METHOD FOR PREPARING PEGYLATED BIOMOLECULE WITH CONTROLLABLE BINDING SITES

Abstract

The present invention discloses a method for preparing a PEGylated biomolecule with controllable binding sites, comprising: (1) PEGylating a biomolecule; (2) binding a barrier to at least one binding site in the PEGylated biomolecule; (3) separating the PEGylated biomolecule not bound to the barrier; and (4) separating the barrier and the PEGylated biomolecule bound thereto. In another aspect, the present invention discloses a method for preparing a PEGylated IL-2 with controllable binding sites, comprising: (1) PEGylating to couple a PEG with IL-2; (2) binding the PEGylated IL-2 to an IL-2α receptor; (3) separating the PEGylated IL-2 not bound to the IL-2α receptor; and (4) separating the IL-2α receptor and the PEGylated IL-2 bound thereto. By regulating the binding sites of IL-2, only 1 or 2 PEGs are added during PEGylation.

Claims

1. A method for preparing a PEGylated biomolecule with controllable binding sites, comprising: (1) PEGylating a biomolecule; (2) binding a barrier to at least one binding site in the PEGylated biomolecule; (3) separating the PEGylated biomolecule not bound to the barrier; and (4) separating the barrier and the PEGylated biomolecule bound thereto; wherein the biomolecule in the step (1) is selected from biological macromolecules and biological micromolecules with specific binding effect to the barrier, and the biomolecule has at least one binding site for the barrier; the barrier and the biomolecule are selected from the following combinations: a ligand and a receptor, a DNA strand and a complementary DNA or RNA strand thereof, an enzyme and a substrate thereof, an enzyme and a competitive inhibitor thereof, an enzyme and a cofactor thereof, a vitamin and a specific binding protein thereof, and a glycoprotein and a corresponding lectin thereof.

2. The method according to claim 1, wherein the DNA strand and the complementary DNA or RNA strand thereof are selected from: a gene probe and a gene sequence of complementary bases; the enzyme and the substrate thereof are selected from: a protease and a protein, an amylase and a starch, a nuclease and a nucleic acid, lactate dehydrogenase and lactic acid, and oxaloacetate decarboxylase and oxaloacetic acid; the enzyme and the competitive inhibitor thereof are selected from: succinate dehydrogenase and malonic acid, dihydrofolate synthetase and a sulfonamide, and cholinesterase and an organophosphorus pesticide; the enzyme and the coenzyme factor thereof are selected from: pyruvate dehydrogenase and Mn.sup.2+, and catalase and oxidoreductase and Fe.sup.2+/Fe.sup.3+; the hormone and the receptor are selected from: estrogen and an estrogen receptor, androgen and an androgen receptor, mineralocorticoid and a mineralocorticoid receptor, thyroid hormone and a thyroid hormone receptor, and progestogen and a progestogen receptor; the drug and the receptor are selected from: insulin, insulin-like growth factor, epithelial growth factor, platelet-derived growth factor or a lymphokine and a receptor with tyrosine kinase activity, and epinephrine, dopamine, 5-hydroxytryptamine, M-acetylcholine, an opioid, a purine, prostaglandin or a polypeptide hormone drug and G protein-coupled receptor; the vitamin and the specific binding protein thereof are selected from: vitamin A and a retinol binding protein, vitamin D and vitamin D binding protein, α-tocopherol and α-tocopherol transport protein, and vitamin K and a lipoprotein; the glycoprotein and the corresponding lectin thereof are selected from: horseradish peroxidase and concanavalin A, lentil lectin and pea lectin, jacalin and galactose, an amaranthin-like lectin and N-acetylgalactosamine, a dimer of chitin-binding lectin and an N-acetylglucosamine oligosaccharide, Dolichos biflorus agglutinin and blood group A substance, and Ulex europaeus agglutinin and blood group O substance 2-L-fucose.

3. The method according to claim 2, wherein the drug and the receptor are selected from: IL-2 and an IL-2 receptor, and the IL-2 receptor includes IL-2α receptor, IL-2β receptor and IL-27 receptor.

4. The method according to claim 1, wherein the step (1) further comprises separating and purifying the PEGylated biomolecule; and the separating in the step (3) and the step (4) includes: one or a combination of two or more of centrifugal separation, precipitation separation, filtration separation, foam separation, extraction separation, membrane separation, chromatographic separation, electrophoretic separation and gradient elution.

5. The method according to claim 1, wherein the PEGylated biomolecule in the step (1) has the following structure: ##STR00037## m is an integer of 1-12, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; X is a linking group between the PEG and the biomolecule selected from: one or a combination of two or more of —(CH.sub.2)a-, —(CR.sub.1R.sub.2)a-, —(CH.sub.2)aNH—, —NHCO(CH.sub.2)a-, —(CH.sub.2)aCONH—, —(CH.sub.2)aCO—, —CO(CH.sub.2)a-, —(CH.sub.2)aCONH(CH.sub.2)a-, —(CH.sub.2)a-S—S—(CH.sub.2)a-, —(CH.sub.2)aCOO(CH.sub.2)a- and —(CH.sub.2)a-S—(CH.sub.2)a-; a is an integer of 0-10; R.sub.1 and R.sub.2 are independently selected from: one or a combination of two or more of —H, a C.sub.1-6 alkyl, —OR′, —NHR′, —N(R′).sub.2, —CN, —F, —Cl, —Br, —I, —COR′, —COOR′, —OCOR′, —CONHR′ and —CON(R′).sub.2; R′ is selected from: —H, a C.sub.1-6 alkyl, —F, —Cl, —Br and —I; the PEG is a linear, Y-shaped or multi-branched polyethylene glycol residue including monomethoxy polyethylene glycol (mPEG), a linear double-ended PEG, a Y-shaped PEG, a 4-arm branched PEG, a 6-arm branched PEG and an 8-arm branched PEG with a molecular weight of 1-100 KDa.

6. The method according to claim 5, wherein a is an integer of 0-5; R.sub.1 and R.sub.2 are independently selected from: one or a combination of two or more of —H, a C.sub.1-3 alkyl, —OH, a C.sub.1-3 alkoxy, —NH.sub.2, —F, —Cl, —Br and —I; R′ is selected from: —H and a C.sub.1-3 alkyl; the PEG is a linear polyethylene glycol residue having a structure of general formula II or III: ##STR00038## wherein p and q are independently selected from an integer of 1-2280; the PEG is a Y-shaped polyethylene glycol residue having a structure of general formula IV or V: ##STR00039## wherein i and h are independently selected from an integer of 1-1140; or the PEG is a multi-branched polyethylene glycol residue having a structure of a general formula VI: ##STR00040## wherein k is an integer of 1-760, and j is an integer of 3-8; Y is a capping group, selected from: —H; a C.sub.1-6 alkyl, specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or n-hexyl; a C.sub.3-6 cycloalkyl, specifically cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; a substituted or unsubstituted C.sub.6-10 aryl, specifically phenyl or naphthyl; and -L-T; L is a linking group between oxygen (O) and an end group T, selected from: one or a combination of two or more of —(CH.sub.2).sub.b—, —(CR.sub.3R.sub.4).sub.b—, —(CH.sub.2).sub.bNH—, —NHCO(CH.sub.2).sub.b—, —(CH.sub.2).sub.bCONH— and —CO(CH.sub.2).sub.b—, wherein b is an integer of 0-10; R.sub.3 and R.sub.4 are independently selected from: one or a combination of two or more of —H, a C.sub.1-6 alkyl, —OR″, —NHR″, —N(R″).sub.2, —CN, —F, —Cl, —Br, —I, —COR′, —COOR″, —OCOR″, —CONHR″ and —CON(R″).sub.2; R″ is selected from: —H, a C.sub.1-6 alkyl, —F, —Cl, —Br and —I; T is an end group, selected from: —H; a C.sub.1-6 alkyl; a C.sub.3-6 cycloalkyl; a substituted or unsubstituted C.sub.6-10 aryl; a residue of a monosaccharide, specifically glucose, fructose, galactose, ribose and deoxyribose; and a residue of an oligosaccharide, specifically disaccharide (sucrose, lactose) and trisaccharide (gentiotriose, raffinose); Q is a residue of a core molecule of a multi-branched polyethylene glycol selected from: pentaerythritol, oligomeric pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol and polyglycerol.

7. The method according to claim 6, wherein T is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, ##STR00041## L is selected from: one or a combination of two or more of —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CONH—, —NH—, —CO—, —CONHCH.sub.2—, —CH.sub.2NH—, —CH.sub.2CONH— and —COCH.sub.2—; Y is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, ##STR00042## Q is selected from: pentaerythritol, dipentaerythritol and tripentaerythritol.

8. The method according to claim 6, wherein the multi-branched polyethylene glycol residue has the following structure: ##STR00043## or the multi-branched polyethylene glycol residue has the following structure: ##STR00044## wherein w is an integer of 1-570, and t is an integer of 1-10; or the multi-branched polyethylene glycol residue has the following structure: ##STR00045## wherein s is an integer of 1-280, and γ is an integer of 1-10.

9. The method according to claim 6, wherein a is an integer of 0-3; R.sub.1 and R.sub.2 are independently selected from: —H, —CH.sub.3, —OH, —OCH.sub.3 and —OCH.sub.2CH.sub.3.

10. The method according to claim 6, wherein X is selected from: one or a combination of two or more of —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CONHCH.sub.2—, —CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2CONHCH.sub.2CH.sub.2NH—, —CH.sub.2CH.sub.2CONHCH.sub.2—, —CH.sub.2CO—, —CH.sub.2CH.sub.2CO—, —CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2NH—, —CH.sub.2CONH—, —COCH.sub.2—, —COCH.sub.2CH.sub.2—, —COCH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2—S—S—CH.sub.2—, —CH.sub.2COOCH.sub.2— and —CH.sub.2—S—CH.sub.2—.

11. The method according to claim 1, wherein the method is a method for preparing a PEGylated IL-2 with controllable binding sites, comprising: (1) PEGylating to couple PEG with IL-2; (2) binding the PEGylated IL-2 to an IL-2α receptor; (3) separating the PEGylated IL-2 not bound to the IL-2α receptor; and (4) separating the IL-2α receptor and the PEGylated IL-2 bound thereto; wherein, the step (1) further comprises separating and purifying the PEG-IL-2 conjugate.

12. The method according to claim 11, wherein the method for preparing the PEGylated IL-2 with controllable binding sites comprises: (1) preparing an IL-2α receptor affinity column; (2) PEGylating to couple PEG with IL-2; (3) separating and purifying the PEG-IL-2 conjugate in the step (2) to give the PEGylated IL-2; (4) binding the PEGylated IL-2 to the IL-2α receptor on the affinity column; (5) separating the PEGylated IL-2 not bound to the IL-2α receptor; and (6) separating the IL-2α receptor and the PEGylated IL-2 bound thereto by gradient elution.

13. A PEGylated IL-2 with controllable binding sites prepared by the method according to claim 11, having the following structure: ##STR00046## wherein: n is 1 or 2, X is a linking group between the PEG and the IL-2 selected from: one or a combination of two or more of —(CH.sub.2)a-, —(CR.sub.1R.sub.2)a-, —(CH.sub.2)aNH—, —NHCO(CH.sub.2)a-, —(CH.sub.2)aCONH—, —(CH.sub.2)aCO—, —CO(CH.sub.2)a-, —(CH.sub.2)aCONH(CH.sub.2)a-, —(CH.sub.2)a-S—S—(CH.sub.2)a-, —(CH.sub.2)aCOO(CH.sub.2)a- and —(CH.sub.2)a-S—(CH.sub.2)a-; a is an integer of 0-10; R.sub.1 and R.sub.2 are independently selected from: one or a combination of two or more of —H, a C.sub.1-6 alkyl, —OR′, —NHR′, —N(R′).sub.2, —CN, —F, —Cl, —Br, —I, —COR′, —COOR′, —OCOR′, —CONHR′ and —CON(R′).sub.2; R′ is selected from: —H, a C.sub.1-6 alkyl, —F, —Cl, —Br and —I; the PEG is a linear, Y-shaped or multi-branched polyethylene glycol residue including monomethoxy polyethylene glycol (mPEG), a linear double-ended PEG, a Y-shaped PEG, a 4-arm branched PEG, a 6-arm branched PEG and an 8-arm branched PEG with a molecular weight of 1-100 KDa.

14.-16. (canceled)

17. A method for the treatment or prevention a disease, comprises administering the PEGylated IL-2 with controllable binding sites prepared by the method according to claim 11, wherein the disease is a tumor, an autoimmune disease, a viral disease or a bacterial disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0138] FIG. 1 is a gel chromatography separation pattern of the PEG-IL-2.

[0139] FIG. 2 shows the SDS-PAGE results of the PEG-IL-2, wherein 1 represents IL-2, 2 represents peak 1, 3 represents peak 2, and 4 represents a protein standard.

DETAILED DESCRIPTION

[0140] The technical schemes in the examples of the present invention will be described clearly and completely below, and it is apparent that the examples described herein are only some examples of the present invention, but not all of them. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Example 1. Preparation of IL-2α Receptor Affinity Column

[0141] S1: 0.8 g of CNBr-activated sehparose (sehparose CNBr) was added to 12 mL of 1 mM HCl, the system was manually and slowly mixed, and incubated in a refrigerator at 4° C. for reaction for 15 min;

[0142] S2: preparation of coupling buffer: 0.1 M NaHCO.sub.3, pH 8.3 was mixed well with 0.5 M NaCl, and 12 mL of the coupling buffer was taken to wash the swollen filler in the previous step;

[0143] S3: 4 vials of IL-2α receptors with a total of 4 mg was taken and dissolved in 2 mL of coupling buffer, and the mixture was added to the swollen filler in the step S2;

[0144] S4: the mixture was slowly shaken at room temperature for 2 h;

[0145] S5: excess receptors were washed out with 5 column volumes of the coupling buffer;

[0146] S6: 0.1 M Tris-HCl, pH 8.0 was added, the mixture was let stand at room temperature for 2 h, and then Tris-HCl was discarded;

[0147] S7: the residue was washed with 5 column volumes of 0.1 M acetic acid/sodium acetate buffer (pH 4.0) containing 0.5 M NaCl and then washed with 5 column volumes of 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl;

[0148] S8: the steps S6 and S7 were repeated 3 times;

[0149] S9: 0.5 mL of 0.1 M Tris-HCl, pH 8.0 was added;

[0150] S10: the sepharose resin was filled into a 5 mL empty column and then stored in a refrigerator at 4° C.

Example 2. Coupling of PEG and IL-2

[0151] 4.9 mg of methoxy polyethylene glycol-succinimidyl propionate (M-SPA-20K) having a molecular weight of 20 KDa was dissolved in 24 μL of 2 mM HCl to prepare a 200 mg/mL solution. 4.7 μL of the solution was taken and added to 420 μL of a 1.1 mg/mL IL-2 solution, and 60 μL of 0.05 M sodium tetraborate, pH 9.77, was added. The mixture was let stand at room temperature for reaction for 1 h. After the reaction was completed, 40 μL of 2 M HAC was added to terminate the reaction to obtain a PEG-IL-2 conjugate.

Example 3. Gel Filtration Separation of PEG-IL-2 Conjugate

[0152] The gel filtration chromatography settings are as follows: mobile phase: 5 mM PBS, flow rate: 0.75 mL/min, column: superdex 200 increase 10/300 GL.

[0153] The separation procedures are as follows:

[0154] S1: the chromatographic system was equilibrated until the UV curve was flat;

[0155] S2: the elution peak was collected after the sample was loaded;

[0156] S3: the fractions was collected, concentrated by ultrafiltration and then detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in FIGS. 1 and 2.

[0157] Results: peak 1 in FIG. 1 was confirmed as PEGylated IL-2 (PEG-IL-2) by SDS-PAGE.

Example 4. IL-2α Receptor Affinity Column Screening for PEGylated IL-2

[0158] S1: about 6 mL of the sample, PEGylated IL-2 (peak 1) prepared in Example 3 by filtration and separation, was injected 3 times;

[0159] S2: an IL-2α receptor affinity column containing 4 mg of receptors prepared in Example 1 was used;

[0160] S3: the mobile phases were prepared, phase A: 5 mM PBS and phase B: 0.2 M HAC+0.2 M NaCl;

[0161] S4: the flow rate of the mobile phase was set as 0.45 mL/min;

[0162] S5: the column was equilibrated with phase A until the UV curve was flat;

[0163] S6: the PEGylated IL-2 was injected 3 times by the injection loop;

[0164] S7: the mixture was subjected to gradient elution, and the flow-through fraction and elution peak were collected after the sample was loaded.

Example 5. Receptor Affinity Assay

[0165] The flow-through fraction and the elution fraction in Example 4 were collected, concentrated and subjected to affinity assay using an SPR system. The receptor proteins were IL-2α receptor and IL-2β receptor. The test results are shown in Table 1:

TABLE-US-00001 TABLE 1 Results of affinity assay for flow-through fraction and elution fraction Flow-through Elution Samples fraction fraction IL-2/IL-2Rα (μM) 15.4 1.89 IL-2/IL-2Rβ (μM) 124900 29.68

[0166] By analyzing the affinity assay results of the flow-through fraction and the elution fraction, we found that the flow-through fraction had an affinity for IL-2β receptor far greater than that for the IL-2α receptor, which indicates that the α receptor binding sites in the PEG-IL-2 in the flow-through fraction were occupied, leading to a stronger binding to the β receptor. Under the same test conditions, IL-2 had an affinity of 0.023 for the α receptor and an affinity of 2.80 for the β receptor. Compared with the receptor affinity of the elution fraction, the elution fraction had an 82-fold affinity for the α receptor of 1.89, and a 10-fold affinity for the β receptor of 29.68. It can be seen that the PEGylated IL-2 with controllable binding sites prepared by the methods according to Examples 1-4 has a significantly increased affinity for the IL-2α receptor. Finally, it should be noted that, the above examples are only used to illustrate the technical schemes of the present invention, but not to limit the present invention. Although the present invention is described in detail with reference to the examples described above, it will be understood by those skilled in the art that, the technical schemes in the examples described above can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements do not make the technical schemes corresponding thereto depart from the scope of the technical solutions in the examples of the present invention.