Method for Removing Coloration of Protein Preparation Active Pharmaceutical Ingredient

Abstract

The present invention addresses the problem of providing: a method for removing color from an active ingredient solution for a protein formulation, particularly, an antibody formulation; a production method that is for an active ingredient solution for a protein formulation and that includes the color removal method as part of the steps thereof; and said formulation active ingredient solution that is highly concentrated and colorless.　The present invention provides a method for removing, by anion exchange chromatography, advanced glycation end-products that cause coloring, from an active ingredient solution for a protein formulation, particularly, an antibody formulation. This method enables provision of a colorless formulation active ingredient solution.

Description

Method for removing color from active pharmaceutical ingredients for protein preparations

[0001] The present invention relates to a method for removing color from a drug substance solution of a protein pharmaceutical, and to the solution produced by a manufacturing process including the method.

[0002] Subcutaneous injection is often the preferred formulation for antibody drugs due to its convenience and patient burden. Subcutaneous administration naturally requires high drug concentrations due to the limited dosage, which can lead to several problems, particularly for antibody drugs. One such problem is the color of the drug substance solution, and the European Pharmacopoeia requires that the drug substance solution be colorless or slightly colored. Furthermore, coloration of the drug substance solution may indicate the presence of impurities and the resulting heterogeneity of the drug substance, which may result in a deterioration in drug performance or an impact on efficacy or safety (Non-Patent Documents 1 and 2).

[0003] One of the causes of such discoloration is the culture environment of the producing cells. In particular, CHO cells, which are widely used for antibody production, are cultured under high-oxygen and high-glucose conditions, which expose the produced proteins to a significant oxidative and glycative environment, resulting in the formation of advanced glycation end products (AGEs), some of which have been reported to be the cause of discoloration (Non-Patent Documents 3, 4, and 5). On the other hand, it has also been reported that such advanced glycation end products can cause a decrease in the efficacy of protein pharmaceuticals. Therefore, removal of the causative substances from discolored drug substance solutions is important for maintaining the quality and performance of the pharmaceutical.

[0004] Anal. Chem. 2014, 86, 9816-9823Biotechnology Progress (2013), 29 (5), 1270-1277European Pharmacopoeia, 8.2 ed.; Council of Europe: Strasbourg, France, 2014.Analytical Chemistry (2013), 85 (23), 11401-11409BMC Ophthalmol. 2006, 6, 10

[0005] An object of the present invention is to provide a method for removing color from a drug substance solution of a protein formulation, and the solution produced by a production process including the method.

[0006] As a result of intensive research to find such a method, the present inventors discovered a method for removing the substances that cause coloration of the drug substance solution by ion exchange chromatography, and completed the present invention.

[0007] That is, the present invention is as follows. [1] A method for separating non-colored proteins from a solution containing non-colored proteins and colored proteins, the method comprising: (i) loading the solution into an ion exchange carrier column and binding the non-colored proteins and colored proteins to the ion exchange carrier using an equilibration buffer having a predetermined initial pH; (ii) flowing an elution buffer, which forms a pH gradient varying from the initial pH to a final pH, through the ion exchange carrier column at a predetermined flow rate under predetermined conditions so that the non-colored proteins and colored proteins are separated; and (iii) collecting a predetermined fraction containing the non-colored proteins eluted in step (ii); [2] A method for separating non-colored proteins from a solution containing non-colored proteins and colored proteins, the method comprising: (i) loading the solution into an ion exchange carrier column; (ii) flowing an equilibration buffer, which has been prepared so that the colored proteins in the solution bind to the ion exchange carrier but the non-colored proteins do not bind to the ion exchange carrier, through the ion exchange carrier column at a predetermined flow rate; (iii) recovering a fraction containing uncolored proteins that flows through in the step (ii) as is; [3] the color density of a solution containing uncolored proteins and colored proteins or a concentrate thereof is (1) darker than that of the reference standard solution BY5 in a visual comparison test in Item 2.65 "Color Comparison Test" of the General Test Methods of the Seventeenth Edition of the Japanese Pharmacopoeia, or (2) a peak area of ​​ultraviolet absorption detection at a wavelength of 280 nm (hereinafter referred to as "UV") calculated based on ultraviolet absorption and fluorescence spectroscopy of the solution or a concentrate thereof.280[4] The separation method according to any one of the preceding items [1] to [3], wherein the ratio of the fluorescence intensity detection peak area (where the excitation wavelength is 380 nm and the detection wavelength is 560 nm; hereinafter, this may be abbreviated as "Em560(ex380)") to the fluorescence intensity detection peak area (where the excitation wavelength is 380 nm and the detection wavelength is 560 nm; hereinafter, this may be abbreviated as "Em / UV*100 value") multiplied by 100 (hereinafter, this may be abbreviated as "Em / UV*100 value") is greater than about 6.3; [4] The separation method according to any one of the preceding items [1] to [3], wherein the ion exchange carrier is an anion exchange carrier; [5] The separation method according to any one of the preceding items [1] to [4], wherein the ion exchange carrier or the anion exchange carrier is in the form of small particles, a membrane, or a monolith; [6] The separation method according to the preceding item [4] or [5], wherein the anion exchange carrier is a strong anion exchange carrier, a weak anion exchange carrier, or a multimode anion exchange carrier; [7] [8] The separation method according to the preceding item [4] or [5], wherein the anion exchange carrier is a weak anion exchange carrier or a multimode anion exchange carrier; [8] The separation method according to the preceding item [7], wherein the weak anion exchange carrier is any one selected from the group consisting of Fractogel (registered trademark) EMD DEAE, Fractogel (registered trademark) EMD DMAE, Capto (registered trademark) DEAE, DEAE Ceramic HyperD (registered trademark) F, Toyopearl (registered trademark) NH2-750F, TOYOPEARL (registered trademark) DEAE-650C, TOYOPEARL (registered trademark) DEAE-650M, TOYOPEARL (registered trademark) DEAE-650S, Cellufine (registered trademark) A-200, Cellufine (registered trademark) A-500, Cellufine (registered trademark) A-800, and Cellufine (registered trademark) MAX DEAE; [9] The separation method according to the above item [7], wherein the multi-mode anion exchange carrier is any one selected from the group consisting of Toyopearl (registered trademark) NH2-750F, Capto (registered trademark) Adhere, Capto (registered trademark) Adhere ImpRes, Capto (registered trademark) MMC, Capto (registered trademark) core 700, and Cellufine (registered trademark) IB (preferably, Toyopearl (registered trademark) NH2-750F);

[10] The strong anion exchange support is selected from the group consisting of Fractogel® EMD TMAE (M), Fractogel® EMD TMAE Medcap (M), Fractogel® EMD TMAE Hicap (M), Eshmuno® Q, Eshmuno® QPX, Eshmuno® QPX Hicap, Capto® Q, Capto® Q ImpRes, Q Sepharose® FF, Q Sepharose® HP, Q Sepharose® XL, Source® 30Q, Capto® Adhere, Capto® Adhere ImpRes, POROS® 50 HQ, POROS® 50 XQ, POROS® 50 PI, Q HyperCel®, Toyopearl® GigaCap Q 650M, and Toyopearl® GigaCap Q.

[11] The separation method according to any one of the preceding items [1] to

[10] , wherein the protein color body has an isoelectric point in the range of about 5.0 to about 9.1 (preferably about 6.0 to about 7.0);

[12] The separation method according to any one of the preceding items [1] to

[10] , wherein the protein color body is any one selected from the group consisting of Cellufine Q-500, Cellufine MAX Qr, Cellufine MAX Qh ...

[13] The separation method according to any one of the preceding items [1] to

[12] , wherein the isoelectric point of the non-colored protein falls within a range selected from the group consisting of about 7.0 or more and less than about 7.5, about 7.5 or more and less than about 8.0, about 8.0 or more and less than about 8.5, about 8.5 or more and less than about 9.0, and about 9.0 or more and less than about 9.5;

[14] The separation method according to any one of the preceding items [1] to

[12] , wherein the total concentration of the non-colored protein and the colored protein input to the ion exchange carrier column is about 2 to about 50 mg / mL (preferably about 10 to about 30 mg / mL);

[15] The separation method according to any one of the preceding items [1] to

[15] , wherein the total concentration is about 5

[15] The separation method according to any one of the preceding items [1] to

[12] , wherein the total load of the non-colored protein and the colored protein loaded onto the ion exchange carrier column is about 2 to about 200 mg / mL;

[16] The separation method according to any one of the preceding items [1] to

[12] , wherein the total load of the non-colored protein and the colored protein loaded onto the ion exchange carrier column is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, and about 50 mg / mL;

[17] The separation method according to any one of the preceding items [1] to

[12] , wherein the total load of the non-colored protein and the colored protein loaded onto the ion exchange carrier column is about 2 to about 200 mg / mL;

[18] The separation method according to any one of the preceding items [1] to

[12] , wherein the total load of the non-colored protein and the colored protein loaded onto the ion exchange carrier column is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg /

[17] The separation method according to any one of the preceding items [1] and [3] to

[16] , wherein the pH gradient range from the initial pH to the final pH is set to sandwich the isoelectric points of the colored form of the protein and / or the non-colored form of the protein;

[18] The separation method according to any one of the preceding items [1] and [3] to

[17] , wherein the pH gradient decreases from the initial pH to the final pH;

[19] The separation method according to any one of the above items [1] and [3] to

[18] , wherein the initial pH is any pH in the range of about 11.0 to about 7.0 (specifically, any pH selected from the group consisting of about 11.0, about 10.8, about 10.6, about 10.4, about 10.2, about 10.0, about 9.8, about 9.6, about 9.4, about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, about 7.4, about 7.2, and about 7.0);

[20] The separation method according to any one of the preceding items [1] and [3] to

[19] , wherein the final pH is any pH in the range of about 4.0 to about 8.2 (specifically, any pH selected from the group consisting of about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, and about 8.2) (provided that the initial pH is higher than the final pH by 1.0 or more).

[21] The pH gradient range is from any one initial pH selected from the group consisting of about 11.0, about 10.8, about 10.6, about 10.4, about 10.2, about 10.0, about 9.8, about 9.6, about 9.4, about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, about 7.4, about 7.2, and about 7.0 to about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5. the separation method according to any one of the preceding items [1] and [3] to

[18] , wherein the final pH is in any one range selected from the group consisting of about 0.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0 and about 8.2 (preferably, the initial pH is higher than the final pH by 2.0 or more);

[22] The separation method according to any one of the preceding items [1] and [3] to

[21] , wherein the pH gradient is a linear gradient or a stepwise gradient;

[23] The separation method according to any one of the preceding items [1] and [3] to

[22] , wherein the elution buffer according to the preceding item [1] consists of a combination of a buffer having the initial pH (hereinafter sometimes abbreviated as "buffer A") and a buffer having the final pH (hereinafter sometimes abbreviated as "buffer B") in any ratio;

[24] The separation method according to the preceding item

[23] , wherein the elution buffer A and / or the elution buffer B consists of a combination of a plurality of Good's buffers each selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS;

[25] The separation method according to the preceding item

[23] , wherein Buffer A and / or Buffer B each comprise a combination of multiple Good's buffers selected from the group consisting of MES, MOPS, TAPS, and CAPSO (preferably a combination of MES, MOPS, TAPS, and CAPSO);

[26] The separation method according to the preceding item

[23] , wherein Buffer A is Thermo Scientific® CX-1 pH Gradient Buffer B (pH 10.2) and Buffer B is Thermo Scientific® CX-1 pH Gradient Buffer A (pH 5.6);

[27] The separation method according to the preceding item

[23] , wherein Buffer A and / or Buffer B each comprise a combination of multiple buffers selected from the group consisting of Tris-acetate, Tris-phosphate, and Tris-citrate;

[28] The separation method according to any one of the above items

[23] to

[27] , wherein a pH gradient is created using the elution buffer by continuously or stepwise changing the ratio of the combination of buffer A and buffer B;

[29] the pH gradient is created over any column volume in the range of about 1 to about 20 CV (specifically, about 1 CV, about 2 CV, about 3 CV, about 4 CV, about 5 CV, about 6 CV, about 7 CV, about 8 CV, about 9 CV, about 10 CV, about 15 CV, and about 20 CV);

[30] the separation method according to any one of the preceding items

[23] to

[28] , wherein the pH gradient is a stepwise gradient performed by increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 0.5% to about 5% (specifically, any percentage selected from the group consisting of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, and about 5%) at intervals corresponding to any column volume in the range of about 1 to about 20 CV (specifically, about 1 CV, about 2 CV, about 3 CV, about 4 CV, about 5 CV, about 6 CV, about 7 CV, about 8 CV, about 9 CV, about 10 CV, about 15 CV, and about 20 CV);The separation method according to any one of the above items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by decreasing the pH of the elution buffer by any value in the range of about 0.1 to about 1.0 (specifically, any value selected from the group consisting of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0) at intervals corresponding to any column volume in the range of about 20 to about 50 CV (specifically, about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[31] The separation method according to any one of the above items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by decreasing the pH of the elution buffer by any value in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[31] the separation method according to any one of the preceding items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 0.5 to about 5% (specifically, any percentage selected from the group consisting of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, and about 5%) in a range corresponding to a column volume in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[32] the separation method according to any one of the preceding items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV); The separation method according to any one of the above items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by decreasing the pH of the elution buffer by any value in the range of about 0.1 to about 1.0 (specifically, any value selected from the group consisting of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0) in a range corresponding to any column volume in the range of about 20 to about 50 CV (specifically, about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[33] The separation method according to any one of the above items

[23] to

[28] , wherein the pH gradient is a stepwise gradient achieved by decreasing the pH of the elution buffer by any value in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[34] The separation method according to any one of the preceding items

[23] to

[28] , wherein the pH gradient is a linear gradient achieved by continuously increasing the proportion (%) of buffer B in the elution buffer within a range corresponding to any column volume in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV);

[35] The separation method according to any one of the preceding items

[23] to

[28] , wherein the pH gradient is a linear gradient achieved by continuously decreasing the pH of the elution buffer within a range corresponding to any column volume in the range of about 20 to about 50 CV (specifically, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV); The separation method according to any one of the above items

[29] ,

[31] and

[33] , wherein the proportion (%) of the buffer B is increased stepwise or continuously from at least any proportion in the range of about 10 to about 100% (specifically, any proportion selected from the group consisting of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100%) to any proportion in the range of about 30% to about 100% (preferably any proportion selected from the group consisting of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100%);

[36] the separation method according to any one of the above items

[29] ,

[31] and

[33] , wherein the pH gradient is (a) from about 4 to about 6 The separation method according to any one of the preceding items

[23] to

[28] , wherein the separation is carried out by (a) increasing the proportion (%) of buffer B in the elution buffer stepwise by any proportion in the range of about 1 to about 2% over a range of at least about 50 to 100% at intervals corresponding to any column volume in the range of CV, or (b) decreasing the pH in the elution buffer stepwise by any pH proportion in the range of about 0.6 to about 1.0 over a range corresponding to a column volume of about 30 to about 40 CV;

[37] The separation method according to any one of the preceding items

[23] to

[28] , wherein the separation is carried out by (a) increasing the proportion (%) of buffer B in the elution buffer stepwise by any proportion in the range of about 1 to about 2% over a range of at least about 50 to 100%, at intervals corresponding to any column volume in the range of CV; or (b) decreasing the pH in the elution buffer stepwise by any pH proportion in the range of about 0.6 to about 1.0 over a range corresponding to a column volume of about 30 to about 40 CV;

[37] The separation method according to any one of the preceding items

[23] to

[28] , wherein the linear flow rates of the elution buffer and the equilibration buffer according to the preceding item [2] are each any linear flow rate in the range of about 100 to about 800 cm / h (specifically, about 100 cm / h, about 150 cm / h, about 200 cm / h, about 250 cm / h, about 300 cm / h, about 350 cm / h, about 400 cm / h, about 450 cm / h, about 500 cm / h, about 600 cm / h, about 700 cm / h, about 800 cm / h, about 900 cm / h, about 1000 cm / h, about 1200

[38] The separation method according to any one of the preceding items [1] to

[36] , wherein the electric conductivity of the equilibration buffer and / or elution buffer according to the preceding item [1] is any electric conductivity in the range of about 1 to about 20 mS / cm (preferably, any electric conductivity in the range of about 2 to about 15 mS / cm, specifically, about 2 mS / cm, about 3 mS / cm, about 4 mS / cm, about 5 mS / cm, about 6 mS / cm, about 7 mS / cm, about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, about 15 mS / cm, about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, about 22 mS / cm, about 23 mS / cm, about 24 mS / cm, about 25 mS / cm, about 26 mS / cm, about 27 mS / cm, about 28 mS / cm, about 29 mS / cm, about 30 mS / cm, about 31 mS / cm, about 32 mS / cm, about 33 mS / cm, about 34 mS / cm, about 35 mS / cm, about 36 mS / cm, about 37 mS / cm, about 38 mS / cm, about 39 mS / cm, about 40 mS / cm, about 41 mS / cm, about 42 mS / cm, about 43 mS / cm, about 44 mS / cm, about 45 mS / cm, about 46 mS / cm, about 47 mS / cm, about 48 mS / cm,

[39] the separation method according to any one of the preceding items [1] and [3] to

[37] , wherein the electrical conductivity of the equilibration buffer according to the preceding item [2] is any electrical conductivity in the range of about 1 to about 20 mS / cm (preferably any electrical conductivity in the range of about 2 to about 15 mS / cm, specifically, an electrical conductivity selected from the group consisting of about 2 mS / cm, about 3 mS / cm, about 4 mS / cm, about 5 mS / cm, about 6 mS / cm, about 7 mS / cm, about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm and about 15 mS / cm, more preferably about 5 to about 7

[40] The separation method according to any one of the preceding items [2] to

[16] and

[39] , wherein the equilibration buffer according to the preceding item [2] has a pH of about 9.2 to about 7.4 (specifically, a pH selected from the group consisting of about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, and about 7.4, preferably a pH of about 8.2 to about 7.8);

[41] The separation method according to any one of the preceding items [1] and [3] to

[38] , wherein the equilibration buffer and the elution buffer according to the preceding item [1] have substantially the same electrical conductivity;

[42] The separation method according to any one of the preceding items [1] to

[41] , wherein one or more of steps (i) to (iii) in the separation method according to the preceding item [1] or one or more of steps (i) to (iii) in the separation method according to the preceding item [2] includes, as appropriate and necessary, a step of washing the ion exchange carrier with a wash buffer;

[43] The separation method according to any one of the preceding items [1] to

[42] , wherein the color intensity of the separated or recovered non-colored protein fraction or a concentrate thereof is substantially the same as or lighter than the color intensity of the comparison standard solution BY5 in a visual comparison test with the BY5 in the "Color Comparison Test Method" of the Japanese Pharmacopoeia;

[44]

[45] The separation method according to any one of the preceding items [1] to

[42] , wherein the color density of the separated or recovered non-colored protein fraction or a concentrate thereof is substantially the same as or lighter than that of the reference standard solution BY6 in the "Color Comparison Test" of the Japanese Pharmacopoeia, in a visual comparison test with the reference standard solution BY7 in the "Color Comparison Test" of the Japanese Pharmacopoeia;

[46] The separation method according to any one of the preceding items [1] to

[42] , wherein the color density of the separated or recovered non-colored protein fraction or a concentrate thereof is substantially the same as that of the reference standard solution BY7 in the "Color Comparison Test" of the Japanese Pharmacopoeia;

[47]

[48] ​​The separation method according to any one of the preceding items [3] and

[43] to

[47] , wherein the protein concentration of the concentrate is equal to or greater than any concentration in the range of about 50 to about 100 mg / mL (specifically, equal to or greater than any concentration selected from the group consisting of about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, and about 100 mg / mL);

[49] The separation method according to any one of the preceding items [1] to

[48] , wherein the protein concentration of the separated or recovered non-colored protein fraction is equal to or greater than any concentration in the range of about 20 to about 300 mg / mL;

[50] The separation method according to any one of the preceding items [1] to

[48] , wherein the protein concentration in the separated or recovered non-colored protein fraction is any concentration in the range of about 100 to about 300 mg / mL;

[51] The separation method according to any one of the preceding items [1] to

[50] , wherein the protein is an antibody or an antibody fragment thereof;

[52] The separation method according to the preceding item

[51] , wherein the antibody is a bispecific antibody;

[53] The separation method according to the preceding item

[51] or

[52] , wherein the antibody isotype is IgG1 or IgG4;

[54] The separation method according to the preceding item

[52] or

[53] , wherein the bispecific antibody is a bispecific antibody capable of specifically binding to PD-1 and CD3, respectively (hereinafter sometimes abbreviated as "anti-PD-1 / CD3 bispecific antibody");

[55] the isolation method according to the preceding item

[54] , wherein the anti-PD-1 / CD3 bispecific antibody is the PD-1 / CD3 bispecific antibody disclosed in the patent application designated by WO2019 / 156199;

[56]

[57] The method for separation according to the preceding item

[55] , wherein the anti-PD-1 / CD3 bispecific antibody comprises a heavy chain and a light chain forming an antigen-binding site that specifically binds to PD-1, and a heavy chain and a light chain forming an antigen-binding site that specifically binds to CD3, wherein (a) the heavy chain of the heavy chain and the light chain forming the antigen-binding site that specifically binds to PD-1 comprises any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, (b) the heavy chain of the heavy chain and the light chain forming the antigen-binding site that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 6, and (c) the light chain of the heavy chain and the light chain forming the antigen-binding site that specifically binds to PD-1, and the light chain of the heavy chain and the light chain forming the antigen-binding site that specifically binds to CD3, both comprise the amino acid sequence of SEQ ID NO: 7 (hereinafter, one or more of these bispecific antibodies may be collectively referred to as "anti-PD-1 / CD3 bispecific antibody A"); the isolation method according to the preceding item

[56] , wherein the heavy chain of the heavy and light chains constituting the antigen-binding site that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5;

[58] The heavy chain constant regions of the heavy chains constituting the antigen-binding site that specifically binds to PD-1 according to the preceding paragraph

[56] or

[57] are substituted with a heavy chain constant region consisting of any one of the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and the heavy chain constant regions of the heavy chains constituting the antigen-binding site that specifically binds to CD3 according to the preceding paragraph

[56] or

[57] are substituted with a heavy chain constant region consisting of any one of the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. the method for separation described in the preceding paragraph

[56] or

[57] , in which the heavy chain constant regions of the anti-PD-1 / CD3 bispecific antibody A described in the preceding paragraph

[56] or

[57] have been substituted with heavy chain constant regions consisting of any one of the amino acid sequences selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 (hereinafter, one or more bispecific antibodies in which the two heavy chain constant regions of anti-PD-1 / CD3 bispecific antibody A described in the preceding paragraph

[56] or

[57] have been substituted with the heavy chain constant regions of this paragraph may be collectively referred to as "anti-PD-1 / CD3 bispecific antibody B");

[59] A method for separating a non-colored protein from a solution containing a non-colored protein and a colored protein, comprising: (1) the separation method comprising: (i) loading the solution into an anion exchange carrier column and using an equilibration buffer having a predetermined initial pH to bind the non-colored protein and the colored protein to the anion exchange carrier; (ii) flowing an elution buffer that forms a pH gradient that descends from the initial pH to a final pH through the anion exchange carrier column at a predetermined flow rate under predetermined conditions so that the non-colored protein and the colored protein are separated; and (iii) collecting a predetermined fraction containing the non-colored protein eluted in the step (ii),(2) The protein is a bispecific antibody that specifically binds to PD-1 and CD3, respectively, and is composed of a heavy chain and a light chain that form an antigen-binding site that specifically binds to PD-1 and a heavy chain and a light chain that form an antigen-binding site that specifically binds to CD3, wherein (a) the heavy chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, (b) the heavy chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 6, and (c) the light chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to PD-1 and the light chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to CD3 both consist of the amino acid sequence of SEQ ID NO: 7, (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F, (4) The initial pH of the equilibration buffer and the elution buffer is any pH in the range of about 10.6 to about 8.4, and the final pH of the elution buffer is any pH in the range of about 7.0 to about 4.0 (provided that the initial pH is at least 2.0 higher than the final pH); (5) The pH gradient is (a) carried out by increasing the percentage (%) of buffer B in the elution buffer stepwise by about 1 to about 2% over a range of at least about 50 to 100% at intervals corresponding to any column volume in the range of about 4 to about 6 CV, or (b) carried out by decreasing the pH in the elution buffer stepwise by any pH in the range of about 0.6 to about 1.0 over a range corresponding to a column volume of about 30 to about 40 CV; (6) The linear flow rate of the elution buffer is any linear flow rate in the range of about 100 to about 800 cm / h; (7) The separation method, wherein the color intensity of the solution containing the separated uncolored protein is (i) substantially the same as or weaker than the color intensity of the reference standard solution BY5 in a visual comparison test with the reference standard solution BY5, or (ii) the Em / UV*100 value of the solution containing the separated uncolored protein is about 6.3 or less;

[60] A method for separating a non-colored protein from a solution containing a non-colored protein and a colored protein, comprising: (1) the separation method comprising: (i) a step of loading the solution into an anion exchange carrier column; (ii) a step of flowing an equilibration buffer, which has been prepared so that the colored protein in the solution binds to the anion exchange carrier but the non-colored protein does not bind, through the anion exchange carrier column at a predetermined flow rate; and (iii) a step of recovering a fraction containing the non-colored protein that flows through in the step (ii), (2) The protein is a bispecific antibody that specifically binds to PD-1 and CD3, respectively, and is composed of a heavy chain and a light chain that form an antigen-binding site that specifically binds to PD-1 and a heavy chain and a light chain that form an antigen-binding site that specifically binds to CD3, wherein (a) the heavy chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, (b) the heavy chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 6, and (c) the light chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to PD-1 and the light chain of the heavy chain and the light chain that form the antigen-binding site that specifically binds to CD3 both consist of the amino acid sequence of SEQ ID NO: 7, (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F, (4) the equilibration buffer has a pH of any value in the range of about 8.2 to about 7.8 and an electric conductivity of any value in the range of about 5 to about 7 mS / cm; (5) the linear flow rate of the equilibration buffer is any value in the range of about 100 to about 400 cm / h; and (6) the color density of the solution containing the separated uncolored protein (i) is substantially the same as or lighter than the color density of the comparison standard solution BY5 in a visual comparison test, or (ii) the Em / UV*100 value of the solution containing the separated uncolored protein is about 6.3 or less;

[0008] [1-1] A method for producing a solution containing an uncolored form of a protein (preferably, an uncolored form of the antibody, bispecific antibody, or anti-PD-1 / CD3 bispecific antibody described in any one of

[51] to

[58] ), the method comprising the separation method (separation step) described in any one of [1] to

[60] ;

[0009] [2-1] A colorless or slightly colored solution comprising the anti-PD-1 / CD3 bispecific antibody of any one of items

[55] to

[58] at a concentration of about 100 to about 300 mg / mL. [2-2] The solution according to the preceding paragraph [2-1], wherein the concentration of the anti-PD-1 / CD3 bispecific antibody is in the range of about 100 to about 200 mg / mL; [2-3] The solution according to the preceding paragraph [2-1], wherein the concentration of the anti-PD-1 / CD3 bispecific antibody is in the range of about 100 to about 150 mg / mL; [2-4] The solution according to the preceding paragraph [2-1], wherein the concentration of the anti-PD-1 / CD3 bispecific antibody is in the range of about 100 to about 120 mg / mL; [2-5] The solution according to any one of the preceding paragraphs [2-1] to [2-4], excluding solutions that are colorless or slightly colored without undergoing any step to reduce or remove coloration; [2-6] [2-7] A colorless or slightly colored solution comprising the anti-PD-1 / CD3 bispecific antibody of any one of the preceding items

[55] to

[58] , produced by a process including the separation method of any one of the preceding items [1] to

[60] ; [2-7] A solution of any one of the preceding items [2-1] to [2-5], produced by a process including the separation method of any one of the preceding items [1] to

[60] ; [2-8] A solution of any one of the preceding items [2-1] to [2-7], produced by further concentrating as necessary; [2-9] A solution of any one of the preceding items [2-1] to [2-8], which, in a visual comparison test with the reference standard solution BY5, is substantially the same as or more diluted than BY5; [2-10] [2-11] The solution according to any one of the preceding paragraphs [2-1] to [2-8], which is substantially the same as or more diluted than the comparative standard solution BY6 in a visual comparison test with the comparative standard solution BY7; [2-12] The solution according to any one of the preceding paragraphs [2-1] to [2-8], which has an Em / UV*100 value of about 6.3 or less; [2-13] The solution according to any one of the preceding paragraphs [2-1] to [2-8] and [2-10], which has an Em / UV*100 value of about 4.1 or less;

[0010] [3-1] A pharmaceutical composition comprising the solution according to any one of the preceding items [2-1] to [2-13]; and [4-1] Use of the pharmaceutical composition according to the preceding item [3-1] in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune disease, graft-versus-host disease (GVHD), or blood cancer.

[0011] The method for removing discoloration from a drug substance for a protein pharmaceutical of the present invention makes it possible to remove discoloration from a protein solution caused by advanced glycation end products.

[0012] The anti-PD-1 / CD3 bispecific antibody was fractionated by strong cation exchange chromatography, and the fractions were concentrated to 100 mg / mL. The highly and less colored fractions were then mixed to prepare a sample corresponding to the reference standard solution BY3.5. This figure shows the chromatogram of reversed-phase HPLC analysis of the sample. The X-axis represents the volume of solution (mL), and the Y-axis represents the UV absorbance detection peak area (UV 280 ) and the fluorescence intensity detection peak area (Em560(ex380)) multiplied by 10 (UV 280 x 10, Em560(ex380) x 10). In the figure, the solid line represents the UV detection chromatogram (UV280_BY3.5), and the dashed line represents the fluorescence detection chromatogram (Fluorescene_BY3.5). The arrow indicates the peak of interest. The anti-PD-1 / CD3 bispecific antibody was fractionated by strong cation exchange chromatography, and the fraction was concentrated to 100 mg / mL. The high-colored fraction and the low-colored fraction were then mixed to prepare a sample corresponding to the reference standard solution BY6.0. This figure shows the chromatogram of reversed-phase HPLC analysis of this sample. The X-axis represents the volume of solution (mL), and the Y-axis represents the UV absorbance detection peak area (UV 280 ) and the fluorescence intensity detection peak area (Em560(ex380)) multiplied by 10 (UV 280The graphs show the Em / UV*100 values ​​(x10, Em560 (ex380) x10), respectively. In the figure, the solid line represents the UV detection chromatogram (UV280_BY6.0), and the dashed line represents the fluorescence detection chromatogram (Fluorescene_BY6.0). The arrows indicate the peaks of interest. The graphs show the results of HPLC analysis of samples corresponding to the comparative standard solutions BY3.5, BY4, BY4.5, BY5, and BY6, which were prepared by mixing the high-colored and low-colored fractions of an anti-PD-1 / CD3 bispecific antibody concentrated to 100 mg / mL. The horizontal axis represents the comparative standard solution number, and the vertical axis represents the Em / UV*100 value. The numbers at the top of each vertical bar represent the Em / UV*100 value of each comparative standard solution. The graphs show pH stepwise gradient elution chromatograms in bind-elute mode for anion exchange using a column packed with Toyopearl® NH2-750F. The sample used in the separation procedure was an antibody. The X-axis shows the volume of the solution (mL), and the Y-axis on the left shows the UV absorption detection peak area (UV 280The right Y-axis represents the electrical conductivity (mS / cm) multiplied by 10. The right Y-axis also represents the percentage of the volume delivered by pump B (%B) of the two mobile phase delivery pumps A and B. In the figure, the solid black line represents the UV detection chromatogram (UV280), the dashed line represents electrical conductivity, and the solid gray line represents the percentage delivered by pump B (%B). The arrows numbered 1 to 4 indicate the position of the purified fractions used for color analysis. This figure shows a chromatogram of anion exchange in flow-through mode using a column packed with Toyopearl® NH2-750F. The sample used for the separation procedure was an antibody. The X-axis, Y-axis, and the solid black, dashed, and gray lines in the figure have the same meanings as in Figure 4. The inverted U-shape represents the range of collected fractions used for analysis after purification. This figure shows a pH linear gradient elution chromatogram of anion exchange using a column packed with Capto® adhere in bind-elute mode. The sample used in the separation was an antibody. The X-axis and Y-axis, as well as the solid black, dashed, and gray lines in the figure, have the same meanings as in Figure 4. The arrows numbered 1 to 5 indicate the positions of the purified fractions used in color analysis. This figure shows a pH linear gradient elution chromatogram of anion exchange using a column packed with Cellufine® MAX IB in bind-elute mode. The sample used in the separation was an antibody. The X-axis and Y-axis, as well as the solid black, dashed, and gray lines in the figure, have the same meanings as in Figure 4. The arrows numbered 1 to 5 indicate the positions of the purified fractions used in color analysis. This figure shows a pH linear gradient elution chromatogram of anion exchange using a column packed with Capto® Q in bind-elute mode. The sample used in the separation was bovine serum albumin (BSA). The X-axis and Y-axis, as well as the black solid line, dashed line, and gray solid line in the figure, have the same meanings as in Figure 4. The arrows numbered 1 to 3 indicate the positions of the purified fractions subjected to color analysis. The amino acid sequences of the heavy and light chains that constitute the antigen-binding site that specifically binds to PD-1 and the heavy and light chains that constitute the antigen-binding site that specifically binds to CD3, which constitute the anti-PD-1 / CD3 bispecific antibody A, are shown.Figure 1 shows the amino acid sequences of the heavy chain constant regions (heavy chain constant regions in the heavy and light chains that form the antigen-binding site that specifically binds to PD-1) constituting the anti-PD-1 / CD3 bispecific antibody B. Figure 2 shows the amino acid sequences of the heavy chain constant regions (heavy chain constant regions in the heavy and light chains that form the antigen-binding site that specifically binds to CD3) constituting the anti-PD-1 / CD3 bispecific antibody B.

[0013] The present invention relates to a method for separating non-colored proteins from a solution containing non-colored proteins and colored proteins (hereinafter referred to as the "Bind-Elute method"), comprising the steps of: (1) loading the solution into an ion exchange carrier column and using an equilibration buffer having a predetermined initial pH to bind the non-colored proteins and colored proteins to the ion exchange carrier; (2) flowing an elution buffer that forms a pH gradient that varies from the initial pH to a final pH through the ion exchange carrier column at a predetermined flow rate so that the non-colored proteins and colored proteins are separated; and (3) isolating a predetermined fraction containing the non-colored proteins eluted in step (2).

[0014] The present invention also includes a method for separating non-colored proteins from a solution containing non-colored proteins and colored proteins, comprising the steps of: (1a) loading the solution into an ion exchange carrier column; (2a) flowing an equilibration buffer, which has been prepared so that the colored proteins in the solution bind to the ion exchange carrier but the non-colored proteins do not bind, through the ion exchange carrier column at a predetermined flow rate; and (3a) recovering a fraction containing the non-colored proteins that flows through in step (2a) (hereinafter referred to as the "Flow-Through method").

[0015] The term "colored protein" as used herein refers to modified proteins that cause the coloration problem. These modifications include several types of advanced glycation end products (hereinafter sometimes abbreviated as "AGEs"). Solutions containing these modifications exhibit a pale yellow to yellow color depending on the amount produced. Advanced glycation end products (AGEs) are a collective term for several types of modified proteins that are produced by the non-enzymatic reaction of free amino groups, such as those in lysine residues, with reducing sugars such as glucose and fructose, followed by repeated irreversible dehydration, condensation, oxidation, and / or reduction. Various AGEs have been confirmed to be produced, including glucose-derived AGEs, glyceraldehyde-derived AGEs, glycolaldehyde-derived AGEs, methylglyoxal-derived AGEs, glyoxal-derived AGEs, and 3-deoxyglucosone-derived AGEs. Some of these AGEs have been reported to contain modified forms that exhibit a yellowish-brown fluorescence. The isoelectric points of these colored protein molecules range from approximately 5 to approximately 9.1, generally ranging from approximately 6 to approximately 7.

[0016] On the other hand, a "non-colored protein" refers to a target protein in which the protein modification is substantially not present or is so slight that the solution is not visually colored. The isoelectric point of the non-colored protein is in the range of about 7 to about 9.5, and more specifically, is in any range selected from the group consisting of about 7.0 or more and less than about 7.5, about 7.5 or more and less than about 8.0, about 8.0 or more and less than about 8.5, about 8.5 or more and less than about 9.0, and about 9.0 or more and less than about 9.5.

[0017] Protein solutions to which the separation method of the present invention is to be applied include those whose color density, either by itself or in a concentrated solution thereof, is darker than that of the BY5 reference standard solution in a visual comparison test under Item 2.65 "Color Comparison Test" in the General Test Methods of the Seventeenth Edition of the Japanese Pharmacopoeia, or whose Em / UV*100 value exceeds approximately 6.3.

[0018] In the present invention, the "equilibration buffer having a predetermined initial pH" refers to a buffer having a pH value substantially equal to the predetermined pH value as the upper limit of the pH gradient formed by the elution buffer used in the present invention, and is prepared in advance so as to have a predetermined electrical conductivity and pH value at which both non-colored and colored protein forms in the solution applied to the ion exchange carrier column can bind to the ion exchange carrier.

[0019] The electrical conductivity of the equilibration buffer and / or elution buffer used in the separation method of the present invention (Bind-Elute method) can be determined in advance depending on the properties of the protein to be used and the type of anion exchange carrier, and can be, for example, any electrical conductivity in the range of about 1 to about 20 mS / cm, preferably any electrical conductivity in the range of about 2 to about 15 mS / cm, specifically, for example, about 2 mS / cm, about 3 mS / cm, about 4 mS / cm, about 5 mS / cm, about 6 mS / cm, about 7 mS / cm, about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, and about 15 mS / cm. In the case of the Bind-Elute method, the electrical conductivities of the equilibration buffer and elution buffer are generally substantially the same.

[0020] The pH and electrical conductivity of the equilibration buffer used in the separation method (Flow-Through method) of the present invention can be adjusted in advance so that the colored protein in the solution binds to the ion exchange carrier but the non-colored protein does not. The pH may be any pH value between about 9.2 and about 7.4, specifically, for example, about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, and about 7.4. Preferably, the pH is any pH value between about 8.6 and about 7.4, more preferably, any pH value between about 8.2 and about 7.4, and even more preferably, any pH value between about 8.2 and about 7.8. The electrical conductivity can be selected from any value in the range of about 1 to about 20 mS / cm, preferably from any value in the range of about 2 to 15 mS / cm, specifically, for example, 2 mS / cm, about 3 mS / cm, about 4 mS / cm, about 5 mS / cm, about 6 mS / cm, about 7 mS / cm, about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, and about 15 mS / cm, preferably from any value in the range of about 5 to 7 mS / cm. A preferred equilibration buffer used in the flow-through method is, for example, HEPES buffer (pH 8.0).

[0021] The wash buffer used in the separation method of the present invention is primarily used to wash the anion exchange carrier before eluting the non-colored form of the protein. Examples of wash buffers that can be used include those having a composition that can remove impurities other than the non-colored form of the target protein and the colored form from the ion exchange carrier, and those having a high pH or low electrical conductivity that is sufficiently outside the elution range of the non-colored form of the target protein.

[0022] The ion exchange carrier in the present invention is preferably an anion exchange carrier. Its form may be, for example, small particles, a membrane, or a monolith, but is preferably in the form of small particles. The ion exchange carrier column in the present invention refers to a column packed with an ion exchange carrier.

[0023] The anion exchange carrier is not limited as long as it exhibits an anion exchange function, and examples thereof include strong anion exchange carriers, weak anion exchange carriers, and multimode anion exchange carriers.

[0024] Examples of strong anion exchange carriers include Fractogel (registered trademark) EMD TMAE (M), Fractogel (registered trademark) EMD TMAE Medcap (M), Fractogel (registered trademark) EMD TMAE Hicap (M), Eshmuno (registered trademark) Q, Eshmuno (registered trademark) QPX, Eshmuno (registered trademark) QPX Hicap, Capto (registered trademark) Q, Capto (registered trademark) Q ImpRes, Q Sepharose (registered trademark) FF, Q Sepharose (registered trademark) HP, Q Sepharose (registered trademark) XL, Source (registered trademark) 30Q, POROS (registered trademark) 50 HQ, POROS (registered trademark) 50 XQ, POROS (registered trademark) 50 PI, Q HyperCel (registered trademark), Toyopearl (registered trademark) GigaCap Q 650M, Toyopearl (registered trademark) GigaCap Q 650S, Toyopearl (registered trademark) Super Q, and YMC (registered trademark) BioPro. Q, Macro-Prep® High Q, Nuvia® Q, UNOsphere® Q, Cellufine® Q-500, Cellufine® MAX Qr and Cellufine® MAX Qh.

[0025] Examples of weak anion exchange carriers include Fractogel (registered trademark) EMD DEAE, Fractogel (registered trademark) EMD DMAE, Capto (registered trademark) DEAE, DEAE Ceramic HyperD (registered trademark) F, Toyopearl (registered trademark) NH2-750F, TOYOPEARL (registered trademark) DEAE-650C, TOYOPEARL (registered trademark) DEAE-650M, TOYOPEARL (registered trademark) DEAE-650S, Cellufine (registered trademark) A-200, Cellufine (registered trademark) A-500, Cellufine (registered trademark) A-800, and Cellufine (registered trademark) MAX DEAE.

[0026] Furthermore, examples of multimode anion exchange carriers include Toyopearl® NH2-750F, Capto® Adhere, Capto® Adhere ImpRes, Capto® MMC, Capto® core 700, and Cellufine® IB. Here, the anion exchange carrier that can be used in the separation method of the present invention is preferably a multimode anion exchange carrier, more preferably Toyopearl® NH2-750F. In the present invention, the "elution buffer that forms a pH gradient that varies from the initial pH to the final pH" refers to an elution solution composed of a combination of two or more buffers that is prepared to form a pH gradient that varies linearly or stepwise in a pH range that includes at least the isoelectric point of the target protein to be eluted. The elution buffer can be prepared, for example, by determining the pH range in advance based on the isoelectric point and its distribution of the target protein, and combining a buffer having an initial pH at the upper limit of the pH range (hereinafter sometimes abbreviated as "buffer A") with a buffer having a final pH at the lower limit of the pH range (hereinafter sometimes abbreviated as "buffer B") in any ratio to achieve a predetermined pH. A pH gradient can be formed in this process by varying the combined ratio of the two buffers continuously or stepwise.

[0027] The pH gradient applied in the separation method of the present invention is a pH gradient that decreases from an initial pH to a final pH, where the initial pH is, for example, any pH value in the range of about 11.0 to about 7.0, specifically, any pH value selected from the group consisting of about 11.0, about 10.8, about 10.6, about 10.4, about 10.2, about 10.0, about 9.8, about 9.6, about 9.4, about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, about 7.4, about 7.2, and about 7.0, and preferably about 10.8 to about 7.2. , about 10.8 to about 7.4, about 10.8 to about 7.6, about 10.8 to about 7.8, about 10.8 to about 8.0, about 10.8 to about 8.2, about 10.8 to about 8.4, about 10.6 to about 7.2, about 10.6 to about 7.4, about 10.6 to about 7.6, about 10.6 to about 7.8, about 10.6 to about 8.0, about 10.6 to about 8.2 or about 10.6 to about 8.4, more preferably any pH value in the range of about 10.6 to about 8.4. On the other hand, the final pH may be, for example, any pH value in the range of about 4.0 to about 8.2, specifically, any pH value selected from the group consisting of about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0 and about 8.2, preferably any pH value in the range of about 4.0 to about 8.0, about 4.0 to about 7.8, about 4.0 to about 7.6, about 4.0 to about 7.4, about 4.0 to about 7.2 or about 4.0 to about 7.0, more preferably any pH value in the range of about 4.0 to about 7.0. However, the initial pH is about 1.0 or more higher than the final pH.

[0028] Furthermore, the range of the pH gradient may be, for example, any one of the initial pHs selected from the group consisting of about 11.0, about 10.8, about 10.6, about 10.4, about 10.2, about 10.0, about 9.8, about 9.6, about 9.4, about 9.2, about 9.0, about 8.8, about 8.6, about 8.4, about 8.2, about 8.0, about 7.8, about 7.6, about 7.4, about 7.2, and about 7.0, to about 4.0, about Examples of final pH ranges include any one selected from the group consisting of 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0 and about 8.2, but preferably the initial pH is at least 2.0 higher than the final pH. Specific examples of the pH gradient range include about 11.0 to about 4.0, about 11.0 to about 4.2, about 11.0 to about 4.4, about 11.0 to about 4.6, about 11.0 to about 4.8, about 11.0 to about 5.0, about 11.0 to about 5.2, about 11.0 to about 5.4, about 11.0 to about 5.6, about 11.0 to about 5.8, about 11.0 to about 6.0, about 11.0 to about 6.2, about 11.0 to about 6.4, about 11.0 to about 6.6, about 11.0 to about 6.8, about 11.0 to about 7.0, about 11.0 to about 7.2, about 11.0 to about 7.4, about 11.0 to about 7.6, about 11.0 to about 7.8, about 11.0 to about 8.0, about 11.0 to about 8.2, about 10.8 to about 4.0, about 10.8 to about 4.2, about 10.8 to about 4.4, about 10.8 to about 4.6, about 10.8 to about 4.8, about 10.8 to about 5.0, about 10.8 to about 5.2, about 10.8 to about 5.4, about 10.8 to about 5.6, about 10.8 to about 5.8, about 10.8 to about 6.0, about 10.8 to about 6.2, about 10.8 to about 6.4, about 10.8 to about 6.6, about 10.8 to about 6.8, about 10.8 to about 7.0, about 10.8 to about 7.2, about 10.8 to about 7.4, about 10.8 to about 7.6, about 10.8 to about 7.8, about 10.8 to about 8.0, about 10.8 to about 8.2, about 10.6 to about 4.0, about 10.6 to about 4.2, about 10.6 to about 4.4, about 10.6 to about 4.6, about 10.6 to about 4.8, about 10.6 to about 5.0, about 10.6 to about 5.2, about 10.6 to about 5.4, about 10.6 to about 5.6, about 10.6 to about 5.8, about 10.6 to about 6.0, about 10.6 to about 6.2, about 10.6 to about 6.4, about 10.6 to about 6.6, about 10.6 to about 6.8, about 10.6 to about 7.0, about 10.6 to about 7.2, about 10.6 to about 7.4, about 10.6 to about 7.6, about 10.6 to about 7.8, about 10.6 to about 8.0, about 10.6 to about 8.2, about 10.4 to about 4.0, about 10.4 to about 4.2, about 10.4 to about 4.4, about 10.4 to about 4.6, about 10.4 to about 4.8, about 10.4 to about 5.0, about 10.4 to about 5.2, about 10.4 to about 5.4, about 10.4 to about 5.6, about 10.4 to about 5.8, about 10.4 to about 6.0, about 10.4 to about 6.2, about 10.4 to about 6.4, about 10.4 to about 6.6, about 10.4 to about 6.8, about 10.4 to about 7.0, about 10.4 to about 7.2, about 10.4 to about 7.4, about 10.4 to about 7.6, about 10.4 to about 7.8, about 10.4 to about 8.0, about 10.4 to about 8.2, about 10.2 to about 4.0, about 10.2 to about 4.2, about 10.2 to about 4.4, about 10.2 to about 4.6, about 10.2 to about 4 0.8, about 10.2 to about 5.0, about 10.2 to about 5.2, about 10.2 to about 5.4, about 10.2 to about 5.6, about 10.2 to about 5.8, about 10.2 to about 6.0, about 10.2 to about 6.2, about 10.2 to about 6.4, about 10.2 to about 6.6, about 10.2 to about 6.8, about 10.2 to about 7.0, about 10.2 to about 7.2, about 10.2 to about 7.4, about 10.2 to about 7.6, about 10.2 to about 7.8, about 10.2 to about 8.0, about 10.2 to about 8.2, about 10.0 to about 4.0, about 10.0 to about 4.2, about 10.0 to about 4.4, about 10.0 to about 4.6, about 10.0 to about 4.8, about 10. 0 to about 5.0, about 10.0 to about 5.2, about 10.0 to about 5.4, about 10.0 to about 5.6, about 10.0 to about 5.8, about 10.0 to about 6.0, about 10.0 to about 6.2, about 10.0 to about 6.4, about 10.0 to about 6.6, about 10.0 to about 6.8, about 10.0 to about 7.0, about 10.0 to about 7 .2, about 10.0 to about 7.4, about 10.0 to about 7.6, about 10.0 to about 7.8, about 10.0 to about 8.0, about 9.8 to about 4.0, about 9.8 to about 4.2, about 9.8 to about 4.4, about 9.8 to about 4.6, about 9.8 to about 4.8, about 9.8 to about 5.0, about 9.8 to about 5.2, about 9.8 to about 5. 4, about 9.8 to about 5.6, about 9.8 to about 5.8, about 9.8 to about 6.0, about 9.8 to about 6.2, about 9.8 to about 6.4, about 9.8 to about 6.6, about 9.8 to about 6.8, about 9.8 to about 7.0, about 9.8 to about 7.2, about 9.8 to about 7.4, about 9.8 to about 7.6, about 9.8 to about 7.8, about 9.6 to about 4.0, about 9.6 to about 4.2, about 9.6 to about 4.4, about 9.6 to about 4.6, about 9.6 to about 4.8, about 9.6 to about 5.0, about 9.6 to about 5.2, about 9.6 to about 5.4, about 9.6 to about 5.6, about 9.6 to about 5.8, about 9.6 to about 6.0, about 9.6 to about 6.2, about 9.6 to about 6. 4, about 9.6 to about 6.6, about 9.6 to about 6.8, about 9.6 to about 7.0, about 9.6 to about 7.2, about 9.6 to about 7.4, about 9.6 to about 7.6, about 9.4 to about 4.0, about 9.4 to about 4.2, about 9.4 to about 4.4, about 9.4 to about 4.6, about 9.4 to about 4.8, about 9.4 to about 5.0, about 9. 4 to about 5.2, about 9.4 to about 5.4, about 9.4 to about 5.6, about 9.4 to about 5.8, about 9.4 to about 6.0, about 9.4 to about 6.2, about 9.4 to about 6.4, about 9.4 to about 6.6, about 9.4 to about 6.8, about 9.4 to about 7.0, about 9.4 to about 7.2, about 9.4 to about 7.4, about 9.2 to about 4. 0, about 9.2 to about 4.2, about 9.2 to about 4.4, about 9.2 to about 4.6, about 9.2 to about 4.8, about 9.2 to about 5.0, about 9.2 to about 5.2, about 9.2 to about 5.4, about 9.2 to about 5.6, about 9.2 to about 5.8, about 9.2 to about 6.0, about 9.2 to about 6.2, about 9.2 to about 6.4, about 9. 2 to about 6.6, about 9.2 to about 6.8, about 9.2 to about 7.0, about 9.2 to about 7.2, about 9.0 to about 4.0, about 9.0 to about 4.2, about 9.0 to about 4.4, about 9.0 to about 4.6, about 9.0 to about 4.8, about 9.0 to about 5.0, about 9.0 to about 5.2, about 9.0 to about 5.4, about 9.0 to about 5. 6, about 9.0 to about 5.8, about 9.0 to about 6.0, about 9.0 to about 6.2, about 9.0 to about 6.4, about 9.0 to about 6.6, about 9.0 to about 6.8, about 9.0 to about 7.0, about 8.8 to about 4.0, about 8.8 to about 4.2, about 8.8 to about 4.4, about 8.8 to about 4.6, about 8.8 to about 4.8, about 8. 8 to about 5.0, about 8.8 to about 5.2, about 8.8 to about 5.4, about 8.8 to about 5.6, about 8.8 to about 5.8, about 8.8 to about 6.0, about 8.8 to about 6.2, about 8.8 to about 6.4, about 8.8 to about 6.6, about 8.8 to about 6.8, about 8.6 to about 4.0, about 8.6 to about 4.2, about 8.6 to about 4. 4, about 8.6 to about 4.6, about 8.6 to about 4.8, about 8.6 to about 5.0, about 8.6 to about 5.2, about 8.6 to about 5.4, about 8.6 to about 5.6, about 8.6 to about 5.8, about 8.6 to about 6.0, about 8.6 to about 6.2, about 8.6 to about 6.4, about 8.6 to about 6.6, about 8.4 to about 4.0, about 8.4 to about 4.2, about 8.4 to about 4.4, about 8.4 to about 4.6, about 8.4 to about 4.8, about 8.4 to about 5.0, about 8.4 to about 5.2, about 8.4 to about 5.4, about 8.4 to about 5.6, about 8.4 to about 5.8, about 8.4 to about 6.0, about 8.4 to about 6.2, about 8.4 to about 6.4, about 8.2 to about 4.0, about 8.2 to about 4.2, about 8.2 to about 4.4, about 8.2 to about 4.6, about 8.2 to about 4.8, about 8.2 to about 5.0, about 8.2 to about 5.2, about 8 0.2 to about 5.4, about 8.2 to about 5.6, about 8.2 to about 5.8, about 8.2 to about 6.0, about 8.2 to about 6.2, about 8.0 to about 4.0, about 8.0 to about 4.2, about 8.0 to about 4.4, about 8.0 to about 4.6, about 8.0 to about 4.8, about 8.0 to about 5.0, about 8.0 to about 5.2, about 8.0 to about 5.4, about 8.0 to about 5.6, about 8.0 to about 5.8, about 8.0 to about 6.0, about 7.8 to about 4.0, about 7.8 to about 4.2, about 7.8 to about 4.4, about 7.8 to about 5.6 0.8 to about 4.6, about 7.8 to about 4.8, about 7.8 to about 5.0, about 7.8 to about 5.2, about 7.8 to about 5.4, about 7.8 to about 5.6, about 7.8 to about 5.8, about 7.6 to about 4.0, about 7.6 to about 4.2, about 7.6 to about 4.4, about 7.6 to about 4.6, about 7.6 to about 4.8, about 7.6 to about 5.0, about 7.6 to about 5.2, about 7.6 to about 5.4, about 7.6 to about 5.6, about 7.4 to about 4.0, about 7.4 to about 4.2, about 7.4 to about 4.4, about 7.6 to about 5.8 These ranges include about 0.4 to about 4.6, about 7.4 to about 4.8, about 7.4 to about 5.0, about 7.4 to about 5.2, about 7.4 to about 5.4, about 7.2 to about 4.0, about 7.2 to about 4.2, about 7.2 to about 4.4, about 7.2 to about 4.6, about 7.2 to about 4.8, about 7.2 to about 5.0, about 7.2 to about 5.2, about 7.0 to about 4.0, about 7.0 to about 4.2, about 7.0 to about 4.4, about 7.0 to about 4.6, about 7.0 to about 4.8, and about 7.0 to about 5.0.

[0029] Buffer A and / or Buffer B can be prepared from a combination of Good's buffers selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS, depending on the individual separation conditions. In particular, when a solution containing an anti-PD-1 / CD3 bispecific antibody of the present invention is subjected to the separation method, both buffers are preferably prepared from a combination of Good's buffers selected from the group consisting of MES, MOPS, TAPS, and CAPSO, with ThermoFisher's Thermo Scientific® CX-1 pH Gradient Buffer A (pH 5.6) and CX-1 pH Gradient Buffer B (pH 10.2) being particularly preferred.

[0030] Alternatively, both buffers may be prepared from a combination of multiple buffering agents selected from the group consisting of, for example, Tris-acetate, Tris-phosphate, and Tris-citrate.

[0031] The pH gradient conditions can be determined in advance depending on the properties of the protein to be subjected to the separation method so that the uncolored and colored forms of the protein are separated. Here, "the uncolored protein and the colored protein are separated" means that the both proteins are separated under predetermined conditions so that a fraction containing the uncolored protein recovered by the separation method or a concentrate thereof has a color density that is "colorless or slightly colored," or that the recovered fraction containing the uncolored protein is substantially free of the colored form, or that the remaining ratio of the colored form is reduced compared to the uncolored form so that the fraction has a color density that is "colorless or slightly colored."

[0032] In the present invention, the color intensity of "colorless or slightly colored" used as a definition of the degree of coloration can be evaluated by qualitative or quantitative analysis. For example, qualitative analysis can be performed by visual comparison with the reference standard solutions listed in the United States Pharmacopoeia 2012 (USP Monograph 631, Color and Achromicity), the European Pharmacopoeia 5.0 (EP Method 2.2.2, Degree of Coloration of Liquids), or the 17th Edition of the Japanese Pharmacopoeia, Section 2.65, "Color Comparison Test Method," of the General Test Methods. For example, with regard to the coloration of the protein in the present invention, among the brownish yellow reference standard solutions BY1 to BY7, if the color is substantially the same as or lighter than BY7, it can be evaluated as "colorless." If the color is darker than BY7 but substantially the same as or lighter than BY5, it can be evaluated as "slightly colored" or "faintly yellow." Specifically, the Clarity, Opalescence and Coloration (COC) assay can be performed by pouring the reference standard solution and the test solution into a flat-bottomed, colorless, transparent, neutral glass test tube with an inner diameter of 15 to 25 mm so that the layers are 40 mm deep, and then comparing the two.

[0033] Quantitative analysis can be carried out by, for example, calculating the Em / UV*100 value, which can be performed by measuring ultraviolet absorption with a spectrophotometer and measuring fluorescence spectra with a spectrofluorometer. In this "color comparison test method," the brownish yellow reference standard solution BY6 corresponds to an Em / UV*100 value of approximately 4.1, and BY5 corresponds to an Em / UV*100 value of approximately 6.3.

[0034] In a visual comparison test with the reference standard solution BY5, the color intensity of the non-colored protein fraction or a concentrate thereof separated or fractionated according to the present invention is preferably evaluated to be substantially the same as or weaker than BY5, more preferably substantially the same as or weaker than BY6, and most preferably substantially the same as BY7. Here, it is preferred that a concentrate of the non-colored protein fraction (having a protein concentration of at least any concentration in the range of about 50 to about 100 mg / mL, preferably at least a concentration selected from the group consisting of about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, and about 100 mg / mL) be evaluated to be substantially the same as or weaker than BY5, more preferably substantially the same as or weaker than BY6, or substantially the same as BY7. The non-colored protein fraction can be concentrated by known methods, such as ultrafiltration, precipitation-redissolution, and freeze-drying-redissolution.

[0035] In the separation method of the present invention, the pH gradient can be a stepwise gradient, for example, by increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 0.5 to about 5%, for example, by any percentage selected from the group consisting of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, and about 5%, at intervals corresponding to any column volume (CV) in the range of about 1 to 20 CV, for example, any column volume selected from the group consisting of about 1 CV, about 2 CV, about 3 CV, about 4 CV, about 5 CV, about 6 CV, about 7 CV, about 8 CV, about 9 CV, about 10 CV, about 15 CV, and about 20 CV, thereby applying a stepwise pH gradient from the initial pH to the final pH. Here, the proportion (%) of buffer B can be increased stepwise from at least any proportion in the range of about 10 to about 100%, for example, any proportion selected from the group consisting of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 70%, about 90%, and about 100%, to any proportion in the range of about 30 to about 100%, for example, any proportion selected from the group consisting of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. Specific ranges for increasing the proportion (%) of buffer B include about 10 to about 30%, about 10 to about 40%, about 10 to about 50%, about 10 to about 60%, about 10 to about 70%, about 10 to about 80%, about 10 to about 90%, about 10 to about 100%, about 20 to about 40%, about 20 to about 50%, about 20 to about 60%, about 20 to about 70%, about 20 to about 80%, about 20 to about 90%, about 20 to about 100%, about 30 to about 50%, about 30 to about 60%, about These ranges include 30 to about 70%, about 30 to about 80%, about 30 to about 90%, about 30 to about 100%, about 40 to about 60%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 40 to about 100%, about 50 to about 70%, about 50 to about 80%, about 50 to about 90%, about 50 to about 100%, about 60 to about 80%, about 60 to about 90%, about 60 to about 100%, about 70 to about 90%, about 70 to about 100% and about 80 to about 100%.The term "column volume (CV)" refers to the volume of the gap between the packing materials packed in the column, i.e., the amount of solvent required to fill the gap, and this volume is expressed as "1 CV."

[0036] In another embodiment of the stepwise gradient pH gradient in the separation method of the present invention, the pH of the elution buffer is decreased by any value in the range of about 0.1 to about 1.0, for example, by any value selected from the group consisting of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0, at intervals corresponding to any column volume in the range of about 1 to about 20 CV, for example, any column volume selected from the group consisting of about 1 CV, about 2 CV, about 3 CV, about 4 CV, about 5 CV, about 6 CV, about 7 CV, about 8 CV, about 9 CV, about 10 CV, about 15 CV, and about 20 CV, thereby forming a stepwise pH gradient from the initial pH to the final pH.

[0037] In yet another embodiment of the stepwise gradient pH gradient in the separation method of the present invention, a stepwise pH gradient can be applied from the initial pH to the final pH by increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 0.5 to about 5%, for example, any percentage selected from the group consisting of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, and about 5%, within a range corresponding to any column volume in the range of about 20 to about 50 CV, for example, any column volume selected from the group consisting of about 20 CV, 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV. Here, the proportion (%) of buffer B can be increased stepwise from at least any proportion in the range of about 10 to about 100%, for example, any proportion selected from the group consisting of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, to any proportion in the range of about 30 to about 100%, for example, any proportion selected from the group consisting of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%.

[0038] Similarly, in yet another embodiment of the stepwise gradient pH gradient in the separation method of the present invention, a stepwise pH gradient can be applied from the initial pH to the final pH by decreasing the pH of the elution buffer by any value in the range of about 0.1 to about 1.0, for example, by any value selected from the group consisting of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0, over a range corresponding to any column volume in the range of about 20 to about 50 CV, for example, any column volume selected from the group consisting of about 20 CV, about 25 CV, about 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV. On the other hand, in a linear embodiment of the pH gradient in the separation method of the present invention, a continuous pH gradient can be applied from the initial pH to the final pH by continuously increasing the proportion (%) of buffer B in the elution buffer in a range corresponding to any column volume in the range of about 20 to about 50 CV, for example, any column volume selected from the group consisting of about 20 CV, 25 CV, 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV. Here, the proportion (%) of buffer B can be continuously increased from at least any proportion in the range of about 10 to about 100%, for example, any proportion selected from the group consisting of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, to any proportion in the range of about 30% to about 100%, for example, any proportion selected from the group consisting of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%.

[0039] In addition, as another embodiment of the linear pH gradient in the separation method of the present invention, a continuous pH gradient can be applied from the initial pH to the final pH by continuously decreasing the pH of the elution buffer over any column volume in the range of about 20 to about 50 CV, for example, a range corresponding to any column volume selected from the group consisting of about 20 CV, 25 CV, 30 CV, about 35 CV, about 40 CV, about 45 CV, and about 50 CV.

[0040] The pH gradient in the separation method of the present invention is preferably carried out, for example, by: (a) increasing the percentage (%) of buffer B in the elution buffer stepwise by any percentage in the range of about 1 to about 2% over a range of at least about 50 to 100% at intervals corresponding to any column volume in the range of about 4 to about 6 CV; or (b) decreasing the pH in the elution buffer stepwise by any pH percentage in the range of about 0.6 to about 1.0 over a range corresponding to any column volume in the range of about 30 to about 40 CV.

[0041] In the separation method of the present invention, the elution buffer can be flowed at an optimal linear flow rate determined in advance depending on the type of ion exchange carrier used and the degree of separation between the colored and non-colored bodies. The linear flow rate can be selected from any linear flow rate in the range of about 100 to about 800 cm / h, for example, from the group consisting of about 100 cm / h, about 150 cm / h, about 200 cm / h, about 250 cm / h, about 300 cm / h, about 350 cm / h, about 400 cm / h, about 450 cm / h, about 500 cm / h, about 550 cm / h, about 600 cm / h, about 650 cm / h, about 700 cm / h, about 750 cm / h, and about 800 cm / h. Here, the linear flow rate refers to the speed at which the elution buffer passes through the cross section of the ion exchange carrier column, and is calculated using the following formula: linear flow rate (cm / h) = flow rate (cm 3 / h) / column cross-sectional area (cm 2 ) is calculated as follows.

[0042] In the separation method of the present invention, the total load (mg / mL) of uncolored and colored proteins applied to the ion exchange carrier column is about 2 to about 200 mg / mL, for example, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, and about 200 mg / mL. Here, the load (mg / mL) refers to the amount of sample applied per volume of the ion exchange carrier column.

[0043] The total concentration of the non-colored protein and the colored protein that can be loaded onto the ion exchange carrier column also depends on the conditions described above, but is usually about 2 to about 50 mg / mL, specifically about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, and about 50 mg / mL, and preferably about 10 to about 30 mg / mL.

[0044] The separation method of the present invention is mainly carried out at room temperature.

[0045] As used herein, the term "about" means that the value may vary below or above the stated value within a range of 10%.

[0046] [Proteins of the Present Invention] The separation method of the present invention can be applied to any protein that is discolored due to modifications resulting from glycation reactions. Protein pharmaceuticals that require high concentrations are particularly susceptible to discoloration due to glycation reactions because they must be produced from cultured cells (e.g., COS cells or CHO cells) under high oxygen and high glucose conditions. Antibodies are particularly suited to the application of this separation method. While the separation method of the present invention can be applied to a wide range of proteins with any molecular weight, it is specifically targeted at proteins in the range of approximately 50 to 750 kDa, and is suitable for separating proteins in the range of approximately 50 to 150 kDa.

[0047] The separation method of the present invention can be applied to a wide range of proteins with essentially any isoelectric point, but preferably targets those with an isoelectric point in the range of about 5 to 9.5. Among these, the target colored proteins are those with an isoelectric point in the range of about 5 to about 9.1, with those generally in the range of about 6 to about 7 being the main target, while the target non-colored proteins are those with an isoelectric point in the range of about 7 to about 9.5, specifically those in any range selected from the group consisting of about 7 or more and less than about 7.5, about 7.5 or more and less than about 8, about 8 or more and less than about 8.5, about 8.5 or more and less than about 9, and about 9 or more and less than about 9.5 being the main target.

[0048] The antibodies to which the separation method of the present invention can be applied may be of any type or form, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and multispecific antibodies (e.g., bispecific antibodies). The species of the antibody is not particularly limited, and may be, for example, antibodies derived from mouse, rat, rabbit, goat, or human. However, for human pharmaceuticals, where strict quality requirements are required, chimeric antibodies, humanized antibodies, or fully human antibodies are preferred. Furthermore, because these monoclonal antibodies are mostly produced from producing cells such as COS cells or CHO cells, discoloration due to glycation reactions can sometimes be a problem.

[0049] In the present invention, the term "antibody fragment" refers to a portion of an antibody molecule that retains its antigen-binding ability, and includes, for example, F(ab')2, Fab', Fab, Fv, and single-chain FV.

[0050] In the present invention, the term "bispecific antibody" refers to an antibody that possesses binding specificities for two different antigen molecules or epitopes in a single molecule. Examples of bispecific antibody forms include diabodies, bispecific sc(Fv)2, bispecific minibodies, bispecific F(ab')2, bispecific hybrid antibodies, covalent diabodies (bispecific DARTs), bispecific (FvCys)2, bispecific F(ab'-zipper)2, bispecific (Fv-zipper)2, bispecific three-chain antibodies, and bispecific mAb2.

[0051] In the present invention, the term "isotype" is used to refer to the antibody class (e.g., IgM or IgG) encoded by the heavy chain constant region gene, and the preferred isotype for bispecific antibodies of the present invention is IgG, more preferably IgG1 or IgG4.

[0052] Examples of antibodies to which the separation method of the present invention can be applied include anti-CD40 antibodies (e.g., Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Ravagalimab, Selicrelumab, Teneliximab, ABBV-428, and APX005M, etc.), anti-CD70 antibodies (e.g., Cusatuzumab, Vorsetuzumab, and ARGX-110, etc.), anti-HER1 antibodies (e.g., Cetuximab, Panitumumab, Necitumumab, Nimotuzumab, Depatuximab, Futuximab, Laprituximab, Matuzumab, Modotuximab, Petosemtamab, Tomuzotuximab, etc.), and anti-HER1 antibodies (e.g., Cetuximab, Panitumumab, Necitumumab, Nimotuzumab, Depatuximab, Futuximab, Laprituximab, Matuzumab, Modotuximab, Petosemtamab, Tomuzotuximab, etc.). Losatuxizumab, Serclutamab, Imgatuzumab, Futuximab, and Zalutumumab, etc.), anti-HER2 antibodies (e.g., Pertuzumab, Disitamab, Gancotamab, Margetuximab, Timigutuzumab, Zanidatamab, Ertumaxomab, Zenocutuzumab, Trastuzumab, MM-111, R48, and ZW33, etc.), anti-HER3 antibodies (e.g., Duligotuzumab, Elgemtumab, Istiratumab, Lumretuzumab, Zenocutuzumab, Patritumab, Seribantumab, and MM-111, etc.), anti-VEGFR1 antibodies (e.g., Icrucumab, etc.), anti-VEGFR2 antibodies (e.g., Ramucirumab, Alacizumab, and Olinvacimab, etc.), anti-CD20 antibodies (e.g., Rituximab, Blontuvetmab, Epitumomab, Ibritumomab, Ocaratuzumab, Ocrelizumab, Nofetumomab, Tositumomab, Veltuzumab, Ofatumumab, Ublituximab, Obinutuzumab, and Nofetumomab, etc.), anti-CD30 antibodies (e.g., Brentuximab and Iratumumab, etc.), anti-CD38 antibodies (e.g., Daratumumab, Isatuximab, Mezagitamab,AT13 / 5 and MOR202, etc.), anti-TNFRSF10B antibodies (e.g., Benufutamab, Conatumumab, Drozitumumab, Lexatumumab, Tigatuzumab, DS-8273a, etc.), anti-TNFRSF10A antibodies (e.g., Mapatumumab, etc.), anti-MUC1 antibodies (e.g., Cantuzumab, Civatuzumab, Epitumomab, Sontuzumab, Gatipotuzumab, Nacolomab, 7F11C7, BrE-3, CMB-401, CTM01, and HMFG1, etc.), anti-MUC5AC antibodies (e.g., Ensituximab, etc.), anti-MUC16 antibodies (e.g., Oregovomab, Abagovomab, Igovomab, and Sofituzumab, etc.), anti-DLL4 antibodies (e.g., Demcizumab, Dilpacimab, Navicizumab, and Enoticumab, etc.), anti-Fucosyl GM1 antibodies (e.g., BMS-986012, etc.), anti-gpNMB antibodies (e.g., Glembatumumab, etc.), anti-Mesothelin antibodies (e.g., Amatuximab, Anet umab, RG7784, and BMS-986148, etc.), anti-MMP9 antibodies (e.g., Andecaliximab, etc.), anti-GD2 antibodies (e.g., Dinutuximab, Naxitamab, 14G2a, MORAb-028, Surek, TRBs07, and ME361, etc.), anti-MET antibodies (e.g., Emibetuzumab, Onartuzumab, and Telisotuzumab, etc.), anti-FOLR1 antibodies (e.g., Farletuzumab and Mirvetuximab, etc.), anti-CD79b antibodies (e.g., Iladatuzumab, and Polatuzumab, etc.), anti-DLL3 antibodies (e.g., Rovalpituzumab, etc.), anti-CD51 antibodies (e.g., Abituzumab, Etaracizumab, and Intetumumab, etc.), anti-EPCAM antibodies (e.g., Adecatumumab, Catumaxomab, Edrecolomab, Oportuzumab, Citatuzumab, and Tucotuzumab, etc.), anti-CEACAM5 antibodies (e.g., Altumomab, Arcitumomab, Cergutuzumab, Labetuzumab,90 Y-cT84.66, AMG211, BW431 / 26, CE25 / B7, COL-1 and T84.66 M5A, etc.), anti-CEACAM6 antibodies (e.g., Tinurilimab, etc.), anti-FGFR2 antibodies (e.g., Aprutumab and Bemarituzumab, etc.), anti-CD44 antibodies (e.g., Bivatuzumab, etc.), anti-PSMA antibodies (e.g., Capromab, 177Lu-J591 and ES414, etc.), anti-Endoglin antibodies (e.g., Carotuximab, etc.), anti-IGF1R antibodies (e.g., Cixutumumab, Figitumumab, Ganitumab, Dalotuzumab, Teprotumumab, Robatumumab, etc.), anti-TNFSF11 antibodies (e.g., Denosumab, etc.), anti-GUCY2C (e.g., Indusatumab, etc.), anti-SLC39A6 antibodies (e.g., Ladiratuzumab, etc.), anti-SLC34A2 antibodies (e.g., Lifastuzumab, etc.), anti-NCAM1 antibodies (e.g., Lorvotuzumab and N901, etc.), anti-gangliosides GD3 antibodies (e.g., Ecromeximab and Mitumomab, etc.), anti-AMHR2 antibodies (e.g., Murlentamab, etc.), anti-CD37 antibodies (e.g., Lilotomab, Naratuximab, Otlertuzumab, etc.), anti-IL1RAP antibodies (e.g., Nidanilimab, etc.), anti-PDGFR2 antibodies (e.g., Olaratumab and Tovetumab, etc.), anti-CD200 antibodies (e.g., Samalizumab, etc.), anti-TAG-72 antibodies (e.g., Anatumomab, Minretu, etc.), momab, Satumomab, CC49, HCC49, and M4, etc.), anti-SLITRK6 antibodies (e.g., Sirtratumab, etc.), anti-DPEP3 antibodies (e.g., Tamrintamab, etc.), anti-CD19 antibodies (e.g., Coltuximab, Denintuzumab, Inebilizumab, Loncastuximab, Obexelimab, Tafasitamab, Taplitumomab, and huAnti-B4, etc.), anti-NOTCH2 / 3 antibodies (e.g., Tarextumab, etc.), anti-tenascin C antibodies (e.g., Tenatumomab, etc.), anti-AXL antibodies (e.g., Enapotamab and Tilvestamab, etc.), anti-STEAP1 antibodies (e.g., Vandortuzumab, etc.), anti-CTAA16 antibodies (e.g., Votumumab, etc.), CLDN18 antibodies (e.g., Zolbetuximab, etc.), anti-GM3 antibodies (e.g., Racotumomab, FCGR1, and H22, etc.), anti-PSCA antibodies (e.g., MK-4721, etc.),Anti-FN extra domain B antibodies (e.g., AS1409, etc.), anti-HAVCR1 antibodies (e.g., CDX-014, etc.), anti-TNFRSF4 antibodies (e.g., MEDI6383, etc.), anti-PD-1 antibodies (e.g., Nivolumab, Cemiplimab-rwlc, Pembrolizumab, Spartalizumab, Tislelizumab, Dostarlimab, Toripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodapolimab, Retifanlimab, Balstilimab, Se anti-PD-L1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab, Manelimab, Paciliimab, Envafolimab, Cosibelimab, Sugemalimab, etc.), anti-CTLA-4 antibodies (e.g., Ipilimumab, Zalifrelimab, Nurulimab, and Trelimab), anti-PD-L1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab, Manelimab, Paciliimab, Envafolimab, Cosibelimab, Sugemalimab, etc.), anti-CTLA-4 antibodies (e.g., Ipilimumab, Zalifrelimab, Nurulimab, and Trelimab), anti-PD-L1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab, Manelimab, Paciliimab, Envafolimab, Cosibelimab, Sugemalimab, etc.), anti-PD-L2 ...1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab, Manelimab, Paciliimab, Envafolimab, Cosibelimab, Sugemalim melimumab, etc.), anti-TIM3 antibodies (e.g., Cobolimab and MBG453, etc.), anti-CD3 antibodies (e.g., Catumaxomab, etc.), anti-CD4 antibodies (e.g., Zanolimumab and IT1208, etc.), anti-CD27 antibodies (e.g., Varlilumab, etc.), anti-CD73 antibodies (e.g., Oleclumab and BMS-986179, etc.), anti-CD80 antibodies (e.g., Galiximab, etc.), anti-CD86 antibodies, anti-CD160 antibodies, CD57 antibodies, CD226 antibodies, CD112 antibodies, CD155 antibodies, anti-OX40 antibodies ( For example, MEDI6469, Ivuxolimab, MEDI0562, MEDI6383, Efizonerimod, GSK3174998, BMS-986178, and MOXR0916, etc.), OX40L antibodies (for example, Oxelumab and Tavolimab, etc.), anti-ICOS antibodies (for example, Vopratelimab and GSK3359609, etc.), anti-4-1BB (CD137) antibodies (for example, Urelumab and Utomilumab, etc.), anti-4-1BBL (CD137L) antibodies, anti-2B4 antibodies, anti-GITR antibodies (for example,MK-4166, INCAGN01876, GWN323, and TRX-518, etc.), anti-B7-H3 antibodies (e.g., Enoblituzumab, Mirzotamab, and Omburtamab, etc.), anti-LAG-3 antibodies (e.g., Relatlimab, Ieramilimab, Fianlimab, Encelimab, and Mavezelimab, etc.), anti-BTLA antibodies, anti-HVEM antibodies, anti-VISTA antibodies (e.g., Onvatilimab, etc.), anti-GITRL antibodies, anti-Galectin-9 antibodies, anti-B7-H4 antibodies, anti-B7-H5 antibodies, PD-L2 antibodies, KLRG-1 antibodies, E-Cadherin antibodies, N-Cadherin antibodies, R-Cadherin antibodies, anti-CSF-1R antibodies (e.g., Cabralizumab, mab, Emactuzumab, LY3022855, Axatilimab, MCS-110, IMC-CS4, AMG820, Pexidartinib, BLZ945, and ARRY-382, etc.), CXCR4 antibodies (e.g., Ulocuplumab, etc.), FLT-3 antibodies, anti-TIGIT antibodies (e.g., Tiragolumab, Etigilimab, Vibostolimab, and BMS-986207, etc.), anti-KIR antibodies (e.g., Lirilumab, IPH2101, LY3321367, and MK-4280, etc.), anti-SLAMF7 antibodies (e.g., Azintuxizumab and Elotuzumab, etc.), anti-CD47 antibodies, and anti-NKG2A antibodies (e.g., Monalizumab, etc.).

[0053] Furthermore, examples of bispecific antibodies to which the separation method of the present invention can be applied include anti-PD-1 / CD3 bispecific antibodies, anti-PD-1 / CD19 bispecific antibodies (preferably bispecific antibodies described in WO2020 / 204152), anti-PD-1 / CD4 bispecific antibodies, anti-HER1-MET bispecific antibodies (e.g., Amivantamab, etc.), anti-EPCAM-CD3 bispecific antibodies (e.g., Solitomab and Catumaxomab, etc.), anti-Ang2-VEGF bispecific antibodies (e.g., Vanucizumab, etc.), anti-HER2-CD3 bispecific antibodies (e.g., Ertumaxomab, etc.), anti-HER3-IGF1R bispecific antibodies (e.g., Istiratumab, etc.), anti-PMSA-CD3 bispecific antibodies (e.g., Pasotuxizumab, etc.), anti-HER1-LGR5 bispecific antibodies (e.g., Petosemtamab, etc.), and anti-SSTR2-CD3 bispecific antibodies (e.g., Ti dutamab, etc.), anti-CD30-CD16A bispecific antibodies (e.g., AFM13, etc.), anti-CEA-CD3 bispecific antibodies (e.g., Cibisatamab and RO6958688, etc.), anti-CD3-CD19 bispecific antibodies (e.g., Duvortuxizumab and Blinatumomab, etc.), anti-IL3RA-CD3 bispecific antibodies (e.g., Flotetuzumab and Vibecotamab, etc.), anti-GPRC5D-CD3 bispecific antibodies (e.g., Talquetamab, etc.), anti-TNFRSF17-CD3 bispecific antibodies (e.g., Teclistamab, etc.), anti-HER2-HER3 bispecific antibodies (e.g., Zenocutuzumab, etc.), and anti-CD20-CD3 bispecific antibodies (e.g., Plamotamab, Odronextamab, Mosunetuzumab, Epcoritamab, Glofitamab, and REGN1979, etc.).

[0054] Antibodies to which the separation method of the present invention may be applied include, for example, anti-PD-1 / CD3 bispecific antibodies. In particular, solutions containing such bispecific antibodies (anti-PD-1 / CD3 bispecific antibody A) comprising heavy and light chains forming the antigen-binding site that specifically binds to PD-1 and heavy and light chains forming the antigen-binding site that specifically binds to CD3, wherein (a) the heavy chain of the heavy and light chains forming the antigen-binding site that specifically binds to PD-1 comprises any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 (preferably the amino acid sequence of SEQ ID NO: 5), (b) the heavy chain of the heavy and light chains forming the antigen-binding site that specifically binds to CD3 comprises the amino acid sequence of SEQ ID NO: 6, and (c) the light chain of the heavy and light chains forming the antigen-binding site that specifically binds to PD-1 and the light chain of the heavy and light chains forming the antigen-binding site that specifically binds to CD3 both comprise the amino acid sequence of SEQ ID NO: 7, are observed to become colored due to the generation of AGEs, and the separation method of the present invention is therefore required.

[0055] The anti-PD-1 / CD3 bispecific antibody A can be produced according to the method disclosed in WO2019 / 156199 and corresponds to the anti-PD-1 / CD3 bispecific antibody clones PD1-1(Bi) to PD1-5(Bi), respectively.

[0056] Furthermore, the separation methods of the present invention are also applicable to anti-PD-1 / CD3 bispecific antibodies (anti-PD-1 / CD3 bispecific antibody B) in which the heavy chain constant regions of the heavy and light chains that constitute the antigen-binding site that specifically binds to PD-1 of the above-mentioned anti-PD-1 / CD3 bispecific antibody A have been substituted with heavy chain constant regions consisting of any one amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and the heavy chain constant regions of the heavy and light chains that constitute the antigen-binding site that specifically binds to CD3 have been substituted with heavy chain constant regions consisting of any one amino acid sequence selected from SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively.

[0057] These anti-PD-1 / CD3 bispecific antibodies B correspond to the antibody clone in which methionine at position 252, according to the EU numbering system, in the heavy chain constant region of the anti-PD-1 / CD3 bispecific antibody clone PD1-5(Bi) (anti-PD-1 / CD3 bispecific antibody A, in which the heavy chain of the heavy and light chains forming the antigen-binding site that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5), has been substituted with glutamic acid, proline, arginine, and aspartic acid, respectively, the antibody clone in which asparagine at position 434 has been substituted with leucine, and the antibody clone in which glutamine at position 438 has been substituted with glutamic acid, respectively, and can be produced by methods in accordance with known techniques for such amino acid substitutions.

[0058] Furthermore, the separation methods of the present invention may also be applied to anti-PD-1 / CD3 bispecific antibodies that cross-compete with anti-PD-1 / CD3 bispecific antibody A for binding to PD-1 and / or CD3, respectively. Here, "cross-compete" means that the bispecific antibody binds to the same or overlapping epitopes on PD-1 and / or CD3 as anti-PD-1 / CD3 bispecific antibody A, thereby inhibiting the binding of anti-PD-1 / CD3 bispecific antibody A to PD-1 and / or CD3, regardless of the extent of such binding. Cross-competition can be assessed by competitive binding assays, such as Biacore analysis, ELISA, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer (FRET), or fluorescence micrometric assay technology (FMAT®).

[0059] Figure 9 shows the amino acid sequences of the heavy and light chains that form the antigen-binding site that specifically binds to PD-1 and the heavy and light chains that form the antigen-binding site that specifically binds to CD3, which constitute anti-PD-1 / CD3 bispecific antibody A. Figures 10 and 11 show the amino acid sequences of the heavy chain constant regions that constitute anti-PD-1 / CD3 bispecific antibody B.

[0060] Here, "heavy and light chains constituting an antigen-binding site that specifically binds to PD-1" refers to a heavy and light chain complex in which the heavy and light chains associate via disulfide bonds to form an antigen-binding site that specifically binds to PD-1. An "antigen-binding site" is the minimum unit for an antibody to have binding activity to its antigen, and "specifically binds to PD-1" means that the heavy and light chains bind to at least 1 x 10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9 This is used as a characteristic of a substance that can directly bind to PD-1 with a binding activity having a higher affinity (dissociation constant (Kd value)) than M, and that does not substantially bind to at least other receptor members belonging to the so-called CD28 family of receptors, such as CD28, CTLA-4, and ICOS.

[0061] Furthermore, "heavy and light chains constituting an antigen-binding site that specifically binds to CD3" refers to a heavy and light chain complex in which the heavy and light chains associate via disulfide bonds to form an antigen-binding site that specifically binds to CD3. An "antigen-binding site" is the minimum unit for an antibody to have binding activity to its antigen, and "specifically binds to CD3" means that the heavy and light chains bind to at least 1 x 10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9 It is used as a characteristic that it can directly bind to CD3 with an affinity (dissociation constant (Kd value)) higher than M and does not substantially bind to other proteins.

[0062] The anti-PD-1 / CD3 bispecific antibodies of the present invention are characterized in that the heavy and light chains constituting the antigen-binding site that specifically binds to PD-1, and the heavy and light chains constituting the antigen-binding site that specifically binds to CD3, have structures consisting of the same amino acid sequence.

[0063] A preferred embodiment of the separation method of the present invention is, for example, a method for separating non-colored proteins from a solution containing non-colored proteins and colored proteins, comprising: (1) the separation method comprising: (i) loading the solution into an anion exchange carrier column and using an equilibration buffer having a predetermined initial pH to bind the non-colored proteins and colored proteins to the anion exchange carrier; (ii) flowing an elution buffer, which forms a pH gradient descending from the initial pH to a final pH, through the anion exchange carrier column at a predetermined flow rate under predetermined conditions so that the non-colored proteins and colored proteins are separated; and (iii) isolating a predetermined fraction containing the non-colored proteins eluted in step (ii); (2) the protein is any one of the anti-PD-1 / CD3 bispecific antibodies A or any one of the anti-PD-1 / CD3 bispecific antibodies B; (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F; (4) The initial pH of the equilibration buffer and the elution buffer is any pH value in the range of about 10.6 to about 8.4, and the final pH of the elution buffer is any pH value in the range of about 7.0 to about 4.0 (provided that the initial pH is at least 2.0 higher than the final pH); (5) The pH gradient is (a) implemented by stepwise increasing the percentage (%) of buffer B in the elution buffer by any percentage in the range of about 1 to about 2% over a range of at least about 50 to 100% at intervals corresponding to any column volume in the range of about 4 to about 6 CV, or (b) implemented by decreasing the pH in the elution buffer by any pH value in the range of about 0.6 to about 1.0 over a range corresponding to any column volume in the range of about 30 to about 40 CV; (6) The linear flow rate of the elution buffer is about 100 to about 800 cm / h; (7) The separation method in which the color intensity of the solution containing the separated uncolored protein is (i) substantially the same as or weaker than the color intensity of the comparison standard solution BY5 in a visual comparison test with the comparison standard solution BY5, or (ii) the Em / UV*100 value of the solution containing the separated uncolored protein is about 6.3 or less.

[0064] Another preferred embodiment of the separation method of the present invention is, for example, a method for separating a non-colored protein from a solution containing the non-colored protein and a colored protein, comprising: (1) the separation method comprising: (i) loading the solution onto an anion exchange carrier column; (ii) flowing an equilibration buffer, which has been prepared so that the colored protein in the solution binds to the anion exchange carrier but the non-colored protein does not bind, through the anion exchange carrier column at a predetermined flow rate; and (iii) recovering a fraction containing the non-colored protein that flows through in the above step; (2) the protein is any antibody of anti-PD-1 / CD3 bispecific antibody A or any antibody of anti-PD-1 / CD3 bispecific antibody B; (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F; (4) the pH of the equilibration buffer is any pH value in the range of about 7.8 to about 8.2, and the electrical conductivity is about 5 to about 7. (5) the linear flow rate of the equilibration buffer is about 100 to about 400 cm / h; and (6) the color density of the solution containing the separated uncolored protein is (i) substantially the same as or lighter than the color density of the comparison standard solution BY5 in a visual comparison test, or (ii) the Em / UV*100 value of the solution containing the separated uncolored protein is about 6.3 or less.

[0065] [Solution Comprising an Anti-PD-1 / CD3 Bispecific Antibody of the Present Invention] The present invention relates to a solution comprising an anti-PD-1 / CD3 bispecific antibody produced by a production method that includes steps corresponding to the separation method of the present invention. Specifically, the solution is colorless or slightly colored, containing either anti-PD-1 / CD3 bispecific antibody A or anti-PD-1 / CD3 bispecific antibody B at a concentration of about 100 to about 300 mg / mL. However, preferred embodiments of the bispecific antibody-containing solution do not include solutions that are colorless or slightly colored at a concentration of about 100 to about 300 mg / mL and that have not undergone any steps to reduce or remove coloration, such as those used in the separation method of the present invention, during any part of the production process.

[0066] A solution containing either anti-PD-1 / CD3 bispecific antibody A or either anti-PD-1 / CD3 bispecific antibody B may be subjected to the separation method of the present invention at a concentration of less than about 100 mg / mL, for example, between about 10 and 50 mg / mL, and then concentrated, if necessary, to produce a purified protein. The uncolored protein fraction can be concentrated using known methods, such as ultrafiltration, precipitation and reconstitution, and lyophilization and reconstitution. The color intensity of the colorless or slightly colored solution can be confirmed by the qualitative or quantitative analysis described above.

[0067] [Pharmaceutical Compositions and Pharmaceutical Uses] Solutions containing anti-PD-1 / CD3 bispecific antibodies produced by a production method that includes, as part thereof, steps corresponding to the separation method of the present invention, are useful for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating autoimmune diseases, graft-versus-host disease (GVHD), or blood cancers.Examples of autoimmune diseases that can be prevented, suppressed in progression, and / or treated by the anti-PD-1 / CD3 bispecific antibody of the present invention include Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic sclerosis, progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult idiopathic arthritis, and idiopathic rheumatoid arthritis. Human Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, genotype 1, genotype 2, genotype 3, genotype 4, genotype 5, genotype 6, genotype 7, genotype 8, genotype 9, genotype 10, genotype 11, genotype 12, genotype 13, genotype 14, genotype 15, genotype 16, genotype 17, genotype 18, genotype 19, genotype 20, genotype 21, genotype 22, genotype 23, genotype 24, genotype 25, genotype 26, genotype 27, genotype 28, genotype 29, genotype 30, genotype 31, genotype 32, genotype 33, genotype 34, genotype 35, genotype 36, genotype 37, geno Primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, bullous pemphigoid, bullous pemphigoid, bullous pemphigoid These include rash, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplant immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, systemic mastocytosis, and inclusion body myositis.

[0068] Furthermore, examples of hematologic cancers that can be prevented, suppressed in progression, and / or treated by the anti-PD-1 / CD3 bispecific antibodies of the present invention include multiple myeloma, malignant lymphomas (e.g., non-Hodgkin's lymphomas (e.g., B-cell non-Hodgkin's lymphomas (e.g., precursor B-cell lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, chronic B-lymphocytic leukemia (e.g., small lymphocytic lymphoma)), B-cell precursor leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, nodal marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma (MALT lymphoma), splenic lymphoma, and leukemia. Marginal zone B-cell lymphoma, hairy cell leukemia, hairy cell leukemia-variant type, follicular lymphoma, childhood follicular lymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma-unspecified type, splenic diffuse red pulp small B-cell lymphoma, lymphoplasmacytic lymphoma, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis, splenic B-cell lymphoma / leukemia-unclassifiable type, monoclonal gammopathy of undetermined significance-IgM type, μ heavy chain disease, λ heavy chain disease, α heavy chain disease Chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma, monoclonal immunoglobulin deposition disease, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, T-cell / histiocytocyte-rich large B-cell lymphoma, primary central nervous system diffuse large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma - leg type, EBV-positive diffuse large B-cell lymphoma - unspecified type, EBV-positive mucocutaneous ulcer, chronic inflammation-associated diffuse large B-cell lymphoma, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, ALK-positive large cell type B-cell lymphoma, plasmablastic lymphoma, primary body cavity effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma, unspecified type, Burkitt-like lymphoma with 11q abnormality, high-grade B-cell lymphoma with MYC and / or BCL2 and / or BCL6 rearrangements, high-grade B-cell lymphoma, unspecified type, and B-cell lymphoma, unclassified type with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, T / NK-cell non-Hodgkin lymphoma (e.g., precursor T-cell lymphoblastic lymphoma, chronic T-lymphocytic leukemia, T-cell large granular lymphocytic leukemia,Large granular NK cell leukemia, rapidly progressive NK cell leukemia, peripheral T cell lymphoma, peripheral T cell lymphoma / unspecified type, unclassifiable peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, undifferentiated large cell (CD30 positive) lymphoma, Angiocentric lymphoma, intestinal T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic γ-δ T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, Hodgkin-like / Hodgkin-related anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma, adult T-cell lymphoma, T-cell prolymphocytic leukemia, chronic NK-cell lymphoproliferative dysplasia, pediatric systemic EBV-positive T-cell lymphoma, variola bullosa-like lymphoproliferative dysplasia, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma, gastrointestinal indolent T-cell lymphoproliferative dysplasia, hepatosplenic T-cell lymphoma, primary cutaneous CD30-positive T-cell lymphoproliferative dysplasia, lymphomatoid papulosis, primary cutaneous anaplastic large cell type Lymphoma, primary cutaneous gamma delta T-cell lymphoma, primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acral CD8-positive T-cell lymphoma, primary cutaneous CD4-positive small / medium-sized T-cell lymphoproliferative disorder, follicular T-cell lymphoma, nodal peripheral T-cell lymphoma with follicular helper T-cell phenotype, anaplastic large cell lymphoma ALK-positive, anaplastic large cell lymphoma ALK-negative, and breast implant-associated anaplastic large cell lymphoma) and Hodgkin's lymphoma lymphomas (e.g., classical Hodgkin's lymphoma (e.g., nodular sclerosing, mixed cell, lymphocyte-rich, and lymphocytopenic) or nodular lymphocyte-predominant Hodgkin's lymphoma)), leukemias (e.g., acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia (lymphoblastic lymphoma), chronic lymphocytic leukemia (small lymphocytic lymphoma), myelodysplastic syndromes, and chronic myeloid leukemia), primary central nervous system malignant lymphoma, and myeloproliferative syndromes, etc.

[0069] Here, "treatment" in the present invention means, for example, curing or ameliorating a certain disease or its symptoms, "prevention" means preventing the onset of a certain disease or symptom or delaying it for a certain period of time, and "suppression of symptom progression" means suppressing the progression or worsening of symptoms to stop the progression of the pathology. Note that "prevention" also includes suppression of recurrence. "Suppression of recurrence" means preventing the recurrence of a certain disease or symptom or reducing the possibility of recurrence.

[0070] Solutions containing the anti-PD-1 / CD3 bispecific antibodies of the present invention are typically administered systemically or locally parenterally as pharmaceutical compositions. Specific administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection methods include, for example, intravenous, intramuscular, and intraperitoneal injections. Intravenous administration is preferably by infusion. The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but typically ranges from 0.1 μg / kg to 300 mg / kg of the anti-PD-1 / CD3 bispecific antibody per adult, and particularly preferably ranges from 0.1 mg / kg to 10 mg / kg of the anti-PD-1 / CD3 bispecific antibody, administered parenterally once or several times a day, or administered intravenously continuously for 30 minutes to 24 hours per day. Of course, as mentioned above, the dosage varies depending on various conditions, so that in some cases a smaller dosage than the above-mentioned dosage is sufficient, and in other cases it is necessary to administer a dosage exceeding the range.

[0071] [Formulations] When a solution containing the anti-PD-1 / CD3 bispecific antibody of the present invention is formulated and used as an injection or infusion solution for intravenous drip, the injection or infusion solution may be in the form of an aqueous solution, suspension, or emulsion. Solvents used in injections or infusion solutions for intravenous drip include, for example, distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). If necessary, the solution may contain a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, and antioxidants. Examples of stabilizers that can be used include various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 20 (registered trademark), Polysorbate 80 (registered trademark), HCO-50, etc.). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and epsilon aminocaproic acid buffer.Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. Examples of antioxidants that can be used include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0072] Injections or infusion solutions for infusion can be produced by sterilizing them in the final step or by aseptic techniques, for example, by sterilizing them by filtration with a filter or the like, and then filling them into sterile containers.

[0073] The contents of all patent and non-patent literature or references explicitly cited in this specification are hereby incorporated by reference in their entirety.

[0074] The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited thereto. Various changes or modifications can be made by those skilled in the art based on the description of the present invention, and these changes or modifications are also included in the present invention.

[0075] [Examples] Example 1: Quantitative analysis of antibody color bodies by reversed-phase HPLC. To quantitatively evaluate the amount of color bodies of anti-PD-1 / CD3 bispecific antibodies, an evaluation system using reversed-phase HPLC was established. The equipment used and the operating conditions are shown in Tables 1 and 2, respectively.

[0076]

[0077]

[0078] Analysis was initiated by loading a solution of 5 μg of anti-PD-1 / CD3 bispecific antibody A onto a column that had been sufficiently stabilized at the designated column temperature with mobile phase A. The target peak was vertically cut from the obtained chromatogram at the position where the detection signal was lowest between the adjacent peaks before and after it, and the peak area detected by ultraviolet light at a wavelength of 280 nm (UV 280 After obtaining the fluorescence intensity detection peak area (Em 560 (ex380)), the Em / UV*100 value was calculated and used as an index for color quantification. Note that, because the system is connected to a fluorescence detector after the UV detector, the fluorescence detection peak appears later.

[0079] Using this method, the Em / UV*100 values ​​were compared with those of the reference standard solution in Section 2.65, "Color Comparison Test," of the General Tests of the Japanese Pharmacopoeia, Seventeenth Edition. Anti-PD-1 / CD3 bispecific antibodies purified under conditions without attention to removing color bodies were fractionated by strong cation exchange chromatography using a salt gradient. The resulting purified fractions were concentrated to 100 mg / mL. The highly and less colored fractions were combined to prepare samples with visually corresponding color to the reference standard solutions BY3.5, BY4, BY4.5, BY5, and BY6, which were then analyzed by HPLC. Comparison of the chromatograms corresponding to BY3.5 (Figure 1) and BY6.0 (Figure 2) clearly shows that the Em / UV*100 value was higher for the sample corresponding to BY3.5. When the comparative standard solution was compared with the HPLC analysis sample, it was confirmed that when the clarity of the target protein is made thinner than the clarity of the comparative standard solution BY5 using the method of the present invention, it is desirable that the Em / UV*100 value be approximately 6 or less (Figure 3).

[0080] Example 2: Anion Exchange Chromatography Purification of Antibody in Bind-Elute Mode (1) To identify separation conditions for reducing the chromophore ratio of anti-PD-1 / CD3 bispecific antibody A of the present invention, pH step gradient anion exchange chromatography was performed in bind-elute mode (Figure 4). The separation column used was a Tricorn column (GE Healthcare) packed with Toyopearl NH2-750F using flow packing. A solution containing anti-PD-1 / CD3 bispecific antibody A purified from clarified CHO cell culture medium by Protein A affinity chromatography was subjected to low pH treatment for virus inactivation, followed by adjustment to pH 10.2. This was used as the sample prior to anion exchange chromatography purification. Other equipment and conditions used are listed in Table 3.

[0081]

[0082] After thoroughly equilibrating the column with commercially available buffer A (ThermoFisher's Thermo Scientific® CX-1 pH Gradient Buffer B (pH 10.2)), which served as mobile phase A, 4.3 mL of the 9.3 mg / mL antibody solution was weighed and loaded to a load of 20 mg IgG / mL resin. The percentage of commercially available buffer B (ThermoFisher's Thermo Scientific® CX-1 pH Gradient Buffer A (pH 5.6)) (%B in Figure 4) was then increased stepwise from 25%. A sharp peak appeared around 26% (%B), and the main peak of the antibody appeared around 20 mL of flow volume. The Em / UV*100 values ​​of the fractions around this peak were 1.4, 1.4, 5.1, and 7.6, respectively (corresponding to arrows 1 to 4 in Figure 4), starting from the weakly retained fractions. The Em / UV*100 value of the antibody sample before purification was 8.6, confirming that the colored body was highly separated by the method of this example.

[0083] Example 3: Anion Exchange Chromatography Purification of Antibody in Flow-Through Mode Furthermore, to find alternative separation conditions for reducing the chromatophore ratio of anti-PD-1 / CD3 bispecific antibody A of the present invention, anion exchange chromatography was performed in flow-through mode using the same separation column system as in Example 1 (Figure 5). Similarly, a solution containing anti-PD-1 / CD3 bispecific antibody A purified by Protein A affinity chromatography from clarified CHO cell culture medium was subjected to a low pH treatment for virus inactivation, followed by adjustment to pH 8.0 to prepare a sample prior to anion exchange chromatography purification. Other equipment used and conditions are listed in Table 4.

[0084]

[0085] After thoroughly equilibrating the column with HEPES buffer (pH 8.0) used as mobile phase A, 3.9 mL of the 10.3 mg / mL antibody solution was measured and loaded to a total load of 20 mg IgG / mL resin. Immediately after loading, a flow-through peak containing the antibody appeared. Subsequently, the pH was continuously decreased with mobile phase B (commercially available buffer B (ThermoFisher's Thermo Scientific® CX-1 pH Gradient Buffer A (pH 5.6))), resulting in the appearance of an elution peak presumably representing adsorbed impurities. The Em / UV*100 value of the fraction containing the flow-through peak was 4.0, while the Em / UV*100 values ​​of the antibody solution before purification and a specific fraction within the elution peak were 9.1 and 22.2, respectively, confirming that the method of this example also achieved high separation of colored bodies.

[0086] Example 4: Antibody Purification by Anion Exchange Chromatography in Bind-Elute Mode (2) To identify additional separation conditions for reducing the chromophore ratio of anti-PD-1 / CD3 bispecific antibodies, anion exchange chromatography was performed in bind-elute mode using a Tricorn column (GE Healthcare) packed with Capto adhere by flow packing (Figure 6). Similarly, a solution containing the anti-PD-1 / CD3 bispecific antibody A purified from clarified CHO cell culture medium by Protein A affinity chromatography was subjected to a low pH treatment for virus inactivation, followed by adjustment to pH 10.2, and this was used as the sample prior to anion exchange chromatography purification. Other equipment and conditions used are listed in Table 5.

[0087]

[0088] After thoroughly equilibrating the column with commercially available buffer A (pH 10.2), which served as mobile phase A, 3.8 mL of the 10.6 mg / mL antibody solution was weighed and loaded to a load of 20 mg IgG / mL resin. The percentage of commercially available buffer B (as mobile phase B, see Figure 6 ) was then increased to 25% (%B) for four column volumes. The percentage of mobile phase B was then gradually increased from 25% (%B). A peak representing the antibody appeared after 40 mL of flow volume. The Em / UV*100 values ​​of the fractions near the peak, from the weakly retained fractions, were 1.7, 1.7, 6.5, 10.8, and 14.4 (corresponding to arrows 1 to 5 in Figure 6 ). Meanwhile, the Em / UV*100 value of the unpurified antibody sample was 9.0. This confirms that the method of this example also achieved high separation of the colored body.

[0089] Example 5: Antibody Purification by Anion Exchange Chromatography in Bind-Elute Mode (3) Subsequently, anion exchange chromatography was performed in bind-elute mode using a separation column consisting of Cellufine MAX IB packed into a Tricorn column (GE Healthcare) using flow packing (Figure 7). Similarly, a solution containing the anti-PD-1 / CD3 bispecific antibody A purified by Protein A affinity chromatography from clarified CHO cell culture medium was subjected to low pH treatment for virus inactivation, followed by adjustment to pH 10.2, and this was used as the sample prior to anion exchange chromatography purification. Other equipment used and conditions are listed in Table 6.

[0090]

[0091] After thoroughly equilibrating the column with commercially available buffer A (pH 10.2), which served as mobile phase A, 3.8 mL of the 10.6 mg / mL antibody solution was weighed and loaded to a load of 20 mg IgG / mL resin. The percentage of commercially available buffer B (%B in Figure 7 ) was then increased to 25% (%B) for four column volumes. The percentage of mobile phase B was then increased continuously, resulting in a peak of the antibody appearing after approximately 30 mL of flow volume. The Em / UV*100 values ​​of the fractions around the peak, from the weakly retained fractions to the unpurified antibody sample, were 1.6, 1.7, 2.1, 3.3, and 8.0 (corresponding to arrows 1 to 5 in Figure 7 ). The Em / UV*100 value of the unpurified antibody sample was 9.0, confirming that the method of this example also achieved high separation of the colored body.

[0092] Example 6: Anion-exchange chromatography purification of BSA To verify whether separation of chromophores from proteins other than antibodies is possible, as in the case of antibodies, anion-exchange chromatography was performed in bind-elute mode on BSA (bovine serum albumin; low salt concentration, Fujifilm Wako Pure Chemical Industries) containing chromophores (Figure 8). A Capto Q Tricorn column (GE Healthcare) was used as the separation column. Other equipment and conditions used are listed in Table 7.

[0093]

[0094] After thoroughly equilibrating the column with citrate-phosphate buffer (pH 7.0), which corresponds to mobile phase A, 157 mL of the 3.0 mg / mL BSA solution was weighed and loaded to a total load of 40 mg BSA / mL resin. The percentage of mobile phase B (%B) was then increased to 70% (%B) for four column volumes, and then the percentage of mobile phase B was continuously increased. A BSA peak appeared at a flow volume of approximately 180 mL or higher. The Em / UV*100 values ​​of the fractions around this peak, starting from the weakly retained fractions, were 7.7, 10.3, and 13.8 (respectively, corresponding to arrows 1-3 in Figure 8 ). The Em / UV*100 value of the unpurified BSA sample was 10.7. These results demonstrate that the method of the present invention can effectively separate colored bodies of not only antibodies but also other proteins.

[0095] The separation method of the present invention can be used to remove color from colorless protein solutions, particularly antibody pharmaceuticals that require strict quality control.

Claims

1. A method for producing a solution containing a protein non-colored body, comprising a step of separating the protein non-colored body from a solution containing the protein non-colored body and a protein colored body, wherein the separation step comprises: (i) introducing the solution into an ion exchange carrier column and binding the protein non-colored body and the protein colored body to the ion exchange carrier using an equilibration buffer having a predetermined initial pH; (ii) flowing an elution buffer that forms a pH gradient varying from the initial pH to the final pH at a predetermined flow rate through the ion exchange carrier column under conditions predetermined such that the protein non-colored body and the protein colored body are separated; and (iii) collecting a predetermined fraction containing the protein non-colored body eluted by the step (ii).

2. A method for producing a solution containing a protein non-colored body, comprising a step of separating the protein non-colored body from a solution containing the protein non-colored body and a protein colored body, wherein the separation step comprises: (i) introducing the solution into an ion exchange carrier column; (ii) flowing an equilibration buffer prepared such that the protein colored body in the solution binds to the ion exchange carrier and the protein non-colored body does not bind thereto through the ion exchange carrier column at a predetermined flow rate; and (iii) collecting a fraction containing the protein non-colored body that flows through as it is in the step (ii).

3. The production method according to claim 1 or 2, wherein the color density of the solution containing the protein non-colored body and the protein colored body or its concentrate is (1) darker than the comparison standard solution BY5 in the color comparison test described in item 2.65 of the general test method in the Seventeenth Revised Japanese Pharmacopoeia by visual comparison test, or (2) the value obtained by multiplying the ratio of the fluorescence intensity detection peak area to the ultraviolet absorption detection peak area at a wavelength of 280 nm (where the excitation wavelength is 380 nm and the detection wavelength is 560 nm) calculated based on the ultraviolet absorption measurement and fluorescence spectrum measurement of the solution or its concentrate by 100 (Em / UV * 100 value) exceeds about 6.

3.

4. The production method according to any one of claims 1 to 3, wherein the ion exchange carrier is an anion exchange carrier.

5. The production method according to claim 4, wherein the anion exchange carrier is a weak anion exchange carrier or a multimode anion exchange carrier.

6. The production method according to claim 5, wherein the weak anion exchange carrier is selected from the group consisting of Fractogel® EMD DEAE, Fractogel® EMD DMAE, Capto® DEAE, DEAE Ceramic HyperD® F, Toyopearl® NH2-750F, TOYOPEARL® DEAE-650C, TOYOPEARL® DEAE-650M, TOYOPEARL® DEAE-650S, Cellufine® A-200, Cellufine® A-500, Cellufine® A-800, and Cellufine® MAX DEAE.

7. The production method according to claim 5, wherein the multimode anion exchange carrier is selected from the group consisting of Toyopearl® NH2-750F, Capto® Adhere, Capto® Adhere ImpRes, Capto® MMC, Capto® core 700, and Cellufine® IB.

8. The production method according to any one of claims 1 to 7, wherein the loading amounts of the protein non-colored body and the protein colored body introduced into the ion exchange carrier column are about 2 to about 200 mg / mL.

9. The production method according to any one of claims 1 and 3 to 8, wherein the range of the pH gradient from the initial pH to the final pH is set so as to sandwich the isoelectric points of the protein colored body and / or the protein non-colored body.

10. The production method according to any one of claims 1 and 3 to 9, wherein the pH gradient decreases from the initial pH to the final pH.

11. The production method according to any one of claims 1 and 3 to 10, wherein the initial pH is any pH value in the range of about 11.0 to about 7.

0.

12. The production method according to any one of claims 1 and 3 to 11, wherein the final pH is any pH value in the range of about 4.0 to about 8.2 (provided that the initial pH is about 1.0 or more higher than the final pH).

13. The production method according to any one of claims 1 and 3 to 12, wherein the pH gradient is a linear gradient or a stepwise gradient.

14. The production method according to any one of claims 1 and 3 to 13, wherein the elution buffer consists of a combination of buffers having the initial pH and buffers having the final pH in any ratio.

15. The production method according to claim 14, wherein the buffer having the initial pH and / or the buffer having the final pH consists of a combination of a plurality of Good buffers each selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS.

16. The production method according to claim 14, wherein the buffer having the initial pH is Thermo Scientific® CX-1 pH Gradient Buffer B (pH 10.2), and the buffer having the final pH is Thermo Scientific® CX-1 pH Gradient Buffer A (pH 5.6).

17. The production method according to any one of claims 14 to 16, wherein a pH gradient with the elution buffer is formed by continuously or stepwise changing the ratio of the combination of the buffer having the initial pH and the buffer having the final pH.

18. The production method according to any one of claims 14 to 17, wherein the pH gradient is formed by: (a) stepwise increasing the percentage of the buffer having the final pH in the elution buffer by an arbitrary percentage in the range of about 1% to about 2% for each interval corresponding to an arbitrary column volume in the range of about 4 to about 6 CV, over a range of at least about 50% to 100%; or (b) stepwise decreasing the pH in the elution buffer by an arbitrary pH in the range of about 0.6 to about 1.0 for a range corresponding to an arbitrary column volume in the range of about 30 to about 40 CV.

19. The production method according to any one of claims 1 to 18, wherein the linear flow rates of the elution buffer according to claim 1 and the equilibration buffer according to claim 2 are each an arbitrary linear flow rate in the range of about 100 to about 800 cm / h.

20. The manufacturing method according to any one of claims 1 and 3 to 19, wherein the electrical conductivity of the equilibration buffer and / or the elution buffer recited in claim 1 is any electrical conductivity in the range of about 1 to about 20 mS / cm.

21. The manufacturing method according to any one of claims 2 to 8 and 19, wherein the electrical conductivity of the equilibration buffer recited in claim 2 is any electrical conductivity in the range of about 1 to about 20 mS / cm.

22. The manufacturing method according to any one of claims 2 to 8, 19, and 21, wherein the pH of the equilibration buffer recited in claim 2 is any pH value in the range of about 9.2 to about 7.

4.

23. In each step included in the separation step according to any one of claims 1 to 22, as appropriate and if necessary, the manufacturing method according to any one of claims 1 to 22, including a step of washing the ion exchange carrier with a washing buffer.

24. The manufacturing method according to any one of claims 1 to 23, wherein the color density of the fraction of non-colored protein or its concentrate obtained by fractionation or recovery is substantially the same as or thinner than BY5 in a visual comparison test with the comparative standard solution BY5 in the "Color Comparison Test Method" of the Japanese Pharmacopoeia.

25. The manufacturing method according to any one of claims 1 to 23, wherein the color density of the fraction of non-colored protein or its concentrate obtained by fractionation or recovery is substantially the same as or thinner than BY6 in a visual comparison test with the comparative standard solution BY6 in the "Color Comparison Test Method" of the Japanese Pharmacopoeia.

26. The manufacturing method according to any one of claims 1 to 23, wherein the color density of the fraction of non-colored protein or its concentrate obtained by fractionation or recovery is substantially the same as BY7 in a visual comparison test with the comparative standard solution BY7 in the "Color Comparison Test Method" of the Japanese Pharmacopoeia.

27. The manufacturing method according to any one of claims 1 to 23, wherein the value of Em / UV * 100 of the fraction of non-colored protein or its concentrate obtained by fractionation or recovery is about 6.3 or less.

28. The manufacturing method according to any one of claims 1 to 23, wherein the value of Em / UV * 100 of the fraction of non-colored protein or its concentrate obtained by fractionation or recovery is about 4.1 or less.

29. The manufacturing method according to any one of claims 1 to 28, wherein the protein concentration in the fraction of non-colored protein obtained by fractionation or recovery is any concentration in the range of about 20 to about 300 mg / mL.

30. The production method according to any one of claims 1 to 29, wherein the protein is an antibody or an antibody fragment thereof.

31. The production method according to claim 30, wherein the antibody is a bispecific antibody.

32. The production method according to claim 31, wherein the bispecific antibody is a bispecific antibody capable of specifically binding to PD-1 and CD3, respectively.

33. The bispecific antibody capable of specifically binding to PD-1 and CD3, respectively, comprises a heavy chain and a light chain constituting an antigen-binding site specifically binding to PD-1 and a heavy chain and a light chain constituting an antigen-binding site specifically binding to CD3, and (a) the heavy chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, (b) the heavy chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to CD3 consists of the amino acid sequence of SEQ ID NO: 6, and (c) both the light chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to PD-1 and the light chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to CD3 consist of the amino acid sequence of SEQ ID NO:

7. The production method according to claim 32, wherein the bispecific antibody is as described above.

34. The production method according to claim 33, wherein the heavy chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to PD-1 consists of the amino acid sequence of SEQ ID NO:

5.

35. The heavy chain constant region of each heavy chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to PD-1 according to claim 33 or 34 is replaced with a heavy chain constant region consisting of any one amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and the heavy chain constant region of each heavy chain in the heavy chain and light chain constituting the antigen-binding site specifically binding to CD3 as described above is replaced with a heavy chain constant region consisting of any one amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO:

19. The production method according to claim 33 or 34.

36. A method for producing a solution containing a protein non-colored body, comprising a step of separating the protein non-colored body from a solution containing a protein non-colored body and a protein colored body, wherein: (1) the separation step comprises: (i) introducing the solution into an anion exchange carrier column and binding the protein non-colored body and the protein colored body to the anion exchange carrier using an equilibration buffer having a predetermined initial pH; (ii) flowing an elution buffer that forms a pH gradient decreasing from the initial pH to the final pH at a predetermined flow rate through the anion exchange carrier column under conditions predetermined such that the protein non-colored body and the protein colored body are separated; and (iii) collecting a predetermined fraction containing the protein non-colored body eluted by the step (ii); (2) the protein consists of a heavy chain and a light chain constituting an antigen-binding site that specifically binds to PD-1 and a heavy chain and a light chain constituting an antigen-binding site that specifically binds to CD3, and is a bispecific antibody that specifically binds to PD-1 and CD3, wherein (a) the heavy chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (b) the heavy chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 6; and (c) both the light chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to PD-1 and the light chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to CD3 consist of the amino acid sequence of SEQ ID NO: 7; (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F; (4) the initial pH of the equilibration buffer and the elution buffer is any pH value from about 10.6 to about 8.4, and the final pH of the elution buffer is any pH value in the range from about 7.0 to about 4.0 (provided that the initial pH is 2.0 or more higher than the final pH).), and (5) the pH gradient is (a) increased stepwise by any proportion in the range of about 1% to about 2% over a range of at least about 50% to 100% of the proportion (%) of buffer B in the elution buffer for each interval corresponding to any column volume in the range of about 4 to about 6 CV, or (b) carried out by stepwise decreasing the pH in the elution buffer by any pH in the range of about 0.6 to about 1.0 in a range corresponding to any column volume in the range of about 30 to about 40 CV, (6) the linear flow rate of the elution buffer is about 100 to about 800 cm / h, and (7) the color density of the solution containing the fractionated protein-free body is (i) substantially the same as or thinner than BY5 in a visual comparison test with the comparison standard solution BY5 in the color comparison test described in item 2.65 of the general test method in the 17th revised Japanese Pharmacopoeia, or (ii) the Em / UV * 100 value of the solution containing the fractionated protein-free body is about 6.3 or less, the production method.

37. A method for producing a solution containing a protein non-colored body, comprising a step of separating the protein non-colored body from a solution containing a protein non-colored body and a protein-colored body, wherein: (1) the separation step comprises: (i) a step of introducing the solution into an anion exchange carrier column; (ii) a step of flowing an equilibration buffer prepared such that the protein-colored body in the solution binds to the anion exchange carrier and the protein non-colored body does not bind, through the anion exchange carrier column at a predetermined flow rate; and (iii) a step of recovering a fraction containing the protein non-colored body that flows through as it is in the step (ii); (2) the protein consists of a heavy chain and a light chain constituting an antigen-binding site that specifically binds to PD-1 and a heavy chain and a light chain constituting an antigen-binding site that specifically binds to CD3, and is a bispecific antibody that specifically binds to PD-1 and CD3, wherein (a) the heavy chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (b) the heavy chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to CD3 consists of the amino acid sequence of SEQ ID NO: 6; and (c) the light chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to PD-1 and the light chain in the heavy chain and light chain constituting the antigen-binding site that specifically binds to CD3 both consist of the amino acid sequence of SEQ ID NO: 7; (3) the anion exchange carrier is Toyopearl (registered trademark) NH2-750F; (4) the pH of the equilibration buffer is any pH value in the range of about 7.8 to about 8.2, and the electrical conductivity is any electrical conductivity in the range of about 5 to about 7 mS / cm; (5) the linear flow rate of the equilibration buffer is about 100 to about 400 cm / h; and (6) the color density of the fractionated solution containing the protein non-colored body is such that (i) in a visual comparison test with the comparison standard solution BY5 in the color comparison test described in item 2.65 of the general test method in the Seventeenth Revision of the Japanese Pharmacopoeia, it is substantially the same as or lighter than the BY5, or (ii) the Em / UV*100 value of the fractionated solution containing the protein non-colored body is about 6.3 or less.

38. A colorless or slightly colored solution containing a bispecific antibody that specifically binds to PD-1 and CD3, respectively, at a concentration of about 100 to about 300 mg / mL, wherein the bispecific antibody that specifically binds to PD-1 and CD3, respectively, consists of a heavy chain and a light chain that constitute an antigen-binding site specifically binding to PD-1 and a heavy chain and a light chain that constitute an antigen-binding site specifically binding to CD3, and (a) the heavy chain in the heavy chain and light chain that constitute the antigen-binding site specifically binding to PD-1 is composed of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, (b) the heavy chain in the heavy chain and light chain that constitute the antigen-binding site specifically binding to CD3 is composed of the amino acid sequence of SEQ ID NO: 6, and (c) the light chain in the heavy chain and light chain that constitute the antigen-binding site specifically binding to PD-1 and the light chain in the heavy chain and light chain that constitute the antigen-binding site specifically binding to CD3 are both composed of the amino acid sequence of SEQ ID NO:

7.

39. The solution according to claim 38, wherein the color density of the solution is (i) substantially the same as or lighter than BY5 in a visual comparison test with the comparative standard solution BY5 in the color comparison test described in item 2.65 of the general test method in the 17th revised Japanese Pharmacopoeia, or (ii) the Em / UV * 100 value of the solution containing the fractionated protein non-coloring substance is about 6.3 or less.

40. The solution according to claim 38 or 39, excluding a solution that is colorless or slightly colored without undergoing any separation step for reducing or removing coloring.

41. A colorless or slightly colored solution containing the bispecific antibody that specifically binds to PD-1 and CD3, respectively, according to any one of claims 33 to 35 and produced by the method according to any one of claims 1 to 37.

42. A pharmaceutical composition comprising the solution according to any one of claims 38 to 41.

43. Use of the pharmaceutical composition according to claim 42 in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune diseases, graft-versus-host disease, or blood cancers.

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