Method for separating modified proteins

Abstract

To provide methods for separating modified proteins from a liquid containing modified proteins and unmodified proteins.SOLUTION: Disclosed is a method for separating a modified protein from a liquid containing the modified protein, which is resultant of a protein modified by modification compound, and an unmodified protein using cation exchange chromatography, the method comprising the steps of: (1) feeding a liquid containing the modified protein and the unmodified protein to a column filled with a cation exchanger; and (2) collecting a first fraction containing the modified protein eluted from the column.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for separating modified proteins.

Background Art

[0002] Since some proteins, such as antibodies, have specific binding properties to target antigens, the idea of labeling such proteins with functional substances (anticancer agents, radionuclides, etc.) for use as a drug delivery system (DDS) has been actively studied (see, for example, Patent Document 1 and Non-Patent Document 1).

[0003] As a standard protein modification method, there is a method of introducing a functional substance by reacting an amine-reactive reagent with a lysine in a protein. After the protein modification reaction, the protein is desalted by a method such as gel filtration chromatography or ultrafiltration to remove unreacted drug, and then used in animal experiments and clinical studies (see Patent Documents 2 and 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of Invention

Problems to be Solved by the Invention

[0006] Conventional separation methods, such as gel filtration or ultrafiltration, rely on the fact that the molecular weight of antibodies is extremely large compared to other coexisting substances, and can remove low-molecular-weight compounds (salts and unreacted functional drugs). However, because there is no significant difference in molecular weight between modified and unmodified antibodies, these methods cannot separate modified and unmodified antibodies. Since unmodified antibodies are not labeled with functional substances and therefore cannot be expected to provide efficacy based on those substances, it is desirable that the dosage administered to patients be strictly controlled. However, conventional technology cannot remove unmodified antibodies, so unmodified antibodies remain in the final product solution, and there is a problem that if a predetermined amount of functional substance antibody is administered to a patient, the unmodified antibodies are also administered to the patient at the same time. Furthermore, similar challenges exist not only for antibodies but also for the modification of proteins in general for medical purposes.

[0007] The present invention aims to provide a method for separating modified proteins from a liquid containing modified and unmodified proteins. [Means for solving the problem]

[0008] The present invention, which solves the above problems, is as follows. A method for separating modified proteins, comprising separating the modified proteins (including antibodies as one of the proteins) and unmodified proteins from a liquid containing the modified proteins by cation exchange chromatography, characterized in that it includes the following steps 1 and 2. Step 1: A step of supplying a liquid containing modified protein and unmodified protein to a column packed with cation exchangers. Step 2: A step of recovering the first fraction containing the modified protein that flows out of the column. [Effects of the Invention]

[0009] According to the method of the present invention, modified proteins can be separated from a liquid containing modified and unmodified proteins. In the examples of this specification, antibodies were used as the proteins. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic diagram showing the reaction equation between a modified antibody and a modified compound. [Figure 2] Figure 2 illustrates the mechanism of ion exchange chromatography for separating modified and unmodified antibodies. [Figure 3] Figure 3 illustrates the steps of the method for separating modified antibodies according to the present invention. [Figure 4] Figure 4 shows an ion exchange chromatogram obtained in an embodiment of the present invention. [Figure 5] Figure 5 shows the relationship between the quantitative ratio of the modified compound to the antibody and the separation rate of the modified antibody in the reaction between the modified compound and the antibody. [Figure 6] Figure 6 shows the process of separating modified antibodies using conventional gel filtration. [Modes for carrying out the invention]

[0011] In this invention, modified antibodies and unmodified antibodies are separated by passing a liquid containing modified and unmodified antibodies through a cation exchange column. To compare the present invention with the prior art, the principle of the prior art gel filtration chromatography will first be explained with reference to Figure 6.

[0012] When a mixture contains molecules a, b, and c of different sizes (molecular size: a > b > c), the following method is used to separate each molecule in the mixture by gel filtration chromatography. As shown in Figure 6A, the liquid containing each molecule is passed through a gel filtration chromatography column. When the mixed solution passes through the mobile phase packed in the column, molecules are discharged at different times according to their molecular weights. Therefore, as shown in B of FIG. 6, first, molecule a passes through the column and is eluted. Next, as shown in C of FIG. 6, molecule b is eluted, and finally, as shown in D of FIG. 6, molecule c is eluted. In this way, separation is performed by collecting each fraction at the timing when each molecule is eluted.

[0013] However, since the modified antibody and the unmodified antibody have almost the same molecular weight and no significant difference in molecular size, it is difficult to separate the two. The present invention is characterized in that the modified antibody and the unmodified antibody are separated by ion exchange chromatography by utilizing the difference in ionic valence between the modified antibody and the unmodified antibody.

[0014] FIG. 1 schematically shows the reaction formula of the reaction when trastuzumab is used as the antibody and this antibody is modified with deferoxamine (DFO) as the modifying compound. Deferoxamine (hereinafter referred to as DFO) is a chelate-forming compound capable of coordinating a radioisotope. A labeled antibody can be obtained by modifying an antibody with DFO and then coordinating a radioisotope to DFO. A radioactive element (for example, radioactive zirconium) can be labeled to an antibody and used as a tracer for imaging by positron emission tomography (PET).

[0015] When trastuzumab is reacted with DFO, as shown in FIG. 1, the reaction solution contains modified antibody, unmodified antibody, and unreacted DFO. Positively charged basic amino acids (such as lysine) are present in proteins. Lysine, which is one of the basic amino acids, has an amino group (-NH3 in its side chain +It has an amino group. The amino group is highly reactive and can be used to introduce functional substances. Known amino group reactive substances include isothiocyanates and N-hydroxysuccinimide, and reagents with these amino group reactive substances introduced at the end are commercially available for many functional substances. When isothiocyanates and N-hydroxysuccinimide react with an amino group, they become thiourea and amide, respectively, and have chemical structures that do not have an electric charge. In other words, lysine, which had a positive charge before the introduction of the functional substance, loses its charge when the functional substance is introduced. In the case of DFO, p-SCN-Bn-Deferoxamine ( C33 H 52 N8O8S2 is commercially available, and when it reacts with an antibody, the reacted lysine loses its positive charge. Therefore, modified antibodies exhibit weaker electrostatic force than unmodified antibodies.

[0016] The mechanism of separation due to electrostatic attraction will be explained based on Figure 2. Modified antibodies have DFO bound to lysine, and because the charge of lysine is lost, their adsorption capacity to the ion exchange column decreases. When a liquid containing modified and unmodified antibodies is passed through a cation exchange column, the basic amino acids (e.g., lysine) in the protein attract the cation exchanger through electrostatic attraction. As a result, unmodified antibodies (proteins rich in basic amino acids) readily adsorb to the column, while modified antibodies (proteins poor in basic amino acids) do not readily adsorb to the column.

[0017] The method for separating modified antibodies according to the present invention will be explained with reference to Figure 3. The method for separating modified antibodies according to the present invention is carried out by the following steps 1 and 2. Step 3 is also performed as needed. Step 1: As shown in Figure 3A, a mixture of modified and unmodified antibodies is passed through a column packed with cation exchange resin. As shown in Figure 3B, modified antibody a and unmodified antibody b are separated by ion exchange chromatography. Step 2: As shown in Figure 3C, once the elution of antibody from the column is detected using an absorbance detector, the eluate is collected and designated as the first fraction. This first fraction contains modified antibodies. Step 3: After collecting the first fraction, an absorbance detector is used to detect the elution of antibodies from the column. The eluate is then collected and designated as the second fraction. The second fraction contains unmodified antibodies.

[0018] The following describes an embodiment of the present invention applied to modified antibodies used when labeling antibodies with radioactive elements. As the antibody used, we employed trastuzumab (an anti-HER2 humanized monoclonal antibody), which is used as an anticancer drug. As the antibody-modifying compound, we used DFO (deferoxamine), a chelate-forming compound that can coordinate radioisotopes.

[0019] First, using a microcentrifuge tube, trastuzumab (1 mg / mL concentration) was reacted with p-SCN-Bn-Deferoxamine in a solvent-substituted 0.1 mol / L sodium bicarbonate buffer (pH 9.0) at a ratio of 15 molecules of deferoxamine to 1 molecule of trastuzumab. The reaction was carried out at 37°C for 1 hour. After the reaction, unreacted p-SCN-Bn-Deferoxamine was removed using a desalting column (Cytiva, PD-10).

[0020] The reaction solution containing the modified and unmodified antibodies obtained above was passed through a column packed with an ion exchanger as shown in Figure 3. As the ion exchange material, we used YMC's "BioPro IEX SF" HiTrap(registered trademark) SP HP, an ion exchange column packed with cation exchange resin. The elution conditions were as follows: Solid phase:BioPro IEX SF Mobile phase A: 10mM MES (pH5.7) Mobile phase B: 10mM MES (pH5.7) + 1M NaCl Fluid flow rate: constant at 0.6 mL / min Gradient: Concentration of mobile phase B 0%(0min)→10%(1min)→12%(30min)

[0021] The ion exchange chromatogram obtained by ion exchange treatment under the above conditions is shown in Figure 4. As shown in Figure 4, the modified antibody DFO-trastuzumab elutes in 15 to 19 minutes, so this fraction is collected.

[0022] The separation rate (%) of modified antibodies was determined by varying the ratio of DFO to antibody. The results are shown in Figure 5. Furthermore, increasing the column length improves the separation rate. In Figure 5, the horizontal axis represents DFO / antibody (how many times the amount of DFO was added relative to the antibody), and the vertical axis represents the separation rate. In this invention, the separation rate (%) is defined as follows: The amount of DFO-trastuzumab injected into the ion exchanger is defined as 100%, and the ratio of DFO-trastuzumab recovered in the fractions from 15 to 19 minutes is defined as the separation rate.

[0023] Examples of the present invention are as follows: (1) A method for separating modified proteins, comprising separating the modified proteins, which are proteins modified by a modifying compound, and unmodified proteins from a liquid by cation exchange chromatography, characterized in that it comprises the following steps 1 and 2. Step 1: A step of supplying a liquid containing modified protein and unmodified protein to a column packed with cation exchangers. Step 2: A step of recovering the first fraction containing the modified protein that flows out of the column. (2) A method for separating the modified protein described in (1) above, further comprising step 3 below. Step 3: A step of recovering the second fraction containing the unmodified protein that flows out of the column after the first fraction. (3) The method for separating a modified protein according to (2) above, further comprising using the second fraction recovered in step 3 as a raw material in a method for producing a modified protein. (4) The method for separating modified proteins according to any one of (1) to (3) above, wherein the cation exchanger is a cation exchange resin. (5) A method for separating a modified protein according to any one of (1) to (4) above, wherein the protein is an antibody and the modifying compound is a chelate-forming compound. (6) The method for separating a modified protein according to (5) above, wherein the antibody is trastuzumab and the chelate-forming compound is p-SCN-Bn-Deferoxamine.

Claims

1. A method for separating modified proteins, comprising separating a modified protein, which is a protein modified by a modifying compound, and an unmodified protein from a liquid, by cation exchange chromatography using an elution method that increases the salt concentration of the mobile phase during separation, wherein the modified protein is separated from the liquid containing the modified protein and an unmodified protein, The modified compound reacts with the amino group of a positively charged basic amino acid in the protein, causing it to lose its positive charge, and the adsorption capacity of the modified protein to the cation exchanger is lower than that of the unmodified protein to the cation exchanger. A method for separating modified proteins, characterized by comprising the following steps 1 to 3. Step 1: A step of supplying a liquid containing modified protein and unmodified protein to a column packed with cation exchangers. Step 2: A step of recovering the first fraction containing the modified protein that flows out of the column. Step 3: A step of recovering the second fraction containing the unmodified protein that flows out of the column after the first fraction.

2. The method for separating a modified protein according to claim 1, wherein the modified compound is a compound in which an isothiocyanate or N-hydroxysuccinimide is introduced at the terminal end.

3. Furthermore, the method for separating a modified protein according to claim 1, wherein the second fraction recovered in step 3 is used as a raw material in a method for producing a modified protein.

4. The method for separating modified proteins according to claim 1, wherein the cation exchanger is a cation exchange resin.

5. The method for separating a modified protein according to claim 1, wherein the protein is an antibody, and the modifying compound is a chelate-forming compound having an isothiocyanate group and capable of coordinating a radioisotope.

6. The method for separating a modified protein according to claim 5, wherein the antibody is trastuzumab and the chelate-forming compound is p-SCN-Bn-Deferoxamine.

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