RU2789476C2 - Production of antibody-drug conjugate and its lyophilization - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to a pharmaceutical composition including (i) an antibody-drug conjugate, (ii) a histidine buffer, (iii) sucrose, and (iv) polysorbate 80 or polysorbate 20, where the antibody-drug conjugate is an antibody-drug conjugate, in which a drug-linker fragment represented by the following formula:

,

where A is a position of binding to the antibody, is conjugated with the antibody through a thioether bond, and where amount of polysorbate 80 or polysorbate 20 is 0.2-0.4 mg per 20 mg of the antibody-drug conjugate. The group of inventions also relates to a method for the production of a pharmaceutical composition, including stages of: (1) obtainment of an aqueous solution including (i) the antibody-drug conjugate, (ii) the histidine buffer, (iii) sucrose, and (iv) polysorbate 80 or polysorbate 20, and then, (2) lyophilization of the aqueous solution. The group of inventions also relates to a method for the production of a pharmaceutical composition, including stages of: (1) obtainment of an aqueous solution including (i) the antibody-drug conjugate, (ii) the histidine buffer, (iii) sucrose, and (iv) polysorbate 80 or polysorbate 20, (2) regulation of pH of the aqueous solution, and then, (3) lyophilization of the aqueous solution.

EFFECT: group of inventions provides reduction in a number of microparticles in a composition containing an antibody-drug conjugate.

49 cl, 18 ex, 17 tbl, 21 dwg

Description

The field of technology to which the invention belongs

[0001]

The present invention relates to a specific composition comprising an antibody-drug conjugate and a method for lyophilization of the composition.

State of the art

[0002]

An antibody-drug conjugate (ADC) containing a drug with cytotoxicity conjugated to an antibody whose antigen is expressed on the surface of cancer cells and which also binds to an antigen capable of cellular internalization and therefore can deliver the drug selectively to cancer cells as expected can thus cause drug accumulation in cancer cells and kill cancer cells (Non-Patent Literature 1-5).

[0003]

As one such antibody-drug conjugate, an antibody-drug conjugate is known comprising an antibody and exatecan, which is a topoisomerase I inhibitor, as its components (Patent Literature 1-8 and Non-Patent Literature 6, 7). Because these antibody-drug conjugates exhibit particularly high antitumor effects and are safe, they are currently in clinical trials.

[0004]

As compositions of such antibody-drug conjugates, known are an antibody-drug conjugate composition comprising a maytansinoid as a component (Patent Literature 9-12), an antibody-drug conjugate composition comprising monomethyl auristatin E as a component (Patent Literature 13-12). 15), an antibody drug conjugate composition including SN-38 as a component (Patent Literature 16), and the like.

List of cited documents

Patent Literature

[0005]

Patent Literature 1: International Publication No. WO 2014/057687

Patent Literature 2: International Publication No. WO 2014/061277

Patent Literature 3: International Publication No. WO 2015/098099

Patent Literature 4: International Publication No. WO 2015/115091

Patent Literature 5: International Publication No. WO 2015/146132

Patent Literature 6: International Publication No. WO 2015/155976

Patent Literature 7: International Publication No. WO 2015/155998

Patent Literature 8: International Publication No. WO 2018/135501

Patent Literature 9: International Publication No. WO 2004/004639

Patent Literature 10: International Publication No. WO 2004/110498

Patent Literature 11: International Publication No. WO 2007/019232

Patent Literature 12: International Publication No. WO 2015/059147

Patent Literature 13: International Publication No. WO 2010/081004

Patent Literature 14: International Publication No. WO 2014/143765

Patent Literature 15: International Publication No. WO 2015/157286

Patent Literature 16: International Publication No. WO 2014/092804

Non-Patent Literature

[0006]

Non-Patent Literature 1: Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13.

Non-Patent Literature 2: Alley, S. C, et al., Current Opinion in Chemical Biology (2010) 14, 529-537.

Non-Patent Literature 3: Damle N. K Expert Opin. Biol. Ther. (2004) 4, 1445-1452.

Non-Patent Literature 4: Senter P. D, et al., Nature Biotechnology (2012) 30, 631-637.

Non-Patent Literature 5: Howard A. et al, J Clin Oncol 29: 398-405.

Non-Patent Literature 6: Ogitani Y. et al., Clinical Cancer Research (2016) 22 (20), 5097-5108.

Non-Patent Literature 7: Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046.

The essence of the invention

Technical task

[0007]

In antibody compositions, the formation of aggregates and the formation of degradation products cause undesirable effects from a medical point of view, for example, they become an immunogenicity factor or cause venous disorders in patients taking such drugs. Thus, when preparing antibody compositions, it is necessary to suppress the formation of aggregates and the formation of degradation products, and therefore, various pharmaceutical compositions (for example, in the form of an aqueous composition for injection and a lyophilized composition for injection) have been investigated.

However, more complex studies are required to prepare antibody-drug conjugate compositions, since not only the specific properties of the antibody as part of the conjugate, but also the specific properties of the drug-linker part must be taken into account.

In addition, when preparing a lyophilized injectable composition from an aqueous solution containing sucrose or trehalose, there are problems such as (1) the primary drying process is long and (2) the lyophilized lozenge tends to dry out.

Thus, the main object of the present invention is to provide, for a specific antibody-drug conjugate, a pharmaceutical composition (especially in the form of an aqueous composition for injection and a lyophilized composition for injection) in which the formation of aggregates and the formation of degradation products are suppressed, and an efficient method for lyophilization of the aqueous solution to obtain a lyophilized composition for injection.

The solution of the problem

[0008]

For a specific antibody-drug conjugate, the present inventors have found a pharmaceutical composition (in particular, in the form of an aqueous composition for injection and a lyophilized composition for injection) in which the formation of aggregates and the formation of degradation products are suppressed, and also found an effective method for lyophilization of an aqueous solution to obtain lyophilized composition for injection.

[0009]

More specifically, the present invention relates to the following.

[1] A pharmaceutical composition comprising

(i) an antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose or trehalose and

(iv) a surfactant,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0010]

[Formula 1]

[0011]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[2] The pharmaceutical composition according to [1], wherein the surfactant is polysorbate 80 or polysorbate 20.

[3] A pharmaceutical composition according to [1] or [2], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 3-80 mmol histidine buffer,

(iii) 24-320 mg sucrose or trehalose and

(iv) 0.05-1.6 mg of polysorbate 80 or polysorbate 20.

[4] A pharmaceutical composition according to any one of [1] to [3], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10-40 mmol histidine buffer,

(iii) 90 mg sucrose or 100 mg trehalose hydrate and

(iv) 0.2-0.4 mg of polysorbate 80 or polysorbate 20.

[5] A pharmaceutical composition according to [1] or [2], comprising

(i) an antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose and

(iv) polysorbate 80 or polysorbate 20.

[0012]

[6] A pharmaceutical composition according to [1], [2] or [5], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 3-80 mmol histidine buffer,

(iii) 24-320 mg sucrose and

(iv) 0.05-1.6 mg of polysorbate 80 or polysorbate 20.

[7] A pharmaceutical composition according to [1], [2], [5] or [6], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10-40 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2-0.4 mg of polysorbate 80 or polysorbate 20.

[8] A pharmaceutical composition according to any one of [1] to [7], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 or 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg of polysorbate 80 or polysorbate 20.

[9] The pharmaceutical composition according to any one of [1] to [8], wherein the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 4.0-7.0.

[10] A pharmaceutical composition according to any one of [1] to [9], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 2 to 8.

[0013]

[11] The pharmaceutical composition according to any one of [1] to [10], wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, or anti-GPR20 antibody.

[12] The pharmaceutical composition according to [11], wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[13] The pharmaceutical composition according to [12], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 1-214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[14] A pharmaceutical composition according to [12] or [13], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[15] A pharmaceutical composition according to [12] - [14], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80,

where the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 5.5.

[0014]

[16] A pharmaceutical composition according to [12] - [14] comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.89 mg L-histidine and 4.04 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

[17] A pharmaceutical composition according to any one of [12] - [14], comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.45 mg L-histidine and 20.2 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[18] The pharmaceutical composition according to [11], wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[19] The pharmaceutical composition according to [18], wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: :4, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[20] A pharmaceutical composition according to [18] or [19], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[0015]

[21] A pharmaceutical composition according to any one of [18] - [20] comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 20,

where the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 5.4.

[22] A pharmaceutical composition according to any one of [18] - [20], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.81 mg L-histidine and 4.14 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 20.

[23] A pharmaceutical composition according to any one of [18] - [20], comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.06 mg L-histidine and 20.7 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 20.

[24] The pharmaceutical composition according to [11], wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[25] The pharmaceutical composition according to [24], wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 21-234 of SEQ ID NO: 6, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[0016]

[26] A pharmaceutical composition according to [24] or [25], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5.

[27] A pharmaceutical composition according to any one of [24] - [26], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 80,

where the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 6.0.

[28] A pharmaceutical composition according to any one of [24] - [26], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.78 mg L-histidine and 1.05 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 80.

[29] A pharmaceutical composition according to any one of [24] - [26], comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.88 mg L-histidine and 5.26 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 80.

[30] The pharmaceutical composition according to [11], wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[0017]

[31] The pharmaceutical composition according to [30], wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-471 of SEQ ID NO: 7 and a light chain consisting of from an amino acid sequence consisting of amino acid residues 21-233 of SEQ ID NO: 8, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[32] A pharmaceutical composition according to [30] or [31], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5.

[33] A pharmaceutical composition according to any one of [30] - [32], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 20,

where the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 5.9.

[34] A pharmaceutical composition according to any one of [30] - [32], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.65 mg L-histidine and 1.22 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 20.

[35] A pharmaceutical composition according to any one of [30] - [32], comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.23 mg L-histidine and 6.12 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 20.

[0018]

[36] The pharmaceutical composition according to [11], wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[37] The pharmaceutical composition according to [36], wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-472 of SEQ ID NO: 9 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 21-234 of SEQ ID NO: 10, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[38] A pharmaceutical composition according to [36] or [37], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[39] A pharmaceutical composition according to any one of [36] - [38], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80,

where the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 5.4.

[40] A pharmaceutical composition according to any one of [36] - [38], comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.32 mg L-histidine and 1.66 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

[0019]

[41] A pharmaceutical composition according to any one of [36] - [38], comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 1.62 mg L-histidine and 8.29 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[42] A pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.89 mg L-histidine and 4.04 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0020]

[Formula 2]

[0021]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[43] A pharmaceutical composition according to [42] comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.45 mg L-histidine and 20.2 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[44] A pharmaceutical composition according to [42] or [43], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[45] The pharmaceutical composition according to any one of [42] to [44], wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0022]

[46] The pharmaceutical composition according to [45], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 1-214 of SEQ ID NO: 2.

[47] The pharmaceutical composition according to [45], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: : 2.

[48] A pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.81 mg L-histidine and 4.14 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 20,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0023]

[Formula 3]

[0024]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[49] A pharmaceutical composition according to [48] comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.06 mg L-histidine and 20.7 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 20.

[50] A pharmaceutical composition according to [48] or [49], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[0025]

[51] The pharmaceutical composition according to any of [48] to [50], wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[52] The pharmaceutical composition according to [51], wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: : 4.

[53] A pharmaceutical composition according to [52], wherein the anti-HER3 antibody does not contain a lysine residue at the carboxy-terminus of the heavy chain.

[54] A pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.78 mg L-histidine and 1.05 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 80,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0026]

[Formula 4]

[0027]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[55] A pharmaceutical composition according to [54] comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.88 mg L-histidine and 5.26 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 80.

[56] A pharmaceutical composition according to [54] or [55], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5.

[57] The pharmaceutical composition according to any one of [54] to [56], wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[58] The pharmaceutical composition according to [57], wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 21-234 of SEQ ID NO: 6.

[59] A pharmaceutical composition according to [58], wherein the anti-TROP2 antibody does not contain a lysine residue at the carboxy-terminus of the heavy chain.

[60] A pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.65 mg L-histidine and 1.22 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 20,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0028]

[Formula 5]

[0029]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[61] A pharmaceutical composition according to [60] comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.23 mg L-histidine and 6.12 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 20.

[62] A pharmaceutical composition according to [60] or [61], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5.

[63] The pharmaceutical composition according to any of [60] to [62], wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[64] The pharmaceutical composition according to [63], wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-471 of SEQ ID NO: 7 and a light chain consisting of from the amino acid sequence consisting of amino acid residues 21-233 of SEQ ID NO: 8.

[65] A pharmaceutical composition according to [64], wherein the anti-B7-H3 antibody does not contain a lysine residue at the carboxy-terminus of the heavy chain.

[66] A pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.32 mg L-histidine and 1.66 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80

where, the antibody-drug conjugate is an antibody-drug conjugate in which the drug-linker moiety is represented by the following formula:

[0030]

[Formula 6]

[0031]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[67] A pharmaceutical composition according to [66] comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 1.62 mg L-histidine and 8.29 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[68] A pharmaceutical composition according to [66] or [67], wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8.

[69] The pharmaceutical composition according to any of [66] to [68], wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[70] The pharmaceutical composition according to [69], wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-472 of SEQ ID NO: 9 and a light chain consisting of an amino acid a sequence consisting of amino acid residues 21-234 of SEQ ID NO: 10.

[0032]

[71] A pharmaceutical composition according to [70], wherein the anti-GPR20 antibody does not contain a lysine residue at the carboxy-terminus of the heavy chain.

[72] A pharmaceutical composition according to any one of [1] to [71], wherein the composition is in the form of an injection.

[73] The pharmaceutical composition according to [72], wherein the composition is in the form of an aqueous composition for injection.

[74] A pharmaceutical composition according to [73], wherein the concentration of the antibody-drug conjugate is 20 mg/mL.

[75] A pharmaceutical composition according to [73] or [74], wherein the composition is frozen.

[0033]

[76] A pharmaceutical composition according to [72], wherein the composition is in the form of a lyophilized composition for injection.

[77] Pharmaceutical composition according to [76], where the composition is stored in a dark vial.

[78] A pharmaceutical composition according to any one of [1] to [77], wherein the composition is for the treatment of cancer.

[79] A method for preparing a pharmaceutical composition according to [76], comprising the steps

(1) obtaining an aqueous solution comprising predetermined amounts

(i) an antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose or trehalose and

(iv) a surfactant,

(2) if necessary, adjust the pH of the aqueous solution, and then

(3) lyophilization of the aqueous solution.

[80] The production method according to [79], wherein the aqueous solution lyophilization step includes an annealing process at a shelf temperature that is close to the eutectic point of the aqueous solution,

where proximity to the eutectic point means the range from a temperature that is 1.5°C below the eutectic point to a temperature that is 1.5°C above the eutectic point.

[0034]

[81] The production method according to [80], wherein the aqueous solution lyophilization step includes an annealing process at a shelf temperature that is equal to the eutectic point of the aqueous solution.

[82] The production method according to [80] or [81], characterized in that the time of the primary drying process is reduced compared to when annealing is carried out at a shelf temperature that is 5° C. below the eutectic point.

[83] The production method according to any one of [80] to [82], characterized in that a lyophilized cake is obtained having less shrinkage compared to that obtained by annealing at a shelf temperature that is 5°C lower than the eutectic points.

[84] The production method according to [79], wherein the aqueous solution lyophilization step includes a method for performing annealing at a shelf temperature of -4 to -1°C.

[85] The production method according to [79], wherein the aqueous solution lyophilization step includes a method for performing annealing at a shelf temperature of -4 to -2°C.

[0035]

[86] The production method according to [79], wherein the aqueous solution lyophilization step includes a method for performing annealing at a shelf temperature of -2.5°C.

[87] The production method according to any of [84] to [86], wherein the aqueous solution lyophilization step also includes a primary drying process at a shelf temperature of −5 to 5°C under a vacuum of 5 to 15 Pa.

[88] The production method according to any of [84] to [86], wherein the aqueous solution lyophilization step also includes a primary drying process at a shelf temperature of 0°C under a vacuum of 10 Pa.

[89] The production method according to [87] or [88], wherein the aqueous solution lyophilization step also includes a secondary drying process at a shelf temperature of 40 to 50°C under a vacuum of 5 to 15 Pa.

[90] The production method according to [87] or [88], wherein the aqueous solution lyophilization step also includes a secondary drying process at a shelf temperature of 45°C under a vacuum of 10 Pa.

[0036]

[91] A kit comprising a pharmaceutical composition according to [42] or [43] and water for injection.

[92] A set according to [91], where water for injection is stored in an ampoule.

[93] A method for producing a lyophilized composition for injection, comprising a process of annealing an aqueous solution containing sucrose or trehalose at a shelf temperature that is close to the eutectic point of the aqueous solution,

where proximity to the eutectic point means the range from a temperature that is 1.5°C below the eutectic point to a temperature that is 1.5°C above the eutectic point.

[94] A method for producing a lyophilized composition for injection, comprising a process of annealing an aqueous solution containing sucrose or trehalose at a shelf temperature that is equal to the eutectic point of the aqueous solution.

Useful effects of the invention

[0037]

The present invention can provide a pharmaceutical composition (especially in the form of an aqueous composition for injection and a lyophilized composition for injection) that inhibits the formation of aggregates and the formation of degradation products, and can also provide an efficient method for lyophilization from an aqueous solution to obtain a lyophilized injectable composition in which a specific antibody-drug conjugate.

Brief description of the drawings

[0038]

[Fig. 1] FIG. 1 is a diagram showing the amino acid sequence of an anti-HER2 antibody heavy chain (SEQ ID NO: 1).

[Fig. 2] FIG. 2 is a diagram showing the amino acid sequence of the light chain of an anti-HER2 antibody (SEQ ID NO: 2).

[Fig. 3] FIG. 3 is a diagram related to the screening of excipients. The abscissa shows the time period for each composition, and the y-axis shows the content of the aggregates.

[Fig. 4] FIG. 4 is a diagram related to buffer screening. The abscissa shows the time period for each composition, and the y-axis shows the content of the aggregates.

[Fig. 5] FIG. 5 is a diagram related to pH screening. The abscissa shows the time period for each composition, and the y-axis shows the content of the aggregates.

[Fig. 6] FIG. 6 is a diagram related to pH screening. The abscissa shows the time period for each composition, and the y-axis shows the NPI content.

[Fig. 7] FIG. 7 is a diagram related to surfactant screening. The abscissa axis shows the time period for each composition, and the y-axis shows the relative content of insoluble microparticles less than 10 mm. In the drawings, "PS" means polysorbate, "PS20" means polysorbate 20, and "PS80" means polysorbate 80.

[Fig. 8] FIG. 8 is a diagram related to vial testing. The abscissa shows the amount of light exposure, and the y-axis shows the content of aggregates.

[Fig. 9] FIG. 9 is a diagram related to vial testing. The abscissa shows the amount of light exposure, and the y-axis shows the IoP content.

[Fig. 10] FIG. 10 shows the contour curve of the temperature of the aqueous solution product (1) by varying the temperature of the shelf and the degree of evacuation of the chamber during the primary drying process.

[0039]

[Fig. 11] FIG. 11 shows the contour curve of the drying time of the aqueous solution (1) by varying the temperature of the shelf and the degree of evacuation of the chamber during the primary drying process.

[Fig. 12] FIG. 12 is a graph showing the temperature of the product (during annealing) and the shape of the lyophilized cakes when annealing at -7 to 0.5°C shelf temperatures and lyophilization is carried out.

[Fig. 13] FIG. 13 is a graph showing the thermal denaturation of the antibody-drug conjugate (1) determined using a capillary differential scanning calorimeter.

[Fig. 14] FIG. 14 is a diagram showing the amino acid sequence of an anti-HER3 antibody heavy chain (SEQ ID NO: 3).

[Fig. 15] FIG. 15 is a diagram showing the amino acid sequence of an anti-HER3 antibody light chain (SEQ ID NO: 4).

[Fig. 16] FIG. 16 is a diagram showing the amino acid sequence of an anti-TROP2 antibody heavy chain (SEQ ID NO: 5).

[Fig. 17] FIG. 17 is a diagram showing the amino acid sequence of the light chain of an anti-TROP2 antibody (SEQ ID NO: 6).

[Fig. 18] FIG. 18 is a diagram showing the amino acid sequence of an anti-B7-H3 antibody heavy chain (SEQ ID NO: 7).

[Fig. 19] FIG. 19 is a diagram showing the amino acid sequence of the light chain of an anti-B7-H3 antibody (SEQ ID NO: 8).

[Fig. 20] FIG. 20 is a diagram showing the amino acid sequence of an anti-GPR20 antibody heavy chain (SEQ ID NO: 9).

[Fig. 21] FIG. 21 is a diagram showing the amino acid sequence of the light chain of an anti-GPR20 antibody (SEQ ID NO: 10).

Description of Embodiments

[0040]

In the following, preferred embodiments of the present invention are described with reference to the drawings. The embodiments described below are presented solely to illustrate one example of a typical embodiment of the present invention and are not intended to limit the scope of the present invention.

[0041]

[Antibody drug conjugate]

The antibody-drug conjugate used in the present invention is an antibody-drug conjugate in which the drug-linker moiety is represented by the following formula:

[0042]

[Formula 7]

[0043]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

[0044]

In the present invention, the linker-drug partial structure in the antibody-drug conjugate is referred to as "drug-linker". The linker drug moiety is linked to a thiol group (in other words, to the sulfur atom of a cysteine residue) formed at the interchain disulfide bond site (two sites between the heavy chains and two sites between the heavy chain and the light chain) of the antibody.

[0045]

The linker drug of the present invention includes exatecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4 -methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline 13-dione, (also chemical name: (1S,9S)-1- amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-10,13(9H,15H)-dione)), which is a topoisomerase I inhibitor, as a component. Exatecan is an antitumor derivative of casptothecine represented by the following formula:

[0046]

[Formula 8]

[0048]

The antibody-drug conjugate used in the present invention can also be represented by the following formula:

[0049]

[Formula 9]

[0050]

wherein the drug-linker portion is conjugated to the antibody via a thioether bond. The value of n is what is called the average number of conjugated drug molecules (DAR; drug-to-antibody ratio), and means the average number of drug-linker units conjugated per antibody molecule.

After it migrates to cancer cells, the antibody drug conjugate used in the present invention releases a compound represented by the following formula:

[0051]

[Formula 10]

[0052]

has a topoisomerase I inhibitory effect (hereinafter referred to as "compound (1)").

It was concluded that the compound (1) is formed by decomposing the aminal structure of the compound represented by the following formula:

[0053]

[Formula 11]

[0054]

which is believed to be formed by cleavage of the linker portion of the antibody-drug conjugate used in the present invention.

[0055]

The antibody-drug conjugate used in the present invention is known to have a non-specific effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).

The non-specific effect is exhibited through a process in which the antibody-drug conjugate used in the present invention is internalized in cancer cells expressing the target, and the released compound (1) then exhibits an antitumor effect also on cancer cells that are present in the environment and do not express the target .

[0056]

[Antibody in an antibody-drug conjugate]

The antibody in the antibody-drug conjugate used in the present invention may be from any species, and is preferably a human, rat, mouse or rabbit antibody. In cases where the antibody is of non-human origin, it is preferably chimerized or humanized in a manner well known in the art. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0057]

The antibody in the antibody-drug conjugate of the present invention is an antibody preferably having a property of being able to target cancer cells, and preferably is an antibody having, for example, a cancer cell recognition property, a cancer cell binding property, an internalization property in the cancer cell and/or cancer cell killing activity.

[0058]

The binding activity of an antibody against cancer cells can be confirmed using flow cytometry. Internalization of the antibody in cancer cells can be confirmed using (1) imaging analysis of the antibody incorporated into the cells using a fluorescent microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761 ), (2) an assay measuring the intensity of fluorescence incorporated into cells using a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3 ) Mab-ZAP assay using an immunotoxin that binds to a therapeutic antibody, wherein the toxin is released upon incorporation into cells to inhibit cell growth (Bio Techniques 28: 162-165, January 2000). The recombinant complex protein of the catalytic region of diphtheria toxin and protein G can be used as an immunotoxin.

[0059]

The antitumor activity of an antibody can be confirmed in vitro by determining inhibitory activity against cell growth. For example, a cancer cell line overexpressing a target protein for an antibody is cultured, and the antibody is added to the culture system at various concentrations to determine inhibitory activity against focus formation, colony formation, and spheroid growth. Antitumor activity can be confirmed in vivo, for example, by injecting a nude mouse antibody transplanted with a cancer cell line highly expressing the target protein and detecting a change in the cancer cell.

[0060]

Since the compound conjugated in the antibody-drug conjugate exhibits an antitumor effect, it is preferable, but not essential, that the antibody itself has an antitumor effect. For the specificity of the antitumor compound and the selective display of cytotoxic activity against cancer cells, it is important and also preferable that the antibody has the property of internalizing to migrate into cancer cells.

The antibody in the antibody-drug conjugate used in the present invention can be obtained using a procedure known in the art. For example, an antibody of the present invention can be obtained using a method usually carried out in the art, which includes immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and animals can be immunized with an antigen derived from a non-human animal such as mouse, rat, and the like. In this case, it is possible to test the cross-reactivity of antibodies binding to the obtained heterologous antigen with human antigens to screen for an antibody applicable to a human disease.

[0061]

Alternatively, antibody-producing cells that produce antibodies against the antigen are fused with myeloma cells according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)), to produce hybridomas, from which monoclonal antibodies can in turn be obtained.

[0062]

The antigen can be genetically engineered to produce host cells that produce the gene encoding the antigenic protein. In particular, vectors are prepared that allow expression of the antigen and transferred to host cells so that the gene is expressed. The antigen expressed in this way can be purified. The antibody can also be obtained by the method of immunizing animals using the genetically engineered antigen-expressing cells or cell line expressing the antigen described above.

[0063]

The antibody in the antibody-drug conjugate used in the present invention is preferably a recombinant antibody obtained by artificial modification to reduce heterologous antigenicity for a human, such as a chimeric antibody or a humanized antibody, or is preferably an antibody containing only the antibody gene sequence human origin, i.e. human antibody. These antibodies can be obtained using a known method.

[0064]

An example of a chimeric antibody is an antibody in which the variable and constant regions of an antibody are from different species, such as a chimeric antibody in which the variable region of an antibody of murine or rat origin is linked to the constant region of an antibody of human origin (Proc. Natl. Acad. Sci U.S.A., 81, 6851-6855, (1984)).

[0065]

Examples of a humanized antibody include an antibody obtained by inserting only the complementarity determining region (CDR) of a heterologous antibody into an antibody of human origin (Nature (1986) 321, pp. 522-525), and an antibody obtained by grafting a portion of the amino acid residues of a framework region a heterologous antibody, as well as the CDR sequences of a heterologous antibody to a human antibody by the CDR grafting method (WO 90/07861), and an antibody humanized using a gene conversion mutagenesis strategy (US Patent No. 5821337).

[0066]

As an example of a human antibody, an antibody produced using a human antibody-producing mouse having a fragment of a human chromosome including the heavy chain and light chain genes of a human antibody can be given (see, e.g., Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 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, pp. 722-727). Alternatively, an antibody obtained by screening a human antibody library by phage display can be exemplified (see, e.g., Wormstone, I. M. 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).

[0067]

The present invention also includes modified variants of the antibody in the antibody-drug conjugate used in the present invention. Modified variant refers to the variant obtained when the antibody of the present invention is subjected to chemical or biological modification. Examples of the chemically modified variant include those involving linking a chemical moiety to an amino acid backbone, options including linking a chemical moiety to an N-linked or O-linked carbohydrate chain, and so on. Examples of a biologically modified variant include those resulting from post-translational modification (such as N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, or methionine oxidation) and variants in which a methionine residue has been added to N-terminus by expression in a prokaryotic host cell. In addition, the term “modified variant” also includes an antibody labeled so as to allow detection or isolation of an antibody or antigen in accordance with the present invention, for example, an enzyme-labeled antibody, a fluorescently labeled antibody, and an affinity-labeled antibody. Such a modified version of an antibody according to the present invention is useful for improving the stability and blood retention of an antibody, reducing its antigenicity, detecting or isolating an antibody or antigen, and so on.

[0068]

In addition, by regulating the modification of the glycan that is associated with the antibody of the present invention (glycosylation, defucosylation, etc.), antibody-dependent cellular cytotoxic activity can be enhanced. Methods for regulating antibody glycan modification are known from WO 99/54342, WO 00/61739, WO 02/31140, etc. However, the methods are not limited to this. The antibody of the present invention also includes antibodies in which glycan modification is regulated.

[0069]

The lysine residue at the carboxy-terminus of the heavy chain of an antibody produced in a cultured mammalian cell is known to be deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) on the carboxy-terminus of the heavy chain of an antibody produced in a cultured mammalian cell is deleted, and the newly appeared proline residue at the carboxy-terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such a deletion and modification of the heavy chain sequence does not affect the antigen binding affinity and effector function (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, an antibody of the present invention also includes antibodies subject to such modification and functional antibody fragments, and also includes deletion variants in which one or two amino acids have been deleted at the carboxy-terminus of the heavy chain, variants obtained by amidation of deletion variants (e.g. , a heavy chain in which the carboxy-terminal proline residue is amidated), and the like. The type of deletion variant, including deletion at the carboxy-terminus of the heavy chain of an antibody according to the present invention, is not limited to the above-described variants, as long as the antigen binding affinity and effector function are maintained. The two heavy chains forming an antibody of the present invention may be of the same type selected from the group consisting of the full length heavy chain and the deletion variant described above, or may be a combination of the two types. The amount of each deletion variant may depend on the type of cultured mammalian cells that produce the antibody of the present invention and culture conditions; however, a preferred example is an antibody in which one amino acid residue at the carboxy terminus is deleted from both of the two heavy chains in the antibody of the present invention.

[0070]

As isotypes of the antibody according to the present invention, for example, IgG (IgG1, IgG2, IgG3, IgG4) can be mentioned, and IgG1 or IgG2 are preferred.

[0071]

Examples of antibodies in the antibody drug conjugate used in the present invention may include, but are not limited to, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody, anti -CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti -FOLR1 antibody, anti-VEGF antibody and anti-GPR20 antibody, and as preferred, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody and anti-GPR20 can be mentioned.

[0072]

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 internalization activity in HER2-expressing cells by binding to HER2.

[0073]

Examples of anti-HER2 antibodies include trastuzumab (US Patent No. 5821337) and pertuzumab (International Publication No. WO 01/00245), and trastuzumab is a preferred example.

[0074]

In the present invention, the term "trastuzumab" means a humanized anti-HER2 monoclonal antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 (Fig. 1) and a light chain consisting of the amino acid sequence , consisting of amino acid residues 1-214 SEQ ID NO: 2 (Fig. 2).

[0075]

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 internalization activity in HER3-expressing cells by binding to HER3 on the surface of HER3 -expressing cells.

[0076]

Examples of anti-HER3 antibodies include Patritumab (U3-1287), U1-59 (International Publication No. WO 2007/077028), MM-121 (seribantumab), anti-ERBB3 antibody described in International Publication No. WO 2008/100624, RG- 7116 (Lumretuzumab) and LJM-716 (Elgemtumab) and Patritumab, and U1-59 is a preferred example.

[0077]

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 internalization activity in TROP2-expressing cells by binding to TROP2.

[0078]

Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. WO 2015/098099).

[0079]

In the present invention, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B-cell antigen #7 homologue 3; PD-L3; CD276) and preferably has internalization activity at B7-H3- expressing cells by binding to B7-H3.

[0080]

Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. WO 2014/057687).

[0081]

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 internalization activity in GPR20-expressing cells by binding to GPR20.

Examples of anti-GPR20 antibodies include h046-H4e/L7 (International Publication No. WO 2018/135501).

[0082]

[Drug-linker intermediate for use in preparing an antibody-drug conjugate]

A drug-linker intermediate for use in preparing an antibody-drug conjugate according to the present invention is represented by the following formula.

[0083]

[Formula 12]

[0084]

The drug-linker intermediate can be prepared as described in International Publication No. WO 2014/057687, International Publication No. WO 2015/098099, International Publication No. WO 2015/115091, International Publication No. WO 2015/155998, etc.

[0085]

[Conjugation between antibody and drug-linker intermediate]

The antibody-drug conjugate used in the present invention can be prepared by reacting the above-described drug-linker intermediate with an antibody containing a thiol group (alternatively referred to as a sulfhydryl group).

[0086]

An antibody containing a sulfhydryl group can be prepared in a manner well known in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, using 0.3-3 molar equivalents of a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) based on interchain disulfide in the antibody and reacting with the antibody in a buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), you can get an antibody containing a sulfhydryl group with partially or completely reduced interchain disulfides in the antibody.

[0087]

In addition, using 2-20 molar equivalents of drug-linker intermediate per antibody containing a sulfhydryl group, an antibody-drug conjugate can be prepared in which 2-8 drug molecules are conjugated per antibody molecule.

[0088]

The average number of conjugated drug molecules per antibody molecule in the resulting antibody-drug conjugate can be determined, for example, by a calculation method based on measuring the UV absorbance of the antibody-drug conjugate and its precursor before conjugation at two wavelengths of 280 nm and 370 nm (UV method), or a calculation method based on quantification using HPLC measurements, for fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0089]

The conjugation reaction between the antibody and the drug-linker intermediate and the calculation of the average number of conjugated drug molecules per antibody molecule of the antibody-drug conjugate can be carried out as described in International Publication No. WO 2014/057687, International Publication No. WO 2015/098099, International Publication No. WO 2015/115091, International Publication No. WO 2015/155998, International Publication No. WO 2018/135501, etc.

[0090]

In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0091]

The anti-HER2 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1-214 of SEQ ID NO: : 2, or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0092]

The average number of conjugated drug-linker units per antibody molecule in the anti-HER2 antibody-drug conjugate used in the present invention is preferably 2-8, more preferably 3-8, even more preferably 7-8, even more preferably 7, 5-8 and even more preferably about 8.

[0093]

The anti-HER2 antibody-drug conjugate used in the present invention can be prepared as described in International Publication No. WO 2015/115091, etc.

[0094]

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

[0095]

The anti-HER3 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4, or an antibody variant in which the lysine residue on the carboxy-terminus of the heavy chain is deleted.

[0096]

The average number of conjugated drug-linker units per antibody molecule in the anti-HER3 antibody-drug conjugate used in the present invention is preferably 2-8, more preferably 3-8, even more preferably 7-8, even more preferably 7, 5-8 and even more preferably about 8.

[0097]

The anti-HER3 antibody-drug conjugate used in the present invention can be prepared as described in International Publication No. WO 2015/155998, etc.

[0098]

In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[0099]

The anti-TROP2 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid sequences consisting of amino acid residues 21-234 of SEQ ID NO: :6, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[0100]

The average number of conjugated drug-linker units per antibody molecule in the anti-TROP2 antibody-drug conjugate used in the present invention is preferably 2-8, more preferably 3-5, even more preferably 3.5-4.5 and more more preferably about 4.

[0101]

The anti-TROP2 antibody-drug conjugate used in the present invention can be prepared as described in International Publication No. WO 2015/098099, etc.

[0102]

In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[0103]

The anti-B7-H3 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20-471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21-233 of SEQ ID NO: 8, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[0104]

The average number of conjugated drug-linker units per antibody molecule in the anti-B7-H3 antibody-drug conjugate used in the present invention is preferably 2-8, more preferably 3-5, even more preferably 3.5-4.5 and even more preferably about 4.

[0105]

The anti-B7-H3 antibody-drug conjugate used in the present invention can be prepared as described in International Publication No. WO 2014/057687, etc.

[0106]

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

[0107]

The anti-GPR20 antibody is preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-472 of SEQ ID NO: 9 and a light chain consisting of an amino acid sequence consisting of amino acid sequences consisting of amino acid residues 21-234 of SEQ ID NO. :10, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted.

[0108]

The average number of conjugated drug-linker units per antibody molecule in the anti-GPR20 antibody-drug used in the present invention is preferably 2-8, more preferably 3-8, even more preferably 7-8, even more preferably 7, 5-8 and even more preferably about 8.

[0109]

The anti-GPR20 antibody-drug conjugate used in the present invention can be prepared as described in International Publication No. WO 2018/135501, etc.

[0110]

[Pharmaceutical compositions]

Next, the pharmaceutical composition according to the present invention will be described.

The pharmaceutical composition according to the present invention comprises

(i) an antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose or trehalose and

(iv) a surfactant,

where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

[0111]

[Formula 13]

[0112]

where A is the position of binding to the antibody,

conjugated to the antibody through a thioether bond.

In the present invention, the term "histidine buffer" means a mixture of histidine and a histidine salt (acid addition product), preferably a mixture of L-histidine and L-histidine hydrochloride (and/or its hydrate). The water in which the histidine buffer in the present invention is dissolved is equivalent to the histidine buffer solution.

[0113]

The amount of histidine buffer is preferably 3-80 mmol per 20 mg antibody-drug conjugate, more preferably 10-80 mmol per 20 mg antibody-drug conjugate, even more preferably 10-40 mmol per 20 mg antibody-drug conjugate, still more preferably 10-25 mmol per 20 mg of antibody-drug conjugate and even more preferably 10 or 25 mmol per 20 mg of antibody-drug conjugate.

[0114]

It should be noted that the pH of the state when the pharmaceutical composition of the present invention is dissolved in water depends on the ratio of the content of histidine and histidine salts in the histidine buffer solution. In other words, by adjusting the ratio of histidine to histidine salts, it is possible to provide a pharmaceutical composition having a desired pH when dissolved in water.

[0115]

The pH of the pharmaceutical composition of the present invention when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 4.0-7.0, preferably 5.0-6.0. In addition, depending on the type of antibody in the antibody-drug conjugate, a more appropriate pH can be selected.

The amount of sucrose or trehalose is preferably 24-320 mg per 20 mg of the antibody-drug conjugate. As for sucrose, the amount is more preferably 90 mg. As for trehalose, based on trehalose hydrate, the amount is more preferably 100 mg. Of sucrose and trehalose, sucrose is more preferably used in the pharmaceutical compositions of the present invention.

In the present invention, the term "surfactant" refers to a substance containing a hydrophilic group and a hydrophobic group, and which is used as one of the constituents of a pharmaceutical composition. The surfactant in the present invention is preferably a polysorbate such as polysorbate 80 (Tween 80), polysorbate 20 (Tween20) and polysorbate 60 (Tween 60), polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene hydrogenated castor oil 60 , or polyoxyethylated castor oil, or sodium lauryl sulfate, and more preferably polysorbate 80 or polysorbate 20.

[0116]

The amount of polysorbate 80 or polysorbate 20 is, per 20 mg of antibody-drug conjugate, preferably 0.05-1.6 mg, more preferably 0.1-1.6 mg, even more preferably 0.2-0.4 mg, even more preferably 0.2-0.3 mg and even more preferably 0.2 or 0.3 mg.

In view of all of the above, the pharmaceutical composition of the invention is preferably

a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 3-80 mmol histidine buffer,

(iii) 24-320 mg sucrose or trehalose and

(iv) 0.05-1.6 mg of polysorbate 80 or polysorbate 20,

more preferably

a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10-40 mmol histidine buffer,

(iii) 90 mg sucrose or 100 mg trehalose hydrate and

(iv) 0.2-0.4 mg of polysorbate 80 or polysorbate 20,

even more preferably

a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 or 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg of polysorbate 80 or polysorbate 20.

In terms of an aqueous solution containing the antibody-drug conjugate at a concentration of 20 mg/ml, the pharmaceutical composition described above can be represented as follows.

[0117]

Namely, the pharmaceutical composition of the present invention is preferably

pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose or trehalose and

(iv) a surfactant and

(v) water,

more preferably

pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 3-80 mM histidine buffer,

(iii) 2.4-32% sucrose or trehalose,

(iv) 0.005-0.16% polysorbate 80 or polysorbate 20 and

(v) water,

even more preferably

pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 10-40 mM histidine buffer,

(iii) 9% sucrose or 10% trehalose,

(iv) 0.02-0.04% polysorbate 80 or polysorbate 20 and

(v) water,

even more preferably

pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 10 or 25 mM histidine buffer,

(iii) 9% sucrose and

(iv) 0.02 or 0.03% polysorbate 80 or polysorbate 20 and

(v) water.

[0118]

In the present invention, "%" content of sucrose and trehalose refers to mass% per 1 ml of water. Thus, for example, "9% sucrose" means that the composition contains 90 mg of sucrose per 1 ml of water, and "10% trehalose hydrate" means that the composition contains 100 mg of trehalose hydrate per 1 ml of water.

[0119]

Similarly, in the present invention, "%", indicating the content of polysorbate 80 and polysorbate 20, refers to mass% per 1 ml of water. Thus, for example, "0.03% polysorbate 80" means that the composition contains 0.3 mg of polysorbate 80 per 1 ml of water, and "0.03% of polysorbate 20" means that the composition contains 0.3 mg of polysorbate 20 per ml of water. 1 ml of water.

[0120]

With regard to the pharmaceutical composition of the present invention, depending on the type of antibody in the antibody-drug conjugate, more suitable compositions can be selected.

[0121]

For example, when the antibody in the antibody-drug conjugate is an anti-HER2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1-214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 2), the pharmaceutical composition of the present invention is preferably a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

In addition, the pH of the pharmaceutical composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is preferably 5.3-5.7, more preferably 5.4-5.6, even more preferably 5.5.

[0122]

When the amount of the histidine buffer is expressed as the content of L-histidine and L-histidine hydrochloride, the above-described pharmaceutical composition, when its pH is 5.5, can be represented as a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.89 mg L-histidine and 4.04 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

[0123]

In addition, in terms of a standard composition comprising 100 mg of the antibody-drug conjugate, the above-described pharmaceutical composition, when its pH is 5.5, can be represented as a pharmaceutical composition comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.45 mg L-histidine and 20.2 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[0124]

Alternatively, in terms of an aqueous solution comprising an antibody-drug conjugate at a concentration of 20 mg/mL, the pharmaceutical composition described above can be represented as a pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 25 mM histidine buffer,

(iii) 9% sucrose,

(iv) 0.03% polysorbate 80 and

(v) water,

where the pH of the composition is 5.5.

[0125]

When the antibody in the antibody-drug conjugate is an anti-HER3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4 , or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted), the pharmaceutical composition of the present invention is preferably a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 25 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 20.

In addition, the pH of the pharmaceutical composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is preferably 5.2-5.6, more preferably 5.3-5.5, even more preferably 5.4.

[0126]

When the amount of histidine buffer is expressed as the content of L-histidine and L-histidine hydrochloride, the above-described pharmaceutical composition, when its pH is 5.4, can be represented as a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.81 mg L-histidine and 4.14 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 20.

[0127]

In addition, in terms of a standard composition comprising 100 mg of the antibody-drug conjugate, the above-described pharmaceutical composition, when its pH is 5.4, can be represented as a pharmaceutical composition comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 4.06 mg L-histidine and 20.7 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 20.

[0128]

Alternatively, in terms of an aqueous solution comprising an antibody-drug conjugate at a concentration of 20 mg/mL, the pharmaceutical composition described above can be represented as a pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 25 mM histidine buffer,

(iii) 9% sucrose,

(iv) 0.03% polysorbate 20 and

(v) water,

where the pH of the composition is 5.4.

[0129]

When the antibody in the antibody-drug conjugate is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5 and a light chain consisting of an amino acid sequence, consisting of amino acid residues 21-234 of SEQ ID NO: 6, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted), the pharmaceutical composition of the present invention is preferably

a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 80.

In addition, the pH of the pharmaceutical composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is preferably 5.8-6.2, more preferably 5.9-6.1, even more preferably 6.0.

[0130]

When the amount of the histidine buffer is expressed as the content of L-histidine and L-histidine hydrochloride, the above-described pharmaceutical composition, when its pH is 6.0, can preferably be presented as a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.78 mg L-histidine and 1.05 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 80.

[0131]

In terms of a standard composition comprising 100 mg of the antibody-drug conjugate, the above-described pharmaceutical composition, when its pH is 6.0, can be represented as a pharmaceutical composition comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.88 mg L-histidine and 5.26 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 80.

[0132]

Alternatively, in terms of an aqueous solution comprising an antibody-drug conjugate at a concentration of 20 mg/mL, the pharmaceutical composition described above can be represented as a pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 10 mM histidine buffer,

(iii) 9% sucrose,

(iv) 0.02 or 0.03% polysorbate 80 and

(v) water,

where the pH of the composition is 6.0.

[0133]

When the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody (preferably, where the antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-471 of SEQ ID NO: 7 and a light chain , consisting of an amino acid sequence consisting of amino acid residues 21-233 of SEQ ID NO: 8, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted), the pharmaceutical composition of the present invention is preferably a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 20.

In addition, the pH of the pharmaceutical composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is preferably 5.7-6.1, more preferably 5.8-6.0, even more preferably 5.9.

[0134]

When the amount of the histidine buffer is expressed as the content of L-histidine and L-histidine hydrochloride, the above-described pharmaceutical composition, when its pH is 5.9, can be represented as a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.65 mg L-histidine and 1.22 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.2 or 0.3 mg polysorbate 20.

[0135]

In addition, in terms of a standard composition comprising 100 mg of the antibody-drug conjugate, the above-described pharmaceutical composition, when its pH is 5.9, can be represented as a pharmaceutical composition comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 3.23 mg L-histidine and 6.12 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.0 or 1.5 mg of polysorbate 20.

[0136]

Alternatively, in terms of an aqueous solution comprising an antibody-drug conjugate at a concentration of 20 mg/mL, the pharmaceutical composition described above can be represented as a pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 10 mM histidine buffer,

(iii) 9% sucrose and

(iv) 0.02 or 0.03% polysorbate 20 and

(v) water,

where the pH of the composition is 5.9.

[0137]

When the antibody in the antibody-drug conjugate is an anti-GPR20 antibody (preferably, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20-472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence, consisting of amino acid residues 21-234 of SEQ ID NO: 10, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted, the pharmaceutical composition of the present invention is preferably a composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 10 mmol histidine buffer,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

In addition, the pH of the pharmaceutical composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is preferably 5.2-5.6, more preferably 5.3-5.5, even more preferably 5.4.

[0138]

When the amount of the histidine buffer is expressed as the content of L-histidine and L-histidine hydrochloride, the above-described pharmaceutical composition, when its pH is 5.4, can preferably be presented as a pharmaceutical composition comprising,

(i) per 20 mg of the antibody-drug conjugate,

(ii) 0.32 mg L-histidine and 1.66 mg L-histidine hydrochloride hydrate,

(iii) 90 mg sucrose and

(iv) 0.3 mg polysorbate 80.

[0139]

In addition, in terms of a standard composition comprising 100 mg of the antibody-drug conjugate, the above-described pharmaceutical composition, when its pH is 5.4, can be represented as a pharmaceutical composition comprising

(i) 100 mg of antibody-drug conjugate,

(ii) 1.62 mg L-histidine and 8.29 mg L-histidine hydrochloride hydrate,

(iii) 450 mg sucrose and

(iv) 1.5 mg polysorbate 80.

[0140]

Alternatively, in terms of an aqueous solution comprising an antibody-drug conjugate at a concentration of 20 mg/mL, the pharmaceutical composition described above can be represented as a pharmaceutical composition comprising

(i) 20 mg/ml antibody-drug conjugate,

(ii) 10 mM histidine buffer,

(iii) 9% sucrose and

(iv) 0.03% polysorbate 80 and

(v) water,

where the pH of the composition is 5.4.

[0141]

The pharmaceutical composition of the present invention is preferably in the form of a composition for injection, more preferably an aqueous composition for injection or a lyophilized composition for injection, even more preferably a lyophilized composition for injection.

[0142]

In the present invention, the term "composition for injection" refers to a pharmaceutical composition for administration by needle into biological tissues, for example intravenously, intradermally, subcutaneously, intramuscularly and intraperitoneally. The injectable composition of the present invention includes not only liquid injectable compositions but also solid injectable compositions which are dissolved in liquids prior to use. For a liquid injectable composition, the solvent may be water or a solvent other than water, or a mixed solvent consisting of water and a solvent other than water.

[0143]

In the present invention, the term "aqueous composition for injection" refers to a pharmaceutical composition that is in the form of a liquid composition for injection, in which the solvent is water (preferably water for injection).

In the present invention, the term "lyophilized injectable composition" is a solid injectable composition that can be used by dissolving on application in water (preferably water for injection), which is obtained by lyophilization of an aqueous solution comprising a predetermined amount of a pharmaceutical component.

When the pharmaceutical composition of the present invention is in the form of an aqueous composition for injection, the concentration of the antibody-drug conjugate is preferably 5-60 mg/ml, more preferably 15-40 mg/ml, even more preferably 20 mg/ml.

When the pharmaceutical composition of the present invention is in the form of an aqueous composition for injection, the pharmaceutical composition of the present invention can preferably be stored frozen.

[0144]

When the pharmaceutical composition of the present invention is in the form of a lyophilized composition for injection, the pharmaceutical composition of the present invention can preferably be stored in a dark vial.

[0145]

When the pharmaceutical composition of the present invention is in the form of a lyophilized composition for injection, a kit comprising the pharmaceutical composition of the present invention and water for injection can be suitably used. In this case, water for injection may preferably be stored in an ampoule.

[0146]

A lyophilized composition for injection wherein the content of the antibody-drug conjugate used in the present invention is 20 mg can be suitably used by redissolving in 1 ml of water for injection. In addition, the lyophilized composition for injection, where the content of the antibody-drug conjugate used in the present invention is 100 mg, can be suitably used by redissolving in 5 ml of water for injection.

[0147]

When the pharmaceutical composition of the present invention is in the form of a lyophilized composition for injection, the method for preparing the pharmaceutical composition of the present invention preferably includes the steps

(1) obtaining an aqueous solution comprising predetermined amounts

(i) an antibody-drug conjugate,

(ii) histidine buffer,

(iii) sucrose or trehalose and

(iv) a surfactant,

(2) if necessary, adjust the pH of the aqueous solution, and then

(3) lyophilization of the aqueous solution.

The aqueous solution lyophilization step preferably includes an annealing process at a shelf temperature close to the eutectic point of the aqueous solution. In this case, "close to the eutectic point" means the range from a temperature that is 1.5°C below the eutectic point to a temperature that is 1.5°C above the eutectic point.

In the present invention, the term "annealing" means the process of growing ice crystals in a frozen substance at a product temperature equal to or above the glass transition temperature (Tg'). As the ice crystals get larger, the passages of water (water vapor) that sublimates upon drying become larger. This reduces sublimation resistance and can be expected to improve the shape of the lyophilized cake and reduce drying time.

In the present invention, the term "shelf temperature" means the temperature in the apparatus (in the system) for the implementation of lyophilization.

In the present invention, the term "product temperature" means the temperature of the object (product) to be lyophilized.

In the present invention, the term "lyophilized cake" means a lyophilized substance (a solid having a porous structure) obtained from a series of lyophilization processes.

Annealing as described above can suitably be carried out from a temperature of 1.5°C below the eutectic point of the aqueous solution to a temperature of 1.5°C above the eutectic point, more preferably can be carried out at a shelf temperature that is equal to the eutectic point of the aqueous solution.

These preparation methods are preferably characterized in that the time for the primary drying process is reduced compared to when the annealing is carried out at a shelf temperature that is 5° C. below the eutectic point. In addition, these preparation methods are preferably characterized in that a lyophilized cake is obtained having less shrinkage than that obtained by annealing at a shelf temperature that is 5° C. below the eutectic point.

[0148]

Since the eutectic point of the pharmaceutical composition of the present invention is in the range of about -3°C to about -1°C, annealing can preferably be carried out at a shelf temperature of -4.5°C to 0.5°C, more preferably at a shelf temperature -4 to -1°C, even more preferably at a shelf temperature of -4 to -2°C, even more preferably at a shelf temperature of -3 to -2°C, and even more preferably at a shelf temperature of -2.5 °C

[0149]

After annealing, the first drying process and the second drying process are carried out, and then the pharmaceutical composition of the present invention is obtained in the form of a lyophilized composition for injection.

[0150]

In the present invention, the term "primary drying process" is one of the processes for obtaining a lyophilized composition for injection, and means the process of sublimation of free water in a frozen product.

In the present invention, the term "secondary drying process" is one of the processes for obtaining a lyophilized composition for injection, and means the process of sublimation of water associated with a solute (bound water).

[0151]

The primary drying process can be carried out preferably at a shelf temperature of -5 to 5°C under a vacuum of 5 to 15 Pa, more preferably at a shelf temperature of -4 to 4°C under a vacuum of 7 to 13 Pa, even more preferably at shelf temperature from -3 to 3°C under vacuum conditions from 8 to 12 Pa, even more preferably at shelf temperature from -2 to 2°C under vacuum conditions from 9 to 11 Pa, even more preferably at shelf temperature from -1 to 1°C under a vacuum of 9 to 11 Pa, and even more preferably at a shelf temperature of 0°C under a vacuum of about 10 Pa.

[0152]

The secondary drying process can be carried out preferably at a shelf temperature of 40 to 50°C under a vacuum of 5 to 15 Pa or no vacuum, more preferably at a shelf temperature of 42 to 48°C under a vacuum of 7 to 13 Pa, even more preferably at at a shelf temperature of 44-46°C under a vacuum of 9 to 11 Pa, and even more preferably at a shelf temperature of 45°C under a vacuum of 10 Pa.

[0153]

The quality of the pharmaceutical compositions of the present invention can be confirmed by determining the storage stability after 1 month, 3 months, 6 months, 12 months, 18 months, 24 months or 36 months at 50°C, at 40°C/75%RH, at 25°C/60%RH or at 5°C using protein concentration, moisture, insoluble microparticles, DAR, monomer, aggregate and fragment content, NPI, IoP, charged isomers and pH as indicators. In addition, depending on the type of antibody in the antibody-drug conjugate, the quality can be determined using as an indicator, for example, the activity or strength of binding (Bioassay) to the antigen. In addition, it is also possible to perform quality evaluation using drug distribution or rCE-SDS as an indicator, and quality evaluation by observing properties (appearance). When the pharmaceutical composition is in the form of an aqueous composition for injection, the quality can be determined using the osmotic pressure as an indicator, and when the pharmaceutical composition is in the form of a lyophilized composition for injection, the quality can be determined using the re-dissolution time as an indicator. Using such quality ratings, it is also possible to confirm the superiority of the pharmaceutical composition of the present invention over existing pharmaceutical compositions.

The lyophilization method of the present invention is expected to have wide applicability, not only for the pharmaceutical composition comprising the antibody-drug conjugate used in the present invention, but also for an aqueous solution containing sucrose or trehalose.

Thus, a process for preparing a lyophilized injectable composition comprising a process of annealing an aqueous solution containing sucrose or trehalose at a shelf temperature close to the eutectic point of the aqueous solution, where "near the eutectic point" means a range from a temperature that is 1.5°C below the eutectic point, up to a temperature that is 1.5°C above the eutectic point, is also included in the scope of the present invention.

In addition, the annealing can suitably be carried out at a shelf temperature equal to the eutectic point of aqueous solutions containing sucrose or trehalose.

These preparation methods are preferably characterized in that the time for the primary drying process is reduced compared to when annealing is carried out at a shelf temperature that is 5° C. below the eutectic point.

[0154]

In addition, these preparation methods are preferably characterized in that a lyophilized cake is obtained having less shrinkage than that obtained by annealing at a shelf temperature that is 5° C. below the eutectic point.

[0155]

[Application of pharmaceutical composition]

The pharmaceutical composition of the present invention can be expected to exhibit a therapeutic effect when used as a systemic therapy for patients and furthermore when applied topically to cancerous tissues.

[0156]

The pharmaceutical composition of the present invention can preferably be used in a mammal, but more preferably it is used in a human.

[0157]

Examples of the route of administration that can be used to administer the pharmaceutical compositions of the present invention include intravenous, intradermal, subcutaneous, intramuscular and intraperitoneal routes, but the preferred route of administration is intravenous.

[0158]

When the pharmaceutical compositions of the present invention are in the form of an aqueous injectable composition, the aqueous injectable composition may preferably be administered intravenously after dilution with a suitable diluent. Examples of the diluent include glucose solution (preferably 5% glucose solution) and saline.

[0159]

When the pharmaceutical composition of the present invention is in the form of a lyophilized injectable composition, the lyophilized injectable composition can be dissolved in water (preferably water for injection) and administered intravenously after dilution with a suitable diluent. Examples of the diluent include glucose solution (preferably 5% glucose solution) and saline.

[0160]

The pharmaceutical composition of the present invention can exhibit a pharmaceutical effect even at a low dosage when the antibody-drug conjugate has a higher affinity for the antigen, that is, expressed as a dissociation constant (Kd value), a higher affinity (lower Kd value) for the antigen for the conjugate antibody drug of the present invention. Thus, the dosage of the pharmaceutical composition of the present invention can be determined in view of the antigen affinity situation. When the pharmaceutical composition of the present invention is administered to a human, for example, about 0.001-100 mg/kg as an antibody-drug conjugate, where "mg/kg" means the dose of the antibody-drug conjugate per 1 kg of human body weight, it can be administered one once or you can enter several parts with an interval of 1-180 days. For example, when the antibody drug conjugate used in the present invention is an anti-HER2 antibody drug conjugate, examples of the method of administering the pharmaceutical composition of the present invention include administering 0.8 mg/kg to 8 mg/kg every three weeks , more preferably 0.8 mg/kg, 1.6 mg/kg, 3.2 mg/kg, 5.4 mg/kg, 6.4 mg/kg, 7.4 mg/kg or 8 mg/kg times at three weeks, even more preferably 5.4 mg/kg, 6.4 mg/kg, 7.4 mg/kg or 8 mg/kg every three weeks, and even more preferably 5.4 mg/kg, 6, 4 mg/kg, 7.4 mg/kg or 8 mg/kg every three weeks, and even more preferably 5.4 mg/kg and 6.4 mg/kg every three weeks.

[0161]

The pharmaceutical composition of the present invention can be used to treat cancer, preferably it can be used to treat at least one cancer selected from the group consisting of breast cancer, gastric cancer (also called gastric adenocarcinoma), colorectal cancer (also called colon cancer). and rectum, including colon and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, adenocarcinoma of the gastroesophageal junction, bile duct cancer, Paget's disease, pancreatic cancer, cancer ovarian cancer, uterine sarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, cancer uterus, kidney cancer, vaginal cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neural epithelial tissue tumors, neural sheath tumors, head and neck cancer, skin cancer, throat cancer, gallbladder cancer, bile duct cancer, mesothelioma and sarcoma. For example, when the antibody drug conjugates used in the present invention are an anti-HER2 antibody drug conjugate, the pharmaceutical compositions of the present invention can more preferably be used to treat at least one cancer selected from the group consisting of breast cancer. gland, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer and uterine sarcoma; more preferably can be used to treat at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, and Paget's disease; and even more preferably can be used to treat breast cancer, gastric cancer, colorectal cancer or non-small cell lung cancer

[0162]

The pharmaceutical composition of the present invention can be selectively used as a drug therapy, which is the main treatment for cancer and, as a result, can delay the development of cancer cells, inhibit their growth, and further kill cancer cells. These effects may enable cancer patients to be free of symptoms caused by cancer, or to achieve an improvement in the quality of life (QOL) of cancer patients and achieve a therapeutic effect, maintaining the life of cancer patients. Even if the pharmaceutical composition and method of treatment of the present invention do not kill cancer cells, they can achieve higher QOL in cancer patients while achieving longer survival by inhibiting or controlling cancer cell growth.

[0163]

In such drug therapy, the pharmaceutical composition of the present invention can be used alone, and furthermore, it can be used in combination with additional therapy in adjuvant therapy, and can be combined with surgery, radiation therapy, hormonal therapy, and the like. In addition, it can also be used for drug therapy in neoadjuvant therapy.

[0164]

In addition to the therapeutic use as described above, for the pharmaceutical composition according to the present invention, for example, a prophylactic effect such as suppressing the growth of small metastatic cancer cells and further killing them can be expected. For example, an effect of inhibiting and killing cancer cells in body fluid during metastasis, or an effect such as inhibiting and killing small cancer cells immediately after implantation in any tissue can be expected. Accordingly, an inhibition of cancer metastasis or a prophylactic effect can be expected, in particular after surgical removal of the cancer.

[0165]

The pharmaceutical composition of the present invention may be administered in combination with other cancer treatments. The anti-cancer effect can be correspondingly enhanced. Examples of other cancer treatments used for such purposes include 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), docetaxel, pemetrexed, sorabenfib, vinblastine , vinorelbine, everolims, tanespimycin, bevacizumab, oxaliplatin, lapatinib, trastuzumab emtansine (T-DM1) or agents described in International Publication No. WO 2003/038043, LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate , estrogen antagonists (tamoxifen, raloxifene, etc.) and aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), but are not limited to these, provided that they are agents having antitumor activity.

Examples

[0166]

The present invention is specifically described using the examples below. However, the present invention is not limited to this. Also, this should in no way be considered a limitation.

[0167]

[Example 1: Preparation of an antibody-drug conjugate (1)]

According to the production method described in International Publication No. WO 2015/115091, using a humanized anti-HER2 antibody (trastuzumab), an antibody-drug conjugate was prepared in which the drug-linker moiety represented by the following formula:

[0168]

[Formula 14]

[0169]

where A is the position of binding to the antibody, conjugated to the antibody through a thioether bond (hereinafter referred to as the antibody-drug conjugate (1)). The antibody-drug conjugate (1) had a DAR value of 7.8.

[0170]

[Example 2: Preparation of the composition of the antibody-drug conjugate (1)]

The method of obtaining the composition (1)

An aqueous solution (pH 5.8) containing antibody-drug conjugate (1) (20 mg/ml), 10 mM histidine buffer, 11.1% trehalose hydrate and 0.02% polysorbate 20 was measured into a dialysis cassette (dialysis membrane, Slide-A-lyzer, MWCO 20,000) and dialyzed with a buffer containing the excipient of the target composition at a volume exceeding the volume of the sample introduced into the dialysis cassette by 50 times or more. Dialysis was carried out twice at 5°C for 5 hours or more. After dialysis, concentration or dilution was carried out, respectively, to obtain a protein concentration of about 20 mg/ml. This solution was filtered through a 0.22 µm filter. The filtered solution was filled into 1 ml glass vials, the vials were loosely closed with rubber stoppers, and lyophilization was carried out under the conditions shown in Table 1. After lyophilization, the vials closed with rubber stoppers were sealed with caps. The filtration and filling procedures were carried out in a clean box.

[0171]

Table 1 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -5 60 6 -5 120 7 -40 60 8 -40 120 Primary drying 9 -20 60 10 10 -20 1200-1800 Secondary drying eleven thirty 120 No vacuum control 12 thirty 900-1800 13 5 60

[0172]

The method of obtaining the composition (2)

An aqueous solution (pH 5.8) containing antibody-drug conjugate (1) (20 mg/ml), 10 mM histidine buffer, 11.1% trehalose hydrate, and 0.02% polysorbate 20 was distributed in a set of UF membranes (AMICON ULTRA-15, 30kDa) and centrifuged at 3000 rpm and 5°C until the concentration decreased by about 2 times. Polysorbate 20 contained in a concentrated solution was removed by adsorption on a hydrophobic adsorbent (Amberlite XAD7HP). The resulting chemical solution after removal of polysorbate 20 was injected into a dialysis cassette (dialysis membrane, Slide-A-lyzer, MWCO 20,000) and dialyzed with a buffer containing the excipient of the target composition at a volume exceeding the volume of the sample introduced into the dialysis cassette by 50 times or more . Dialysis was carried out twice at 5°C for 5 hours or more. After dialysis, respectively, concentration or dilution was carried out to obtain a protein concentration of about 20 mg/ml, and then polysorbate 20 or polysorbate 80 was added to the resulting solution and mixed. This solution was filtered through a 0.22 µm filter. The filtered solution was filled into 1 ml glass vials, the vials were loosely closed with rubber stoppers, and lyophilization was carried out under the conditions shown in Table 2. After lyophilization, the vials closed with rubber stoppers were sealed with caps. The filtration and filling procedures were carried out in a clean box.

[0173]

table 2 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -7 60 6 -7 120 7 -40 60 8 -40 120 Primary drying 9 -20 60 No vacuum control 10 -20 2400 Secondary drying eleven 40 120 12 40 900 13 5 60

[0174]

[Example 3: Stability test]

Lyophilized compositions for injection were stored at 40°C/75% RH for 3 months. Their stability was compared and evaluated using protein concentration (measured by UV), moisture (measured by Karl Fischer), insoluble microparticles (measured by Micro Flow Imaging), drug to antibody ratio (DAR) (measured by HPLC), relative content of monomers, aggregates and fragments (measured by SE-HPLC (SEC)), non-protein impurities (NPI) (measured by HPLC), payload contamination (IoP) (measured by HPLC), charged isomers (measured by capillary zone electrophoresis ( CZE)), pH and HER2 binding activity (measured by ELISA).

[0175]

[Example 4: Screening of excipients]

The excipient of the lyophilized compositions for injection was chosen from 10% trehalose hydrate, 9% sucrose and 5% sorbitol, which are actually used in biopharmaceuticals and at these concentrations make the osmotic pressure essentially isotonic. The other components in the lyophilized formulations for injection typically included antibody-drug conjugate (1) (20 mg/ml), 40 mM histidine buffer and 0.02% polysorbate 20, and the pH of the formulations was adjusted to 5.8.

[0176]

The results are shown in Table 3. Aggregate formation was found to be less when 9% sucrose or 10% trehalose hydrate was used compared to when 5% sorbitol was used. In particular, it was found that the formation of aggregates becomes the least when using 9% sucrose (Fig. 3).

[0177]

Table 3 Tested indicators Composition with 10% trehalose Composition with 9% sucrose Composition with 5% sorbitol 40°C/75%RH 40°C/75%RH 40°C/75%RH Initial values 1M 2m 3M Initial values 1M 2m 3M Initial values 1M 2m 3M Protein Content(mg/ml) 21.3 21.9 20.6 17.9 21.9 22.0 19.9 20.1 23.1 22.8 21.6 20.9 Humidity (%) 0.35 0.64 0.77 1.28 0.40 0.61 1.39 1.33 0.22 0.44 1.49 1.08 Insoluble microparticles
(particles/ml) ≥10 µm 19 5 83 86 26 17 167 12 N.A. 63 44 31 ≥25 µm 2 0 2 12 2 0 28 0 N.A. 5 10 0 DAR 7.8 7.7 7.7 7.8 7.8 7.8 7.7 7.8 7.8 7.8 7.7 7.8 SEC (%) Monomers 97.3 97.3 98.0 98.1 97.4 97.5 98.2 98.2 97.5 96.9 97.9 97.7 Aggregates 0.8 0.9 0.9 1.0 0.8 0.8 0.7 0.8 0.8 1.2 1.1 1.3 Fragments 1.9 1.7 1.1 1.0 1.7 1.7 1.0 1.0 1.6 1.9 1.0 1.0 NPI (%) 0.65 0.69 0.70 0.66 0.64 0.66 0.66 0.57 0.60 0.60 0.66 0.61 IoP (%) 1.02 1.02 0.99 1.29 0.92 0.99 0.85 1.71 1.00 2.84 1.10 1.88 Charged isomers (main peak %) 54.5 N.A. N.A. 56.7 53.4 N.A. N.A. 56.7 51.1 N.A. N.A. 51.3

[0178]

[Example 5: Screening buffers]

The buffer of the lyophilized formulations for injection was selected from histidine, citric acid, phosphoric acid, succinic acid and glutamic acid, which are used in biopharmaceuticals and have a buffering capacity at about pH 4 -pH 7. The concentration of these buffers was the same 25 mM. Other components in lyophilized formulations for injection typically included antibody-drug conjugates (1) (20 mg/ml), 10% trehalose hydrate and 0.02% polysorbate 20, and the pH of the formulations was adjusted to 5.8.

[0179]

The results are shown in Table 4. It was found that the formation of aggregates becomes the least when using histidine as a buffer (Fig. 4).

[0180]

Table 4 Tested indicators Composition with histidine Composition with citric acid Composition with phosphoric acid Composition with succinic acid Composition with glutamic acid 40°C/75%RH 40°C/75%RH 40°C/75%RH 40°C/75%RH 40°C/75%RH Initial values 3M Initial values 3M Initial values 3M Initial values 3M Initial values 3M Protein Content(mg/ml) 19.6 19.0 22.9 20.8 22.4 20.5 19.6 20.0 22.7 19.9 Humidity (%) 0.26 1.12 0.43 1.19 0.45 1.31 0.37 1.30 0.47 1.15 Insoluble microparticles
(particles/ml) ≥10 µm 10 19 5 24 12 34 10 12 N.A. 7 ≥25 µm 2 2 0 0 7 7 0 2 N.A. 0 DAR 7.8 7.7 7.8 7.8 7.8 7.8 7.7 7.8 7.8 7.8 SEC (%) Monomers 97.6 98.2 97.4 97.7 97.3 97.6 97.2 97.6 97.4 97.9 Aggregates 0.7 1.1 0.9 1.5 1.0 1.6 0.9 1.6 0.9 1.4 Fragments 1.7 0.7 1.6 0.7 1.7 0.8 1.8 0.7 1.7 0.7 NPI (%) 0.71 0.84 0.64 0.78 0.62 0.83 0.73 0.79 0.63 0.76 IoP (%) 1.10 1.00 1.54 2.36 1.15 2.29 1.27 2.96 1.35 0.94 Charged isomers (main peak %) 53.4 57.9 58.8 58.4 58.2 58.7 58.6 56.3 55.9 58.4

[0180]

[Example 6: pH Screening]

For the pH of the reconstituted freeze-dried formulations for injection, the pH was selected from pH 4 - pH 7 considering the use of a histidine buffer, which was selected by buffer screening, and biocompatibility and safety, eg, irritation when administered by intravenous drip infusion. Other components in lyophilized formulations for injection typically included antibody-drug conjugates (1) (20 mg/ml), 9% sucrose and 25 mM histidine buffer, and 0.02% polysorbate 20. Samples were obtained by the method of preparing composition (1).

[0182]

The results are shown in Table 5. It was found that with increasing pH, the number of aggregates increased (Fig. 5), and with decreasing pH, the content of NPI increased (Fig. 6). Therefore, it has been found that it is possible to suppress the increase in the content of aggregates and to suppress the increase in the content of NPI with a good balance when the pH is 5.5.

[0183]

Table 5 Tested indicators Composition with pH 4.0 Composition with pH 5.0 Composition with pH 5.5 Composition with pH 6.0 Composition with pH 7.0 40°C/75%RH 40°C/75%RH 40°C/75%RH 40°C/75%RH 40°C/75%RH Initial values 3M Initial values 3M Initial values 3M Initial values 3M Initial values 3M Protein Content(mg/ml) 18.1 18.4 18.2 19.0 18.7 19.1 18.4 19.1 18.9 19.2 Humidity (%) 0.64 1.35 0.41 1.26 0.54 1.28 N.D. 1.29 0.63 1.29 Insoluble microparticles
(particles/ml) ≥10 µm 36 7 0 10 17 7 38 14 19 15 ≥25 µm 2 0 0 2 0 0 5 0 2 0 DAR 7.7 7.7 7.7 7.7 7.8 7.7 7.7 7.7 7.7 7.7 SEC (%) Monomers 99.0 98.8 98.9 98.7 98.8 98.7 98.8 98.6 98.5 98.0 Aggregates 0.5 0.7 0.6 0.7 0.7 0.8 0.7 0.9 1.1 1.4 Fragments 0.5 0.5 0.4 0.5 0.5 0.5 0.5 0.5 0.4 0.5 NPI (%) 0.82 1.02 0.77 0.92 0.78 0.83 0.67 0.78 0.63 0.63 IoP (%) 1.18 0.77 0.74 0.82 0.84 0.96 0.94 1.27 0.90 2.12 Charged isomers (main peak %) 61.6 62.9 62.4 62.9 60.4 62.6 59.1 61.0 51.5 57.1

[0184]

[Example 7: Buffer Concentration Test]

The concentration of histidine buffer, which was chosen as the buffer of the lyophilized compositions for injection, was chosen from the range of 10 mm - 40 mm. Other components in lyophilized injectable formulations typically included antibody-drug conjugates (1) (20 mg/ml), 9% sucrose and 0.02% polysorbate 20, and the pH of the formulations was 5.5. Sample preparation was carried out in accordance with the method for obtaining composition (1).

[0185]

The results are shown in Table 6. Any of the samples showed comparable stability when the concentration of histidine buffer was 10 mm - 40 mm.

[0186]

Table 6 Tested indicators Histidine buffer concentration 10mM 25mM 40mM 40°C/75%RH 40°C/75%RH 40°C/75%RH Initial values 3M Initial values 3M Initial values 3M Protein Content(mg/ml) 19.2 19.2 18.7 19.1 18.5 19.0 pH 5.59 5.55 5.52 5.47 5.56 5.51 Humidity (%) 0.44 1.27 0.54 1.28 0.50 1.28 Insoluble microparticles
(particles/ml) ≥10 µm 10 19 17 7 14 17 ≥25 µm 0 0 0 0 0 5 DAR 7.7 7.7 7.8 7.7 7.8 7.7 SEC (%) Monomers 98.9 98.7 98.8 98.7 98.8 98.7 Aggregates 0.6 0.8 0.7 0.8 0.7 0.8 Fragments 0.5 0.5 0.5 0.5 0.5 0.5 NPI (%) 0.72 0.84 0.78 0.83 0.73 0.86 IoP (%) 0.81 1.01 0.84 0.96 0.90 0.98 Charged isomers (main peak %) 59.6 61.6 60.4 62.6 60.4 61.9

[0187]

[Example 8: Surfactant Screening]

In a lyophilized injectable formulation, the surfactant is expected to serve as the interfacial protectant of the protein concentration layer during the freezing process, or as an interfacial protectant against microbubbles generated during the re-dissolution of the lyophilized preparation. With this in mind, 0.02% - 0.04% polysorbate 20 (Tween 20) and polysorbate 80 (Tween 80) were investigated as surfactants. Other components in lyophilized formulations for injection typically included antibody-drug conjugates (1) (20 mg/ml), 9% sucrose and 25 mM histidine buffer, and the pH of the formulations was adjusted to 5.5. The preparation of samples was carried out in accordance with the method for obtaining the composition (2).

[0188]

The results are shown in Tables 7 and 8 (where "PS" means polysorbate, "PS20" means polysorbate 20 and "PS80" means polysorbate 80. Reduction in fines was observed when using 0.02% - 0.04% polysorbate 20 or 80, and it was found that the use of 0.02% - 0.04% polysorbate 80 especially reduces the number of microparticles (Fig. 7).

[0189]

Table 7 Tested indicators 0% PS 0.02% PS20 0.04% PS20 40°C/75%RH 40°C/75%RH 40°C/75%RH Initial values 1M 2m 3M Initial values 1M 2m 3M Initial values 1M 2m 3M Protein Content(mg/ml) 17.8 18.5 18.3 17.9 18.2 18.6 18.6 18.4 18.8 18.9 19.1 18.8 pH 5.6 5.6 5.5 5.5 5.6 5.6 5.5 5.5 5.6 5.5 5.5 5.5 Humidity (%) 0.4 0.6 0.9 1.1 0.3 0.6 0.8 1.1 0.4 0.7 0.9 1.1 Insoluble microparticles
(particles/ml) <10 µm 132 3120 3788 7570 314 2832 2214 3419 154 2810 1334 1107 ≥10 µm 0 81 138 72 2 0 83 61 2 16 89 44 ≥25 µm 0 22 28 0 0 0 6 eleven 0 0 eleven 17 DAR 7.7 7.7 7.6 7.7 7.7 7.7 7.6 7.6 7.7 7.7 7.6 7.7 SEC (%) Monomers 98.8 98.8 98.8 98.8 98.8 98.7 98.7 98.8 98.8 98.8 98.8 98.8 Aggregates 0.7 0.8 0.8 0.8 0.7 0.8 0.8 0.8 0.7 0.8 0.8 0.8 Fragments 0.5 0.4 0.4 0.4 0.5 0.5 0.4 0.4 0.5 0.4 0.4 0.4 NPI (%) 0.00 0.05 0.07 0.09 0.00 0.05 0.07 0.09 0.00 0.05 0.07 0.10 IoP (%) 0.78 0.79 0.94 0.83 0.78 0.79 0.98 0.81 0.79 0.83 0.99 0.82 HER2 binding activity (%) 86 n.t. n.t. 83 82 n.t. n.t. 78 n.t. n.t. n.t. n.t.

[0190]

Table 8 Tested indicators 0.02% PS80 0.04% PS80 40°C/75%RH 40°C/75%RH Initial values 2m 3M Initial values 1M 2m 3M Protein Content(mg/ml) 18.7 18.8 19.3 18.7 18.7 18.9 19.2 18.7 pH 5.6 5.5 5.5 5.6 5.6 5.6 5.6 5.5 Humidity (%) 0.4 0.6 0.9 1.2 0.4 0.8 0.9 1.1 Insoluble microparticles
(particles/ml) <10 µm 918 2663 476 1068 134 2411 694 1030 ≥10 µm 7 38 28 28 5 22 eleven 83 ≥25 µm 5 0 0 17 0 6 6 33 DAR 7.7 7.7 7.7 7.6 7.7 7.7 7.6 7.6 SEC (%) Monomers 98.8 98.8 98.8 98.8 98.8 98.8 98.8 98.8 Aggregates 0.7 0.8 0.8 0.8 0.7 0.8 0.8 0.8 Fragments 0.5 0.4 0.4 0.4 0.5 0.4 0.4 0.4 NPI (%) 0.00 0.05 0.07 0.09 0.00 0.05 0.07 0.09 IoP (%) 0.75 0.86 0.99 0.82 0.79 0.82 0.97 0.85 HER2 binding activity (%) 86 n.t. n.t. 87 n.t. n.t. n.t. n.t.

[0191]

[Example 9: Vial Examination]

Each of the dark glass vials and the clear glass vials were loaded with the lyophilized composition for injection and compared and examined the change in the quality of the composition when exposed to light under a 1000 lux white fluorescent lamp. The components of lyophilized injectable formulations typically included antibody-drug conjugate (1) (20 mg/ml), 9% sucrose, 25 mM histidine buffer and 0.02% polysorbate 20, and the pH of the formulations was adjusted to 5.5. The preparation of samples was carried out in accordance with the method for obtaining the composition (1).

The results are shown in Table 9. Aggregation (FIG. 8) and IoP (FIG. 9) were found to be more suppressed when dark glass vials were used compared to when clear glass vials were used.

[0192]

Table 9 Tested indicators Transparent bottle Dark Flask white fluorescent lamp 1000 lux white fluorescent lamp 1000 lux 0h 8h 24h 48h 72h 144h 168h 0h 72h 168h Protein Content(mg/ml) 18.9 19.2 19.3 19.4 18.7 19.1 19.3 18.7 18.9 18.9 pH 5.54 5.49 5.52 5.51 5.51 5.50 5.47 5.52 5.50 5.47 DAR 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.8 7.6 7.7 SEC (%) Monomers 98.5 96.5 95.2 94.0 93.1 91.7 91.6 98.8 97.3 96.8 Aggregates 0.7 2.8 4.1 5.3 6.2 7.6 7.7 0.7 2.1 2.7 Fragments 0.7 0.7 0.6 0.6 0.6 0.6 0.6 0.5 0.6 0.5 NPI (%) 0.84 0.72 0.71 0.74 0.74 0.79 0.74 0.78 0.70 0.75 IoP (%) 0.98 1.95 2.07 2.33 2.55 3.53 3.20 0.84 1.28 1.15

[0193]

[Example 10: Establishment of the lyophilization method]

Important physical properties as indicators in determining the method of lyophilization of compositions for injection are the glass transition temperature at which freezing (Tg') of the drug-containing aqueous solution to be lyophilized takes place, and the breaking temperature (Tc) of the obtained cake having a porous structure, which is obtained in the lyophilization process. By drying at a product temperature equal to or less than Tg' or Tc, a lyophilized mass is obtained having a porous cake-like structure. The drying process mainly consists of a first drying step for sublimating free water in the frozen substance and a secondary drying step for sublimating water associated with solutes (bound water). In the primary drying process, it is expedient that free water sublimates at a product temperature equal to or less than Tg', and it is also possible to sublimate at a product temperature equal to or less than Tc.

For an aqueous solution comprising antibody-drug conjugate (1) (20 mg/ml), 9% sucrose, 25 mM histidine buffer and 0.03% polysorbate 80 and having a pH of 5.5 (hereinafter referred to as "aqueous solution (1 )"), Tg' is -31°C, and Tc is -27°C.

The study of lyophilization methods was carried out by introducing 5.2-5.5 ml of an aqueous solution (1) into glass vials having a volume of about 10 ml.

[0194]

(1) Investigation of the parameters of the primary drying process

The most important parameters of the primary drying process are the temperature of the shelf and the degree of evacuation of the chamber. The shape (stability) and re-dissolution capacity of the lyophilized cake and the drying time significantly depend on these parameters, thus the shelf temperature in the range of -20°C to 0°C and the vacuum degree of the chamber in the range of 5 Pa to 15 Pa were studied with using a specially developed experimental method. Lyophilization was carried out using the parameters shown in Table 9 (shelf temperature at annealing was -7°C).

[0195]

Table 10 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -7 60 6 -7 120 7 -40 60 8 -40 120 Primary drying 9 -20 to 0 60 5-15 10 -20 to 0 2400-4800 Secondary drying eleven thirty 60-120 No vacuum control 12 thirty 480-600 unloading 13 5 60

[0196]

Fig. 10 shows the contour curve of the temperature of the aqueous solution product (1) by varying the temperature of the shelf and the degree of evacuation of the chamber during the primary drying process. The temperature of the product showed a general trend in product temperature, characterized by the fact that under conditions of low shelf temperature and high vacuum of the chamber, the temperature of the product during the primary drying process becomes lower, while under conditions of high temperature of the shelf and low vacuum of the chamber, the temperature of the product becomes higher. At a shelf temperature of -20°C - 0°C and a chamber vacuum degree of 5 Pa - 15 Pa, the product temperature showed Tc or less. However, it has been found that partial failures occur in the porous structure of the lyophilized cake at parameters outside of the triangulated portion and the appearance of the lyophilized cake becomes compressed (shrinkage).

Fig. 11 shows the contour curve of the drying time of the aqueous solution (1) by varying the temperature of the shelf and the degree of evacuation of the chamber during the primary drying process. The drying time showed a general trend in the drying speed, characterized by the fact that under conditions of low temperature of the shelf and a high vacuum degree of the chamber, the primary drying time becomes longer, while under the conditions of high temperature of the shelf and low degree of vacuum of the chamber, the drying time becomes shorter. A relationship was observed between drying time and product temperature. With regard to the parameters in the triangle area of the product temperature contour plot, it has been found that a good shaped lyophilized cake can be obtained, but this requires a long primary drying process.

Since it is desirable that the form of the lyophilized cake does not have a shrinkage state in terms of appearance quality, based on the results of this study, it became apparent that the primary drying process takes at least 58 hours. Thus, it was concluded that the total lyophilization time is 81 hours, since the freezing process requires at least 11 hours, and the secondary drying and unloading processes require at least 12 hours. In other words, it was calculated that the lyophilization process required 3 days or more and that the manufacturing time required 5 days when adding the start date and end date of lyophilization.

However, for lyophilization, a short time or a short period is desirable in terms of production efficiency and manufacturing cost. Thus, the need for a long freeze-drying process was considered a problem.

In order to solve this problem and obtain a lyophilized cake having less shrinkage even when the primary drying process time is shortened, an annealing parameter study was carried out focusing on the annealing step, which can be expected to improve the pore structure of the lyophilized cake.

[0197]

(2) Setting the annealing parameters

An important parameter in the annealing process is the shelf temperature. By changing the temperature of the shelf, the temperature of the product of the frozen substance can be adjusted.

In this study, the annealing shelf temperature was optimized to take into account the porous structure of the lyophilized cake resulting from the sublimation of ice in ice crystals. The annealing shelf temperature has traditionally been set so that there is a margin of safety in terms of eutectics. With this in mind, the annealing was carried out at a shelf temperature of -7°C, since the eutectic point of the aqueous solution (1) is -2.5°C. In addition, in order to grow ice crystals in the frozen substance and reduce the resistance of ice to sublimation so that drying proceeds smoothly and then to obtain a porous structure having a small specific surface area that causes almost no shrinkage in the lyophilized cake, attempts have also been made to anneal at a temperature shelves above -7°C. Accordingly, by setting the temperature range of the annealing shelf from -7° C. to 0.5° C. to obtain a porous structure having a small specific surface area, improvement in appearance of the lyophilized cake was investigated. Lyophilization in this study was carried out using the parameters shown in Table 11, and the primary drying time was set to a total of about 42 hours.

[0198]

Table 11 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -7.0 to 0.5 60 6 -7.0 to 0.5 120 7 -40 60 8 -40 120 Primary drying 9 -15 60 5 10 -15 300 eleven -10 thirty 12 -10 300 13 -5 thirty 14 -5 300 15 0 thirty 16 0 1500 Secondary drying 17 40 60 5 18 40 600 unloading 19 5 60 5

[0199]

Annealing was carried out at a shelf temperature in the range from -7°C to 0.5°C. The product temperature (at annealing) and the shape of the lyophilized cake when lyophilization was performed are shown in FIG. 12. When the product temperature at annealing was in the range of -7.0°C to -4.6°C, strong shrinkage of the lyophilized cake was observed from the bottom of the vial in the lower direction, and when the product temperature was in the range of -4.2 °C to -3.4°C, slight shrinkage was observed. However, when the temperature of the product was in the range of -2.6°C to -1.5°C, a lyophilized cake having no shrinkage was obtained. Based on these results, it was found that when annealing is carried out at a shelf temperature closer to the eutectic point, shrinkage of the lyophilized cake is reduced, and when annealing is carried out at a shelf temperature near the eutectic point, a lyophilized cake having no shrinkage can be obtained. In addition, it was found that even when the temperature of the product is raised to -1.5°C, i.e. above the eutectic point, the solid state is maintained and a normal shaped lyophilized cake can be obtained.

Although annealing was generally carried out at a shelf temperature about 5° C. below the eutectic point, the present study showed that by annealing at a temperature close to the eutectic point, a lyophilized cake having a better appearance can be obtained. Based on the results of this study, the annealing temperature was set to -4°C to -2°C, which is a temperature close to the eutectic point.

In addition, based on the results of this study, it was found that by annealing at a temperature close to the eutectic point, the time of the primary drying process can be reduced by about 40 hours. Based on the results shown in FIG. 10 and 11, a shelf temperature of -5°C and a chamber vacuum of 7 Pa were set as the primary drying parameters, as this was seen as a condition to allow drying at a relatively low product temperature and is expected to reduce the drying time by about 18 hours.

[0200]

(3) Setting the parameters of the secondary drying process

The secondary drying process is a process for drying water associated with a solute (bound water), and it is generally known that the degree of drying after secondary drying greatly affects the storage stability of a lyophilized composition for injection. Although the amount of bound water can be reduced by drying at higher temperatures, the antibody portion of the antibody-drug conjugate is generally heat-labile. In view of this, the onset of thermal denaturation of proteins was measured using a capillary differential scanning calorimeter. As shown in FIG. 13, the onset of thermal denaturation of the antibody-drug conjugate (1) occurred at 52-53°C, so dryness (residual moisture) was evaluated when the secondary drying shelf temperature was set to 50°C or less.

Dryness evaluation was carried out at the temperature of the shelf of the secondary drying process in the range from 30 to 50°C. Lyophilization in this study was performed using the parameters shown in Table 12.

[0201]

Table 12 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -7 60 6 -7 120 7 -40 60 8 -40 120 Primary drying 9 -10 to 0 60 7.5-15 10 -10 to 0 2400 Secondary drying eleven 30 to 50 60 No vacuum control 12 30 to 50 600 unloading 13 5 60

[0202]

The results of the analysis of residual moisture in the lyophilized compositions for injection at each temperature of the secondary drying rack are shown in Table 13. It was confirmed that the stability is maintained until the residual moisture in the lyophilized composition for injection is 5%. However, in order to achieve a higher level of stability, the residual moisture in the composition was set to 1% or less. Although the residual moisture is more or less dependent on the primary drying conditions, it has been confirmed that the residual moisture content becomes 1% or less when the temperature of the secondary drying shelf is in the range of 40°C to 50°C. Based on the onset of thermal denaturation and the results of this study, an intermediate value of 45°C with a range of 40°C to 50°C was established as the temperature of the shelf in the secondary drying.

[0203]

Table 13 Secondary drying shelf temperature (°C) Residual Moisture(%) Primary drying Shelf temperature (°C) Chamber vacuum degree (Pa) thirty 1.1 -10 7.5 1.2 0 7.5 1.7 -10 15 1.9 0 15 40 0.8 -10 7.5 1.1 -10 15 0.9 -5 10 1.0 0 7.5 1.2 0 15 50 0.4 -10 7.5 0.7 0 7.5 0.6 -10 15 0.9 0 15

[0204]

(4) Conclusion

Excipients in lyophilized injectable biopharmaceutical formulations (protein pharmaceuticals) have a cryoprotective effect on proteins, and non-reducing disaccharides such as sucrose and trehalose, which do not have the risk of Maillard reaction during storage, are usually chosen for pharmaceutical formulations. These disaccharides have lower Tg' and Tc than other saccharides, so they require drying at low shelf temperatures to keep the product temperature below the Tg' and Tc. Thus, there are problems that the lyophilization process becomes longer and that the lyophilized cake tends to shrink. It was also believed that the aqueous solution (1) has the same problems, therefore, in order to solve these problems, studies were carried out on the first drying, the annealing process, and the secondary drying process, and based on the results of these studies, the lyophilization method of the aqueous solution (1) was determined as shown in Table 14. A feature of this lyophilization method is the annealing parameters, and it has been found that by annealing at a temperature close to the eutectic point, a lyophilized cake having significantly reduced shrinkage can be obtained in a short period of time.

[0205]

Table 14 Process Stage No. Shelf temperature (°C) Time (min) Vacuum degree of the chamber (Pa) Freezing 1 5 thirty without vacuum 2 5 120 3 -40 60 4 -40 120 5 -4 to -2 60 6 -4 to -2 120 7 -40 60 8 -40 120 Primary drying 9 -5 60 7 10 -5 2400 Secondary drying eleven 45 180 7 12 45 480 unloading 13 5 60

[0206]

For lyophilized injectable compositions prepared by the lyophilization process of the present invention, storage stability after 1 month at 50°C, 3 months at 40°C/75% RH, 3 months at 25°C/60% RH, and 3 months at 5 °C was evaluated using protein concentration, moisture, insoluble microparticles, DAR, monomer, aggregate and fragment content, NPI, IoP, charged isomers, and pH as indicators. As a result, it was found that no significant changes in quality were observed.

[0207]

[Example 11: Evaluation of long-term storage stability]

For the lyophilized injectable composition prepared in Example 10, storage stability after 6 months at 40°C/75% RH, 12 months at 25°C/60% RH, and 18 months at 5°C was evaluated using concentration protein, moisture, amount of insoluble microparticles, DAR, content of monomers, aggregates and fragments, NPI, IoP, charged isomers and pH. As a result, it was found that no significant changes in quality were observed.

[0208]

[Example 12: Additional study of the lyophilization method]

(1) Setting the annealing parameters

As a lyophilization method for aqueous solutions (1) for growing ice crystals in a frozen substance, an annealing method was used while maintaining the shelf temperature at -2.5°C for 120 minutes. However, it was found that within 120 minutes it was not possible to raise the temperature at the top of the frozen product to -2.5°C, thus, the growth of ice crystals becomes insufficient and the freeze-dried cake shrinks slightly. In addition, it was found that when the annealing time is increased to 240 minutes, the temperature of the top of the frozen product reaches -2.5°C, so the set annealing time was changed to 4 hours. It was found that when the annealing time is changed to 4 hours, the growth of ice crystals becomes sufficient, so the rate of water sublimation increases, and the primary drying time is reduced.

[0209]

(2) Setting the parameters of the primary drying process

Annealing was carried out at -2.5°C for 4 hours and the parameters of the primary drying process were investigated for shelf temperatures from -10°C to 15°C and the degree of vacuum chamber from 4 Pa to 30 Pa. Lyophilization was carried out using the parameters shown in Table 15.

[0210]

Table 15 Process Stage No. Shelf temperature (°C) Time (min) Vacuum degree of the chamber (Pa) Freezing 1 5 120 without vacuum 2 -40 60 3 -40 120 4 -2.5 60 5 -2.5 240 6 -40 60 7 -40 120 Primary drying 8 -10 to 15 60 4-30 9 -10 to 15 2100-3840 Secondary drying 10 45 180 4-30 eleven 45 480 unloading 12 5 60 4-30 13 5 3600

[0211]

When the shelf temperature was in the range of -10°C to 10°C and the vacuum degree of the chamber was 4 Pa to 30 Pa, the product temperature was Tc or less, and the lyophilized cake was shaped without any shrinkage or breakage.

[0212]

In particular, a shelf temperature of -5°C to 5°C and a chamber vacuum degree of 5 Pa to 15 Pa were considered particularly safe ranges, and, considering the drying time, a shelf temperature of 0°C and a degree of vacuum chamber 10 Pa, which are intermediate values in these ranges.

[0213]

(3) Conclusion

Based on the above results, the aqueous solution lyophilization method (1) was set as shown in Table 16.

[0214]

Table 16 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 120 without vacuum 2 -40 60 3 -40 120 4 -2.5 60 5 -2.5 240 6 -40 60 7 -40 120 Primary drying 8 0 60 10 9 0 2400 Secondary drying 10 45 180 10 eleven 45 480 unloading 12 5 60 10

[0215]

For the lyophilized injectable composition obtained by the lyophilization method of the present invention, the storage stability after 3 months at 40°C/75% RH was evaluated using protein concentration, moisture content, amount of insoluble microparticles, DAR, content of monomers, aggregates and fragments as indicators. , NPI, IoP, charged isomers and pH. Significant changes in quality from the initial point in time were not observed.

[0216]

[Example 13: Study of other compositions and lyophilization conditions]

(1) Getting the composition

Active pharmaceutical ingredients (antibody-drug conjugate (1) (20 mg/ml), 25 mM histidine, 9% sucrose, pH 5.5) were distributed in a set of UF membranes (AMICON ULTRA-15, 30 kDa) and centrifuged at 3000 rpm /min and 5°C until the concentration decreases by about 3 times. The resulting concentrated active pharmaceutical ingredients were injected into a dialysis cassette (dialysis membrane, Slide-A-lyzer, MWCO 20,000) and dialyzed with target composition buffer to 50 times or more the volume of the sample injected into the dialysis cassette. Dialysis was carried out twice for more than 5 hours at 5°C. After dialysis, respectively, concentration or dilution was carried out so that the protein concentration was about 5 mg/ml or about 50 mg/ml, and then polysorbate 80 was added and mixed to obtain an aqueous solution containing an antibody-drug conjugate (1) (5 mg/ml ), 8% sucrose, 20 mM histidine buffer and 0.04% polysorbate 80 and having a pH of 4.0 (hereinafter referred to as "aqueous solution (2)"); an aqueous solution containing the antibody-drug conjugate (1) (50 mg/ml), 6% sucrose, 40 mM histidine buffer and 0.04% polysorbate 80 and having a pH of 7.0 (hereinafter referred to as "aqueous solution (3) "); an aqueous solution containing the antibody-drug conjugate (1) (5 mg/ml), 8% trehalose hydrate, 20 mM histidine buffer and 0.04% polysorbate 80 and having a pH of 4.0 (hereinafter referred to as "aqueous solution (4 )"); and an aqueous solution containing the antibody-drug conjugate (1) (50 mg/ml), 6% trehalose hydrate, 40 mM histidine buffer and 0.04% polysorbate 80 and having a pH of 7.0 (hereinafter referred to as "aqueous solution ( 5)").

Based on 20 mg of the antibody-drug conjugate (1), the aqueous solution (2) includes 320 mg of sucrose, 80 mm histidine buffer and 1.6 mg of polysorbate 80; aqueous solution (3) includes 24 mg of sucrose, 16 mm histidine buffer and 0.16 mg of polysorbate 80; the aqueous solution (4) comprises 320 mg trehalose hydrate, 80 mM histidine buffer and 1.6 mg polysorbate 80; and aqueous solution (5) includes 24 mg trehalose hydrate, 16 mM histidine buffer and 0.16 mg polysorbate 80.

These solutions were filtered through a 0.22 µm filter. 5 ml of each filtered solution was added to a dark glass vial and, in the case of a lyophilized composition, the vials were loosely sealed with rubber stoppers and lyophilization was carried out under the conditions shown in Table 17. After lyophilization, the vials sealed with rubber stoppers were sealed with caps.

[0217]

Table 17 Process Stage No. Shelf temperature (°C) Time (min) Chamber vacuum degree (Pa) Freezing 1 5 thirty without vacuum 2 -40 60 3 -40 120 4 -1 60 5 -1 240 6 -40 60 7 -40 120 Primary drying 8 from -20 to 10 60 4-30 9 from -20 to 10 1800-3600 Secondary drying 10 45 180 4-30 eleven 45 780 unloading 12 5 60 4-30 13 5 3600

[0218]

(2) Stability test

For the lyophilized injectable compositions obtained by the lyophilization method of the present invention, storage stability at 40°C/75% RH was evaluated using protein concentration, moisture content, amount of insoluble microparticles, DAR, content of monomers, aggregates and fragments, NPI, IoP, charged isomers and pH.

[0219]

[Example 14: Study for the preparation and formulation of the composition of the antibody-drug conjugate (2)]]

Guided by the description of the production method in International Publication No. WO 2015/155998, using an anti-HER3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4) an antibody-drug conjugate was prepared in which the drug-linker moiety represented by the following formula:

[0220]

[Formula 15]

[0221]

where A is the position of binding to the antibody,

conjugated to an antibody via a thioether bond (hereinafter referred to as "antibody-drug conjugate (2)"). The DAR of the antibody-drug conjugate (2) is 7.7.

[0222]

For antibody-drug conjugate (2) screening compositions (20 mg/ml antibody-drug conjugate (2), 9% sucrose, 25 mM histidine buffer, 0.01-0.1% polysorbate 20, pH 4.9- 5.9) was carried out in the same way as the methods of Examples 2-8. As a result, it was found that an aqueous solution comprising the antibody-drug conjugate (2) (20 mg/ml), 9% sucrose, 25 mM histidine buffer and 0.03% polysorbate 20 and having a pH of 5.4 (hereinafter referred to as "aqueous solution (6)") is the preferred composition.

[0223]

[Example 15: Study on the preparation and formulation of the composition of the antibody-drug conjugate (3)]

Guided by the description of the production method in International Publication No. WO 2015/098099, using an anti-TROP2 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5, and a light chain consisting of amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 6) an antibody-drug conjugate was obtained in which the drug-linker moiety represented by the following formula:

[0224]

[Formula 16]

[0225]

where A is the position of binding to the antibody,

conjugated to an anti-TROP2 antibody via a thioether bond (hereinafter referred to as "antibody-drug conjugate (3)"). The DAR of the antibody-drug conjugate (3) is 4.3.

[0226]

For antibody-drug conjugate (3), composition screening (antibody-drug conjugate (3) (20 mg/ml), sucrose (3-9%), histidine buffer (3-15 mM), polysorbate 20 or polysorbate 80 ( 0.01-0.1%), pH 5-7, or antibody-drug conjugate (3) (15-40 mg/ml), 9% sucrose, 10 mM histidine buffer, 0.02% polysorbate 80, pH 6.0) was carried out in the same manner as the methods of Examples 2-8. As a result, it was found that an aqueous solution comprising the antibody-drug conjugate (3) (20 mg/ml), 9% sucrose, 10 mM histidine buffer and 0.02% polysorbate 80 and having a pH of 6.0 (hereinafter referred to as "aqueous solution (7)"); as well as an aqueous solution containing the antibody-drug conjugate (3) (20 mg/ml), 9% sucrose, 10 mm histidine buffer and 0.03% polysorbate 80 and having a pH of 6.0 (hereinafter referred to as "aqueous solution ( 8)") are the preferred compositions.

[0227]

[Example 16: Study on the preparation and formulation of the composition of the antibody-drug conjugate (4)]

Guided by the description of the production method in International Publication No. WO 2014/057687, using an anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 7, and a light chain, consisting of the amino acid sequence consisting of amino acid residues 21-233 of SEQ ID NO: 8), an antibody-drug conjugate was obtained in which the drug-linker fragment represented by the following formula:

[0228]

[Formula 17]

[0229]

where A is the position of binding to the antibody,

conjugated to an anti-B7-H3 antibody via a thioether linkage (hereinafter referred to as "antibody-drug conjugate (4)"). The DAR of the antibody-drug conjugate (4) is 4.6.

[0230]

For antibody-drug conjugate (4), composition screening (antibody-drug conjugate (4) (20 mg/mL), 9% sucrose or 10% trehalose hydrate, 10 mM histidine buffer or 10 mM succinate buffer, polysorbate 20 or polysorbate 80 (0.005-0.04%), pH 5.2-6.2; or antibody-drug conjugate (4) (20-60 mg/ml), 9% sucrose, 10 mM histidine buffer, polysorbate 20 (0.005 -0.04%), pH 5.2-6.2) was carried out in the same way as the methods of Examples 2-8. As a result, it was found that an aqueous solution comprising the antibody-drug conjugate (4) (20 mg/ml), 9% sucrose, 10 mM histidine buffer and 0.02% polysorbate 20 and having a pH of 5.9 (hereinafter referred to as "aqueous solution (9)"); and an aqueous solution containing the antibody-drug conjugate (4) (20 mg/ml), 9% sucrose, 10 mM histidine buffer and 0.03% polysorbate 20 and having a pH of 5.9 (hereinafter referred to as "aqueous solution ( 10)") are the preferred compositions.

[0231]

[Production Example 17: Study on the preparation and formulation of an antibody-drug conjugate composition (5)]

Guided by the description of the production method in International Publication No. WO 2018/135501, using an anti-GPR20 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 9, and a light chain consisting of amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 10), an antibody-drug conjugate was obtained in which the drug-linker moiety represented by the following formula:

[0232]

[Formula 18]

[0233]

where A is the position of binding to the antibody,

conjugated to an anti-GPR20 antibody via a thioether linkage (hereinafter referred to as "antibody-drug conjugate (5)"). The DAR of the antibody-drug conjugate (5) is 7.8.

[0234]

For antibody-drug conjugate (5) composition screening (antibody-drug conjugate (5) (20-60 mg/ml), 9% sucrose, 10 mM histidine buffer and polysorbate 80 (0.01-0.1%) , pH 5.0-6.5) was carried out in the same way as the methods of Examples 2-8. As a result, it was found that an aqueous solution comprising the antibody-drug conjugate (5) (20 mg/ml), 9% sucrose, 10 mM histidine buffer and 0.03% polysorbate 80 and having a pH of 5.4 (hereinafter referred to as "aqueous solution (11)") is the preferred composition.

[0235]

[Example 18: Preparation of lyophilized composition for injection and stability test]

Aqueous solutions (6) - (11), adjusted in Examples 14-17, were lyophilized in the same manner as described in Examples 10 and 12, respectively, to obtain lyophilized compositions for injection.

For these lyophilized injectable formulations, storage stability after 1 month and 3 months at 40° C./75% RH was evaluated using protein concentration, moisture, insoluble microparticles, DAR, monomers, aggregates and fragments, NPI, IoP, charged isomers and pH. Significant changes in quality from the initial point in time were not observed.

Sequence listing text in natural language

[0236]

SEQ ID NO: 1 - Anti-HER2 antibody heavy chain amino acid sequence

SEQ ID NO: 2 - Anti-HER2 antibody light chain amino acid sequence

SEQ ID NO: 3 - Anti-HER3 antibody heavy chain amino acid sequence

SEQ ID NO: 4 - Anti-HER3 antibody light chain amino acid sequence

SEQ ID NO: 5 - Anti-TROP2 antibody heavy chain amino acid sequence

SEQ ID NO: 6 - Anti-TROP2 antibody light chain amino acid sequence

SEQ ID NO: 7 - Anti-B7-H3 antibody heavy chain amino acid sequence

SEQ ID NO: 8 - Anti-B7-H3 antibody light chain amino acid sequence

SEQ ID NO: 9 - Anti-GPR20 antibody heavy chain amino acid sequence

SEQ ID NO: 10 - Anti-GPR20 antibody light chain amino acid sequence

--->

SEQUENCE LIST

<110> DAIICHI SANKYO COMPANY, LIMITED

<120> PRODUCTION OF ANTIBODY-DRUG CONJUGATE AND ITS

LYOPHILIZATION

<130> DSPCT-FP1822

<150>JP2017-160071

<151> 2017-08-23

<160> 10

<170>PatentIn version 3.5

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

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Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

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Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

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Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

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Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

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Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

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Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

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Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

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Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

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Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

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Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

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Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

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Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

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Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

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Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

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Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala

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Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr Ser

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Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val

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Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp

115 120 125

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

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Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

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Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

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Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

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Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

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Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

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Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

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Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

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Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

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Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

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Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

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Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

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Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr

100 105 110

Ile Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

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<211> 471

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Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

130 135 140

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

145 150 155 160

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

165 170 175

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

180 185 190

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

195 200 205

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

210 215 220

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

225 230 235 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

370 375 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 8

<211> 233

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-B7-H3 antibody light chain

<400> 8

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg

35 40 45

Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

50 55 60

Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser

100 105 110

Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu

130 135 140

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 9

<211> 472

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-GPR20 antibody heavy chain

<400> 9

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Lys Tyr Met Gly Phe Ile Asn Pro Gly Ser Gly His Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Thr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Ala Gly Gly Phe Leu Arg Ile Ile Thr Lys

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

145 150 155 160

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

210 215 220

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280 285

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

340 345 350

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr

370 375 380

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

420 425 430

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

435 440 445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

450 455 460

Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 10

<211> 234

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-GPR20 antibody light chain

<400> 10

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser

35 40 45

Val Ser Thr Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Gln Pro

50 55 60

Lys Leu Leu Ile Tyr Ser Ala Gly Asn Leu Glu Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Asn Tyr Tyr Cys Gln Gln Ile Asn

100 105 110

Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<---

Claims (113)

1. Pharmaceutical composition comprising (i) an antibody-drug conjugate, (ii) histidine buffer, (iii) sucrose and (iv) polysorbate 80 or polysorbate 20, where the antibody-drug conjugate is an antibody-drug conjugate wherein the drug-linker moiety is represented by the following formula:

where A is the position of binding to the antibody, conjugated to an antibody through a thioether bond, and wherein the amount of Polysorbate 80 or Polysorbate 20 is 0.2-0.4 mg per 20 mg of the antibody drug conjugate. 2. The pharmaceutical composition according to claim 1, wherein the amount of polysorbate 80 or polysorbate 20 is 0.2-0.3 mg per 20 mg of the antibody-drug conjugate. 3. The pharmaceutical composition of claim 2, wherein the amount of polysorbate 80 or polysorbate 20 is 0.2 or 0.3 mg per 20 mg of the antibody drug conjugate. 4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the amount of sucrose is 24-320 mg per 20 mg of the antibody-drug conjugate. 5. The pharmaceutical composition of claim 4, wherein the amount of sucrose is 90 mg per 20 mg of the antibody drug conjugate. 6. Pharmaceutical composition according to any one of claims 1 to 5, wherein the histidine buffer is a mixture of L-histidine and L-histidine hydrochloride. 7. The pharmaceutical composition according to claim 6, wherein the histidine buffer is a mixture of L-histidine and L-histidine hydrochloride hydrate. 8. A pharmaceutical composition according to any one of claims 1-7, wherein the pH of the composition when the antibody-drug conjugate is dissolved in water at a concentration of 20 mg/ml is 4.0-7.0. 9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, or an anti-GPR20 antibody. 10. The pharmaceutical composition of claim 9, wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody. 11. The pharmaceutical composition according to claim 10, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of from amino acid residues 1-214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2. 12. The pharmaceutical composition of claim 10 or 11, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8. 13. Pharmaceutical composition according to any one of claims 10-12, including (i) 20 mg antibody-drug conjugate, (ii) 0.89 mg L-histidine and 4.04 mg L-histidine hydrochloride hydrate, (iii) 90 mg sucrose and (iv) 0.3 mg polysorbate 80. 14. Pharmaceutical composition according to any one of claims 10-12, including (i) 100 mg of antibody-drug conjugate, (ii) 4.45 mg L-histidine and 20.2 mg L-histidine hydrochloride hydrate, (iii) 450 mg sucrose and (iv) 1.5 mg polysorbate 80. 15. The pharmaceutical composition of claim 9, wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody. 16. The pharmaceutical composition according to claim 15, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain has been deleted. 17. The pharmaceutical composition of claim 15 or 16, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8. 18. Pharmaceutical composition according to any one of claims 15-17, including (i) 20 mg antibody-drug conjugate, (ii) 0.81 mg L-histidine and 4.14 mg L-histidine hydrochloride hydrate, (iii) 90 mg sucrose and (iv) 0.3 mg polysorbate 20. 19. Pharmaceutical composition according to any one of claims 15-17, including (i) 100 mg of antibody-drug conjugate, (ii) 4.06 mg L-histidine and 20.7 mg L-histidine hydrochloride hydrate, (iii) 450 mg sucrose and (iv) 1.5 mg polysorbate 20. 20. The pharmaceutical composition of claim 9, wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody. 21. The pharmaceutical composition according to claim 20, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5 and a light chain consisting of an amino acid sequence consisting of from amino acid residues 21-234 of SEQ ID NO: 6, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted. 22. The pharmaceutical composition of claim 20 or 21, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5. 23. Pharmaceutical composition according to any one of claims 20-22, including (i) 20 mg antibody-drug conjugate, (ii) 0.78 mg L-histidine and 1.05 mg L-histidine hydrochloride hydrate, (iii) 90 mg sucrose and (iv) 0.2 or 0.3 mg polysorbate 80. 24. Pharmaceutical composition according to any one of claims 20-22, including (i) 100 mg of antibody-drug conjugate, (ii) 3.88 mg L-histidine and 5.26 mg L-histidine hydrochloride hydrate, (iii) 450 mg sucrose and (iv) 1.0 or 1.5 mg of polysorbate 80. 25. The pharmaceutical composition of claim 9, wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody. 26. The pharmaceutical composition according to claim 25, wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20-471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence , consisting of amino acid residues 21-233 of SEQ ID NO: 8, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted. 27. The pharmaceutical composition of claim 25 or 26, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 3 and 5. 28. Pharmaceutical composition according to any one of claims 25-27, including (i) 20 mg antibody-drug conjugate, (ii) 0.65 mg L-histidine and 1.22 mg L-histidine hydrochloride hydrate, (iii) 90 mg sucrose and (iv) 0.2 or 0.3 mg polysorbate 20. 29. Pharmaceutical composition according to any one of claims 25-27, including (i) 100 mg of antibody-drug conjugate, (ii) 3.23 mg L-histidine and 6.12 mg L-histidine hydrochloride hydrate, (iii) 450 mg sucrose and (iv) 1.0 or 1.5 mg of polysorbate 20. 30. The pharmaceutical composition of claim 9, wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody. 31. The pharmaceutical composition according to claim 30, wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-472 of SEQ ID NO: 9 and a light chain consisting of an amino acid sequence consisting of from amino acid residues 21-234 of SEQ ID NO: 10, or an antibody variant in which the lysine residue at the carboxy-terminus of the heavy chain is deleted. 32. The pharmaceutical composition of claim 30 or 31, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is between 7 and 8. 33. Pharmaceutical composition according to any one of claims 30-32, including (i) 20 mg antibody-drug conjugate, (ii) 0.32 mg L-histidine and 1.66 mg L-histidine hydrochloride hydrate, (iii) 90 mg sucrose and (iv) 0.3 mg polysorbate 80. 34. Pharmaceutical composition according to any one of claims 30-32, including (i) 100 mg of antibody-drug conjugate, (ii) 1.62 mg L-histidine and 8.29 mg L-histidine hydrochloride hydrate, (iii) 450 mg sucrose and (iv) 1.5 mg polysorbate 80. 35. A pharmaceutical composition according to any one of claims 1 to 34, wherein the composition is in the form of an injection composition. 36. The pharmaceutical composition of claim 35, wherein the composition is in the form of an aqueous composition for injection. 37. The pharmaceutical composition of claim 36, wherein the concentration of the antibody-drug conjugate is 20 mg/mL. 38. The pharmaceutical composition according to claim 36 or 37, wherein the composition is frozen. 39. The pharmaceutical composition of claim 35, wherein the composition is in the form of a lyophilized composition for injection. 40. The pharmaceutical composition according to claim 39, wherein the composition is stored in a dark vial. 41. A pharmaceutical composition according to any one of claims 1 to 40, wherein the composition is for the treatment of cancer. 42. A method for producing a pharmaceutical composition according to claim 39, including the steps: (1) obtaining an aqueous solution comprising (i) an antibody-drug conjugate, (ii) histidine buffer, (iii) sucrose and (iv) polysorbate 80 or polysorbate 20, and then (2) lyophilization of the aqueous solution. 43. A method for producing a pharmaceutical composition according to claim 39, including the steps: (1) obtaining an aqueous solution comprising (i) an antibody-drug conjugate, (ii) histidine buffer, (iii) sucrose and (iv) polysorbate 80 or polysorbate 20, (2) adjusting the pH of the aqueous solution, and then (3) lyophilization of the aqueous solution. 44. The production method according to claim 42 or 43, where the step of lyophilizing the aqueous solution includes an annealing process at a shelf temperature that is close to the eutectic point of the aqueous solution, where proximity to the eutectic point means the range from a temperature that is 1.5°C below the eutectic point to a temperature that is 1.5°C above the eutectic point. 45. The production method according to claim 44, characterized in that the time of the primary drying process is reduced compared to when annealing is carried out at a shelf temperature that is 5°C below the eutectic point. 46. The production method according to claim 44 or 45, characterized in that a lyophilized cake is obtained having a lower shrinkage compared to that obtained when annealing is carried out at a shelf temperature that is 5°C below the eutectic point. 47. The production method according to claim 42 or 43, wherein the step of freeze-drying the aqueous solution comprises a method for performing annealing at a shelf temperature of -4 to -1°C. 48. The production method according to claim 47, wherein the aqueous solution lyophilization step also includes a primary drying process at a shelf temperature of −5 to 5° C. under a vacuum of 5 to 15 Pa. 49. The production method according to claim 48, wherein the aqueous solution lyophilization step also includes a secondary drying process at a shelf temperature of 40 to 50°C under a vacuum of 5 to 15 Pa.

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