Effective method for producing antibody-drug conjugates

Abstract

To provide a production method for a specific antibody-drug conjugate including a refining process for effectively removing by-products while reducing the generation of an aggregate.SOLUTION: A production method for a specific antibody-drug conjugate in which a drug linker and an antibody are bonded by thioether bond includes: (i) a process of reducing an antibody with a reducing agent; (ii) a process of allowing a drug linker intermediate to react with the antibody reduced in the process (i); (iii) a process of adding a reagent having a thiol group and allowing it to react with the drug linker intermediate remaining from the process (ii); and then (iv) a process of removing by-products derived from the drug linker intermediate by ultrafiltration using a buffer containing a salt consisting of strong acid and strong base.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an antibody-drug conjugate, which includes a purification step for effectively removing by-products while reducing the formation of aggregates, and a method for producing a pharmaceutical composition comprising the antibody-drug conjugate. [Background technology]

[0002] Antibody-drug conjugates (ADCs), which combine a cytotoxic drug with an antibody that can bind to an antigen expressed on the surface of cancer cells and be internalized into the cells, are expected to be able to selectively deliver the drug to cancer cells, thereby accumulating the drug within the cancer cells and killing the cancer cells (Non-Patent Documents 1 to 5).

[0003] Methods for producing antibody-drug conjugates are described in the Examples of Patent Documents 1 to 9. These methods include a purification step by chromatography such as hydrophobic chromatography or gel filtration chromatography.

[0004] As an example of an antibody-drug conjugate, an antibody-drug conjugate containing an antibody and a derivative of exatecan, a topoisomerase I inhibitor, as components, is known (Patent Documents 10 to 14, Non-Patent Documents 6 to 9). These antibody-drug conjugates have excellent antitumor effects and safety, and therefore clinical trials are currently underway.

[0005] Patent Documents 10 to 14 describe, as methods for producing the above-mentioned antibody-drug conjugates, methods for conjugating an antibody with a drug linker intermediate and methods for purifying the obtained antibody-drug conjugates.

[0006] The examples of Patent Documents 11 to 13 state that the above antibody-drug conjugates were purified by ultrafiltration using a sorbitol-containing acetate buffer. Patent Document 14 also states that the above antibody-drug conjugates can be purified by ultrafiltration using an acetate buffer, a histidine buffer, or a phosphate buffer. However, there is a demand for the development of an industrially superior method for producing antibody-drug conjugates. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2002 / 098883 [Patent Document 2] International Publication No. 2005 / 037992 [Patent Document 3] International Publication No. 2005 / 084390 [Patent Document 4] International Publication No. 2006 / 086733 [Patent Document 5] International Publication No. 2007 / 024536 [Patent Document 6] International Publication No. 2010 / 141566 [Patent Document 7] International Publication No. 2011 / 039724 [Patent Document 8] International Publication No. 2012 / 135517 [Patent Document 9] International Publication No. 2015 / 104359 [Patent Document 10] International Publication No. 2014 / 057687 [Patent Document 11] International Publication No. 2015 / 098099 [Patent Document 12] International Publication No. 2015 / 115091 [Patent Document 13] International Publication No. 2015 / 155998 [Patent Document 14] International Publication No. WO2017 / 002776

Non-Patent Literature

[0008]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Non-Patent Literature 7

Non-Patent Literature 8

Non-Patent Literature 9

Summary of the Invention

Problems to be Solved by the Invention

[0009] The antibody-drug conjugate according to the production method of the present invention is represented by the formula (1):

[0010] [ka]

[0011] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond.

[0012] A method for producing such an antibody-drug conjugate includes the following steps: (i) reducing the antibody with a reducing agent; (ii) Formula (2)

[0013] [ka]

[0014] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding a reagent having a thiol group and reacting it with the compound represented by formula (2) remaining in step (ii).

[0015] However, in order to produce such antibody-drug conjugates, particularly antibody-drug conjugates in which the average number of drug linkers bound per antibody is in the range of 7 to 8, a large amount of the compound represented by formula (2) is required, which may result in the production of a large amount of by-products derived from the compound represented by formula (2).In addition, in producing antibody-drug conjugates, it is necessary to suppress the formation of aggregates, and it is necessary to consider reaction and purification conditions taking into account the physical properties specific to each of the antibody portion and the drug linker portion.

[0016] One object of the present invention is to provide an industrially excellent method for producing an antibody-drug conjugate, which includes a purification step for effectively removing by-products derived from the compound represented by formula (2) and suppresses the formation of aggregates. Another object of the present invention is to establish an industrially excellent method for producing a pharmaceutical composition containing the antibody-drug conjugate. [Means for solving the problem]

[0017] The present inventors have conducted extensive research to solve the above problems, and have found that by-products derived from the compound represented by formula (2) can be effectively removed by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base in the purification step of an antibody-drug conjugate. Furthermore, they have found reaction and purification conditions that can suppress the formation of aggregates in the production and purification steps of the antibody-drug conjugate. This has provided an industrially excellent method for producing a pharmaceutical composition containing the antibody-drug conjugate.

[0018] That is, the present invention provides the following [1] to

[0410] . [1] Formula (1)

[0019] [ka]

[0020] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond, (i) reducing the antibody with a reducing agent; (ii) Formula (2)

[0021] [ka]

[0022] reacting the compound represented by the formula: with the antibody reduced in step (i); (iii) adding a reagent having a thiol group to react with the compound represented by formula (2) remaining from step (ii); and then (iv) removing, by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base, a compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) and a compound in which the reagent having a thiol group used in step (iii) is added to the maleimidyl group of the compound represented by formula (2); A manufacturing method comprising the steps of: [2] The method according to [1], wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine or a salt thereof. [3] The method according to [1], wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride. [4] The method according to any one of [1] to [3], wherein step (i) is carried out in a buffer solution. [5] The method according to [4], wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate. [6] The method according to [4] or [5], wherein the buffer solution is an acetate buffer solution. [7] The method according to any one of [1] to [6], wherein step (i) is carried out in the presence of a chelating agent. [8] The method according to [7], wherein the chelating agent is ethylenediaminetetraacetic acid. [9] The manufacturing method according to any one of [4] to [8], wherein the buffer used in step (i) contains a surfactant.

[10] The method according to [9], wherein the surfactant is polysorbate 20.

[11] The method according to [9], wherein the surfactant is polysorbate 80.

[12] The method according to any one of [1] to

[11] , wherein the reagent having a thiol group used in step (iii) is N-acetylcysteine.

[13] The method according to any one of [1] to

[12] , wherein the pH of the buffer solution used in step (iv) is about 5.

[14] The method according to any one of [1] to

[12] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[15] The method according to any one of [1] to

[12] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[16] The method according to any one of [1] to

[12] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[17] The method according to any one of [1] to

[12] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[18] The method according to any one of [1] to

[17] , wherein the buffer used in step (iv) is a histidine buffer.

[19] The method according to any one of [1] to

[18] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0023]

[20] The method according to any one of [1] to

[18] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv). [twenty one] The method according to any one of [1] to

[18] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv). [twenty two] The method according to any one of [1] to

[18] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv). [twenty three] The method according to any one of [1] to

[22] , wherein the salt of a strong acid and a strong base used in step (iv) is sodium chloride. [twenty four] Following step (iv), (v) removing salts of strong acids and strong bases by ultrafiltration using a buffer solution; The method for producing a product according to any one of [1] to

[23] , comprising: [twenty five] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[26] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is about 5.

[27] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[28] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[29] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[30] The method according to

[24] , wherein the pH of the buffer solution used in step (v) is 5.0.

[31] The method according to any one of

[24] to

[30] , wherein the buffer used in step (v) is a histidine buffer.

[32] The method for production according to any one of [1] to

[31] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[33] The method for production according to any one of [1] to

[31] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[34] The method for production described in any one of [1] to

[33] , wherein the antibody is an anti-HER2 antibody.

[35] The method for producing an anti-HER2 antibody described in

[34] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[36] The method for producing an anti-HER2 antibody described in

[34] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[37] The method for production described in any one of [1] to

[33] , wherein the antibody is an anti-HER3 antibody.

[38] The method for producing an anti-HER3 antibody described in

[37] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[39] The method for production described in

[38] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0024]

[40] The method for production according to any one of [1] to

[33] , wherein the antibody is an anti-GPR20 antibody.

[41] The method for producing an anti-GPR20 antibody described in

[40] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[42] The production method described in

[41] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-GPR20 antibody is deleted.

[43] The method of any one of [1] to

[33] , wherein the antibody is an anti-CDH6 antibody.

[44] The method for producing an anti-CDH6 antibody according to

[43] , wherein the anti-CDH6 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[45] The production method described in

[44] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[46] The method according to any one of [1] to

[45] , characterized in that it does not include a purification step by chromatography.

[47] The method according to

[46] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[48]

[47] , comprising: producing an antibody-drug conjugate by the production method according to any one of [1] to

[47] ; and (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[49] The method according to

[48] , wherein the buffer is a histidine buffer.

[50] The manufacturing method according to

[48] or

[49] , wherein the excipient is sucrose.

[51] The method according to

[48] or

[49] , wherein the excipient is trehalose.

[52]

[48] ​​to

[51] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[53] The method according to

[52] , wherein the surfactant is polysorbate 80.

[54] The method according to

[52] , wherein the surfactant is polysorbate 20.

[55] Formula (1)

[0025] [ka]

[0026] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond, (i) reducing the antibody with tris(2-carboxyethyl)phosphine hydrochloride; (ii) Formula (2)

[0027] [ka]

[0028] reacting the compound represented by the formula: with the antibody reduced in step (i); (iii) adding N-acetylcysteine ​​to react with the compound of formula (2) remaining from step (ii); and then (iv) By ultrafiltration using a histidine buffer containing sodium chloride,

[0029] [ka]

[0030] and a compound represented by formula (4)

[0031] [ka]

[0032] A step of removing the compound represented by A manufacturing method comprising the steps of:

[56] The method according to

[55] , wherein step (i) is carried out in a buffer solution.

[57] The method according to

[56] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[58] The method according to

[56] or

[57] , wherein the buffer is an acetate buffer.

[59] The method according to any one of

[55] to

[58] , wherein step (i) is carried out in the presence of a chelating agent.

[60] The method according to

[59] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[61] The manufacturing method described in any one of

[56] to

[60] , wherein the buffer solution used in step (i) contains a surfactant.

[62] The method according to

[61] , wherein the surfactant is polysorbate 20.

[63] The method according to

[61] , wherein the surfactant is polysorbate 80.

[64] The method according to any one of

[55] to

[63] , wherein the pH of the buffer solution used in step (iv) is about 5.

[65] The method according to any one of

[55] to

[63] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[66] The method according to any one of

[55] to

[63] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[67] The method according to any one of

[55] to

[63] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[68] The method according to any one of

[55] to

[63] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[69] The method according to any one of

[55] to

[68] , wherein the concentration of sodium chloride used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0033]

[70] The method according to any one of

[55] to

[68] , wherein the concentration of sodium chloride used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[71] The method according to any one of

[55] to

[68] , wherein the concentration of sodium chloride used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[72] The method according to any one of

[55] to

[68] , wherein the concentration of sodium chloride used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[73] Following step (iv), (v) removing sodium chloride by ultrafiltration using a histidine buffer; The method for producing a product according to any one of

[55] to

[72] , comprising:

[74] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[75] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is about 5.

[76] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[77] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[78] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[79] The method according to

[73] , wherein the pH of the buffer solution used in step (v) is 5.0.

[80] The method for production according to any one of

[55] to

[79] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[81] The method for production according to any one of

[55] to

[79] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[82] The method of any one of

[55] to

[81] , wherein the antibody is an anti-HER2 antibody.

[83] The method for producing an anti-HER2 antibody described in

[82] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[84] The method for producing an anti-HER2 antibody described in

[82] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[85] The method of any one of

[55] to

[81] , wherein the antibody is an anti-HER3 antibody.

[86] The method for producing an anti-HER3 antibody described in

[85] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[87] The method for production described in

[86] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[88] The method of any one of

[55] to

[81] , wherein the antibody is an anti-GPR20 antibody.

[89] The method for producing an anti-GPR20 antibody described in

[88] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[0034]

[90] The production method described in

[89] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-GPR20 antibody is deleted.

[91] The method of any one of

[55] to

[81] , wherein the antibody is an anti-CDH6 antibody.

[92] The method for producing an anti-CDH6 antibody described in

[91] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[93] The production method described in

[92] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[94] The method according to any one of

[55] to

[93] , characterized in that it does not include a purification step by chromatography.

[95] The method according to

[94] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[96]

[55] to

[95] , and further comprising: (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[97] The method according to

[96] , wherein the buffer is a histidine buffer.

[98] The method according to

[96] or

[97] , wherein the excipient is sucrose.

[99] The method according to

[96] or

[97] , wherein the excipient is trehalose.

[0100]

[96] to

[99] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of

[96] to

[99] , and further comprising: (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0101] The manufacturing method described in

[0100] , wherein the surfactant is polysorbate 80.

[0102] The manufacturing method described in

[0100] , wherein the surfactant is polysorbate 20.

[0103] Formula (1)

[0035] [ka]

[0036] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond, (i) reducing the antibody with a reducing agent; (ii) Formula (2)

[0037] [ka]

[0038] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding a reagent having a thiol group to the solution containing the unpurified or roughly purified antibody-drug conjugate. (iv) removing, by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base, a compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) and a compound in which the reagent having a thiol group used in step (iii) is added to the maleimidyl group of the compound represented by formula (2); The production method according to claim 1, further comprising purifying the product by the steps of:

[0104] The method for producing the compound according to

[0103] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine or a salt thereof.

[0105] The method for producing the compound according to

[0103] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride.

[0106] A manufacturing method described in any one of

[0103] to

[0105] , wherein step (i) is carried out in a buffer solution.

[0107] The manufacturing method according to

[0106] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0108] A manufacturing method described in

[0106] or

[0107] , wherein the buffer solution is an acetate buffer solution.

[0109] A method for producing a compound according to any one of claims 0103 to 0108, wherein step (i) is carried out in the presence of a chelating agent.

[0110] The manufacturing method described in

[0109] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0111] A manufacturing method described in any one of

[0106] to

[0110] , wherein the buffer solution used in step (i) contains a surfactant.

[0112] The manufacturing method described in

[0111] , wherein the surfactant is polysorbate 20.

[0113] The manufacturing method described in

[0111] , wherein the surfactant is polysorbate 80.

[0114] A method for producing a compound according to any one of claims 1 to 3, wherein the reagent having a thiol group used in step (iii) is N-acetylcysteine.

[0115] A manufacturing method described in any one of

[0103] to

[0114] , wherein the pH of the buffer used in step (iv) is about 5.

[0116] The method for producing according to any one of

[0103] to

[0114] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0117] The method for producing according to any one of the items

[0103] to

[0114] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0118] The method for producing according to any one of the items

[0103] to

[0114] , wherein the pH of the buffer used in step (iv) is in the range of 4.9 to 5.1.

[0119] A method for producing a product described in any one of

[0103] to

[0114] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0039]

[0120] A manufacturing method described in any one of

[0103] to

[0119] , wherein the buffer used in step (iv) is a histidine buffer.

[0121] The method for producing a compound according to any one of claims

[0103] to

[0120] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0122] A manufacturing method described in any one of

[0103] to

[0120] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0123] The method for producing a liquid crystal display according to any one of claims

[0103] to

[0120] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0124] A manufacturing method described in any one of

[0103] to

[0120] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0125] A method for producing a compound according to any one of claims 0103 to 0124, wherein the salt of a strong acid and a strong base used in step (iv) is sodium chloride.

[0126] Following step (iv), (v) removing salts of strong acids and strong bases by ultrafiltration using a buffer solution; A manufacturing method according to any one of

[0103] to

[0125] , comprising:

[0127] The method for producing the present invention described in

[0126] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0128] A manufacturing method described in

[0126] , wherein the pH of the buffer used in step (v) is about 5.

[0129] The method for producing the present invention described in

[0126] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0130] The method for producing the present invention described in

[0126] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0131] The method for producing the present invention described in

[0126] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0132] The method for producing the present invention described in

[0126] , wherein the pH of the buffer solution used in step (v) is 5.0.

[0133] A manufacturing method described in any one of

[0126] to

[0132] , wherein the buffer used in step (v) is a histidine buffer.

[0134] A manufacturing method described in any one of

[0103] to

[0133] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0135] A manufacturing method described in any one of

[0103] to

[0133] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0136] A manufacturing method described in any one of

[0103] to

[0135] , wherein the antibody is an anti-HER2 antibody.

[0137] The method for producing the anti-HER2 antibody described in

[0136] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0138] The method for producing an anti-HER2 antibody described in

[0136] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0139] A manufacturing method described in any one of

[0103] to

[0135] , wherein the antibody is an anti-HER3 antibody.

[0040]

[0140] The method for producing the anti-HER3 antibody described in

[0139] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0141] A manufacturing method described in

[0140] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0142] A manufacturing method described in any one of

[0103] to

[0135] , wherein the antibody is an anti-GPR20 antibody.

[0143] The method for producing the anti-GPR20 antibody described in

[0142] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[0144] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 4, wherein the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0145] A manufacturing method described in any one of

[0103] to

[0135] , wherein the antibody is an anti-CDH6 antibody.

[0146] The method for producing an anti-CDH6 antibody described in

[0145] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0147] A production method described in

[0146] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0148] A manufacturing method described in any one of

[0103] to

[0147] , characterized in that it does not include a chromatographic purification step.

[0149] The manufacturing method described in

[0148] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0150]

[0103] to

[0149] , an antibody-drug conjugate is produced by the production method according to any one of the above, (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0151] A manufacturing method described in

[0150] , wherein the buffer is a histidine buffer.

[0152] A manufacturing method described in

[0150] or

[0151] , wherein the excipient is sucrose.

[0153] A manufacturing method described in

[0150] or

[0151] , wherein the excipient is trehalose.

[0154]

[0150] To

[0153] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above, (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0155] The manufacturing method described in

[0154] , wherein the surfactant is polysorbate 80.

[0156] The manufacturing method described in

[0154] , wherein the surfactant is polysorbate 20.

[0157] Formula (1)

[0041] [ka]

[0042] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond, (i) reducing the antibody with tris(2-carboxyethyl)phosphine hydrochloride; (ii) Formula (2)

[0043] [ka]

[0044] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding N-acetylcysteine ​​to the solution containing the unpurified or crude antibody-drug conjugate obtained by the above step, (iv) By ultrafiltration using a histidine buffer containing sodium chloride,

[0045] [ka]

[0046] and a compound represented by formula (4)

[0047] [ka]

[0048] A step of removing the compound represented by The production method according to claim 1, further comprising purifying the product by the steps of:

[0158] A manufacturing method described in

[0157] , in which step (i) is carried out in a buffer solution.

[0159] The manufacturing method according to

[0158] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0160] A manufacturing method described in

[0158] or

[0159] , wherein the buffer solution is an acetate buffer solution.

[0161] A manufacturing method described in any one of

[0157] to

[0160] , wherein step (i) is carried out in the presence of a chelating agent.

[0162] The manufacturing method described in

[0161] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0049]

[0163] A manufacturing method described in any one of

[0158] to

[0162] , wherein the buffer solution used in step (i) contains a surfactant.

[0164] The manufacturing method described in

[0163] , wherein the surfactant is polysorbate 20.

[0165] The manufacturing method described in

[0163] , wherein the surfactant is polysorbate 80.

[0166] A manufacturing method described in any one of

[0157] to

[0165] , wherein the pH of the buffer used in step (iv) is about 5.

[0167] A method for producing a product according to any one of items

[0157] to

[0165] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0168] The method for producing according to any one of

[0157] to

[0165] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0169] A method for producing a product according to any one of items

[0157] to

[0165] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[0170] A method for producing a product described in any one of

[0157] to

[0165] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0171] The method for producing a product according to any one of items

[0157] to

[0170] , wherein the concentration of sodium chloride used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0172] A manufacturing method described in any one of items

[0157] to

[0170] , wherein the concentration of sodium chloride used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0173] The method for producing according to any one of items

[0157] to

[0170] , wherein the concentration of sodium chloride used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0174] A manufacturing method described in any one of items

[0157] to

[0170] , wherein the concentration of sodium chloride used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0175] Following step (iv), (v) removing sodium chloride by ultrafiltration using a histidine buffer; A manufacturing method described in any one of

[0157] to

[0174] , comprising:

[0176] The method for producing the present invention described in

[0175] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0177] A manufacturing method described in

[0175] , wherein the pH of the buffer used in step (v) is about 5.

[0178] The method for producing the present invention described in

[0175] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0179] The method for producing the present invention described in

[0175] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0050]

[0180] The method for producing the present invention described in

[0175] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0181] The manufacturing method described in

[0175] , wherein the pH of the buffer used in step (v) is 5.0.

[0182] A manufacturing method described in any one of

[0157] to

[0181] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0183] A manufacturing method described in any one of

[0157] to

[0181] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0184] A manufacturing method described in any one of

[0157] to

[0183] , wherein the antibody is an anti-HER2 antibody.

[0185] The method for producing the anti-HER2 antibody described in

[0184] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0186] The method for producing an anti-HER2 antibody described in

[0184] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0187] A manufacturing method described in any one of

[0157] to

[0183] , wherein the antibody is an anti-HER3 antibody.

[0188] The method for producing an anti-HER3 antibody described in

[0187] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0189] A manufacturing method described in

[0188] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0051]

[0190] A manufacturing method described in any one of

[0157] to

[0183] , wherein the antibody is an anti-GPR20 antibody.

[0191] The method for producing the anti-GPR20 antibody described in

[0190] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[0192] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 4, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0193] A manufacturing method described in any one of

[0157] to

[0183] , wherein the antibody is an anti-CDH6 antibody.

[0194] The method for producing an anti-CDH6 antibody described in

[0193] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0195] A production method described in

[0194] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0196] A manufacturing method described in any one of

[0157] to

[0195] , characterized in that it does not include a chromatographic purification step.

[0197] The manufacturing method described in

[0196] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0198]

[0157] to

[0197] , an antibody-drug conjugate is produced by the production method according to any one of the above, (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0199] A manufacturing method described in

[0198] , wherein the buffer is a histidine buffer.

[0052]

[0200] A manufacturing method described in

[0198] or

[0199] , wherein the excipient is sucrose.

[0201] A manufacturing method described in

[0198] or

[0199] , wherein the excipient is trehalose.

[0202]

[0198] to

[0201] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0203] The manufacturing method described in

[0202] , wherein the surfactant is polysorbate 80.

[0204] The manufacturing method described in

[0202] , wherein the surfactant is polysorbate 20.

[0205] Formula (6)

[0053] [ka]

[0054] (In the formula, the drug linker is bound to the antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) A method for producing an antibody-drug conjugate represented by the following formula: (i) reducing the antibody with a reducing agent; (ii) Formula (2)

[0055] [ka]

[0056] reacting the compound represented by the formula: with the antibody reduced in step (i); (iii) adding a reagent having a thiol group to react with the compound represented by formula (2) remaining from step (ii); and then (iv) removing, by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base, a compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) and a compound in which the reagent having a thiol group used in step (iii) is added to the maleimidyl group of the compound represented by formula (2); A manufacturing method comprising the steps of:

[0206] The method for producing the compound according to

[0205] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine or a salt thereof.

[0207] The method for producing the compound according to

[0205] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride.

[0208] A manufacturing method described in any one of

[0205] to

[0207] , wherein step (i) is carried out in a buffer solution.

[0209] The manufacturing method according to

[0208] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0210] A manufacturing method described in

[0208] or

[0209] , wherein the buffer solution is an acetate buffer solution.

[0211] A manufacturing method described in any one of

[0205] to

[0210] , wherein step (i) is carried out in the presence of a chelating agent.

[0212] The manufacturing method described in

[0211] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0213] A manufacturing method described in any one of

[0208] to

[0212] , wherein the buffer solution used in step (i) contains a surfactant.

[0214] The manufacturing method described in

[0213] , wherein the surfactant is polysorbate 20.

[0215] The manufacturing method described in

[0213] , wherein the surfactant is polysorbate 80.

[0216] A method for producing a compound according to any one of claims

[0205] to

[0215] , wherein the reagent having a thiol group used in step (iii) is N-acetylcysteine.

[0217] A manufacturing method described in any one of

[0205] to

[0216] , wherein the pH of the buffer used in step (iv) is about 5.

[0218] A method for producing according to any one of items

[0205] to

[0216] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0219] A method for producing a product according to any one of items

[0205] to

[0216] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0057]

[0220] A method for producing according to any one of items

[0205] to

[0216] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[0221] A manufacturing method described in any one of

[0205] to

[0216] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0222] A manufacturing method described in any one of

[0205] to

[0221] , wherein the buffer used in step (iv) is a histidine buffer.

[0223] The manufacturing method according to any one of

[0205] to

[0222] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0224] A manufacturing method described in any one of items

[0205] to

[0222] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0225] The manufacturing method according to any one of

[0205] to

[0222] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0226] A manufacturing method described in any one of items

[0205] to

[0222] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0227] A manufacturing method described in any one of

[0205] to

[0226] , wherein the salt of a strong acid and a strong base used in step (iv) is sodium chloride.

[0228] Following step (iv), (v) removing salts of strong acids and strong bases by ultrafiltration using a buffer solution; A manufacturing method described in any one of

[0205] to

[0227] , comprising:

[0229] The method for producing the present invention described in

[0228] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0058]

[0230] A manufacturing method described in

[0228] , wherein the pH of the buffer used in step (v) is about 5.

[0231] The method for producing the present invention described in

[0228] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0232] The method for producing the present invention described in

[0228] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0233] The method for producing the present invention described in

[0228] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0234] The manufacturing method described in

[0228] , wherein the pH of the buffer used in step (v) is 5.0.

[0235] A manufacturing method described in any one of

[0228] to

[0234] , wherein the buffer used in step (v) is a histidine buffer.

[0236] A manufacturing method described in any one of

[0205] to

[0235] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0237] A manufacturing method described in any one of

[0205] to

[0235] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0238] A manufacturing method described in any one of

[0205] to

[0237] , wherein the antibody is an anti-HER2 antibody.

[0239] The method for producing the anti-HER2 antibody described in

[0238] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0059]

[0240] The method for producing the anti-HER2 antibody described in

[0238] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0241] A manufacturing method described in any one of

[0205] to

[0237] , wherein the antibody is an anti-HER3 antibody.

[0242] The method for producing the anti-HER3 antibody described in

[0241] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0243] A manufacturing method described in

[0242] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0244] A manufacturing method described in any one of

[0205] to

[0237] , wherein the antibody is an anti-GPR20 antibody.

[0245] The method for producing the anti-GPR20 antibody described in

[0244] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence described in amino acid numbers 20 to 472 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence described in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[0246] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 4, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0247] A manufacturing method described in any one of

[0205] to

[0237] , wherein the antibody is an anti-CDH6 antibody.

[0248] The method for producing the anti-CDH6 antibody described in

[0247] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0249] A production method described in

[0248] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0060]

[0250] A manufacturing method described in any one of

[0205] to

[0249] , characterized in that it does not include a chromatographic purification step.

[0251] The manufacturing method described in

[0250] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0252]

[0205] to

[0251] , an antibody-drug conjugate is produced by the production method according to any one of the above, (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0253] A manufacturing method described in

[0252] , wherein the buffer is a histidine buffer.

[0254] A manufacturing method described in

[0251] or

[0252] , wherein the excipient is sucrose.

[0255] A manufacturing method described in

[0251] or

[0252] , wherein the excipient is trehalose.

[0256]

[0252] to

[0255] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0257] The manufacturing method described in

[0256] , wherein the surfactant is polysorbate 80.

[0258] The manufacturing method described in

[0256] , wherein the surfactant is polysorbate 20.

[0259] Formula (6)

[0061] [ka]

[0062] (In the formula, the drug linker is bound to the antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) A method for producing an antibody-drug conjugate represented by the following formula: (i) reducing the antibody with tris(2-carboxyethyl)phosphine hydrochloride; (ii) Formula (2)

[0063] [ka]

[0064] reacting the compound represented by the formula: with the antibody reduced in step (i); (iii) adding N-acetylcysteine ​​to react with the compound of formula (2) remaining from step (ii); and then (iv) By ultrafiltration using a histidine buffer containing sodium chloride,

[0065] [ka]

[0066] and a compound represented by formula (4)

[0067] [ka]

[0068] A step of removing the compound represented by A manufacturing method comprising the steps of:

[0260] A manufacturing method described in

[0259] , in which step (i) is carried out in a buffer solution.

[0261] The manufacturing method described in

[0260] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0262] A manufacturing method described in

[0260] or

[0261] , wherein the buffer solution is an acetate buffer solution.

[0263] A manufacturing method described in any one of

[0259] to

[0262] , wherein step (i) is carried out in the presence of a chelating agent.

[0264] The manufacturing method described in

[0263] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0265] A manufacturing method described in any one of

[0260] to

[0264] , wherein the buffer solution used in step (i) contains a surfactant.

[0266] The manufacturing method described in

[0265] , wherein the surfactant is polysorbate 20.

[0267] The manufacturing method described in

[0265] , wherein the surfactant is polysorbate 80.

[0268] A manufacturing method described in any one of

[0259] to

[0267] , wherein the pH of the buffer used in step (iv) is about 5.

[0269] A method for producing a product described in any one of items

[0259] to

[0267] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0069]

[0270] A method for producing a product described in any one of items

[0259] to

[0267] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0271] A method for producing a product described in any one of items

[0259] to

[0267] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[0272] A manufacturing method described in any one of

[0259] to

[0267] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0273] The method for producing according to any one of items

[0259] to

[0272] , wherein the concentration of sodium chloride used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0274] A manufacturing method described in any one of items

[0259] to

[0272] , wherein the concentration of sodium chloride used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0275] The manufacturing method according to any one of

[0259] to

[0272] , wherein the concentration of sodium chloride used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0276] A manufacturing method described in any one of items

[0259] to

[0272] , wherein the concentration of sodium chloride used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0277] Following step (iv), (v) removing sodium chloride by ultrafiltration using a histidine buffer; A manufacturing method described in any one of

[0259] to

[0276] , comprising:

[0278] The method for producing the present invention described in

[0277] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0279] A manufacturing method described in

[0277] , wherein the pH of the buffer used in step (v) is about 5.

[0070]

[0280] The method for producing the present invention described in

[0277] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0281] The method for producing the present invention described in

[0277] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0282] The method for producing the present invention described in

[0277] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0283] The manufacturing method described in

[0277] , wherein the pH of the buffer used in step (v) is 5.0.

[0284] A manufacturing method described in any one of

[0259] to

[0283] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0285] A manufacturing method described in any one of

[0259] to

[0283] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0286] A manufacturing method described in any one of

[0259] to

[0285] , wherein the antibody is an anti-HER2 antibody.

[0287] The method for producing the anti-HER2 antibody described in

[0286] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0288] The method for producing an anti-HER2 antibody described in

[0286] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0289] A manufacturing method described in any one of

[0259] to

[0285] , wherein the antibody is an anti-HER3 antibody.

[0290] The method for producing an anti-HER3 antibody described in

[0289] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0291] A manufacturing method described in

[0290] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0292] A manufacturing method described in any one of

[0259] to

[0285] , wherein the antibody is an anti-GPR20 antibody.

[0293] The method for producing the anti-GPR20 antibody described in

[0292] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[0294] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 4, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0295] A manufacturing method described in any one of

[0259] to

[0285] , wherein the antibody is an anti-CDH6 antibody.

[0296] The method for producing the anti-CDH6 antibody described in

[0295] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0297] A production method described in

[0296] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0298] A manufacturing method described in any one of

[0259] to

[0297] , characterized in that it does not include a chromatographic purification step.

[0299] The manufacturing method described in

[0298] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0071]

[0300]

[0259] to

[0299] , and further comprising: (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0301] A manufacturing method described in

[0300] , wherein the buffer is a histidine buffer.

[0302] A manufacturing method described in

[0300] or

[0301] , wherein the excipient is sucrose.

[0303] A manufacturing method described in

[0300] or

[0301] , wherein the excipient is trehalose.

[0304]

[0300] To

[0303] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0305] A manufacturing method described in

[0304] , in which the surfactant is polysorbate 80.

[0306] A manufacturing method described in

[0304] , in which the surfactant is polysorbate 20.

[0307] Formula (6)

[0072] [ka]

[0073] (In the formula, the drug linker is bound to the antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) A method for producing an antibody-drug conjugate represented by the following formula: (i) reducing the antibody with a reducing agent; (ii) Formula (2)

[0074] [ka]

[0075] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding a reagent having a thiol group to the solution containing the unpurified or roughly purified antibody-drug conjugate. (iv) removing, by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base, a compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) and a compound in which the reagent having a thiol group used in step (iii) is added to the maleimidyl group of the compound represented by formula (2); The production method according to claim 1, further comprising purifying the product by the steps of:

[0308] The method for producing the compound according to

[0307] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine or a salt thereof.

[0309] The method for producing the compound according to

[0307] , wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride.

[0310] A manufacturing method described in any one of

[0307] to

[0309] , wherein step (i) is carried out in a buffer solution.

[0311] The manufacturing method according to

[0310] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0312] A manufacturing method described in

[0310] or

[0311] , wherein the buffer solution is an acetate buffer solution.

[0313] A method for producing a compound according to any one of claims

[0307] to

[0312] , wherein step (i) is carried out in the presence of a chelating agent.

[0314] The manufacturing method described in

[0313] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0315] A manufacturing method described in any one of

[0310] to

[0314] , wherein the buffer solution used in step (i) contains a surfactant.

[0316] The manufacturing method described in

[0315] , wherein the surfactant is polysorbate 20.

[0317] The manufacturing method described in

[0315] , wherein the surfactant is polysorbate 80.

[0318] A method for producing a compound according to any one of claims

[0307] to

[0317] , wherein the reagent having a thiol group used in step (iii) is N-acetylcysteine.

[0319] A manufacturing method described in any one of items

[0307] to

[0318] , wherein the pH of the buffer used in step (iv) is about 5.

[0076]

[0320] A method for producing according to any one of items

[0307] to

[0318] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0321] A method for producing according to any one of items

[0307] to

[0318] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0322] A method for producing according to any one of items

[0307] to

[0318] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[0323] A manufacturing method described in any one of items

[0307] to

[0318] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0324] A manufacturing method described in any one of

[0307] to

[0323] , wherein the buffer used in step (iv) is a histidine buffer.

[0325] The method for producing a compound according to any one of items

[0307] to

[0324] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0326] A manufacturing method described in any one of items

[0307] to

[0324] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0327] The method for producing a compound according to any one of items

[0307] to

[0324] , wherein the concentration of the salt of a strong acid and a strong base used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0328] A manufacturing method described in any one of items

[0307] to

[0324] , wherein the concentration of the salt consisting of a strong acid and a strong base used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0329] A manufacturing method described in any one of

[0307] to

[0328] , wherein the salt of a strong acid and a strong base used in step (iv) is sodium chloride.

[0330] Following step (iv), (v) removing salts of strong acids and strong bases by ultrafiltration using a buffer solution; A manufacturing method described in any one of

[0307] to

[0329] , comprising:

[0331] The method for producing the present invention described in

[0330] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0332] A manufacturing method described in

[0330] , wherein the pH of the buffer used in step (v) is about 5.

[0333] The method for producing the present invention described in

[0330] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0334] The method for producing the present invention described in

[0330] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0335] The method for producing the present invention described in

[0330] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0336] The manufacturing method described in

[0330] , wherein the pH of the buffer used in step (v) is 5.0.

[0337] A manufacturing method described in any one of

[0330] to

[0336] , wherein the buffer used in step (v) is a histidine buffer.

[0338] A manufacturing method described in any one of

[0307] to

[0337] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0339] A manufacturing method described in any one of

[0307] to

[0337] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0077]

[0340] A manufacturing method described in any one of

[0307] to

[0339] , wherein the antibody is an anti-HER2 antibody.

[0341] The method for producing the anti-HER2 antibody described in

[0340] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0342] A method for producing an anti-HER2 antibody described in

[0340] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0343] A manufacturing method described in any one of

[0307] to

[0339] , wherein the antibody is an anti-HER3 antibody.

[0344] The method for producing an anti-HER3 antibody described in

[0343] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0345] A manufacturing method described in

[0344] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0346] A manufacturing method described in any one of

[0307] to

[0339] , wherein the antibody is an anti-GPR20 antibody.

[0347] The method for producing the anti-GPR20 antibody described in

[0346] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence described in amino acid numbers 20 to 472 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence described in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[0348] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 3, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0349] A manufacturing method described in any one of

[0307] to

[0339] , wherein the antibody is an anti-CDH6 antibody.

[0078]

[0350] The method for producing the anti-CDH6 antibody described in

[0349] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0351] A production method described in

[0350] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0352] A method for producing a compound according to any one of claims

[0307] to

[0351] , characterized in that it does not include a purification step by chromatography.

[0353] The manufacturing method described in

[0352] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0354]

[0307] to

[0353] , an antibody-drug conjugate is produced by the production method according to any one of the above, (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0355] A manufacturing method described in

[0354] , wherein the buffer is a histidine buffer.

[0356] A manufacturing method described in

[0354] or

[0355] , wherein the excipient is sucrose.

[0357] A manufacturing method described in

[0354] or

[0355] , wherein the excipient is trehalose.

[0358]

[0354] To

[0357] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0359] The manufacturing method described in

[0358] , wherein the surfactant is polysorbate 80.

[0360] The manufacturing method described in

[0358] , wherein the surfactant is polysorbate 20.

[0361] Formula (6)

[0079] [ka]

[0080] (In the formula, the drug linker is bound to the antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) A method for producing an antibody-drug conjugate represented by the following formula: (i) reducing the antibody with tris(2-carboxyethyl)phosphine hydrochloride; (ii) Formula (2)

[0081] [ka]

[0082] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding N-acetylcysteine ​​to the solution containing the unpurified or crude antibody-drug conjugate obtained by the above step, (iv) By ultrafiltration using a histidine buffer containing sodium chloride,

[0083] [ka]

[0084] and a compound represented by formula (4)

[0085] [ka]

[0086] A step of removing the compound represented by The production method according to claim 1, further comprising purifying the product by the steps of:

[0362] A manufacturing method described in

[0361] , in which step (i) is carried out in a buffer solution.

[0363] The manufacturing method described in

[0362] , wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

[0364] A manufacturing method described in

[0362] or

[0363] , wherein the buffer solution is an acetate buffer solution.

[0365] A manufacturing method described in any one of

[0361] to

[0364] , wherein step (i) is carried out in the presence of a chelating agent.

[0366] A manufacturing method described in

[0365] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0367] A manufacturing method described in any one of

[0362] to

[0366] , wherein the buffer solution used in step (i) contains a surfactant.

[0368] The manufacturing method described in

[0367] , wherein the surfactant is polysorbate 20.

[0369] The manufacturing method described in

[0367] , wherein the surfactant is polysorbate 80.

[0370] A manufacturing method described in any one of items

[0361] to

[0369] , wherein the pH of the buffer used in step (iv) is about 5.

[0371] A method for producing a product described in any one of items

[0361] to

[0369] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.7 to 5.3.

[0372] A method for producing a product according to any one of items

[0361] to

[0369] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.8 to 5.2.

[0373] A method for producing a product described in any one of items

[0361] to

[0369] , wherein the pH of the buffer solution used in step (iv) is in the range of 4.9 to 5.1.

[0374] A manufacturing method described in any one of items

[0361] to

[0369] , wherein the pH of the buffer solution used in step (iv) is 5.0.

[0375] The method for producing according to any one of items

[0361] to

[0374] , wherein the concentration of sodium chloride used in step (iv) is 0.2 to 1% by weight relative to the buffer solution used in step (iv).

[0376] A manufacturing method described in any one of items

[0361] to

[0374] , wherein the concentration of sodium chloride used in step (iv) is about 0.5% by weight relative to the buffer solution used in step (iv).

[0377] The method for producing according to any one of items

[0361] to

[0374] , wherein the concentration of sodium chloride used in step (iv) is 0.4 to 0.6% by weight relative to the buffer solution used in step (iv).

[0378] A manufacturing method described in any one of items

[0361] to

[0374] , wherein the concentration of sodium chloride used in step (iv) is 0.5% by weight relative to the buffer solution used in step (iv).

[0379] Following step (iv), (v) removing sodium chloride by ultrafiltration using a histidine buffer; A manufacturing method described in any one of

[0361] to

[0378] , comprising:

[0087]

[0380] The method for producing the present invention described in

[0379] , wherein the pH of the buffer solution used in step (v) is in the range of 4 to 6.

[0381] A manufacturing method described in

[0379] , wherein the pH of the buffer used in step (v) is about 5.

[0382] The method for producing the present invention described in

[0379] , wherein the pH of the buffer solution used in step (v) is in the range of 4.7 to 5.3.

[0383] The method for producing the present invention described in

[0379] , wherein the pH of the buffer solution used in step (v) is in the range of 4.8 to 5.2.

[0384] The method for producing the present invention described in

[0379] , wherein the pH of the buffer solution used in step (v) is in the range of 4.9 to 5.1.

[0385] The manufacturing method described in

[0379] , wherein the pH of the buffer used in step (v) is 5.0.

[0386] A manufacturing method described in any one of

[0361] to

[0385] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0387] A manufacturing method described in any one of

[0361] to

[0385] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0388] A manufacturing method described in any one of

[0361] to

[0387] , wherein the antibody is an anti-HER2 antibody.

[0389] The method for producing the anti-HER2 antibody described in

[0388] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[0088]

[0390] The method for producing an anti-HER2 antibody described in

[0388] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0391] A manufacturing method described in any one of

[0361] to

[0387] , wherein the antibody is an anti-HER3 antibody.

[0392] The method for producing an anti-HER3 antibody described in

[0391] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0393] A manufacturing method described in

[0392] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0394] A manufacturing method described in any one of

[0361] to

[0387] , wherein the antibody is an anti-GPR20 antibody.

[0395] The method for producing the anti-GPR20 antibody described in

[0394] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[0396] A method for producing an anti-GPR20 antibody according to any one of claims 1 to 3, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0397] A manufacturing method described in any one of

[0361] to

[0387] , wherein the antibody is an anti-CDH6 antibody.

[0398] The method for producing an anti-CDH6 antibody described in

[0397] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0399] A production method described in

[0398] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0089]

[0400] A manufacturing method described in any one of

[0361] to

[0399] , characterized in that it does not include a chromatographic purification step.

[0401] The manufacturing method described in

[0400] , wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0402]

[0361] to

[0401] , an antibody-drug conjugate is produced by the production method according to any one of the above. (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

[0403] A manufacturing method described in

[0402] , wherein the buffer solution is a histidine buffer solution.

[0404] A manufacturing method described in

[0401] or

[0402] , wherein the excipient is sucrose.

[0405] A manufacturing method described in

[0401] or

[0402] , wherein the excipient is trehalose.

[0406]

[0402] To

[0405] , a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, and an excipient is produced by the production method according to any one of the above. (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

[0407] A manufacturing method described in

[0406] , wherein the surfactant is polysorbate 80.

[0408] A manufacturing method described in

[0406] , wherein the surfactant is polysorbate 20.

[0409] An antibody-drug conjugate produced by the production method described in any one of the items [1] to

[47] ,

[55] to

[95] ,

[0103] to

[0149] ,

[0157] to

[0197] ,

[0205] to

[0251] ,

[0259] to

[0299] ,

[0307] to

[0353] , and

[0361] to

[0401] .

[0410] A pharmaceutical composition manufactured by the manufacturing method described in any one of the following items

[48] to

[54] ,

[96] to

[0102] ,

[0150] to

[0156] ,

[0198] to

[0204] ,

[0252] to

[0258] ,

[0300] to

[0306] ,

[0354] to

[0360] , and

[0402] to

[0408] . Effect of the Invention

[0090] The present invention provides a method for producing an antibody-drug conjugate, which includes a purification step for effectively removing by-products derived from the compound represented by formula (2) while suppressing the formation of aggregates, and further provides an industrially excellent method for producing a pharmaceutical composition containing the antibody-drug conjugate. [Brief description of the drawings]

[0091] [Figure 1] FIG. 1 shows the contents of compound (3) and compound (4) when ultrafiltration was performed using histidine buffer (pH 5.0) or histidine buffer (pH 5.0) containing 0.5% sodium chloride. [Diagram 2] FIG. 1 shows the aggregate content when ultrafiltration was performed using a histidine buffer solution (pH 5.0 or pH 5.8) or a histidine buffer solution (pH 5.0 or pH 5.8) containing 0.4% sodium chloride. [Diagram 3] FIG. 1 shows the amino acid sequence of the anti-HER2 antibody heavy chain (SEQ ID NO:1). [Figure 4] FIG. 2 shows the amino acid sequence of the anti-HER2 antibody light chain (SEQ ID NO:2). [Diagram 5] FIG. 2 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 3). [Figure 6] FIG. 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 4). [Figure 7] FIG. 1 shows the amino acid sequence of the anti-GPR20 antibody heavy chain (SEQ ID NO:5). [Figure 8] FIG. 1 shows the amino acid sequence of the anti-GPR20 antibody light chain (SEQ ID NO: 6). [Figure 9] FIG. 1 shows the amino acid sequence of the anti-CDH6 antibody heavy chain (SEQ ID NO: 7). [Figure 10] FIG. 1 shows the amino acid sequence of the anti-CDH6 antibody light chain (SEQ ID NO: 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. Note that the embodiment described below shows one example of a typical embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly by this embodiment.

[0093] 1. Antibody-drug conjugates The antibody-drug conjugate produced by the present invention comprises: Formula (1)

[0094] [ka]

[0095] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond.

[0096] In the present invention, a partial structure of an antibody-drug conjugate consisting of a linker and a drug is referred to as a "drug linker". This drug linker is bonded to a thiol group (in other words, a sulfur atom of a cysteine ​​residue) generated at the disulfide bond site between antibody chains (two sites between heavy chains and two sites between heavy chains and light chains).

[0097] The drug linker of the antibody-drug conjugates produced by the present invention is comprised of the topoisomerase I inhibitor exatecan. Formula (5)

[0098] [ka]

[0099] It is a camptothecin derivative having an antitumor effect, represented by the following formula:

[0100] The antibody-drug conjugate produced by the present invention comprises: Formula (6)

[0101] [ka]

[0102] It can also be expressed as:

[0103] Here, the drug linker is bound to the antibody via a thioether bond, and n is synonymous with the so-called average drug binding number (DAR; Drug-to-Antibody Ratio), and indicates the average number of drug linkers bound per antibody.

[0104] The antibody-drug conjugate produced by the present invention, after being transferred into cancer cells, Formula (7)

[0105] [ka]

[0106] This liberates a compound represented by the formula:

[0107] The compound represented by formula (7) is considered to be the main component of the antitumor activity of the antibody-drug conjugate produced by the present invention, and has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0108] The compound represented by formula (7) is believed to be generated by cleavage of the linker moiety of the antibody-drug conjugate produced by the present invention, and is represented by formula (8):

[0109] [ka]

[0110] This is believed to occur due to decomposition of the aminal structure of the compound represented by the following formula:

[0111] The antibody-drug conjugate produced by the present invention is also known to have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). This bystander effect is exerted when the antibody-drug conjugate produced by the present invention is internalized into a target-expressing cancer cell, and then the compound represented by formula (7) is released, exerting an antitumor effect on nearby cancer cells that do not express the target.

[0112] 2. Antibodies used in the production of antibody-drug conjugates The antibody used in the preparation of the antibody-drug conjugate of the present invention may be derived from any species, but is preferably derived from human, rat, mouse, and rabbit. If the antibody is derived from a species other than human, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

[0113] The antibody used in the production of the antibody-drug conjugate of the present invention preferably has the property of targeting cancer cells, and preferably has the property of recognizing cancer cells, the property of binding to cancer cells, the property of being taken up and internalized within cancer cells, and / or cytocidal activity against cancer cells.

[0114] The binding of the antibody to the cancer cells can be confirmed by flow cytometry. The uptake of the antibody into the cancer cells can be confirmed by (1) an assay in which the antibody taken up into the cells is visualized by a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which the amount of fluorescence taken up into the cells is measured using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released when the antibody is taken up into the cells, thereby suppressing cell proliferation (Bio Techniques 28:162-165, January 2000). A recombinant complex protein of the catalytic domain of diphtheria toxin and protein G can also be used as the immunotoxin.

[0115] The antitumor activity of an antibody can be confirmed in vitro by measuring the inhibitory activity against cell proliferation. For example, a cancer cell line that overexpresses the antibody's target protein is cultured, and the antibody is added to the culture system at various concentrations to measure the inhibitory activity against focus formation, colony formation, and spheroid proliferation. In vivo, the antitumor activity can be confirmed, for example, by administering the antibody to nude mice transplanted with a cancer cell line that highly expresses the target protein, and measuring the changes in the cancer cells.

[0116] It is preferable that the antibody itself has an antitumor effect, but since the antibody-drug conjugate is bound to a compound that exerts an antitumor effect, the antibody itself does not necessarily have an antitumor effect. For the purpose of exerting the cytotoxicity of the antitumor compound specifically and selectively in cancer cells, it is important and preferable that the antibody has the property of being internalized and transferred into the cancer cells.

[0117] The antibody used for producing the antibody-drug conjugate of the present invention can be obtained by known means. For example, it can be obtained by immunizing an animal with an antigenic polypeptide, and collecting and purifying the antibody produced in the body using a method commonly used in this field. The origin of the antigen is not limited to humans, and an animal can also be immunized with an antigen derived from an animal other than humans, such as a mouse or a rat. In this case, an antibody applicable to a human disease can be selected by testing the cross-reactivity of the obtained antibody that binds to a heterologous antigen with a human antigen.

[0118] Alternatively, a hybridoma can be established by fusing an antibody-producing cell that produces an antibody against an antigen with a myeloma cell according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)), thereby obtaining a monoclonal antibody.

[0119] The antigen can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing an antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0120] The antibody used in the production of the antibody-drug conjugate of the present invention is preferably a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as a chimeric antibody or a humanized antibody, or is preferably an antibody having only the genetic sequence of an antibody derived from humans, i.e., a human antibody. These antibodies can be produced using known methods.

[0121] Chimeric antibodies include antibodies whose variable and constant regions are of different species, such as chimeric antibodies in which the variable region of an antibody derived from a mouse or rat is joined to a constant region derived from a human (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0122] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody has been incorporated into an antibody of human origin (Nature (1986) 321, p. 522-525), antibodies in which not only the CDR sequence of a heterologous antibody but also some framework amino acid residues of the heterologous antibody have been grafted onto a human antibody by CDR grafting (WO 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0123] Examples of human antibodies include antibodies produced using human antibody-producing mice having a human chromosome fragment containing the heavy and light chain genes of a human antibody (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be used (see, for example, Wormstone, IM et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109 (3), p. 427-431).

[0124] The antibody used in the production of the antibody-drug conjugate of the present invention also includes modified antibodies. The modified antibody means an antibody according to the present invention that has been chemically or biologically modified. Chemical modifications include those having a chemical moiety bonded to the amino acid backbone, or a chemical moiety bonded to an N- or O-linked carbohydrate chain. Biological modifications include those that have been post-translationally modified (e.g., addition of an N- or O-linked sugar chain, processing of the N- or C-terminus, deamidation, isomerization of aspartic acid, oxidation of methionine, etc.), and those that have been expressed using a prokaryotic host cell to add a methionine residue to the N-terminus. In addition, those labeled to enable detection or isolation of the antibody or antigen according to the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled, are also included in the meaning of such modified antibodies. Such modified antibodies according to the present invention are useful for improving the stability and blood retention of the antibody, reducing antigenicity, and detecting or isolating the antibody or antigen, etc.

[0125] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by regulating the sugar chain modification bound to the antibody of the present invention (glycosylation, defucosylation, etc.). Techniques for regulating the sugar chain modification of antibodies are known, including, but not limited to, those disclosed in International Publication Nos. 99 / 54342, 00 / 61739, and 02 / 31140. The antibody of the present invention also includes an antibody in which the sugar chain modification has been regulated.

[0126] It is known that antibodies produced in cultured mammalian cells have a deletion of the lysine residue at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)), and also have a deletion of two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain, and amidation of the proline residue located at the carboxyl terminus (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibody according to the present invention also includes antibodies and functional fragments of the antibodies that have been modified in this way, and also includes deletion products in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, and deletion products that have been amidated (for example, heavy chains in which the proline residue at the carboxyl terminus has been amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above deletions, or may be a combination of any two types. The quantitative ratio of each deletion may be influenced by the type and culture conditions of the cultured mammalian cells producing the antibody according to the present invention, but the antibody according to the present invention is preferably one in which one amino acid residue is deleted at the carboxyl terminus of both of the two heavy chains.

[0127] The isotype of the antibody according to the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), etc., and preferably IgG1 or IgG2.

[0128] The antibody that can be used for producing the antibody-drug conjugate of the present invention is not particularly limited, and examples thereof include an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-CD3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD56 antibody, an anti-CD98 antibody, an anti-DR5 antibody, an anti-EGFR antibody, an anti-EPHA2 antibody, an anti-FGFR2 antibody, an anti-FGFR4 antibody, an anti-FOLR1 antibody, an anti-VEGF antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD70 antibody, an anti-PSMA antibody, an anti-CEA antibody, an anti-Mesothelin antibody, Examples of antibodies include anti-A33 antibody, anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, and preferred examples include anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, and more preferred examples include anti-HER2 antibody, anti-HER3 antibody, anti-GPR20 antibody, and anti-CDH6 antibody.

[0129] In the present invention, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2; ErbB-2), and preferably has the activity of being internalized into HER2-expressing cells upon binding to HER2.

[0130] Examples of anti-HER2 antibodies include trastuzumab (US Pat. No. 5,821,337) and pertuzumab (International Publication No. WO 01 / 00245), and preferably trastuzumab.

[0131] In the present invention, the term "anti-HER3 antibody" refers to an antibody that specifically binds to HER3 (Human Epidermal Growth Factor Receptor Type 3; ErbB-3) and preferably has the activity of being internalized into HER3-expressing cells upon binding to HER3.

[0132] Examples of anti-HER3 antibodies include Patritumab (U3-1287), U1-59 (WO 2007 / 077028), MM-121 (Seribantumab), the anti-ERBB3 antibodies described in WO 2008 / 100624, RG-7116 (Lumretuzumab), and LJM-716 (Elgemtumab), and preferred examples include Patritumab and U1-59.

[0133] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1), and preferably has the activity of being internalized into TROP2-expressing cells by binding to TROP2.

[0134] An example of an anti-TROP2 antibody is hTINA1-H1L1 (WO 2015 / 098099).

[0135] In the present invention, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen #7 homolog 3; PD-L3; CD276), and preferably has the activity of being internalized into B7-H3-expressing cells through binding to B7-H3.

[0136] An example of an anti-B7-H3 antibody is M30-H1-L4 (WO 2014 / 057687).

[0137] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (G protein-coupled receptor 20), and preferably has the activity of being internalized into GPR20-expressing cells through binding to GPR20.

[0138] An example of an anti-GPR20 antibody is h046-H4e / L7 (International Publication No. 2018 / 135501).

[0139] In the present invention, the term "anti-CDH6 antibody" refers to an antibody that specifically binds to CDH6 (Cadherin-6), and preferably has the activity of being internalized into CDH6-expressing cells through binding to CDH6.

[0140] An example of an anti-CDH6 antibody is H01L02 (International Publication No. 2018 / 212136).

[0141] 3. Drug-Linker Intermediates Used in the Preparation of Antibody-Drug Conjugates The drug linker intermediate used in the preparation of the antibody-drug conjugate of the present invention is It is a compound represented by formula (2).

[0142] [ka]

[0143] The compound represented by formula (2) can be produced with reference to the descriptions in International Publication No. 2014 / 057687, International Publication No. 2015 / 098099, International Publication No. 2015 / 115091, International Publication No. 2015 / 155998, and International Publication No. 2019 / 044947.

[0144] 4. Conjugation of Antibody and Drug-Linker Intermediate In the production of the antibody-drug conjugate of the present invention, the conjugation of the antibody with the drug linker intermediate is carried out by: (i) reducing the antibody with a reducing agent; (ii) reacting the compound represented by formula (2) with the antibody reduced in step (i); (iii) adding a reagent having a thiol group to react with the compound represented by formula (2) remaining from step (ii); Includes.

[0145] The reducing agent used in step (i) is not particularly limited as long as it can reduce the interchain disulfide of the antibody. For example, tris(2-carboxyethyl)phosphine or a salt thereof, dithiothreitol, or 2-mercaptoethanol can be used. Preferably, tris(2-carboxyethyl)phosphine or a salt thereof can be used. More preferably, tris(2-carboxyethyl)phosphine hydrochloride can be used.

[0146] The equivalent amount of the reducing agent used in step (i) per antibody molecule (hereinafter, in the present invention, "equivalent amount" means molar equivalent amount) can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody.

[0147] For example, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), the reducing agent can be preferably used in an amount of 4.1 to 5.1 equivalents, more preferably 4.4 to 4.8 equivalents, and even more preferably about 4.6 equivalents per antibody molecule. Here, "about 4.6 equivalents" is preferably 4.5 to 4.7 equivalents, and more preferably 4.6 equivalents.

[0148] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), the reducing agent can be preferably used in an amount of 5.5 to 6.5 equivalents, more preferably 5.8 to 6.2 equivalents, and even more preferably about 6 equivalents per antibody molecule. Here, "about 6 equivalents" preferably means 5.9 to 6.1 equivalents, and more preferably 6.0 equivalents.

[0149] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-GPR20 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the reducing agent can be preferably used in an amount of 4.3 to 5.3 equivalents, more preferably 4.6 to 5 equivalents, and even more preferably about 4.8 equivalents per antibody molecule. Here, "about 4.8 equivalents" preferably means 4.7 to 4.9 equivalents, more preferably 4.8 equivalents.

[0150] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-CDH6 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), the reducing agent can be preferably used in an amount of 4.3 to 5.3 equivalents, more preferably 4.6 to 5 equivalents, and even more preferably about 4.8 equivalents per antibody molecule. Here, "about 4.8 equivalents" preferably means 4.7 to 4.9 equivalents, more preferably 4.8 equivalents.

[0151] Step (i) may be preferably carried out in a buffer solution.

[0152] The pH of the buffer used in step (i) is preferably 6 to 8, more preferably 6.5 to 7.5, even more preferably 6.9 to 7.4, and even more preferably 7.0 to 7.3.

[0153] The buffer used in step (i) is not particularly limited as long as it can be used during the reduction of interchain disulfides in an antibody. For example, an acetate buffer, a histidine buffer, a phosphate buffer, a piperazine-1,4-bis(2-ethanesulfonic acid) (hereinafter also referred to as "PIPES") buffer, or a 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (hereinafter also referred to as "HEPES") buffer can be used.

[0154] For example, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), preferably, an acetate buffer adjusted to pH 6 to 8 can be used, more preferably, an acetate buffer adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate can be used, even more preferably, an acetate buffer adjusted to pH 6.8 to 7.8 with an aqueous solution of disodium hydrogen phosphate can be used, and even more preferably, an acetate buffer adjusted to pH about 7.3 with an aqueous solution of disodium hydrogen phosphate can be used. Here, "about 7.3" preferably means a range of 7.1 to 7.5, more preferably a range of 7.2 to 7.4, and even more preferably, 7.3.

[0155] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody), preferably, an acetate buffer adjusted to pH 6 to 8 can be used, more preferably, an acetate buffer adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate can be used, even more preferably, an acetate buffer adjusted to pH 6.5 to 7.5 with an aqueous solution of disodium hydrogen phosphate can be used, and even more preferably, an acetate buffer adjusted to pH about 7 with an aqueous solution of disodium hydrogen phosphate can be used. Here, "about 7" preferably means a range of 6.8 to 7.2, more preferably a range of 6.9 to 7.1, and even more preferably 7.0.

[0156] The use of such a buffer can minimize the formation of aggregates. The buffer used in step (i) may contain a buffer derived from the antibody production.

[0157] Step (i) is preferably carried out in the presence of a chelating agent. The chelating agent is not particularly limited as long as it can be used in reducing interchain disulfides of an antibody, and examples of the chelating agent that can be used include ethylenediaminetetraacetic acid (hereinafter also referred to as "EDTA"), diethylenetriaminepentaacetic acid, and glycoletherdiaminetetraacetic acid, and preferably ethylenediaminetetraacetic acid.

[0158] The chelating agent can be used preferably in an amount of 1 to 20 equivalents per antibody molecule, more preferably in an amount of 3 to 8 equivalents per antibody molecule, even more preferably in an amount of 4 to 6 equivalents per antibody molecule, and even more preferably in an amount of 5 equivalents per antibody molecule.

[0159] The buffer used in step (i) may contain a surfactant. In the present invention, the term "surfactant" refers to a substance that has a hydrophilic group and a hydrophobic group and can be used as one of the components of a pharmaceutical preparation. Examples of such surfactants include polysorbates (including polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and polysorbate 60 (Tween 60)), polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene castor oil, and sodium lauryl sulfate, and more preferably polysorbate 20 or polysorbate 80.

[0160] Whether or not the buffer used in step (i) contains a surfactant, and the type of surfactant can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody.

[0161] For example, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), the buffer used in step (i) preferably does not contain a surfactant.

[0162] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), the buffer used in step (i) contains polysorbate 20 and can be preferably used.

[0163] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-GPR20 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO:6, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), the buffer used in step (i) contains polysorbate 80 and can be preferably used.

[0164] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-CDH6 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), the buffer used in step (i) contains polysorbate 80 and can be preferably used.

[0165] The step (i) can be preferably carried out at an internal temperature of 25 to 50°C.

[0166] When the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), step (i) can be preferably performed at an internal temperature of 30 to 40°C, more preferably at an internal temperature of about 35°C. Here, "about 35°C" is preferably 33°C to 37°C, more preferably 34°C to 36°C, and even more preferably 35°C.

[0167] When the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), step (i) can be preferably carried out at an internal temperature of 30 to 40° C., more preferably at about 35° C. Here, "about 35° C." preferably means 33 to 37° C., more preferably 34 to 36° C., and even more preferably 35° C.

[0168] When the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-GPR20 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), step (i) can be preferably carried out at an internal temperature of 25 to 35°C, more preferably at about 30°C. Here, "about 30°C" is preferably 28°C to 32°C, more preferably 29°C to 31°C, and even more preferably 30°C.

[0169] When the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-CDH6 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain), step (i) can be preferably performed at an internal temperature of 25 to 35° C., more preferably at about 30° C. Here, "about 30° C." preferably means 28° C. to 32° C., more preferably 29° C. to 31° C., and even more preferably 30° C.

[0170] The reaction time in step (i) is preferably 1 to 4 hours.

[0171] The equivalent amount of the compound represented by formula (2) used in step (ii) per antibody molecule can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody.

[0172] For example, when the average number of drug linkers bound per antibody of the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), the compound represented by formula (2) can be preferably used in an amount of 8 to 10 equivalents, more preferably 8.2 to 9.2 equivalents, and even more preferably about 8.7 equivalents per antibody molecule. Here, "about 8.7 equivalents" is preferably 8.5 to 8.9 equivalents, more preferably 8.6 to 8.8 equivalents, and even more preferably 8.7 equivalents.

[0173] In addition, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the compound represented by formula (2) can be used preferably in an amount of 8 to 10 equivalents, more preferably 9 to 10 equivalents, and even more preferably about 9.5 equivalents per antibody molecule. Here, "about 9.5 equivalents" is preferably 9.3 to 9.7 equivalents, more preferably 9.4 to 9.6 equivalents, and even more preferably 9.5 equivalents.

[0174] In addition, when the average number of drug linkers bound per antibody of the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-GPR20 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the compound represented by formula (2) can be preferably used in an amount of 8 to 10 equivalents, more preferably 8.3 to 9.3 equivalents, and even more preferably about 8.8 equivalents per antibody molecule. Here, "about 8.8 equivalents" is preferably 8.6 to 9.0 equivalents, more preferably 8.7 to 8.9 equivalents, and even more preferably 8.8 equivalents.

[0175] In addition, when the average number of drug linkers bound per antibody of the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-CDH6 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the compound represented by formula (2) can be preferably used in an amount of 8 to 10 equivalents, more preferably 8.6 to 9.6 equivalents, and even more preferably about 9.1 equivalents per antibody molecule. Here, "about 9.1 equivalents" is preferably 8.9 to 9.3 equivalents, more preferably 9.0 to 9.2 equivalents, and even more preferably 9.1 equivalents.

[0176] The compound represented by formula (2) can be preferably added to the reaction solution obtained in step (i) in a state dissolved in a solvent. Such a solvent is not particularly limited as long as it can be used in a binding reaction with an antibody, but preferably, dimethyl sulfoxide, an aqueous dimethyl sulfoxide solution, acetone, or an aqueous acetone solution can be used, more preferably, an aqueous dimethyl sulfoxide solution can be used, and even more preferably, an 80% aqueous dimethyl sulfoxide solution can be used. Furthermore, these solvents can be preferably used in a state containing acetic acid. The acetic acid can be preferably used in an amount of 9 to 10 equivalents per antibody molecule.

[0177] The step (ii) can be preferably carried out at an internal temperature of 5 to 25° C., more preferably at an internal temperature of 10 to 20° C., and even more preferably at an internal temperature of about 15° C. Here, "about 15° C." preferably means 13 to 17° C., more preferably 14 to 16° C., and even more preferably 15° C.

[0178] The reaction time in step (ii) is preferably 0.5 to 2 hours.

[0179] The reagent having a thiol group used in step (iii) can be used to react with the compound represented by formula (2) remaining in step (ii) In other words, the reagent having a thiol group used in step (iii) can be used to quench the excess compound represented by formula (2).

[0180] The reagent having a thiol group used in step (iii) is not particularly limited as long as it can react with the maleimidyl group of the compound represented by formula (2). For example, N-acetylcysteine ​​and cysteine ​​can be used, and preferably N-acetylcysteine ​​can be used.

[0181] The thiol group-containing reagent used in step (iii) can be used preferably in an amount of 10 to 50 equivalents, and more preferably 10 to 30 equivalents, per antibody molecule.

[0182] The step (iii) can be preferably carried out at an internal temperature of 5 to 25° C., more preferably at an internal temperature of 10 to 20° C., and even more preferably at an internal temperature of about 15° C. Here, "about 15° C." preferably means 13 to 17° C., more preferably 14 to 16° C., and even more preferably 15° C.

[0183] The reaction time in the step (iii) is preferably 0.5 to 2 hours.

[0184] 5. Purification of Antibody-drug Conjugates The method for producing the antibody-drug conjugate of the present invention includes the steps of: (i) reducing an antibody with a reducing agent; (ii) Formula (2)

[0185] [ka]

[0186] reacting the compound represented by the formula (I) with the antibody reduced in step (i); and then (iii) adding a reagent having a thiol group to the solution containing the unpurified or roughly purified antibody-drug conjugate. (iv) a step of removing a compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) and a compound in which the reagent having a thiol group used in step (iii) is added to the maleimidyl group of the compound represented by formula (2) by ultrafiltration using a buffer solution containing a salt of a strong acid and a strong base. The purification is characterized by the above.

[0187] In the present invention, the term "unpurified or roughly purified antibody-drug conjugate" refers to an antibody-drug conjugate in an unpurified state immediately after steps (i) to (iii) are performed, or an antibody-drug conjugate in a roughly purified state by a procedure other than chromatography (e.g., simple filtration other than ultrafiltration). An example of simple filtration other than ultrafiltration is microfiltration (membrane filter filtration).

[0188] Furthermore, the above-mentioned "unpurified or roughly purified antibody-drug conjugate" may be one whose pH has been adjusted so that the buffer used in step (iv) has an appropriate pH. Such pH adjustment can be preferably performed using an aqueous acetic acid solution, more preferably a 10% aqueous acetic acid solution.

[0189] In the present invention, "ultrafiltration" refers to a purification method for separating large solute molecules from small solute molecules, or solute molecules from solvent molecules, by filtering through a membrane (ultrafiltration membrane) having a pore size ranging from about 0.001 μm to about 0.05 μm. Ultrafiltration membranes generally have a molecular weight cut-off (MWCO) ranging from 1 kDa to 1000 kDa. MWCO is generally defined as the molecular weight of a spherical solute that is generally retained by the membrane at 90%. The ultrafiltration of the present invention can be preferably performed using an ultrafiltration membrane having a MWCO of 1 kDa to 100 kDa, more preferably an ultrafiltration membrane having a MWCO of 30 kDa. Examples of materials for the ultrafiltration membrane include regenerated cellulose, cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyethersulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, nylon, and ceramics. The ultrafiltration of the present invention can be preferably carried out using an ultrafiltration membrane made of regenerated cellulose, but is not limited thereto. Examples of the ultrafiltration membrane used in the present invention include Pellicon (registered trademark) XL Cassette Ultracel (registered trademark) (manufactured by Merck), Pellicon (registered trademark) 2 Ultracel (registered trademark) (manufactured by Merck), and Pellicon (registered trademark) 3 Ultracel (registered trademark) (manufactured by Merck). In the narrow sense, ultrafiltration may mean a method of forced filtration by pressure operation or centrifugation operation, while a method of filtration by passive diffusion may generally be called "diafiltration", but any method using an ultrafiltration membrane is included in the "ultrafiltration" of the present invention.

[0190] In the present invention, "aggregates" refer to fine particles consisting of associations of protein molecules and other optional components that are stable under a wide range of pH and electrical conductivity conditions. The formation of aggregates in antibody-drug conjugate formulations may have undesirable medical effects, such as causing immunogenicity or venous disorders in patients receiving the formulation. Therefore, in formulating antibody-drug conjugates, it is necessary to suppress the formation of aggregates.

[0191] When the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride, the compound in which the reducing agent used in step (i) is added to the maleimidyl group of the compound represented by formula (2) is represented by formula (3):

[0192] [ka]

[0193] It is a compound represented by the formula:

[0194] When the reagent having a thiol group used in the step (iii) is N-acetylcysteine, the compound in which the reagent having a thiol group used in the step (iii) is added to the maleimidyl group of the compound represented by formula (2) is represented by formula (4):

[0195] [ka]

[0196] It is a compound represented by the formula:

[0197] By carrying out step (iv), the compound represented by formula (3) and the compound represented by formula (4) can be preferably removed by 97%, more preferably by 98%, even more preferably by 99%, and even more preferably by 100%, compared to the state before carrying out step (iv).

[0198] The pH of the buffer used in step (iv) is about 5. Here, "about 5" preferably means a range of 4.7 to 5.3, more preferably a range of 4.8 to 5.2, even more preferably a range of 4.9 to 5.1, and even more preferably 5.0. By carrying out ultrafiltration at such a pH, it is possible to suppress the formation of aggregates. The amount of aggregates after this step is preferably 4% or less, more preferably 2% or less. Note that aggregates are contained to a certain extent in the raw antibody itself, and the amount of aggregates detected in this step is the total amount including these.

[0199] Examples of the buffer solution used in step (iv) include a histidine buffer solution containing a salt of a strong acid and a strong base, an acetate buffer solution containing a salt of a strong acid and a strong base, or a phosphate buffer solution containing a salt of a strong acid and a strong base, and preferably a histidine buffer solution containing a salt of a strong acid and a strong base.

[0200] The concentration of the salt consisting of a strong acid and a strong base is preferably 0.2 to 1 wt %, more preferably 0.3 to 0.9 wt %, even more preferably 0.3 to 0.8 wt %, even more preferably 0.4 to 0.7 wt %, and even more preferably about 0.5 wt %, based on the buffer solution used in step (iv). Here, "about 0.5 wt %" is preferably 0.4 to 0.6 wt %, and even more preferably 0.5 wt %.

[0201] The salt of a strong acid and a strong base contained in the buffer solution used in step (iv) is not particularly limited as long as it exhibits the effects of the present invention. For example, it is at least one salt selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, or a combination of two or more salts, and is preferably sodium chloride.

[0202] The production method of the present invention preferably includes, following the step (iv), a step (v) of removing a salt formed from a strong acid and a strong base by ultrafiltration using a buffer solution.

[0203] The pH of the buffer solution used in step (v) is not particularly limited as long as it exhibits the effects of the present invention, but is, for example, in the range of 4 to 6, and preferably about 5. Here, "about 5" preferably means in the range of 4.7 to 5.3, more preferably in the range of 4.8 to 5.2, even more preferably in the range of 4.9 to 5.1, and even more preferably 5.0.

[0204] The buffer used in step (v) is not particularly limited as long as it exhibits the effects of the present invention, but a histidine buffer is preferably used, which does not substantially contain salts of strong acids and strong bases.

[0205] According to the above-mentioned method, by-products derived from the compound represented by formula (2) can be removed by ultrafiltration, and further, the formation of aggregates can be minimized, so that an industrially excellent purification method can be provided that does not require purification by chromatography (e.g., at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography).

[0206] 6. Calculation of the average number of drugs bound to antibody-drug conjugates The average number of drugs bound per antibody molecule in the produced antibody-drug conjugate can be calculated, for example, by a method in which the UV absorbance of the antibody-drug conjugate and its conjugation precursor are measured at two wavelengths, 280 nm and 370 nm (UV method), or by a method in which the antibody-drug conjugate is treated with a reducing agent, and each of the resulting fragments is quantified by HPLC measurement and then calculated (HPLC method).

[0207] The average number of drugs bound per antibody molecule of an antibody-drug conjugate can be calculated with reference to the descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, WO 2018 / 135501, WO 2018 / 212136, and the like.

[0208] The HPLC method can be carried out, for example, as follows. (1) Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates) The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to cleave the disulfide between the chains of the antibody-drug conjugate, and the resulting sample is used for HPLC analysis. (2)HPLC analysis The HPLC analysis can be performed under measurement conditions that suit the properties of each antibody.

[0209] For example, when the antibody is an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2) or an anti-HER3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4), HPLC analysis can be performed under the following measurement conditions.

[0210] HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1 x 50 mm, 8 μm, 1000 Å; Agilent Technologies, P / N PL1912-1802) Column temperature: 80℃ Mobile phase A: 0.04% trifluoroacetic acid (TFA) in water Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program: 29%-36% (0 min-12.5 min), 36%-42% (12.5 min-15 min), 42%-29% (15 min-15.1 min), 29%-29% (15.1 min-25 min) Sample injection volume: 15μL Furthermore, when the antibody is an anti-GPR20 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO:6) or an anti-CDH6 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO:7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO:8), HPLC analysis can be performed under the following measurement conditions.

[0211] HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 x 50 mm, 1.7 μm, 130 Å; Waters, P / N 186002884) Column temperature: 80℃ Mobile phase A: 0.10% trifluoroacetic acid (TFA), 15% 2-propanol in water Mobile phase B: 0.075% TFA, 15% 2-propanol in acetonitrile Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17 min), 80%-14% (17 min-17.01 min), 14% (17.01 min-25 min) Sample injection volume: 10 μL (3) Data analysis The light chains (L 0 ) and heavy chain (H 0 ) and the light chain with one drug bound to it (light chain with one drug bound to it: L 1 ) and heavy chain (heavy chain with one drug bound: H 1 , heavy chain with two drugs bound: H 2 , heavy chain with three drugs bound: H 3 ) increases in hydrophobicity in proportion to the number of bound drugs, and the retention time increases. 0 , L 1 , H 0 , H 1 , H2 , H 3 They are eluted in the order of L 0 and H 0 The detected peaks were identified by comparing the retention times with L 0 , L 1 , H 0 , H 1 , H 2 , H 3 can be assigned to either

[0212] Since the drug linker has UV absorption, the peak area value is corrected according to the molar extinction coefficients of the light chain, heavy chain, and drug linker according to the number of drug linkers bonded, according to the following formula.

[0213]

number

[0214]

number

[0215] Here, the molar extinction coefficient (280 nm) of the light chain and heavy chain of each antibody can be a value estimated from the amino acid sequence of the light chain and heavy chain of each antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423).

[0216] For example, when the antibody is an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2), the molar absorption coefficient of the light chain can be estimated as 26150, and the molar absorption coefficient of the heavy chain can be estimated as 81290.

[0217] Furthermore, when the antibody is an anti-HER3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4), the molar absorption coefficient of the light chain can be estimated as 34,690, and the molar absorption coefficient of the heavy chain can be estimated as 95,000.

[0218] Furthermore, when the antibody is an anti-GPR20 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6), the molar absorption coefficient of the light chain can be estimated as 26210, and the molar absorption coefficient of the heavy chain can be estimated as 68990.

[0219] Furthermore, when the antibody is an anti-CDH6 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8), the molar absorption coefficient of the light chain can be estimated as 31,710, and the molar absorption coefficient of the heavy chain can be estimated as 79,990.

[0220] The molar absorption coefficient (280 nm) of the drug linker can be determined by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine ​​to convert the maleimidyl group to a succinimide thioether, and using the measured molar absorption coefficient (280 nm) of the compound.

[0221] The ratio (%) of each chain peak area to the total corrected peak area is calculated according to the following formula.

[0222]

number

[0223] The average drug binding number in the antibody-drug conjugate is calculated according to the following formula:

[0224] Average number of drug bonds = (L 0 Peak area ratio x 0+L 1 Peak area ratio x 1 + H 0 Peak area ratio x 0+H 1 Peak area ratio x 1 + H 2 Peak area ratio x 2 + H 3 Peak area ratio x 3) / 100 x 2 In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate produced by the present invention, in which the antibody is an anti-HER2 antibody.

[0225] The anti-HER2 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0226] The average number of drug linker bonds per antibody in the anti-HER2 antibody-drug conjugate is preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0227] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate produced by the present invention, in which the antibody is an anti-HER3 antibody.

[0228] The anti-HER3 antibody is preferably an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 26 to 35 of SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 50 to 65 of SEQ ID NO: 3, and CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 98 to 106 of SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 24 to 39 of SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 56 to 62 of SEQ ID NO: 4, and CDRL3 consisting of the amino acid sequence set forth in amino acid numbers 95 to 103 of SEQ ID NO: 4, More preferably, the antibody comprises a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 117 of SEQ ID NO: 3, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 113 of SEQ ID NO: 4, Even more preferably, the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0229] The average number of drug linker bonds per antibody in the anti-HER3 antibody-drug conjugate is preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0230] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate produced by the present invention, in which the antibody is an anti-GPR20 antibody.

[0231] The anti-GPR20 antibody is preferably an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 45 to 54 of SEQ ID NO:5, CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 78 of SEQ ID NO:5, and CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 131 of SEQ ID NO:5, and a light chain comprising CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 of SEQ ID NO:6, CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 of SEQ ID NO:6, and CDRL3 consisting of the amino acid sequence set forth in amino acid numbers 109 to 117 of SEQ ID NO:6, More preferably, the antibody comprises a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 142 of SEQ ID NO:5, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 of SEQ ID NO:6, Even more preferably, the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:6, or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0232] The average number of drug linker bonds per antibody in the anti-GPR20 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0233] In the present invention, the term "anti-CDH6 antibody-drug conjugate" refers to an antibody-drug conjugate produced by the present invention in which the antibody is an anti-CDH6 antibody.

[0234] The anti-CDH6 antibody is preferably an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 45 to 54 of SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 78 of SEQ ID NO: 7, and CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 130 of SEQ ID NO: 7, and a light chain comprising CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 of SEQ ID NO: 8, CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 of SEQ ID NO: 8, and CDRL3 consisting of the amino acid sequence set forth in amino acid numbers 109 to 116 of SEQ ID NO: 8, More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 of SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 128 of SEQ ID NO: 8, Even more preferably, the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8, or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0235] The average number of drug linker bonds per antibody in the anti-CDH6 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0236] 7. Preparation of pharmaceutical compositions The pharmaceutical composition of the present invention is a pharmaceutical composition comprising an antibody-drug conjugate of the present invention, a buffer, and an excipient.

[0237] Such pharmaceutical compositions further comprise an antibody-drug conjugate produced (including purification) by the above-mentioned method, (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; The present invention can be produced by carrying out a process including the steps of:

[0238] The buffer added in step (vi) is preferably the same as the buffer used in step (v), and therefore, the buffer used in step (vi) can preferably be a histidine buffer.

[0239] The adjustment of pH in step (viii) can be preferably carried out using an aqueous histidine solution when the buffer used in step (vi) is a histidine buffer.

[0240] In the present invention, the term "excipient" refers to a substance added to make a pharmaceutical product have a certain size or concentration in order to improve the convenience of molding, handling, and taking the drug, etc. Such excipients are not particularly limited as long as they exhibit the effects of the present invention, and examples thereof include sucrose, trehalose, and sorbitol.

[0241] As the excipient added in step (ix), sucrose can be suitably used.

[0242] Furthermore, the pharmaceutical composition of the present invention preferably further comprises a surfactant, i.e., the pharmaceutical composition of the present invention is more preferably a pharmaceutical composition comprising an antibody-drug conjugate, a buffer solution, an excipient, and a surfactant.

[0243] Such a pharmaceutical composition can be produced by carrying out a step including the step of (x) adding a surfactant after the above step (ix).

[0244] As the surfactant added in step (x), polysorbate 80 or polysorbate 20 can be suitably used.

[0245] The concentrations of the buffer solution, excipient, surfactant, and antibody-drug conjugate in the pharmaceutical composition, as well as the pH of the pharmaceutical composition, can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody.

[0246] For example, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), preferably, histidine buffer (preferably 25 mM histidine buffer), sucrose (preferably 9% sucrose), and polysorbate 80 (preferably 0.03% polysorbate 80) can be used as the buffer, excipient, and surfactant in the pharmaceutical composition, the concentration of the antibody-drug conjugate in the pharmaceutical composition is preferably 20 mg / mL, and the pH of the pharmaceutical composition is preferably 5.5.

[0247] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain is deleted), preferably, histidine buffer (preferably 25 mM histidine buffer), sucrose (preferably 9% sucrose), and polysorbate 20 (preferably 0.03% polysorbate 20) can be used as the buffer, excipient, and surfactant in the pharmaceutical composition, the concentration of the antibody-drug conjugate in the pharmaceutical composition is preferably 20 mg / mL, and the pH of the pharmaceutical composition is preferably 5.4.

[0248] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate produced is in the range of 7 to 8, and the antibody is an anti-GPR20 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), preferably, histidine buffer (preferably 10 mM histidine buffer), sucrose (preferably 9% sucrose), and polysorbate 80 (preferably 0.03% polysorbate 80) can be used as the buffer, excipient, and surfactant in the pharmaceutical composition, the concentration of the antibody-drug conjugate in the pharmaceutical composition is preferably 20 mg / mL, and the pH of the pharmaceutical composition is preferably 5.4.

[0249] Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-CDH6 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), preferably, histidine buffer (preferably 10 mM histidine buffer), sucrose (preferably 9% sucrose), and polysorbate 80 (preferably 0.03% polysorbate 80) can be used as the buffer, excipient, and surfactant in the pharmaceutical composition, the concentration of the antibody-drug conjugate in the pharmaceutical composition is preferably 20 mg / mL, and the pH of the pharmaceutical composition is preferably 5.4.

[0250] 8. Use of the pharmaceutical composition The pharmaceutical composition of the present invention can be applied to patients as a systemic therapy, and can also be applied locally to cancer tissues to be expected to have a therapeutic effect.

[0251] The pharmaceutical composition of the present invention can be preferably used for mammals, and more preferably for humans.

[0252] Examples of introduction routes that can be used to administer the pharmaceutical composition of the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, with intravenous being preferred.

[0253] When the pharmaceutical composition of the present invention is an aqueous injection, it can be preferably administered intravenously after diluting with a suitable diluent, such as a glucose solution or saline, preferably a glucose solution, more preferably a 5% glucose solution.

[0254] When the pharmaceutical composition of the present invention is a lyophilized injection, it is preferably dissolved in water for injection, and then diluted with a required amount of an appropriate diluent, and then administered by intravenous drip infusion. The diluent may be a glucose solution, a physiological saline solution, etc., preferably a glucose solution, and more preferably a 5% glucose solution.

[0255] In terms of the affinity of the antibody-drug conjugate of the present invention to the antigen, that is, the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value), the more effective the pharmaceutical composition of the present invention can be even at a smaller dose. Therefore, the dose of the pharmaceutical composition of the present invention can also be set based on the affinity with the antigen. When the pharmaceutical composition of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg (here, "mg / kg" means the dose of the antibody-drug conjugate per kg of human body weight) of the antibody-drug conjugate may be administered once or multiple times at intervals of once every 1 to 180 days, and preferably, 0.8 mg / kg to 8 mg / kg may be administered once every three weeks.

[0256] The dosage and administration interval of the antibody-drug conjugate can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody. For example, when the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced is in the range of 7 to 8, and the antibody is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2), the dosage and administration interval of the antibody-drug conjugate can be appropriately selected depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate to be produced and the type of antibody. A preferred embodiment of the present invention is a method of administering 1.6 mg / kg, 3.2 mg / kg, 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, or 8 mg / kg of the antibody-drug conjugate once every three weeks, more preferably a method of administering 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, or 8 mg / kg of the antibody-drug conjugate once every three weeks, and even more preferably a method of administering 5.4 mg / kg or 6.4 mg / kg of the antibody-drug conjugate once every three weeks. Furthermore, when the average number of drug linkers bound per antibody in the antibody-drug conjugate produced is in the range of 7 to 8, and the antibody is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody in which a lysine residue is deleted at the carboxyl terminus of the heavy chain of the antibody), preferred methods include a method of administering 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 5.6 mg / kg, 6.4 mg / kg, 8.0 mg / kg, 9.6 mg / kg, or 12.8 mg / kg of the antibody-drug conjugate once every three weeks, and more preferred methods include a method of administering 4.8 mg / kg, 5.6 mg / kg, or 6.4 mg / kg of the antibody-drug conjugate once every three weeks.

[0257] The pharmaceutical composition of the present invention can be used for the treatment of cancer, and can be preferably used for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer (sometimes referred to as gastric adenocarcinoma), colon cancer (sometimes referred to as colorectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), gastroesophageal junction adenocarcinoma, biliary tract cancer (including bile duct cancer), Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, and melanoma.

[0258] The pharmaceutical composition of the present invention can be selected and used as a drug for drug therapy, which is the main method of cancer treatment, and as a result, it can delay the growth of cancer cells, suppress their proliferation, and even destroy them. These actions can relieve cancer patients from symptoms caused by cancer and improve their QOL, and the therapeutic effect can be achieved while preserving the life of the cancer patient. Even if the destruction of cancer cells is not achieved, the inhibition and control of the proliferation of cancer cells can help cancer patients achieve a higher QOL and a longer life.

[0259] In addition to the use of the drug alone in such drug therapy, the pharmaceutical composition of the present invention can also be used in adjuvant therapy in combination with other therapies, such as surgery, radiation therapy, hormone therapy, etc. Furthermore, it can also be used as drug therapy in neoadjuvant therapy.

[0260] In addition to the above therapeutic uses, the pharmaceutical composition of the present invention can be expected to have a preventive effect of suppressing and even destroying the proliferation of minute metastatic cancer cells. For example, it can be expected to have an effect of suppressing and destroying cancer cells in body fluids during the metastasis process, and an effect of suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it can be expected to have an effect of suppressing and preventing cancer metastasis, especially after surgical removal of cancer.

[0261] The pharmaceutical composition of the present invention can be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. The other cancer therapeutic agents used for such a purpose may be administered to an individual simultaneously with the pharmaceutical composition of the present invention, separately, or consecutively, or may be administered at different administration intervals.Such cancer therapeutic agents are not limited as long as they have antitumor activity, and examples thereof include irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, paclitaxel, docetaxel, doxorubicin, epirubicin, cyclophosphamide, mitomycin C, and the like. C), Tegafur-Gimeracil-Oteracil combination drug, Cetuximab, Panitumumab, Bevacizumab, Ramucirumab, Regorafenib, Trifluridine-Tipiracil combination drug, Gefitinib, Erlotinib, Afatinib, Methotrexate, Pemetrexed, Trastuzumab emtansine At least one selected from the group consisting of emtansin, trastuzumab, pertuzumab, tamoxifen, toremifene, fulvestrant, leuprorelin, goserelin, letrozole, anastrozole, and progesterone formulations may be mentioned. EXAMPLES

[0262] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0263] [Example 1] Examination of buffer solutions A solution containing an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2) was diluted with various buffer solutions (10 mM acetate buffer, 10 mM phosphate buffer, or 20 mM PIPES buffer), and a 0.5 mol / L EDTA aqueous solution was added, followed by addition of various weak alkaline aqueous solutions (0.3 mol / L disodium hydrogen phosphate aqueous solution (hereinafter referred to as "aq.Na 2 HPO 4 "), saturated aqueous sodium acetate solution (hereafter referred to as "aq.CH 3 COONa), or 0.5 mol / L sodium bicarbonate solution (hereafter referred to as aq.NaHCO 3 ") was added and the pH was adjusted to 7. An aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (hereinafter also referred to as "TCEP") (1 mg / mL) was added in an amount of 4.8 equivalents or 4.9 equivalents per antibody molecule with stirring at 37°C, and the interchain disulfides of the antibody were reduced.

[0264] The resulting reaction solution was stirred at an internal temperature of 15° C. or 25° C. while adding 9.2 or 9.8 equivalents of a compound represented by the formula (2) per antibody molecule to the antibody.

[0265] [ka]

[0266] A compound represented by the formula (also referred to as compound (2)) was dissolved in an 80% aqueous dimethyl sulfoxide solution and added to the antibody, and allowed to bind to the antibody. Next, a 50 mmol / L aqueous solution of N-acetylcysteine ​​was added to quench the excess compound (2). This resulted in the formation of a compound represented by the formula

[0267] [ka]

[0268] (wherein A represents the binding site to the antibody) An anti-HER2 antibody-drug conjugate was obtained in which the drug linker represented by the formula: The DAR of the obtained anti-HER2 antibody-drug conjugate was measured by HPLC, and the aggregate content was measured by SEC.

[0269] The results are shown in Table 1.

[0270] [Table 1]

[0271] Among the results in Table 1, the lowest aggregate content was observed when an acetate buffer solution whose pH had been adjusted with an aqueous solution of disodium hydrogen phosphate was used.

[0272] [Example 2] Study of ultrafiltration process (1) A solution containing an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2) was diluted with 10 mM acetate buffer (pH 5.5), and a 0.5 mol / L aqueous EDTA solution (5 equivalents per antibody molecule) was added, followed by the addition of a 0.3 mol / L aqueous disodium hydrogen phosphate solution to adjust the pH to 7.4.

[0273] A 0.010 mol / L aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (6 equivalents per antibody molecule) was added at 35 to 39°C with stirring, and the mixture was stirred at an internal temperature of 35 to 39°C for 2.5 hours to reduce the interchain disulfide of the antibody.

[0274] The resulting reaction solution was stirred at an internal temperature of 13 to 17°C, and compound (2) (12.5 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution was added to bind to the antibody. Next, a 0.1 mol / L N-acetylcysteine ​​aqueous solution (35 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 1 hour to quench excess compound (2). The reaction solution was then adjusted to pH 5.0 using a 10% acetic acid aqueous solution. This resulted in the formation of the compound (2) of the formula

[0275] [ka]

[0276] (wherein A represents the binding site to the antibody) A reaction solution containing an anti-HER2 antibody-drug conjugate in which the drug linker represented by the formula:

[0277] The obtained reaction solution was circulated by adding 26 mM histidine buffer (pH 5.0) or 26 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride using an ultrafiltration membrane Pellicon (registered trademark) XL Cassette Ultracel (registered trademark) (manufactured by Merck) with a roller pump, and ultrafiltration was performed. 26 mM histidine buffer (pH 5.0) and 26 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride were used in amounts of 5 times, 10 times, or 15 times the amount of the obtained reaction solution, respectively, and the content of by-products derived from compound (2) relative to the antibody-drug conjugate after ultrafiltration was measured. In addition, for comparison, the content of by-products derived from compound (2) in the case where ultrafiltration was not performed was measured (in such cases, the amount of buffer used was expressed as 0).

[0278] Here, the by-product derived from compound (2) is a compound in which tris(2-carboxyethyl)phosphine is added to the maleimidyl group of compound (2), i.e., Formula (3)

[0279] [ka]

[0280] (also referred to as compound (3)) and a compound in which N-acetylcysteine ​​is added to the maleimidyl group of compound (2), i.e., Formula (4)

[0281] [ka]

[0282] The content of the by-product derived from compound (2) was measured by measuring the content of each of compound (3) and compound (4) relative to the antibody-drug conjugate by HPLC. The results are shown in Table 2 and FIG. 1.

[0283] [Table 2]

[0284] The results in Table 2 show that, when compared at the same buffer volume / reaction volume, the contents of compound (3) and compound (4) were lower when the sodium chloride-containing histidine buffer was used than when the histidine buffer was used.

[0285] [Example 3] Study of ultrafiltration process (2) A solution containing an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2) was diluted with 10 mM acetate buffer, and a 0.5 mol / L aqueous solution of EDTA was added, followed by addition of a 0.3 mol / L aqueous solution of disodium hydrogen phosphate to adjust the pH to 7.3. A 1 mg / mL aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride was added and stirred to reduce the interchain disulfide of the antibody.

[0286] Compound (2) was added to the reaction solution in a state of being dissolved in 80% dimethyl sulfoxide aqueous solution, and bound to the antibody. Next, a 50 mmol / L N-acetylcysteine ​​aqueous solution was added to quench the excess compound (2). This resulted in the formation of the compound represented by the formula

[0287] [ka]

[0288] (wherein A represents the binding site to the antibody) A reaction solution containing an anti-HER2 antibody-drug conjugate in which the drug linker represented by the formula:

[0289] The obtained reaction solution was circulated by using a Pellicon (registered trademark) XL Cassette Ultracel (registered trademark) (manufactured by Merck) with a roller pump while adding histidine buffer (pH 5.0 or pH 5.8) or 0.4% sodium chloride-containing histidine buffer (pH 5.0 or pH 5.8), and ultrafiltration was performed. The histidine buffer had a concentration of 10.8 mM. The histidine buffer (pH 5.0 and pH 5.8) was used in an amount 15 times that of the obtained reaction solution. The histidine buffer (pH 5.0 and pH 5.8) containing 0.4% sodium chloride was used in an amount 10 times that of the obtained reaction solution. The content of aggregates after ultrafiltration was measured by SEC. For comparison, the content of aggregates was measured when ultrafiltration was not performed (in such cases, the amount of buffer used was expressed as 0). The results are shown in Table 3 and FIG. 2.

[0290] [Table 3]

[0291] In the results in Table 3, when ultrafiltration was performed using a buffer solution of pH 5.8, the aggregate content was higher when a histidine buffer solution containing sodium chloride was used than when a histidine buffer solution was used. On the other hand, when ultrafiltration was performed using a buffer solution of pH 5.0, the aggregate content was approximately the same whether a histidine buffer solution or a histidine buffer solution containing sodium chloride was used.

[0292] [Example 4] Preparation of pharmaceutical composition containing anti-HER2 antibody-drug conjugate (1) A solution (5.4 kg liquid weight: equivalent to 120 g antibody) containing an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in amino acid numbers 1 to 214 in SEQ ID NO: 2) was placed in a glass reaction vessel, and 0.01 mol / L acetate buffer (4.3 L, pH 5.5) was further added. A 0.5 mol / L EDTA aqueous solution (8.3 mL; 5 equivalents to the antibody) was added to this solution, and then a 0.3 mol / L disodium hydrogen phosphate aqueous solution was added to adjust the pH to 7.3. A 0.010 mol / L tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (390 mL; 4.7 equivalents to one antibody molecule) was added under stirring at 35 to 39°C, and the mixture was stirred for 2 hours at an internal temperature of 35 to 39°C to reduce the interchain disulfide of the antibody.

[0293] The resulting reaction solution was cooled, and while stirring at an internal temperature of 13 to 17°C, compound (2) (8.2 g; 9.7 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (900 mL) was added over 20 minutes, and the mixture was stirred at the same temperature for 1 hour to bind to the antibody. Next, a 0.1 mol / L N-acetylcysteine ​​aqueous solution (210 mL; 25 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 1 hour to quench the excess compound (2), and the pH was adjusted to 5.0 using a 10% acetic acid aqueous solution. As a result, the compound of the formula

[0294] [ka]

[0295] (wherein A represents the binding site to the antibody) A solution containing an anti-HER2 antibody-drug conjugate in which the drug linker represented by the formula:

[0296] The resulting solution was placed in a Pellicon (registered trademark) 2 Mini Cassette Ultracel (registered trademark) (Merck, 0.1 ml 2 ) was used, and ultrafiltration was performed by circulating the solution while adding 26 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride using a roller pump, to remove by-products derived from compound (2). Furthermore, ultrafiltration was performed by circulating the solution while adding 26 mM histidine buffer (pH 5.0) to remove sodium chloride. Next, the solution was adjusted to pH 5.5 with an aqueous histidine solution, and further concentrated to obtain about 4 L of a solution containing an anti-HER2 antibody-drug conjugate.

[0297] Furthermore, 364 g of sucrose was added to 3.86 kg of the above solution and dissolved. Further, 9% sucrose-containing histidine buffer (pH 5.5) was added to adjust the protein concentration to about 20 mg / mL, and a pharmaceutical composition (5.7 kg) containing an anti-HER2 antibody-drug conjugate was obtained. The obtained pharmaceutical composition had a protein concentration of 20.4 mg / mL, a protein yield of 112 g, and an average number of drugs bound per antibody molecule (n): 7.7.

[0298] [Example 5] Preparation of pharmaceutical composition containing anti-HER2 antibody-drug conjugate (2) A solution (89 g liquid weight: equivalent to 2.0 g antibody) containing an anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214) was placed in a glass reaction vessel, and 0.01 mol / L acetate buffer (72 mL, pH 5.5) was further added. After adding a 0.5 mol / L EDTA aqueous solution (0.14 mL; 5 equivalents to the antibody) to this solution, a 0.3 mol / L disodium hydrogen phosphate aqueous solution was added to adjust the pH to 7.3. A 0.010 mol / L tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (6.3 mL; 4.6 equivalents to one antibody molecule) was added under stirring at 35 to 39°C, and the mixture was stirred for 2 hours at an internal temperature of 35 to 39°C to reduce the interchain disulfide of the antibody.

[0299] The resulting reaction solution was cooled, and while stirring at an internal temperature of 13 to 17°C, compound (2) (140 mg; 9.6 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (13 mL) was added, and the mixture was stirred at the same temperature for 1 hour to bind to the antibody. Next, a 0.1 mol / L N-acetylcysteine ​​aqueous solution (3.4 mL; 25 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 40 minutes to quench the excess compound (2), and the pH was adjusted to 5.0 using a 10% acetic acid aqueous solution. As a result, the compound of the formula

[0300] [ka]

[0301] (wherein A represents the binding site to the antibody) A solution containing an anti-HER2 antibody-drug conjugate in which the drug linker represented by the formula:

[0302] 1 g of sodium chloride was added to the resulting solution, and then the solution was centrifuged using a Pellicon® XL Ultracel® (Merck, 50 cm 2) was used, and ultrafiltration was performed by circulating the solution while adding 26 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride using a roller pump, to remove by-products derived from compound (2). Furthermore, ultrafiltration was performed by circulating the solution while adding 26 mM histidine buffer (pH 5.0) to remove sodium chloride. Next, the solution was concentrated while adjusting the pH to 5.5 with an aqueous histidine solution, to obtain about 65 g (64.5 g, 64.1 mL, protein concentration: 28.5 mg / mL, protein yield: 1.83 g) of a solution containing an anti-HER2 antibody-drug conjugate.

[0303] Further, 19 mL of histidine buffer (pH 5.5) containing 7.7 g of sucrose was added to 64 g of the above solution, and then 9% sucrose-containing histidine buffer (pH 5.5) was added to adjust the protein concentration to about 20 mg / mL, to obtain a pharmaceutical composition (93 g) containing an anti-HER2 antibody-drug conjugate. The obtained pharmaceutical composition had a protein concentration of 20.3 mg / mL, a protein yield of 1.8 g, and an average number of drugs bound per antibody molecule (n): 7.8.

[0304] [Example 6] Preparation of pharmaceutical composition containing anti-HER3 antibody-drug conjugate A solution (liquid weight 43.95 kg: equivalent to 3.00 kg of antibody) containing an anti-HER3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4) was placed in a 400 L single-use reactor, and 0.01 mol / L acetate buffer (pH 5.5, 255 kg) containing polysorbate 20 (60.0 g) and 0.5 mol / L EDTA aqueous solution (224.5 g; 5 equivalents to the antibody) was further added. A 0.3 mol / L aqueous disodium hydrogen phosphate solution was added to this solution to adjust the pH to 7.0. An aqueous solution (12.3 kg) containing tris(2-carboxyethyl)phosphine hydrochloride (35.1 g; 6.0 equivalents to one antibody molecule) was added under stirring at 33 to 37 °C, and the mixture was stirred at an internal temperature of 33 to 37 °C for 2.5 hours to reduce the interchain disulfide of the antibody.

[0305] The resulting reaction solution was cooled, and while stirring at an internal temperature of 12 to 17°C, compound (2) (200.5 g; 9.5 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (21.1 kg) containing 10% acetic acid aqueous solution (116.4 g) was added over 50 minutes, and the mixture was stirred at the same temperature for 0.5 hours to bind to the antibody. Next, a 0.1 mol / L N-acetylcysteine ​​aqueous solution (3.11 kg; 15 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 0.5 hours to quench the excess compound (2), and the pH was adjusted to 5.0 using a 10% acetic acid aqueous solution. As a result, the compound (2) of the formula

[0306] [ka]

[0307] (wherein A represents the binding site to the antibody) A solution containing an anti-HER3 antibody-drug conjugate in which the drug linker represented by the formula: was bound to the anti-HER3 antibody via a thioether bond was obtained.

[0308] The resulting solution was then transferred to a Pellicon® 2 Ultracel® (Merck, 2.5 ml 2 ) was used, and ultrafiltration was performed by circulating the solution while adding 26.5 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride in an automatic ultrafiltration device, to remove by-products derived from compound (2). Furthermore, ultrafiltration was performed by circulating the solution while adding 26.5 mM histidine buffer (pH 5.0) to remove sodium chloride. Next, concentration was performed while adjusting the pH to 5.4 with 26.5 mM histidine aqueous solution, and 97.4 kg (96.5 L, protein concentration: 30.5 mg / mL, protein yield: 2.94 kg) of a solution containing an anti-HER3 antibody-drug conjugate was obtained.

[0309] Further, to 48.6 kg of the above solution, histidine buffer solution (pH 5.4, 24.3 kg) containing 6.29 kg of sucrose was added, and further 9% sucrose-containing histidine buffer solution (pH 5.4, 4.25 kg) containing polysorbate 20 (7.6 g) was added to adjust the protein concentration to about 20 mg / mL, and a pharmaceutical composition (77.1 kg) containing an anti-HER3 antibody-drug conjugate was obtained. The obtained pharmaceutical composition had a protein concentration of 19.9 mg / mL, a protein yield of 1.49 kg, and an average number of drugs bound per antibody molecule (n): 7.6.

[0310] [Example 7] Preparation of pharmaceutical composition containing anti-GPR20 antibody-drug conjugate A solution (liquid weight 24.2 kg: equivalent to 0.94 kg of antibody) containing an anti-GPR20 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO:6) was placed in a 200 L reactor, and a 26 mM histidine aqueous solution (57.6 kg) containing polysorbate 80 (14.4 g) was further added. The temperature was raised to 30°C, and an aqueous solution (3.17 kg) containing tris(2-carboxyethyl)phosphine hydrochloride (9.06 g; 4.9 equivalents per antibody molecule) was added, and the mixture was stirred at an internal temperature of 30°C for 3 hours to reduce the interchain disulfide of the antibody.

[0311] The resulting reaction solution was cooled, and while stirring at an internal temperature of 15°C, compound (2) (63.5 g; 9.0 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (7.26 kg) containing acetic acid (3.7 g) was added over 55 minutes, and the mixture was stirred at the same temperature for 0.5 hours to bind to the antibody. Next, a 0.1 M N-acetylcysteine ​​aqueous solution (1.00 kg; 15 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 0.5 hours to quench the excess compound (2), and the pH was adjusted to 5.0 using a 10% acetic acid aqueous solution. This resulted in the formation of the compound of the formula

[0312] [ka]

[0313] (wherein A represents the binding site to the antibody) A solution containing an anti-GPR20 antibody-drug conjugate in which the drug linker represented by the formula: was bound to the anti-GPR20 antibody via a thioether bond was obtained.

[0314] The resulting solution was then transferred to a Pellicon® 2 Ultracel® (Merck, 2.5 ml 2 ) was used, and ultrafiltration was performed by circulating the solution while adding 11 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride in an ultrafiltration device, to remove by-products derived from compound (2). Ultrafiltration was further performed by circulating the solution while adding 11 mM histidine buffer (pH 5.0) to remove sodium chloride. Next, concentration was performed while adjusting the pH to 5.4 with an 11 mM histidine aqueous solution, to obtain a solution containing an anti-GPR20 antibody-drug conjugate (31.8 kg, protein concentration: 29.9 mg / mL, protein yield: 0.94 kg).

[0315] Further, to the above solution, histidine buffer (pH 5.4, 14.9 kg) containing 4.00 kg of sucrose was added, and further 9% sucrose-containing histidine buffer (pH 5.4, 3.00 kg) containing polysorbate 80 (4.5 g) was added to adjust the protein concentration to about 20 mg / mL, and a pharmaceutical composition (49.3 kg) containing an anti-GPR20 antibody-drug conjugate was obtained. The obtained pharmaceutical composition had a protein concentration of 19.9 mg / mL, a protein yield of 0.94 kg, and an average number of drugs bound per antibody molecule (n): 7.8.

[0316] [Example 8] Preparation of pharmaceutical composition containing anti-CDH6 antibody-drug conjugate A solution (2.56 kg liquid weight: equivalent to 100 g antibody) containing an anti-CDH6 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, amino acid numbers 20 to 471, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 8, amino acid numbers 21 to 233) was placed in a 14 L reactor, and a 100 mM histidine aqueous solution (6.00 kg) containing polysorbate 80 (1.50 g) and 0.5 M EDTA aqueous solution (7.54 g; 5 equivalents to the antibody) was added. The temperature was raised to 30°C, and an aqueous solution (330 g) containing tris(2-carboxyethyl)phosphine hydrochloride (945 mg; 4.8 equivalents to one antibody molecule) was added, and the mixture was stirred at an internal temperature of 30°C for 3 hours to reduce the interchain disulfide of the antibody.

[0317] The resulting reaction solution was cooled, and while stirring at an internal temperature of 15°C, compound (2) (6.96 g; 9.1 equivalents per antibody molecule) dissolved in 80% dimethyl sulfoxide aqueous solution (687 g) containing 10% acetic acid aqueous solution (3.89 g) was added over 41 minutes, and stirred at the same temperature for 0.4 hours to bind to the antibody. Next, a 0.1 M N-acetylcysteine ​​aqueous solution (103 g; 15 equivalents per antibody molecule) was added, and the mixture was further stirred at the same temperature for 0.6 hours to quench excess compound (2), and the pH was adjusted to 5.0 using a 10% acetic acid aqueous solution. This resulted in the formation of the compound (2) of the formula

[0318] [ka]

[0319] (wherein A represents the binding site to the antibody) A solution containing an anti-CDH6 antibody-drug conjugate in which the drug linker represented by the formula (I) was bound to the anti-CDH6 antibody via a thioether bond was obtained.

[0320] The resulting solution was then transferred to a Pellicon® 2 Ultracel® (Merck, 0.5 ml 2) was used, and ultrafiltration was performed by circulating the solution while adding 11 mM histidine buffer (pH 5.0) containing 0.5% sodium chloride in an ultrafiltration device, to remove by-products derived from compound (2). Ultrafiltration was further performed by circulating the solution while adding 11 mM histidine buffer (pH 5.0) to remove sodium chloride. Next, concentration was performed while adjusting the pH to 5.4 with an 11 mM histidine aqueous solution, to obtain a solution containing an anti-CDH6 antibody-drug conjugate (2.97 kg, protein concentration: 31.8 mg / mL, protein yield: 93.7 g).

[0321] Further, to the above solution, histidine buffer (pH 5.4, 1.67 kg) containing sucrose (401 g) was added, and then 9% sucrose-containing histidine buffer (pH 5.4, 276 g) containing polysorbate 80 (437 mg) was added to adjust the protein concentration to about 20 mg / mL, and a pharmaceutical composition (4.81 kg) containing an anti-CDH6 antibody-drug conjugate was obtained. The obtained pharmaceutical composition had a protein concentration of 20.2 mg / mL, a protein yield of 93.2 g, and an average number of drugs bound per antibody molecule (n): 7.8. [Sequence List Free Text]

[0322] SEQ ID NO: 1: Amino acid sequence of the anti-HER2 antibody heavy chain SEQ ID NO: 2: Amino acid sequence of the anti-HER2 antibody light chain SEQ ID NO: 3: Amino acid sequence of the anti-HER3 antibody heavy chain SEQ ID NO: 4: Amino acid sequence of the anti-HER3 antibody light chain SEQ ID NO: 5: Amino acid sequence of the anti-GPR20 antibody heavy chain SEQ ID NO: 6: Amino acid sequence of the anti-GPR20 antibody light chain SEQ ID NO: 7: Amino acid sequence of the anti-CDH6 antibody heavy chain SEQ ID NO: 8: Amino acid sequence of the anti-CDH6 antibody light chain

Claims

1. Formula (1) 【Chemistry 1】 (In the formula, A represents the binding site with the antibody.) and an antibody via a thioether bond, the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8; (i) reducing the antibody with tris(2-carboxyethyl)phosphine or a salt thereof; (ii) Formula (2) 【Chemistry 2】 reacting a compound represented by the formula: with the antibody reduced in step (i); (iii) adding N-acetylcysteine ​​or cysteine ​​to react with the compound represented by formula (2) remaining from step (ii); and then (iv) ultrafiltration using a buffer containing at least one salt or a combination of two or more salts selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate; Formula (3) 【Chemistry 3】 A compound represented by the formula: Formula (4) 【Chemistry 4】 A step of removing the compound represented by A manufacturing method comprising the steps of:

2. The process according to claim 1, wherein the reducing agent used in step (i) is tris(2-carboxyethyl)phosphine hydrochloride.

3. The method according to claim 1 or 2, wherein step (i) is carried out in a buffer solution.

4. The method according to claim 3, wherein the buffer solution is a buffer solution adjusted to pH 6 to 8 with an aqueous solution of disodium hydrogen phosphate.

5. The method according to claim 3, wherein the buffer is an acetate buffer.

6. The method according to claim 3, wherein the buffer is a histidine buffer.

7. The process according to any one of claims 1 to 6, wherein step (i) is carried out in the presence of a chelating agent.

8. The process according to claim 7, wherein the chelating agent is ethylenediaminetetraacetic acid.

9. The method according to any one of claims 3 to 8, wherein the buffer used in step (i) contains a surfactant.

10. The method according to claim 9, wherein the surfactant is polysorbate 20 or polysorbate 80.

11. The method according to any one of claims 1 to 10, wherein the reagent used in step (iii) is N-acetylcysteine.

12. The method according to any one of claims 1 to 11, wherein the pH of the buffer used in step (iv) is in the range of 4.7 to 5.

3.

13. The method according to any one of claims 1 to 12, wherein the buffer used in step (iv) is a histidine buffer.

14. The method according to any one of claims 1 to 13, wherein the concentration of the salt used in step (iv) is 0.2 to 1% by weight based on the buffer solution used in step (iv).

15. The method according to any one of claims 1 to 13, wherein the concentration of the salt used in step (iv) is 0.4 to 0.6% by weight based on the buffer solution used in step (iv).

16. The process according to any one of claims 1 to 15, wherein the salt used in step (iv) is sodium chloride.

17. Following step (iv), (v) removing salts by ultrafiltration using a buffer; The method of any one of claims 1 to 16, comprising:

18. The method according to claim 17, wherein the pH of the buffer used in step (v) is in the range of 4 to 6.

19. The method according to claim 17, wherein the pH of the buffer used in step (v) is in the range of 4.7 to 5.

3.

20. The method according to any one of claims 17 to 19, wherein the buffer used in step (v) is a histidine buffer.

21. The method according to any one of claims 1 to 20, wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

22. The method according to any one of claims 1 to 21, wherein the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody.

23. The method of claim 22, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:

2.

24. The method for producing the anti-HER3 antibody according to claim 22, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a modified version of said antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

25. The method for producing the anti-GPR20 antibody described in claim 22, wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6, or a modified version of said antibody in which a lysine residue at the carboxyl terminus of the heavy chain is deleted.

26. The method according to claim 22, wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8, or a modified form of said antibody in which a lysine residue at the carboxyl terminus of the heavy chain is deleted.

27. The process according to any one of claims 1 to 26, characterized in that it does not include a chromatographic purification step.

28. The method according to claim 27, wherein the chromatography is at least one selected from the group consisting of gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

29. An antibody-drug conjugate is produced by the production method according to any one of claims 1 to 28, and further comprising: (vi) adding a buffer solution to the solution containing the antibody-drug conjugate; (vii) concentrating the solution containing the antibody-drug conjugate; and (viii) adjusting the solution containing the antibody-drug conjugate to a predetermined pH; At least one step selected from the group consisting of: and, (ix) adding an excipient to the solution containing the antibody-drug conjugate; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient.

30. 30. The method of claim 29, wherein the buffer is a histidine buffer.

31. 31. The method of claim 29 or 30, wherein the excipient is sucrose or trehalose.

32. A pharmaceutical composition comprising an antibody-drug conjugate, a buffer, and an excipient is produced by the production method according to any one of claims 29 to 31, and further comprising: (x) adding a surfactant to the pharmaceutical composition; By carrying out a process including A method for making a pharmaceutical composition comprising an antibody-drug conjugate, a buffer, an excipient, and a surfactant.

33. The method of claim 32, wherein the surfactant is polysorbate 80 or polysorbate 20.

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