Method for producing modified protein, copolymer for protein modification, copolymer, and pharmaceutical composition

Abstract

A copolymer for protein modification comprising a polymer part having a -(RAO)- structure as a repeating unit (wherein RA represents an alkylene group having 2-5 carbon atoms) and an adsorption part derived from a basic compound having an unshared electron pair on sp2 hybrid orbitals is brought into contact with a protein to form a complex of the protein and the copolymer for protein modification.

Description

Method for producing modified proteins, copolymer for protein modification, copolymer, pharmaceutical composition

[0001] This disclosure relates to a method for producing modified proteins, a copolymer for protein modification, a copolymer, and a pharmaceutical composition.

[0002] By modifying biologically active proteins such as peptides, antibodies or their fragments, and enzymes with polyethylene glycol chains, it is possible to stabilize and improve the function of these proteins. This modification of proteins with polyethylene glycol chains is called pegylation. In particular, in pharmaceuticals, pegylation increases the molecular weight and prevents degradation by proteolytic enzymes, so a significant improvement in the blood half-life can be expected. Furthermore, it is known that pegylation of proteins also brings benefits such as avoiding renal excretion and improving solubility.

[0003] Generally, protein pegylation can be achieved by introducing a maleimide group to the end of polyethylene glycol and reacting the maleimide group with the thiol group in the protein (maleimide method), or by introducing an azide group to the end of polyethylene glycol and causing a click reaction between the alkyne group introduced in the protein and the azide group. However, these methods have problems such as the covalent bonding between the protein and polyethylene glycol, which can lead to a decrease in function due to structural changes in the protein.

[0004] Incidentally, Japanese Patent Publication No. 2012-097211 discloses a copolymer that facilitates the handling of metal complexes as liquid preparations, comprising a monomer having a polydentate ligand such as bipyridine, dipicolylamine, and terpyridine that coordinates to a metal atom, and a hydrophilic monomer. The copolymer disclosed in Japanese Patent Publication No. 2012-097211 has a polyethylene glycol chain made of the above-mentioned hydrophilic monomer. It is stated that by using the copolymer disclosed in Japanese Patent Publication No. 2012-097211, a platinum chain polymer complex and micelles made of the complex could be formed.

[0005] Furthermore, International Publication No. 2011 / 071167 discloses the use of polyethylene glycol having a pyridine skeleton instead of a cationic surfactant (e.g., cetyltrimethylammonium bromide) when preparing gold-silver core-shell nanorod particles in which a silver shell layer is coated on the surface of gold nanorod particles.

[0006] As mentioned above, while copolymers having polyethylene glycol chains disclosed in Patent Document 1 and those having polyethylene glycol chains disclosed in International Publication No. 2011 / 071167 are known, there was no knowledge regarding the interaction between these copolymers and proteins. This disclosure aims to explore new polymer compounds that modify proteins using bonding modes other than covalent bonding, and to provide a method for producing modified proteins that can maintain protein function, a copolymer for protein modification, a copolymer, and a pharmaceutical composition.

[0007] As a result of diligent consideration in order to achieve the above-mentioned objective, sp ２ We have discovered that a site derived from a basic compound having a lone pair of electrons in its hybrid orbital physically adsorbs to a protein, and have completed this disclosure. This disclosure includes the following: <1>-(R Ａ O) A polymer portion having a repeating structure (wherein R Ａ (represents an alkylene group with 2 to 5 carbon atoms) and sp ２A method for producing a modified protein, comprising the step of contacting a protein with a protein to form a complex of the protein and the protein-modifying copolymer, wherein the protein-modifying copolymer comprises an adsorption portion derived from a basic compound having a lone pair of electrons on its hybrid orbitals. <2> The method for producing a modified protein according to <1>, wherein the basic compound is pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative. <3> The method for producing a modified protein according to <1> or <2>, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. <4> The method for producing a modified protein according to any one of <1> to <3>, wherein the polymer portion is polyethylene glycol or poly(oligoethylene glycol) methacrylate. <5> The method for producing a modified protein according to any one of <1> to <4>, wherein the weight-average molecular weight of the specific copolymer is 1,000 to 1,000,000. <6> The method for producing a modified protein according to any one of <1> to <5>, wherein the specific copolymer is at least one copolymer selected from the group consisting of the following formulas (1) to (4). [In formulas (1) to (4) above, n is an integer from 5 to 600, in formulas (1) to (2) above, m is an integer from 20 to 2000, in formulas (3) to (4) above, p is an integer from 5 to 1000, and in formulas (3) to (4) above, q is an integer from 1 to 15.] <7> The protein is an active ingredient in disease treatment, a method for producing the modified protein described in any one of <1> to <6> <8>-(R Ａ O) A polymer portion having a repeating structure (wherein R Ａ (represents an alkylene group with 2 to 5 carbon atoms) and sp ２A copolymer for protein modification comprising an adsorption portion derived from a basic compound having a lone pair of electrons in its hybrid orbitals. <9> The copolymer for protein modification according to <8>, wherein the basic compound is pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative. <10> The copolymer for protein modification according to <8> or <9>, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. <11> The copolymer for protein modification according to any one of <8> to <10>, wherein the polymer portion is polyethylene glycol or poly(oligoethylene glycol) methacrylate. <12> The copolymer for protein modification according to any one of <8> to <11>, wherein the weight-average molecular weight is 1,000 to 1,000,000. <13> The copolymer for protein modification according to any one of <8> to <12>, wherein the copolymer is at least one copolymer selected from the group consisting of the following formulas (1) to (4). [In formulas (1) to (4) above, n is an integer from 5 to 600, in formulas (1) to (2) above, m is an integer from 20 to 2000, in formulas (3) to (4) above, p is an integer from 5 to 1000, and in formulas (3) to (4) above, q is an integer from 1 to 15.] <14> A polymer portion having poly(oligoethylene glycol) methacrylate and sp ２ A copolymer comprising an adsorbent portion derived from a basic compound having lone pairs of electrons in its hybrid orbitals. <15> The copolymer according to <14>, wherein the basic compound is pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative. <16> The copolymer according to <14> or <15>, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. <17> The copolymer according to any one of <14> to <16>, having a weight-average molecular weight of 1,000 to 1,000,000. <18> The copolymer according to any one of <14> to <17>, having the following formula (3) or formula (4). [In the above formulas (3) to (4), n is an integer from 5 to 600, p in the above formulas (3) to (4) is an integer from 5 to 1000, and q in the above formulas (3) to (4) is an integer from 1 to 15.] <19> A pharmaceutical composition comprising a protein that is an active ingredient for treating a disease and a copolymer for protein modification described in any one of <8> to <13> adsorbed to the protein. <20> The pharmaceutical composition according to <19>, wherein the protein and the copolymer for protein modification are bound by physical adsorption.

[0008] According to the present disclosure, a method for producing a modified protein capable of maintaining the function of a protein, a copolymer for protein modification, a copolymer, and a pharmaceutical composition can be provided.

[0009] FIG. 1 is a characteristic diagram showing the １ 1H NMR spectrum of PyMA synthesized in the examples. FIG. 2 is the １ 1H NMR spectrum of DPA-OH synthesized in the examples. FIG. 3 is the ２ 1H NMR spectrum of DPAMA synthesized in the examples. FIG. 4 is the １ 1H NMR spectrum of PEG(4k)-macro CTA synthesized in the examples. FIG. 5 is the １ 1H NMR spectrum of pOEGMA synthesized in the examples. FIG. 6 is the １ 1H NMR spectrum of PEG(4k)-b-pPyMA synthesized in the examples. FIG. 7 is the １ 1H NMR spectrum of PEG(4k)-b-pDPAMA synthesized in the examples. FIG. 8 is the １ 1H NMR spectrum of pOEGMA-b-pPyMA synthesized in the examples. FIG. 9 is the １ 1H NMR spectrum of pOEGMA-b-pDPAMA synthesized in the examples. FIG. 10 is the １Figure 11 is a characteristic diagram showing the H NMR spectrum. Figure 11 is a characteristic diagram showing the relationship between the equilibrium concentration of Lys and the number of adsorbed Lys when Lysozyme is adsorbed onto silica particles. Figure 12 is a characteristic diagram showing the results of measuring the zeta potential when Lysozyme is adsorbed onto silica particles. Figure 13 is a characteristic diagram showing the results of calculating the adsorption isotherm when a protein modification copolymer is applied to silica particles adsorbed with Lysozyme. Figure 14 is a characteristic diagram showing the results of measuring the zeta potential when a protein modification copolymer is applied to silica particles adsorbed with Lysozyme. Figure 15 is PEG(4k)-b-pPyMA １４ This is a characteristic diagram showing the Languir plot prepared for PEG(4k)-b-pDPAMA. Figure 16 shows PEG(4k)-b-pDPAMA. １３ This is a characteristic diagram showing the Languir plot prepared for PEG(4k)-b-pPyMA. Figure 17 is a characteristic diagram showing PEG(4k)-b-pPyMA １４ This is a characteristic diagram showing the adsorption isotherm (30°C) in PEG(4k)-b-pPyMA. Figure 18 shows PEG(4k)-b-pPyMA １４ This is a characteristic diagram showing the adsorption isotherm (35°C) in PEG(4k)-b-pPyMA. Figure 19 shows PEG(4k)-b-pPyMA １４ This is a characteristic diagram showing the Langmuir plot (30°C) at PEG(4k)-b-pPyMA. Figure 20 shows the PEG(4k)-b-pPyMA １４ This is a characteristic diagram showing the Langmuir plot (35°C) in the region. Figure 21 is PEG(4k)-b-pDPAMA １３ This is a characteristic diagram showing the adsorption isotherm (30°C) in PEG(4k)-b-pDPAMA. Figure 22 shows PEG(4k)-b-pDPAMA. １３ This is a characteristic diagram showing the adsorption isotherm (35°C) in PEG(4k)-b-pDPAMA. Figure 23 shows PEG(4k)-b-pDPAMA. １３ This is a characteristic diagram showing the Langmuir plot (30°C) in the following location. Figure 24 shows PEG(4k)-b-pDPAMA １３ This is a characteristic diagram showing the Langmuir plot (35°C) at PEG(4k)-b-pPyMA. Figure 25 is a characteristic diagram showing PEG(4k)-b-pPyMA １４ and PEG(4k)-b-pDPAMA １３This is a characteristic diagram plotting lnA, the natural logarithm of the adsorption coefficient A, against the reciprocal of temperature, 1 / T. Figure 26 is a schematic diagram showing a QCM chip and a SAM film formed on the QCM chip. Figure 27 is a schematic diagram showing the process of immobilizing Lysozyme (Lys) on a SAM film formed on a QCM chip. Figure 28 is a characteristic diagram showing the adsorption density of a protein modification copolymer to the Lys surface immobilized on the SAM film. Figure 29 is a characteristic diagram showing the lytic activity when various protein modification copolymer solutions are mixed at various concentrations. Figure 30 is a characteristic diagram showing the amount of gold adsorbed by proteins modified using various protein modification copolymers.

[0010] The following describes embodiments that are examples of this disclosure. These descriptions and embodiments are illustrative and do not limit the scope of the invention. In the following embodiments, the components (including elemental steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges and do not limit this disclosure. For example, this disclosure allows for additions, omissions, substitutions, and changes to numbers, quantities, positions, ratios, materials, configurations, types, and order, etc., without departing from the spirit of the invention.

[0011] In this disclosure, numerical ranges indicated using "~" include the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. When multiple types of the substance corresponding to each component exist in a composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified.

[0012] [Copolymer for protein modification] The protein modification copolymer of this disclosure is -(R ＡO) A polymer portion having a repeating structure (wherein R Ａ (represents an alkylene group with 2 to 5 carbon atoms) and sp ２ The copolymer includes an adsorption site derived from a basic compound having a lone pair of electrons in its hybrid orbitals. The protein-modifying copolymer of this disclosure may be a block copolymer or a graft copolymer, but a block copolymer is preferred. The adsorption site of the protein-modifying copolymer of this disclosure physically adsorbs to the protein to be modified and forms a complex with the protein. Even if the protein forms a complex with the protein-modifying copolymer of this disclosure, it will not undergo any structural changes, or the structural changes will be extremely minimal compared to the reaction between a maleimide group and a thiol group or an alkyne group and an azide group. Therefore, the protein-modifying copolymer of this disclosure can form a complex that maintains the function of the protein to be modified. The principle is not clear, but it is presumed that a so-called Lewis base-Lewis acid chemical bond is formed between the lone pair of electrons in the adsorption site of the protein-modifying copolymer and the protein, thus enabling the formation of a complex without inducing structural changes in the protein.

[0013] <Polymer portion> In the polymer copolymer for protein modification of this disclosure, the polymer portion is -(R Ａ O) - It has a repeating structure as a unit. In the formula, R Ａ R represents an alkylene group having 2 to 5 carbon atoms, but it is preferable that it be an alkylene group having 2 to 3 carbon atoms. Ａ When the adsorbent is a C2 alkylene group, the polymer portion becomes a polyethylene glycol chain. Therefore, in the form in which the adsorbent portion has a polyethylene glycol chain, protein modification using the protein modification copolymer of this disclosure is equivalent to pegation.

[0014] In the polymer portion - (R Ａ O) Having a structure as a repeating unit means that - (R Ａ O) - When the main chain has a structure, the - (R Ａ O) - When the side chain has a structure, the - (R ＡO) - A case in which the structure is present in the main chain and side chains can be cited. - (R Ａ O) - When the main chain has a structure, - (R Ａ O) - The number of repeating structures is not particularly limited, but can be 5 to 2000, 10 to 1000, 20 to 500, 40 to 250, or 50 to 200. Also, - (R Ａ O) - When the side chain has a structure, the - (R Ａ O) - The number of repeating structures is not particularly limited, but can be 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5. Also, - (R Ａ O) When the structure is present in the side chains, the number of side chains attached to the main chain can be 5 to 2000, 10 to 1000, 20 to 500, 40 to 250, or 50 to 200.

[0015] Furthermore, the polymer portion is -(R Ａ O) - May also contain polymerizable monomers, including polymerizable monomers that contain the structure as a repeating unit. The polymerizable monomer is a polymerizable monomer and has polymerizable groups in its structure. The type of polymerizable group is not particularly limited, and examples include vinyl groups, allyl groups, styryl groups, methacryloyl groups, acryloyl groups, etc. Through this polymerizable group, the adsorbent sp ２ It can polymerize with basic compounds that have lone pairs of electrons in their hybrid orbitals.

[0016] Specifically, the polymer portion can be polyethylene glycol or poly(oligoethylene glycol) methacrylate. That is, one form of the polymer portion is -(C ２ H ４ O) - The main chain can be polyethylene glycol. Another form of the polymer part is one in which methyl methacrylate is the main chain and - (C ２ H ４O) A poly(oligoethylene glycol) methacrylate having a - structure in its side chain can be used. This poly(oligoethylene glycol) methacrylate is also called poly-[oligo-(ethylene glycol) methyl ether methacrylate] (POEGMA).

[0017] Note - (R Ａ O) When the side chain has a structure, the monomers constituting the main chain are not particularly limited, but for example, (meth)acrylamide, methylol(meth)acrylamide, methoxymethyl(meth)acrylamide, ethoxymethyl(meth)acrylamide, propoxymethyl(meth)acrylamide, butoxymethoxymethyl(meth)acrylamide, N-methylol(meth)acrylamide, N-hydroxymethyl(meth)acrylamide, 2-hydroxyethyl(meth)acrylamide, 2-hydroxypropyl(meth)acrylamide, 2-hydroxybutyl(meth)acrylamide, (meth)acrylic acid, fumaric acid, maleic acid, maleic anhydride, itaconic acid, itaconic anhydride, cyto Examples include laconic acid, citraconic anhydride, crotonic acid, methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 2-hydroxybutyl (meth)acrylate, 2-phenoxy-2-hydroxypropyl (meth)acrylate, 2-(meth)acryloyloxy-2-hydroxypropyl phthalate, glycerin mono (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, dimethylamino (meth)acrylate, glycidyl (meth)acrylate, etc. These monomers may be used individually or in combination of two or more to constitute the main chain.

[0018] <Adsorption portion> In the protein modification copolymer of this disclosure, the adsorption portion is sp ２It is derived from a basic compound having a lone pair of electrons in its hybrid orbitals. Derived from the basic compound means that the compound binds to the polymer portion to form an adsorption portion. Therefore, the protein modification copolymer of this disclosure is based on the said compound. ２ These compounds have lone pairs of electrons in their hybrid orbitals. Examples of such compounds include pyridine, bipyridine, dipicolylamine, tris(2-pyridylmethyl)amine, 2,2'-dipicolylamine, 3,3'-dipicolylamine, terpyridine, pyridazine, pyrazine, pyrazole, imidazole, oxazole, thiazole, thiophene, and derivatives of these compounds. In particular, it is preferable to use pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative as the compound.

[0019] In particular, the compound forming the adsorption site is preferably one that has multiple lone pairs of electrons. Such compounds are not particularly limited and include, for example, bipyridine, Schiff bases, phenanthrines, orthobenzoquinone derivatives, bidentate ligands such as nucleic acid bases, dipicolylamines, terpyridine, diethylenetriamine, triazacycloalkanes, tetrakis(2'-aminoethyl)-1,2-diaminopropane, porphyrins and their derivatives, phthalocyanines and their derivatives, tetraazacycloalkanes, aminoalkyl tetraazacycloalkanes, tri(aminoalkyl) triazacycloalkanes, pentadentate ligands such as 1,14-diamino-3,6,9,12-tetraazatetradecane, tri(aminoalkyl) triazacycloalkanes, and 1,14-diamino-3,6,9,12-tetraazatetradecane. Among these, it is preferable that at least one is selected from the group consisting of bipyridine, dipicolylamine, and terpyridine.

[0020] Derivatives are, sp ２This refers to a group of basic compounds having lone pairs of electrons in hybrid orbitals, in which some of the atoms constituting the compound are substituted with functional groups. Examples of functional groups include substituted or unsubstituted alkyl groups, cycloalkyl groups, heterocyclic groups, alkenyl groups, cycloalkenyl groups, alkynyl groups, aryl groups, and heteroaryl groups.

[0021] The compound forming the adsorption site may have polymerizable functional groups in its structure. The polymerizable functional groups are not particularly limited and include, for example, vinyl groups, allyl groups, styryl groups, methacryloyl groups, and acryloyl groups. The compound may also copolymerize with the polymerized portion described above via these polymerizable functional groups. Examples of compounds forming the adsorption site include pyridine methacrylate and dipicolylamine methacrylate.

[0022] <Structural Example> As described above, the protein modification copolymer of this disclosure, which includes a polymer portion and an adsorption portion, can be, for example, at least one copolymer selected from the group consisting of the following formulas (1) to (4). In formulas (1) to (4), "Me" represents "methyl". [In equations (1) to (4) above, n is an integer between 5 and 600; in equations (1) to (2) above, m is an integer between 20 and 2000; in equations (3) to (4) above, p is an integer between 5 and 1000; and in equations (3) to (4) above, q is an integer between 1 and 15.]

[0023] Furthermore, the weight-average molecular weight (measured by GPC with polystyrene as the standard substance) of the protein-modifying copolymer of this disclosure is not particularly limited, but is preferably 1,000 to 100,000, more preferably 1,000 to 20,000, and even more preferably 1,000 to 10,000. If the weight-average molecular weight of the protein-modifying copolymer is within the above range, the protein to be modified can be stabilized while maintaining its function. The weight-average molecular weight was determined using a gel permeation chromatography (GPC) analyzer with a configuration of two gels linked in this order: "TSKgel SuperH4000 (particle size 3 μm, inner diameter 6.0 mm × length 15 cm)" manufactured by Tosoh Corporation and "TSKgel SuperH3000 (particle size 3 μm, inner diameter 6.0 mm × length 15 cm)" manufactured by Tosoh Corporation. The weight-average molecular weight was detected using a differential refractometer with THF (tetrahydrofuran) as the solvent, and the molecular weight was converted using polystyrene as the standard substance. N,N-dimethylformamide containing 10 mM lithium chloride was used as the eluent, and the measurement was performed at a flow rate of 0.3 mL / min.

[0024] [Method for Producing Protein Modification Copolymers] The protein modification copolymers of this disclosure are block copolymers or graft copolymers obtained by polymerizing at least the polymer portion and the adsorption portion described above. The polymerization method for the protein modification copolymers of this disclosure is not particularly limited, and conventionally known methods can be used, but living radical polymerization methods such as addition-cleavage chain transfer (RAFT) polymerization and atom transfer radical polymerization (ATRP) are preferred. Living radical polymerization methods allow for control of the molecular weight and molecular weight distribution of the synthesized copolymer.

[0025] First, we will illustrate the production of a polymer copolymer for protein modification using RAFT. The polymer portion consists of -(R Ａ O) A polymerizable monomer containing a - structure, a chain transfer agent, and a polymerization initiator are dissolved in a predetermined solvent, and after completely removing the oxygen in the reaction vessel containing dissolved oxygen, the polymerizable monomer is polymerized by heating at a temperature above the temperature at which the polymerization initiator cleaves, but below 100°C, for 24 to 48 hours. - (R ＡO) A macrochain transfer agent is synthesized in which a chain transfer agent is introduced at the end of a polymer portion having a repeating structure. Next, this macrochain transfer agent and a polymerizable monomer constituting the adsorption portion are dissolved in a predetermined solvent and heated for 24 to 300 hours at a temperature above the temperature at which the polymerization initiator cleaves, but below 100°C, thereby synthesizing the protein modification copolymer of the present disclosure in which the polymer portion and the adsorption portion are polymerized.

[0026] Also, the side chain has -(R Ａ O) The polymer portion having a structure consists of polymerizable groups constituting the main chain and -(R Ａ O) A monomer having a - structure, a chain transfer agent, and a polymerization initiator are dissolved in a predetermined solvent and heated at a temperature above the temperature at which the polymerization initiator cleaves, but below 100°C, for 24 to 300 hours. This causes - (R) to form in the main chain. Ａ O) A polymer moiety can be synthesized in which the structure is linked in a comb-like manner as side chains. Here, the polymerization initiator is not particularly limited and examples include azo polymerization initiators such as 2,2'-azobisisobutyronitrile (AIBN) and 2,2'-azobis(2-methylbutyronitrile), sulfate polymerization initiators such as ammonium persulfate and potassium persulfate, and organic peroxide polymerization initiators such as benzoyl peroxide and lauroyl peroxide.

[0027] The preferred amount of polymerization initiator to use is 0.1% to 10% by mass relative to the amount of monomer. In the case of graft copolymers, chain transfer agents such as mercaptoacetic acid, mercaptopropionic acid, 2-propanethol, and 1-butanethiol may be added to adjust the molecular weight. The polymerization temperature and polymerization time are not particularly limited, but are preferably 30°C to 90°C, more preferably 50°C to 70°C, and 1 to 96 hours.

[0028] [Method for producing modified proteins] The method for producing modified proteins according to this disclosure includes the step of contacting the protein modification copolymer described above with a protein to form a complex of the protein and the protein modification copolymer. The protein modification copolymer described above is sp ２Because it has a lone pair of electrons in its hybrid orbital, this lone pair of electrons can physically adsorb to a protein, allowing it to form a complex with the protein.

[0029] The protein modification copolymer and the protein can be brought into contact in a buffer solution. Suitable buffer solutions include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer, Tris (Tris(Hydroxymethyl)aminomethyl) buffer, and borate buffer.

[0030] When contacting the protein modification copolymer with the protein, the temperature conditions are not particularly limited, but can be, for example, 10°C to 50°C, particularly preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C. Similarly, when contacting the protein modification copolymer with the protein, the time conditions are not particularly limited, but can be, for example, 1 hour to 48 hours, particularly preferably in the range of 5 hours to 40 hours, and more preferably in the range of 12 hours to 36 hours.

[0031] Here, the term "protein to be modified" is not particularly limited and encompasses all compounds having a structure in which amino acids are linked by peptide bonds. Therefore, the proteins to be modified are not limited to macromolecular compounds, but also include oligopeptides in which 2 to 20 amino acids are linked by peptide bonds. Furthermore, the proteins to be modified may be proteins with unknown functions, enzymes with specific activity, antibodies, or antibody fragments that specifically bind to specific antigens.

[0032] The term "antibody" refers to an immunoglobulin molecule that corresponds to a tetramer containing four polypeptide chains (two heavy chains (H chains) and two light chains (L chains)) linked together by disulfide bonds. Furthermore, antibodies can be either monoclonal or polyclonal antibodies. Monoclonal antibodies can be human antibodies, chimeric antibodies, or humanized antibodies.

[0033] Furthermore, the term "antibody fragment" refers to an antibody fragment that has the ability to react with a specific antigen. Such fragments include Fab fragments (e.g., by papain digestion), Fab' fragments (e.g., by pepsin digestion and partial reduction), F(ab')2 fragments (e.g., by pepsin digestion), Facb (e.g., by plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reassembly) fragments, and also include scFv (single-chain Fv; e.g., by molecular biological techniques) fragments.

[0034] In particular, the protein-modifying copolymers of this disclosure are preferably used to stabilize proteins that are active ingredients in disease treatment. That is, the pharmaceutical compositions of this disclosure include a protein that is an active ingredient in disease treatment and the protein-modifying copolymers of this disclosure adsorbed to the protein. In particular, the protein-modifying copolymers of this disclosure can stabilize the protein that is an active ingredient in disease treatment by physically adsorbing to it, while maintaining the protein's function as an active ingredient in disease treatment. As a result, the pharmaceutical compositions of this disclosure can improve regimens to reduce the burden on patients, such as by reducing the dosage or extending the dosing interval.

[0035] The present disclosure will be described in more detail below with reference to examples, but the technical scope of the present disclosure is not limited to the following examples.

[0036] [1. Monomer Synthesis] 1-1. Synthesis of Pyridine Methacrylate (PyMA) The synthesis scheme for PyMA is shown below.

[0037] The reagents used in this synthesis example are listed below.

[0038]

[0039] Place 2.988 g (21.78 mmol) of 4-pyridine propanol (1) into a 100 mL two-necked round-bottom flask and super-dehydrate CH4 under a nitrogen atmosphere. ２ Cl ２10 mL of 2.879 g of TEA (28.45 mmol, 1.306 equivalents per (1)) was added. This was designated as Solution I. In another 100 mL round-bottom flask, 2.982 g of methacrylloyl chloride (28.52 mmol, 1.309 equivalents per (1)) was added, and super-dehydrated CH4 under a nitrogen atmosphere and ice bath. ２ Cl ２ 8 mL was added. Solution I was then added dropwise, and the mixture was stirred at room temperature for 24 hours in a light-shielded place. Afterwards, the mixture was filtered by suction and concentrated to obtain 20 mL saturated Na. ２ CO ３ The organic layer was extracted by washing it three times with aqueous solution and once with 20 mL of saline solution, and the resulting organic layer was extracted using MgSO4. ４ Dehydration was performed. Suction filtration and concentration were carried out, and separation and purification were performed by column chromatography (eluent: EtOAc). Subsequently, concentration and vacuum drying were performed, and nuclear magnetic resonance (CNC) was performed using a Bruker Advance 400 Hz (Bruker). １ 1H NMR spectrum measurement (CDCl ３ ) was performed.

[0040] Figure 1 shows １ The results of the 1H NMR spectrum measurement are shown. The yield was 1.580 g (7.698 mmol), and the yield was 35.34%.

[0041] 1-2. Synthesis of Dipicolylamine-OH (DPA-OH) The synthesis scheme for DPA-OH is shown below.

[0042]

[0043] The reagents used in this synthesis example are listed below.

[0044]

[0045] In a 2L round-bottom flask, combine 40.13g (244.7 mmol, 3.0 equivalents relative to (2)) of 2-chloromethyl-pyridine hydrochloride (1), 1.322g (4.101 mmol, 0.050 equivalents relative to (2)), and K ２ CO ３112.65 g (815.1 mmol, 10 equivalents relative to (2)) was added to 500 mL of acetonetrile and mixed by stirring. At this point, the solution turned pale pink. 6.2 mL (81.7 mmol) of 3-aminopropan-1-ol (2) was added and stirred under reflux at 98°C for 60 hours. After reflux, the supernatant solution, which had turned dark brown, was collected by suction filtration, thoroughly washed with EtOAc, and then concentrated to obtain crude. The crude was separated by column chromatography (eluent: EtOAc / MeOH = 9 / 1) and concentrated. After concentration, the yield of the purified product was measured and measured using a nuclear magnetic resonance spectrometer (Bruker Advance 400 Hz (Bruker)). １ H NMR measurement (CDCl ３ ) was performed.

[0046] Figure 2 shows １ The results of the 1H NMR spectrum measurement are shown. The yield was 1.580 g and 14.993 g (58.26 mmol), and the yield was 71.50%. 1-3. Synthesis of Dipicolylamine Methacrylate (DPAMA) The synthesis scheme for DPAMA is shown below.

[0047]

[0048] The reagents used in this synthesis example are listed below.

[0049]

[0050] Place 2.043 g (7.939 mmol) of DPA-OH(1) into a 100 mL round-bottom flask and dissolve it in CH under a nitrogen atmosphere. ２ Cl ２ 3 mL of TEA (3) and 1.40 mL (10.1 mmol, 1.27 equivalents relative to (1)) were added. This was Solution I. Next, 1.081 g (10.34 mmol, 1.31 equivalents relative to (1)) of methacrylloyl chloride (2) was added to a 100 mL round-bottom flask with a two-neck, and the mixture was steeped under a nitrogen atmosphere and ice bath. ２ Cl ２ 1 mL was added. Solution I was added to this reaction solution, and the mixture was protected from light and stirred at room temperature for 24 hours. After that, the mixture was filtered by suction and concentrated, and CH ２ Cl ２15 mL was added. It was washed three times with 20 mL of saturated NaHCO ３ aqueous solution and once with 20 mL of brine for extraction. The obtained organic layer was dehydrated with MgSO ４ . Suction filtration and concentration were carried out, and it was separated and purified by column chromatography (developing solvent: EtOAc / MeOH = 9 / 1). Then, concentration and vacuum drying were performed, and 1H NMR spectrum measurement (using a nuclear magnetic resonance apparatus (Bruker Advance 400 Hz, manufactured by Bruker)) was carried out in １ CDCl ３ .

[0051] The 1H NMR spectrum measurement results are shown in Fig. 3. Also, the yield was 1.425 g (4.379 mmol), and the yield was １ 55.16%.

[0052] [2. Polymer synthesis] 2-1. Synthesis of polyethylene glycol chain (PEG(4k)-macro CTA) The synthesis scheme of PEG(4k)-macro CTA is shown below.

[0053]

[0054] The reagents used in this synthesis example are shown below.

[0055]

[0056] 4.004 g (1.00 mmol) of MeO-PEG-OH(4k)(1) was placed in a 300 mL two-necked eggplant flask and dissolved in 3 mL of chloroform, and then 17 mL of benzene was added. Freeze-drying was carried out for 4 hours, and 60 mL of THF and 0.352 g (3.48 mmol, 3.48 equivalents relative to (1)) of TEA(3) were added under N ２ atmosphere. This solution was designated as Solution I. Subsequently, 0.365 g (3.14 mmol, 3.14 equivalents relative to (1)) of Ms-Cl(2) was placed in a 300 mL two-necked eggplant flask, vacuum dried, and 10 mL of THF was added under N ２ atmosphere, and Solution I was added while cooling with water. Then, it was stirred at room temperature overnight. Suction filtration and concentration were carried out, and reprecipitation with ethyl ether was carried out to obtain a white precipitate.

[0057] The precipitate was washed with NH ３The solution was completely dissolved in approximately 300 mL of aqueous solution and stirred at room temperature for 120 hours. After concentration, it was transferred to a dialysis membrane (1 kDa), and the external solution was changed twice with 0.01 M HCl (1 hour each time) and three times with MilliQ water (1 hour, 1 hour, and overnight). After dialysis, freeze-drying was performed for 1 day to obtain a white solid (yield: 3.509 g, yield: 87.64%).

[0058] Of the obtained white solid, (4) 1002.00 mg (0.2387 mmol) was dissolved in 20 mL of MeOH in a 100 mL vial, and then 206.69 mg (0.7469 mmol, 3.13 equivalents relative to (1)) of DMTMM (6) and 75.50 mg (0.7461 mmol, 3.13 equivalents relative to (1)) were added and dissolved, and the mixture was protected from light. In this state, 198.11 mg (0.7091 mmol, 2.97 equivalents relative to (1)) of CTA-COOH (5) was added and the mixture was stirred at room temperature for 24 hours. After 24 hours, 199.32 mg (0.7203 mmol, 3.02 equivalents relative to (1)) of DMTMM (6) was added and the mixture was stirred at room temperature for another 24 hours under light protection. The resulting pink solution was subjected to two reprecipitations using ethyl ether, followed by vacuum drying, and then subjected to nuclear magnetic resonance (NMU) testing using a Bruker Advance 400 Hz (Bruker). １ H NMR measurement (CDCl ３ ) was performed.

[0059] Figure 4 １ The results of the 1H NMR spectrum measurement are shown. The yield was 0.949 g (0.213 mmol), and the yield was 89.2%.

[0060] 2-2. Synthesis of Poly(Oligoethylene Glycol) Methyl Ester Methacrylate (pOEGMA) The synthesis scheme for pOEGMA is shown below.

[0061]

[0062] The reagents used in this synthesis example are listed below.

[0063]

[0064] Weighed 3278.2 mg of OEGMA into a 100 mL Meyer, and dissolved it in 30 mL of acetone. Added 1008.3 mg of activated carbon here, and stirred for 2 h in the light-shielded state. Then, the activated carbon was removed by gravity filtration, and OEGMA was purified by concentration and vacuum drying. (Yield: 2977 mg, Yield rate: 90.08%) Subsequently, 67.68 mg (0.2481 mmol) of CDB(1), 2977 mg (9.923 mmol, 40 equivalents relative to (1)) of OEGMA, and 18.85 mL of 1,4-dioxane (monomer concentration: 0.5 M) were added to a 100 mL eggplant flask, １ The charging ratio was confirmed by 1H NMR. Subsequently, 8.15 mg (0.0496 mmol, 0.2 equivalent relative to (1)) of AIBN(3) and 1.000 mL of 1,4-dioxane were added, and freeze degassing was performed 3 times, nitrogen substitution and freeze degassing were performed 2 times, and polymerization was carried out at the temperature of 60 °C for 16.5 h. After the reaction was completed, MeOH was added to the reaction solution and diluted approximately twice, and all the solution was transferred to a dialysis membrane (MWCO: 3.5 kDa), and dialysis was performed (outer solution; MeOH, exchange time; 1, 1, 2, 2, 18 h). Then, the inner solution was recovered and concentrated, and benzene freeze drying was performed to obtain a peach-colored product. Finally １ 1H NMR spectrum measurement (CDCl ３ ), gel permeation chromatography (GPC measurement) (mobile phase: 10 mM LiCl in DMF, column: TSKgel SuperH, temperature: 40 °C, flow rate: 0.3 mL / min) were performed.

[0065] The actual charging ratio in this synthesis example was CDB:OEGMA:AIBN = 1:43:0.2. Also, the conversion rate was 59.31%. The yield was 1691 mg, and the recovery rate was 92.24%. Also, the number of chains in the synthesized pOEGMA was 25 (Mn; 7772.42 g / mol, polyethylene glycol chain; 113, sometimes denoted as pOEGMA ２５ hereinafter). Figure 5 shows the １ 1H NMR measurement results of the synthesized pOEGMA.

[0066] 2-3. Synthesis of Protein Modification Copolymer 1 (PEG(4k)-b-pPyMA) Using the PyMA synthesized in "1-1" and the PEG(4k)-macro CTA synthesized in "2-1", protein modification copolymer 1 (PEG(4k)-b-pPyMA: Example 1) was synthesized according to the following scheme.

[0067]

[0068] The reagents used in this synthesis example are listed below.

[0069]

[0070] Add PEG(4k)-macro CTA(3) 708.9 mg (0.1590 mmol) to the polymerization tube and add CHCl3. ３ Dissolve in 2 mL of benzene and 12 mL of benzone, and freeze-dry for 3 hours. Add 978.65 mg of PyMA(2) (4.769 mmol, 30 equivalents relative to (3)) and 9.34 mL of DMF to this polymerization tube and confirm the starting ratio (monomer concentration: 0.5 M). Subsequently, 5.22 mg of AIBN(3) (3.18 × 10) －２ mmol (0.2 equivalents relative to (3)), 0.200 mL of DMF was added. The polymerization reaction was carried out at 60°C for 48 hours, after three freeze-degassing cycles and three nitrogen purging and freeze-degassing cycles. Then, reprecipitation (good solvent: CHCl3) was performed. ３ The process was repeated three times using a poor solvent (ethylene hydroxide). Then, freeze-drying with benzene was performed for 3 hours to obtain a brown product. １ 1H NMR spectrum measurement (CDCl ３ ) was performed.

[0071] In this synthesis example, the actual input ratio was PEG(CTA):PyMA:AIBN = 1:25:0.2. The conversion rate was 48.00%. The yield was 891 mg, and the recovery rate was 75.59%. The number of chains in the synthesized pPyMA was approximately 14 (hereafter, PEG(4k)-b-pPyMA). １４ (It may also be written as follows.) Figure 6 shows the synthesized PEG(4k)-b-pPyMA (Example 1). １ The 1H NMR measurement results are shown.

[0072] 2-4. Synthesis of Protein Modification Copolymer 2 (PEG(4k)-b-pDPAMA) Using the pDPAMA synthesized in "1-3" and the PEG(4k)-macro CTA synthesized in "2-1", protein modification copolymer 2 (PEG(4k)-b-pDPAMA: Example 2) was synthesized according to the following scheme. The reagents used in this synthesis example are listed below.

[0073]

[0074] PEG(4k)-macro CTA(1) 363.91 mg (7.982 x 10) in polymerization tubes －２ Add mmol and CHCl ３ 0.5 mL and 3 mL of benzene were added to dissolve the mixture, and then freeze-dried. 410.65 mg of DPAMA(2) (1.596 mmol, 20 equivalents relative to (1)) and 3.49 mL of DMF were added to this polymerization tube, and the charging ratio was confirmed (monomer concentration: 0.5 M). Subsequently, 2.62 mg of AIBN(3) (1.60 × 10⁻¹⁰) was added. －２ mmol (0.2 equivalents per (1)) and 0.500 mL of DMF were added. The polymerization reaction was carried out at 60°C for 48 hours, with three freeze-degassing cycles and three nitrogen purging and freeze-degassing cycles. Afterwards, reprecipitation was performed (good solvent: CHCl3). ３ The process of adding a poor solvent (ethylene ether) was repeated twice. Then, freeze-drying was performed to obtain a brown product. Finally, １ 1H NMR spectrum measurement (CDCl ３ ) was performed.

[0075] In this synthesis example, the actual input ratio was PEG(CTA):DPAMA:AIBN = 1:14:0.2. The conversion rate was 92.28%. The yield was 550.6 mg, and the recovery rate was 74.12%. The number of chains in the synthesized PEG(4k)-b-pDPAMA was approximately 13 (hereafter, PEG(4k)-b-pDPAMA). １３ (It may also be written as follows.) Figure 7 shows the synthesized PEG(4k)-b-pDPAMA (Example 2). １ The 1H NMR measurement results are shown.

[0076] Furthermore, the GPC measurement results for protein modification copolymer 1 and protein modification copolymer 2 are shown in Table 8 below.

[0077]

[0078] 2-5. Synthesis of Protein Modification Copolymer 3 (pOEGMA-b-pPyMA) Using the PyMA synthesized in "1-1" and the pOEGMA synthesized in "2-2", protein modification copolymer 3 (pOEGMA-b-pPyMA: Example 3) was synthesized according to the following scheme.

[0079] The reagents used in this synthesis example are listed below.

[0080]

[0081] pOEGMA in polymerization tube ２５ (1) 679.4mg (8.741×10 －２ The mixing ratio was confirmed by adding 549.0 mg of PyMA(2) (2.714 mmol, 30 equivalents relative to (1)) and 10.49 mL of DMF. To this, 2.87 mg of AIBN (1.75 × 10) was added. －２ mmol (0.2 equivalents per (1)), 0.200 mL of DMF (monomer concentration: 0.25 M) was added, and polymerization was carried out at 60°C after three freeze-degassing cycles, nitrogen purging, and two freeze-degassing cycles. A few drops of the reaction solution were taken at 3, 6, 10, and 24 hours. １ ¹H NMR and GPC measurements were performed. After 24 hours, the sample was reprecipitation twice using ethyl hydroxide, and then freeze-dried with benzene to obtain an orange-brown product. １ 1H NMR spectrum measurement (CDCl ３ ), GPC measurements were performed.

[0082] In this synthesis example, the actual input ratio was pOEGMA(CTA):DPAMA:AIBN = 1:22:0.2. The conversion rate was 70.92%. The yield was 809.4 mg, and the recovery rate was 76.05%. The number of chains in the synthesized pOEGMA-b-pPyMA was approximately 17 (hereinafter, pOEGMA-b-pPyMA). １７(It may also be written as follows). Figure 8 shows the synthesized pOEGMA-b-pPyMA (Example 3). １ The 1H NMR measurement results are shown.

[0083] 2-6. Synthesis of Protein Modification Copolymer 4 (pOEGMA-b-pDPAMA) Using the pDPAMA synthesized in "1-2" and the pOEGMA synthesized in "2-2", protein modification copolymer 4 (pOEGMA-b-pDPAMA: Example 4) was synthesized according to the following scheme.

[0084] The reagents used in this synthesis example are listed below.

[0085]

[0086] pOEGMA in polymerization tube ２５ (4) 484.9mg (6.239×10 －２ (5) 413.7 mg (1.271 mmol, 20 equivalents relative to (2)) and DPAMA (polymerization group conversion rate 100%) were added to confirm the charging ratio. To this, 1.02 mg of AIBN (6.24 × 10) was added. －３ mmol (0.2 equivalents per (2)), 0.200 mL of DMF (monomer concentration: 0.25 M) was added, and polymerization was carried out at 60°C after three freeze-degassing cycles, nitrogen purging, and two freeze-degassing cycles. After 48 hours, reprecipitation was performed twice with ethyl ether, and benzene freeze-drying was performed to obtain a pink product. Finally １ 1H NMR spectrum measurement (CDCl ３ ), GPC measurements were performed.

[0087] In this synthesis example, the actual input ratio was pOEGMA(CTA):DPAMA:AIBN = 1:16:0.1. The conversion rate was 84.87%. The yield was 414 mg, and the recovery rate was 49.52%. The number of chains in the synthesized pOEGMA-b-pDPAMA was approximately 16 (hereinafter referred to as pOEGMA-b-pDPAMA). １６ (It may also be written as follows.) Figure 9 shows the synthesized pOEGMA-b-pDPAMA (Example 4). １ The 1H NMR measurement results are shown.

[0088] Furthermore, the GPC measurement results for protein modification copolymer 3 and protein modification copolymer 4 are shown in Table 11 below.

[0089]

[0090] 2-7. To compare PEG(4k)-b-PLA with copolymers 1 to 4 for protein modification, a copolymer (PEG(4k)-b-PLA: comparative example) in which lactide was bonded to a polyethylene glycol chain was synthesized according to the following scheme.

[0091] The reagents used in this synthesis example are listed below.

[0092]

[0093] 4420 mg of DL-Lactide was weighed into a 50 mL round-neck flask, 15 mL of EtOAc was added, and the mixture was heated to 70°C. Undissolved material was removed by suction filtration, and the filtrate was recrystallized by cooling in an ice bath. The precipitated white solid was recovered by suction filtration and vacuum-dried (recovery amount: 2857 mg, recovery rate: 64.64%). Subsequently, 1079.6 mg (0.2511 mmol) of MeO-PEG(4k)-OH(1) and 906.8 mg (6.292 mmol, 25 equivalents relative to (1)) of DL-Lactide(2) were added to a 500 mL two-neck flask, dissolved with methanol and benzene, and freeze-dried. Then, 80.68 mL of super-dehydrated toluene was added under a nitrogen atmosphere. Subsequently, Sn(Oct) ２ (3) 50.87 mg (0.1256 mmol, 0.5 equivalents relative to (1)) of toluene was added to 10 mL (Dl-Lactide concentration: 10 mg / mL). This solution was heated under reflux to 120°C and stirred for 48 hours. After the reaction, the solution was allowed to cool, concentrated, and then reprecipitation was performed (good solvent: THF, poor solvent: ethyl ether). The obtained precipitate was collected and freeze-dried to obtain a white solid. １ Analysis was performed using 1H NMR and GPC.

[0094] In this synthesis example, the yield was 1.026 g, the conversion rate was 15.54%, and the recovery rate was 84.10%. Furthermore, the number of chains in the synthesized PEG(4k)-b-PLA was approximately 8 (hereinafter, PEG(4k)-b-PLA...). ８ (It may also be written as follows). Figure 10 shows the synthesized PEG(4k)-b-PLA (comparative example). １ The 1H NMR measurement results are shown. Additionally, the GPC measurement results are shown in Table 13 below.

[0095]

[0096] [3. Adsorption Test 1] 3-1. Adsorption of Lysozyme onto Silica Particles (Determination of Adsorption Concentration) In this example, the following reagents were used.

[0097]

[0098] Twelve vials were prepared, and approximately 20 mg of silica particles were weighed into each vial. 4 mL of Lys solution of various concentrations (0–0.7 mg / mL) was added to each vial. The silica particles were dispersed by sonication for 10 min, followed by shaking at 25°C for 2 hours. Each solution was then transferred to a 15 mL centrifuge tube and centrifuged twice (4000 rpm, 10 min). 4 mL of the supernatant was collected, and the amount of adsorbed Lys was quantified by measuring the UV-Vis spectrum of the supernatant at each concentration using a UV-Vis spectrophotometer (JASCO V-650 spectrometer, manufactured by JASCO Corporation) (calibration curve concentrations: 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 mg / mL). Furthermore, the zeta potential of the obtained silica particles was measured using a Zetasizer Nano ZS (Malvern Panalogical). (Solvent: 1 mM HEPES, pH 7.01, Temperature: 25°C).

[0099] Regarding the silica particles used in this example, based on the average particle diameter (300 nm) and specific gravity (2.2), the total surface area of ​​20 mg of silica particles is calculated as 1.82 × 10⁻⁶. ３ cm ２This was determined. Using this value, the amount of Lys adsorbed per unit area was plotted. The absorbance of the supernatant after centrifugation at 278 nm was measured and extrapolated to the calibration curve to determine the weight of Lys contained in the supernatant. The weight of Lys adsorbed on the silica particles was calculated by subtracting the weight of Lys in the supernatant from the weight of Lys contained in 4 mL of the Lys solution at the time of adsorption. Using the above, the concentration of Lys in the supernatant (Lys equilibrium concentration) was plotted on the x-axis and the number of Lys adsorbed per unit area of ​​silica particles was plotted on the y-axis. The results are shown in Figure 11.

[0100] Furthermore, the zeta potential measurement results are shown in Figure 12. In Figure 12, the horizontal axis represents the weight concentration of Lys when adsorbed onto silica particles, and 0 mg / mL represents the zeta potential of silica particles alone without adsorbed Lys. The zeta potential of silica particles alone was -47 mV, but the positive charge increased as Lys was adsorbed, and there was no change after 0.13 mg / mL, becoming approximately 23 mV. From the adsorption isotherm and the zeta potential measurement results, Lys was adsorbed onto silica particles under the weight ratio condition of 0.3 mg / mL thereafter.

[0101] 3-2. PEG-b-pPyMA １４ and PEG-b-pDPAMA １３ Adsorption of Lys-immobilized silica particles (25°C) PEG(4k)-b-pPyMA synthesized in this example １４ Dissolve in HEPES and prepare a 50 μM PEG(4k)-b-pPyMA １４ A solution was prepared. Similarly, PEG(4k)-b-pDPAMA １３ Dissolve in HEPES to make 50 μM PEG(4k)-b-pDPAMA １３Solutions were prepared. Next, approximately 230 mg of Lys silica-modified silica particles were weighed, approximately 23 mL of HEPES was added, and sonication was performed for 1 minute to prepare a 10 mg / mL Lys silica particle dispersion. These solutions and 1 mM HEPES were added to 5 mL vials in the mixing ratios shown in Table 15, and shaken at 25°C for 24 hours. Calibration solutions were prepared at 3, 6, 10, 15, 20, and 35 μM concentrations and shaken under the same conditions. The solutions were then transferred to centrifuge tubes and centrifuged (4000 rpm, 10 min, twice). After centrifugation, the supernatant was collected, and the unadsorbed polymer was quantified by UV-Vis measurement. In addition, 1 mL of 1 mM HEPES was added to the precipitate to redisperse it, and the zeta potential was measured. These experiments were repeated three times.

[0102]

[0103] The results of the adsorption isotherm are shown in Figure 13, and the zeta potential measurement results are shown in Figure 14. From these results, it was confirmed that the zeta potential shifted in the negative direction with the adsorption of the protein modification copolymer. This is thought to be because the protein modification copolymer shields the potential of the silica particle surface. Furthermore, for each result, the formula is: [c / A] = [c / A] ｍ ] + [1 / aA ｍ A Langmuir plot was created from the following. In the formula, c is the equilibrium concentration (μM), and A is the adsorption density (chains / nm). ２ ), A ｍ is the saturation adsorption density, and a is the adsorption coefficient. From this, PEG(4k)-b-pPyMA １４ The Languir plot prepared for PEG(4k)-b-pDPAMA is shown in Figure 15. １３ The Languir plot created for this purpose is shown in Figure 16. The calculated parameters are shown in Table 16.

[0104]

[0105] 3-3. Adsorption and van't Hoff plots at 30°C and 35°C PEG(4k)-b-pPyMA １４ Dissolve in HEPES and prepare a 50 μM PEG(4k)-b-pPyMA １４A solution was prepared. Similarly, PEG(4k)-b-pDPAMA １３ Dissolve in HEPES to make 50 μM PEG(4k)-b-pDPAMA １３ Solutions were prepared. Next, approximately 230 mg of Lys silica-modified silica particles were weighed, approximately 23 mL of HEPES was added, and sonication was performed for 1 minute to prepare a 10 mg / mL Lys silica particle dispersion. These solutions and 1 mM HEPES were added to 5 mL vials in the mixing ratios shown in Table 15 above, and shaken at 30°C or 35°C for 24 hours. Calibration solutions were prepared at 3, 6, 10, 15, 20, and 35 μM concentrations and shaken under the same conditions. The solutions were then transferred to centrifuge tubes and centrifuged (4000 rpm, 10 min, twice). After centrifugation, the supernatant was collected, and the unadsorbed polymer was quantified by UV-Vis measurement. Outlier values ​​were removed.

[0106] First, PEG(4k)-b-pPyMA １４ The adsorption isotherm (30°C) in Figure 17 is shown, and the adsorption isotherm (35°C) is shown in Figure 18. Also, PEG(4k)-b-pPyMA １４ The Langmuir plot (30°C) is shown in Figure 19, and the Langmuir plot (35°C) is shown in Figure 20. Similarly, PEG(4k)-b-pDPAMA １３ The adsorption isotherm (30°C) in Figure 21 is shown, and the adsorption isotherm (35°C) in Figure 22. Also, PEG(4k)-b-pDPAMA １３ The Langmuir plot (30°C) is shown in Figure 23, and the Langmuir plot (35°C) is shown in Figure 24. The results of the adsorption experiments at each temperature shown above are presented in PEG(4k)-b-pPyMA １４ Table 17 summarizes the information regarding PEG(4k)-b-pDPAMA. １３ This is summarized in Table 18.

[0107]

[0108]

[0109] Based on these results, a van't Hoff plot was created. The result of plotting lnA, which is the natural logarithm of the adsorption coefficient A against the reciprocal of the temperature 1 / T, from the following equation, is shown in Figure 25 and Table 19.

[0110]

[0111]

[0112] Based on the results so far, the Gibbs free energy change ΔG associated with adsorption was negative in all temperature ranges for the two protein modification copolymers tested. From this, it was confirmed that the two protein modification copolymers tested have adsorption affinity for proteins. Here, it was confirmed that enthalpy change is dominant in adsorption. Among them, PEG-b-pDPAMA １３ The enthalpy change of adsorption due to PEG-b-pPyMA １４ It was found to be larger than sp ２ We hypothesized that DPA, which has two lone pairs of electrons in its hybrid orbitals, may be advantageous in adsorption. Although this hypothesis does not include detailed experimental results, in adsorption inhibition experiments using 4-methylpyridine, PEG-b-pDPAMA １３ is PEG-b-pPyMA １４ It is also supported because it is less likely to be inhibited than other methods.

[0113] [4. Adsorption Test 2] In Adsorption Test 2, a self-assembled monolayer (SAM film) was formed on a quartz crystal microbalance (hereinafter referred to as QCM) chip, and the adsorption capacity of the protein modification copolymer to the enzyme immobilized on the SAM film was verified. 4-1. Formation of SAM film on QCM chip As shown in Figure 26, a SAM film was formed on a QCM chip (QCM sensor chip QA-A9M AU(M), Freq. 8.94MHz ± 30kHz, Lot. 13327 (Seiko Easy & Good Co., Ltd.)). The following reagents were used in this example.

[0114]

[0115] First, both sides of the QCM tip were washed with piranha solution for one minute each, and this was repeated twice. Furthermore, UV-O ３ The cleaning process was performed for 30 minutes, and the contact angle and frequency f were measured. ０ The following measurements were taken. A simple portable QCM measuring instrument (THQ-100P-SW model, Tama Device Co., Ltd.) was used for the measurements. Next, a 1 mM SAM-COOH ethanol solution was placed in a vial and the QCM tip was immersed in it. This was left at 25°C for 2 hours. After that, the contact angle θ and frequency were measured, and the modification density was calculated from the frequency change.

[0116] Table 21 summarizes the frequency changes after SAM formation, as well as the SAM modification density and contact angle. The SAM frequency change was calculated using the following Sauerbrey equation.

[0117]

[0118] In the equation, Δf is the frequency change, and F ｏ is the chip frequency, A is the electrode area, and μ is the recommended shear stress (2.947 × 10⁻⁶). １０ (kgms), where ρ is the density of quartz (2648 kg / m³). ３ ) and Δm is the change in mass.

[0119]

[0120] 4-2. Immobilization of Lys onto SAM membrane As shown in Figure 27, the protein Lysozyme (Lys) was immobilized onto a SAM membrane. The reagents used are as follows.

[0121]

[0122] The QCM tip with the SAM membrane formed on it was immersed for 7 minutes in a 200 mM MEDC / 50 mM NHS solution (100 mM acetate buffer, pH 4.67). At 3.5 minutes, a cycle of 5 seconds of sonication followed by a 15-second rest was repeated three times. After immersion, the tip was washed by immersing it 10 times in Milli-Q water, and then immersed for 7 minutes in a 100 μg / mL Lys solution (1 mM phosphate buffer solution, pH 7.40). Similarly, at 3.5 minutes, a cycle of 5 seconds of sonication followed by a 15-second rest was repeated. After that, it was washed 10 times with Milli-Q water droplets, and then N ２ Air drying is performed, and the frequency change Δf １ The following measurements were taken. Then, the sample was immersed for 7 minutes in a 1 M aminoethanol solution adjusted to pH 8.5 with 1 M HCl, and at 3.5 minutes, a cycle of 5 seconds of ultrasound followed by a 15-second rest was repeated. Finally, after washing 10 times with Milli-Q water droplets, N ２ Air drying is performed, and the frequency change Δf ２ We measured it.

[0123] Table 23 shows the frequency changes after immobilization of Lys onto the SAM film and immersion in aminoethanol. Here, the Lys modification density was calculated from the frequency changes after immersion in aminoethanol.

[0124]

[0125] The adsorption density is 0.0910 ± 0.0109 [molecules / nm]. ２ The area occupied by one molecule is 11.5 ± 1.35 [nm]. ２ The molecule was identified as [Lys]. Lys is a protein measuring 4.5 nm × 3.0 nm × 3.0 nm. Based on the area occupied by one molecule, it is thought that two orientations, End-On and Side-On, are immobilized on the SAM membrane in a mixed state. From this, it was inferred that Lys was immobilized on the sensor in a densely packed state.

[0126] 4-3. Adsorption amount of protein modification copolymer to immobilized Lys The adsorption amount of protein modification copolymer to Lysozyme (Lys) immobilized on a SAM membrane was measured. The reagents used are as follows.

[0127]

[0128] First, PEG-b-pPyMA at a concentration of 93.8 μM, which is 10 times that of cmc. １４ (1 mM phosphate buffer solution, pH 7.40), or 85.8 μM PEG-b-pDPAMA １３ It was immersed in (1 mM phosphate buffer solution, pH 7.40). For comparison, PEG-b-PLA at 1 mg / mL was also administered. ８ The instrument was also immersed in a 1 mM phosphate buffer solution (pH 7.40). After immersion, a cycle of 5 seconds of ultrasound followed by a 15-second rest was repeated every 15 minutes, and the frequency change was measured every 30 minutes. Before frequency measurement, the instrument was washed 10 times with Milli-Q water droplets, and then N ２ The samples were air-dried. Measurements were taken until no more frequency changes were observed (three times).

[0129] Figure 28 shows the adsorption density of the protein modification copolymer onto the Lys surface, calculated from the frequency change at the end of the measurement. In Figure 28, the horizontal axis represents PEG-b-pPyMA, PyMA, and PLA, respectively. １４ , PEG-b-pDPAMA １３ , and PEG-b-PLA ８ This indicates that.

[0130] As shown in Figure 28, PEG-b-PLA ８ In comparison with PEG-b-pPyMA １４ and PEG-b-pDPAMA １３ It was confirmed that the adsorption density increased when using PEG-b-pPyMA. From these results, it was found that PEG-b-pPyMA １４ and PEG-b-pDPAMA １３ This suggests that it possesses not only hydrophobic interactions but also a unique adsorption mechanism.

[0131] [5. Effects of protein modification copolymers on enzyme activity] 5-1. Lysolytic activity of the protein modification copolymer Lys The enzyme activity of Lysozyme was investigated when the protein modification copolymer was adsorbed onto Lysozyme. Lysozyme is an example of an active ingredient that makes up a pharmaceutical composition, as it was approved as a drug for indications related to "chronic sinusitis," "bronchitis," "bronchial asthma," and "bronchiectasis." The reagents used in this example are as follows.

[0132]

[0133] First, Lys was weighed into vials and dissolved in each solvent to prepare a 20 μM (0.292 mg / mL) Lys solution. Also, PEG(4k)-b-pPyMA １４ , PEG(4k)-b-pDPAMA １３ , pOEGMA25-b-pPyMA １７ , pOEGMA25-b-pDPAMA １６ A 1000 μM solution was prepared for this as well, and this was diluted 10-fold and 5-fold to prepare 100 μM and 20 μM solutions of the protein-modifying copolymer. 1 mL of each of these protein-modifying copolymers was mixed with 1 mL of Lys solution and shaken at 25°C for 24 hours (final Lys concentration 10 μM (0.146 mg / mL), 50 equivalents, 5 equivalents, and 1 equivalent relative to the protein-modifying copolymer).

[0134] Next, Micrococcus lysodeikticus was weighed into a vial, and HEPES was added to prepare a 0.15 mg / mL suspension. After adding 800 μL of the suspension to the measurement cell, 80 μL of the mixed solution of the protein modification copolymer solution and Lys solution prepared above was added, and after three pipetting steps, the change in absorbance at 450 nm was measured at 25°C for 5 minutes. Absorbance A at 0 seconds. ｉｎｉｔｉａｌ Absorbance A at 300 seconds ｆｉｎａｌ Difference ΔA ４５０ The following was calculated (n=3). In addition, a solution containing only each protein modification copolymer was used for the blank, and the ΔA of the blank was calculated. ｂｌａｎｋ The enzyme activity was compared by subtracting the result from each measurement.

[0135] The lytic activity of the Lys solution, which was not mixed with the protein-modifying copolymer solution, was set to 100%. Figure 29 shows the results of the lytic activity when various protein-modifying copolymer solutions were mixed at various concentrations. As shown in Figure 29, it was confirmed that Lys maintained high activity for all the protein-modifying copolymers tested. Furthermore, it was confirmed that Lys maintained high activity at all the mixing ratios tested. The results of this experiment suggest that the four protein-modifying copolymers synthesized in this example can modify the surface by physical adsorption while maintaining the structure of Lys.

[0136] [6. Biocompatibility of proteins modified with protein modification copolymers] In this example, PEG(4k)-b-pPyMA with a chain number of 13 was used. １３ And, pOEGMA29-b-pPyMA with a chain count of 22 ２２ The following was used: PEG(4k)-b-pPyMA １３ is PEG (4k)-macro CTA (3) 700.4 mg, CHCl ３ The compound was synthesized in the same manner as described in "2-3" above, except that it consisted of 2 mL of benzene, 12 mL of PyMA(2), 965.1 mg of DMF, 9.6 mL of DMF, and 5.15 mg of AIBN(3). pOEGMA29-b-pPyMA ２２ This was synthesized in the same manner as described in "2-5" above, except that it contained 683.1 mg of pOEGMA25, 552.0 mg of PyMA(2), 11.5 mL of DMF, and 2.77 mg of AIBN.

[0137] In this example, these protein-modifying copolymers were used to modify four different proteins with varying molecular weights and isoelectric points. The four proteins were bovine serum albumin (BSA, molecular weight: 68 kDa, isoelectric point: 4.8), myoglobin (molecular weight: 17.6 kDa, isoelectric point: 6.8), lysozyme (molecular weight: 14.3 kDa, isoelectric point: 10.9), and aprotinin (molecular weight: 6.5 kDa, isoelectric point: 10).

[0138] For each of these four types of proteins, PEG(4k)-b-pPyMA １３ or pOEGMA29-b-pPyMA２２ High-concentration (20 μM) and low-concentration (5 μM) modified proteins were prepared using [a specific method / tool]. The adsorption of the obtained modified proteins to the gold surface of the QCM sensor was investigated. The measurement conditions were a fundamental resonance frequency of 5 MHz, a temperature of 27°C, running buffer of PBS (150 mM NaCl), and an overtone number of 3 (15 MHz). For the measurement, a modified protein at a concentration of 1.0 mg / ml was reacted with the gold surface of the QCM sensor for 10 minutes, and then the amount of protein adsorbed was measured under the above conditions.

[0139] The results are shown in Figure 30. In Figure 30, PEG(4k)-b-pPyMA １３ High-concentration modified PEG was referred to as "high-concentration PEG," and low-concentration modified PEG was referred to as "low-throat PEG." Similarly, pOEGMA29-b-pPyMA ２２ We referred to highly modified pOEG as "high-concentration pOEG" and low-concentration modified pOEG as "low-concentration pOEG". As can be seen from Figure 30, pOEGMA29-b-pPyMA ２２ When highly modified using PEG(4k)-b-pPyMA, １３ Compared to cases where high-concentration modification is used, the amount adsorbed to gold is lower, indicating higher biocompatibility. Furthermore, when comparing with low-concentration modification, PEG(4k)-b-pPyMA １３ Using pOEGMA29-b-pPyMA results in less adsorption to gold and higher biocompatibility. This is thought to be due to the high affinity of the PEG chain for water. ２２ When this material was used, a significant improvement in biocompatibility was observed by modifying the protein at high density. As shown in this example, it was found that when modifying proteins using a copolymer for protein modification, there are molecular structures and densities that are excellent for biocompatibility. High biocompatibility can offer advantages such as low immune response during intravenous injection, a lower probability of recognition as an antigen by immune cells, and lower antibody production.

[0140] The disclosure of Japanese Patent Application No. 2024-156975, filed on 10 September 2024, is incorporated herein by reference in its entirety. Furthermore, all documents, patent applications, and technical standards described herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

- (R Ａ O) A polymer portion having a repeating structure (wherein R Ａ (represents an alkylene group with 2 to 5 carbon atoms) and sp ２ A method for producing a modified protein, comprising the step of contacting a protein with a protein to form a complex of the protein and the protein-modifying copolymer, the copolymer comprising an adsorption portion derived from a basic compound having lone pairs of electrons in hybrid orbitals. 　 The method for producing a modified protein according to claim 1, wherein the basic compound is pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative. 　 The method for producing a modified protein according to claim 1, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. 　 The method for producing a modified protein according to claim 1, wherein the polymer portion is polyethylene glycol or poly(oligoethylene glycol) methacrylate. 　 The method for producing a modified protein according to claim 1, wherein the weight-average molecular weight of the protein-modifying copolymer is 1,000 to 1,000,000. 　 The method for producing a modified protein according to claim 1, wherein the copolymer for protein modification is at least one copolymer selected from the group consisting of the following formulas (1) to (4). [In equations (1) to (4) above, n is an integer between 5 and 600; in equations (1) to (2) above, m is an integer between 20 and 2000; in equations (3) to (4) above, p is an integer between 5 and 1000; and in equations (3) to (4) above, q is an integer between 1 and 15.] 　 The method for producing the modified protein according to claim 1, wherein the protein is an active ingredient in the treatment of a disease. - (R Ａ O) A polymer portion having a repeating structure (wherein R Ａ (represents an alkylene group with 2 to 5 carbon atoms) and sp ２ A copolymer for protein modification, comprising an adsorption portion derived from a basic compound having lone pairs of electrons in its hybrid orbitals. 　 The protein modification copolymer according to claim 8, wherein the basic compound is pyridine or a pyridine derivative or dipicolylamine or a dipicolylamine derivative. 　 The protein-modifying copolymer according to claim 8, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. 　 The polymer portion is polyethylene glycol or poly(oligoethylene glycol) methacrylate, as described in claim 8, for use as a protein-modifying copolymer. The protein-modifying copolymer according to claim 8, wherein the weight-average molecular weight is 1,000 to 100,000. The protein modification copolymer according to claim 8, which is at least one copolymer selected from the group consisting of the following formulas (1) to (4). [In equations (1) to (4) above, n is an integer between 5 and 600; in equations (1) to (2) above, m is an integer between 20 and 2000; in equations (3) to (4) above, p is an integer between 5 and 1000; and in equations (3) to (4) above, q is an integer between 1 and 15.] A polymer portion having poly(oligoethylene glycol) methacrylate, and sp ２ A copolymer comprising an adsorbent portion derived from a basic compound having lone pairs of electrons in its hybrid orbitals. 　 The copolymer according to claim 14, wherein the basic compound is pyridine or a pyridine derivative, or dipicolylamine or a dipicolylamine derivative. 　 The copolymer according to claim 14, wherein the basic compound is pyridine methacrylate or dipicolylamine methacrylate. The copolymer according to claim 14, wherein the weight-average molecular weight is 1,000 to 100,000. 　 The copolymer according to claim 14, wherein the copolymer is of the following formula (3) or formula (4). [In equations (3) and (4) above, n is an integer between 5 and 600, p is an integer between 5 and 1000, and q is an integer between 1 and 15.] A pharmaceutical composition comprising a protein that is an active ingredient for treating a disease, and a protein modification copolymer according to any one of claims 8 to 13 that is adsorbed to the protein. 　 The pharmaceutical composition according to claim 19, wherein the protein and the protein modification copolymer are bound together by physical adsorption.

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