sequence after codon optimization is:
- the bacterial cells are broken by high-pressure homogenization or ultrasonication, and after centrifugation, they are further purified by anion exchange to obtain a protein with a purity of > 95% for subsequent characterization and analysis.
- the amino acid sequence of the protease was obtained by DNA sequencing.
- SEQ ID NO: 6 was generated, designated AG-02, and the sequence is as follows:
- the N-terminal methionine can be removed by Met aminopeptidase, depending on the size of the second amino acid residue after the methionine, if the second position is Typical cleavage occurs for smaller residues, no cleavage if the second position is a bulky residue, or Met amino if the second position is neither bulky nor particularly small Peptidases can cleave the moiety, creating inhomogeneity. Therefore, in order to eliminate the incomplete cleavage of the first amino acid caused by prokaryotic expression, the second threonine (T) in the amino acid sequence of SEQ ID NO: 4 was mutated to alanine (A), resulting in SEQ ID NO: 4. NO: 8, named AG-00-2A.
- Polyethylene glycol (PEG)-modified Therapeutic proteins can be randomly attached to selected protein residues (such as lysine side chains) or specific for specific sites. Whereas, site-specific PEGylation allows for better control and yields highly homogeneous PEGylated products with defined biological activities.
- site-specific attachment the most widely used is conjugation to unpaired cysteine residues, which requires the introduction of one or more free cysteines into the protein sequence Residues.
- the C-terminal truncated AG-00-1 molecular sequence (SEQ ID NO: 3) does not contain cysteine, so cysteine can be introduced by amino acid mutation.
- Determination of uricase activity using a phosphate buffer system (pH 7.3).
- the activity measurement system refers to the above method (Example 2) to measure the absorbance value of residual uric acid at 292 nm.
- the substrate (uric acid) concentration gradient 600 micromolar, 500 micromolar, 400 micromolar, 200 micromolar, 100 micromolar, 50 micromolar, 25 micromolar, 12.5 micromolar; first add 50ul of the above uric acid The solution and 30ul of the above Tris-HCl buffer were placed in a 96-well plate, incubated at 37°C for 20min, and then 20ul of appropriately diluted uricase solution (0.004mg/mL) was quickly added to the 96-well plate.
- Setting program 10min reaction, read data every 20s, start reaction. The results are shown in Figure 4 and Table 3.
- Control uricase MEDI4945 The uricase disclosed in International Application No. PCT/US2016/032415.
- Km Michaelis constant
- Vmax maximum reaction rate
- Kcat conversion number, which refers to the number of substrates converted by a single enzyme molecule in one second.
- uricase A-2C01-2-PEG
- concentrations of about 10 mg/mL and 20 mg/mL.
- the samples were subjected to ultrafiltration and concentration exchange using Millipore Pellicon3 0.11m 2 membrane. After exchange, the samples were in the corresponding prescription, and the samples were placed in a closed centrifuge tube for buffer screening.
- the buffers were screened for phosphate buffer and Tris hydrochloric acid buffer, pH from 7.5 and 8.0; the stabilizers were screened for sodium chloride, arginine hydrochloride, mannitol, sucrose and trehalose for comparative testing (as shown in Table 4). ).
- Embodiment 7 Compatibility stability experiment
- Uricase AG-2C01-2-PEG
- Table 8 The stability of six formulations of FS3-1 to 6 in Example 2 after being diluted by two media, 0.9% sodium chloride injection and 5% glucose injection, was evaluated.
- Uricase AG-2C01-2-PEG
- Table 8 The stability of six formulations of FS3-1 to 6 in Example 2 after being diluted by two media, 0.9% sodium chloride injection and 5% glucose injection, was evaluated.
- Uricase AG-2C01-2-PEG was diluted to different concentrations in the corresponding medium according to the following scheme (Table 8), placed at 25°C for 24h, and then detected, and different medium pairs were investigated.
- Evaluation indicators include: 1. Visual appearance; 2. Protein concentration (UV spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and fragments. The results are shown in Table 9.
- the uricase of the present invention has good stability in the concentration range of 0.9% sodium chloride injection from 0.2 mg/ml to 4 mg/ml. Fragmentation increases in SEC at low concentrations of 0.2 mg/ml in 5% glucose injection.
- the isoelectric point of the uricase of the present invention is in the range of 5.5 to 6.3, and the precipitation phenomenon occurred at pH 7.0 in the early preliminary experiment, the pH was finally increased to 7.8 to increase its solubility as far as possible from the pI, and the pH range was 7.5 ⁇ 8.1.
- mice On the next day after administration, except for the blank control group, the other groups of experimental mice were injected with uric acid (250 mg/kg, 10 ml/kg) intraperitoneally for modeling, and blood samples were collected from animals 1 to 10 in each group at 30 minutes and 3 hours after modeling. , 11 to 20 animals collected 1.5h and 6h blood samples.
- uric acid 250 mg/kg, 10 ml/kg
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Abstract
Provided are a uricase, a pharmaceutical composition thereof and the use thereof. The uricase comprises an amino acid sequence as shown in SEQ ID NO: 9 or a variant thereof. The pharmaceutical composition thereof comprises a buffer solution and a uricase, wherein the pH value of the pharmaceutical composition is about 6.0-8.5, preferably about 7.5-8.1.
Description
本发明涉及治疗性药物领域,具体涉及尿酸酶、其药物组合物及其用途。The present invention relates to the field of therapeutic drugs, in particular to uricase, its pharmaceutical composition and its use.
痛风和高尿酸血症是目前危害人类健康的常见疾病,据统计高尿酸血症的发病率已经达到了人群的10%。痛风是由于血尿酸水平过高而造成尿酸盐结晶在关节中沉淀而导致的一种急性关节炎。一方面,是随着人们饮食和生活习惯的变化,高蛋白和高嘌呤食物的摄取增加,痛风患者的数量逐年增加;另一方面,某些恶性肿瘤,特别是白血病和淋巴瘤患者放化疗之后,在短期内将有大量的尿酸沉积于肾脏而引起的剂型肾功能衰竭。近几十年来,有很多科学家致力于将其他种属来源的尿酸酶用于人类高尿酸血症相关疾病的治疗。但是,异种蛋白应用于人体内最大的问题之一就是蛋白的免疫原性引起的抗蛋白反应和变态性反应。后来科学家采用乙二醇(PEG)对蛋白进行共价修饰,证明能够降低蛋白免疫原性,增加蛋白溶解度并延长蛋白的半衰期。Gout and hyperuricemia are common diseases that endanger human health. According to statistics, the incidence of hyperuricemia has reached 10% of the population. Gout is an acute arthritis caused by the deposition of urate crystals in the joints due to high blood uric acid levels. On the one hand, with the changes in people's diet and living habits, the intake of high-protein and high-purine foods has increased, and the number of gout patients has increased year by year; on the other hand, some malignant tumors, especially leukemia and lymphoma patients after radiotherapy and chemotherapy , In a short period of time, there will be a large amount of uric acid deposited in the kidneys and the dosage form will cause renal failure. In recent decades, many scientists have devoted themselves to the use of uricase from other species for the treatment of human hyperuricemia-related diseases. However, one of the biggest problems in the application of xenogeneic proteins in the human body is the anti-protein reaction and allergic reaction caused by the immunogenicity of the protein. Later, scientists used ethylene glycol (PEG) to covalently modify proteins, which proved to reduce protein immunogenicity, increase protein solubility and prolong protein half-life.
目前临床上批准的尿酸酶类药物有两种,其一是Krystexxa,另一个是Elitek。Krystexxa(聚乙二醇重组尿酸酶(pegloticase))是经批准的用于治疗常规疗法难治的成人患者的慢性痛风的聚乙二醇化尿酸酶,其是高聚乙二醇化的猪和狒狒尿酸酶序列的嵌合蛋白,可通过静脉内注射在2小时内给药完毕。Krystexxa含有针对过敏性反应和输液反应的黑框警告。而Elitek(拉布立酶)是被指示用于初始管理患有白血病、淋巴瘤和实体瘤恶性肿瘤的儿童和成人患者的血浆尿酸水平的修饰的重组黄曲霉尿酸酶,这些患者接受预期导致肿瘤溶解和血浆尿酸随后升高的抗癌治疗。但是Elitek在体内具有16-21小时的半衰期,并且必须经由静脉输注每天给药。同样的,Elitek也具有针对过敏性反应和溶血的黑框警告。There are currently two clinically approved uricase drugs, one is Krystexxa and the other is Elitek. Krystexxa (pegloticase) is an approved pegylated uricase for the treatment of chronic gout in adult patients refractory to conventional therapy, which is a highly pegylated porcine and baboon uric acid The chimeric protein of the enzymatic sequence can be administered by intravenous injection within 2 hours. Krystexxa contains a Boxed Warning for allergic reactions and infusion reactions. While Elitek (rasburicase) is a modified recombinant A. flavus uricase indicated for the initial management of plasma uric acid levels in pediatric and adult patients with leukemia, lymphoma, and solid tumor malignancies who are receiving tumors expected to cause Anticancer therapy for lysis and subsequent elevation of plasma uric acid. But Elitek has a half-life in the body of 16-21 hours and must be administered daily via intravenous infusion. Likewise, Elitek has a boxed warning for anaphylaxis and hemolysis.
鉴于以上情况,本领域迫切需要开发用于治疗高尿酸血症的更安全、更方便、且具有更低免疫原性的药物。In view of the above situation, there is an urgent need in the art to develop safer, more convenient and less immunogenic drugs for the treatment of hyperuricemia.
发明内容SUMMARY OF THE INVENTION
本发明一方面提供了一种尿酸酶,其包含如SEQ ID NO:9所示的氨基酸序列或其保留了酶活性的变体,其中所述变体相对于SEQ ID NO:9的突变包括:N末端17个以内 氨基酸残基替换和/或C末端10个以内氨基酸残基替换。One aspect of the present invention provides a uricase comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof that retains enzymatic activity, wherein the mutation of the variant relative to SEQ ID NO: 9 comprises: Replacement of amino acid residues within 17 N-terminal and/or within 10 C-terminal amino acid residues.
在一些方案中,所述变体相对于SEQ ID NO:9的突变包括:N末端氨基酸序列MTATAETSTGTKVVLGQ替换为MANIILGK,和/或C末端氨基酸序列SRADHPIWSN替换为C。。In some aspects, the mutation of the variant relative to SEQ ID NO: 9 comprises: the N-terminal amino acid sequence MTATAETSTGTKVVLGQ is replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C. .
在一些方案中,所述尿酸酶分别包含如SEQ ID NO:10或SEQ ID NO:11所示的序列。In some aspects, the uricase comprises the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11, respectively.
在一些方案中,所述变体相对于SEQ ID NO:9的突变还包括在选自35、82、142、194、216、287和288位的1、2、3、4或5个位置中引入1、2、3、4或5个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the variant is further comprised of mutations relative to SEQ ID NO: 9 at 1, 2, 3, 4, or 5 positions selected from positions 35, 82, 142, 194, 216, 287, and 288 1, 2, 3, 4 or 5 surface accessible cysteine residues were introduced, where the position number is relative to that of SEQ ID NO:3.
在一些方案中,所述变体相对于SEQ ID NO:9的突变还包括在选自35、82、142、194、216、287和288位的2个位置上引入2个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the mutation of the variant relative to SEQ ID NO: 9 further comprises the introduction of 2 surface accessible halves at 2 positions selected from positions 35, 82, 142, 194, 216, 287 and 288 cystine residues, wherein the position numbering is relative to that of SEQ ID NO:3.
在一些方案中,所述变体相对于SEQ ID NO:9的突变还包括在以下两个位置中包含半胱氨酸残基:142和216位,35和288位,35和142位,216和288位,35和194位,或82和287位,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the variant relative to SEQ ID NO: 9 further comprises a mutation of a cysteine residue in the following two positions: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, where the position number is relative to that of SEQ ID NO:3.
在一些方案中,所述变体相对于SEQ ID NO:9的突变还包括:第205位和/或第208位的取代突变,其中位置编号是相对于SEQ ID NO:3的位置编号。优选地,所述取代突变为第205位的S被取代成D或E、第208位的G被取代成R、K或S。在一些实施方式中,所述取代突变为S205D或G208R或其组合。In some aspects, the mutation of the variant relative to SEQ ID NO:9 further comprises: a substitution mutation at position 205 and/or position 208, wherein the position numbering is relative to the position numbering of SEQ ID NO:3. Preferably, the substitution mutation is that S at position 205 is replaced by D or E, and G at position 208 is replaced by R, K or S. In some embodiments, the substitution mutation is S205D or G208R or a combination thereof.
在一些方案中,本发明所述的尿酸酶包含如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列。In some aspects, the uricase of the present invention comprises as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, The amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
在又一个方面,本发明一方面提供了一种尿酸酶偶联物(即修饰的尿酸酶),其含有本文所述的尿酸酶和与该尿酸酶共价连接或融合表达的聚合物。在一些实施方式中,所述聚合物选自聚乙二醇(PEG)、磷酰胆碱聚合物、重复肽或“X-TEN”序列的聚合物和基于碳水化合物的聚合物。在一些实施方式中,所述聚合物与所述尿酸酶的N-末端和/或C-末端融合表达,或通过所述尿酸酶上存在的半胱氨酸残基进行共价连接。In yet another aspect, the present invention provides, in one aspect, a uricase conjugate (ie, a modified uricase) comprising a uricase as described herein and a polymer covalently linked or fused to the uricase expressed. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), phosphorylcholine polymers, polymers of repeating peptides or "X-TEN" sequences, and carbohydrate-based polymers. In some embodiments, the polymer is expressed fused to the N-terminus and/or C-terminus of the uricase, or covalently linked through a cysteine residue present on the uricase.
在一些方案中,所述聚合物为PEG,其通过所述尿酸酶上存在的半胱氨酸残基进行共价连接。优选地,尿酸酶在选自35、82、142、194、216、287和288位的1、2、3、4或5个位置中具有1、2、3、4或5个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号,所述PEG通过这些位置上的半胱氨酸残基进行共价连接。更优选地,所述的尿酸酶分别在以下两个位置中包含半胱氨酸残基:142和216位,35 和288位,35和142位,216和288位,35和194位,或82和287位,其中位置编号是相对于SEQ ID NO:3的位置编号,所述PEG通过这些位置上的半胱氨酸残基进行共价连接。In some aspects, the polymer is PEG covalently linked through cysteine residues present on the uricase. Preferably, the uricase has 1, 2, 3, 4 or 5 surface accessible uricases in 1, 2, 3, 4 or 5 positions selected from positions 35, 82, 142, 194, 216, 287 and 288 Cysteine residues, where the position numbers are relative to those of SEQ ID NO:3, through which the PEG is covalently attached. More preferably, the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to that of SEQ ID NO: 3, and the PEG is covalently attached through cysteine residues at these positions.
在一些方案中,所述的尿酸酶在第288位置中包含半胱氨酸残基,所述PEG通过该位置上的半胱氨酸残基进行共价连接,其中位置编号是相对于SEQ ID NO:10的位置编号。In some aspects, the uricase comprises a cysteine residue at position 288 through which the PEG is covalently attached, wherein position numbering is relative to SEQ ID NO: The position number of 10.
在一些方案中,所述尿酸酶的氨基酸序列如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20所示,所述PEG分别通过所述氨基酸序列的第142和216、35和288、35和142、216和288、35和288、35和194、82和287、35和194以及142和216位共价连接所述氨基酸序列,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the amino acid sequence of the uricase is such as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 17, ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, the PEG passes through the 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 288, Positions 35 and 194, 82 and 287, 35 and 194, and 142 and 216 are covalently linked to the amino acid sequence, wherein the position numbers are relative to SEQ ID NO:3.
在一些方案中,所述尿酸酶的氨基酸序列如SEQ ID NO:13、SEQ ID NO:15和SEQ ID NO:16所示,所述PEG还通过所述氨基酸序列的第288位共价连接所述氨基酸序列,其中位置编号是相对于SEQ ID NO:10的位置编号。In some aspects, the amino acid sequence of the uricase is set forth in SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 16, and the PEG is also covalently linked to the uricase at position 288 of the amino acid sequence. Said amino acid sequence, wherein the position number is relative to the position number of SEQ ID NO:10.
在一些方案中,所述尿酸酶的氨基酸序列如SEQ ID NO:17所示,所述PEG通过35和194的半胱氨酸残基进行共价连接,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the amino acid sequence of the uricase is set forth in SEQ ID NO: 17, and the PEG is covalently linked through cysteine residues 35 and 194, wherein position numbering is relative to SEQ ID NO: 3 position number.
在又一个方面,本发明提供了一种药物组合物,包含:(1)缓冲液;(2)如本文任一实施方案所述的尿酸酶或尿酸酶偶联物。In yet another aspect, the present invention provides a pharmaceutical composition comprising: (1) a buffer; (2) the uricase or uricase conjugate of any of the embodiments herein.
在一些方案中,所述尿酸酶偶联物为本发明所述的PEG修饰的尿酸酶。In some embodiments, the uricase conjugate is the PEG-modified uricase of the present invention.
在一些方案中,上述药物组合物的pH为约6.0-8.5,优选约为7.0-8.5,优选为约7.5~8.1。In some aspects, the pH of the above pharmaceutical composition is about 6.0-8.5, preferably about 7.0-8.5, preferably about 7.5-8.1.
在一些方案中,上述药物组合物的尿酸酶分别包含如SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:17所示的氨基酸序列。In some aspects, the uricase of the above pharmaceutical composition comprises the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:17, respectively.
在一些方案中,所述药物组合物中尿酸酶或尿酸酶偶联物的浓度约为1-300mg/mL,优选约为1-250mg/mL,优选约为1-200mg/mL,优选约为5-80mg/mL,更优选5-40mg/mL;更优选5-20mg/mL;更优选地,上述尿酸酶浓度约为5mg/mL,6mg/mL,7mg/mL,8mg/mL,9mg/mL,10mg/mL,12mg/mL,14mg/mL,16mg/mL,18mg/mL,20mg/mL,25mg/mL,30mg/mL,35mg/mL,40mg/mL,50mg/mL,60mg/mL,70mg/mL,80mg/mL,90mg/mL,100mg/mL,110mg/mL,120mg/mL,130mg/mL或140mg/mL,优选约为8mg/mL,10mg/mL或20mg/mL。In some aspects, the concentration of uricase or uricase conjugate in the pharmaceutical composition is about 1-300 mg/mL, preferably about 1-250 mg/mL, preferably about 1-200 mg/mL, preferably about 5-80mg/mL, more preferably 5-40mg/mL; more preferably 5-20mg/mL; more preferably, the above-mentioned uricase concentration is about 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL mL, 10mg/mL, 12mg/mL, 14mg/mL, 16mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL or 140 mg/mL, preferably about 8 mg/mL, 10 mg/mL or 20 mg/mL.
在一些方案中,上述缓冲液选自磷酸盐缓冲液、Tris盐酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液;优选地,所述缓冲液为磷酸盐缓冲液或Tris盐酸缓冲液;更优选地,所述 缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液。In some schemes, the above-mentioned buffer is selected from phosphate buffer, Tris hydrochloric acid buffer, citrate buffer and histidine buffer; preferably, the buffer is phosphate buffer or Tris hydrochloric acid buffer; more Preferably, the buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
在一些方案中,上述缓冲液为磷酸盐缓冲液,优选地,所述磷酸盐缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液。在一些方案中,磷酸盐缓冲液由1-30mM的磷酸氢二钠和1-30mM的磷酸二氢钠制成。在一些方案中,磷酸盐缓冲液由17mM的磷酸氢二钠和3mM的一水磷酸二氢钠制成。在一些方案中,磷酸盐缓冲液由18.5mM的磷酸氢二钠和1.5mM的一水磷酸二氢钠制成。In some solutions, the above buffer is a phosphate buffer, preferably, the phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some protocols, the phosphate buffer is made up of 1-30 mM disodium phosphate and 1-30 mM sodium dihydrogen phosphate. In some protocols, the phosphate buffer is made up of 17 mM disodium phosphate and 3 mM sodium dihydrogen phosphate monohydrate. In some protocols, the phosphate buffer is made up of 18.5 mM disodium phosphate and 1.5 mM sodium dihydrogen phosphate monohydrate.
在一些方案中,上述缓冲液为Tris盐酸(Tris-HCl)缓冲液。在一些方案中,Tris盐酸缓冲液由1-30mM的HCl和1-30mM的Tris(三羟甲基氨基甲烷)制成。在一些方案中,Tris盐酸缓冲液由11.8mM的Tris-HCl和8.2mM的Tris制成。在一些方案中,Tris盐酸缓冲液由16.5mM的Tris-HCl和3.5mM的Tris制成。在一些方案中,Tris盐酸缓冲液由20mM Tris,添加浓盐酸调节PH到7.5或8.0制成。In some aspects, the above buffer is Tris hydrochloric acid (Tris-HCl) buffer. In some protocols, the Tris hydrochloride buffer is made up of 1-30 mM HCl and 1-30 mM Tris (tris. In some protocols, the Tris HCl buffer is made up of 11.8 mM Tris-HCl and 8.2 mM Tris. In some protocols, the Tris HCl buffer is made up of 16.5 mM Tris-HCl and 3.5 mM Tris. In some protocols, Tris hydrochloric acid buffer is made from 20 mM Tris, with the addition of concentrated hydrochloric acid to adjust the pH to 7.5 or 8.0.
在一些方案中,上述缓冲液为组氨酸缓冲液,优选地,所述组氨酸缓冲液选自组氨酸-盐酸盐缓冲液或组氨酸-醋酸盐缓冲液。In some schemes, the above buffer is histidine buffer, preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer.
在一些方案中,上述缓冲液为柠檬酸缓冲液,优选地,所述柠檬酸缓冲液为柠檬酸-柠檬酸钠缓冲液。In some solutions, the above buffer is a citrate buffer, preferably, the citrate buffer is a citric acid-sodium citrate buffer.
在一些方案中,上述缓冲液的浓度约为1-200mM,优选约为5-200mM,优选约为10-50mM,优选约为10-30mM;优选约为20-30mM,上述缓冲液浓度非限制性实施例约为5mM,10mM,15mM,20mM,25mM,30mM、40mM、45mM、50mM、60mM、70mM、80mM、90mM、100mM、105mM、110mM、115mM、120mM、130mM、140mM、150mM、160mM、170mM或180mM或这些范围内任意两个数值作为端点形成的范围,优选为10mM、15mM、20mM或30mM。In some embodiments, the concentration of the above buffer is about 1-200 mM, preferably about 5-200 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 20-30 mM, the concentration of the above buffer is not limited Typical examples are about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM or 180 mM or a range formed by any two values within these ranges as endpoints, preferably 10 mM, 15 mM, 20 mM or 30 mM.
在一些方案中,上述缓冲液的pH约为6.0-8.5,优选约为6.5-8.5,优选约为7.0-8.5,优选约为7.5-8.5,优选约为7.5-8.1,上述缓冲液的pH非限制性实施例约为6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,优选约为7.5,7.6,7.7,7.8,7.9,8.0或8.1,更优选7.8。In some aspects, the pH of the above buffer is about 6.0-8.5, preferably about 6.5-8.5, preferably about 7.0-8.5, preferably about 7.5-8.5, preferably about 7.5-8.1, and the pH of the above buffer is not A limiting example is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, preferably about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or 8.1, more preferably 7.8.
在一些方案中,上述的药物组合物还包括稳定剂,所述稳定剂选自盐酸精氨酸、氯化钠、甘露醇、蔗糖和海藻糖中的一种或多种;优选地,所述稳定剂选自甘露醇、海藻糖、甘露醇与氯化钠的组合或海藻糖与氯化钠的组合。In some schemes, the above-mentioned pharmaceutical composition further comprises a stabilizer, the stabilizer is selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose and trehalose; preferably, the The stabilizer is selected from mannitol, trehalose, a combination of mannitol and sodium chloride or a combination of trehalose and sodium chloride.
在一些方案中,上述稳定剂的浓度为约10mM~400mM,优选20mM~300mM,更优选50mM~200mM。In some aspects, the concentration of the aforementioned stabilizer is about 10 mM to 400 mM, preferably 20 mM to 300 mM, more preferably 50 mM to 200 mM.
在一些方案中,上述稳定剂为浓度约30-200mM的氯化钠;或所述稳定剂为浓度约100-300mM的甘露醇;或所述稳定剂为浓度约100-300mM的蔗糖;或所述稳定剂为浓度 约100-300mM的海藻糖;或所述稳定剂为浓度约30-200mM的盐酸精氨酸。In some aspects, the above-mentioned stabilizer is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or The stabilizer is trehalose with a concentration of about 100-300 mM; or the stabilizer is arginine hydrochloride with a concentration of about 30-200 mM.
在一些方案中,上述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的海藻糖的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合。In some aspects, the above stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the stabilizer is about 30-200 mM sodium chloride and about 30-200 mM trehalose or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose.
在一些方案中,上述稳定剂为氯化钠。在一些方案中,上述稳定剂为浓度约30-200mM的氯化钠,上述氯化钠的浓度优选约为50-190mM,优选约为100-180mM,优选约为120-170mM,优选约为130-150mM,上述氯化钠浓度的非限制性实施例为约100mM,110mM,120mM,125mM,130mM,135mM,140mM,145mM,150mM,155mM,160mM,170mM,180mM,190mM,200mM,优选135mM或140mM。In some aspects, the above stabilizer is sodium chloride. In some embodiments, the above-mentioned stabilizer is sodium chloride at a concentration of about 30-200 mM, and the concentration of the above-mentioned sodium chloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about 130 mM -150mM, non-limiting examples of the above sodium chloride concentrations are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM .
在一些方案中,上述稳定剂为甘露醇。在一些方案中,上述稳定剂为浓度约100-300mM的甘露醇,上述甘露醇的浓度优选约为150-300mM,优选约为200-300mM,上述甘露醇浓度的非限制性实施例为约200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM,优选为240mM。In some aspects, the aforementioned stabilizer is mannitol. In some embodiments, the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-300 mM, and a non-limiting example of the aforementioned mannitol concentration is about 200 mM , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
在一些方案中,上述稳定剂为蔗糖。在一些方案中,上述稳定剂为浓度约100-300mM的蔗糖,上述蔗糖的浓度优选约为150-300mM,优选约为200-300mM,上述蔗糖浓度的非限制性实施例为约200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM,优选为220mM。In some aspects, the aforementioned stabilizer is sucrose. In some embodiments, the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-300 mM, non-limiting examples of the aforementioned sucrose concentrations are about 200 mM, 210 mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220mM.
在一些方案中,上述稳定剂为海藻糖。在一些方案中,上述稳定剂为浓度约100-300mM的海藻糖,上述海藻糖的浓度优选约为150-300mM,优选约为200-300mM,上述海藻糖浓度的非限制性实施例为约180mM,200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM,优选为220mM。In some aspects, the aforementioned stabilizer is trehalose. In some embodiments, the above-mentioned stabilizer is trehalose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-300 mM, and a non-limiting example of the above-mentioned trehalose concentration is about 180 mM , 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220mM.
在一些方案中,上述稳定剂为盐酸精氨酸。在一些方案中,上述稳定剂为浓度约30-200mM的盐酸精氨酸,上述盐酸精氨酸的浓度优选约为50-190mM,优选约为100-180mM,优选约为120-170mM,优选约为130-150mM,上述盐酸精氨酸浓度的非限制性实施例为约100mM,110mM,120mM,125mM,130mM,135mM,140mM,145mM,150mM,155mM,160mM,170mM,180mM,190mM,200mM,优选135mM或140mM。In some aspects, the above stabilizer is arginine hydrochloride. In some embodiments, the above-mentioned stabilizer is arginine hydrochloride at a concentration of about 30-200 mM, and the concentration of the above-mentioned arginine hydrochloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about 130-150mM, non-limiting examples of the above arginine hydrochloride concentrations are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM.
在一些方案中,上述稳定剂为氯化钠与甘露醇的组合。在一些方案中,上述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合,优选约30-150mM的氯化钠与约40-200mM的甘露醇的组合,优选约30-100mM的氯化钠与约100-180mM的甘露醇的组合,上述稳定剂的非限制性实施例约为50mM的氯化钠与约140mM的甘露醇的组合。In some aspects, the above stabilizer is a combination of sodium chloride and mannitol. In some aspects, the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM mannitol, preferably about 30-150 mM sodium chloride in combination with about 40-200 mM mannitol, preferably about 30-200 mM mannitol. A combination of 30-100 mM sodium chloride and about 100-180 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 50 mM sodium chloride and about 140 mM mannitol.
在一些方案中,上述稳定剂为氯化钠与蔗糖的组合。在一些方案中,上述稳定剂为约 30-200mM的氯化钠与约30-200mM的蔗糖的组合,优选约30-150mM的氯化钠与约40-200mM的蔗糖的组合,优选约30-100mM的氯化钠与约100-180mM的蔗糖的组合,上述稳定剂的非限制性实施例约为50mM的氯化钠与约140mM的蔗糖的组合。In some aspects, the above stabilizer is a combination of sodium chloride and sucrose. In some aspects, the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM sucrose, preferably about 30-150 mM sodium chloride in combination with about 40-200 mM sucrose, preferably about 30- A combination of 100 mM sodium chloride and about 100-180 mM sucrose, a non-limiting example of the above stabilizer is about 50 mM sodium chloride and about 140 mM sucrose.
在一些方案中,上述稳定剂为氯化钠与海藻糖的组合。在一些方案中,上述稳定剂为约30-200mM的氯化钠与约30-200mM的海藻糖的组合,优选约30-150mM的氯化钠与约40-200mM的海藻糖的组合,优选约30-100mM的氯化钠与约100-180mM的海藻糖的组合,上述稳定剂的非限制性实施例约为50mM的氯化钠与约140mM的海藻糖的组合。In some aspects, the above stabilizer is a combination of sodium chloride and trehalose. In some aspects, the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM trehalose, preferably about 30-150 mM sodium chloride in combination with about 40-200 mM trehalose, preferably about 30-200 mM trehalose A combination of 30-100 mM sodium chloride and about 100-180 mM trehalose, non-limiting examples of the above stabilizers are about 50 mM sodium chloride and about 140 mM trehalose.
在一些方案中,上述药物组合物还包括表面活性剂,所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20或泊洛沙姆188。In some aspects, the above pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188.
在一些方案中,上述表面活性剂选自聚山梨醇酯80。In some aspects, the above-mentioned surfactant is selected from polysorbate 80.
在一些方案中,上述表面活性剂选自聚山梨醇酯20。In some aspects, the above-mentioned surfactant is selected from polysorbate 20.
在一些方案中,以w/v计算,上述表面活性剂浓度约为0.001%-0.1%,优选约为0.01%-0.1%,优选约为0.02%-0.08%;作为非限制性实施例,上述表面活性剂的浓度约为0.02%,0.04%或0.08%,优选0.02%。In some embodiments, on a w/v basis, the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.02%-0.08%; as a non-limiting example, the above-mentioned surfactant concentration The concentration of surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.
在一些方案中,本文药物组合物包含如下(1)-(5)任一项所示的组分,或分别由(1)-(5)任一项所示的组分组成:In some aspects, the pharmaceutical compositions herein comprise the components set forth in any one of (1)-(5) below, or consist of the components set forth in any one of (1)-(5), respectively:
(1)(a)约5mg/mL-40mg/mL的尿酸酶或尿酸酶偶联物;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约100-300mM的甘露醇;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(1) (a) about 5 mg/mL-40 mg/mL uricase or uricase conjugate; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 100-300 mM mannitol; and (d) about 0.01%-0.1% polysorbate 80; or
(2)(a)约5mg/mL-40mg/mL的尿酸酶或尿酸酶偶联物;(b)约10-30mM Tris盐酸缓冲液,pH约为7.5-8.1;(c)约100-300mM的海藻糖;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(2) (a) about 5mg/mL-40mg/mL uricase or uricase conjugate; (b) about 10-30mM Tris hydrochloric acid buffer, pH about 7.5-8.1; (c) about 100-300mM and (d) about 0.01%-0.1% polysorbate 80; or
(3)(a)约5mg/mL-40mg/mL的尿酸酶或尿酸酶偶联物;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约100-300mM的海藻糖;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(3) (a) about 5 mg/mL-40 mg/mL uricase or uricase conjugate; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 100-300 mM and (d) about 0.01%-0.1% polysorbate 80; or
(4)(a)约5mg/mL-40mg/mL的尿酸酶或尿酸酶偶联物;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约30-200mM的氯化钠与约30-200mM的甘露醇的组合;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(4) (a) about 5 mg/mL-40 mg/mL uricase or uricase conjugate; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 30-200 mM of sodium chloride in combination with about 30-200 mM mannitol; and (d) about 0.01%-0.1% polysorbate 80; or
(5)(a)约5mg/mL-40mg/mL的尿酸酶或尿酸酶偶联物;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约30-200mM的氯化钠与约30-200mM的海藻糖的组合;以及(d)约0.01%-0.1%的聚山梨醇酯80。(5) (a) about 5 mg/mL-40 mg/mL uricase or uricase conjugate; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 30-200 mM of sodium chloride in combination with about 30-200 mM trehalose; and (d) about 0.01%-0.1% polysorbate 80.
优选地,所述尿酸酶如本文任一实施方案所述。Preferably, the uricase is as described in any of the embodiments herein.
在一些方案中,药物组合物包含如下(1)-(5)任一项所示的组分,或分别由(1)-(5)任一项所示的组分组成:In some aspects, the pharmaceutical composition comprises the components set forth in any one of (1)-(5) below, or consists of the components set forth in any one of (1)-(5), respectively:
(1)(a)约8mg/mL的尿酸酶或尿酸酶偶联物;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约240mM的甘露醇;以及(d)约0.02%的聚山梨醇酯80;或(1) (a) about 8 mg/mL uricase or uricase conjugate; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 240 mM mannitol; and (d) about 0.02% polysorbate 80; or
(2)(a)约8mg/mL的尿酸酶或尿酸酶偶联物;(b)约20mM Tris盐酸缓冲液,pH约为7.5-8.0;(c)约220mM的海藻糖;以及(d)约0.02%的聚山梨醇酯80;或(2) (a) about 8 mg/mL uricase or uricase conjugate; (b) about 20 mM Tris hydrochloric acid buffer, pH about 7.5-8.0; (c) about 220 mM trehalose; and (d) about 0.02% polysorbate 80; or
(3)(a)约8mg/mL的尿酸酶或尿酸酶偶联物;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约220mM的海藻糖;以及(d)约0.02%的聚山梨醇酯80;或(3) (a) about 8 mg/mL uricase or uricase conjugate; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 220 mM trehalose; and (d) about 0.02% polysorbate 80; or
(4)(a)约8mg/mL的尿酸酶或尿酸酶偶联物;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约50mM的氯化钠与约140mM的甘露醇的组合;以及(d)约0.02%的聚山梨醇酯80;或(4) (a) about 8 mg/mL uricase or uricase conjugate; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 50 mM sodium chloride with about 140 mM a combination of mannitol; and (d) about 0.02% polysorbate 80; or
(5)(a)约8mg/mL的尿酸酶或尿酸酶偶联物;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约50mM的氯化钠与约140mM的海藻糖的组合;以及(d)约0.02%的聚山梨醇酯80。(5) (a) about 8 mg/mL uricase or uricase conjugate; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 50 mM sodium chloride with about 140 mM A combination of trehalose; and (d) about 0.02% polysorbate 80.
优选地,所述尿酸酶或尿酸酶偶联物如本文任一实施方案所述。Preferably, the uricase or uricase conjugate is as described in any of the embodiments herein.
在一些方案中,所述药物组合物为液体制剂或冻干制剂。In some aspects, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
在一些方案中,所述药物组合物为液体制剂。In some aspects, the pharmaceutical composition is a liquid formulation.
在一些方案中,上述液体制剂或冻干制剂于2-8℃稳定至少2个月,3个月,至少6个月,至少12个月,至少18个月或至少24个月。In some embodiments, the above-described liquid or lyophilized formulations are stable at 2-8°C for at least 2 months, 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.
在一些方案中,上述水溶液或冻干制剂于23-27℃稳定至少2周,至少4周或至少8周。In some embodiments, the aqueous or lyophilized formulations described above are stable at 23-27°C for at least 2 weeks, at least 4 weeks, or at least 8 weeks.
在一些方案中,上述水溶液或冻干制剂于40℃稳定至少7天,至少14天或至少28天。In some embodiments, the aqueous or lyophilized formulations described above are stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
本发明还提供一种注射剂,其含有本发明任一实施方案所述的药物组合物与0.9wt%氯化钠溶液或5wt%葡萄糖水溶液;优选地,所述含0.9wt%氯化钠溶液的注射剂中,所述尿酸酶的浓度为0.2~4mg/mL,所述含5wt%葡萄糖水溶液的注射剂中,所述尿酸酶的浓度为1~4mg/mL;优选地,所述注射剂的pH为7.5~8.1。The present invention also provides an injection, which contains the pharmaceutical composition according to any embodiment of the present invention and 0.9wt% sodium chloride solution or 5wt% aqueous glucose solution; preferably, the 0.9wt% sodium chloride solution containing In the injection, the concentration of the uricase is 0.2-4 mg/mL, and in the injection containing the 5wt% aqueous glucose solution, the concentration of the uricase is 1-4 mg/mL; preferably, the pH of the injection is 7.5 ~8.1.
本发明还提供了上述尿酸酶、尿酸酶偶联物或药物组合物在制备降低哺乳动物体液和组织中尿酸水平或治疗高尿酸血症的药物中的用途。The present invention also provides the use of the above uricase, uricase conjugate or pharmaceutical composition in the preparation of a medicament for reducing the level of uric acid in mammalian body fluids and tissues or for treating hyperuricemia.
图1:各种尿酸酶的酶比活性对比。Figure 1: Comparison of specific enzyme activities of various uricases.
图2:各种尿酸酶的热稳定性实验。Figure 2: Thermostability experiments for various uricases.
图3:各种尿酸酶热稳定性研究。3a:蛋白分子4℃PR-HPLC分析;3b:蛋白分子4℃SEC-HPLC分析。Figure 3: Various uricase thermostability studies. 3a: 4°C PR-HPLC analysis of protein molecules; 3b: 4°C SEC-HPLC analysis of protein molecules.
图4:PEG化修饰的尿酸酶酶活动力学研究评估。Figure 4: Kinetic study assessment of PEGylation-modified uricase enzymatic activity.
图5:PEG化修饰的尿酸酶热稳定性评估。Figure 5: Assessment of thermal stability of PEGylated uricases.
图6:PEG化修饰的尿酸酶体内药效评估。Figure 6: In vivo efficacy evaluation of PEGylated uricase.
定义和说明Definition and Explanation
为了更容易理解本发明,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。应理解本发明不限于具体的方法、试剂、化合物、组合物或生物系统,当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式,并不旨在进行限制。For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
除非该内容被另外明确说明,否则本说明书以及所附权利要求中所用的单数形式"一个"、"一种"和"该"包括复数指代。因此,例如,提及"一种多肽"包括了两种或更多种多肽等的组合。As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides, and the like.
术语"药物组合物"或“制剂”表示含有一种或多种本文所述抗体与其他组分的混合物,所述其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药,利于活性成分的吸收进而发挥生物活性。The term "pharmaceutical composition" or "formulation" refers to a mixture comprising one or more of the antibodies described herein in admixture with other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
术语“液体制剂”是指处于液体状态下的制剂,且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定,并且其稳定性不依赖于冻干(或其他状态改变方法,例如喷雾干燥)。The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable on storage, and their stability is independent of lyophilization (or other state-changing methods, such as spray drying).
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一些方案中,水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定性)和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚合物。The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure. Examples of common excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
本申请所用的"约"在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20%或±10%的变化,包括±5%、±1%和±0.1%,因为这些变化适于进行所公开的方法。As used herein, "about" when referring to a measurable value (eg, amount, duration, etc.) is intended to encompass a variation of ±20% or ±10% from a particular value, including ±5%, ±1%, and ±0.1 % as these changes are suitable for carrying out the disclosed method.
术语"缓冲液pH约为6.0-8.5"是指这样的试剂,通过其酸/碱共轭组分的作用使得包含 该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约6.0至约8.5范围内的pH、或约7.0至约8.5范围内的pH、或约7.5至约8.1范围内的pH。The term "buffer pH of about 6.0-8.5" refers to an agent which, through the action of its acid/base conjugate component, renders a solution containing the agent resistant to pH changes. The buffer used in the formulations of the present invention may have a pH in the range of about 6.0 to about 8.5, or a pH in the range of about 7.0 to about 8.5, or a pH in the range of about 7.5 to about 8.1.
在本文中,将pH控制在该范围内的“缓冲液”实例包括乙酸盐(例如乙酸钠)、Tris盐酸、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组氨酸盐酸盐、甲硫氨酸、柠檬酸盐、磷酸盐、柠檬酸盐/磷酸盐、咪唑、醋酸、醋酸盐、枸橼酸盐、其组合和其他有机酸缓冲剂。Herein, examples of "buffers" that control the pH within this range include acetate (eg, sodium acetate), Tris hydrochloric acid, succinate (eg, sodium succinate), gluconic acid, histidine, histidine Hydrochloride, methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof and other organic acid buffers.
“磷酸盐缓冲液”是包括磷酸根离子的缓冲液。磷酸盐缓冲液的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钾-磷酸二氢钾等。优选的磷酸盐缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液缓冲液。A "phosphate buffer" is a buffer that includes phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, dipotassium hydrogen phosphate-potassium dihydrogen phosphate, and the like. The preferred phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
“Tris盐酸缓冲液”是三羟甲基氨基甲烷盐酸盐缓冲液,也为Tris-HCl缓冲液。Tris盐酸缓冲液包括Tris和HCl。"Tris hydrochloride buffer" is Tris-HCl buffer, also Tris-HCl buffer. Tris hydrochloric acid buffers include Tris and HCl.
"组氨酸缓冲液"为包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸和组氨酸的盐,如组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐等,如含有组氨酸与组氨酸盐酸盐的组氨酸缓冲液。"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like, such as histidine-containing acid with histidine hydrochloride in histidine buffer.
“柠檬酸盐缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸纳、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲液为柠檬酸-柠檬酸纳缓冲液。A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like. A preferred citrate buffer is a citric acid-sodium citrate buffer.
术语“稳定剂”表示药学上可接受的赋形剂,其在制造,储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于如以下定义的糖,氨基酸,盐,多元醇和他们的代谢产物,例如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖、海藻糖、精氨酸或其盐(如盐酸精氨酸)、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)、丙氨奥品、章鱼碱、甘氨奥品(strombine))和三甲胺的N-氧化物(TMAO)、人血清白蛋白(hsa)、牛血清白蛋白(bsa)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶菌酶、肌红蛋白、卵清蛋白和RNAaseA。部分稳定剂,如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖等也可起到控制渗透压的作用。在本发明中具体地使用的稳定剂选自多元醇、氨基酸、盐、糖中的一种或多种。优选的盐为氯化钠,优选的糖为蔗糖和海藻糖,优选的多元醇为甘露醇。优选的氨基酸为精氨酸或其盐(如盐酸精氨酸)、甘氨酸、脯氨酸。优选的稳定剂为氯化钠、甘露醇、蔗糖、海藻糖、盐酸精氨酸、氯化钠-甘露醇、氯化钠-蔗糖和氯化钠-海藻糖。The term "stabilizer" means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine, or their metabolites, as defined below. Salts (such as arginine hydrochloride), glycine, alanine (alpha-alanine, beta-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline acid, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanopine, octopine, glycine strombine), and trimethylamine's N- oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), alpha-casein, globulin, alpha-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. The preferred salt is sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyol is mannitol. Preferred amino acids are arginine or a salt thereof (eg arginine hydrochloride), glycine, proline. Preferred stabilizers are sodium chloride, mannitol, sucrose, trehalose, arginine hydrochloride, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose.
术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示 例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨醇酯20和聚山梨醇酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆,Pluronic)、十二烷基硫酸钠(SDS)。The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibody or to minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (eg, polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polysorbate Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
术语"等渗"是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmolarity of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.
术语“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100%的化学结构或生物功能,医药制剂也可以是稳定的。在某些情况下,在经过一定时间储存之后,能维持约90%、约95%、约96%、约97%、约98%或约99%的抗体结构或功能,也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的,并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247-301,Vincent Lee主编,Marcel Dekker,Inc.,New York,N.Y.,Pubs.(1991)),和Jones,A.(1993)Adv.Drug Delivery Rev.10:29-90中(二者引入作为参考)。The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. A pharmaceutical formulation may be stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage. In certain instances, an antibody that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after storage for a certain period of time may also be considered "" stable". Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, edited by Vincent Lee, Marcel Dekker, Inc. ., New York, N.Y., Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).
制剂在一定温度下经过一定时间的储存之后,通过测定其中剩余的天然抗体的百分比(及其它方法),可以测量其稳定性。除其它方法外,天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SEC-HPLC])来测量,“天然的”指未聚集的和未降解的。在一些方案中,蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一些方案中,制剂可以在室温、约25-30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月,或更长,最多不超过约6%、5%、4%、3%、2%、1%、0.5%,或0.1%聚集形式的抗体。The stability of a formulation can be measured by determining the percentage (among other methods) of native antibody remaining in a formulation after storage at a given temperature for a given period of time. Among other methods, the percentage of native antibody can be measured by size exclusion chromatography (eg, size exclusion high performance liquid chromatography [SEC-HPLC]), with "native" meaning unaggregated and undegraded. In some embodiments, protein stability is determined as the percentage of monomeric protein in solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein. In some regimens, the formulations are stable for storage at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or Longer, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.
通过测定在离子交换期间在此抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法),可以测量稳定性,其中稳定性与酸性形式抗体的百分比成反比。除其它方法外,“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一些实施方式中,可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后,其中可检测出的酸性形式的抗体最多不超过约49%、45%、40%、35%、30%、25%、20%、15%、10%、5%、4%、3%、2%、1%、0.5%或0.1%。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18 个月、至少24个月,或更长。当评估稳定性时,容许储存医药制剂的一定温度可以是约-80℃至约45℃范围内的任何温度,例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2-8℃、约5℃、约25℃,或约40℃。Stability can be measured by determining (among other methods) the percentage of antibody ("acidic form") that migrates in the more acidic fraction of this antibody main fraction ("predominantly charged form") during ion exchange, where stability is related to The percentage of antibody in the acidic form is inversely proportional. Among other methods, the percentage of "acidified" antibody can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]). In some embodiments, an acceptable degree of stability means that upon storage of the formulation at a certain temperature and for a certain period of time, the detectable acidic form of the antibody therein does not exceed at most about 49%, 45%, 40%, 35% %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. The certain period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When assessing stability, the temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象,则所述抗体在该药物组合物中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。If the antibody exhibits substantially no evidence of, for example, aggregation, precipitation and/or denaturation upon visual inspection of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography, the antibody is in the drug combination "maintain its physical stability". Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估,包括测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质,并且通常通过比浊法来测量,并用比浊法浊度单位(NTU)来量度。The stability of the formulation, eg, physical stability, can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements correlate with the turbidity of the formulation. The turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution, and is typically measured by nephelometric methods and measured in nephelometric turbidity units (NTU).
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia),第四版,“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM),Strasbourg,France)。根据《欧洲药典》标准,澄清溶液被定义为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射,其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。The level of turbidity as a function of, for example, the concentration of one or more components in solution (eg, protein and/or salt concentration) is also referred to as the "opaque" or "opaque appearance" of the formulation. Turbidity levels can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of pharmaceutical compositions can be based on the European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, "Directorate for the Quality of Medicine"). of the Council of Europe) (EDQM), Strasbourg, France). According to the European Pharmacopoeia standard, a clear solution is defined as a solution with a turbidity lower than or equal to that of a reference suspension according to the European Pharmacopoeia standard of about 3. Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of associative or non-ideal effects, which typically vary linearly with concentration. Other methods for assessing physical stability are known in the art.
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物活性,则所述抗体在药物组合物中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短),其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生),其可以通过例如离子交换层析来评估。An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody. Chemical changes can include size changes (eg, clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (eg occur as a result of deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.
如果药物组合物中的抗体对于其预期目的来说是生物活性的,则所述抗体在药物组合物中“保持其生物活性”。例如,如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后,该制剂所含尿酸酶与TIGIT结合的亲和力为所述储存之前抗体结合亲和力的至少90%、95%或以上,则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。An antibody in a pharmaceutical composition "retains its biological activity" in a pharmaceutical composition if it is biologically active for its intended purpose. For example, if the preparation is stored at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (eg, 1 to 12 months), the binding affinity of uricase contained in the preparation to TIGIT is the same as the binding affinity of the antibody prior to said storage. At least 90%, 95% or more, the formulations of the present invention are considered stable. Binding affinity can also be determined using techniques such as ELISA or plasmon resonance.
在本发明的情形中,在药理学意义上,抗体的“治疗有效量”或“有效量”是指在抗体可 以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。本发明中,药物的“治疗有效量”或“治疗有效剂量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物,所述疾病消退通过疾病症状的严重性的降低,疾病无症状期的频率和持续时间的增加,或由疾病痛苦引起的损伤或失能的预防来证明。药物促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价,比如在临床试验期间的人受试者中,在预测人类功效的动物模型系统中,或通过在体外测定法中测定所述药剂的活性。药物治疗有效量包括“预防有效量”,即当单独或如与其它治疗药物组合给与处于患病风险的受试者或患病复发的受试者时,抑制疾病的发展或复发的任何量的药物。In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody in the pharmacological sense refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, so The disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by the suffering of the disease. The ability of a drug to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by in vitro assays The activity of the agent is determined. A pharmacologically effective amount includes a "prophylactically effective amount," that is, any amount that inhibits the progression or recurrence of a disease when administered to a subject at risk for the disease or to a subject relapsed with the disease, alone or as in combination with other therapeutic agents medicine.
术语“受试者”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物,例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中,受试者是人类。The terms "subject" or "patient" are intended to include mammalian organisms. Examples of subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the invention, the subject is a human.
术语“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送系统中的任意一种将包含治疗剂的组合物引入受试者。抗PD-1抗体的给药途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径,比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式,包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to modes of administration other than enteral or topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular , intra-frame, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion and in vivo electroporation.
“有效量”包括足以改善或预防医学病症的症状或体征的量。有效量还意指足以允许或便于诊断的量。特定患者或兽医对象的有效量可根据例如待治疗的病况、患者的总体健康状况、给药的方法途径和剂量及副作用的严重程度等因素而变化。有效量可以是避免明显副作用或毒性作用的最大剂量或给药方案。结果可导致诊断测量或参数改进至少5%、通常为至少10%、更常为至少20%、最常为至少30%、优选至少40%、更优选至少50%、最优选至少60%、理想为至少70%、更理想为至少80%、最理想为至少90%,其中100%定义为正常受试者显示的诊断参数(参见例如Maynard等(1996)A Handbook of SOPs for Good Clinical Practice,Interpharm Press,Boca Raton,FL;Dent(2001)Good Laboratory and Good ClinicalPractice,Urch Publ.,London,UK)。An "effective amount" includes an amount sufficient to ameliorate or prevent symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending upon factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The results may result in an improvement in a diagnostic measure or parameter of at least 5%, usually at least 10%, more often at least 20%, most often at least 30%, preferably at least 40%, more preferably at least 50%, most preferably at least 60%, ideally is at least 70%, more ideally at least 80%, and most ideally at least 90%, with 100% defined as the diagnostic parameters exhibited by normal subjects (see e.g. Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).
尿酸酶uricase
尿酸氧化酶(尿酸酶)Uric acid oxidase (uricase)
尿酸氧化酶(尿酸酶)是一类酶,这类酶催化尿酸氧化,氧化产物是更易于溶解的尿囊素。尿囊素属于嘌呤类代谢物,更易于排泄。其是由四个相同的34KDa亚基组成的同源四聚体酶,该酶作为开始一系列反应的起始物,将尿酸转化为更易溶解和更易排泄的产 物(尿囊素),即尿酸酶催化尿酸(UA)与O
2和H
2O反应而形成5-羟基-异尿酸酯(HIU)和H
2O
2的释放。HIU是一种经历非酶水解生成2-氧代-4-羟基-5-脲基咪唑(OHCU)且随后自发的脱羧基从而形成外消旋尿囊素的不稳定的产物。在含有功能性尿酸酶的物种中表达两种额外的酶(HIU水解酶和OHCU脱羧酶),这两种额外的酶能更快的催化这些反应从而产生(s)-尿囊素。功能性尿酸酶在很多种生物体中发现,比如古细菌、细菌和真核生物。但是,在人和一些灵长类动物中并不表达尿酸酶。这是由于在密码子33处、密码子187处出现了无义突变,以及在内含子2中的剪接受体位点处的突变造成的。总之,在人体中尿酸酶表达的缺乏导致了高的全身UA水平,且在一些情况下导致高尿酸血症,如痛风和肿瘤溶解综合征的发生。据保守估计,中国现有的高尿酸血症患者达到了1.2亿,高尿酸血症已成为继高血压、高血糖、高血脂之后的第四高。痛风的定义是血清UA水平超过体液中的UA溶解度的一种炎症性关节炎,高于6.8mg/dL的血清UA水平可导致组织中UA结晶形成,从而引起急性炎性反应。痛风导致慢性疼痛、在工作和家庭生活中的功能障碍、以及健康相关的生活质量受损等。肿瘤溶解综合征(TLS)是指肿瘤细胞短期内大量溶解释放细胞内代谢产物,引起以高尿酸血症、高血钾、高血磷、低血钙和急性肾衰竭为主要表现的一组临床综合征。
Uric acid oxidase (uricase) is a class of enzymes that catalyze the oxidation of uric acid to the more readily soluble allantoin. Allantoin is a purine metabolite that is more easily excreted. It is a homotetrameric enzyme composed of four identical 34KDa subunits, which serves as the starting material to initiate a series of reactions that convert uric acid into a more soluble and excreted product (allantoin), uric acid The enzyme catalyzes the reaction of uric acid (UA) with O 2 and H 2 O to form 5-hydroxy-isourate (HIU) and the release of H 2 O 2 . HIU is an unstable product that undergoes non-enzymatic hydrolysis to 2-oxo-4-hydroxy-5-ureidoimidazole (OHCU) and subsequent spontaneous decarboxylation to form racemic allantoin. Two additional enzymes (HIU hydrolase and OHCU decarboxylase) were expressed in species containing functional uricase, which catalyzed these reactions more rapidly to produce (s)-allantoin. Functional uricases are found in a wide variety of organisms, such as archaea, bacteria and eukaryotes. However, uricase is not expressed in humans and some primates. This is due to a nonsense mutation at codon 33, codon 187, and a mutation at the splice acceptor site in intron 2. In conclusion, the lack of uricase expression in humans results in high systemic UA levels and, in some cases, hyperuricemia, such as gout and the development of tumor lysis syndrome. According to conservative estimates, there are 120 million patients with hyperuricemia in China, and hyperuricemia has become the fourth highest after hypertension, hyperglycemia, and hyperlipidemia. Gout is defined as an inflammatory arthritis in which serum UA levels exceed the solubility of UA in body fluids. Serum UA levels higher than 6.8 mg/dL can lead to the formation of UA crystals in tissues, resulting in an acute inflammatory response. Gout causes chronic pain, functional impairment in work and family life, and impaired health-related quality of life. Tumor lysis syndrome (TLS) refers to a group of clinical manifestations mainly manifested by hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia and acute renal failure due to the massive lysis of tumor cells and the release of intracellular metabolites in a short period of time. syndrome.
本发明的尿酸酶包含如SEQ ID NO:9所示的氨基酸序列或其保留了酶活性的变体,其中所述变体相对于SEQ ID NO:9的突变包括:N末端17个以内氨基酸残基替换和/或C末端10个以内氨基酸残基替换。本发明尿酸酶的变体相对于SEQ ID NO:9的突变还可包括在选自35、82、142、194、216、287和288位的1、2、3、4或5个位置中引入1、2、3、4或5个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号。本发明尿酸酶的变体相对于SEQ ID NO:9的突变还可包括:第205位和/或第208位的取代突变,其中位置编号是相对于SEQ ID NO:3的位置编号。The uricase of the present invention comprises the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof that retains the enzymatic activity, wherein the mutation of the variant relative to SEQ ID NO: 9 includes: amino acid residues within 17 N-terminal Base substitution and/or amino acid residue substitution within 10 C-terminal. The variants of the uricase of the invention may also include mutations relative to SEQ ID NO: 9 introduced at 1, 2, 3, 4 or 5 positions selected from the group consisting of positions 35, 82, 142, 194, 216, 287 and 288 1, 2, 3, 4 or 5 surface accessible cysteine residues, wherein the position numbering is relative to that of SEQ ID NO:3. Mutations of the variants of the uricase of the present invention relative to SEQ ID NO:9 may also include: substitution mutations at positions 205 and/or 208, wherein the position numbering is relative to the position numbering of SEQ ID NO:3.
在优选的实施方案中,所述变体相对于SEQ ID NO:9的突变包括:N末端氨基酸序列MTATAETSTGTKVVLGQ替换为MANIILGK,和/或C末端氨基酸序列SRADHPIWSN替换为C。In a preferred embodiment, the mutation of the variant relative to SEQ ID NO: 9 comprises: the N-terminal amino acid sequence MTATAETSTGTKVVLGQ is replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C.
在优选的实施方案中,所述尿酸酶变体相对于SEQ ID NO:9的突变还可包括在选自35、82、142、194、216、287和288位的2个位置上引入2个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号。在进一步优选的实施方案中,所述变体在以下两个位置中包含半胱氨酸残基:142和216位,35和288位,35和142位,216和288位,35和194位,或82和287位,其中位置编号是相对于SEQ ID NO:3的位置编号。In a preferred embodiment, the mutation of the uricase variant relative to SEQ ID NO:9 may further comprise the introduction of 2 at 2 positions selected from the group consisting of positions 35, 82, 142, 194, 216, 287 and 288 Surface accessible cysteine residues, wherein the position numbering is relative to that of SEQ ID NO:3. In a further preferred embodiment, the variant comprises cysteine residues in the following two positions: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194 , or positions 82 and 287, where the position number is relative to that of SEQ ID NO:3.
在优选的实施方案中,所述尿酸酶的变体相对于SEQ ID NO:9的突变还包括第205 位的S被取代成D或E、第208位的G被取代成R、K或S。在一些实施方式中,所述取代突变为S205D或G208R或其组合。In a preferred embodiment, the variant of the uricase with respect to the mutation of SEQ ID NO: 9 further comprises that the S at position 205 is substituted with D or E, and the G at position 208 is substituted with R, K or S . In some embodiments, the substitution mutation is S205D or G208R or a combination thereof.
在优选的实施方案中,所述尿酸酶的变体的氨基酸序列分别包含如SEQ ID NO:10-20中任一所示的序列,其中,SEQ ID NO:12-20的氨基酸序列如下所示:In a preferred embodiment, the amino acid sequences of the uricase variants respectively comprise the sequences shown in any one of SEQ ID NOs: 10-20, wherein the amino acid sequences of SEQ ID NOs: 12-20 are shown below :
AG-2C12/AG-2C12-PEG(SEQ ID NO:12,Cys突变基于SEQ ID NO:9)AG-2C12/AG-2C12-PEG (SEQ ID NO: 12, Cys mutation based on SEQ ID NO: 9)
AG-2C13/AG-2C13-PEG(SEQ ID NO:13,Cys突变基于SEQ ID NO:10)AG-2C13/AG-2C13-PEG (SEQ ID NO: 13, Cys mutation based on SEQ ID NO: 10)
AG-2C16(SEQ ID NO:14,Cys突变基于SEQ ID NO:9)AG-2C16 (SEQ ID NO: 14, Cys mutation based on SEQ ID NO: 9)
AG-2C17(SEQ ID NO:15,Cys突变基于SEQ ID NO:10)AG-2C17 (SEQ ID NO: 15, Cys mutation based on SEQ ID NO: 10)
AG-2C13-1(SEQ ID NO:16,Cys突变基于SEQ ID NO:10)AG-2C13-1 (SEQ ID NO: 16, Cys mutation based on SEQ ID NO: 10)
AG-2C01-2/AG-2C01-2-PEG(SEQ ID NO:17,Cys突变基于SEQ ID NO:11)AG-2C01-2/AG-2C01-2-PEG (SEQ ID NO: 17, Cys mutation based on SEQ ID NO: 11)
AG-2C05-1(SEQ ID NO:18,Cys突变基于SEQ ID NO:9)AG-2C05-1 (SEQ ID NO: 18, Cys mutation based on SEQ ID NO: 9)
AG-2C01-1(SEQ ID NO:19,Cys突变基于SEQ ID NO:11)AG-2C01-1 (SEQ ID NO: 19, Cys mutation based on SEQ ID NO: 11)
AG-2C12-1(SEQ ID NO:20,Cys突变基于SEQ ID NO:9)AG-2C12-1 (SEQ ID NO:20, Cys mutation based on SEQ ID NO:9)
尿酸酶偶联物(修饰的尿酸酶)Uricase Conjugates (Modified Uricases)
本发明的尿酸酶偶联物含有本文所述的尿酸酶和与该尿酸酶共价连接或融合表达的聚合物。在一些实施方式中,所述聚合物选自聚乙二醇(PEG)、磷酰胆碱聚合物、重复肽或“X-TEN”序列的聚合物和基于碳水化合物的聚合物。在一些实施方式中,所述聚合 物与所述尿酸酶的N-末端和/或C-末端融合表达,或通过所述尿酸酶上存在的半胱氨酸残基进行共价连接。The uricase conjugates of the present invention comprise a uricase as described herein and a polymer covalently linked or fused to the uricase. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), phosphorylcholine polymers, polymers of repeating peptides or "X-TEN" sequences, and carbohydrate-based polymers. In some embodiments, the polymer is expressed fused to the N-terminus and/or C-terminus of the uricase, or covalently linked through a cysteine residue present on the uricase.
本文中,聚乙二醇(PEG)是具有结构H-(O-CH
2-CH
2)
n-OH的聚醚化合物。最通常用于蛋白质共价连接的PEG试剂是具有结构CH
3-O-(CH
2-CH
2-O)
n-X的单甲氧基聚(乙二醇)衍生物,其中X含有线性接头和反应性官能团(线性PEG),n为110-18200的整数。在某些情况下,X可以含有支化元件,这样使得PEG试剂含有一个反应性官能团和多于一个PEG聚合物链(支化PEG)或多于一个反应性官能团和PEG聚合物链(叉状PEG)。PEG试剂可以包含约5、10、20、40、60和80KDa的总PEG聚合物。
Herein, polyethylene glycol (PEG) is a polyether compound having the structure H-(O- CH2 - CH2 ) n -OH. The PEG reagents most commonly used for covalent attachment of proteins are monomethoxypoly(ethylene glycol) derivatives with the structure CH3 -O-( CH2 - CH2 -O) n -X, where X contains a linear linker and reactive functional groups (linear PEG), n is an integer from 110 to 18200. In some cases, X may contain branching elements such that the PEG reagent contains one reactive functional group and more than one PEG polymer chain (branched PEG) or more than one reactive functional group and PEG polymer chain (branched PEG) PEG). PEG reagents can comprise total PEG polymers of about 5, 10, 20, 40, 60 and 80 KDa.
在一些实施例中,硫醇反应性PEG可以用于与至少一个半胱氨酸上的硫醇基团反应。例如,可以使用PEG-马来酰亚胺,以及PEG-邻吡啶基二硫化物,PEG-乙烯基砜和PEG-碘乙酰胺。在其他实施方式中,硫醇反应性PEG可以是带有单个硫醇反应性部分的直链或支链结构,或者可以是每个PEG分子带有两个或两个以上反应性基团的叉状结构。In some embodiments, a thiol-reactive PEG can be used to react with a thiol group on at least one cysteine. For example, PEG-maleimide can be used, as well as PEG-o-pyridyl disulfide, PEG-vinyl sulfone, and PEG-iodoacetamide. In other embodiments, the thiol-reactive PEG can be a linear or branched structure with a single thiol-reactive moiety, or can be a fork with two or more reactive groups per PEG molecule like structure.
本领域已知多种方法可以PEG化尿酸酶中的一个或多个半胱氨酸。本发明均可采用。Various methods are known in the art to PEGylate one or more cysteines in a uricase. The present invention can be used.
在优选的实施方案中,所述聚合物为PEG,本发明所述的尿酸酶偶联物为PEG修饰的尿酸酶,本文中也表示为PEG-尿酸酶。PEG可通过所述尿酸酶上存在的半胱氨酸残基进行共价连接。优选地,尿酸酶在选自35、82、142、194、216、287和288位的1、2、3、4或5个位置中具有1、2、3、4或5个表面可及的半胱氨酸残基,其中位置编号是相对于SEQ ID NO:3的位置编号,所述PEG通过这些位置上的半胱氨酸残基进行共价连接。更优选地,所述的尿酸酶分别在以下两个位置中包含半胱氨酸残基:142和216位,35和288位,35和142位,216和288位,35和194位,或82和287位,其中位置编号是相对于SEQ ID NO:3的位置编号,所述PEG通过这些位置上的半胱氨酸残基进行共价连接。In a preferred embodiment, the polymer is PEG, and the uricase conjugate of the present invention is a PEG-modified uricase, also referred to herein as PEG-uricase. PEG can be covalently attached through cysteine residues present on the uricase. Preferably, the uricase has 1, 2, 3, 4 or 5 surface accessible uricases in 1, 2, 3, 4 or 5 positions selected from positions 35, 82, 142, 194, 216, 287 and 288 Cysteine residues, where the position numbers are relative to those of SEQ ID NO:3, through which the PEG is covalently attached. More preferably, the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to that of SEQ ID NO: 3, and the PEG is covalently attached through cysteine residues at these positions.
在一些方案中,所述尿酸酶的氨基酸序列如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20所示,所述PEG分别通过所述氨基酸序列的第142和216、35和288、35和142、216和288、35和288、35和194、82和287、35和194以及142和216位共价连接所述氨基酸序列,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the amino acid sequence of the uricase is such as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 17, ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, the PEG passes through the 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 288, Positions 35 and 194, 82 and 287, 35 and 194, and 142 and 216 are covalently linked to the amino acid sequence, wherein the position numbers are relative to SEQ ID NO:3.
在一些方案中,所述尿酸酶的氨基酸序列如SEQ ID NO:17所示,所述PEG通过35和194的半胱氨酸残基进行共价连接,其中位置编号是相对于SEQ ID NO:3的位置编号。In some aspects, the amino acid sequence of the uricase is set forth in SEQ ID NO: 17, and the PEG is covalently linked through cysteine residues 35 and 194, wherein position numbering is relative to SEQ ID NO: 3 position number.
疾病及其治疗或预防Diseases and their treatment or prevention
本发明还提供了用本发明的尿酸酶或尿酸酶偶联物(如PEG-尿酸酶)治疗或预防哺乳动物体液或组织中高尿酸血症的方法。该方法通常包括给与需要的对象治疗或预防有效量的本发明任一实施方式所述的改进的尿酸酶或尿酸酶偶联物(如PEG-尿酸酶),或含有所述尿酸酶或尿酸酶偶联物(如PEG-尿酸酶)的药物组合物。可根据实际情况选择合适的给药方式,包括但不限于口服、静脉内、皮下、肌内、动脉内、通过吸入、气雾剂递送。The present invention also provides methods of treating or preventing hyperuricemia in mammalian body fluids or tissues using the uricases or uricase conjugates (eg, PEG-uricase) of the present invention. The method generally comprises administering to a subject in need thereof a therapeutically or prophylactically effective amount of the improved uricase or uricase conjugate (eg, PEG-uricase) of any embodiment of the present invention, or containing the uricase or uricase Pharmaceutical compositions of enzyme conjugates such as PEG-uricase. Appropriate modes of administration can be selected according to the actual situation, including but not limited to oral, intravenous, subcutaneous, intramuscular, intraarterial, inhalation, and aerosol delivery.
受试者通常患有与尿酸升高相关的疾病,即受益于尿酸的降低的疾病。通常,与尿酸升高相关的疾病,包括高尿酸血症,比如痛风和肿瘤溶解综合征。The subject typically suffers from a disease associated with elevated uric acid, ie, a disease that benefits from a reduction in uric acid. Often, conditions associated with elevated uric acid include hyperuricemia, such as gout and tumor lysis syndrome.
医药制剂Pharmaceutical preparations
本发明所述的药物组合物是一种含有尿酸酶或尿酸酶偶联物的高稳定性药物组合物。本发明发现海藻糖能明显提高药物组合物的稳定性。The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing uricase or a uricase conjugate. The present invention finds that trehalose can obviously improve the stability of the pharmaceutical composition.
本发明所述的药物组合物是一种具有高稳定性的液体性制剂。特别地,本发明发现含有磷酸盐缓冲液的制剂稳定性明显高于其它缓冲液。The pharmaceutical composition of the present invention is a liquid preparation with high stability. In particular, the present inventors found that formulations containing phosphate buffers were significantly more stable than other buffers.
本发明提供了一种药物组合物,包含:(1)缓冲液;(2)本发明任一实施方案所述的尿酸酶或尿酸酶偶联物。The present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) the uricase or uricase conjugate according to any embodiment of the present invention.
本发明药物组合物中的尿酸酶或尿酸酶偶联物可以是包含如SEQ ID NO:3-20中任一所示的氨基酸序列的尿酸酶或其偶联物,尤其是PEG修饰的尿酸酶。The uricase or uricase conjugate in the pharmaceutical composition of the present invention can be a uricase comprising the amino acid sequence shown in any of SEQ ID NOs: 3-20 or a conjugate thereof, especially a PEG-modified uricase .
本发明的药物组合物中,尿酸酶或尿酸酶偶联物的浓度约为1-300mg/mL,优选约为1-250mg/mL,优选约为1-200mg/mL,优选约为5-80mg/mL,更优选5-40mg/mL;更优选5-20mg/mL;更优选地,上述尿酸酶浓度约为5mg/mL,6mg/mL,7mg/mL,8mg/mL,9mg/mL,10mg/mL,12mg/mL,14mg/mL,16mg/mL,18mg/mL,20mg/mL,25mg/mL,30mg/mL,35mg/mL,40mg/mL,50mg/mL,60mg/mL,70mg/mL,80mg/mL,90mg/mL,100mg/mL,110mg/mL,120mg/mL,130mg/mL或140mg/mL,优选约为8mg/mL,10mg/mL或20mg/mL。In the pharmaceutical composition of the present invention, the concentration of uricase or uricase conjugate is about 1-300 mg/mL, preferably about 1-250 mg/mL, preferably about 1-200 mg/mL, preferably about 5-80 mg /mL, more preferably 5-40mg/mL; more preferably 5-20mg/mL; more preferably, the above-mentioned uricase concentration is about 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg /mL, 12mg/mL, 14mg/mL, 16mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL , 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL or 140mg/mL, preferably about 8mg/mL, 10mg/mL or 20mg/mL.
本发明药物组合物中的缓冲液可选自磷酸盐缓冲液和Tris盐酸缓冲液,用以为本发明的药物组合物提供6.0-8.5,优选约为6.5-8.5,优选约为7.0-8.5,优选约为7.5-8.5,优选约为7.5-8.1,更优选为约7.8的pH。另一方面,用于本发明药物组合物中的缓冲液的pH可为6.0-8.5,优选约为6.5-8.5,优选约为7.0-8.5,优选约为7.5-8.5,优选约为7.5-8.1,更优选为约7.8。The buffer in the pharmaceutical composition of the present invention can be selected from phosphate buffer and Tris hydrochloric acid buffer to provide the pharmaceutical composition of the present invention with 6.0-8.5, preferably about 6.5-8.5, preferably about 7.0-8.5, preferably A pH of about 7.5-8.5, preferably about 7.5-8.1, more preferably about 7.8. On the other hand, the pH of the buffer used in the pharmaceutical composition of the present invention may be 6.0-8.5, preferably about 6.5-8.5, preferably about 7.0-8.5, preferably about 7.5-8.5, preferably about 7.5-8.1 , more preferably about 7.8.
本发明药物组合物中特别优选的缓冲液是磷酸盐缓冲液。优选地,该磷酸盐缓冲液的pH为约7.0到约8.5,优选约为7.5-8.5,更优选为约7.5-8.1。优选地,所述磷酸盐缓冲液 为磷酸氢二钠-磷酸二氢钠缓冲液。在一些方案中,磷酸盐缓冲液由1-30mM的磷酸氢二钠和1-30mM的磷酸二氢钠制成。在一些方案中,磷酸盐缓冲液由17mM的磷酸氢二钠和3mM的一水磷酸二氢钠制成。在一些方案中,磷酸盐缓冲液由18.5mM的磷酸氢二钠和1.5mM的一水磷酸二氢钠制成。A particularly preferred buffer in the pharmaceutical compositions of the present invention is a phosphate buffer. Preferably, the pH of the phosphate buffer is about 7.0 to about 8.5, preferably about 7.5-8.5, more preferably about 7.5-8.1. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some protocols, the phosphate buffer is made up of 1-30 mM disodium phosphate and 1-30 mM sodium dihydrogen phosphate. In some protocols, the phosphate buffer is made up of 17 mM disodium phosphate and 3 mM sodium dihydrogen phosphate monohydrate. In some protocols, the phosphate buffer is made up of 18.5 mM disodium phosphate and 1.5 mM sodium dihydrogen phosphate monohydrate.
本发明药物组合物中的缓冲液可以是Tris盐酸缓冲液。在一些方案中,Tris盐酸缓冲液由1-30mM的HCl和1-30mM的Tris(三羟甲基氨基甲烷)制成。在一些方案中,Tris盐酸缓冲液由11.8mM的Tris-HCl和8.2mM的Tris制成。在一些方案中,Tris盐酸缓冲液由16.5mM的Tris-HCl和3.5mM的Tris制成。在一些方案中,Tris盐酸缓冲液由20mM Tris,添加浓盐酸调节PH到7.5或8.0制成。The buffer in the pharmaceutical composition of the present invention can be Tris hydrochloric acid buffer. In some protocols, the Tris hydrochloride buffer is made up of 1-30 mM HCl and 1-30 mM Tris (tris. In some protocols, the Tris HCl buffer is made up of 11.8 mM Tris-HCl and 8.2 mM Tris. In some protocols, the Tris HCl buffer is made up of 16.5 mM Tris-HCl and 3.5 mM Tris. In some protocols, Tris hydrochloric acid buffer is made from 20 mM Tris, with the addition of concentrated hydrochloric acid to adjust the pH to 7.5 or 8.0.
因此,本发明的药物组合物中,缓冲液可以是pH为7.5-8.1的磷酸盐缓冲液,其在药物组合物中的浓度为10-30mM;且所述药物组合物含有5-40mg/mL的前文任一实施方案所述的尿酸酶或修饰的尿酸酶,尤其是本文所述的PEG修饰的尿酸酶AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG。Therefore, in the pharmaceutical composition of the present invention, the buffer can be a phosphate buffer with a pH of 7.5-8.1, and its concentration in the pharmaceutical composition is 10-30 mM; and the pharmaceutical composition contains 5-40 mg/mL The uricases or modified uricases of any of the preceding embodiments, especially the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG described herein.
在一些方案中,本发明的药物组合物还含有稳定剂。优选地,所述稳定剂选自盐酸精氨酸、氯化钠、甘露醇、蔗糖和海藻糖中的一种或多种;优选地,所述稳定剂选自甘露醇、海藻糖、甘露醇与氯化钠的组合或海藻糖与氯化钠的组合;更优选海藻糖或海藻糖与氯化钠的组合。本发明的药物组合物中稳定剂的浓度为约10mM-400mM,优选20mM-300mM,更优选50mM-250mM。在一些方案中,稳定剂为浓度约30-200mM、优选100-180的氯化钠;或所述稳定剂为浓度约100-300mM、优选200-300mM的甘露醇;或所述稳定剂为浓度约100-300mM、优选200-300mM的蔗糖;或所述稳定剂为浓度约100-300mM、优选200-300mM的海藻糖;或所述稳定剂为浓度约30-200mM、优选约100-180mM的盐酸精氨酸。在一些方案中,所述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇、优选为约30-100mM的氯化钠与约100-180mM的甘露醇的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖、优选为约30-100mM的氯化钠与约100-180mM的蔗糖的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的海藻糖、优选为约30-100mM的氯化钠与约100-180mM的海藻糖的组合。In some aspects, the pharmaceutical compositions of the present invention also contain stabilizers. Preferably, the stabilizer is selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose and trehalose; preferably, the stabilizer is selected from mannitol, trehalose, mannitol A combination with sodium chloride or a combination of trehalose and sodium chloride; more preferably trehalose or a combination of trehalose and sodium chloride. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10 mM to 400 mM, preferably 20 mM to 300 mM, more preferably 50 mM to 250 mM. In some aspects, the stabilizer is sodium chloride at a concentration of about 30-200 mM, preferably 100-180; or the stabilizer is mannitol at a concentration of about 100-300 mM, preferably 200-300 mM; or the stabilizer is a concentration of about 100-300 mM, preferably 200-300 mM sucrose; or the stabilizer is trehalose at a concentration of about 100-300 mM, preferably 200-300 mM; or the stabilizer is about 30-200 mM, preferably about 100-180 mM Arginine hydrochloride. In some aspects, the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol, preferably about 30-100 mM sodium chloride and about 100-180 mM mannitol; or The stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably about 30-100 mM sodium chloride and about 100-180 mM sucrose; or the stabilizer is about 30-200 mM of sodium chloride and about 30-200 mM trehalose, preferably about 30-100 mM sodium chloride and about 100-180 mM trehalose.
因此,在一些实施方案中,本发明的药物组合物含有:(a)10-30mM的缓冲液,其为pH为7.5-8.1的磷酸盐缓冲液;(b)5-40mg/mL的前文任一实施方案所述的尿酸酶或修饰的尿酸酶,尤其是本文所述的PEG修饰的尿酸酶AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG;以及(c)20mM-300mM的稳定剂,优选为浓度约200-300mM的甘露醇,或为约200-300mM的海藻糖,或为约30-100mM的氯化钠与约100-180mM的海藻糖的组合,或为约30-100mM的氯化钠与约100-180mM的甘露醇的组合。在一些实施方 案中,所述稳定剂为约200-300mM的海藻糖。在一些实施方案中,所述稳定剂为约30-100mM的氯化钠与约100-180mM的海藻糖的组合。Thus, in some embodiments, the pharmaceutical compositions of the present invention contain: (a) 10-30 mM buffer, which is a phosphate buffer at pH 7.5-8.1; (b) 5-40 mg/mL of any of the foregoing The uricases or modified uricases of one embodiment, especially the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG, and AG-2C12-PEG described herein; and (c) 20 mM - 300 mM stabilizer, preferably mannitol at a concentration of about 200-300 mM, or about 200-300 mM trehalose, or about 30-100 mM sodium chloride in combination with about 100-180 mM trehalose, or about About 30-100 mM sodium chloride in combination with about 100-180 mM mannitol. In some embodiments, the stabilizer is about 200-300 mM trehalose. In some embodiments, the stabilizer is about 30-100 mM sodium chloride in combination with about 100-180 mM trehalose.
在一些方案中,本发明的药物组合物还包括表面活性剂。优选的表面活性剂选自聚山梨醇酯80、聚山梨醇酯20和泊洛沙姆188。最优选的表面活性剂是聚山梨醇酯80。以w/v计,本发明药物组合物中表面活性剂的浓度约为0.001%-0.1%,优选约为0.02%-0.08%,优选约为0.02%-0.04%。作为非限制性实施例,本发明药物组合物中表面活性剂的浓度约为0.02%,0.04%或0.08%,优选0.02%。In some aspects, the pharmaceutical compositions of the present invention further include a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. The concentration of surfactant in the pharmaceutical composition of the present invention is about 0.001%-0.1%, preferably about 0.02%-0.08%, preferably about 0.02%-0.04%, on a w/v basis. By way of non-limiting example, the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04% or 0.08%, preferably 0.02%.
因此,在一些实施方案中,本发明的药物组合物含有:缓冲液,其为pH为7.5-8.1的磷酸盐缓冲液,其在药物组合物中的浓度为10-30mM;5-40mg/mL的前文任一实施方案所述的尿酸酶或修饰的尿酸酶,尤其是本文所述的PEG修饰的尿酸酶AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG;100mM-300mM的稳定剂,优选地,所述稳定剂为约30-100mM的氯化钠与约100-180mM的海藻糖的组合,或为约200-300mM的海藻糖;以及以w/v计0.02%-0.04%的聚山梨醇酯80。Thus, in some embodiments, the pharmaceutical composition of the present invention contains: a buffer, which is a phosphate buffer at pH 7.5-8.1, at a concentration of 10-30 mM in the pharmaceutical composition; 5-40 mg/mL The uricases or modified uricases of any of the preceding embodiments, especially the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG described herein; 100 mM- 300 mM stabilizer, preferably about 30-100 mM sodium chloride in combination with about 100-180 mM trehalose, or about 200-300 mM trehalose; and 0.02% w/v - 0.04% polysorbate 80.
本发明的药物组合物可以是液体制剂,或者是冻干制剂。The pharmaceutical composition of the present invention can be a liquid preparation or a lyophilized preparation.
医药用途和方法Medicinal uses and methods
本发明还提供了用于降低哺乳动物体液和组织中尿酸水平或治疗或预防高尿酸血症的本发明任一实施方案所述的尿酸酶或尿酸酶偶联物或药物组合物,本发明任一实施方案所述的尿酸酶或尿酸酶偶联物或药物组合物在制备降低哺乳动物体液和组织中尿酸水平或治疗高尿酸血症的药物中的用途,以及给予需要的个体或患者治疗有效量的本发明任一实施方案所述的尿酸酶或尿酸酶偶联物或药物组合物以降低哺乳动物体液和组织中尿酸水平或治疗高尿酸血症。The present invention also provides the uricase or uricase conjugate or pharmaceutical composition according to any embodiment of the present invention for reducing the level of uric acid in body fluids and tissues of mammals or treating or preventing hyperuricemia. Use of the uricase or uricase conjugate or pharmaceutical composition described in one embodiment in the preparation of a medicament for reducing the level of uric acid in mammalian body fluids and tissues or treating hyperuricemia, and administering to an individual or patient in need of treatment effective amount of the uricase or uricase conjugate or pharmaceutical composition of any of the embodiments of the present invention to reduce uric acid levels in body fluids and tissues of mammals or to treat hyperuricemia.
下文将以具体实施例的方式阐述本发明。应理解,这些实施例仅仅是阐述性的,并非意图限制本发明的范围。下述实施例将使用到以下方法。The invention will hereinafter be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The following examples will use the following methods.
蛋白含量检测方法protein content detection method
蛋白浓度使用紫外分光光度计来检测。将百分比消光系数(E1%)设定在1.221(g/ml)-1cm-
1。使用BIO MATE 3S仪器。每个样品平行测定2份溶液,每份溶液重复测定3次,取平均值。通过消光系数及OD值计算出对应检品的浓度。
Protein concentration was detected using a UV spectrophotometer. The percent extinction coefficient (E1%) was set at 1.221 (g/ml)-1 cm- 1 . Use a BIO MATE 3S instrument. Two solutions were measured in parallel for each sample, and each solution was repeated three times, and the average value was taken. The concentration of the corresponding sample was calculated from the extinction coefficient and OD value.
体积排阻色谱尺寸排阻色谱(SEC-HPLC)纯度检测方法Size Exclusion Chromatography Size Exclusion Chromatography (SEC-HPLC) Purity Detection Method
SEC-HPLC纯度采用安装了SEC色谱柱(TOSOH G4000sxl SEC 7.8mm ID x 30cm, 8μm)的HPLC(Waters e2695仪器)进行检测。流动相组成为50mM Tris,150mM NaCl,pH 7.4。采用面积归一化法计算主峰,高聚物和低聚物的相对百分比。详细见下表:SEC-HPLC purity was checked using HPLC (Waters e2695 instrument) fitted with an SEC column (TOSOH G4000sxl SEC 7.8mm ID x 30cm, 8μm). The mobile phase composition was 50 mM Tris, 150 mM NaCl, pH 7.4. Area normalization was used to calculate the relative percentages of major peaks, polymers and oligomers. See the table below for details:
色谱条件Chromatographic conditions | 色谱参数Chromatographic parameters |
检测器波长Detector wavelength | 280nm280nm |
自动进样器温度Autosampler temperature |
25±3℃25±3 |
柱温column temperature | 5±3℃5±3℃ |
流速flow rate | 0.50ml/min0.50ml/min |
进样体积Injection volume | 25μl25μl |
运行模式operating mode | 等度模式isocratic mode |
运行时间operation hours | 30.00min30.00min |
反相高效液相色谱(RP-HPLC)纯度检测方法Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Purity Detection Method
RP-HPLC纯度采用安装了反相色谱柱(Advancebio RP-mAb C4 4.6mm ID x 150mm,3.5μm)的HPLC(Agilent 1260)进行检测。流动相组成为A相:0.1%TFA水溶液;B相:0.1%TFA ACN。采用面积归一化法计算双PEG修饰蛋白峰,单PEG修饰蛋白峰,裸蛋白的相对百分比。详细见下表:RP-HPLC purity was checked using HPLC (Agilent 1260) fitted with a reversed phase column (Advancebio RP-mAb C4 4.6 mm ID x 150 mm, 3.5 μm). The mobile phase composition is A phase: 0.1% TFA aqueous solution; B phase: 0.1% TFA ACN. The area normalization method was used to calculate the relative percentages of double-PEG-modified protein peaks, single-PEG-modified protein peaks, and naked protein. See the table below for details:
非还原十二烷基硫酸钠毛细管电泳(NR-CE-SDS)纯度检测方法Non-reducing sodium dodecyl sulfate capillary electrophoresis (NR-CE-SDS) purity detection method
NR-CE-SDS纯度采用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)进行检测。供试品溶液(1mg/mL)95μL加入0.8M碘乙酰胺5μL,70℃孵育变性热处理5min,使用毛细管电泳仪(Maurice)检测。以蛋白、单PEG修饰蛋白与双PEG修饰蛋白的修正峰面积分别占所有修正峰面积之和的百分比分别计算蛋白、单PEG修饰蛋白峰与双PEG修饰蛋白峰的纯度。The NR-CE-SDS purity was checked using a capillary electrophoresis apparatus (Maurice Instruments) equipped with a CE-SDS cartridge. Add 5 μL of 0.8M iodoacetamide to 95 μL of the test solution (1 mg/mL), incubate at 70° C. for denaturation and heat treatment for 5 min, and use capillary electrophoresis (Maurice) to detect. The purity of protein, single-PEG-modified protein peak and double-PEG-modified protein peak were calculated as the percentage of corrected peak area of protein, single-PEG-modified protein and double-PEG-modified protein respectively to the sum of all corrected peak areas.
酶活检测方法Enzyme activity detection method
将尿酸溶液用缓冲液稀释至300μM,250μL/孔加至
平底黑色微孔板中,将微孔板放入预先设定为30℃的Multi-Mode Microplate Reader酶标仪中,平衡10min后将稀释至8.0μg/mL的样品以50μL/孔的量快速转移至加入了尿酸溶液的微孔板中,震动15s后,在292nm条件下每2min读数一次,共读20min。
Dilute the uric acid solution to 300 μM with buffer, add 250 μL/well to In a flat-bottomed black microplate, put the microplate into a Multi-Mode Microplate Reader microplate reader pre-set at 30 °C, equilibrate for 10 minutes, and quickly transfer the sample diluted to 8.0 μg/mL at 50 μL/well. To the microplate with uric acid solution added, after shaking for 15s, read once every 2min under the condition of 292nm for a total of 20min.
缓冲液:35.3mM Tris碱;14.7mM Tris HCl;150mM NaCl,1.3mM EDTA(乙二胺四乙酸);0.02%聚山梨酯80;220mM蔗糖,最终pH为8.4±0.2。Buffer: 35.3 mM Tris base; 14.7 mM Tris HCl; 150 mM NaCl, 1.3 mM EDTA (ethylenediaminetetraacetic acid); 0.02% polysorbate 80; 220 mM sucrose, final pH 8.4 ± 0.2.
实施例中所用到的其它方法和材料,除非另有说明,否则为本领域的常规方法和材料。Other methods and materials used in the examples, unless otherwise stated, are conventional methods and materials in the art.
实施例Example
实施例1:尿酸酶表达载体的构建Example 1: Construction of uricase expression vector
合成尿酸酶核苷酸序列(苏州金唯智生物科技科技有限公司)并构建到PET28a表达载体上。该构建目的基因的5'酶切位点是NcoI,3'酶切位点是XhoI。The uricase nucleotide sequence was synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.) and constructed into the PET28a expression vector. The 5' enzyme cleavage site of the constructed target gene is NcoI, and the 3' enzyme cleavage site is XhoI.
以AG-2C13分子构建为例,密码子优化后核苷酸序列为:Taking the construction of AG-2C13 molecule as an example, the nucleotide sequence after codon optimization is:
尿酸酶的表达:Expression of uricase:
通过大肠杆菌BL21(DE3)(Merck)转化实验,并在LB/Kana平板上挑取转化后单克隆菌落。使含有尿酸酶表达载体的大肠杆菌在5ml的LB液体培养基(含有50um/ml的卡那霉素)中37℃培养过夜,将上述种子液接种到含有200ml LB培养基的摇瓶中,接种比例为3%,37摄氏度培养。当菌体生长至OD 600为0.6-0.8后,培养温度降至25摄氏度。加入IPTG使其终浓度为0.3mM诱导表达目标尿酸酶蛋白。The transformation experiment was carried out by Escherichia coli BL21(DE3) (Merck), and the transformed monoclonal colonies were picked on LB/Kana plates. Make the Escherichia coli containing uricase expression vector in 5ml LB liquid culture medium (containing 50um/ml kanamycin) in 37 DEG C and cultivate overnight, the above-mentioned seed liquid is inoculated in the shake flask containing 200ml LB culture medium, inoculate The ratio is 3% and cultured at 37 degrees Celsius. When the cells grew to an OD 600 of 0.6-0.8, the culture temperature was lowered to 25 degrees Celsius. Add IPTG to a final concentration of 0.3 mM to induce the expression of the target uricase protein.
尿酸酶的纯化:Purification of uricase:
菌体用高压匀浆法或者超声波法破碎,经过离心处理后,用阴离子交换的方法进行进一步纯化,得到纯度>95%的蛋白,进行后续的表征分析。通过DNA测序,获得蛋白酶的氨基酸序列。The bacterial cells are broken by high-pressure homogenization or ultrasonication, and after centrifugation, they are further purified by anion exchange to obtain a protein with a purity of > 95% for subsequent characterization and analysis. The amino acid sequence of the protease was obtained by DNA sequencing.
以AG-00为例,验证其与优化后DNA序列一致,氨基酸序列为SEQ ID NO:1,如下所示:Take AG-00 as an example to verify that it is consistent with the optimized DNA sequence, and the amino acid sequence is SEQ ID NO: 1, as shown below:
对照尿酸酶的合成Synthesis of control uricase
采用与以上相似的方法合成对照尿酸酶MEDI4945,其氨基酸序列为SEQ ID NO:2。MEDI4945是国际申请号为PCT/US2016/032415中公开的尿酸酶,其氨基酸序列如下所示:The control uricase MEDI4945 was synthesized by a method similar to the above, and its amino acid sequence is SEQ ID NO: 2. MEDI4945 is the uricase disclosed in the international application number PCT/US2016/032415, and its amino acid sequence is as follows:
实施例2:微球菌属尿酸酶序列进化Example 2: Micrococcus uricase sequence evolution
A.优化C-末端A. Optimizing the C-terminus
将SEQ ID NO:1截短C-末端5个氨基酸,以便消除C-末端Cys,从而产生SEQ ID NO:3。即通过与实施例1相似的方法,设计DNA特异性引物,以AG-00序列为模板,通过PCR扩增删除其C末端的第298-302位氨基酸,并将构建产物进行DNA测序验证,得到的分子命名为:AG-00-1,序列如下所示:SEQ ID NO: 1 was truncated by 5 amino acids at the C-terminal end in order to eliminate the C-terminal Cys, resulting in SEQ ID NO: 3. That is, by a method similar to Example 1, DNA-specific primers were designed, and the AG-00 sequence was used as a template, and the 298-302nd amino acids at the C-terminus were deleted by PCR amplification, and the constructed product was subjected to DNA sequencing verification to obtain The molecule is named: AG-00-1, and the sequence is as follows:
B.N-末端氨基酸替换B. N-terminal amino acid substitutions
使用大于15种比对的尿酸酶数据库,发现SEQ ID NO:1中的第1-17位与同种属尿酸酶不是高度保守的,因此,通过与实施例1相似的方法,设计特异性引物,以AG-00为模板,通过PCR扩增用氨基酸序列(MTNIILGK)替换AG-00尿酸酶分子的第1-17位氨基酸,并将构建产物进行DNA测序进行验证,产生SEQ ID NO:4,分子命名为:AG-00-2,序列如下所示:Using a uricase database of more than 15 alignments, it was found that positions 1-17 in SEQ ID NO: 1 are not highly conserved with conspecific uricases, therefore, by a method similar to Example 1, specific primers were designed , using AG-00 as a template, replace the 1-17th amino acid of AG-00 uricase molecule with the amino acid sequence (MTNIILGK) by PCR amplification, and carry out the DNA sequencing of the construction product to verify, resulting in SEQ ID NO: 4, The molecule is named: AG-00-2 and the sequence is as follows:
C.定点突变C. Site-directed mutagenesis
通过与实施例1相同的方法,在AG-00序列的基础上,设计DNA特异性引物,通过搭桥PCR技术将第258位谷氨酰胺(Q)突变为丝氨酸(S),并将构建产物进行DNA测序验证,产生SEQ ID NO:5,命名为AG-01,序列如下所示:By the same method as in Example 1, on the basis of the AG-00 sequence, DNA-specific primers were designed, and the 258th glutamine (Q) was mutated to serine (S) by bridging PCR technology. DNA sequencing verification, resulting in SEQ ID NO: 5, named AG-01, the sequence is as follows:
采用相似的方法,将第175位谷氨酰胺(Q)突变为脯氨酸(P),178位苏氨酸(T)突变为谷氨酸(E),并将构建产物进行DNA测序验证,产生SEQ ID NO:6,命名为AG-02,序列如下所示:Using a similar method, the 175th glutamine (Q) was mutated to proline (P), and the 178th threonine (T) was mutated to glutamic acid (E), and the constructed product was verified by DNA sequencing, SEQ ID NO: 6 was generated, designated AG-02, and the sequence is as follows:
采用相似的方法,将第175位谷氨酰胺(Q)突变为脯氨酸(P)、第178位苏氨酸(T)突变为谷氨酸(E)和第258位谷氨酰胺(Q)突变为丝氨酸(S),并将构建产物进行DNA测序验证,产生SEQ ID NO:7,命名为AG-05,序列如下所示:Using a similar method, glutamine (Q) at position 175 was mutated to proline (P), threonine (T) at position 178 to glutamic acid (E), and glutamine at position 258 (Q ) was mutated to serine (S), and the construction product was verified by DNA sequencing, resulting in SEQ ID NO: 7, named AG-05, the sequence is as follows:
D.优化N-末端D. Optimizing the N-terminus
已知,当蛋白质在大肠杆菌中表达时,N-末端甲硫氨酸可以通过Met氨基肽酶除去,这取决于甲硫氨酸后的第二个氨基酸残基的大小,如果第二位是较小的残基则发生典型的裂解,如果第二位是体积较大的残基,则不发生裂解,或者如果第二位的残基既不是体积大的也不是特别小的,则Met氨基肽酶可裂解部分,从而产生不均一性。因此,为了消除原核生物表达导致的第一位氨基酸的不完全剪切,将SEQ ID NO:4的氨基酸序列中第二位苏氨酸(T)突变为丙氨酸(A),产生SEQ ID NO:8,命名为AG-00-2A。It is known that when the protein is expressed in E. coli, the N-terminal methionine can be removed by Met aminopeptidase, depending on the size of the second amino acid residue after the methionine, if the second position is Typical cleavage occurs for smaller residues, no cleavage if the second position is a bulky residue, or Met amino if the second position is neither bulky nor particularly small Peptidases can cleave the moiety, creating inhomogeneity. Therefore, in order to eliminate the incomplete cleavage of the first amino acid caused by prokaryotic expression, the second threonine (T) in the amino acid sequence of SEQ ID NO: 4 was mutated to alanine (A), resulting in SEQ ID NO: 4. NO: 8, named AG-00-2A.
SEQ ID NO:8SEQ ID NO: 8
E.优化C-末端、定点突变E. Optimized C-terminal, site-directed mutagenesis
通过与实施例1相同的方法,在AG-00序列的基础上,设计DNA特异性引物,通过PCR扩增删除其C末端的第298-302位氨基酸,并将第175位谷氨酰胺(Q)突变为脯氨酸(P)、第178位苏氨酸(T)突变为谷氨酸(E)和第258位谷氨酰胺(Q)突变为丝氨酸(S),将构建产物进行DNA测序验证,产生SEQ ID NO:9,命名为AG-1,序列如下所示:By the same method as in Example 1, on the basis of the AG-00 sequence, a DNA-specific primer was designed, the amino acids 298-302 of its C-terminal were deleted by PCR amplification, and the 175th glutamine (Q ) to proline (P), threonine (T) at position 178 to glutamic acid (E), and glutamine (Q) at position 258 to serine (S), and the constructed product was subjected to DNA sequencing Verification, resulting in SEQ ID NO: 9, named AG-1, the sequence is as follows:
AG-1(SEQ ID NO:9)AG-1 (SEQ ID NO: 9)
F.进一步优化C-末端、定点突变F. Further optimization of C-terminal, site-directed mutagenesis
通过与实施例1相似的方法,设计DNA特异性引物,以AG-00序列为模板,通过PCR扩增删除其C末端的第288-301位氨基酸(共14个氨基酸),进一步精简蛋白结构,减少异源蛋白可能带来的免疫原性,同时,保留末端Cys,作为后续PEG修饰位点之一;将第175位谷氨酰胺(Q)突变为脯氨酸(P)、第178位苏氨酸(T)突变为谷氨酸(E)和第258位谷氨酰胺(Q)突变为丝氨酸(S),将构建产物进行DNA测序验证,产生SEQ ID NO:10,命名为AG-2,序列如下所示:By a method similar to Example 1, DNA-specific primers were designed, and the AG-00 sequence was used as a template, and the amino acids 288-301 of its C-terminus (14 amino acids in total) were deleted by PCR amplification to further simplify the protein structure, Reduce the possible immunogenicity of heterologous proteins, and at the same time, retain the terminal Cys as one of the subsequent PEG modification sites; mutate the 175th glutamine (Q) to proline (P), the 178th threonine Amino acid (T) was mutated to glutamic acid (E) and glutamine (Q) at position 258 was mutated to serine (S), and the constructed product was verified by DNA sequencing, resulting in SEQ ID NO: 10, named AG-2 , the sequence looks like this:
AG-2(SEQ ID NO:10)AG-2 (SEQ ID NO: 10)
G.优化C/N-末端、定点突变G. Optimization of C/N-terminal, site-directed mutagenesis
通过与实施例1相同的方法,在AG-00序列的基础上,设计DNA特异性引物,通过PCR扩增删除其C末端的第298-302位氨基酸,用氨基酸序列MANIILGK替换AG-00尿酸酶分子的第1-17位氨基酸,将第175位谷氨酰胺(Q)突变为脯氨酸(P)、第178位苏氨酸(T)突变为谷氨酸(E)和第258位谷氨酰胺(Q)突变为丝氨酸(S),将构建产物进行DNA测序验证,产生SEQ ID NO:11,命名为AG-3,序列如下所示:By the same method as in Example 1, on the basis of the AG-00 sequence, DNA-specific primers were designed, the amino acids 298-302 of the C-terminal were deleted by PCR amplification, and the amino acid sequence MANIILGK was used to replace the AG-00 uricase. Amino acids 1-17 of the molecule, mutating glutamine (Q) at position 175 to proline (P), threonine (T) at position 178 to glutamic acid (E) and glutamic acid at position 258 Amino amide (Q) was mutated to serine (S), and the constructed product was verified by DNA sequencing, resulting in SEQ ID NO: 11, named AG-3, and the sequence is as follows:
SEQ ID NO:11SEQ ID NO: 11
H.评估修饰的微球菌属尿酸酶的酶活性H. Assessing the Enzymatic Activity of Modified Micrococcus uricases
使用Tris-HCl缓冲体系(pH 8.5),先加入0.25mL尿酸溶液和0.375mL Tris-HCl缓冲液于试管中,于37℃水浴中保温10min,加入0.125ml适当稀释的上述尿酸酶溶液(0.004mg/mL)到试管中,准确反应10min后,加入0.05mL 20%KOH终止反应。测定剩余尿酸在292nm处吸光值,评价经修饰的微球菌属尿酸酶的酶活性,结果如图1所示,可见引入点突变并未影响尿酸酶的酶活性。Using Tris-HCl buffer system (pH 8.5), first add 0.25mL of uric acid solution and 0.375mL of Tris-HCl buffer to the test tube, incubate in a 37°C water bath for 10min, and add 0.125ml of appropriately diluted uricase solution above (0.004mg /mL) into a test tube, and after accurate reaction for 10 min, 0.05 mL of 20% KOH was added to terminate the reaction. The absorbance value of residual uric acid at 292 nm was measured, and the enzymatic activity of the modified Micrococcus uricase was evaluated. The results are shown in Figure 1. It can be seen that the introduction of point mutation did not affect the enzymatic activity of uricase.
尿酸氧化酶活性定义为:在37℃、pH8.5下,每分钟催化1umol尿酸分解所需要的酶量定义为1个酶活力单位。Uric acid oxidase activity was defined as: the amount of enzyme required to catalyze the decomposition of 1 umol of uric acid per minute at 37°C and pH 8.5 was defined as 1 unit of enzyme activity.
I.评估修饰的微球菌属尿酸酶的热稳定I. Assessing the Thermostability of Modified Micrococcus uricases
将经修饰的上述尿酸酶分子在50℃,60℃,70℃条件下水浴放置30分钟。37℃条件下,平衡20分钟,然后加入20ul的0.004mg/ml尿酸酶溶液后立即使用酶标仪进行读数,反应10分钟。测尿酸酶剩余酶活,结果以吸光度差值反应。以4℃放置尿酸酶酶活作归一化处理,结果如图2所示。测活体系:50ul浓度800uM的尿酸,30ul缓冲液。The modified uricase molecules were placed in a water bath at 50°C, 60°C, and 70°C for 30 minutes. Under the condition of 37°C, equilibrate for 20 minutes, then add 20ul of 0.004mg/ml uricase solution, immediately use a microplate reader for reading, and react for 10 minutes. The residual enzyme activity of uricase was measured, and the result was reacted by the difference in absorbance. The uricase enzyme activity was placed at 4°C for normalization, and the results are shown in Figure 2. Viability assay system: 50ul uric acid with a concentration of 800uM, 30ul buffer.
AG-00在60℃热处理半小时后,酶活保持47%,而AG-01、AG-02和AG-05在相同条件下处理,酶活保持率分别为60%,75%和78%。在70℃条件下处理,AG-00酶活保持为6%,尿酸酶AG-01、AG-02和AG-05的酶活保持率分别为17%,20%和15%。可见,点突变的引入提高了酶活热稳定性。After AG-00 was heat-treated at 60°C for half an hour, the enzyme activity retained 47%, while AG-01, AG-02, and AG-05 were treated under the same conditions, and the enzyme activity retention rates were 60%, 75%, and 78%, respectively. Under the condition of 70℃, the enzyme activity of AG-00 remained at 6%, and the retention rates of uricase AG-01, AG-02 and AG-05 were 17%, 20% and 15%, respectively. It can be seen that the introduction of point mutation improves the thermal stability of enzyme activity.
J.评估修饰的微球菌属尿酸酶的Tm值J. Assessing the Tm of Modified Micrococcus uricases
使用蛋白质Thermo Shift检测试剂盒评估蛋白分子的Tm值。用缓冲液(Na
2HPO
42.34g/L;NaH
2PO
4·H
2O 0.48g/L;蔗糖75.31g/L;Tween80 0.2g/L;pH 7.6±2)将上述尿酸酶稀释至0.5715mg/mL;将试剂盒中1000×荧光染料用超纯水稀释为8×荧光染料;取70μL的0.5715mg/mL尿酸酶与10μL的8×荧光染料在1.5mL EP管中混合均匀并分装至8联PCR管或96孔PCR板中,分装体积为30μL,共分装2个孔。另有空白对照样品由70μL制剂缓冲液与10μL的8×荧光染料混合而成,并分装至8联PCR管或96孔PCR板中,分装体积为30μL,共分装2个孔。另有系统空白对照由70μL超纯水、10μL的8×荧光染料混合而成,分装至8联PCR管或96孔PCR板中,分装体积为30μL,共分装2个孔。
The Tm values of protein molecules were evaluated using the Protein Thermo Shift Detection Kit. Dilute the above uricase to 0.5715 with buffer (Na 2 HPO 4 2.34g/L; NaH 2 PO 4 ·H 2 O 0.48g/L; sucrose 75.31g/L; Tween80 0.2g/L; pH 7.6±2) mg/mL; dilute the 1000× fluorescent dye in the kit with ultrapure water to 8× fluorescent dye; take 70 μL of 0.5715mg/mL uricase and 10 μL of 8× fluorescent dye in a 1.5 mL EP tube and mix them evenly and aliquot them. Dispense into 8 PCR tubes or 96-well PCR plates, with a volume of 30 μL, for a total of 2 wells. Another blank control sample was mixed with 70 μL of preparation buffer and 10 μL of 8× fluorescent dye, and was dispensed into 8 PCR tubes or 96-well PCR plates with a volume of 30 μL, and a total of 2 wells were dispensed. Another system blank control is mixed with 70μL of ultrapure water and 10μL of 8× fluorescent dye, and is dispensed into 8 PCR tubes or 96-well PCR plates with a volume of 30μL, and a total of 2 wells are dispensed.
结果如下表1示。The results are shown in Table 1 below.
可见,AG-01、AG-02和AG-05和AG-00-2的Tm D值(第一个蛋白变性的温度)均高于野生型尿酸酶分子AG-00和对照MEDI4945。可见,点突变的引入提高了尿酸酶的热稳定性。It can be seen that the Tm D values (the temperature of the first protein denaturation) of AG-01, AG-02, AG-05 and AG-00-2 were all higher than those of the wild-type uricase molecule AG-00 and the control MEDI4945. It can be seen that the introduction of point mutation improves the thermostability of uricase.
表1Table 1
序号serial number | 样品名称sample name | Tm D(℃)Tm D(℃) |
11 | MEDI4945MEDI4945 | 53.4553.45 |
22 | AG-00AG-00 | 58.1258.12 |
33 | AG-00-1AG-00-1 | 56.5956.59 |
44 | AG-00-2AG-00-2 | 61.9161.91 |
55 | AG-01AG-01 | 61.4061.40 |
66 | AG-02AG-02 | 58.9258.92 |
77 | AG-05AG-05 | 60.3860.38 |
实施例3:特异性PEG化Example 3: Specific PEGylation
聚乙二醇(PEG)修饰的治疗性蛋白质可随机附接至所选择的蛋白质残基(比如赖氨 酸侧链)或者对于特点位点具有特异性。而对于特定位点特异性的PEG化可以更好的控制并产生具有确定的生物活性的高度均匀的聚乙二醇化产物。在用于位点特异性附接的方法中,最广泛使用的是与未配对的半胱氨酸残基进行偶联,而这需要在蛋白质序列中引入一个或多个游离的半胱氨酸残基。而经C-末端截短的AG-00-1分子序列(SEQ ID NO:3)中不含有半胱氨酸,因此可通过氨基酸突变来引入半胱氨酸。Polyethylene glycol (PEG)-modified Therapeutic proteins can be randomly attached to selected protein residues (such as lysine side chains) or specific for specific sites. Whereas, site-specific PEGylation allows for better control and yields highly homogeneous PEGylated products with defined biological activities. Among the methods for site-specific attachment, the most widely used is conjugation to unpaired cysteine residues, which requires the introduction of one or more free cysteines into the protein sequence Residues. However, the C-terminal truncated AG-00-1 molecular sequence (SEQ ID NO: 3) does not contain cysteine, so cysteine can be introduced by amino acid mutation.
Cys突变引入Cys mutation introduced
为了选择AG-00-1序列中Cys残基引入的潜在位点,考虑以下几个因素:To select potential sites for introduction of Cys residues in the AG-00-1 sequence, several factors were considered:
(1)位点必须远离酶活性位点,以避免影响活性的风险;(1) The site must be far away from the active site of the enzyme to avoid the risk of affecting the activity;
(2)位点彼此不可靠近,以避免空间位阻使相邻Cys难以PEG化;(2) The sites should not be close to each other to avoid steric hindrance making adjacent Cys difficult to PEGylate;
(3)位点必须位于蛋白表面,以确保与硫醇反应性PEG试剂能发生有效的反应;(3) The site must be located on the surface of the protein to ensure an effective reaction with the thiol-reactive PEG reagent;
(4)位点必须不在结构区或保守区。(4) The site must not be in the structural region or the conserved region.
经过分析,发现满足以上要求的位点包括:A35、F82、S142、T194、E216、G287、和S288,因此,选择以上几个位点作为Cys突变的位点(以上位点相对于SEQ ID NO:3)。After analysis, it is found that the sites that meet the above requirements include: A35, F82, S142, T194, E216, G287, and S288. Therefore, the above sites are selected as the sites of Cys mutation (the above sites are relative to SEQ ID NO. : 3).
采用与实施例1相似的方法,在AG-1、AG-2和AG-3的基础上,分别引入Cys突变,制备如下分子:AG-2C12、AG-2C13、AG-2C16、AG-2C17、AG-2C13-1、AG-2C01、AG-2C05-1、AG-2C01-1和AG-2C12-1,其氨基酸序列分别如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20所示。Using a method similar to Example 1, on the basis of AG-1, AG-2 and AG-3, Cys mutations were introduced respectively to prepare the following molecules: AG-2C12, AG-2C13, AG-2C16, AG-2C17, AG-2C13-1, AG-2C01, AG-2C05-1, AG-2C01-1 and AG-2C12-1, whose amino acid sequences are respectively as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
蛋白稳定性研究Protein Stability Studies
各种改进尿酸酶在磷酸盐缓冲液中,pH7.0,1mM DTT,4℃条件下放置,使用SEC-HPLC和RP-HPLC比较各蛋白放置稳定性,结果如图3所示。Various improved uricases were placed in phosphate buffer, pH 7.0, 1 mM DTT, at 4°C, and SEC-HPLC and RP-HPLC were used to compare the placement stability of each protein. The results are shown in Figure 3.
RP-HPLC(图3,3a)结果显示:AG-2C13,AG-2C13-1,AG-2C12,AG-2C01-2纯度较好。SEC-HPLC(图3,3b)结果显示:AG-2C13,AG-2C17,AG-2C12,AG-2C01-2纯度较好。综合SEC-HPLC和RP-HPLC的结果,以AG-2C13,AG-2C12,AG-2C01-2分子作为优选。The results of RP-HPLC (Fig. 3, 3a) showed that: AG-2C13, AG-2C13-1, AG-2C12, AG-2C01-2 had better purity. The results of SEC-HPLC (Fig. 3, 3b) showed that: AG-2C13, AG-2C17, AG-2C12, AG-2C01-2 had better purity. Based on the results of SEC-HPLC and RP-HPLC, AG-2C13, AG-2C12 and AG-2C01-2 molecules are preferred.
尿酸酶PEG修饰Uricase PEGylation
向2mg的尿酸酶(100mM PBS缓冲液,pH=7.0,150mM NaCl)中依次加入TCEP.HCl(0.5mg,三(2-羧乙基)膦盐酸盐)、M-MAL-PEG-10K(27.5mg,马来酰亚胺聚乙二醇,分子量10000Da),室温反应2小时。反应液使用离子交换层析纯化即可得到聚乙二醇化的尿酸酶。To 2 mg of uricase (100 mM PBS buffer, pH=7.0, 150 mM NaCl), TCEP.HCl (0.5 mg, tris(2-carboxyethyl) phosphine hydrochloride), M-MAL-PEG-10K ( 27.5mg, maleimide polyethylene glycol, molecular weight 10000Da), react at room temperature for 2 hours. The reaction solution is purified by ion exchange chromatography to obtain PEGylated uricase.
尿酸酶活性测定方法:使用磷酸盐缓冲体系(pH 7.3)。活性测定体系参考上述方法(实施例2),测定剩余尿酸在292nm处吸光值。Determination of uricase activity: using a phosphate buffer system (pH 7.3). The activity measurement system refers to the above method (Example 2) to measure the absorbance value of residual uric acid at 292 nm.
将上述选择的Cys突变位点进行两两组合后进行PEG化修饰及尿酸酶活性评估。结果见表2。The Cys mutation sites selected above were combined in pairs to carry out PEGylation modification and evaluation of uricase activity. The results are shown in Table 2.
表2Table 2
分子名称Molecular name | 位点突变site mutation | 序列编号serial number | 单亚基个数Number of single subunits | 酶活保持Enzyme activity is maintained | 修饰后酶活U/mgEnzyme activity after modification U/mg |
AG-2C12AG-2C12 |
S142C/E216CS142C/ |
1212 | 22 | 98%98% | 2.02.0 |
AG-2C13AG-2C13 |
A35C/S288CA35C/ |
1313 | 22 | 90%90% | 1.951.95 |
AG-2C16AG-2C16 |
A35C/S142CA35C/ |
1414 | 22 | 98%98% | 1.691.69 |
AG-2C17AG-2C17 |
E216C/S288CE216C/ |
1515 | 22 | 90%90% | 1.851.85 |
AG-2C13-1AG-2C13-1 |
A35C/S288CA35C/ |
1616 | 22 | 113%113% | 2.332.33 |
AG-2C01-2AG-2C01-2 |
A35C/T194CA35C/ |
1717 | 22 | 99%99% | 1.551.55 |
AG-2C05-1AG-2C05-1 |
F82C/G287CF82C/ |
1818 | 22 | 44%44% | 0.670.67 |
AG-2C01-1AG-2C01-1 |
A35C/T194CA35C/ |
1919 | 22 | 92%92% | 1.431.43 |
AG-2C12-1AG-2C12-1 |
S142C/E216CS142C/ |
2020 | 22 | 102%102% | 1.651.65 |
*注:与AG-2C13、AG-2C01-2和AG-2C12相比,AG-2C13-1、AG-2C01-1和AG-2C12-1还分别包括以下突变:S205D和G208R。*Note: Compared with AG-2C13, AG-2C01-2 and AG-2C12, AG-2C13-1, AG-2C01-1 and AG-2C12-1 also include the following mutations: S205D and G208R, respectively.
表2所列的分子中引入的两个半胱氨酸都能很好的被PEG修饰,证明该引入位点暴露在分子表面。并且尿酸酶的单个亚基被mPEG双修饰后,酶活保持率从44%到113%不等。此外,在205和208位上引入突变能进一步提高酶活。The two cysteines introduced into the molecules listed in Table 2 can be well modified by PEG, which proves that the introduction site is exposed on the surface of the molecule. And after the single subunit of uricase was double-modified with mPEG, the enzyme activity retention rate varied from 44% to 113%. In addition, the introduction of mutations at positions 205 and 208 can further improve the enzymatic activity.
实施例4:PEG化修饰的尿酸酶活性评估Example 4: Evaluation of PEGylation-modified uricase activity
A.PEG化尿激酶的酶活动力学研究A. Kinetics of the enzymatic activity of PEGylated urokinase
使用Tris-HCl缓冲体系(pH 8.5),尿酸酶工作溶液的母液为2mM尿酸,4℃保存。酶活性测定过程:Tris-HCl buffer system (pH 8.5) was used, and the stock solution of uricase working solution was 2mM uric acid and stored at 4°C. Enzyme activity determination process:
设置温度37度,底物(尿酸)浓度梯度:600微摩尔、500微摩尔、400微摩尔、200 微摩尔、100微摩尔、50微摩尔、25微摩尔、12.5微摩尔;先加入50ul上述尿酸溶液和30ul上述Tris-HCl缓冲液于96孔板,37℃保温20min,然后快速加入20ul适当稀释的尿酸酶溶液(0.004mg/mL)到96孔板。设置程序:反应10min,每间隔20s读取数据一次,启动反应。结果如图4和表3所示。Set the temperature to 37 degrees, the substrate (uric acid) concentration gradient: 600 micromolar, 500 micromolar, 400 micromolar, 200 micromolar, 100 micromolar, 50 micromolar, 25 micromolar, 12.5 micromolar; first add 50ul of the above uric acid The solution and 30ul of the above Tris-HCl buffer were placed in a 96-well plate, incubated at 37°C for 20min, and then 20ul of appropriately diluted uricase solution (0.004mg/mL) was quickly added to the 96-well plate. Setting program: 10min reaction, read data every 20s, start reaction. The results are shown in Figure 4 and Table 3.
对照尿酸酶MEDI4945:国际申请号为PCT/US2016/032415中公开的尿酸酶。Control uricase MEDI4945: The uricase disclosed in International Application No. PCT/US2016/032415.
结果显示,尿酸酶分子的最大反应速率依次为AG-2C01-2-PEG、AG-2C12-PEG、AG-2C13-PEG和MEDI4945。The results showed that the maximum reaction rates of uricase molecules were AG-2C01-2-PEG, AG-2C12-PEG, AG-2C13-PEG and MEDI4945.
表3table 3
分子名称Molecular name | KmKm | VmaxVmax | KcatKcat |
AG-2C01-2-PEGAG-2C01-2-PEG | 451.4451.4 | 13.8413.84 | 5.45.4 |
AG-2C12-PEGAG-2C12-PEG | 355.5355.5 | 13.2413.24 | 5.35.3 |
AG-2C13-PEGAG-2C13-PEG | 167.7167.7 | 9.49.4 | 3.63.6 |
MEDI4945MEDI4945 | 101.5101.5 | 6.86.8 | 2.62.6 |
注:Km:米氏常数;Vmax:最大反应速率;Kcat:转化数,指单个酶分子在一秒内转化底物的数量。Note: Km: Michaelis constant; Vmax: maximum reaction rate; Kcat: conversion number, which refers to the number of substrates converted by a single enzyme molecule in one second.
B.PEG修饰的尿激酶的加速稳定性评估(37℃)B. Accelerated Stability Assessment of PEG-Modified Urokinase (37°C)
将PEG修饰的尿酸酶放置在37℃恒温箱中,缓冲体系为磷酸盐缓冲液体系,pH7.0。SEC-HPLC结果如图5所示。可见,在37℃条件下,AG-2C01-2-PEG的稳定性优于AG-2C12-PEG和AG-2C13-PEG。The PEG-modified uricase was placed in a 37°C incubator, and the buffer system was a phosphate buffer system, pH 7.0. The SEC-HPLC results are shown in FIG. 5 . It can be seen that the stability of AG-2C01-2-PEG is better than that of AG-2C12-PEG and AG-2C13-PEG at 37 °C.
实施例5:药物组合物缓冲液体系、pH和稳定剂筛选实验Example 5: Pharmaceutical Composition Buffer System, pH and Stabilizer Screening Experiment
液体型药物组合物中,缓冲液体系和pH密切影响抗体的稳定性,每种具有独特理化性质的抗体都具有最适宜的缓冲液的种类和pH。本实施例旨在筛选一种最佳缓冲液体系和pH,使本发明公开的尿酸酶具有最佳的稳定性以适宜临床应用。In liquid pharmaceutical compositions, the buffer system and pH closely affect the stability of antibodies, and each antibody with unique physicochemical properties has the most suitable buffer type and pH. The purpose of this example is to screen an optimal buffer system and pH, so that the uricase disclosed in the present invention has the best stability and is suitable for clinical application.
5.1实验步骤5.1 Experimental steps
本实施例以约10mg/mL和20mg/mL浓度的尿酸酶(AG-2C01-2-PEG)进行。样品使用Millipore Pellicon3 0.11m
2膜进行超滤浓缩换液,换液后样品处于相应的处方中,样品放置在密闭的离心管中进行缓冲液筛选。缓冲液筛选了磷酸盐缓冲液和Tris盐酸缓冲液,pH从7.5和8.0;稳定剂筛选了氯化钠、盐酸精氨酸、甘露醇、蔗糖和海藻糖进行了比较测试(如表4所示)。
This example was performed with uricase (AG-2C01-2-PEG) at concentrations of about 10 mg/mL and 20 mg/mL. The samples were subjected to ultrafiltration and concentration exchange using Millipore Pellicon3 0.11m 2 membrane. After exchange, the samples were in the corresponding prescription, and the samples were placed in a closed centrifuge tube for buffer screening. The buffers were screened for phosphate buffer and Tris hydrochloric acid buffer, pH from 7.5 and 8.0; the stabilizers were screened for sodium chloride, arginine hydrochloride, mannitol, sucrose and trehalose for comparative testing (as shown in Table 4). ).
将样品放置在25±2℃或5±3℃环境下,分别在第0周、第2周、第4周、第8周(仅 5±3℃)和3个月(仅5±3℃)取出进行分析检测。评价指标包括:1、目测外观;2、蛋白浓度(紫外分光光度法);3、SEC-HPLC(尺寸排阻色谱法)测定蛋白单体、聚集和碎片的百分比;4、RP-HPLC(反相高效液相色谱)测定蛋白纯度;5、NR-CE-SDS(非还原十二烷基硫酸钠毛细管电泳)检测蛋白的纯度;6、酶活分析检测。考察不同缓冲液体系和pH对尿酸酶稳定性的影响,结果见表5-1~5-6所示。Place the samples at 25±2°C or 5±3°C at week 0, week 2, week 4, week 8 (only 5±3°C) and 3 months (only 5±3°C) ) for analysis. Evaluation indicators include: 1. Visual appearance; 2. Protein concentration (UV spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and fragments; 4. RP-HPLC (reverse 5. NR-CE-SDS (non-reducing sodium dodecyl sulfate capillary electrophoresis) to detect the protein purity; 6. Enzyme activity analysis to detect. The effects of different buffer systems and pH on the stability of uricase were investigated, and the results are shown in Tables 5-1 to 5-6.
表4:缓冲液体系、pH和稳定剂筛选实验中的处方信息Table 4: Prescribing Information for Buffer Systems, pH, and Stabilizer Screening Experiments
5.2实验结果5.2 Experimental results
5.2.1外观和蛋白含量5.2.1 Appearance and protein content
根据表5-1中的结果,经加速条件或长期条件,所有样品的蛋白含量均未出现显著变化。According to the results in Table 5-1, the protein content of all samples did not change significantly after accelerated or long-term conditions.
根据表5-2中的结果,所有样品在T0时均未发现明显可见异物,无明显乳光。经加速条件下放置4周,FS2-8和FS2-3处方在浓度为20mg/ml时出现蛋白沉淀(FS2-8终止后续检测),其余处方均无显著变化;经长期条件下放置3个月,处方FS2-8在浓度20mg/ml和FS2-13处方出现蛋白沉淀,其他样品外观均未出现显著变化。According to the results in Table 5-2, no obvious visible foreign bodies and no obvious opalescence were found in all samples at T0. After being placed under accelerated conditions for 4 weeks, FS2-8 and FS2-3 prescriptions showed protein precipitation at a concentration of 20 mg/ml (FS2-8 terminated the follow-up detection), and the rest of the prescriptions had no significant changes; after 3 months of long-term placement , the formula FS2-8 showed protein precipitation at the concentration of 20mg/ml and the formula FS2-13, and the appearance of other samples did not change significantly.
表5-1:蛋白含量检测数据Table 5-1: Protein content detection data
注:“/”表示:终止检测。Note: "/" means: stop detection.
表5-2:外观检测数据Table 5-2: Appearance Inspection Data
注:“/”表示:终止检测。Note: "/" means: stop detection.
5.2.2 SEC纯度5.2.2 SEC purity
根据表5-3中的SEC纯度结果,FS2-8(20mg/ml)组终止检测;经加速条件下放置4周,含有盐酸精氨酸的处方(FS2-2和FS2-7)均出现显著的聚体增加;经长期条件放置下放置3个月,所有处方均未出现SEC纯度的显著降低。According to the SEC purity results in Table 5-3, the FS2-8 (20mg/ml) group terminated the detection; after being placed under accelerated conditions for 4 weeks, the formulations containing arginine hydrochloride (FS2-2 and FS2-7) showed significant The aggregates increased; after 3 months under long-term conditions, all formulations did not show a significant decrease in SEC purity.
表5-3:SEC-HPLC单体、聚体和片段含量数据Table 5-3: SEC-HPLC monomer, aggregate and fragment content data
注:“/”表示:终止检测。Note: "/" means: stop detection.
5.2.3 RP-HPLC纯度5.2.3 RP-HPLC purity
根据表5-4中的RP-HPLC纯度结果,FS2-8(20mg/ml)组终止检测;经加速条件下放置4周,FS2-2、FS2-7、FS2-12和FS2-13四组纯度降低速度较快;经长期条件放置下放置3M,FS2-3(10/20mg/ml),FS2-4和FS2-5四组纯度降低速度较慢。According to the RP-HPLC purity results in Table 5-4, the FS2-8 (20 mg/ml) group terminated the detection; after being placed under accelerated conditions for 4 weeks, the four groups of FS2-2, FS2-7, FS2-12 and FS2-13 The purity decreased rapidly; the four groups of 3M, FS2-3 (10/20mg/ml), FS2-4 and FS2-5 were placed under long-term conditions to decrease the purity slower.
表5-4:RP-HPLC蛋白纯度筛选数据Table 5-4: RP-HPLC protein purity screening data
注:“/”表示:终止检测。Note: "/" means: stop detection.
5.2.4 NR-CE-SDS纯度5.2.4 Purity of NR-CE-SDS
根据表5-5中的NR-CE-SDS纯度结果,FS2-8(20mg/ml)组终止检测;经加速条件下放置4周或经长期条件放置下放置3M,所有样品均未出现纯度的显著变化。According to the NR-CE-SDS purity results in Table 5-5, the FS2-8 (20mg/ml) group was terminated for detection; after being placed under accelerated conditions for 4 weeks or placed under long-term conditions for 3M, all samples did not appear pure Significant changes.
表5-5:NR-CE-SDS纯度筛选数据Table 5-5: NR-CE-SDS Purity Screening Data
注:“/”表示:终止检测。Note: "/" means: stop detection.
5.2.5酶活5.2.5 Enzyme activity
根据表5-6中的酶活结果,经加速条件或长期条件下,除FS2-8(20mg/ml)外,所有处方均未出现酶活降低。According to the enzymatic activity results in Tables 5-6, under accelerated conditions or long-term conditions, all formulations except FS2-8 (20 mg/ml) showed no decrease in enzymatic activity.
表5-6:酶活筛选数据Table 5-6: Enzyme activity screening data
注:“/”表示:终止检测。Note: "/" means: stop detection.
5.3第一轮处方筛选结论5.3 Conclusions of the first round of prescription screening
从蛋白含量、酶活和NR-CE-SDS纯度结果来看,所有处方未出现显著差异。从外观来看,FS2-8(20mg/ml)外观结果较差;从SEC-HPLC结果来看,FS2-2和FS2-7出现聚体峰增加;从RP-HPLC结果来看,FS2-3(10/20mg/ml),FS2-4和FS2-5四组结果优于其余组。From the results of protein content, enzymatic activity and NR-CE-SDS purity, there was no significant difference among all formulations. From the appearance point of view, FS2-8 (20mg/ml) has poor appearance results; from the SEC-HPLC results, FS2-2 and FS2-7 have increased aggregate peaks; from the RP-HPLC results, FS2-3 (10/20mg/ml), the four groups of FS2-4 and FS2-5 were better than the other groups.
综上所述,FS2-3、FS2-4和FS2-5三个处方较好。因此选择20mM磷酸盐缓冲体系(pH7.5)作为缓冲体系进入下一轮筛选实验;并在下一轮实验中对蔗糖、海藻糖和甘露醇三种稳定剂进行进一步的评估。To sum up, the three prescriptions FS2-3, FS2-4 and FS2-5 are better. Therefore, 20 mM phosphate buffer system (pH7.5) was selected as the buffer system to enter the next round of screening experiments; and three stabilizers, sucrose, trehalose and mannitol, were further evaluated in the next round of experiments.
实施例6:稳定剂和表面活性剂筛选Example 6: Stabilizer and Surfactant Screening
6.1实验步骤6.1 Experimental steps
将尿酸酶(AG-2C01-2-PEG)使用Millipore Pellicon3 0.11m
2膜进行换液,换液后样品处于相应的处方中,最终蛋白浓度约8mg/ml(如表6),无菌灌装到2R西林瓶2.0ml/瓶,进行稳定性放样和检测。
The uricase (AG-2C01-2-PEG) was exchanged using Millipore Pellicon3 0.11m 2 membrane. After the exchange, the sample was in the corresponding prescription, and the final protein concentration was about 8 mg/ml (as shown in Table 6). Aseptic filling To 2R vials of 2.0ml/bottle for stability stakeout and testing.
1)将样品放置在25±2℃或5±3℃环境下,分别在第0周、第1周、第2周、第4周和第8周取出进行分析检测;2)将样品进行3次冻融循环;3)将样品进行3天振摇。评价指标包括:1、目测外观;2、蛋白浓度(紫外分光光度法);3、SEC-HPLC(尺寸排阻色谱法)测定蛋白单体、聚集和碎片的百分比;4、RP-HPLC(反相高效液相色谱)测定蛋白纯度;5、NR-CE-SDS(十二烷基硫酸钠毛细管电泳)检测蛋白的纯度;6、酶活分析检测。结果见表7-1~7-6。1) Place the samples at 25±2°C or 5±3°C, and take them out for analysis at week 0, week 1, week 2, week 4, and week 8, respectively; 2) The sample is subjected to 3 freeze-thaw cycles; 3) the samples were shaken for 3 days. Evaluation indicators include: 1. Visual appearance; 2. Protein concentration (UV spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and fragments; 4. RP-HPLC (reverse 5. NR-CE-SDS (sodium dodecyl sulfate capillary electrophoresis) to detect the protein purity; 6. Enzyme activity analysis to detect. The results are shown in Tables 7-1 to 7-6.
表6:稳定剂筛选实验方案Table 6: Stabilizer Screening Protocol
注:“/”表示:未添加。Note: "/" means: not added.
6.2实验结果6.2 Experimental results
6.2.1外观和蛋白浓度6.2.1 Appearance and protein concentration
根据表7-1和7-2中的结果,经加速条件和长期条件下放置8周,所有样品的蛋白含量均未出现显著变化;所有样品在T0时均未发现明显可见异物,无明显乳光。经加速条件或长期条件下放置8周,FS3-1、FS3-3、FS3-4和FS3-6四组处方未见外观变化,其余处方出现乳光增加;经3次冻融循环,3天振摇样品均未发现明显可见异物,无明显乳光。According to the results in Tables 7-1 and 7-2, the protein content of all samples did not change significantly after being placed under accelerated conditions and long-term conditions for 8 weeks; no obvious foreign body was found in all samples at T0, and no obvious milk was found. Light. After being placed under accelerated conditions or long-term conditions for 8 weeks, the four groups of prescriptions FS3-1, FS3-3, FS3-4 and FS3-6 showed no change in appearance, and the other prescriptions showed increased opalescence; after 3 freeze-thaw cycles, 3 days There was no obvious visible foreign body and no obvious opalescence after shaking the sample.
表7-1:蛋白含量筛选数据Table 7-1: Protein Content Screening Data
注:“/”表示:未检测。Note: "/" means: not detected.
表7-2:外观筛选数据Table 7-2: Appearance Screening Data
注:“/”表示:未检测。Note: "/" means: not detected.
6.2.2 SEC纯度6.2.2 SEC purity
根据表7-3中的SEC纯度结果,所有样品经加速条件或长期条件下放置8周,均未出现SEC纯度的显著变化;经3次冻融循环,3天振摇均未出现SEC纯度的显著变化。According to the SEC purity results in Table 7-3, all samples were placed under accelerated conditions or long-term conditions for 8 weeks, and there was no significant change in SEC purity; after 3 freeze-thaw cycles, no SEC purity appeared after 3 days of shaking. Significant changes.
表7-3:SEC-HPLC单体、聚体和片段含量数据Table 7-3: SEC-HPLC monomer, aggregate and fragment content data
注:“/”表示:未检测。Note: "/" means: not detected.
6.2.3 RP-HPLC纯度6.2.3 RP-HPLC purity
根据表7-4中的RP-HPLC纯度结果,经加速条件下放置8周,所有样品均出现纯度下降,但未表现出组间差异;经长期条件下放置8周,均未出现纯度的显著变化;经3次冻融循环,3天振摇均未出现纯度的显著变化。According to the RP-HPLC purity results in Table 7-4, after being placed under accelerated conditions for 8 weeks, all samples showed a decrease in purity, but there was no difference between groups; after being placed under long-term conditions for 8 weeks, no significant change in purity appeared change; after 3 freeze-thaw cycles and 3 days of shaking, there was no significant change in purity.
表7-4:RP-HPLC纯度筛选数据Table 7-4: RP-HPLC Purity Screening Data
注:“/”表示:未检测。Note: "/" means: not detected.
6.2.4 NR-CE-SDS纯度6.2.4 Purity of NR-CE-SDS
根据表7-5中的NR-CE-SDS纯度结果,经加速条件下放置8周,所有样品均出现纯度下降,但未表现出组间差异;经长期条件下放置8周,均未出现纯度的显著变化。According to the NR-CE-SDS purity results in Table 7-5, after 8 weeks of storage under accelerated conditions, all samples showed a decrease in purity, but showed no difference between groups; after 8 weeks of storage under long-term conditions, no purity appeared significant changes.
表7-5:NR-CE-SDS纯度筛选数据Table 7-5: NR-CE-SDS Purity Screening Data
6.2.5酶活结果6.2.5 Enzyme activity results
根据表7-6中的酶活结果,经加速条件或长期条件下放置8周,所有样品均未出现酶活的显著变化;经3次冻融循环,3天振摇均未出现酶活的显著变化。According to the enzymatic activity results in Table 7-6, no significant changes in enzymatic activity occurred in all samples after being placed under accelerated conditions or long-term conditions for 8 weeks; Significant changes.
表7-6:酶活筛选数据Table 7-6: Enzyme activity screening data
注:“/”表示:未检测。Note: "/" means: not detected.
6.3第二轮处方筛选结论6.3 Conclusions of the second round of prescription screening
从蛋白含量、酶活和NR-CE-SDS纯度、SEC-HPLC纯度和RP-HPLC纯度结果来看,所有处方未出现显著差异。从外观结果来看,FS3-1、FS3-3、FS3-4和FS3-6四组结果较优。From the results of protein content, enzymatic activity and purity of NR-CE-SDS, SEC-HPLC and RP-HPLC, there were no significant differences among all formulations. From the appearance results, the four groups of FS3-1, FS3-3, FS3-4 and FS3-6 have better results.
综上所述,FS3-1、FS3-3、FS3-4和FS3-6三个处方较好。考虑到甘露醇冷冻易形成晶体,存在冻融风险,选择FS3-3(20mM磷酸盐,pH7.5,含220mM海藻糖和0.02%聚山梨酯80)作为优选处方。In conclusion, the three prescriptions of FS3-1, FS3-3, FS3-4 and FS3-6 are better. Considering that mannitol is easy to form crystals in freezing and has the risk of freezing and thawing, FS3-3 (20 mM phosphate, pH 7.5, containing 220 mM trehalose and 0.02% polysorbate 80) was selected as the preferred formulation.
实施例7:配伍稳定性实验Embodiment 7: Compatibility stability experiment
7.1实验步骤7.1 Experimental steps
评估实施例2中FS3-1~6六个处方经0.9%氯化钠注射液和5%葡萄糖注射液两种介质稀释后的稳定性。尿酸酶(AG-2C01-2-PEG)按如下方案稀释至相应介质不同浓度(表8),放置于25℃24h后检测,考察不同介质对。The stability of six formulations of FS3-1 to 6 in Example 2 after being diluted by two media, 0.9% sodium chloride injection and 5% glucose injection, was evaluated. Uricase (AG-2C01-2-PEG) was diluted to different concentrations in the corresponding medium according to the following scheme (Table 8), placed at 25°C for 24h, and then detected, and different medium pairs were investigated.
评价指标包括:1、目测外观;2、蛋白浓度(紫外分光光度法);3、SEC-HPLC(尺寸排阻色谱法)测定蛋白单体、聚集和碎片的百分比。结果见表9。Evaluation indicators include: 1. Visual appearance; 2. Protein concentration (UV spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and fragments. The results are shown in Table 9.
表8:相容性方案Table 8: Compatibility Scheme
7.2实验结果7.2 Experimental results
本发明尿酸酶在0.9%氯化钠注射液0.2mg/ml~4mg/ml浓度范围都有良好的稳定性。5%葡萄糖注射液中低浓度0.2mg/ml SEC出现片段增加。The uricase of the present invention has good stability in the concentration range of 0.9% sodium chloride injection from 0.2 mg/ml to 4 mg/ml. Fragmentation increases in SEC at low concentrations of 0.2 mg/ml in 5% glucose injection.
表9:相容性检测结果Table 9: Compatibility Test Results
综上所述,通过对不同缓冲体系,不同pH条件、不同蛋白浓度和不同辅料组成进行考察,探索研究尿酸酶的稳定性,并确定了最佳液体制剂配方。根据上述实验,本发明尿酸酶选择pH7.5的磷酸盐缓冲液来调节pH,海藻糖调节制剂渗透压,添加聚山梨酯80来增加制剂溶解性。In conclusion, the stability of uricase was studied by investigating different buffer systems, different pH conditions, different protein concentrations and different excipient compositions, and the optimal liquid formulation was determined. According to the above experiments, the uricase of the present invention selects a pH 7.5 phosphate buffer to adjust the pH, trehalose adjusts the osmotic pressure of the preparation, and polysorbate 80 is added to increase the solubility of the preparation.
配伍相容性实验显示,本发明尿酸酶最终处方经0.9%氯化钠注射液稀释,在蛋白浓度0.2~4mg/ml的范围内,室温放置24小时未见显著降解。Compatibility experiments showed that the final formulation of the uricase of the present invention was diluted with 0.9% sodium chloride injection, within the range of protein concentration of 0.2-4 mg/ml, and no significant degradation was found in room temperature for 24 hours.
考虑到本发明尿酸酶的等电点在5.5~6.3范围内,且在早期预实验中在pH7.0时出现沉淀现象,最终将pH增加至7.8以尽量远离pI增加其溶解度,pH范围7.5~8.1。Considering that the isoelectric point of the uricase of the present invention is in the range of 5.5 to 6.3, and the precipitation phenomenon occurred at pH 7.0 in the early preliminary experiment, the pH was finally increased to 7.8 to increase its solubility as far as possible from the pI, and the pH range was 7.5~ 8.1.
实施例8:体内药效学研究Example 8: In vivo pharmacodynamic studies
通过单次静脉注射(i.v.)给药的方式,在腹腔注射(i.p.)外源性尿酸诱导的高尿酸血症小鼠模型上评价受试物对血清尿酸水平的影响。把120只正常小鼠(ICR-male)随机分成6组,每组20只。第1组、第2组分别作空白对照和溶剂对照给予溶剂,第3组作为阳性对照给予MEDI4945 0.1mg/kg,第4、5、6组作为治疗组分别给予0.1mg/kg剂量的AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG(处方FS3-3)。于给药后次日,除空白对照组外,其余各组实验小鼠均腹腔注射尿酸造模(250mg/kg,10ml/kg),每组1~10号动物采集造模后30min和3h血样,11~20号动物采集1.5h和6h血样。The effects of test substances on serum uric acid levels were evaluated in a mouse model of hyperuricemia induced by intraperitoneal injection (i.p.) of exogenous uric acid by single intravenous (i.v.) administration. 120 normal mice (ICR-male) were randomly divided into 6 groups with 20 mice in each group. Groups 1 and 2 were given solvent as blank control and solvent control, respectively, group 3 was given MEDI4945 0.1 mg/kg as positive control, and groups 4, 5 and 6 were given 0.1 mg/kg dose of AG- 2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG (formulation FS3-3). On the next day after administration, except for the blank control group, the other groups of experimental mice were injected with uric acid (250 mg/kg, 10 ml/kg) intraperitoneally for modeling, and blood samples were collected from animals 1 to 10 in each group at 30 minutes and 3 hours after modeling. , 11 to 20 animals collected 1.5h and 6h blood samples.
结果如图6所示。0.1mg/kg剂量的AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG治疗组动物血清尿酸水平在造模后0.5h~6h各个时间点均显著低于溶剂对照组(p<0.01), 于造模后0.5h~1.5h,3个治疗组的动物血清尿酸水平均低于检测下限(AG-2C13-PEG组除外,在1.5h时为8.35μmol/l)。在所测剂量下(0.1mg/kg),单次注射给予AG-2C13-PEG、AG-2C01-2-PEG和AG-2C12-PEG后,在腹腔注射外源性尿酸诱导的ICR小鼠高尿酸血症模型上对血清尿酸水平有显著降低作用。在此剂量下,尿激酶AG-2C13-PEG、AG-2C01-2-PEG以及AG-2C12-PEG在造模后各时间点效果无明显差异,但在3h后药效均优于MEDI4945。The results are shown in Figure 6. 0.1mg/kg dose of AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG treatment group of animals serum uric acid levels were significantly lower than the solvent control group at each time point 0.5h ~ 6h after modeling ( p<0.01), at 0.5h to 1.5h after modeling, the serum uric acid levels in the three treatment groups were lower than the detection limit (except for the AG-2C13-PEG group, which was 8.35μmol/l at 1.5h). At the dose tested (0.1 mg/kg), following a single injection of AG-2C13-PEG, AG-2C01-2-PEG, and AG-2C12-PEG, the ICR induced by intraperitoneal injection of exogenous uric acid in mice was high The serum uric acid level was significantly reduced in the uricemia model. At this dose, the effects of urokinase AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG had no significant difference at each time point after modeling, but they were better than MEDI4945 after 3 hours.
Claims (20)
- 一种尿酸酶,其包含如SEQ ID NO:9所示的氨基酸序列或其保留了酶活性的变体,其中所述变体相对于SEQ ID NO:9的突变包括:N末端17个以内氨基酸残基替换和/或C末端10个以内氨基酸残基替换。A uricase comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof that retains enzymatic activity, wherein the mutation of the variant relative to SEQ ID NO: 9 comprises: amino acids within 17 N-terminal Residue substitutions and/or amino acid residue substitutions within the C-terminal 10.
- 如权利要求1所述的尿酸酶,其中所述变体相对于SEQ ID NO:9的突变包括:N末端氨基酸序列MTATAETSTGTKVVLGQ替换为MANIILGK,和/或C末端氨基酸序列SRADHPIWSN替换为C;优选地,所述尿酸酶分别包含如SEQ ID NO:10或SEQ ID NO:11所示的序列。The uricase of claim 1, wherein the mutation of the variant relative to SEQ ID NO: 9 comprises: the N-terminal amino acid sequence MTATAETSTGTKVVLGQ is replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C; preferably, The uricase comprises the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11, respectively.
- 如权利要求1或2所述的尿酸酶,其中所述尿酸酶相对于SEQ ID NO:9的突变还包括:在选自35、82、142、194、216、287和288的位置上引入1、2、3、4或5个表面可及的半胱氨酸残基,其中所述位置编号是相对于SEQ ID NO:3的位置编号。The uricase of claim 1 or 2, wherein the mutation of the uricase relative to SEQ ID NO: 9 further comprises: introducing 1 at a position selected from the group consisting of 35, 82, 142, 194, 216, 287 and 288 , 2, 3, 4 or 5 surface accessible cysteine residues, wherein the position numbering is relative to the position numbering of SEQ ID NO:3.
- 如权利要求3所述的尿酸酶,其中所述尿酸酶分别在以下两个位置中包含半胱氨酸残基:142和216位,35和288位,35和142位,216和288位,35和194位,或82和287位,其中所述位置编号是相对于SEQ ID NO:3的位置编号。The uricase of claim 3, wherein the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, Positions 35 and 194, or 82 and 287, wherein the position numbers are relative to SEQ ID NO:3.
- 如权利要求1-4中任一项所述的尿酸酶,其中所述尿酸酶相对于SEQ ID NO:9的突变还包括:第205位和/或第208位上的取代突变,优选地,所述取代突变为S205D或G208R或其组合,其中所述位置编号是相对于SEQ ID NO:3的位置编号。The uricase of any one of claims 1-4, wherein the mutation of the uricase relative to SEQ ID NO: 9 further comprises: a substitution mutation at position 205 and/or position 208, preferably, The substitution mutation is S205D or G208R or a combination thereof, wherein the position numbering is relative to that of SEQ ID NO:3.
- 如权利要求1-5中所述的尿酸酶,其中所述尿酸酶分别包含如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列。The uricase of claims 1-5, wherein the uricase comprises as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, respectively , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or the amino acid sequence shown in SEQ ID NO: 20.
- 一种修饰的尿酸酶,其中所述修饰的尿酸酶为聚乙二醇(PEG)修饰的权利要求1-6中任一项所述的尿酸酶;优选地,所述PEG与所述尿酸酶的N-末端和/或C-末端共价连接,和/或通过所述尿酸酶上存在的半胱氨酸残基进行共价连接。A modified uricase, wherein the modified uricase is the uricase of any one of claims 1-6 modified by polyethylene glycol (PEG); preferably, the PEG and the uricase Covalently linked to the N-terminus and/or C-terminus of the uricase, and/or via the cysteine residue present on the uricase.
- 一种药物组合物,其包含:A pharmaceutical composition comprising:(1)缓冲液;和(1) Buffer; and(2)如权利要求1-6中任一项所述的尿酸酶或权利要求7所述的修饰的尿酸酶。(2) The uricase of any one of claims 1-6 or the modified uricase of claim 7.
- 如权利要求8所述的药物组合物,其中所述缓冲液选自磷酸盐缓冲液、Tris盐酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液;优选地,所述缓冲液为磷酸盐缓冲液或Tris盐酸缓冲液;更优选地,所述缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液。The pharmaceutical composition of claim 8, wherein the buffer is selected from the group consisting of phosphate buffer, Tris hydrochloric acid buffer, citrate buffer and histidine buffer; preferably, the buffer is phosphate buffer solution or Tris hydrochloric acid buffer; more preferably, the buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
- 如权利要求9所述的药物组合物,其中所述缓冲液的浓度约为10~50mM;优选地,所述缓冲液的浓度约为10~30mM。The pharmaceutical composition of claim 9, wherein the concentration of the buffer is about 10-50 mM; preferably, the concentration of the buffer is about 10-30 mM.
- 如权利要求9所述的药物组合物,其中所述缓冲液的pH约为6.0-8.5,优选约为7.0-8.5,优选为约7.5~8.1。The pharmaceutical composition of claim 9, wherein the pH of the buffer is about 6.0-8.5, preferably about 7.0-8.5, preferably about 7.5-8.1.
- 如权利要求8所述的药物组合物,其中所述药物组合物还包括稳定剂,所述稳定剂选自盐酸精氨酸、氯化钠、甘露醇、蔗糖和海藻糖中的一种或多种;优选地,所述稳定剂选自甘露醇、海藻糖、甘露醇与氯化钠的组合或海藻糖与氯化钠的组合。The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose and trehalose Preferably, the stabilizer is selected from mannitol, trehalose, a combination of mannitol and sodium chloride or a combination of trehalose and sodium chloride.
- 如权利要求12所述的药物组合物,其中所述稳定剂的浓度为约20mM-300mM,优选50mM-250mM;优选地,所述稳定剂为浓度约30-200mM的氯化钠;或所述稳定剂为浓度约30-200mM的盐酸精氨酸;或所述稳定剂为浓度约100-300mM的甘露醇;或所述稳定剂为浓度约100-300mM的蔗糖;或所述稳定剂为浓度约100-300mM的海藻糖;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的海藻糖的组合;或所述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合;优选地,所述稳定剂为约100-300mM的海藻糖、约100-300mM的甘露醇、约30-200mM的氯化钠与约30-200mM的甘露醇的组合或约30-200mM的氯化钠与约30-200mM的海藻糖的组合;更优选为约100-300mM的海藻糖或约30-200mM的氯化钠与约30-200mM的海藻糖的组合。The pharmaceutical composition of claim 12, wherein the concentration of the stabilizer is about 20mM-300mM, preferably 50mM-250mM; preferably, the stabilizer is sodium chloride at a concentration of about 30-200mM; or the The stabilizer is arginine hydrochloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is a concentration of about 100-300 mM trehalose; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the stabilizer is about 30-200 mM sodium chloride and about 30 - a combination of 200 mM trehalose; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; preferably, the stabilizer is about 100-300 mM trehalose, about 100 -300 mM mannitol, about 30-200 mM sodium chloride in combination with about 30-200 mM mannitol or about 30-200 mM sodium chloride in combination with about 30-200 mM trehalose; more preferably about 100- 300 mM trehalose or about 30-200 mM sodium chloride in combination with about 30-200 mM trehalose.
- 如权利要求8所述的药物组合物,其中所述药物组合物还包括表面活性剂,所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20或泊洛沙姆188,优选地,所述表面活性剂为聚山梨醇酯80。The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188, preferably , the surfactant is polysorbate 80.
- 如权利要求14所述的药物组合物,其中所述表面活性剂浓度约为0.01%-0.1%,优选地,所述表面活性剂浓度约为0.02%-0.08%,优选约为0.02%-0.05%。The pharmaceutical composition of claim 14, wherein the surfactant concentration is about 0.01%-0.1%, preferably the surfactant concentration is about 0.02%-0.08%, preferably about 0.02%-0.05 %.
- 如权利要求8所述的药物组合物,其中所述尿酸酶或修饰的尿酸酶的浓度约为1-200mg/mL,优选约为5-80mg/mL,更优选约为5-40mg/mL。The pharmaceutical composition of claim 8, wherein the concentration of the uricase or modified uricase is about 1-200 mg/mL, preferably about 5-80 mg/mL, more preferably about 5-40 mg/mL.
- 如权利要求8所述的药物组合物,其包含如下(1)-(5)任一项所示的组分:The pharmaceutical composition of claim 8, comprising the components shown in any one of the following (1)-(5):(1)(a)约5mg/mL-40mg/mL的尿酸酶或修饰的尿酸酶;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约100-300mM的甘露醇;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(1) (a) about 5 mg/mL-40 mg/mL uricase or modified uricase; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 100-300 mM mannitol; and (d) about 0.01%-0.1% polysorbate 80; or(2)(a)约5mg/mL-40mg/mL的尿酸酶或修饰的尿酸酶;(b)约10-30mM Tris盐酸缓冲液,pH约为7.5-8.1;(c)约100-300mM的海藻糖;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(2) (a) about 5mg/mL-40mg/mL uricase or modified uricase; (b) about 10-30mM Tris hydrochloric acid buffer, pH about 7.5-8.1; (c) about 100-300mM and (d) about 0.01%-0.1% polysorbate 80; or(3)(a)约5mg/mL-40mg/mL的尿酸酶或修饰的尿酸酶;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约100-300mM的海藻糖;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(3) (a) about 5 mg/mL-40 mg/mL uricase or modified uricase; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 100-300 mM and (d) about 0.01%-0.1% polysorbate 80; or(4)(a)约5mg/mL-40mg/mL的尿酸酶或修饰的尿酸酶;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约30-200mM的氯化钠与约30-200mM的甘露醇的组合;以及(d)约0.01%-0.1%的聚山梨醇酯80;或(4) (a) about 5 mg/mL-40 mg/mL uricase or modified uricase; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 30-200 mM sodium chloride in combination with about 30-200 mM mannitol; and (d) about 0.01%-0.1% polysorbate 80; or(5)(a)约5mg/mL-40mg/mL的尿酸酶或修饰的尿酸酶;(b)约10-30mM磷酸盐缓冲液,pH约为7.5-8.1;(c)约30-200mM的氯化钠与约30-200mM的海藻糖的组合;以及(d)约0.01%-0.1%的聚山梨醇酯80。(5) (a) about 5 mg/mL-40 mg/mL uricase or modified uricase; (b) about 10-30 mM phosphate buffer, pH about 7.5-8.1; (c) about 30-200 mM sodium chloride in combination with about 30-200 mM trehalose; and (d) about 0.01%-0.1% polysorbate 80.
- 如权利要求17所述的药物组合物,其包含如下(1)-(5)任一项所示的组分:The pharmaceutical composition of claim 17, comprising the components shown in any one of the following (1)-(5):(1)(a)约8mg/mL的尿酸酶或修饰的尿酸酶;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约240mM的甘露醇;以及(d)约0.02%的聚山梨醇酯80;或(1) (a) about 8 mg/mL uricase or modified uricase; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 240 mM mannitol; and (d) about 0.02% polysorbate 80; or(2)(a)约8mg/mL的尿酸酶或修饰的尿酸酶;(b)约20mM Tris盐酸缓冲液,pH约为7.5-8.0;(c)约220mM的海藻糖;以及(d)约0.02%的聚山梨醇酯80;或(2) (a) about 8 mg/mL uricase or modified uricase; (b) about 20 mM Tris hydrochloric acid buffer, pH about 7.5-8.0; (c) about 220 mM trehalose; and (d) about 220 mM trehalose 0.02% polysorbate 80; or(3)(a)约8mg/mL的尿酸酶或修饰的尿酸酶;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约220mM的海藻糖;以及(d)约0.02%的聚山梨醇酯80;或(3) (a) about 8 mg/mL uricase or modified uricase; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 220 mM trehalose; and (d) about 0.02% polysorbate 80; or(4)(a)约8mg/mL的尿酸酶或修饰的尿酸酶;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约50mM的氯化钠与约140mM的甘露醇的组合;以及(d)约0.02%的聚山梨醇酯80;或(4) (a) about 8 mg/mL uricase or modified uricase; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 50 mM sodium chloride and about 140 mM mannose a combination of alcohols; and (d) about 0.02% polysorbate 80; or(5)(a)约8mg/mL的尿酸酶或修饰的尿酸酶;(b)约20mM磷酸盐缓冲液,pH约为7.5-7.8;(c)约50mM的氯化钠与约140mM的海藻糖的组合;以及(d)约0.02%的聚山梨醇酯80。(5) (a) about 8 mg/mL uricase or modified uricase; (b) about 20 mM phosphate buffer, pH about 7.5-7.8; (c) about 50 mM sodium chloride and about 140 mM seaweed a combination of sugars; and (d) about 0.02% polysorbate 80.
- 一种注射剂,其含有权利要求8-18中任一项所述的药物组合物与0.9wt%氯化钠溶液或5wt%葡萄糖水溶液;优选地,所述含0.9wt%氯化钠溶液的注射剂中,所述尿酸酶的浓度为0.2~4mg/mL,所述含5wt%葡萄糖水溶液的注射剂中,所述尿酸酶的浓度为1~4mg/mL;优选地,所述注射剂的pH为7.5~8.1。An injection containing the pharmaceutical composition according to any one of claims 8-18 and 0.9wt% sodium chloride solution or 5wt% aqueous glucose solution; preferably, the injection containing 0.9wt% sodium chloride solution Among them, the concentration of the uricase is 0.2-4 mg/mL, and in the injection containing 5wt% aqueous glucose solution, the concentration of the uricase is 1-4 mg/mL; preferably, the pH of the injection is 7.5- 8.1.
- 如权利要求1-6中任一项所述的尿酸酶或权利要求7所述的修饰的尿酸酶或权利要求8-18中任一项所述的药物组合物在制备降低哺乳动物体液和组织中尿酸水平或治疗高尿酸血症的药物中的用途。The uricase of any one of claims 1-6 or the modified uricase of claim 7 or the pharmaceutical composition of any one of claims 8-18 is used in the preparation of reducing mammalian body fluids and tissues Use in uric acid levels or in a drug for the treatment of hyperuricemia.
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