METHOD OF PREPARING PEGYLATED BIOMOLECULES HAVING CONTROLLABLE BINDING SITES

Abstract

The present invention discloses a method of preparing PEGylated biomolecules having controllable binding sites, including the following steps: (1) binding a blocker to a biomolecule; (2) PEGylating the biomolecule; and (3) separating the blocker from the biomolecule. In another aspect, the present invention discloses a method for preparing PEGylated IL-2 having controllable binding sites, including the following steps: (1) binding IL-2 to an IL-2α receptor, closing the a binding site of the IL-2; (2) PEGylating, coupling PEG with the IL-2; and (3) separating the IL-2 from the IL-2α receptor. By regulating IL-2 binding sites and a PEGylation process only adding one or two polyethylene glycols, the IL-2 is caused to selectively bind to an IL-2R βγ-type receptor.

Claims

1. A method of preparing PEGylated biomolecules having controllable binding sites, comprising the following steps: (1) binding a blocker to at least one binding site in a biomolecule; (2) PEGylating the biomolecule; and (3) separating the blocker from the biomolecule; wherein the biomolecule in the step (1) is selected from large biomolecules or small biomolecules having specific binding to the blocker, and the biomolecule has at least one binding site for the blocker; the blocker and the biomolecule are selected from the following combinations: a ligand and a receptor, DNA and complementary DNA or RNA thereof, an enzyme and a substrate thereof, an enzyme and a competitive inhibitor thereof, an enzyme and a co-enzyme factor thereof, vitamin and a specific binding protein thereof, and glycoprotein and corresponding lectin thereof.

2. The preparation method according to claim 1, wherein the DNA and the complementary DNA or RNA thereof are selected from: a gene probe and a base-complementary gene sequence; the enzyme and the substrate thereof are selected from: protease and protein, amylase and starch, nuclease and nucleic acid, lactate dehydrogenase and lactic acid, and oxaloacetic decarboxylase and oxaloacetic acid; the enzyme and the competitive inhibitor thereof are selected from: succinate dehydrogenase and malonic acid, dihydrofolate synthetase and sulfonamides, cholinesterase and an organophosphorus pesticide, and a sulfhydryl enzyme and lewisite; the enzyme and the co-enzyme factor thereof are selected from: pyruvate dehydrogenase and Mn2+, catalase, and oxidordeuctase and Fe2+/Fe3+; the hormone and the receptor are selected from: oestrogen and an oestrogen receptor, androgen and an androgen receptor, mineralocorticoid and a mineralocorticoid receptor, thyroid hormone and a thyroid hormone receptor, and progesterone and a progesterone receptor; the drug and the receptor are selected from: insulin, an insulin-like growth factor, an epidermal growth factor, a platelet-derived growth factor, and lymphokine, and a receptor having a tyrosine kinase activity, and epinephrine, dopamine, 5-hydroxytryptamine, M-acetylcholine, opiates, purines, prostaglandin, and polypeptide hormone drugs, and a 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 a vitamin D binding protein, α-tocopherol and an α-tocopherol transport protein, and vitamin K and lipoprotein; and the glycoprotein and the corresponding lectin thereof are selected from: horseradish peroxidase and concanavalin A, Lens culinaris agglutinin and Pisum sativum agglutinin, jacalin and galactose, an amaranthus lectin and N-acetylgalactosamine, a dimer chitin binding lectin and N-acetylglucosamine oligosaccharide, dolichos bifows agglutinin and a blood group substance A, and Ulex europaeus agglutinin and a blood group substance O (2-L-fucose).

3. The preparation 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 comprises an IL-2α receptor, an IL-2β receptor and an IL-2γ receptor.

4. The preparation method according to claim 1, wherein a method of separating the blocker from the biomolecule in the step (3) comprises: one or a combination of two or more of centrifugal separation, precipitation separation, filtering separation, foam separation, extraction separation, membrane separation, chromatographic separation, electrophoretic separation, and gradient elution.

5. The preparation method according to claim 1, wherein the biomolecule PEGlyated in the step (2) has a following structure: ##STR00037## m is an integer in the range of 1-12, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; X is a linking group between PEG and the biomolecule and is selected from: a combination of one or more of —(CH2)a-, —(CR1R2)a-, —(CH2)aNH—, —NHCO(CH2)a-, —(CH2)aCONH—, —(CH2)aCO—, —CO(CH2)a-, —(CH2)aCONH(CH2)a-, —(CH2)a-S—S—(CH2)a-, —(CH2)aCOO(CH2)a- and —(CH2)a-S—(CH2)a-; a is an integer in the range of 0-10; R1 and R2 are independently selected from: a combination of one or more of —H, C1-6 alkyl, —OR′, —NHR′, —N(R′)2, —CN, —F, —Cl, —Br, —I, —COR′, —COOR′, —OCOR′, —CONHR′ or —CON(R′)2; R′ is selected from: —H, C1-6 alkyl, —F, —Cl, —Br or —I; and the PEG is a linear, Y-type and multi-branch polyethylene glycol residue, for example comprising monomethoxypolyethylene glycol (mPEG), linear double-terminated PEG, Y-type PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG, and a molecular weight of the PEG is in the range of 1-100 KDa.

6. The preparation method according to claim 5, wherein the a is an integer in the range of 0-5; R1 and R2 are independently selected from: a combination of one or more of H, C1-3 alkyl, —OH, C1-3 alkoxy, —NH2, —F, —Cl, —Br and —I; R′ is selected from: —H and C1-3 alkyl; the PEG is a linear polyethylene glycol residue and has a structure as shown in a general formula II or III: ##STR00038## wherein p and q are independently selected from integers in the range of 1-2280; or the PEG is a Y-type polyethylene glycol residue and has a structure as shown in a general formula IV or V: ##STR00039## wherein i and h are independently selected from integers in the range of 1-1140; or the PEG is a multi-branch polyethylene glycol residue and has a structure as shown in a general formula VI: ##STR00040## wherein k is an integer in the range of 1-760, and j is an integer in the range of 3-8; Y is a capping group and is selected from: H, C1-6 alkyl (specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc.), C3-6 cycloalkyl (specifically, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.), C6-10 aryl (specifically, phenyl, naphthyl, etc.) and -L-T; L is a linking group between oxygen (O) and a terminal group T and is selected from: a combination of one or more of —(CH2)b-, —(CR3R4)b-, —(CH2)bNH—, —NHCO(CH2)b-, —(CH2)bCONH— and —CO(CH2)b-, and b is an integer in the range of 0-10; R3 and R4 are independently selected from: a combination of one or more of —H, C1-6 alkyl, —OR″, —NHR″, —N(R″)2, —CN, —F, —Cl, —Br, —I, —COR′, —COOR″, —OCOR″, —CONHR″ and —CON(R″)2; R″ is selected from: —H, C1-6 alkyl, —F, —Cl, —Br and —I; T is a terminal group and is selected from: H, C1-6 alkyl, C3-6 cycloalkyl, C6-10 aryl, monosaccharide (specifically, glucose, fructose, galactose, ribose, deoxyribose, etc.), and oligosaccharide (specifically, residues of disaccharides including sucrose, lactose, etc. and trisaccharides including gentianose, raffinose, etc.); and Q is a core molecule of multi-branch polyethylene glycol, and Q is selected from: pentaerythritol, oligomerized pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol and polyglycerol residues.

7. The preparation method according to claim 6, wherein the T is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, ##STR00041## L is selected from: a combination of one or more of —CH2-, —CH2CH2-, —CH2CH2CH2-, —CONH—, —NH—, —CO—, —CONHCH2-, —CH2NH—, —CH2CONH— and —COCH2-; the Y is selected from: methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, ##STR00042## and Q is selected from: pentaerythritol, dipentaerythritol and tripentaerythritol.

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

9. The preparation method according to claim 6, wherein the a is an integer in the range of 0-3; and R1 and R2 are independently selected from: H, —CH3, —OH, —OCH3 and —OCH2CH3.

10. The preparation method according to claim 6, wherein the X is selected from: a combination of one or more of —CH2-, —CH2CH2-, —CH2CH2CH2-, —CH2CONHCH2-, —CH2CONHCH2CH2-, —CH2CONHCH2CH2NH—, —CH2CH2CONHCH2-, —CH2CO—, —CH2 CH2CO—, —CH2CH2CONHCH2CH2-, —CH2NH—, —CH2CONH—, —COCH2-, —COCH2CH2-, —COCH2CH2CH2-, —CH2-S—S—CH2-, —CH2COOCH2- and —CH2-S—CH2-.

11. The preparation method according to claim 1, wherein a method for preparing PEGylated IL-2 having controllable binding sites comprises the following steps: (1) binding IL-2 to an IL-2α receptor; (2) PEGylating, coupling PEG with the IL-2; and (3) separating the IL-2 from the IL-2α receptor.

12. The preparation method according to claim 11, wherein the method for preparing PEGylated IL-2 having controllable binding sites comprises the following steps: (1) preparing an IL-2α receptor affinity column; (2) binding IL-2 to an IL-2α receptor on the affinity column; (3) PEGylating, coupling PEG with the IL-2; and (4) separating the IL-2 from the IL-2α receptor by gradient elution.

13. PEGylated IL-2 having controllable binding sites prepared by the preparation method according to claim 11, having a following structure: ##STR00046## defined scopes of PEG and X are as described in claims 5-10, and n is 1 or 2.

14. A pharmaceutical composition prepared from the PEGylated IL-2 having controllable binding sites prepared by the preparation method according to claim 11 and a pharmaceutically acceptable carrier.

15-16. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0135] FIG. 1 is a chromatograph of PEGylation of IL-2 on a receptor affinity column;

[0136] FIG. 2 shows an electrophoresis result of PEG-IL-2, wherein 1 represents the PEG-IL-2 and 2 represents a marker;

[0137] FIG. 3 is a chromatograph of gel chromatographic separation of PEG-IL-2; and

[0138] FIG. 4 shows an SDS-PAGE result of PEG-IL-2, wherein 1 represents a marker, 2 represents IL-2, 3 represents a peak 2, and 4 represents a peak 1.

DETAILED DESCRIPTION

[0139] The technical solutions in the embodiments of the present invention will be clearly and completely described below. It is obvious that the described embodiments are only a part of embodiments of the present invention, but not all embodiments of the present invention. All the other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts all belong to the protection scope of the present invention.

Embodiment 1 Preparation of IL-2α Receptor Affinity Column

[0140] S1: 0.8 g of CNBr activated sepharose (sepharose CNBr) was weighed, and added into 12 ml of 1 mM HCl, manually and slowly mixed well, and put into a refrigerator at 4° C. to be subjected to a reaction for 15 min;

[0141] S2: A coupling buffer was prepared: 0.1 M NaHCO.sub.3 (pH=8.3) and 0.5 M NaCl were well mixed, and 12 ml of coupling buffer was taken to wash a filler swollen in the last step;

[0142] S3: Four bottles, 4 mg in total, of IL-2α receptor was taken, dissolved in 2 ml of coupling buffer, and added to the filler swollen in the step 2;

[0143] S4: Slow rocking-turn was conducted at room temperature for 2 h;

[0144] S5: An excess receptor was washed away with 5 column volumes of coupling buffer;

[0145] S6: 0.1 M tris(hydroxymethyl)aminomethane (Tris-HCl) (pH=8.0) was added, the mixture was placed at room temperature for 2 h, and the Tris-HCl was poured away;

[0146] S7: Washing was conducted with 5 column volumes of 0.1 M acetic acid/sodium acetate buffer (pH=4.0) containing 0.5 M NaCl, and then washing was conducted with 5 column volumes of 0.1 M Tris-HCl buffer (pH=8.0) containing 0.5 M NaCl;

[0147] S8: The step 6 and step 7 were repeated for three times;

[0148] S9: 0.5 ml of 0.1 M Tris-HCl (pH=8.0) was added; and

[0149] S10: A 5 ml empty column was filled with sepharose resin, and preserved in the refrigerator at 4° C.

Embodiment 2 PEGylation of IL-2 on Receptor Affinity Column

[0150] S1: A 5 ml receptor affinity column was mounted on a protein purification system (AKTA purifier);

[0151] S2: Mobile phases, including a phase A: 5 mM PBS (pH=7.0), and a phase B: a 0.2 M acetic acid solution containing 0.2 M NaCl, were prepared;

[0152] S3: A mobile phase flow rate was set to be 0.45 ml/min;

[0153] S4: The phase A was balanced to a stable UV curve;

[0154] S5: An IL-2 sample was fed through a sampling loop, and an a receptor binding site on IL-2 was bound to an IL-2α receptor;

[0155] S6: A proper amount of methoxyl polyethylene glycol-succinimide propionate (M-SPA-20K) with a molecular weight of 20 KDa was taken, and dissolved in 2.5 mM PB (pH=8.0) to a final concentration of 40 mg/ml, 2 ml of M-SPA-20K was sucked, and fed through a sampling loop, and balanced to a stable UV curve;

[0156] S7: The step 6 was repeated for three times; and

[0157] S8: Gradient elution was conducted, a chromatograph was as shown in FIG. 1, elution peaks were collected, ultrafiltrated, concentrated, and detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Embodiment 3 Gel Filtration Chromatographic Separation of PEG-IL-2

[0158] Gel filtration chromatography settings include: a mobile phase: 5 mM PBS, a flow rate: 0.75 ml/min, and a column: superdex 200 increase 10/300 GL.

[0159] Separation includes: S1: a chromatographic system was balanced to a stable UV curve; S2: elution peaks were collected after sampling; and S3: the collected components were ultrafiltrated, concentrated, and detected by SDS-PAGE, and results were as shown in FIG. 3 and FIG. 4.

[0160] Detection results: after confirmation by SDS-PAGE, in FIG. 4, a peak 1 represents PEG-IL-2 and a peak 2 represents IL-2.

Embodiment 4 Test on Receptor Affinities

[0161] Samples at the peak 1 (PEG-IL-2) and the peak 2 (IL-2) in FIG. 4 were collected, respectively. Test on affinities was carried out by an SPR instrument after concentration. Receptor proteins included an IL-2α receptor and an IL-2β receptor. Test results were as below:

TABLE-US-00001 Sample PEG-IL-2 IL-2 IL-2/IL-2Rα (μM) 0.34 0.023 IL-2/IL-2Rβ (μM) 31.96 2.80

[0162] Results in the above table show that the PEG-IL-2 prepared by the method of the present invention has a relatively strong binding force to the IL-2β receptor.

[0163] Finally, it should be noted that the above embodiments are merely used to describe the technical solutions of the present invention, but not intended to limit the present invention. Although the present invention is described in detail with reference to the preceding embodiments, it should be appreciated that those of ordinary skill in the art may still make modifications of the technical solutions described in the preceding embodiments or make equivalent replacements to some or all technical features. Such modifications or replacements do not make the nature of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.