Method for removing color from drug substance of protein preparation

A method for preparing a solution containing non-colored proteins comprising a process for separating non-colored proteins from a solution containing non-colored proteins and colored proteins,

• (1) wherein the separation process comprises
• (i) a step for loading the solution containing non-colored proteins and colored proteins onto an anion-exchange carrier column, and applying an equilibration buffer to the anion-exchange carrier column at a predetermined initial pH, resulting the non-colored proteins and colored proteins bind to anion-exchange carriers,
• (ii) a step for flowing an elution buffer, forming a pH gradient which descends from the initial pH to a terminal pH, through the anion-exchange carrier column at a predetermined flow rate, under a condition predetermined to separate the non-colored proteins and colored proteins;
• and
• (iii) a step for collecting a predetermined fraction containing the non-colored proteins eluted by the preceding step (ii) to a give collected solution containing the non-colored proteins,
• (2) wherein the non-colored proteins and colored-proteins are a bispecific antibody specifically binding to PD-1 and CD3, respectively, comprising a heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and a heavy chain and light chain constituting an antigen-binding site specifically binding to CD3, and wherein (a) the heavy chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 comprises the amino acid sequence set forth in any one 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 light chain constituting an antigen-binding site specifically binding to CD3 comprises the amino acid sequence set forth in SEQ ID NO:
• 6, and (c) both of the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to CD3 comprise the amino acid sequence set forth in SEQ ID NO:
• 7,
• (3) wherein anion-exchange carrier in the anion-exchange carrier column is TOYOPEARL (registered trademark) NH2-750F,
• (4) wherein the initial pH of the equilibration buffer and elution buffer is an arbitrary pH in the range of from about 10.6 to about 8.4, and the terminal pH of the elution buffer is an arbitrary pH in the range of from about 7.0 to about 4.0 (provided that the initial pH is at least 2.0 higher than the terminal pH),
• (5) wherein the pH gradient is performed (a) by increasing the percentage (%) of the buffer B in the elution buffer step-by-step by an arbitrary percentage in the range of from about 1 to about 2%, at an interval corresponding to an arbitrary column volume in the range of from about 4 to about 6 CV, over at least the range of from about 50 to 100%, or (b) by decreasing the pH of the elution buffer step-by-step by an arbitrary pH in the range of from about 0.6 to about 1.0, within a range corresponding to an arbitrary column volume of from about 30 to about 40 CV,
• (6) wherein the linear flow rate of the elution buffer is from about 100 to about 800 cm/h, and
• (7) wherein (i) a color concentration of the collected solution containing the non-colored proteins is substantially the same as or lighter than that of BY5, according to a visual comparison test with the reference standard solution BY5 of the color comparison test described in item 2.65 of the general tests of the Japanese Pharmacopoeia, 17th Edition or (ii) the Em/UV*100 value of the collected solution containing the non-colored proteins is about 6.3 or less.

A method for preparing a solution containing non-colored proteins comprising a process for separating non-colored proteins from a solution containing non-colored proteins and colored proteins,

• (1) wherein the separation process comprises
• (i) a step for loading the solution containing non-colored proteins and colored proteins onto an anion-exchange carrier column,
• (ii) a step for flowing an equilibration buffer prepared so that the colored proteins in the solution bind to anion-exchange carriers and the non-colored proteins do not, through the anion-exchange carrier column at a predetermined flow rate, and
• (iii) a step for collecting the fraction containing the non-colored proteins flowed through in the preceding step (ii) as it is to give a collected solution containing the non-colored proteins,
• (2) wherein the non-colored proteins and colored-proteins are a bispecific antibody specifically binding to PD-1 and CD3, respectively, comprising a heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and a heavy chain and light chain constituting an antigen-binding site specifically binding to CD3, and wherein (a) the heavy chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 comprises the amino acid sequence set forth in any one 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 light chain constituting an antigen-binding site specifically binding to CD3 comprises the amino acid sequence set forth in SEQ ID NO:
• 6, and (c) both of the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to CD3 comprise the amino acid sequence set forth in SEQ ID NO:
• 7,
• (3) wherein anion-exchange carrier in the anion-exchange carrier column is TOYOPEARL (registered trademark) NH2-750F,
• (4) wherein the pH of the equilibration buffer is an arbitrary pH in the range of from about 7.8 to about 8.2, and the electrical conductivity thereof is an arbitrary electrical conductivity from about 5 to about 7 mS/cm,
• (5) wherein the linear flow rate of the equilibration buffer is from about 100 to about 400 cm/h, and
• (6) wherein a color concentration of the collected solution containing the non-colored proteins is substantially the same as or lighter than that of BY5, according to a visual comparison test with the reference standard solution BY5 of the color comparison test described in item 2.65 of the general tests of the Japanese Pharmacopoeia, 17th Edition, or (ii) the Em/UV*100 value of the collected solution containing the non-colored protein is about 6.3 or less.

A colorless or slightly colored solution comprising a bispecific antibody specifically binding to PD-1 and CD3, respectively, at a concentration of from about 100 to about 300 mg/mL,

• wherein the bispecific antibody specifically binding to PD-1 and CD3, respectively, comprises the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and the heavy chain and light chain constituting an antigen-binding site specifically binding to CD3, and wherein (a) the heavy chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 comprises the amino acid sequence set forth in any one 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 light chain constituting an antigen-binding site specifically binding to CD3 comprises the amino acid sequence set forth in SEQ ID NO:
• 6, and (c) both of the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to PD-1 and the light chain of the heavy chain and light chain constituting an antigen-binding site specifically binding to CD3 comprise the amino acid sequence set forth in SEQ ID NO:
• 7.