WO2024017379A1 - 一种活性多肽或蛋白的聚乙二醇缀合物的制备方法 - Google Patents

一种活性多肽或蛋白的聚乙二醇缀合物的制备方法 Download PDF

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WO2024017379A1
WO2024017379A1 PCT/CN2023/108707 CN2023108707W WO2024017379A1 WO 2024017379 A1 WO2024017379 A1 WO 2024017379A1 CN 2023108707 W CN2023108707 W CN 2023108707W WO 2024017379 A1 WO2024017379 A1 WO 2024017379A1
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preparation
concentration
polyethylene glycol
buffer
protein
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French (fr)
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徐敏
张映辉
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派格生物医药(苏州)股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)

Definitions

  • the present invention relates to the field of biotechnology, and specifically relates to a method for preparing a polyethylene glycol conjugate of an active polypeptide or protein.
  • PEGylation is a commonly used method to improve the pharmaceutical properties of active ingredients. It has the effects of improving the water solubility of active ingredients, reducing immunogenicity, reducing renal filtration, prolonging the half-life of circulation in the body, and changing tissue distribution.
  • This technology generally covalently links polyethylene glycol (PEG) molecules to active molecules through chemical or enzymatic reactions. Prior to this, it is usually necessary to carry out appropriate chemical modifications of PEG and introduce specific functional groups to adapt to the subsequent covalent modification process of active molecules.
  • the technical problem to be solved by the present invention is the problem in the prior art that the reaction conversion rate is not high when polyethylene glycol is used to modify polypeptides or proteins, thereby providing a preparation of polyethylene glycol conjugates of active polypeptides or proteins. method.
  • the technical solution of the present invention to solve the above technical problems is: a method for preparing an active polypeptide or protein polyethylene glycol conjugate, which includes the step of mixing and reacting the active polypeptide or protein with polyethylene glycol, wherein the polyethylene glycol conjugate is Ethylene glycol contains a single reactive functional group and has not been connected to a non-polyethylene glycol original functional structure in advance.
  • the total concentration of the conjugate acid-base pair is 0.13-5.5M.
  • the original functional structure of polyethylene glycol includes: active functional groups, ethylene glycol repeating units and end-capping groups, and the rest are It is a "non-polyethylene glycol pro-functional structure", and the non-polyethylene glycol pro-functional structure is preferably a polypeptide or protein.
  • polyethylene glycol mentioned in the present invention has the meaning commonly understood by those of ordinary skill in the art. It can be linear, branched, bifurcated or composed of multiple arms. Different polyethylene glycols can have different The polymer chain length and polymer structure.
  • the "polyethylene glycol original functional structure” mainly includes (CH 2 CH 2 O) n repeating units, and may also include active functional groups and end-capping groups.
  • the reactive functional groups have high reactivity and are used for conjugation with active peptides or proteins.
  • Optional reactive functional groups include halogen, vinyl sulfone, disulfide bond, sulfhydryl group, aldehyde group, carbonyl group, O-substituted hydroxylamine, active ester, Alkenyl, alkynyl, azido or other activated groups, specifically including but not limited to aldehyde derivatives such as propionaldehyde and butyraldehyde, maleimide derivatives, succinimide derivatives and aryl groups Ester derivatives, etc.
  • the succinimide derivatives include succinimide carboxymethyl ester, succinimide valerate, succinimide butyrate methyl ester, succinimide propionate methyl ester, and succinimide butyric acid.
  • SPA succinimide propionate
  • SC Succinimide carbonate
  • NPC nitrophenyl carbonate
  • the end-capping group can be a hydroxyl group or other groups, including but not limited to alkoxy, cycloalkoxy, cycloalkyloxy, alkenyl, aryloxy or aralkyloxy, etc.
  • the end-capping group is an alkoxy group, preferably a methoxy group.
  • Non-polyethylene glycol original functional structures are preferably polypeptides or proteins.
  • polypeptide refers to a linear amino acid chain formed by the dehydration and condensation of 3 or more amino acid molecules.
  • the molecular weight is usually less than 10,000 Da.
  • polypeptides with certain physiological functions in organisms are called active polypeptides.
  • Polypeptides with They have a common structure and have various physiological activities.
  • the active polypeptide plays a role in regulating gene expression and physiological functions, correcting abnormal pathological conditions due to lack or excessive secretion of substances involved in functional regulation in the body, and may include common protein therapeutic agents.
  • Active peptides include immunoactive peptides, neuroactive peptides, other active peptides, etc.
  • active peptides include: cholesterol peptides, mineral absorption-promoting peptides (CPPS), enzyme regulators (such as trypsin), hormonal peptides such as growth hormone releasing factor (GRFS), albumin insulin potentiating peptides, antibacterial peptides (such as nisin, rubberin), anti-cancer peptides (such as tumor cell necrosis factor, cyclohexanide), anti-AIDS peptides (such as GLQ protein), etc.
  • CPPS mineral absorption-promoting peptides
  • enzyme regulators such as trypsin
  • hormonal peptides such as growth hormone releasing factor (GRFS), albumin insulin potentiating peptides
  • antibacterial peptides such as nisin, rubberin
  • anti-cancer peptides such as tumor cell necrosis factor, cyclohexanide
  • anti-AIDS peptides such as GLQ protein
  • Protein refers to a substance with a certain spatial structure (including secondary structure, tertiary structure or quaternary structure) formed by twisting and folding of a polypeptide chain composed of amino acids through dehydration and condensation. Proteins can have only one polypeptide chain, or they can be composed of multiple polypeptide chains, and they usually have biological activity. According to the shape of protein molecules, they can be divided into three categories: 1. Globular proteins; the molecular shape is close to spherical, has good water solubility, and can perform a variety of biological functions. 2. Fibrous protein molecules; rod-shaped or fibrous in shape, most of which are insoluble In water, it is an important structural component of organisms or plays a protective role in organisms. 3. Membrane proteins; generally folded into a nearly spherical shape and inserted into the biological membrane, some are bound to the surface of the biological membrane through non-covalent bonds or covalent bonds. Most functions of biological membranes are achieved through membrane proteins.
  • Proteins have various functions in organisms, such as catalytic functions, motor functions, transport functions, mechanical support and protection functions, immune and defense functions, and regulatory functions. Proteins with catalytic function are called enzymes. From the flagellar movement of the lowest bacteria to the muscle contraction of higher animals, proteins are used. In the process of life activities, the transportation of many small molecules and ions is completed by various specialized proteins. For example, plasma albumin in the blood transports small molecules, and hemoglobin in red blood cells transports oxygen and carbon dioxide. In order to maintain their own survival, organisms have various types of defense means, many of which are performed by proteins. For example, antibodies can recognize and bind foreign substances that invade the organism and eliminate their harmful effects.
  • the single reactive functional group means that the polyethylene glycol contains only one reactive functional group, and the reactive functional group is conventional in this field, such as aldehyde derivatives, maleimide derivatives, and succinimide derivatives.
  • the total concentration of the conjugate acid-base pairs is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M , 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9 M, 3.0M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5.0M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M.
  • the total concentration of the conjugate acid-base pair is 0.15-3M, preferably 0.2-2.5M.
  • the conjugate acid-base pair includes a buffer solution, or a buffer solution and an additional salt.
  • the buffer solution is conventional in the art.
  • the buffer solution is selected from boric acid buffer, phosphate buffer, carbonic acid buffer, acetate buffer, citrate buffer, PBS, Tris and one or more of HEPES.
  • the buffer solution is selected from one or more of boric acid buffer, phosphate buffer and carbonic acid buffer.
  • the buffer solution is phosphate buffer.
  • the conjugate acid-base pair includes a buffer solution, and the concentration of the buffer solution is 0.15-3M.
  • the concentration of the buffer is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M.
  • the concentration of the buffer is preferably 0.2-2M; more preferably 0.25-1M; for example, 0.3-0.8M.
  • the conjugate acid-base pair includes a buffer solution and an additional salt.
  • concentration of the buffer solution is 0.02-0.3M, preferably 0.03-0.2M, and more preferably 0.05 -0.15M.
  • the additional salt added in the present invention can be conventional in the art.
  • the salt can be selected from sodium chloride, sodium acetate, sodium sulfate, sodium phosphate, carbonic acid.
  • the additional salt added is selected from NaCl, KCl, NH 4 Cl, MgCl 2 , CaCl 2 , Na 2 CO 3 , NaHCO 3 , CH 3 COONa, NaH 2 PO 4 , One or more of Na 2 HPO 4 , KH 2 PO 4 , K 2 HPO 4 , Na 2 SO 4 , K 2 SO 4 , (NH 4 ) 2 SO 4 , MgSO 4 and CaSO 4 .
  • the additional added salt is selected from the group consisting of NaCl, KCl, NH 4 Cl, K 2 HPO 4 , CH 3 COONa, Na 2 SO 4 , K 2 SO 4 , (NH 4 ) 2 One or more of SO 4 .
  • the concentration of the additional salt added is 0.02-5M.
  • the concentration of the additional salt added is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2 M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M , 4.7M, 4.8M, 4.9M, 5.0M.
  • the concentration of the additional salt is preferably 0.05-4M; more preferably 0.1-3M, for example, preferably 0.5-2M.
  • the additional salt added is Na 2 SO 4 with a concentration of 0.02-1M, preferably 0.1-0.8M, and more preferably 0.2-0.5M.
  • the additional salt added is NaCl, and the concentration is 0.05-5.0M, preferably 0.1-4.0M, more preferably 0.2-3M, even more preferably 0.5-2M.
  • the additional salt added is CH 3 COONa, and the concentration is 0.5-2.5M, preferably 0.9-1.8M, preferably 1.8M; or the additional salt added is KCl. , the concentration is 0.2-2.0M, preferably 0.7-1.6M, preferably 1.6M; or, the additional added salt is K 2 HPO 4 , the concentration is 0.05-1.0M, preferably 0.1-0.5M, preferably 0.45M .
  • the active polypeptide or protein can be selected from uricase, lysozyme, human growth hormone, growth hormone-releasing hormone, ghrelin, interferon, interferon receptor, colony stimulating hormone Factors, glucagon-like peptides (GLP-1, etc.), oxyntomodulin, Gprotein-coupledreceptor, interleukin, interleukin receptor, enzymes, interleukin binding Protein, cytokine binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitory factor, necrotic glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor Inhibitory factor, transforming growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, erythropoiesis factor, hyperglycosylated erythropoiesis factor, angiopoietin
  • the active polypeptide or protein is an enzyme, such as uricase or lysozyme.
  • the uricase in the present invention can be derived from humans, mammals, plants, microorganisms or hybrids of human and mammalian uricases. It is preferably derived from microorganisms, more preferably derived from fungi and bacteria, such as Arthrobacter globus, Aspergillus flavus, protein Candida, Bacillus fastidiosa, etc.
  • the uricase is derived from Arthrobacter globiformis, and the amino acid sequence of the uricase is preferably as shown in SEQ ID NO: 1, or the amino acid sequence of the uricase is the same as SEQ ID NO :1 have at least 90%, at least 95% or at least 99% sequence identity compared to each other, while at least retaining the function of the original sequence.
  • the concentration of the active polypeptide or protein in the reaction solution is 0.5-2.5 mg/mL, for example, 1.0-2.0 mg/mL.
  • the active polypeptide or protein is uricase, and the concentration is 1.5-2.5 mg/mL, such as 2.0 mg/mL.
  • the active polypeptide or protein is lysozyme, and the concentration is 0.5-1.5 mg/mL, such as 1.0 mg/mL.
  • the molecular weight of the polyethylene glycol is 1 kDa-50 kDa, preferably 5 kDa-30 kDa.
  • the molecular weight of polyethylene glycol is 5kDa, 10kDa, 20kDa, 30kDa or 50kDa.
  • the end-capping polyethylene glycol is a methoxy group; and/or the active functional group of the polyethylene glycol is an aldehyde derivative, a maleimide derivative or
  • the succinimide derivative is preferably N succinimide propionate, succinimide carbonate or nitrophenyl carbonate.
  • the polyethylene glycol is mPEG-SPA-5K, mPEG-SPA-10K, mPEG-SC-5K and/or mPEG-NPC-10K.
  • the mass ratio of the polyethylene glycol and the active polypeptide or protein in the reaction solution is 2:1 to 50:1, preferably 3:1 to 30:1, and more preferably It is 5:1 ⁇ 25:1.
  • the temperature of the reaction is 5-30°C, preferably 20-25°C, and more preferably 25°C.
  • the pH value of the reaction is 6.0-10.0, preferably 6.5-9.5, more preferably 7.0-9.0, even more preferably 7.5-8.7, more preferably 8.7.
  • the active functional group of polyethylene glycol can be combined with the N-terminal amino group, Lys side chain amino group, His side chain imidazolyl group and Ser or Tyr side chain hydroxyl group of the active polypeptide or protein. reaction, but is not limited to this.
  • the conversion rate refers to the conversion rate of the reaction substrate; for polypeptides or proteins randomly modified with polyethylene glycol, the conversion rate refers to the average degree of modification.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the degree of improvement in the reaction conversion rate is dependent on the ionic strength. As the ionic strength of the reaction solution gradually increases, the reaction conversion rate gradually increases.
  • the addition of various types of salt can improve the degree of modification, and the greater the salt concentration, the greater the improvement in degree of modification.
  • PEGylation modification will reduce the activity of active molecules to varying degrees.
  • the degree of modification is increased by increasing the ionic strength, and the activity of the polypeptide or protein is not significantly reduced due to the increase in the degree of modification.
  • Figure 1 shows the SDS-PAGE results of samples with reaction numbers 3-1 to 3-4.
  • Figure 2 shows the SDS-PAGE results of samples with reaction numbers 5-1 to 5-18.
  • Figure 3 shows the SDS-PAGE results of samples with reaction numbers 5-19 to 5-30.
  • Figure 4 is a correlation curve diagram between salt concentration and average modification degree in a series of reactions with different feed ratios.
  • Figure 5 is a graph showing the correlation between enzyme specific activity and average degree of modification and salt concentration.
  • Figure 6 shows the comparison of the experimental values of the average modification degree and the predicted values of the equation.
  • Figure 7 shows the SDS-PAGE results of samples with reaction numbers 9-1 to 9-6.
  • modification degree refers to the number of conjugate molecules coupled to the modified molecule.
  • Average degree of modification refers to the ratio of the amount of substance of the conjugate molecule divided by the amount of substance of the modified molecule for a mixture formed from a group of molecules with different degrees of modification.
  • the “salt” in the present invention refers to a type of compound in which metal ions or ammonium ions (NH 4 + ) are combined with acid ions. It is not sodium chloride in the narrow sense. Solutions and molten salts of soluble salts are conductive, so they can as an electrolyte.
  • polyethylene glycol generally refers to various polyethylene glycols and their derivatives, not specifically H-(OCH 2 CH 2 )n-OH, but “-(OCH 2 CH 2 )n- ” is a type of compound with the main structure.
  • mPEG-SC mPEG-Succinimidyl Carbonate, monomethoxy polyethylene glycol succinimide carbonate
  • mPEG monomethoxy polyethylene glycol
  • mPEG-SPA mPEG-Succinimidyl Propionnate, monomethoxy polyethylene glycol succinyl Imide propionate
  • mPEG monomethoxypolyethylene glycol
  • mPEG-NPC mPEG-Nitrophenyl Carbonate, methoxy-polyethylene glycol-nitrophenyl carbonate
  • reaction conversion rate includes two meanings: the degree and proportion of the reaction products converted into products; or the degree of modification of the modified molecule. If the reaction conversion rate is high, the proportion of reactants converted into products is large; or the degree of modification is high.
  • ionic strength refers to the ionic strength calculated based on the volume molar concentration of ions, in mol/L.
  • uricase is uricase (UniProtKB-DOVWQ1) of Arthrobacter globiformis, and its amino acid sequence is as shown in SEQ ID NO: 1:
  • lysozyme is egg lysozyme, Lysozyme, purchased from SIGMA-ALDRICH, product number 62971-10G-F, CAS: 12650-88-3.
  • N02 refers to a polypeptide with the sequence Ac-LGGSKGGSWG (SEQ ID NO: 2), and Ac represents N-terminal acetylation.
  • Tris is trishydroxymethylaminomethane, CAS: 77-86-1; EDTA is ethylenediaminetetraacetic acid, CAS: 60-00-4; UA is uric acid, CAS: 69-93-2; DMSO It is dimethyl sulfoxide, CAS: 67-68-5.
  • Uricase is purified after fermentation expression in Escherichia coli, and the concentration is measured by UV280nm absorbance method. 4.0mg/mL uricase in 200mM pH 8.7 boric acid buffer solution.
  • the solution additionally contains the salts shown in Table 3 below.
  • PEGi% The percentage of ingredients with degree of modification i.
  • Example 3 Enzyme activity determination of samples obtained from Example 2
  • Buffer (BF8) 50mM pH8.0 Tris-HCl buffer solution, additionally containing 2.0mM disodium EDTA;
  • Uric acid solution 100 ⁇ M, use BF8 to dissolve;
  • Test temperature about 25°C
  • Example 2 Each sample obtained in Example 2 was diluted to an appropriate concentration using BF8. Take 50 ⁇ L sample dilution, add 950 ⁇ L UA100, mix quickly and then measure the UV290nm light absorption, and record the decrease in the absorption value in the first 120s. The test results are shown in Table 5 below.
  • Example 2 After the reaction is completed, analysis is performed according to the method of Example 2.
  • the electrophoresis results are shown in Figure 2 and Figure 3.
  • M is the protein molecular weight standard, and the number represents the reaction number.
  • the product composition percentage and average modification degree of each reaction are shown in Table 7 below. and Figure 4. The results show that in a series of reactions with different feed ratios, as the salt concentration increases, the modification degree shows a trend of increasing.
  • Example 5 Enzyme activity determination of samples obtained from Example 4
  • Buffer (BF8) 50mM pH8.0 Tris-HCl buffer solution, additionally containing 2.0mM disodium EDTA;
  • Uric acid solution 100 ⁇ M, use BF8 to dissolve;
  • Test temperature about 25°C
  • Example 4 Each sample obtained in Example 4 was diluted to an appropriate concentration using BF8. Take 100 ⁇ L sample dilution, add 900 ⁇ L UA100, mix quickly and then measure the UV290nm light absorption, record the light absorption time curve, and calculate the enzyme specific activity based on the curve. The results of 10 times the feed ratio are shown in Table 8 and Figure 5 below. Similar to the results in Example 3, the increase in salt concentration increased the average modification degree, but the product activity did not significantly decrease.
  • the solution additionally contains the following table 10 Salt shown.
  • mPEG-SPA 10 kDa, purchased from Beijing Jiankai
  • the degree of improvement in the reaction conversion rate is dependent on the ionic strength; as the ionic strength of the reaction solution increases, the reaction conversion rate increases.
  • the addition of various types of salt can improve the degree of modification, and the greater the salt concentration, the greater the improvement in degree of modification.
  • N02 (polypeptide with the amino acid sequence of SEQ ID NO: 2) was prepared into a concentrated solution of 5 mg/mL with DMSO, and diluted to 1 mg/mL with pH 9.5 100 mM sodium carbonate buffer. In addition, the following Table 14 was added to the solution: indicated salt. Take 0.5mL N02 solution, add 5mg mPEG-SC (20kDa, purchased from Beijing Jiankai), and react for 2 hours according to the conditions shown in Table 14. After the reaction is completed, use RP-HPLC to analyze the reaction conversion rate.

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Abstract

一种活性多肽或蛋白的聚乙二醇缀合物的制备方法，包括将活性多肽或蛋白与聚乙二醇混合反应的步骤，其中，聚乙二醇含有单活性官能团且未事先与非聚乙二醇原功能结构连接，在反应的反应液中，共轭酸碱对的总浓度为0.13-5.5M。通过增加离子强度提高修饰度，多肽或蛋白的活性并未因修饰度的提高而显著降低。

Description

一种活性多肽或蛋白的聚乙二醇缀合物的制备方法
本申请要求申请日为2022/7/22的中国专利申请2022108686822的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物技术领域，具体涉及一种活性多肽或蛋白的聚乙二醇缀合物的制备方法。
背景技术
活性多肽及蛋白因其独特的生物学效应，被广泛用于各类疾病的治疗，但这类药物容易被血液中的蛋白酶水解并通过肾脏或肝脏被排除。为了维持多肽或蛋白药物在血液中的有效浓度，往往需要频繁地向患者注射药物，这给患者带来了很大的痛苦，并且会产生高昂的治疗费用。此外，外源多肽及蛋白所引起的免疫原性同样是用药时需要考虑的安全问题。
聚乙二醇化是一种常用的提高活性成分成药性的方法，具有提高活性成分的水溶性、减少免疫原性、降低肾脏滤过作用、延长体内循环半衰期、改变组织分布等效果。该技术一般通过化学反应或酶促反应将聚乙二醇(PEG)分子共价连接到活性分子上。在此之前，通常需要对PEG进行适当的化学改造，引入特定的官能团，以适应之后的对活性分子的共价修饰过程。
在PEG化技术中，高质量的PEG原料制备工艺较复杂，生产成本较高。因此，在偶联反应中提高反应转化率对减少物料损耗和降低生产成本具有十分重要的意义。
发明内容
本发明所要解决的技术问题是现有技术中用聚乙二醇修饰多肽或蛋白时的反应转化率不高的问题，从而提供了一种活性多肽或蛋白的聚乙二醇缀合物的制备方法。
本发明解决上述技术问题的技术方案为：一种活性多肽或蛋白聚乙二醇缀合物的制备方法，其包括将活性多肽或蛋白与聚乙二醇混合反应的步骤，其中，所述聚乙二醇含有单活性官能团且未事先与非聚乙二醇原功能结构连接，在所述反应的反应液中，共轭酸碱对的总浓度为0.13-5.5M。
所述聚乙二醇原功能结构包括：活性官能团、乙二醇重复单元和封端基团，其余均 为“非聚乙二醇原功能结构”，所述非聚乙二醇原功能结构优选多肽或蛋白。
本发明所述“聚乙二醇”具有本领域普通技术人员通常理解的含义，其可以为线性的、分枝的、分叉的或由多个臂组成，不同的聚乙二醇可以具有不同的聚合链长度和聚合结构。
“聚乙二醇原功能结构”主要包括(CH2CH2O)n重复单元，还可以包括活性官能团和封端基团。活性官能团具有高反应活性，用于和活性多肽或蛋白进行缀合，可选的活性官能团包括卤素、乙烯基砜、二硫键、巯基、醛基、羰基、O-取代羟氨、活性酯、烯基、炔基、叠氮基或其他活化基团，具体来说，包括但不限于丙醛基、丁醛基等醛衍生物、马来酰亚胺衍生物、琥珀酰亚胺衍生物及芳基酯类衍生物等。所述琥珀酰亚胺衍生物可使用琥珀酰亚胺羧基甲酯、琥珀酰亚胺戊酸酯、琥珀酰亚胺丁酸甲酯、琥珀酰亚胺丙酸甲酯、琥珀酰亚胺丁酸酯、琥珀酰亚胺丙酸酯、羟基琥珀酰亚胺或琥珀酰亚胺碳酸酯；在一些实施方案中，所述活性官能团可以为活性酯，包括琥珀酰亚胺丙酸酯(SPA)、琥珀酰亚胺碳酸酯(SC)、硝基苯基碳酸酯(NPC)等。封端基团可以为羟基，也可以为其他基团，包括但不限于烷氧基、环烷氧基、环烷基氧基、烯基、芳基氧基或芳烷基氧基等，在一些实施方案中，所述封端基团为烷氧基，优选为甲氧基。
除前述(CH2CH2O)n重复单元、活性官能团和封端基团外，其他与聚乙二醇所发挥生物学效应无关的结构为“非聚乙二醇原功能结构”，所述非聚乙二醇原功能结构优选多肽或蛋白。
本发明所述“多肽”指由3个或3个以上氨基酸分子脱水缩合形成的线性氨基酸链，分子量通常低于10,000Da，其中生物体中具有某种生理功能的多肽称为活性多肽，拥有多肽结构的共同点，且具有各种生理活性。所述活性多肽发挥调整基因表达及生理功能,当因在体内参与功能调节的物质缺乏或过度分泌而呈现不正常的病态时进行纠正的作用，可包含通常的蛋白治疗剂。活性多肽包括免疫活性肽、神经活性肽、其他活性肽等。其他活性肽包括：胆固醇肽、促进矿物质吸收的肽(CPPS)、酶调节剂(如促胰酶肽)、激素肽如生长激素释放因子(GRFS)、白蛋白胰岛素增效肽、抗菌多肽(如乳酸链球菌素、橡胶素)、抗癌多肽(如肿瘤细胞坏死因子、环已肽)、抗艾滋病肽(如GLQ蛋白)等。
本发明所述“蛋白质”指由氨基酸以脱水缩合的方式组成的多肽链经过盘曲折叠形成的具有一定空间结构(包括二级结构、三级结构或四级结构)的物质。蛋白质可以只具有一条多肽链，也可以是由多条多肽链构成，具有通常都具有生物学活性。根据蛋白质分子的外形，可以将其分作3类：1.球状蛋白质；分子形状接近球形，水溶性较好，可行使多种多样的生物学功能。2.纤维状蛋白质分子；外形呈棒状或纤维状，大多数不溶 于水，是生物体重要的结构成分，或对生物体起保护作用。3.膜蛋白质；一般折叠成近球形，插入生物膜，也有一些通过非共价键或共价键结合在生物膜的表面。生物膜的多数功能是通过膜蛋白实现的。
蛋白质在生物体中有多种功能，例如催化功能、运动功能、运输功能、机械支持和保护功能、免疫和防御功能和调节功能等。有催化功能的蛋白质称酶。从最低等的细菌鞭毛运动到高等动物的肌肉收缩都是通过蛋白质实现的。在生命活动过程中，许多小分子及离子的运输是由各种专一的蛋白质来完成的，例如在血液中血浆白蛋白运送小分子、红细胞中的血红蛋白运送氧气和二氧化碳等。生物体为了维持自身的生存，拥有多种类型的防御手段，其中不少是靠蛋白质来执行的，例如抗体能识别和结合侵入生物体的外来物质，消除其有害作用。
所述单活性官能团指的是所述聚乙二醇仅含有一个活性官能团，所述活性官能团为本领域常规，例如醛衍生物、马来酰亚胺衍生物和琥珀酰亚胺衍生物等。
在本发明的一些优选实施方案中，所述共轭酸碱对的总浓度为0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M、0.9M、1.0M、1.1M、1.2M、1.3M、1.4M、1.5M、1.6M、1.7M、1.8M、1.9M、2.0M、2.2M、2.3M、2.4M、2.5M、2.6M、2.7M、2.8M、2.9M、3.0M、3.1M、3.2M、3.3M、3.4M、3.5M、3.6M、3.7M、3.8M、3.9M、4.0M、4.1M、4.2M、4.3M、4.4M、4.5M、4.6M、4.7M、4.8M、4.9M、5.0M、5.1M、5.2M、5.3M、5.4M、5.5M。
如本发明技术方案所述的制备方法，所述共轭酸碱对的总浓度为0.15-3M，优选为0.2-2.5M。
如本发明技术方案所述的制备方法，所述共轭酸碱对包括缓冲溶液，或缓冲溶液和额外添加的盐。
所述缓冲溶液为本领域常规，在本发明的较佳实施方案中，所述缓冲溶液选自硼酸缓冲液、磷酸缓冲液、碳酸缓冲液、醋酸缓冲液、枸橼酸缓冲液、PBS、Tris和HEPES中的一种或多种。
在本发明的更佳实施方案中，所述缓冲溶液选自硼酸缓冲液、磷酸缓冲液和碳酸缓冲液中的一种或多种。
在本发明的进一步更佳实施方案中，所述缓冲溶液为磷酸缓冲液。
如本发明技术方案所述的制备方法，所述共轭酸碱对包括缓冲溶液，所述缓冲溶液的浓度为0.15-3M。
在本发明的一些优选实施方案中，所述缓冲液的浓度为0.2M、0.3M、0.4M、0.5M、 0.6M、0.7M、0.8M、0.9M、1.0M、1.1M、1.2M、1.3M、1.4M、1.5M、1.6M、1.7M、1.8M、1.9M、2.0M。
在本发明的具体实施方案中，所述缓冲液的浓度优选为0.2-2M；更优选为0.25-1M；例如为0.3-0.8M。
如本发明技术方案所述的制备方法，所述共轭酸碱对包括缓冲溶液和额外添加的盐，所述缓冲溶液的浓度为0.02-0.3M，优选为0.03-0.2M，更优选为0.05-0.15M。
本发明所额外添加的盐可为本领域常规，例如所述盐可选自由氯化钠(sodium chloride)、乙酸钠(sodium acetate)、硫酸钠(sodium sulfate)、磷酸钠(sodium phosphate)、碳酸钠(sodium carbonate)、氰化钠(sodium cyanide)、柠檬酸钠(sodium citrate)、硝酸钠(sodium nitrate)、氯化钾(potassium chloride)、乙酸钾(potassium acetate)、硫酸钾(potassium sulfate)、磷酸钾(potassium phosphate)、碳酸钾(potassium carbonate)、氰化钾(potassium cyanide)、柠檬酸钾(potassium citrate)、硝酸钾(potassium nitrate)、氯化镁(magnesium chloride)、乙酸镁(magnesium acetate)、硫酸镁(magnesium sulfate)、磷酸镁(magnesium phosphate)、碳酸镁(magnesium carbonate)、氰化镁(magnesium cyanide)、柠檬酸镁(magnesium citrate)、硝酸镁(magnesium nitrate)、氯化铵(ammonium chloride)、乙酸铵(ammonium acetate)、硫酸铵(ammonium sulfate)、磷酸铵(ammonium phosphate)、碳酸铵(ammonium carbonate)、氰化铵(ammonium cyanide)、柠檬酸铵(ammonium citrate)、硝酸铵(ammonium nitrate)、氯化钙(calcium chloride)、乙酸钙(calcium acetate)、硫酸钙(calcium sulfate)、磷酸钙(calcium phosphate)、碳酸钙(calcium carbonate)、氰化钙(calcium cyanide)、柠檬酸钙(calcium citrate)或硝酸钙(calcium nitrate)所构成的群组，但并不限制于此。
如本发明技术方案所述的制备方法，所述额外添加的盐选自NaCl、KCl、NH4Cl、MgCl2、CaCl2、Na2CO3、NaHCO3、CH3COONa、NaH2PO4、Na2HPO4、KH2PO4、K2HPO4、Na2SO4、K2SO4、(NH4)2SO4、MgSO4和CaSO4中的一种或多种。
在本发明的较佳实施方案中，所述额外添加的盐选自NaCl、KCl、NH4Cl、K2HPO4、CH3COONa、Na2SO4、K2SO4、(NH4)2SO4中的一种或多种。
如本发明技术方案所述的制备方法，所述额外添加的盐的浓度为0.02-5M。
在本发明的一些优选实施方案中，所述额外添加的盐的浓度为0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M、0.9M、1.0M、1.1M、1.2M、1.3M、1.4M、1.5M、1.6M、1.7M、1.8M、1.9M、2.0M、2.2M、2.3M、2.4M、2.5M、2.6M、2.7M、2.8M、2.9M、3.0M、3.1M、3.2M、3.3M、3.4M、3.5M、3.6M、3.7M、3.8M、3.9M、4.0M、4.1M、4.2M、4.3M、4.4M、4.5M、4.6M、4.7M、4.8M、4.9M、5.0M。
在本发明的具体实施方案中，所述额外添加的盐的浓度优选为0.05-4M；更优选为0.1-3M，例如优选为0.5-2M。
如本发明技术方案所述的制备方法，所述额外添加的盐为Na2SO4，浓度为0.02-1M，优选为0.1-0.8M，更优选为0.2-0.5M。
如本发明技术方案所述的制备方法，所述额外添加的盐为NaCl，浓度为0.05-5.0M，优选为0.1-4.0M，更优选为0.2-3M，甚至更优选为0.5-2M。
如本发明技术方案所述的制备方法，所述额外添加的盐为CH3COONa，浓度为0.5-2.5M，优选为0.9-1.8M，优选1.8M；或，所述额外添加的盐为KCl，浓度为0.2-2.0M，优选为0.7-1.6M，优选1.6M；或，所述额外添加的盐为K2HPO4，浓度为0.05-1.0M，优选为0.1-0.5M，优选0.45M。
如本发明技术方案所述的制备方法，所述活性多肽或蛋白可选自由尿酸酶、溶菌酶、人生长激素、生长激素释放激素、生长激素释放肽、干扰素、干扰素受体、集落刺激因子、类胰高血糖素肽(GLP-1等)、胃泌酸调节素、G蛋白偶联受体(Gprotein-coupledreceptor)、白细胞介素、白细胞介素受体、酶类、白细胞介素结合蛋白、细胞因子结合蛋白、巨噬细胞活化因子、巨噬细胞肽、B细胞因子、T细胞因子、蛋白质A、变态反应抑制因子、细胞坏死糖蛋白、免疫毒素、淋巴毒素、肿瘤坏死因子、肿瘤抑制因子、转化生长因子、α-1抗胰蛋白酶、白蛋白、α-乳白蛋白、载脂蛋白E、促红细胞生成因子、高糖基化促红细胞生成因子、血管生成素、血红素、凝血酶、凝血酶受体活化肽、血栓调节素、血液因子VII、VIIa、VIII、IX与XIII、纤溶酶原活性因子、纤维蛋白肽、尿激酶、链球菌激酶、水蛭素、蛋白质C、C-反应蛋白、肾素抑制剂、胶原酶抑制剂、超氧化岐化酶、瘦蛋白、血小板衍生生长因子、上皮细胞生长因子、表皮细胞生长因子、血管抑制素、血管收缩素、骨生长因子、促骨生成蛋白、降血钙素、胰岛素、心钠素、软骨诱导因子、依降钙素、结缔组织活化因子、组织因子途径抑制剂、卵泡刺激素、黄体生成素、黄体生成素释放激素、神经生长因子、甲状旁腺激素、松弛素、分泌素、生长调节素、类胰岛素生长因子、肾上腺皮质激素、胰高血糖素、胆囊收缩素、胰腺多肽、促胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺刺激激素、自毒素、乳铁蛋白、肌肉抑制素、细胞表面抗原、来自病毒的疫苗抗原、单克隆抗体、多克隆抗体及抗体片段所构成的群组，但并不限制于此。
在本发明的具体实施方案中，所述活性多肽或蛋白为酶，例如尿酸酶或溶菌酶。
本发明中的尿酸酶可来源于人、哺乳动物、植物、微生物或人与哺乳动物尿酸酶的杂合体，优选来源于微生物，更优选来源于真菌和细菌，例如球形节杆菌、黄曲霉、产朊 假丝酵母、苛求芽孢杆菌等。
在本发明的具体实施方案中，所述尿酸酶来源于球形节杆菌Arthrobacter globiformis，所述尿酸酶的氨基酸序列优选如SEQ ID NO:1所示，或所述尿酸酶的氨基酸序列与SEQ ID NO:1相比具有至少90％、至少95％或者至少99％的序列同一性，同时至少保留原本序列的功能。
如本发明技术方案所述的制备方法，反应液中所述活性多肽或蛋白的浓度为0.5-2.5mg/mL，例如为1.0-2.0mg/mL。
在本发明的优选实施方案中，所述活性多肽或蛋白为尿酸酶，浓度为1.5-2.5mg/mL，例如2.0mg/mL。或所述活性多肽或蛋白为溶菌酶，浓度为0.5-1.5mg/mL，例如1.0mg/mL。
如本发明技术方案所述的制备方法，所述聚乙二醇的分子量为1kDa-50kDa，优选为5kDa-30kDa。例如所述聚乙二醇的分子量为5kDa、10kDa、20kDa、30kDa或50kDa。
在本发明的较佳实施方案中，所述聚乙二醇的封端为甲氧基；和/或，所述聚乙二醇的活性官能团为醛衍生物、马来酰亚胺衍生物或琥珀酰亚胺衍生物，优选为N琥珀酰亚胺丙酸酯、琥珀酰亚胺碳酸酯或硝基苯基碳酸酯。
在本发明的更佳实施方案中，所述聚乙二醇为mPEG-SPA-5K、mPEG-SPA-10K、mPEG-SC-5K和/或mPEG-NPC-10K。
如本发明技术方案所述的制备方法，反应液中所述聚乙二醇和所述活性多肽或蛋白的质量比为2:1～50:1，优选为3:1～30:1，更优选为5:1～25:1。
如本发明技术方案所述的制备方法，所述反应的温度为5～30℃，优选为20～25℃，更优选为25℃。
如本发明技术方案所述的制备方法，所述反应的pH值为6.0～10.0，优选为6.5～9.5，更优选为7.0～9.0，甚至更优选为7.5～8.7，更优选为8.7。
如本发明技术方案所述的制备方法，所述聚乙二醇的活性官能团可与所述活性多肽或蛋白的N末端氨基、Lys侧链氨基、His侧链咪唑基以及Ser或Tyr侧链羟基反应，但并不限制于此。
在本发明中，对于聚乙二醇定点修饰的多肽或蛋白，转化率指反应底物的转化率；对于聚乙二醇随机修饰的多肽或蛋白，转化率指平均修饰度。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明人发现在活性多肽或蛋白与聚乙二醇缀合的反应中，增加反应液离子强度即可以提高反应转化率。
反应转化率的提高程度具有离子强度依赖性，随着反应液离子强度的逐步提高，反应转化率逐步提高。多种类型的盐的添加都能提高修饰度，且盐浓度越大，修饰度提高的幅度越大。
一般来说，PEG化修饰会使活性分子的活性不同程度的下降，修饰度越高活性下降幅度越大。但在本发明中，通过增加离子强度提高修饰度，多肽或蛋白的活性并未因修饰度的提高而显著降低。
附图说明
图1为反应编号3-1～3-4的样品SDS-PAGE结果图。
图2为反应编号5-1～5-18的样品SDS-PAGE结果图。
图3为反应编号5-19～5-30的样品SDS-PAGE结果图。
图4为不同投料比的系列反应中盐浓度和平均修饰度的相关性曲线图。
图5为酶比活和平均修饰度与盐浓度之间相关性的曲线图。
图6为平均修饰度的实验值和方程预测值的对比。
图7为反应编号9-1～9-6的样品SDS-PAGE结果图。
具体实施方式
术语解释：
本发明中，“修饰度”指的是被修饰分子上偶联的缀合物分子的数量。“平均修饰度”指的是对于由一群具有不同修饰度的分子形成的混合物，将缀合物分子的物质的量除以被修饰分子的物质的量，得到的比值。
本发明中的“盐”是指一类金属离子或铵根离子(NH4 +)与酸根离子结合的化合物，不是狭义的氯化钠，可溶盐的溶液及熔盐有导电性，因此可作为电解质。
本发明中，聚乙二醇(PEG)泛指各种聚乙二醇和其衍生物，非特指H-(OCH2CH2)n-OH，而是以“-(OCH2CH2)n-”为主要结构的一类化合物。
本发明中，mPEG-SC(mPEG-Succinimidyl Carbonate，单甲氧基聚乙二醇琥珀酰亚胺碳酸酯)为羟基末端活化为琥珀酰亚胺碳酸酯的单甲氧基聚乙二醇(mPEG)修饰剂。
本发明中，mPEG-SPA(mPEG-Succinimidyl Propionnate，单甲氧基聚乙二醇琥珀酰 亚胺丙酸酯)为羟基末端活化为琥珀酰亚胺丙酸酯的单甲氧基聚乙二醇(mPEG)修饰剂。
mPEG-NPC(mPEG-Nitrophenyl Carbonate,甲氧基-聚乙二醇-硝基苯基碳酸酯)为一种线本发明中性单官能团甲基醚PEG，带有活性硝基苯碳酸酯或NPC基团。
本发明中，“反应转化率”包含两层含义：反应物转化为产物的程度和比例；或者被修饰分子的修饰度。反应转化率高，则反应物转化成产物的比例大；或者修饰度高。
本发明中，“离子强度”指的是以离子的体积摩尔浓度计算所得的离子强度，单位mol/L。
本发明中，尿酸酶为球形节杆菌(Arthrobacter globiformis)的尿酸酶(UniProtKB-D0VWQ1)，其氨基酸序列如SEQ ID NO:1所示：
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC，其中，BL21(DE3)菌株表达时第一位氨基酸M会被切除。
本发明中，溶菌酶为鸡蛋溶菌酶，Lysozyme，采购自SIGMA-ALDRICH，货号62971-10G-F，CAS：12650-88-3。
本发明中，N02指的是多肽，序列Ac-LGGSKGGSWG(SEQ ID NO:2)，Ac代表N末端乙酰化。
本发明中，Tris为三羟甲基氨基甲烷，CAS：77-86-1；EDTA为乙二胺四乙酸，CAS：60-00-4；UA为尿酸，CAS：69-93-2；DMSO为二甲亚砜，CAS：67-68-5。
查化学手册获得各种盐的溶解度信息，见下表1。
表1

下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1尿酸酶对盐浓度的耐受性
尿酸酶通过大肠杆菌发酵表达后经提纯所得，浓度通过UV280nm吸光度法测定。4.0mg/mL尿酸酶在200mM pH 8.7硼酸缓冲溶液中。
按下表2混合各溶液，测试尿酸酶对盐浓度的耐受情况，目测是否形成不溶性物质。测试显示尿酸酶对多种类型的盐在相当高的浓度下都保持稳定。
表2
实施例2 mPEG-SPA(5kDa)修饰尿酸酶
2.0mg/mL尿酸酶在100mM pH8.7硼酸缓冲溶液中，此外溶液中额外含有如下表3所示的盐。取蛋白溶液0.5mL，加入10mg mPEG-SPA(5kDa，采购自日本油脂)，25℃反应2h。
表3
反应完毕后SDS-PAGE变性电泳，考马斯亮蓝染色分析反应结果，如图1，M是蛋白分子量标准品，U是尿酸酶，3-1～3-4代表反应编号3-1～3-4。结果显示随着盐浓度的逐步提高，平均修饰度不断提高。电泳图使用FIJI软件分析，获得修饰产物各成分百分 比的定量数据，如表4所示。PEG0、PEG1、PEG2、PEG3、PEG4、PEG5代表不同的修饰度的产物，其中PEG代表聚乙二醇分子，数字代表修饰度。PEG0代表未被修饰的尿酸酶。平均修饰度的计算公式如下：
M：平均修饰度；
i：修饰度；
PEGi％：修饰度为i的成分的百分比。
表4
实施例3从实施例2中获得的样品的酶活测定
酶活测定条件：
缓冲液(BF8)：50mM pH8.0Tris-HCl缓冲溶液，另含2.0mM EDTA二钠；
尿酸溶液(UA100)：100μM，使用BF8溶解；
测试温度：约25℃；
实施例2中获得的各样品，使用BF8稀释到合适浓度。取50μL样品稀释液，加入950μL UA100，迅速混匀后测定UV290nm的光吸收，记录最初的120s吸收值的下降幅度。测试结果如下表5。
数据显示各修饰产物活性保留程度很高(70-90％)。一般来说，PEG化修饰会使活性分子的活性不同程度的下降，修饰度越高活性下降幅度越大。但有趣的是反应3-2～3-4的修饰度虽然显著高于反应3-1，但活性保留比例并未显著低于反应3-1，甚至还不同程度的有所提高。
表5

实施例4 mPEG-SPA(10kDa)修饰尿酸酶
2.0mg/mL尿酸酶在100mM pH8.7硼酸缓冲溶液中，此外溶液中额外含有如下表6所示的盐。取蛋白溶液适量，加入计算量的mPEG-SPA(10kDa，采购自北京键凯)，25℃反应2h。
表6
反应完毕后，按照实施例2的方式进行分析处理。电泳结果如图2、图3所示，M是蛋白分子量标准品，数字代表反应编号。各反应的产物组成百分比和平均修饰度见下表7 和图4。结果显示，在各种不同投料比的系列反应中，伴随盐浓度的提高，修饰度都呈现趋势性上升。
表7
实施例5从实施例4中获得的样品的酶活测定
酶活测定条件：
缓冲液(BF8)：50mM pH8.0 Tris-HCl缓冲溶液，另含2.0mM EDTA二钠；
尿酸溶液(UA100)：100μM，使用BF8溶解；
测试温度：约25℃；
实施例4中获得的各样品，使用BF8稀释到合适浓度。取100μL样品稀释液，加入900μL UA100，迅速混匀后测定UV290nm的光吸收，记录光吸收时间曲线，根据曲线计算酶比活。10倍投料比的结果如下表8和图5。与实施例3的结果类似，盐浓度的上升提高了平均修饰度，但产物活性并未因此发生显著降低。
表8
实施例6修饰度方程
根据上述实施例4的实验数据，进行二元二次方程拟合，获得如下方程(R2＝0.9831)；并根据方程比较平均修饰度的方程计算值和实验值的差异，如下表9和图6所示。
M＝-0.001273×N2-0.3372×C2+0.01936×N×C+0.1351×N
+0.9055×C+0.4218
R2：0.9831
M：平均修饰度，≥0；
N：投料比，PEG/尿酸酶(m/m)，≥0；
C：氯化钠浓度，单位mol/L，≥0.
表9
实施例7改变盐的类型
2.0mg/mL尿酸酶在100mM pH8.7硼酸缓冲溶液中，此外溶液中额外含有如下表10 所示的盐。取蛋白溶液0.25mL，加入7.5mg的mPEG-SPA(10kDa，采购自北京键凯)，25℃反应2h。反应转化率的提高程度具有离子强度依赖性；随着反应液离子强度的提高，反应转化率提高。多种类型的盐的添加都能提高修饰度，且盐浓度越大，修饰度提高的幅度越大。
表10
实施例8改变修饰反应浓度
1.0mg/mL尿酸酶在100mM pH8.7硼酸缓冲溶液中，此外溶液中额外含有如下表11所示的盐。取尿酸酶0.5mL，加入10mg mPEG-SPA(10kDa，采购自北京键凯)，25℃反应2h。
表11
反应完毕后，按照实施例2的方式进行分析处理。电泳结果如图7，数字代表反应编号。试验结果同实施例2和4保持一致，随着盐浓度的提高，修饰度上升。
实施例9改变pH值
2.0mg/mL尿酸酶在不同浓度的pH7.5的磷酸钾缓冲溶液中。取各尿酸酶0.5mL，加入15mg mPEG-SPA(10kDa，采购自北京键凯)，25℃反应2h。反应完毕后，按照实施例2的方式进行分析处理。结果显示，随着磷酸钾缓冲盐浓度升高，平均修饰度提高， 如下表12。本实施例说明：①增加离子强度提高反应转化率的效果在不同的pH值条件下都起效；②仅通过提高缓冲溶液的浓度来提高反应液的离子强度，也能达到增加反应转化率的效果。
表12
实施例10 mPEG-SC(5kDa)和mPEG-NPC(10kDa)修饰尿酸酶
2.0mg/mL尿酸酶在50mM pH8.7硼酸缓冲溶液中，此外溶液中额外含有如下表13所示的盐。按表中配比进行反应，25℃反应2h。反应完毕后，按照实施例2的方式进行分析处理。结果显示，无论是mPEG-SC-5K(5kDa，采购自日本油脂)，还是mPEG-NPC-10K(10kDa，采购自日本油脂)，平均修饰度都随着盐浓度的提高而提高。
表13
实施例11不同温度下聚乙二醇修饰Lys侧链氨基
N02(氨基酸序列为SEQ ID NO:2的多肽)用DMSO配置成5mg/mL的浓液，并用pH9.5 100mM碳酸钠缓冲液稀释到1mg/mL，此外在溶液中还额外添加如下表14所示的盐。取0.5mL N02溶液，加入5mg mPEG-SC(20kDa，采购自北京键凯)，按表14所示条件反应2h。反应完毕后，使用RP-HPLC分析反应转化率，色谱柱：C18-300A-4.6*250mm(Phenomenex)；流动相A：0.05％TFA in H2O；流动相B：0.05％TFA in CAN；柱温：25℃；流速：1mL/min。如表14，无论在5℃条件下反应，还是在室温(约25℃)条件下反应，在反应液中添加盐都有利于提高反应转化率。
表14
实施例12聚乙二醇化溶菌酶
1.0mg/mL溶菌酶在50mM pH8.5硼酸缓冲溶液中，此外溶液中额外含有如下表15所示的盐。取酶溶液5mL，加入20mg PEG室温反应，见下表15。反应结束后使用实施例2的方法分析平均修饰度。结果显示硫酸钠浓度的增大提高了平均修饰度。
表15
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (14)

  1. 一种活性多肽或蛋白的聚乙二醇缀合物的制备方法，其包括将活性多肽或蛋白与聚乙二醇混合反应的步骤，其特征在于，所述聚乙二醇含有单活性官能团且未事先与非聚乙二醇原功能结构连接，在所述反应的反应液中，共轭酸碱对的总浓度为0.13-5.5M。
  2. 如权利要求1所述的制备方法，其特征在于，所述共轭酸碱对的总浓度为0.15-3M，优选为0.2-2.5M。
  3. 如权利要求1或2所述的制备方法，其特征在于，所述共轭酸碱对包括缓冲溶液，或缓冲溶液和额外添加的盐；
    较佳地，所述缓冲溶液选自硼酸缓冲液、磷酸缓冲液、碳酸缓冲液、醋酸缓冲液、枸橼酸缓冲液、PBS、Tris和HEPES中的一种或多种；
    更佳地，所述缓冲溶液选自硼酸缓冲液、磷酸缓冲液和碳酸缓冲液中的一种或多种。
  4. 如权利要求3所述的制备方法，其特征在于，所述共轭酸碱对包括缓冲溶液，所述缓冲溶液的浓度为0.15-3M；优选为0.2-2M；更优选为0.25-1M；甚至更优选为0.3-0.8M。
  5. 如权利要求3所述的制备方法，其特征在于，所述共轭酸碱对包括缓冲溶液和额外添加的盐，所述额外添加的盐选自NaCl、KCl、NH4Cl、MgCl2、CaCl2、Na2CO3、NaHCO3、CH3COONa、NaH2PO4、Na2HPO4、KH2PO4、K2HPO4、Na2SO4、K2SO4、(NH4)2SO4、MgSO4和CaSO4中的一种或多种；
    较佳地，所述额外添加的盐选自NaCl、KCl、NH4Cl、K2HPO4、CH3COONa、Na2SO4、K2SO4、(NH4)2SO4中的一种或多种。
  6. 如权利要求5所述的制备方法，其特征在于，所述额外添加的盐的浓度为0.02-5M；优选为0.05-4M；更优选为0.1-3M，甚至更优选为0.5-2M。
  7. 如权利要求6所述的制备方法，其特征在于，所述额外添加的盐为Na2SO4，浓度为0.02-1M，优选为0.1-0.8M，更优选为0.2-0.5M。
  8. 如权利要求6所述的制备方法，其特征在于，所述额外添加的盐为NaCl，浓度为0.05-5.0M，优选为0.1-4.0M，更优选为0.2-3M，甚至更优选为0.5-2M。
  9. 如权利要求6所述的制备方法，其特征在于，所述额外添加的盐为CH3COONa，浓度为0.5-2.5M，优选为0.9-1.8M；或，
    所述额外添加的盐为KCl，浓度为0.2-2.0M，优选为0.7-1.6M；或，
    所述额外添加的盐为K2HPO4，浓度为0.05-1.0M，优选为0.1-0.5M。
  10. 如权利要求5所述的制备方法，其特征在于，所述缓冲溶液的浓度为0.02-0.3M，优选为0.03-0.2M，更优选为0.05-0.15M。
  11. 如权利要求1～10任一项所述的制备方法，其特征在于，所述活性多肽或蛋白为酶，例如尿酸酶或溶菌酶；
    较佳地，所述尿酸酶来源于微生物，优选细菌；
    更佳地，所述尿酸酶来源于球形节杆菌(Arthrobacter globiformis)，所述尿酸酶的氨基酸序列优选如SEQ ID NO:1所示。
  12. 如权利要求1～11任一项所述的制备方法，其特征在于，反应液中所述活性多肽或蛋白的浓度为0.5-2.5mg/mL。
  13. 如权利要求1～12任一项所述的制备方法，其特征在于，所述聚乙二醇的分子量为1kDa-50kDa，优选为5kDa-30kDa。
  14. 如权利要求1～13任一项所述的制备方法，其特征在于，反应液中所述聚乙二醇和所述活性多肽或蛋白的质量比为2:1～50:1，优选为3:1～30:1，更优选为5:1～25:1；和/或，
    所述反应的pH值为6.0～10.0，优选为6.5～9.5，更优选为7.0～9.0，甚至更优选为7.5～8.7。
PCT/CN2023/108707 2022-07-22 2023-07-21 一种活性多肽或蛋白的聚乙二醇缀合物的制备方法 WO2024017379A1 (zh)

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* Cited by examiner, † Cited by third party
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CN1561390A (zh) * 2001-08-02 2005-01-05 菲尼克斯药物股份有限公司 Peg修饰的尿酸酶
CN101928704A (zh) * 2010-03-04 2010-12-29 杭州北斗生物技术有限公司 黄曲霉尿酸氧化酶的聚乙二醇修饰物及其制备方法
CN105820232A (zh) * 2016-04-08 2016-08-03 昂德生物药业有限公司 单修饰聚乙二醇重组人促红素的制备方法及其制品和应用
CN114438047A (zh) * 2020-11-05 2022-05-06 重庆派金生物科技有限公司 制备聚乙二醇修饰的尿酸氧化酶的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1561390A (zh) * 2001-08-02 2005-01-05 菲尼克斯药物股份有限公司 Peg修饰的尿酸酶
CN101928704A (zh) * 2010-03-04 2010-12-29 杭州北斗生物技术有限公司 黄曲霉尿酸氧化酶的聚乙二醇修饰物及其制备方法
CN105820232A (zh) * 2016-04-08 2016-08-03 昂德生物药业有限公司 单修饰聚乙二醇重组人促红素的制备方法及其制品和应用
CN114438047A (zh) * 2020-11-05 2022-05-06 重庆派金生物科技有限公司 制备聚乙二醇修饰的尿酸氧化酶的方法

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