Method for producing intermediate of radioactive pharmaceutical composition, and kit for purifying intermediate of radioactive pharmaceutical composition

Abstract

Provided is a method for producing a higher-purity radioactive antibody pharmaceutical intermediate. This production method comprises: an adsorption step in which a composition containing an intermediate is adsorbed to a stationary phase for hydrophobic chromatography; and a purification step for eluting the intermediate from the stationary phase. The intermediate is a complex composed of 3, 6, 9, 15-tetraazabicyclo [9.3. 1] pentadecane-1 (15), 11, 13-triene-3, 6, 9-triacetic acid (PCTA) and an anti-epithelial growth factor receptor antibody (an anti-EGFR antibody), and the preparation method is simple and easy to operate.

Description

Technical Field

[0001] The present invention relates to a method for producing an intermediate of a radiopharmaceutical composition, a method for producing a radiopharmaceutical composition comprising a step of producing an intermediate by the method for producing an intermediate, and a kit for purifying an intermediate of a radiopharmaceutical composition. Background Art

[0002] Research and development of radioactive antibody drugs by labeling with radioactive nuclides using bifunctional chelating agents such as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) is progressing. For example, non-patent documents 1 to 5 describe radioimmunotherapy and diagnostic methods using radioactive antibody drugs.

[0003] (Prior art literature)

[0004] Non-patent literature 1: Yoshii et al., Sci Rep 2020; 10: 4143

[0005] Non-patent literature 2: Matsumoto et al., Pharmaceutics. 2022; 14: 67

[0006] Non-patent literature 3: Matsumoto et al., Pharmaceuticals. 2021; 14: 950

[0007] Non-patent literature 4: Song IH et al., Oncotarget. 2017; 8: 92090-92105

[0008] Non-patent literature 5: Song IH et al., J Nucl Med. 2016; 57: 1105-11 Summary of the invention

[0009] (Problems to be solved by the invention)

[0010] Radioactive antibody drugs can be produced by coordinating a radioactive metal with an antibody-ligand complex formed by binding an antibody and a bifunctional ligand. After production, antibodies and antibody-ligand complexes, which are raw materials for radioactive antibody drugs, undergo a purification process based on ultrafiltration or ion exchange chromatography, and are then provided for the production of radioactive antibody drugs.

[0011] However, these antibodies and antibody-ligand complexes also have the following concerns: similar substances will be formed during storage, resulting in a decrease in the quality of radioactive antibody pharmaceuticals and hindering practical application. As examples of such similar substances, macromolecular similar substances such as aggregated dimers or polymers and small molecule similar substances produced by the rupture of intramolecular bonds of antibodies can be cited. However, in the past, purification processes based on ultrafiltration or ion exchange chromatography were unable to separate and purify the target substance, i.e., antibodies and antibody-ligand complexes, from these similar substances. Therefore, it is desirable to develop a technology for manufacturing higher purity radioactive antibody pharmaceutical raw materials.

[0012] One aspect of the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to realize a technology for producing a radioactive antibody pharmaceutical intermediate with a higher purity.

[0013] (Technical means to solve the problem)

[0014] The present inventors have conducted intensive studies and have found that a high-purity radioactive antibody pharmaceutical intermediate can be obtained stably and at low cost by purifying the radioactive antibody pharmaceutical intermediate by hydrophobic chromatography using a buffer solution containing a stabilizer as a mobile phase, thereby completing the present invention.

[0015] That is, in order to solve the above-mentioned problems, a method for producing an intermediate of a radioactive pharmaceutical composition according to one aspect of the present invention comprises: an adsorption step of adsorbing a composition containing the intermediate onto a stationary phase for hydrophobic chromatography; and a purification step of passing an acetic acid buffer as an eluent through the stationary phase adsorbed with the composition to elute the intermediate from the stationary phase, wherein the acetic acid buffer contains at least one stabilizer selected from neutral amino acids and polysorbates, and the intermediate is a complex composed of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and an anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

[0016] In one aspect of the present invention, a method for producing a radioactive pharmaceutical composition comprises the steps of: producing an intermediate by the method for producing an intermediate; and labeling the intermediate with a radioactive nuclide coordinated to PCTA.

[0017] A kit for purifying an intermediate of a radioactive pharmaceutical composition according to one aspect of the present invention comprises: a stationary phase for hydrophobic chromatography; and an eluent, wherein the eluent comprises at least one stabilizer selected from neutral amino acids and polysorbates, and the intermediate is a complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and an anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

[0018] (Effects of the Invention)

[0019] According to one aspect of the present invention, a radioactive antibody pharmaceutical intermediate with a higher purity can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a result diagram of analyzing PCTA-cetuximab by size exclusion chromatography before separation and purification by hydrophobic chromatography in an embodiment of the present invention.

[0021] Figure 2 This is a diagram showing the results of analyzing the fraction 2 of PCTA-cetuximab obtained by hydrophobic chromatography by size exclusion chromatography in an example of the present invention.

[0022] Figure 3 This is a diagram showing the results of analyzing fraction 9 of PCTA-cetuximab obtained by hydrophobic chromatography by size exclusion chromatography in an example of the present invention.

[0023] Figure 4 This is a diagram showing the results of analyzing the fraction 16 of PCTA-cetuximab obtained by hydrophobic chromatography by size exclusion chromatography in an example of the present invention.

[0024] Figure 5 This is a diagram showing the results of analyzing PCTA-cetuximab separated and purified by hydrophobic chromatography by size exclusion chromatography at the beginning of refrigerated storage in an example of the present invention.

[0025] Figure 6 This is a diagram showing the results of analyzing PCTA-cetuximab separated and purified by hydrophobic chromatography by size exclusion chromatography after being stored in a refrigerator for one month in an example of the present invention.

[0026] Figure 7 This is a diagram showing the results of analyzing PCTA-cetuximab separated and purified by hydrophobic chromatography by size exclusion chromatography after refrigerated storage for 3 months in an example of the present invention.

[0027] Figure 8 This is a diagram showing the results of analyzing PCTA-cetuximab separated and purified by hydrophobic chromatography by size exclusion chromatography after refrigerated storage for 6 months in an example of the present invention. DETAILED DESCRIPTION

[0028] [Method for producing intermediate of radioactive pharmaceutical composition]

[0029] In the method for producing an intermediate of a radioactive pharmaceutical composition according to one aspect of the present invention (hereinafter sometimes referred to as "the method for producing an intermediate according to the present embodiment"), similar substances of the intermediate of the radioactive pharmaceutical composition can be separated and removed by the adsorption step and purification step described later, thereby obtaining a high-purity intermediate.

[0030] (Radiopharmaceutical compositions and intermediates thereof)

[0031] In this specification, a radiopharmaceutical composition refers to a complex consisting of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and an anti-epidermal growth factor receptor antibody (anti-EGFR antibody) after being labeled with a radionuclide. The radiopharmaceutical composition can be used for diagnosis or treatment of a disease. In addition, in this specification, an intermediate of a radiopharmaceutical composition refers to a pharmaceutical composition before being labeled with a radionuclide (i.e., a complex consisting of PCTA and an anti-EGFR antibody that has not been labeled with a radionuclide). The radionuclide will be described later.

[0032] (PCTA)

[0033] PCTA is a bifunctional chelating agent. By introducing an isocyanate group or an N-hydroxysuccinimide group into PCTA and allowing it to act on the side chain amino group or the N-terminal amino group of the antibody's lysine residue, a chelating site is introduced into the antibody. Then, by complexing the radionuclide to the chelating site, an antibody labeled with a radionuclide can be obtained.

[0034] (Anti-EGFR Antibody)

[0035] About the example of anti-EGFR antibody, can enumerate the antibody etc. that specifically binds with cancer. About the specific example of anti-EGFR antibody, can enumerate Cetuximab (Cetuximab), Panitumumab (Panitumumab) and Necitumumab (Necitumumab) etc. Anti-EGFR antibody can be mouse antibody, chimeric antibody, humanized antibody or human antibody. In addition, anti-EGFR antibody can be monoclonal antibody or polyclonal antibody. As anti-EGFR antibody, commercially available anti-EGFR antibody can be used, and anti-EGFR antibody synthesized by known method can also be used.

[0036] As the intermediate, an intermediate synthesized by a known method can be used. For example, as shown in the Examples, an intermediate can be obtained by mixing a solution containing PCTA and a solution containing an anti-EGFR antibody and incubating them at 1 to 60°C for 1 to 24 hours.

[0037] Examples of analogous substances of the intermediate of the radiopharmaceutical composition include macromolecular analogous substances such as dimers and polymers of the intermediate, and small molecular analogous substances produced by cleavage of intramolecular bonds of antibodies in the intermediate.

[0038] (Adsorption process)

[0039] In the adsorption step, the composition containing the intermediate is adsorbed on the stationary phase for hydrophobic chromatography. Examples of the composition containing the intermediate include a composition containing a newly synthesized intermediate, a composition containing an intermediate stored for a certain period of time after synthesis, etc. The composition containing the intermediate may contain, in addition to the intermediate, a similar substance to the intermediate, etc.

[0040] For the stationary phase used for hydrophobic chromatography, for example, a hydrophilic polymer substrate with a cyclic or linear fat-soluble functional group can be used. About the example of a hydrophilic polymer substrate, known hydrophilic polymer substrates (such as synthetic resins such as polysaccharides and vinyl polymers, silicon oxide, etc.) used in hydrophobic chromatography can be cited. The hydrophilic polymer substrate can be porous or non-porous. In addition, the shape of the hydrophilic polymer substrate is not particularly limited, for example, it can be spherical. The fat-soluble functional group can be directly bonded to the hydrophilic polymer or indirectly bonded via a linker.

[0041] The carbon number of the above-mentioned cyclic fat-soluble functional group can be 6 or more, or it can be 20 or less. In addition, the cyclic fat-soluble functional group can be an aryl group. From the viewpoint of obtaining an intermediate with higher purity, an aryl group with a carbon number of 6 or more and 20 or less is preferred. Examples of aryl groups with a carbon number of 6 or more and 20 or less include phenyl and naphthyl. Examples of hydrophilic polymer substrates bonded with an aromatic group with a carbon number of 6 or more and 20 or less include TOYOPEARL (registered trademark) Phenyl, HiPrep (trademark) Phenyl FF, POROS (trademark) Benzyl, POROS (trademark) Benzyl Ultra, etc.

[0042] The carbon number of the above-mentioned straight-chain fat-soluble functional group can be more than 1 or less than 10. In addition, the straight-chain fat-soluble functional group can also be an alkyl group. Examples of straight-chain fat-soluble functional groups with carbon numbers of more than 1 and less than 10 include methyl, ethyl, propyl, isopropyl, butyl, hexyl and octyl groups. Examples of hydrophilic polymer substrates bonded with straight-chain alkyl groups with carbon numbers of more than 1 and less than 10 include TOYOPEARL (registered trademark) Butyl, TOYOPEARL (registered trademark) Hexyl, HiPrep (trademark) Butyl FF, HiPrep (trademark) Octyl FF, etc.

[0043] The adsorption of the composition containing the intermediate onto the stationary phase can be carried out, for example, by passing a solution containing the composition through a column filled with the stationary phase to bring the composition into contact with the stationary phase.

[0044] (Purification process)

[0045] In the purification step, a buffer solution (mobile phase) as an eluent is passed through the stationary phase on which the composition is adsorbed, thereby eluting the intermediate from the stationary phase.

[0046] The buffer used as the eluent in the purification step is an acetate buffer, and the pH value of the acetate buffer is preferably 5 to 8.

[0047] The eluent contains at least one stabilizer selected from the group consisting of neutral amino acids and polysorbate.

[0048] From the viewpoint of being able to have free radical capture activity, the eluent preferably contains a neutral amino acid as a stabilizer. From the viewpoints of increasing the stability of the antibody eluted into the eluent, a neutral amino acid with high solubility is more preferably adopted among the neutral amino acids. About the example of a neutral amino acid with high solubility, asparagine, glutamine, serine, threonine, cysteine, alanine or glycine can be cited. The neutral amino acid contained in the above-mentioned eluent can be 1 kind, or it can be more than 2 kinds.

[0049] From the viewpoint of increasing the stability of the antibody eluted into the eluate, the concentration of the neutral amino acid contained in the eluate is preferably 1 μM or more, more preferably 10 μM or more, and further preferably 50 μM or more. In addition, the concentration of the neutral amino acid contained in the eluate is preferably 260 mM or less, more preferably 200 mM or less, and further preferably 150 mM or less.

[0050] From the viewpoints of being able to capture free radicals and suppressing the aggregation of antibodies (proteins) to stably elute the intermediates into the eluent, the eluent preferably contains polysorbate. Examples of polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80. The polysorbate contained in the eluent may be one or more.

[0051] From the viewpoint of being able to suppress protein aggregation and stably elute the intermediate into the eluent, the concentration of polysorbate contained in the eluent is preferably 1 μM or more, more preferably 10 μM or more, and further preferably 50 μM or more. In addition, the concentration of polysorbate contained in the eluent is preferably 400 mM or less, more preferably 200 mM or less, and further preferably 100 mM or less.

[0052] The eluent more preferably contains at least one neutral amino acid and at least one polysorbate. When the eluent as the acetic acid buffer contains neutral amino acids and polysorbates, the synergistic effect of the acetic acid buffer, neutral amino acids and polysorbates can be exerted. Therefore, the aggregation of proteins is further suppressed, and the intermediate can be stably eluted into the eluent. In addition, the radiopharmaceutical composition prepared by the intermediate eluted into the eluent can be inhibited from decomposing due to radioactivity.

[0053] The method for producing an intermediate of the present embodiment can remove similar substances from the intermediate by a simple isocratic elution method without changing the composition of the eluent.

[0054] It is also possible to make the above-mentioned eluent containing salt flow through the stationary phase adsorbed with the composition after the above-mentioned eluent flows through it. By making the above-mentioned eluent containing salt flow through the stationary phase further, the components adsorbed by the stationary phase (components other than the intermediate) can be recovered. As the above-mentioned salt, for example, sodium chloride can be cited. The eluent containing salt can be flowed through at a certain concentration, or a mixture of the above-mentioned eluent and the above-mentioned eluent containing salt can be flowed through in a manner of changing the mixing ratio.

[0055] (Purity measurement process)

[0056] The method for producing an intermediate of the present embodiment may further include a purity measurement step of measuring the purity of the intermediate after the purification step. The purity measurement method may be a known method, such as a method of measuring by size exclusion chromatography.

[0057] (Save process)

[0058] The method for producing the intermediate of the present embodiment may further include: after the above-mentioned purification step, a storage step of storing the above-mentioned eluent containing the intermediate for more than 24 hours. By using the eluent as a storage liquid, it is possible to suppress the generation of similar substances of the intermediate during the storage process. The storage time of the intermediate can be more than 48 hours, more than 1 week, or more than 1 month. In addition, the storage time of the intermediate can be less than 3 years, less than 2 years, or less than 12 months.

[0059] From the viewpoint of the stability of the intermediate, the storage temperature of the intermediate in the storage step is preferably 1° C. or higher, more preferably 3° C. or higher. The storage temperature of the intermediate is preferably 8° C. or lower, more preferably 5° C. or lower.

[0060] The method for producing an intermediate of the present embodiment may include other steps. Examples of other steps include a step of adjusting the concentration of the intermediate using an ultrafiltration membrane or the like.

[0061] The acetic acid buffer used in the eluent can inhibit protein aggregation, thereby enabling the intermediate to be stored for a desired period of time. In addition, the stabilizer contained in the eluent can improve the stability of the intermediate stored in the liquid. Therefore, the eluent containing the intermediate obtained through the purification process can store the intermediate without solvent replacement.

[0062] In the prior art, as a purification method for intermediates, for example, purification is performed by ultrafiltration or ion exchange chromatography. However, such methods cannot separate the target intermediate from similar substances such as dimers of the intermediate, so there is a problem of high risk of not meeting the quality specifications of radioactive antibody pharmaceuticals in practical application. The method for manufacturing the intermediate of the present embodiment can efficiently remove similar substances such as agglomerates and molecular fragments through one step (purification step). Therefore, by using the method for manufacturing the intermediate of the present embodiment, the risk that the intermediate as the target does not meet the quality specifications can be reduced, and the stored product can be separated, purified and used for a long time. In addition, there is no need for solvent replacement after purification, and stable and low-cost radioactive antibody pharmaceuticals can be manufactured. In addition, the storage method of the intermediate of the present embodiment can also comply with the concept of pharmaceutical GMP (Good Manufacturing Practice).

[0063] [Kit for purification of intermediates of radioactive pharmaceutical compositions]

[0064] A purification kit for purifying the intermediate of this embodiment is also included in one aspect of the present invention. The purification kit comprises: the stationary phase for the hydrophobic chromatography described above; and an eluent comprising at least one stabilizer selected from neutral amino acids and polysorbates.

[0065] In addition to the stationary phase and the eluent, the purification kit may also include other materials such as salts such as sodium chloride and a column for filling the stationary phase. The stationary phase may be pre-filled in the column. In addition, the eluent may be the following two types of eluents: an eluent containing at least one stabilizer selected from neutral amino acids and polysorbates; and an eluent containing salts such as sodium chloride.

[0066] In addition, the purification kit may also include an instruction book recording the steps for purifying the intermediate. It may be written or printed on paper or other media, or attached to a magnetic tape, a computer-readable storage disk, or an electronic medium such as a CD-ROM. An audio file or an audio-visual file explaining the intermediate purification steps may also be included instead of the instruction book. It may be attached to a magnetic tape, a computer-readable storage disk, or an electronic medium such as a CD-ROM, or it may be stored in an accessible cloud server and referenced via the Internet.

[0067] [Method for producing radioactive pharmaceutical composition]

[0068] The method for producing a radiopharmaceutical composition according to one embodiment of the present invention comprises a labeling step of labeling the intermediate produced by the above production method with a radioactive nuclide coordinated to PCTA.

[0069] (Marking process)

[0070] The radionuclide used in the labeling step is a radionuclide coordinated with PCTA. Examples of such radionuclides include 64 Cu and 225 Ac et al.

[0071] The intermediate can be labeled with a radionuclide by a known method. For example, as shown in the Examples, a storage solution containing the intermediate can be mixed with a solution containing the radionuclide and incubated at 1 to 60° C. for 5 minutes to 12 hours to label the intermediate with the radionuclide. The solution containing the radionuclide is preferably an acetic acid buffer, more preferably an acetic acid buffer having the same pH as the storage solution.

[0072] The method for producing a radiopharmaceutical composition of the present embodiment may include other steps in addition to the above-mentioned labeling step. Examples of other steps include a step of measuring the radiochemical purity of the radiopharmaceutical composition.

[0073] 〔Summarize〕

[0074] A method for producing an intermediate of a radioactive pharmaceutical composition according to one embodiment of the present invention comprises: an adsorption step of adsorbing a composition containing the intermediate onto a stationary phase for hydrophobic chromatography; and a purification step of allowing an acetic acid buffer as an eluent to flow through the stationary phase onto which the composition is adsorbed to elute the intermediate from the stationary phase, wherein the acetic acid buffer contains at least one stabilizer selected from neutral amino acids and polysorbates, and the intermediate is a complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and an anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

[0075] In the method for producing an intermediate according to one aspect of the present invention, the stabilizer may be at least one neutral amino acid and at least one polysorbate.

[0076] In the method for producing an intermediate according to one aspect of the present invention, the stationary phase may be a hydrophilic polymer substrate to which a cyclic or linear lipophilic functional group is bonded.

[0077] In the method for producing an intermediate according to one aspect of the present invention, after the eluent has been passed, the eluent containing sodium chloride may be further passed through the stationary phase.

[0078] In the method for producing an intermediate according to one aspect of the present invention, the anti-EGFR antibody may be cetuximab.

[0079] The method for producing an intermediate according to one aspect of the present invention may further include a purity measurement step of measuring the purity of the intermediate by size exclusion chromatography after the purification step.

[0080] The method for producing an intermediate according to one aspect of the present invention may further include a storage step of storing the eluate containing the intermediate for 24 hours or more after the purification step.

[0081] A method for producing a radioactive pharmaceutical composition according to one aspect of the present invention comprises the steps of: producing an intermediate by the method for producing an intermediate; and labeling the intermediate with a radioactive nuclide coordinated to PCTA.

[0082] A kit for purifying an intermediate of a radioactive pharmaceutical composition according to one aspect of the present invention comprises a stationary phase for hydrophobic chromatography and an eluent, wherein the eluent comprises at least one stabilizer selected from neutral amino acids and polysorbates, and the intermediate is a complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and an anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

[0083] The following examples are provided to illustrate the embodiments of the present invention in more detail. Of course, the present invention is not limited to the following examples, and the technical details can be various ways. Furthermore, the present invention is not limited to the above-mentioned embodiments, and various changes can be made within the scope of the technical concept shown in the specification, and the embodiments obtained by appropriately combining the technical means of each disclosure are also included in the technical scope of the present invention. In addition, the documents recorded in this specification are all cited as references.

[0084] [Example]

[0085] In the following examples, % means mass %.

[0086] 〔Materials and methods〕

[0087] [Synthesis of PCTA-Cetuximab]

[0088] Cetuximab, an anti-EGFR antibody, is obtained by a known method. Specifically, a DNA chain encoding cetuximab is obtained using a cetuximab gene sequence, the DNA chain is transfected into Chinese hamster ovary (CHO) cells, and cetuximab biosynthesized by the CHO cells is purified.

[0089] According to Nucl Med Biol 43(11):685-691, the 64 Cu. 3,6,9,15-Tetraazabicyclo[9.3.1]pentadecan-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA, Macrocyclics, Plano, TX, USA) was used for subsequent 64 Cu coordinates and binds to cetuximab. The sterilizing water used is of pharmaceutical grade, and the other chemical substances used are of chemical grade.

[0090] The method described in the following literature was modified to form a complex with cetuximab and PCTA. Literature: Cancer Sci 103(3):600-605; PLoS One 8(4):e61230; PLoS One 10(4):e0123761. Briefly, a borate buffer solution (0.05 M, pH 8.5) (2 mg / mL) of cetuximab was prepared by buffer exchange using a Vivaspin ultrafiltration device (Sartorius, Aubagne, France). The borate buffer solution of cetuximab was mixed with a dimethyl sulfoxide solution of p-SCN-Bn-PCTA (0.42 mg / mL) at a ratio of 10:1 (cetuximab: p-SCN-Bn-PCTA; v / v). Then, the mixture was reacted at 37°C for 24 hours to prepare PCTA-cetuximab. The prepared PCTA-cetuximab was subjected to solution replacement with 0.1 M acetate buffer (pH 6.0) containing 100 mM glycine and 76.3 μM polysorbate 80 using an ultrafiltration centrifugal concentrator to obtain a 2 mg / mL solution.

[0091] [Analysis method for chemical purity of PCTA-cetuximab]

[0092] By size exclusion chromatography (SEC-HPLC), the chemical purity of the PCTA-cetuximab prepared above was analyzed. As a column, TSKgel G3000 SWXL (300 × 7.8mm, 5μm, Tosoh Corporation) was used, and as a guard column, Security Guard Cartridges GFC-3000 (Phenomex Corporation) was used. As a mobile phase, a potassium phosphate buffer containing 250mM KCl was used. Analyzed with a mobile phase flow rate of 0.5mL / min, wherein the detection wavelength was set to 280nm.

[0093] Representative results are shown in Figure 1 For the prepared PCTA-cetuximab, the target substance (PCTA-cetuximab) was detected at a retention time of 15.871 minutes. On the other hand, molecular analogs larger than the target substance were detected at 11.018 minutes and 13.566 minutes, and molecular analogs smaller than the target substance were detected at 16.864 minutes and 19.474 minutes.

[0094] (Example 1) Method for removing analogs from PCTA-cetuximab by hydrophobic chromatography

[0095] The prepared PCTA-cetuximab was separated and purified by hydrophobic chromatography. As a column, TOYOPEARL (registered trademark) Phenyl FT-750F (1.0cm×6.0cm, tube bed 4.7mL, manufactured by Tosoh Corporation) was used. As mobile phase A, 0.1M acetate buffer (pH6.0) containing 100mM glycine and 76.3μM polysorbate 80 as a stabilizer was used. As mobile phase B, a mobile phase formed by adding 1M sodium chloride to mobile phase A was used. 5mL of PCTA-cetuximab solution (2mg / mL) was injected into the column. Then, mobile phase A was shunted by 10 column volumes, and then 8 column volumes were spent to increase the proportion of mobile phase B to 100%. The flow rate was set to 2.5mL / min (linear flow rate 191cm / h), the detection wavelength was set to 280nm, and the fraction division amount was set to 5mL.

[0096] The chemical purity of PCTA-cetuximab contained in each fraction was analyzed by size exclusion chromatography. The analysis conditions were the same as those described in the above section "[Analysis method for chemical purity of PCTA-cetuximab]".

[0097] Representative results are shown in Figures 2 to 4 . Figure 2 This is a diagram showing the results of size exclusion chromatography analysis of fraction 2 obtained by hydrophobic chromatography of the produced PCTA-cetuximab. Figure 3 This is a diagram showing the results of size exclusion chromatography analysis of fraction 9 obtained by hydrophobic chromatography of the produced PCTA-cetuximab. Figure 4 This is a result diagram of analyzing the fraction 16 obtained by hydrophobic chromatography of the prepared PCTA-cetuximab by size exclusion chromatography. For fractions 1 to 3, the target substance and molecular analogs smaller than the target substance were detected. For fractions 4 to 9, only the target substance containing almost no analogs was detected. For each fraction after fraction 10, the target substance and molecular analogs larger than the target substance were detected. Therefore, it is judged that by collecting fractions 4 to 9, PCTA-cetuximab containing almost no analogs can be obtained by separation and purification.

[0098] (Comparative Examples 1 to 4)

[0099] The prepared PCTA-cetuximab was separated and purified by hydrophobic chromatography. In each comparative example, TOYOPEARL (registered trademark) Phenyl FT-750F (1.0 cm × 6.0 cm, tube bed 4.7 mL, manufactured by Tosoh Corporation) was used as a column, and the four combinations shown in Table 1 were used as mobile phases. In each comparative example, mobile phase B and mobile phase A were injected into the column in the order of mobile phase B and mobile phase A, and the sample was introduced into a resin equilibrated in a high salt concentration, so that the target was recovered to the circulation tube. A setting (circulation mode) was adopted to elute the aggregate by reducing the salt concentration. As a result, in Comparative Examples 1 to 4, the target and the aggregate were eluted into the circulation tube in an unseparated manner. According to the comparison of the results between the embodiment and Comparative Example 3, it can be considered that the presence or absence of polysorbate is related to the retention of the aggregate in the column for a certain period of time.

[0100] [Table 1]

[0101] Table 1: Separation and purification conditions of PCTA-cetuximab by chromatography (flow mode, comparative examples 1 to 4)

[0102]

[0103] (Comparative Examples 5 to 7)

[0104] PCTA-cetuximab was separated and purified by anion exchange chromatography. HiTrap QFF (1.6×2.5cm, tube bed 5mL, manufactured by Cytiva) was used as a column, and the two combinations shown in Table 2 were used as mobile phases. In each comparative example, mobile phase A and mobile phase B were injected into the column in the order of mobile phase A and mobile phase B, and the sample was introduced into a resin equilibrated at a low salt concentration, so that the target substance was recovered into the flow tube. A setting (desorption mode) was adopted to elute the aggregates by increasing the salt concentration. As a result, in comparative examples 5 and 6, the target substance and the aggregates were eluted into the flow tube and could not be separated. In comparative example 7, under the gradient division of salt concentration, the target substance and the aggregates were eluted in an unseparated manner.

[0105] [Table 2]

[0106] Table 2: Separation and purification conditions of PCTA-cetuximab based on chromatography (desorption mode, comparative examples 5 to 7)

[0107]

[0108] 〔Stability of purified PCTA-cetuximab〕

[0109] The stability of PCTA-cetuximab separated and purified by the method described in Example 1 was confirmed. The chemical purity at the beginning of storage, the chemical purity after 1 month of storage, the chemical purity after 3 months of storage, and the chemical purity after 6 months of storage were analyzed by size exclusion chromatography to evaluate the stability. The analysis conditions were the same as those described in the aforementioned "[Analysis Method for Chemical Purity of PCTA-cetuximab]" column.

[0110] The results are shown in Figures 5 to 8 . It was found that similar substances with larger molecules than the corresponding peak of PCTA-cetuximab tended to increase slightly over time, but it was also found that even after 6 months of storage, the chemical purity of PCTA-cetuximab was maintained at more than 96%. Therefore, the PCTA-cetuximab separated and purified by the hydrophobic chromatography method described in Example 1 was stable during the 6-month refrigerated storage. Therefore, it was concluded that the separation and purification method is practically useful.

[0111] [Industrial Applicability]

[0112] The present invention can be used for the stable and low-cost production of radiopharmaceutical composition intermediates.

Claims

1. A method for producing an intermediate of a radioactive pharmaceutical composition, characterized in that: include: an adsorption step of adsorbing the composition containing the intermediate onto a stationary phase for hydrophobic chromatography; as well as a purification step of allowing an acetic acid buffer as an eluent to flow through a stationary phase adsorbed with the composition to elute the intermediate from the stationary phase, wherein the acetic acid buffer contains at least one stabilizer selected from neutral amino acids and polysorbates, The intermediate is a complex composed of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

2. The method for producing an intermediate according to claim 1, wherein: The stabilizer is at least one neutral amino acid and at least one polysorbate.

3. The method for producing an intermediate according to claim 1, wherein: The stationary phase is a hydrophilic polymer substrate to which cyclic or linear lipid-soluble functional groups are bonded.

4. The method for producing an intermediate according to claim 3, wherein: The cyclic fat-soluble functional group is a functional group having 6 to 20 carbon atoms.

5. The method for producing an intermediate according to claim 3, wherein: The linear fat-soluble functional group is a functional group having 1 to 10 carbon atoms.

6. The method for producing an intermediate according to claim 1, wherein: After the eluent has passed through, the eluent containing sodium chloride is further passed through the stationary phase.

7. The method for producing an intermediate according to claim 1, wherein: The anti-EGFR antibody is cetuximab.

8. The method for producing an intermediate according to claim 1, further comprising: A purity determination step is performed after the purification step to determine the purity of the intermediate by size exclusion chromatography.

9. The method for producing an intermediate according to claim 2, further comprising: A storage step of storing the eluate containing the intermediate for 24 hours or more after the purification step.

10. A method for producing a radioactive pharmaceutical composition, characterized in that: Including the following processes: An intermediate is produced by the method for producing an intermediate according to any one of claims 1 to 9; and The intermediate is labeled with a radionuclide coordinated to PCTA.

11. A kit for purifying an intermediate of a radioactive pharmaceutical composition, characterized in that: Include: Stationary phases for hydrophobic chromatography; and An eluent, wherein the eluent comprises at least one stabilizer selected from neutral amino acids and polysorbates, The intermediate is a complex composed of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and anti-epidermal growth factor receptor antibody (anti-EGFR antibody).

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