A method for pegylation of proteins, non-covalently pegylated proteins, protein drugs containing the same, and applications thereof

Abstract

The present invention discloses a method for PEGylating a protein, a non-covalent PEGylated protein, a protein drug containing the same, and an application thereof. The present invention develops a new strategy for non-covalent PEGylation of a protein based on site-specific in situ polymerization-induced co-assembly, wherein the site-specific modification can improve the in vitro activity retained by the protein, increase the yield and simplify the purification by in situ polymerization, and prolong the in vivo circulation half-life of the protein by co-assembly, thereby greatly improving the overall therapeutic effect. In addition, the non-covalent PEGylation method of the present invention has good versatility and is suitable for the long-acting modification of protein drugs of different properties.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for pegylation of proteins, non-covalent pegylation proteins, and protein drugs containing the same and applications thereof. Background Art

[0002] Compared with small molecule drugs, protein drugs have advantages such as high specificity, potency and efficacy, and play an irreplaceable role in the diagnosis and treatment of serious diseases such as malignant tumors, autoimmune diseases and viral infections. Statistics show that in 2021, five of the top ten drugs sold worldwide are protein-based drugs. However, despite these advantages, proteins, as complex biological macromolecules with multi-level structures, still have inherent defects such as poor solubility, instability, short in vivo circulation half-life and potential immunogenicity, which hinder the further development of protein drugs. In order to address these defects, various long-acting protein technologies (such as PEGylation, Fc fusion and albumin fusion) have emerged, among which PEGylation is the most common and mature.

[0003] PEGylation was first proposed by Abuchowski et al. in 1977, which involves coupling hydrophilic polyethylene glycol (PEG) to proteins to significantly reduce their immunogenicity. After more than 40 years of development, PEGylation has been widely used in protein drugs, and more than 16 PEGylated protein drugs have been approved by the FDA for the treatment of various clinical serious diseases, such as viral diseases and cancer.

[0004] Currently, PEGylation mainly relies on non-selective covalent coupling methods based on the "grafting" strategy. However, this approach has several disadvantages. First, due to the non-specificity of the PEG binding site on the protein, the final product is composed of various positional isomers, each with different biological activities, which poses a major challenge to the subsequent purification and quality control. Second, in the traditional "grafting" strategy, both PEG and protein are large molecules, and their coupling efficiency is low due to steric hindrance, resulting in a complicated purification process and increased costs.

[0005] To address these limitations and challenges, some studies have attempted to develop non-covalent PEGylation strategies aimed at improving coupling efficiency, simplifying downstream purification processes, and avoiding any adverse effects of covalent chemical reactions on protein structure and activity. For example, various non-covalent PEGylation strategies based on host-guest interactions, histidine-metal ion coordination interactions, hydrophobic interactions, and ionic interactions. However, these methods still require complex design and optimization for proteins with different properties (e.g., differences in hydrophobic domain density, surface charge, and specific amino acid content), and even require protein engineering to achieve, resulting in poor versatility. Therefore, it is crucial to develop new non-covalent PEGylation strategies. Summary of the invention

[0006] In order to solve at least some of the technical problems in the prior art, the present invention provides a general method specifically for non-covalent PEGylation of proteins, which is achieved by connecting hydrophobic blocks of proteins and PEG respectively to site-specific in-situ co-assembly. It is worth noting that this method retains the biological activity of protein drugs while enhancing their pharmacokinetic properties. Specifically, the present invention includes the following contents.

[0007] According to a first aspect of the present invention, a non-covalently PEGylated protein is provided, comprising a polyethylene glycol molecule and a protein, wherein the polyethylene glycol molecule comprises a hydrophilic block and a first hydrophobic block, the protein comprises a hydrophilic peptide segment and a second hydrophobic block, and the first hydrophobic block and the second hydrophobic block are capable of binding the polyethylene glycol molecule and the protein via non-covalent bonds, thereby forming a non-covalently PEGylated protein.

[0008] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the first hydrophobic block and the second hydrophobic block are the same or different.

[0009] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the hydrophobic block comprises acrylic acid units, methacrylic acid units and / or hydroxypropyl methacrylate units.

[0010] In certain embodiments, the non-covalently PEGylated protein according to the present invention has a spherical, worm-like or vesicle-like nanostructure.

[0011] In certain embodiments, the non-covalently PEGylated protein according to the present invention has an average size of 10-200 nm.

[0012] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the molar ratio of the hydrophobic block to the hydrophilic portion is (1-20):1.

[0013] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the molecular weight of polyethylene glycol or its derivative in the hydrophilic block is 200-30K.

[0014] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the molecular weight of polyethylene glycol or its derivative in the hydrophilic block is 1000-10000.

[0015] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the molecular weight of polyethylene glycol or its derivative in the hydrophilic block is 4000-6000.

[0016] In certain embodiments, according to the non-covalently PEGylated protein of the present invention, the peptide segment is derived from a protein, a polypeptide, a peptide, an antibody, an antigen or an enzyme.

[0017] A second aspect of the present invention provides a method for pegylation of a protein, comprising:

[0018] (1) preparing a first hydrophilic block unit containing halogen, polyethylene glycol or its derivatives;

[0019] (2) preparing a second hydrophilic block unit containing a halogen, a protein or a polypeptide;

[0020] (3) In situ co-assembly of a hydrophobic molecule with the first hydrophilic block unit and the second hydrophilic block unit in the presence of a catalyst system to form a non-covalently PEGylated protein.

[0021] In certain embodiments, according to the method for pegylation of proteins of the present invention, the molar ratio of the first hydrophilic block unit to the second hydrophilic block unit is (1-20):1; the molar ratio of the hydrophobic molecule to the hydrophilic block unit is (100-1500):1.

[0022] In certain embodiments, according to the method for PEGylating a protein of the present invention, the catalyst system comprises 0.01-1 mmol of a copper ion solution and 0.01-1 mmol of pentamethyldiethylenetriamine or hexamethyltriethylenetetramine, preferably 1,1,4,7,10,10-hexamethyltriethylenetetramine.

[0023] In certain embodiments, according to the method for PEGylating a protein of the present invention, the non-covalently PEGylated protein has a spherical, worm-like or vesicle-like nanostructure.

[0024] In certain embodiments, according to the method for PEGylating a protein of the present invention, the average size of the non-covalently PEGylated protein is 10-200 nm.

[0025] In certain embodiments, according to the method for PEGylating a protein of the present invention, the hydrophobic molecule comprises an acrylic acid unit, a methacrylic acid unit and / or a hydroxypropyl methacrylate unit.

[0026] In certain embodiments, according to the method for PEGylating a protein according to the present invention, the polyethylene glycol or a derivative thereof is reacted with a first initiator via an acylation catalyst to obtain a first hydrophilic block unit, wherein the first initiator is selected from alkyl halides or aryl sulfonyl halide compounds, preferably benzyl halides, bromoesters, halogenated nitriles, halogenated amides, halogenated acyl groups, aryl sulfonyl chlorides or aryl sulfonyl bromides, and further preferably α-bromoisobutyryl bromide or 2-bromopropionyl bromide.

[0027] In certain embodiments, according to the method for PEGylating a protein of the present invention, the protein or polypeptide is subjected to transamination to obtain an intermediate whose N-terminal amino group is converted, which is then coupled with a second initiator to obtain a second hydrophilic block unit with a halogen connected to the N-terminus, preferably the intermediate contains a carbonyl group, and the second initiator has a functionalized group capable of bonding to the carbonyl group; or

[0028] The protein or polypeptide is reacted with a third initiator in the presence of a biological enzyme, preferably a transpeptidase, to obtain a second hydrophilic block unit with a halogen connected to its C-terminus.

[0029] The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned PEGylated protein assembly, combination, conjugate or complex.

[0030] The fourth aspect of the present invention provides the use of the above-mentioned protein assembly, binding substance, conjugate or complex in the preparation of a drug for improving or treating a disease or condition.

[0031] The method of the present invention can effectively assemble PEG and protein in a non-covalent manner, and the resulting nanostructure not only improves the in vitro activity retention of the protein, but also prolongs its in vivo circulation half-life, thereby improving the overall therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram showing the preparation of non-covalently PEGylated proteins by site-specific in situ polymerization-induced co-assembly.

[0033] Figure 2 The physicochemical properties of PEG / Mb-PHPMA are shown. (a) The macroinitiator PEG-Br1 H-NMR. (b) SDS-PAGE analysis of PEG / Mb-PHPMA formed by site-specific in situ polymerization-induced co-assembly. Lane 1: protein marker; Lane 2: Mb; Lane 3: Mb-CHO; Lane 4: Mb-Br; Lane 5: purified PEG / Mb-PHPMA. (c) Representative TEM images of PEG / Mb-PHPMA under negative staining. (d) DLS analysis of Mb, Mb-Br and the formation of PEG / Mb-PHPMA. (e) CD spectra of Mb, Mb-Br and PEG / Mb-PHPMA. (f) Normalized peroxidase activity of Mb, Mb-Br and PEG / Mb-PHPMA.

[0034] Figure 3 Representative TEM images of PEG / Mb-PHPMA with different HPMA / initiator ratios are shown. (a) Spherical PEG / Mb-PHPMA200. (b) PEG / Mb-PHPMA500. (c) PEG / Mb-PHPMA1000. The results show that at different HPMA / initiator ratios, PEG / Mb-PHPMA can be in situ co-assembled into nanostructures with adjustable morphologies from spheres to worms to vesicles.

[0035] Figure 4 The in vitro physical and chemical characterization of PEG / IFN-PHPMA is shown. (a) SDS-PAGE analysis of PEG / IFN-PHPMA formed by site-specific in situ polymerization-induced co-assembly. Lane 1: protein marker; Lane 2: purified PEG / IFN-PHPMA; Lane 3: IFN. (b) Representative TEM images of PEG / IFN-PHPMA under negative staining. (c) DLS analysis of IFN and the formation of PEG / IFN-PHPMA. (d) CD spectra of IFN, PEGASYS and PEG / IFN-PHPMA.

[0036] Figure 5 The biological properties of PEG / IFN-PHPMA are shown. (a) MTT assay for antiproliferative activity of IFN, PEGASYS, and PEG / IFN-PHPMA. (b) OAS1 levels in liver tissues of mice injected intravenously with equivalent doses of IFN, PEGASYS, and PEG / IFN-PHPMA (n=3, Student t test was used to determine P values. *P<0.05, ns indicates no significance).

[0037] Figure 6 The performance test results of the co-assembled proteins prepared at different PEG and protein ratios are shown.

[0038] Figure 7 The figure shows the effects of different PEGylation methods on the activity and therapeutic efficacy of proteins with the same molecular weight.

[0039] Figure 8 Dynamic light scattering nanoparticle size detection results of non-nanospheres are shown. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] Methods for PEGylating proteins

[0044] In a first aspect of the present invention, a method for pegylation of proteins is provided, and the method of the present invention can be used for modification of proteins or assembly of proteins to form suitable assemblies, conjugates, conjugates or complexes. The "protein" in the subject matter of the present invention should be understood in a broad sense, and refers to a macromolecular organic compound formed by amino acids linked together by peptide bonds to form a certain spatial structure. In some cases, "protein", "polypeptide" and "peptide" can be used interchangeably.

[0045] In the present invention, specific examples of proteins and polypeptides include, but are not limited to, insulin, hemoglobin, myoglobin, albumin, cytochrome, interferon, antigen, antibody, erythropoietin, growth hormone, interleukin, colony stimulating factor and other conditional growth factors.

[0046] Unless otherwise specified, the proteins mentioned in this application refer to therapeutic proteins or protein drugs.

[0047] Unless otherwise stated, "halogen" as mentioned herein includes fluorine, chlorine, bromine or iodine, especially bromine.

[0048] Unless otherwise specified, the molecular weight of polyethylene glycol in this application refers to its number average molecular weight.

[0049] In step (1) of the present invention, a first hydrophilic block unit containing halogen, polyethylene glycol or a derivative thereof (sometimes referred to herein as a "macroinitiator containing polyethylene glycol") is first provided. The preparation of the first hydrophilic block unit is not particularly limited, and any method known in the art can be used to connect the terminal hydroxyl group of polyethylene glycol or a derivative thereof to the halogen. The terms "polyethylene glycol" and "PEG" can be used interchangeably and refer to polyethylene glycol or a derivative thereof known in the art. Generally, "PEG" is a linear polymer having a terminal hydroxyl group and having a molecular formula of HO-CH2CH2-(CH2CH2O) n -CH2CH2-OH, wherein n is about 8 to about 4000. It should be noted that the PEG of the present invention is not covalently bonded to the protein or polypeptide, that is, the PEG of the present invention is not bonded to any residue of the protein. In a preferred embodiment, the PEG is not directly bonded to the protein or polypeptide via a covalent bond, but is non-covalently bonded to each other.

[0050] In the present invention, PEG derivatives can be products in which the terminal hydrogen of PEG is replaced by a protecting group such as an alkyl or alkanol group. Such derivatives generally include monomethoxy polyethylene glycol (mPEG), dihydroxy polyethylene glycol (HO-PEG-OH) or functionalized PEG. Functionalized PEG can contain at least one functional group of carboxyl, amino, sulfhydryl, etc. PEG derivatives further include high molecular weight long-chain PEG.

[0051] A variety of chemical modifications can be used to prepare active PEG derivatives with functional groups (e.g., active carbonates, active esters, aldehydes, monomethoxylation). The active PEG derivatives can then be covalently bound to halogens by catalytic end groups. There are many PEG forms that can be used in the present invention. There are a large number of PEG derivatives in the art and are all suitable for use in the present invention. The PEG molecules covalently bound to halogens are not intended to be limited to specific types or sizes. However, considering the possibility of PEG affecting the activity and pharmacokinetics or drug efficacy of proteins, in the present invention, the PEG molecular weight is preferably from about 0.1k to about 30k daltons, and is also preferably from about 0.1k to about 20k daltons, and further preferably from about 0.1k to about 8000 daltons, more preferably 4000 to about 6000 daltons, such as 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000 daltons. The research results of the present invention show that the non-covalently PEGylated protein prepared by PEG or its derivatives within the above molecular weight range can retain the activity and efficacy of the therapeutic protein to the maximum extent. PEG can have a linear structure or a branched structure, and the halogen can have at least one, most preferably one, PEG molecule covalently bonded thereto.

[0052] In the present invention, the chain length of the PEG derivative can be 500 to 2000 Daltons, 2000 to 5000 Daltons, greater than 5000 Daltons to 10000 Daltons, or greater than 10000 to 20000 Daltons, greater than 20000 to 150000 Daltons.

[0053] As a non-limiting example of step (1) of the present invention, polyethylene glycol or its derivatives can be reacted with a first initiator through an acylation catalyst to obtain a first hydrophilic block unit, wherein the first initiator is selected from alkyl halides or aryl sulfonyl halides, preferably benzyl halides, bromoesters, halogenated nitrile, halogenated amides, halogenated acyl groups, aryl sulfonyl chlorides or aryl sulfonyl bromides, and also preferably α-bromoisobutyryl bromide (2-bromo-2-methylpropionyl) or 2-bromopropionyl bromide. Therefore, the catalyst involved in the reaction can be any known catalyst capable of acylation of hydroxyl groups. In a preferred embodiment, the catalyst is selected from one or more of diethylamine, triethylamine, pyridine, lutidine or sodium acetate.

[0054] In addition to the above-mentioned introduction of acyl groups between PEG and halogen, those skilled in the art can select appropriate reaction reagents as needed to covalently link PEG and halogen.

[0055] In a preferred embodiment of step (1), the polyethylene glycol derivative used in the present invention is polyethylene glycol monomethyl ether, and its molecular weight (Mw) is 1000-10000, preferably 2000-6000, and more preferably 4000-6000. Its concentration in the organic solvent is 0.01-1 g / mL, preferably 0.01-0.08 g / mL, and more preferably 0.02-0.06 g / mL. The volume ratio of triethylamine to 2-bromo-2-methylpropionyl added is (0.5-1.5):1, preferably (0.9-1.2):1, and more preferably 1:1.

[0056] Step (2) of the present invention is to provide a second hydrophilic block unit containing a halogen, a protein or a polypeptide (sometimes also referred to as a "protein-containing macroinitiator"). Various methods for connecting a protein or a polypeptide to a halogen are known in the art. In one embodiment, the present invention uses the N-terminus of a protein or a polypeptide as a connection site to connect the halogen to the N-terminus. In another embodiment, the present invention uses the C-terminus of a protein or a polypeptide as a connection site to connect the halogen to the C-terminus.

[0057] When the N-terminus is selected as the attachment site of the halogen, the protein or polypeptide can be subjected to transamination to obtain an intermediate whose N-terminal amino group is converted, and then coupled with a second initiator to obtain a second hydrophilic block unit with a halogen attached to the N-terminus. The intermediate can have any modification group suitable for covalent attachment of the halogen, and is not particularly limited to this. Preferably, the intermediate contains a carbonyl group, and the second initiator has any functionalized group capable of bonding to an aldehyde group, preferably an aldehyde group.

[0058] The amino group of an amino acid can be converted to an aldehyde group by transamination reagents known in the art, non-limiting examples of which include pyridoxal 5-phosphate.

[0059] In the present invention, the second initiator is preferably a hydroxylamine functionalized halogenated propionate compound, so that the halogens are covalently linked to each other by forming an oxime bond in the subsequent reaction. The halogenated propionate compound includes a halogenated methyl propionate compound, and a non-limiting example of a halogenated methyl propionate compound includes (2-(aminooxy)ethyl)2-bromo-2-methylpropionate (ABM).

[0060] When the C-terminus is selected as the attachment site of the halogen, the protein or polypeptide transpeptidase can be coupled to the third initiator to obtain a second hydrophilic block unit to which the C-terminus is connected to the halogen. The third initiator and the second initiator can be the same or different. Preferably, the third initiator includes a halogenated propionate compound without or with functionalization. A non-limiting example of the third initiator includes 2-bromo-2-methylpropionic acid 2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl ester hydrochloride (AEBM).

[0061] In the present invention, the protein or polypeptide may be bound to at least one or more than one halogen, and preferably the N-terminus and / or C-terminus of the protein or polypeptide is bound to one halogen.

[0062] In a preferred embodiment of step (2), the concentration of the transamination reagent 5'-pyridoxal phosphate used in the present invention is 10-80 mM, preferably 20-70 mM, more preferably 30-60 mM, and more preferably 40-60 mM. The concentration of the initiator used in step (2) is 0.1-10 mM, preferably 0.5-8 mM, more preferably 1-5 mM, and more preferably 1-3 mM.

[0063] In another preferred embodiment of step (2), the synthesis system used in the present invention comprises: 50-250 mM, preferably 80-200 mM, also preferably 80-150 mM, further preferably 80-120 mM IFN-LPETGGH6; 10-100 mM, preferably 20-80 mM, also preferably 30-70 mM, further preferably 40-60 mM transpeptidase; 0.1-10 mM, preferably 0.5-8 mM, also preferably 1-5 mM, further preferably 1-3 mM AEBM; and pH near neutral (pH 6-8) 10-100mM, preferably 20-80mM, also preferably 30-70mM, further preferably 40-60mM Tris·HCl; 50-250mM, preferably 80-200mM, also preferably 120-180mM, further preferably 140-160mM NaCl; 1-50mM, preferably 5-20mM, also preferably 8-15mM, further preferably 8-12mM CaCl2.

[0064] Step (3) of the present invention is a co-assembly process, in which the hydrophobic molecule is in situ co-assembled with the first hydrophilic block unit and the second hydrophilic block unit in the presence of a catalyst system to obtain a protein PEGylated product.

[0065] Considering the stability of PEG and protein formation and the effect of PEG on protein activity and ultimately on the efficacy of therapeutic proteins. The ratio between PEG and protein and the ratio between hydrophobic molecules and PEG and the hydrophilic part of the protein can be maintained within a suitable range. The results of the present invention show that if the content of PEG is too low, the stability of the assembly cannot be maintained. On the other hand, if the content of PEG is too high, the activity and efficacy of the protein will be affected. In a preferred embodiment, the molar ratio of the first hydrophilic block unit and the second hydrophilic block unit is (1-20): 1, preferably (2-18): 1, also preferably (3-15): 1, and further preferably (5-12): 1, for example 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1.

[0066] In the co-assembly process of the present invention, the inventors found that the ratio of hydrophobic molecules to the above-mentioned hydrophilic block units affects the activity of proteins, the stability of the assembly, and the pharmacokinetics and efficacy of therapeutic drugs. Studies have found that excessively high content of hydrophobic molecules reduces the efficacy of the assembly, and even loses activity and therapeutic efficacy. In a preferred embodiment, the molar ratio of the hydrophobic molecules to the above-mentioned hydrophilic block units is (100-1500):1, preferably (100-1000):1. The molar ratio of the hydrophobic molecules to the above-mentioned hydrophilic block units is preferably (100-500):1, further preferably (100-400):1, also preferably (100-300):1, and most preferably (100-200):1, for example, 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, the resulting spherical nanostructure assembly has excellent stability and therapeutic efficacy.

[0067] In another preferred embodiment of step (3), the catalyst system used in the present invention comprises 0.01-1 mmol of copper ion solution (CuCl and / or CuCl2), preferably 0.01-0.5 mmol of CuCl and CuCl2, further preferably 0.01-0.4 mmol of CuCl and CuCl2, further preferably 0.01-0.1 mmol of CuCl and CuCl2; 0.01-1 mmol, preferably 0.02-0.5 mmol, further preferably 0.03-0.1 mmol, further preferably 0.03-0.08 mmol, most preferably 0.03-0.05 mmol of pentamethyldiethylenetriamine or hexamethyltriethylenetetramine, particularly preferably 1,1,4,7,10,10-hexamethyltriethylenetetramine.

[0068] In order to obtain a polymeric structure in which the core is a hydrophobic molecule and the surface is a protein assembly, a combination, a conjugate or a complex of a hydrophilic protein and polyethylene glycol that are non-covalently linked to each other. The hydrophobic molecule used in the present invention contains an acrylic acid unit, a methacrylic acid unit and / or a hydroxypropyl methacrylate unit. In a preferred embodiment, the hydrophobic molecule used in the present invention is polyhydroxypropyl methacrylate (HPMA).

[0069] Unless otherwise specified, the organic solvent used in the present invention can be selected from one or more of dichloromethane, ethyl acetate, diethyl ether, N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0070] The method of the present invention improves the in vitro activity retention of proteins due to site-specific modification. The determination of the retained biological activity can be determined by activity determination data for different proteins. It is well known in the art how to determine the activity of different proteins. The term "activity retention" refers to that the non-covalently PEGylated protein can still retain all or part of the function of the protein compared to the untreated protein or its derivatives. Typically, the non-covalently PEGylated protein retains at least 50% of the activity of the protein. Under more preferred conditions, the non-covalently PEGylated protein can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the protein.

[0071] In the present invention, the polymerization reaction is carried out under anaerobic conditions at a low temperature, preferably in an ice water bath for 0.5-5, preferably 0.5-4, and more preferably 1-3 hours.

[0072] Protein assembly, combination, conjugate or complex

[0073] The present invention provides a protein assembly, a conjugate, a conjugate or a complex, which is obtained by the above-mentioned preparation method, and includes a polyethylene glycol molecule and a protein, wherein the polyethylene glycol molecule includes a hydrophilic block and a first hydrophobic block, and the protein includes a hydrophilic peptide segment and a second hydrophobic block, and the first hydrophobic block and the second hydrophobic block can bind the polyethylene glycol molecule and the protein through non-covalent bonds, thereby co-assembling to form a non-covalent PEGylated protein. It should be noted that the binding here refers to the process in which the polyethylene glycol molecule and the protein are close to each other but not connected by a covalent bond. "Co-assembly" refers to the process in which at least two active ingredients (PEG and protein) form an assembly.

[0074] The protein assembly, conjugate, conjugate or complex of the present invention has a spherical, worm-like or vesicle-like nanostructure. In a preferred embodiment, the protein assembly, conjugate, conjugate or complex has an average particle size of 10-200 nm, preferably 10-100 nm, and also preferably 10-80 nm, such as 10, 20, 30, 40, 50, 60, 70, 80 nm.

[0075] Pharmaceutical composition

[0076] The present invention also provides a pharmaceutical composition, which includes the above-mentioned protein assembly, combination, conjugate or complex, which may further include another therapeutic protein that can be covalently or non-covalently linked to the protein assembly, combination, conjugate or complex of the present invention, and an optional pharmaceutically acceptable carrier.

[0077] Pharmaceutically acceptable carriers are well known in the art and include any carrier that does not itself induce the production of any carrier that is harmful to the subject receiving the composition. One of ordinary skill in the art can determine that it meets clinical criteria. Pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, wetting agents, emulsifiers, pH buffer substances, and the like. Diluents include, but are not limited to, water, sterile pyrogen-free water, saline, phosphate buffered saline, glycerol, and the like.

[0078] The pharmaceutical composition of the present invention can be any suitable dosage form. For example, injection, suspension, emulsifier, etc. The pharmaceutical composition of the present invention can be applied to the body by known means. For example, delivered to the tissue of interest by intramuscular injection, optionally administered via intravenous, percutaneous, intranasal, oral, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered herein can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0079] application

[0080] The present invention also provides the use of protein assemblies, binding substances, conjugates or complexes in the preparation of drugs for improving or treating diseases or conditions, wherein the drugs are protein-based drugs and the diseases or conditions refer to any indications suitable for the selected protein drugs.

[0081] As used herein, the term "improvement or treatment" refers to improving a condition before or after the onset of a disease or disorder. This relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique compared to an untreated control group under the same conditions. In the present invention, the term "treatment" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or disorders. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation (whether partial or complete). Those in need of treatment include those who already have a disease or disorder, as well as those who are susceptible to a disease or disorder, or those who need to prevent the disease or disorder.

[0082] Example

[0083] The following is a method for site-specific in situ polymerization-induced co-assembly-mediated non-covalent PEGylation of proteins.

[0084] 1. Synthesis of PEG-Br

[0085] 2g of polyethylene glycol monomethyl ether (mPEG) with a molecular weight of 5000 was dissolved in 50mL of dichloromethane, and 300μL of triethylamine was added thereto. 300μL of 2-bromo-2-methylpropionyl was dissolved in 5mL of dichloromethane and added dropwise within 10 minutes. The reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed by rotary evaporation. 50mL of ethyl acetate was added to the obtained white solid residue, and the mixture was quickly stirred at room temperature for 30 minutes to dissolve the product. The solution was filtered to remove insoluble matter, and the filter cake was washed with 25mL of water and then with 25mL of ether. The product obtained was a white powdery solid.

[0086] 20 mg of purified PEG-Br powder was dissolved and recorded 1 H-NMR spectrum.

[0087] 2. Synthesis of PEG / Mb-PHPMA

[0088] The synthesis of Mb-Br is as follows: myoglobin Mb (25 mL 100 μM solution, pH 6.5 in 25 mM phosphate buffer) and 5'-pyridoxal phosphate (PLP) (25 mL 50 mM solution, pH 6.5 in 25 mM phosphate buffer) are loaded into a 50 mL Eppendorf tube. After a brief stirring to ensure adequate mixing, the mixture is incubated at 37 ° C for 12 hours without additional stirring. Unreacted PLP is removed from the reaction mixture using a desalting column (Cytiva). The purified mixture (50 μM solution, pH 5.5 in 50 mM phosphate buffer) is treated with ATRP initiator ABM (2-bromo-2-methylpropionic acid-2 aminooxyethyl ester) (25 mL 2 mM solution, pH 5.5 in 50 mM phosphate buffer) and left to stand for 12 h without stirring. Subsequently, the reaction solution is similarly purified by a desalting column.

[0089] 0.5mL PBS solution containing obtained Mb-Br (0.01 μmol), PEG-Br (0.1 μmol) and purified HPMA polyhydroxypropyl methacrylate (22 μmol) is added to Schlenk tube. The mixture is bubbling with nitrogen to remove oxygen. Meanwhile, in another vial, the catalyst solution in 0.5mL MilliQ water containing 0.01mmol CuCl, 0.01mmol CuCl2 and 0.04mmol 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) is deoxygenated by bubbling with nitrogen. Subsequently, the deoxygenated catalyst solution is transferred to the first deoxygenated Schlenk tube by cannula. The Schlenk tube is sealed and, before being quenched in air, polymerization is carried out in an ice-water bath for 2 hours. During the reaction, the solution undergoes a transition from transparent to slightly opaque, which indicates the formation of in-situ PEG / Mb-PHPMA assemblies. The assembly was additionally purified and isolated from the reaction mixture using a desalting column followed by a Sephacryl S-400 gel column (Cytiva). In addition, increasing the feed ratio between HPMA monomer and macroinitiator to 500:1 and 1000:1 produced worm-like and vesicle-like nanostructured assemblies.

[0090] 3. Synthesis of PEG / IFN-PHPMA

[0091] IFN-Br was synthesized as follows: a mixture of 50mM Tris HCl, 150mM NaCl, 10mM CaCl2, pH 7.4 containing 100mM IFN-LPETGGH6, 50mM Sortase A and 2mM ATRP initiator (AEBM) was incubated overnight at room temperature without stirring. Subsequently, the reaction mixture was diluted and applied to a HiTrap Capto Q column (Cytiva). Subsequently, 20mM Tris HCl, pH 7.4 was used as an equilibrium buffer, and 20mM Tris-HCl, 1M NaCl, pH 7.4 was used as an eluent for gradient chromatography. The peak containing the desired product mixture (IFN-Br) was collected, and the solution of IFN-Br was then exchanged into a buffer of 10mM PBS, 10% glycerol, pH 7.4 using a desalting column and concentrated by ultrafiltration.

[0092] 0.5mL PBS solution containing IFN-Br (0.01μmol), PEG-Br (0.1μmol) and purified HPMA (22μmol) obtained is added to Schlenk tube.Mixture is bubbling with nitrogen to remove oxygen.Meanwhile, in another vial, the catalyst solution in 0.5mL MilliQ water containing 0.01mmol CuCl, 0.01mmol CuCl2 and 0.04mmol 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) is deoxygenated by bubbling with nitrogen.The deoxygenated catalyst solution is subsequently transferred to the first deoxygenated Schlenk tube by cannula.The Schlenk tube is sealed and, before being exposed to quenching in air, polymerization is carried out in an ice-water bath for 2 hours.During the reaction, the solution has undergone a transition from transparent to slightly opaque, which shows the formation of original situ PEG / IFN-PHPMA assemblies. The assembly was additionally purified and isolated from the reaction mixture using a desalting column followed by a Sephacryl S-400 gel column (Cytiva).

[0093] Test Case

[0094] 1. Determination method

[0095] 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0096] The samples were dissolved in SDS-PAGE loading buffer supplemented with 2-mercaptoethanol and bromophenol blue. Subsequently, they were heat denatured at 95°C for 5 minutes and loaded onto a precast 12% polyacrylamide gel. Electrophoresis was performed at 90-120V using a protein gel apparatus (Bio-Rad) for 90 minutes and then stained with Coomassie blue.

[0097] 2. Dynamic Light Scattering (DLS)

[0098] Dynamic light scattering (DLS) analysis was performed using a Malvern Zetasizer Nano-zs90 instrument. Prior to analysis, the sample was filtered through a filter with a pore size of 0.22 mm. The instrument was operated at a laser wavelength of 633 nm and a scattering angle of 90° at 20°C. Data processing was performed using Zetasizer version 6.32 software.

[0099] 3. Transmission Electron Microscopy

[0100] The morphology of the assembled nanostructures was studied by transmission electron microscopy at an operating voltage of 100 kV. The samples were deposited onto copper grids coated with a 230 mesh carbon film by immersing the grids in the sample solution for 2-3 minutes. Subsequently, the grids were immersed in a 2% (w / v) phosphotungstic acid solution for 20 seconds for negative staining and then air-dried at room temperature.

[0101] 4. Circular Dichroism (CD)

[0102] CD spectra were recorded in the far UV region (190-260 nm) on a Jasco J-1500 instrument. The samples were diluted in H2O to a concentration of 0.15 mg / mL.

[0103] 5. Critical micelle concentration (CMC) measurement

[0104] To prepare the dilution solution, 45 μL of Nile Red stock solution (0.88 mg / ml in ethanol) was added to 100 ml of PBS solution (pH 7.4) to a final concentration of 1.25 μM. For CMC measurement, the samples were serially diluted 2-fold with the dilution solution. After the mixture was allowed to equilibrate for 15 minutes, 100 μL of each solution was loaded onto a 96-well plate. The fluorescence emission spectrum of each well was then scanned and a graph of the maximum emission intensity versus concentration was plotted to determine the CMC.

[0105] 6. Myoglobin (Mb) activity assay

[0106] 100 μL of 2,2'-azidobis(3-ethyl-benzothiol-6-sulfonic acid) diammonium salt (ABTS), 10 μL of Mb and 100 μL of H2O2 were mixed in PBS and the absorption at 734 nm was measured every 15 s for 5 min. The obtained data (absorption-time curve) were linearly fitted.

[0107] 7. Pharmacokinetics

[0108] Female BALB / c nude mice (10 weeks old) were used for pharmacokinetic evaluation. These mice were randomly divided into three groups (n=3 in each group) and IFN, PEGASYS (pegylated interferon) and PEG / IFN-PHPMA were intravenously administered at a dose of 1 mg IFN equivalent / kg body weight through the tail vein. Blood samples (5 μL) were collected from the tail vein at specified time intervals (1, 5, 15, 30 minutes; 1, 3, 6, 24, 48 and 72 hours). After incubation at 4°C for 30 minutes, the samples were centrifuged at 4000g for 15 minutes, plasma was collected and stored at -80°C. According to the instructions provided by the human IFN-α2 ELISA kit, the concentration of IFN was measured using an ELISA assay. The pharmacokinetic parameters were fitted using a two-compartment model.

[0109] 8. In vitro antiproliferative activity

[0110] The in vitro activity evaluation procedure was as follows: Daudi B cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Hyclone) at 37°C in a humidified atmosphere containing 5% CO2. For the bioassay, cells were seeded in 96-well plates at a density of 7500 cells (50 μL) per well, and serial dilutions of samples prepared in culture medium (ranging from 0.0565 to 10000 pg / mL) were added to the test wells (50 μL per well in triplicate). Background and control wells consisted of culture medium alone and culture medium-treated cells, respectively, and cell viability was defined as 0% and 100%. After a 96-hour incubation period, cell proliferation was assessed using the MTS assay according to the instructions of the cell proliferation assay kit. Data analysis, including curve fitting and IC50 determination, was performed using GraphPad Prism 5.0 software.

[0111] 9. Determination of antiviral 2',5'-oligoadenylate synthetase 1 (OAS1)

[0112] Female BALB / c nude mice aged 5 weeks were randomly divided into groups (n=3). Each group received a single intravenous injection of IFN or IFN conjugate at a dose equivalent to 1 mg IFN per kg body weight, or an equal volume of PBS. On the third day after injection, perfused liver tissue was extracted by exposure to CO2. Liver tissue (0.1 g) was lysed using lysis buffer (1 mL) and then centrifuged to collect the supernatant for OAS1 measurement. The level of mouse OAS1 was quantified using a mouse OAS1 ELISA kit (DLdevelop).

[0113] 10. Statistical Analysis

[0114] Unless otherwise specified, data are presented as mean ± SD. All experiments were repeated at least three times. Two groups were compared using unpaired two-tailed t-test. For comparisons between three or more independent groups, one-way analysis of variance (ANOVA) was performed using Tukey's multiple comparison test. Statistical analysis was performed using GraphPad Prism 7. Differences were considered statistically significant when P < 0.05.

[0115] 2. Experimental Results

[0116] First, the present invention synthesizes a PEG macro-initiator, which is used to co-initiate polymerization with a protein macro-initiator to promote co-assembly. By mixing polyethylene glycol monomethyl ether (mPEG) with α-bromoisobutyryl bromide and slowly adding triethylamine as a catalyst, the terminal hydroxyl group of mPEG is converted into α-bromoisobutyrate through an acylation reaction to obtain a PEG macro-initiator PEG-Br. Nuclear magnetic resonance (NMR) spectroscopy is used to characterize the successful preparation of the PEG-Br and quantitatively evaluate the conversion rate of the reaction ( Figure 2 a). By calculating the integrated areas of the chemical shifts of the two hydrogen atoms -C2H4O- and -C(Br)(CH3)CH3, it was determined that the conversion rate of the reaction was as high as 97.6%.

[0117] Subsequently, as an exemplary embodiment of the present invention, the feasibility of site-specific in-situ co-assembly of PEG and protein by hydrophobic blocks was explored with myoglobin (Mb), a model protein derived from horse skeletal muscle, and verified. First, the N-terminus of Mb was modified by a combination of N-terminal transamination and oxime formation reaction with an ATRP initiator. Briefly, using a biomimetic transamination reagent, 5-pyridoxal phosphate (PLP) converts the amino group of the N-terminal glycine of myoglobin into an aldehyde group to form Mb-CHO, and then with a hydroxylamine-functionalized ATRP initiator (2-(aminooxy)ethyl) 2-bromo-2-methylpropionate (ABM) through oxime formation coupling to obtain Mb-Br. The conditions of this two-step reaction are mild, and the resulting product can be quickly and easily purified by dialysis.

[0118] Next, PHPMA polymerization was initiated simultaneously by in situ ATRP reaction on two macroinitiators (PEG-Br and Mb-Br, molar ratio of 10:1). HPMA is a water-soluble methacrylate monomer, and its polymer is insoluble in water. Due to the hydrophobicity of PHPMA, and PEG and Mb as hydrophilic ends, PEG-PHPMA and Mb-PHPMA co-assembled during the polymerization process to form PEG / Mb-PHPMA-assembly. SDS-PAGE showed successful polymerization, showing typical polymer smearing bands in the high molecular weight region ( Figure 2b). The final PEG / Mb-PHPMA assemblies were purified by size exclusion chromatography. These results indicate that the site-specific in situ polymerization-induced co-assembly strategy of the present invention can successfully prepare PEG / Mb-PHPMA assemblies in high yield and in an easily purified manner. In theory, the morphology of the diblock copolymer co-assemblies depends on the volume ratio of the hydrophilic block and the hydrophobic block. After optimization, when the feed molar ratio of HPMA monomer to macroinitiator was controlled at 200:1, uniform spherical nanostructures were obtained. In addition, increasing the feed ratio between HPMA monomer and macroinitiator to 500:1 and 1000:1 can produce worm-like and vesicle-like nanostructured assemblies ( Figure 3 ). In order to better perform biomedical applications in vivo, assemblies with spherical nanostructures were selected for subsequent studies.

[0119] Transmission electron microscopy (TEM) characterization showed that PEG / Mb-PHPMA formed uniform spherical assemblies with an average size of 45 nm ( Figure 2 c), while dynamic light scattering (DLS) showed a narrow single peak, indicating an intensity-averaged hydration radius of 29 nm ( Figure 2 d). In addition, the critical micelle concentration (CMC) of PEG / Mb-PHPMA was determined to be approximately 0.15 μM by Nile red assay, indicating that PEG / Mb-PHPMA may decompose into a single-chain structure at low concentrations. In addition, circular dichroism (CD) spectroscopy was used to characterize the secondary structure of the protein in PEG / Mb-PHPMA ( Figure 2 e). The results showed that both Mb-Br and PEG / Mb-PHPMA showed no significant changes in the protein secondary structure compared with unmodified Mb.

[0120] The present invention further evaluated the peroxidase-like activity of Mb to verify the preservation of protein biological activity in the PEG / Mb-PHPMA assembly. The peroxidase-like activity of Mb in catalyzing the reaction between hydrogen peroxide and ABTS was determined by monitoring the absorbance change of ABTS at 734 nm using a UV-visible spectrophotometer. The results clearly showed that PEG / Mb-PHPMA retained 97% of the activity compared to Mb itself, which may be due to the small molecular size of the substrate, which is less affected by the flanking PEG ( Figure 2 f). Therefore, these data indicate that the co-assembly strategy of the present invention does not impair the biological activity of Mb.

[0121] Based on these findings, the present invention also uses a key protein drug, recombinant human interferon-α (IFN), to further study the universality of this method and illustrate its potential application in enhancing the pharmacology of protein drugs. IFN is a pleiotropic cytokine with multiple physiological effects, such as antiviral, antitumor and immunomodulatory activities, and is widely used in the clinic to treat viral hepatitis and various tumors, such as chronic lymphocytic leukemia, renal cell carcinoma, melanoma, Kaposi's sarcoma, and chronic myeloid leukemia. However, its short half-life remains a limitation, and long-acting interferons approved by the U.S. Food and Drug Administration, such as PEGASYS, still have low binding efficiency, difficulty in purification, and significantly reduced activity. Therefore, the present invention uses an ATRP initiator mediated by Sortase A to modify the C-terminus of interferon to obtain IFN-Br, which is then co-assembled with PEG-Br through the site-specific in situ polymerization-induced co-assembly strategy of the present invention to obtain a PEG / IFN-PHPMA assembly.

[0122] SDS-PAGE analysis showed that the polymerization was successful, showing a typical polymer dispersion band in the high molecular weight region ( Figure 4 a). TEM characterization showed uniform spherical assemblies of PEG / IFN-PHPMA with an average size of 42 nm ( Figure 4 b), while DLS showed a narrow single peak, indicating an intensity-averaged hydration radius of 28 nm ( Figure 4 c). CD spectra showed that the secondary structure of the protein in PEG / IFN-PHPMA did not change significantly compared with unmodified IFN and long-acting PEGASYS ( Figure 4 d).

[0123] Then, the effect of PEG / IFN-PHPMA on the proliferation of human lymphoma Daudi B cells was evaluated. Compared with PEGASYS (6.53%), PEG / IFN-PHPMA showed a higher in vitro activity retention rate (21.8%), which increased by 3.34 times ( Figure 5 This highlights the advantage of the co-assembly strategy of the present invention in minimizing the impact on protein activity.

[0124] In addition, the pharmacokinetics of PEG / IFN-PHPMA were studied in a mouse model. After intravenous administration, the plasma concentration of IFN decreased rapidly, while the plasma concentration of PEG / IFN-PHPMA decreased much more slowly. In addition, the pharmacokinetic parameters were derived by fitting the data to a two-compartment model. The clearance rate (CL) of PEG / IFN-PHPMA (2.36 mL / h) was 59.7 times that of IFN (141 mL / h). It is worth noting that the elimination half-life (t 1 / 2=4.40 hours) is 4.86 times the elimination half-life of IFN (t 1 / 2 =0.906 hours), although it is shorter than that of PEGASYS. In addition, the AUC value (area under the curve) of PEG / IFN-PHPMA (8.48 mg / L·h) is 59.7 times that of IFN (0.142 mg / L·hour) (Table 2). These results collectively demonstrate that the site-specific in situ polymerization-induced co-assembly of the present invention significantly improves the pharmacokinetics of IFN compared with free IFN. Subsequently, the in vivo antiviral activity of PEG / IFN-PHPMA was further explored by evaluating the expression level of OAS1 in mouse liver tissue. OAS1 is an enzyme that synthesizes 2',5'-oligoadenylic acid, and IFN upregulates OAS1 and inhibits viral replication by initiating RNase L to degrade double-stranded RNA. After intravenous injection of equimolar doses of PEG / IFN-PHPMA, PEGASYS and IFN, the expression level of OAS1 in mouse liver tissue was measured 72 hours later. Compared with free IFN, PEG / IFN-PHPMA resulted in a significant increase in OAS1 levels (1.39-fold), even comparable to PEGASYS ( Figure 5 b), which is attributed to the combined effect of its high bioactivity retention and improved pharmacokinetic properties. Table 1 summarizes the in vitro bioactivity, in vivo pharmacokinetic half-life and in vivo antiviral activity of PEG / IFN-PHPMA, PEGASYS and IFN. These results show that the co-assembly strategy of the present invention can improve the in vitro activity retention rate, in vivo half-life and in vivo efficacy of therapeutic proteins, providing a promising alternative for long-term protein modification.

[0125] Table 1 Overview of pharmacological parameters of interferon and its conjugates

[0126]

[0127] Table 2 Pharmacokinetic parameters of interferon and its conjugates

[0128]

[0129] In summary, the present invention has developed a new strategy for non-covalent PEGylation of proteins based on site-specific in situ polymerization-induced co-assembly, which achieves: 1) improved in vitro activity retention of proteins due to site-specific modification, 2) increased yield and simplified purification through in situ polymerization, and 3) extended in vivo circulation half-life of proteins through co-assembly, thereby improving the overall therapeutic effect. This new strategy not only enriches the non-covalent PEGylation methods, but also has good versatility and is suitable for long-acting modification of protein drugs of different properties. In addition, this supramolecular co-assembly system is expected to be applied to protein drugs with multivalent interactions. Therefore, the method of the present invention can expand the application of polymerization-induced co-assembly technology in the field of biomedical applications.

[0130] Comparative Example 1

[0131] The present application further studies and evaluates the effects of different amounts of proteins and PEG used in the protein PEGylation method on the activity and therapeutic efficacy of the co-assembled protein. The following is an exemplary description using IFN as the protein.

[0132] Different from the above examples, the ratio of PEG:IFN was set to 0:1, 2:1, 5:1, 10:1 and 20:1 respectively. Figure 6 As shown, the results indicate that in the experimental groups with PEG:IFN ratios of 0:1, 2:1 and 5:1, in some cases, due to the high protein content, the stability of the product was poor and it was completely precipitated within 2 hours and could not be used for subsequent activity testing and in vivo use. The stability of the co-assembled protein in the experimental groups with PEG:IFN ratios of 10:1 and 20:1 was better. Surprisingly, the activity retention rate of the co-assembled protein in the experimental group with PEG:IFN ratio of 10:1 was higher than that in the experimental group with PEG:IFN ratio of 20:1. Therefore, PEG:protein of 10:1 can be selected for subsequent activity testing and in vivo use.

[0133] Comparative Example 2

[0134] The present application further studies and evaluates the effects of different PEGylation methods on the activity and therapeutic efficacy of proteins under the same molecular weight conditions in the protein PEGylation method. The following is an exemplary description using IFN as the protein.

[0135] Different from the above examples, PEG with molecular weight of 5000 and 40K was covalently linked to IFN by conventional methods, and the antiviral activity of the resulting assembled proteins was compared in vivo. Figure 7The results of in vivo antiviral activity showed that the in vivo antiviral activity of the PEG-IFN sample prepared by covalent coupling with PEG with a molecular weight of 5000 was not significantly different from that of the IFN original drug and the control group, while the in vivo antiviral activity of the PEG / IFN-PHPMA used in the present invention was significantly increased by 1.39 times, wherein the molecular weight of the PEG used in the present invention was 5000. In addition, the comparison results of the commercial product named PEGASYS, which uses 40K PEG to covalently link IFN, and the PEG / IFN-PHPMA with a molecular weight of 5000 of the present PEG, can be found in Figure 4-5 .

[0136] Comparative Example 3

[0137] This application further evaluates the stability and therapeutic efficacy of non-nanosphere co-assembled proteins, see Figure 8 . Figure 8 Dynamic light scattering nanoparticle size detection of non-nanospheres is shown. Dynamic light scattering nanoparticle size detection shows that the hydration radius of the co-assembled proteins of non-nanospheres (such as worm-like and vesicle-like) is greater than 110nm, and cannot pass through the 220nm sterilization filter membrane and cannot be used for subsequent cell and animal experiments.

[0138] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A non-covalently PEGylated protein, characterized in that The invention comprises polyethylene glycol molecules and proteins, wherein the polyethylene glycol molecules comprise a hydrophilic block and a first hydrophobic block, the protein comprises a hydrophilic peptide segment and a second hydrophobic block, the first hydrophobic block and the second hydrophobic block can bind the polyethylene glycol molecules and the protein in the presence of a catalyst system through non-covalent bonds, thereby forming a non-covalently PEGylated protein, the catalyst system comprises a copper ion solution and pentamethyldiethylenetriamine or hexamethyltriethylenetetramine, the hydrophobic block comprises an acrylic acid unit, a methacrylic acid unit and / or a hydroxypropyl methacrylate unit, and the molar ratio of the hydrophobic block to the hydrophilic portion is (1-20):

1.

2. The non-covalently PEGylated protein according to claim 1, characterized in that The first hydrophobic block and the second hydrophobic block are the same or different.

3. The non-covalently PEGylated protein according to claim 1, characterized in that They have spherical, worm-like or vesicle-like nanostructures.

4. The non-covalently PEGylated protein according to claim 1, characterized in that They have an average size of 10-200 nm.

5. The non-covalently PEGylated protein according to claim 1, characterized in that The molecular weight of polyethylene glycol or its derivative in the hydrophilic block is 200-30K.

6. The non-covalently PEGylated protein according to claim 1, characterized in that The molecular weight of the polyethylene glycol or its derivative in the hydrophilic block is 1000-10000.

7. The non-covalently PEGylated protein according to claim 1, characterized in that The molecular weight of polyethylene glycol or its derivative in the hydrophilic block is 4000-6000.

8. The non-covalently PEGylated protein according to claim 1, characterized in that The peptide segment is from at least one of insulin, hemoglobin, myoglobin, albumin, cytochrome, interferon, antigen, antibody, erythropoietin, growth hormone, interleukin, colony stimulating factor and enzyme.

9. A pharmaceutical composition, characterized in that The non-covalently PEGylated protein comprises the non-covalently PEGylated protein according to any one of claims 1 to 8.

10. A method for pegylation of a protein, characterized in that include: (1) preparing a first hydrophilic block unit containing halogen and polyethylene glycol or its derivative, wherein the polyethylene glycol or its derivative reacts with a first initiator through an acylation catalyst to obtain the first hydrophilic block unit, wherein the first initiator is selected from alkyl halides or aryl sulfonyl halide compounds; (2) preparing a second hydrophilic block unit containing a halogen and a protein or polypeptide, wherein the protein or polypeptide is subjected to transamination to obtain an intermediate containing a carbonyl group whose N-terminal amino group is converted, and then coupled with a second initiator to obtain a second hydrophilic block unit having a halogen connected to the N-terminus, wherein the second initiator has a functionalized group capable of bonding to the carbonyl group, or the protein or polypeptide is reacted with a third initiator in the presence of a biological enzyme to obtain a second hydrophilic block unit having a halogen connected to the C-terminus, wherein the third initiator and the second initiator are the same or different; (3) allowing the hydrophobic molecule to be in situ co-assembled with the first hydrophilic block unit and the second hydrophilic block unit in the presence of a catalyst system to form a non-covalently pegylated protein, wherein the molar ratio of the first hydrophilic block unit to the second hydrophilic block unit is (1-20):1, the molar ratio of the hydrophobic molecule to the hydrophilic block unit is (100-1500):1, the catalyst system comprises a copper ion solution and pentamethyldiethylenetriamine or hexamethyltriethylenetetramine, and the hydrophobic molecule comprises an acrylic acid unit, a methacrylic acid unit and / or a hydroxypropyl methacrylate unit.

11. The method for PEGylating a protein according to claim 10, characterized in that The catalyst system comprises 0.01-1 mmol of copper ion solution and 0.01-1 mmol of pentamethyldiethylenetriamine or hexamethyltriethylenetetramine.

12. The method for pegylation of a protein according to claim 10, characterized in that The catalyst system includes 1,1,4,7,10,10-hexamethyltriethylenetetramine.

13. The method for pegylation of a protein according to claim 10, characterized in that The non-covalently PEGylated protein has a spherical, worm-like or vesicle-like nanostructure.

14. The method for PEGylating a protein according to claim 10, characterized in that The average size of the non-covalently PEGylated proteins was 10-200 nm.

15. The method for pegylation of a protein according to claim 10, characterized in that The first initiator is selected from benzyl halides, bromoesters, halogenated nitriles, halogenated amides, halogenated acyl groups, arylsulfonyl chlorides or arylsulfonyl bromides.

16. The method for pegylation of a protein according to claim 10, characterized in that The first initiator includes α-bromoisobutyryl bromide or 2-bromopropionyl bromide.

17. Use of the non-covalently PEGylated protein according to any one of claims 1 to 8 in the preparation of a drug for improving or treating a disease or condition.

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