CN115724903A - Protein purification method - Google Patents

Abstract

The invention discloses a protein purification method, which comprises the following steps: adjusting the pH of a protein sample to be purified to a first pH; loading the protein sample onto a cationic chromatography column; and, performing a linear gradient elution with an eluent comprising a first buffer and a second buffer. The method can effectively reduce the alkaline peak and simultaneously improve the ratio of the main peak, the whole separation and purification process is simple, the buffer solution has simple components and low price, and the method can well meet the requirements in industrial application.

Description

Protein purification method

Technical Field

The present disclosure belongs to the technical field of protein separation and purification. In particular, the present disclosure relates to a method of purifying a protein.

Background

Antibodies expressed by recombinant techniques are complex multimeric glycoproteins, including, for example, dimers, tetramers, and the like. The expressed antibodies often exhibit heterogeneity of properties, including charge, hydrophobicity, morphology, and the like. The heterogeneity of charges due to the difference in charge of antibody molecules is called charge isomers, and can be generally classified into acidic isomers and basic isomers. In general, acidic isomers have a lower isoelectric point relative to the target protein, while basic isomers have a higher isoelectric point relative to the target protein.

The main causes of charge isomers include C-terminal lysine truncation of proteins, N-terminal glutamine/glutamate cyclization, N-glycosylation, oxidation, heterogeneity due to cysteine residues, molecular fragments and multimerization, and the like. The charge isomer of the antibody may affect the structural stability, biological activity, immunogenicity, pharmacokinetics, etc. of the antibody drug, thereby having a great influence on the effectiveness, safety, shelf life, etc. of the antibody drug in clinical use.

The methods for removing the charge isomers commonly used at present generally comprise an anion exchange chromatography technology and a cation exchange chromatography technology. These techniques usually require complicated buffers or require additional pre-washing steps for the purpose of removing the charge isomers.

Thus, there is a need for a simple purification process that can separate charge isomers in protein products, particularly expressed antibody products.

Disclosure of Invention

In order to solve one of the above technical problems in the prior art, the present disclosure provides a method for purifying a protein, and particularly provides a method for removing basic isomers from a protein.

According to one aspect of the present disclosure, there is provided a protein purification method, characterized in that the method comprises: adjusting the pH of a protein sample to be purified to a first pH; loading the protein sample onto a cationic chromatography column; and performing linear gradient elution by using an eluent comprising a first buffer solution and a second buffer solution, wherein the pH value of the first buffer solution is the first pH value. According to some embodiments of the disclosure, the protein has an isoelectric point = first pH + n, n being 1 to 2.

According to some embodiments of the disclosure, the first pH is 5.0 to 6.8. According to some embodiments of the disclosure, the first pH is 5.5 to 6.5. According to some embodiments of the disclosure, the first pH is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

According to some embodiments of the disclosure, the first buffer is selected from 10 to 100mM disodium phosphate-citric acid buffer, 10 to 100mM sodium dihydrogen phosphate-disodium phosphate buffer, or 10 to 100mM citric acid-phosphoric acid-Tris buffer.

According to some embodiments of the disclosure, the pH of the second buffer is greater than the isoelectric point of the protein. According to some embodiments of the present disclosure, the second buffer is selected from 10 to 100mM disodium hydrogen phosphate-citric acid buffer, 10 to 100mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, or 10 to 100mM citric acid-phosphoric acid-Tris buffer.

According to some embodiments of the disclosure, the protein has an isoelectric point between 6.5 and 8.0. According to some embodiments of the disclosure, the protein has an isoelectric point between 6.5 and 7.6, for example, may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

According to some embodiments of the disclosure, the protein is a fusion protein or a monoclonal antibody. According to some embodiments of the disclosure, the protein is a monoclonal antibody, which may include a murine antibody, a rabbit antibody, a human antibody, or a fully human antibody. According to some embodiments of the disclosure, the protein is a monospecific or bispecific antibody, for example, infliximab (Infliximab), panitumumab (Panitumumab), pembrolizumab (Pembrolizumab), rituximab (resizumab), cetuximab (catuzumab).

According to some embodiments of the present disclosure, the packing of the cationic chromatography column comprises sulfonic acid groups (SO) ₃ ^2- ) Strong cation exchange of functional groups such as Sulfopropyl (SP) and phosphate (P)And (4) changing the reagent. According to some embodiments of the present disclosure, the packing of the cationic chromatography column comprises a mixture comprising Carboxymethyl (CM), carboxyl (COO) ^- ) Weak cation exchangers of plasma exchange groups. According to some embodiments of the present disclosure, the packing of the cationic chromatography column is a strong cation exchanger containing sulfopropyl groups. According to some embodiments of the present disclosure, the packing of the cationic chromatography column is High SP High Performance.

According to some embodiments of the present disclosure, the method further comprises performing affinity chromatography on the protein sample to be purified prior to adjusting the pH of the protein sample to be purified to a first pH value. According to some embodiments of the disclosure, the affinity medium of the affinity chromatography is protein a.

According to some embodiments of the present disclosure, the method further comprises subjecting the protein sample to be purified to anion chromatography prior to adjusting the pH of the protein sample to be purified to a first pH value. According to some embodiments of the disclosure, the packing material for anion chromatography comprises Q-sepharose, a resin containing quaternary aminoethyl or quaternary amine functional groups. According to some embodiments of the present disclosure, the packing material for anion chromatography may be Capto Q.

According to some embodiments of the disclosure, the protein is expressed by a prokaryotic cell or a eukaryotic cell. According to some embodiments of the disclosure, the protein is expressed by a eukaryotic cell. According to some embodiments of the disclosure, the protein is expressed by a mammalian cell. For example, the protein is expressed by Chinese Hamster Ovary (CHO) cells.

According to some embodiments of the disclosure, the expressed protein sample is subjected to affinity chromatography and the elution peak at UV 280nm is collected.

According to some embodiments of the disclosure, the eluted protein sample is virus inactivated. According to some embodiments of the disclosure, the protein sample is virus inactivated by adjusting the pH to an acidic pH. For example, the protein sample may be virus inactivated by adjusting the pH to about 3 to about 5, for example, to about 3.5 to 3.7. According to some embodiments of the disclosure, the pH of the protein sample is adjusted using citric acid.

According to some embodiments of the present disclosure, the method further comprises, prior to loading the protein sample onto the cationic chromatography column, equilibrating the cationic chromatography column with the first buffer.

According to some embodiments of the present disclosure, prior to performing the linear gradient elution, the method further comprises re-equilibrating the cationic chromatography column with the first buffer.

According to another aspect of the present disclosure, there is provided a purified protein obtained by the method of the present disclosure. The purified proteins obtained by the methods of the present disclosure contain lower basic isomers.

The "linear gradient elution" used in the present invention refers to an elution mode in which the pH value is linearly increased from the start to the end of chromatography by gradually changing the pH value of the mobile phase or the elution buffer during the chromatography by changing the volume ratio of the first buffer to the second buffer at the same rate. Therefore, in a sample with complex components, the components with larger property difference can achieve good separation according to the respectively proper pH or isoelectric point.

The detection of charge heterogeneity is based on the differential charge of the antibodies. Common detection methods include electro-focusing gel electrophoresis (IEF), cation exchange Chromatography (CEX), anion exchange chromatography, capillary isoelectric focusing (CIEF), imaging capillary isoelectric focusing (iCIEF), and the like. In the expressed antibody sample, the charge isomers generally correspond to an acidic peak and a basic peak, as compared to the main peak corresponding to the protein of interest.

Proteins are net charged due to the presence of their surface ionized side chains. Since these side chains are titratable, there is a pH for each protein at which the net surface charge of the protein is zero, i.e. the Isoelectric point (pI). The protein has amphoteric ionization phenomenon in solution, when pI = pH, the number of positive and negative ions dissociated from the polar group of the protein is equal, the net charge is 0, and the pH value of the solution is the pI value of the protein. The pI size of a protein is specific, and is related to the structure of the protein, but not to the pH of the environment.

As used herein, "isoelectric focusing" is the process of placing a carrier ampholyte in an electrophoresis chamber, wherein the ampholyte forms a pH gradient that increases from anode to cathode when a direct current is applied. When proteins are placed in this system, positively charged proteins migrate to the negative electrode and negatively charged proteins migrate to the positive electrode. When they migrate to a pH position corresponding to their isoelectric point, the electrostatic charge of the protein is zero and no longer migrates, a process known as isoelectric focusing.

As used herein, "capillary isoelectric focusing (CIEF)" refers to isoelectric focusing performed within a capillary.

The Infliximab (Infliximab) used in the invention is a purified chimeric human-mouse IgG monoclonal antibody, can resist tumor necrosis factor alpha (TNF-alpha), and can be used for treating or researching autoimmune diseases.

The Panitumumab used in the present invention is a fully humanized monoclonal antibody that targets Epidermal Growth Factor Receptor (EGFR) and can be used for the treatment of metastatic colorectal cancer after chemotherapy failure.

"Pembrolizumab" as used herein is a monoclonal antibody that binds to and blocks programmed cell death protein 1 (PD-1) located on lymphocytes.

"Ritizumab (Reslizumab)" used in the present invention is a monoclonal antibody targeting interleukin 5 (IL-5), and is a humanized rat IgG 4. Kappa. Antibody.

The 'Katumaxomab' used in the invention is a bispecific antibody drug targeting CD3 and EpCAM and is used for targeted combination immunotherapy of cancerous ascites.

The term "about" as used herein means within ± 20% of the value following. In some embodiments, the term "about" means a range of ± 10% of the value following. In some embodiments, the term "about" means a range of ± 5% of the value following.

The purification method can effectively reduce the alkaline peak, simultaneously improve the ratio of the main peak, has simple whole separation and purification process and simple buffer solution component and low price, and can well meet the requirements in industrial application.

Drawings

In order that the disclosure may be more readily and clearly understood, the following detailed description of the disclosure is provided in connection with specific embodiments thereof and with the accompanying drawings in which:

figure 1 shows a chromatogram of a purified protein under pH linear elution conditions at pH 5.5 to 8.0.

Figure 2 shows a chromatogram of purified protein under linear elution conditions from pH 6.5 to pH 8.0.

FIG. 3 shows a chromatogram of a protein purified under pH 5.5 loading and linear elution conditions using 500mM sodium chloride.

FIG. 4 shows a chromatogram of a purified protein at pH 6.3 loading and using a 500mM sodium chloride linear elution.

FIG. 5 shows a chromatogram of a protein purified under pH 6.8 loading and linear elution conditions using 300mM sodium chloride.

FIG. 6 shows a chromatogram of a protein purified under pH 7.0 loading and linear elution conditions using 300mM sodium chloride.

Detailed Description

In order to make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure is further described in detail below with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the disclosure and do not constitute any limitation on the disclosure. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure.

All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. It is noted that the terms used herein should be interpreted as having a meaning that is consistent with the context of this specification and should not be interpreted in an idealized or overly formal sense.

The raw materials and consumables used in the examples were commercially available or prepared by a known method.

Example 1 expression and purification of cetuximab (Catumaxomab).

This example was performed using cetuximab as the target protein on an AKTA Avant 25 protein purification instrument. The used cetuximab was prepared using the following procedure.

And (3) carrying out cytosol separation on the supernatant expressed by the CHO cells in a deep filtration mode, and collecting the filtered supernatant.

Affinity chromatography: carrying out protein A chromatography on the collected supernatant, and collecting an elution peak at A280;

virus inactivation: adjusting the pH of the elution peak collected by A280 to 3.5-3.7 by using citric acid for virus inactivation;

anion chromatography: adjusting the pH value of the sample solution after virus inactivation to 5.3-5.7, conducting for 5-6 ms/cm, and carrying out anion chromatography (the filler of the anion chromatography is Capto Q). When A280 reached 50mAU, the transudate was collected. This is the sample of cetuximab used in the subsequent studies.

The peaks of the acid and base of the sample of cetuximab are shown in table 1

Table 1.

Example 2: antibody samples were purified under pH linear elution conditions at pH 5.5 to 8.0.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:50mM disodium phosphate-citric acid buffer, pH 5.5;

and (3) buffer solution B:50mM disodium phosphate-citric acid buffer, pH 8.0.

The sample of cetuximab obtained in example 1 was adjusted to a conductance of < 6ms/cm with purified water while controlling the pH at 5.5, giving sample 1.

A chromatographic column HiTrap SP HP (Cytiva, 17108703) CV 4.2ml, 10cm column height was used. The column was equilibrated with buffer A, 5CV. Sample 1 was loaded onto the column with a contact time of 6min and a Dynamic Binding Capacity (DBC) < 7mg/ml. The column was re-equilibrated with buffer A, 3CV. Then, a mixed solution of buffer A and buffer B was used at 10CV to elute. During elution, the volume of buffer B in total volume increased linearly from 0 to 100%. The main peak at UV 280nm was collected. Wherein the collection parameters of the main peak are as follows: 50mAU was collected initially and 50mAU was terminated. Peaks 1 to 3 were collected in order. The chromatogram is shown in FIG. 1.

The used column was washed with 0.5M sodium hydroxide at 40cm/h for 3CV. The pH was equilibrated with buffer A until the pH was stabilized, and the column was then stored with 20% ethanol.

The collected fractions from peak 1 to peak 3 were subjected to acid-base peak detection using icIEF, and the results are shown in table 2.

TABLE 2

Example 3: antibody samples were purified under linear elution conditions from pH 6.5 to pH 8.0.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:50mM phosphate-citrate buffer, pH 6.5;

and (3) buffer solution B:50mM phosphate-citrate buffer, pH 8.0.

The sample of cetuximab obtained in example 1 was adjusted to a conductance of < 6ms/cm with purified water while controlling the pH at 6.5, giving sample 2.

A chromatography column HiTrap SP HP CV 4.2ml, column height 10cm was used. The column was equilibrated with buffer A, 5CV. Sample 2 was loaded onto the column with a contact time of 6min and DBC < 7mg/ml. The column was reequilibrated with buffer A, 3CV. Then, elution was performed using 10CV of a mixed solution of buffer A and buffer B. In the elution process, the volume of the buffer solution B in the mixed solution of the buffer solution A and the buffer solution B is 0-100%, and the linear increase is realized. The main peak at 280nm of UV was collected. Wherein the collection parameter of the main peak is that 300mAU starts to be collected, and 300mAU stops to be collected. The chromatogram is shown in FIG. 2.

Regenerating the used chromatographic column, washing with 0.5M sodium hydroxide at 40cm/h for 3CV, balancing with balancing solution until pH is stable, and storing with 20% ethanol.

The peak 1 fractions were collected, and the acid-base peaks of the fractions were measured using icIEF, and the results are shown in table 3.

TABLE 3

As can be seen from the results in tables 2 and 3, the loading pH of the present invention is 5.5 to 6.5, the conductance is < 6ms/cm, the peak collection parameter is 400 to 950mAU, the alkaline peak can be effectively reduced by about 10% while the main peak proportion is increased by about 7%. Moreover, the whole separation and purification process is simple, the buffer solution has simple components and low price, and the requirements in industrial application can be well met.

Comparative example 1: antibody samples were loaded using pH 5.5 and purified using 500mM sodium chloride linear elution conditions.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:50mM phosphate-citrate buffer, pH 5.5;

and (3) buffer solution B:50mM phosphate-citrate buffer, 500mM sodium chloride, pH 5.5.

The sample of cetuximab obtained in example 1 was adjusted to a conductance of < 6ms/cm with purified water while controlling the pH at 5.5, giving control sample 1.

A chromatography column HiTrap SP HP CV 4.2ml, column height 10cm was used. The column was equilibrated with buffer A, 5CV. Control sample 1 was loaded onto the column with a contact time of 6min and DBC < 7mg/ml. Column 3CV was reequilibrated with buffer A. Then, a mixed solution of buffer a and buffer B was used, and elution was performed with 15 CV. During elution, the volume of buffer B in the total volume increased linearly from 0 to 100%. Collecting a main peak at the ultraviolet 280nm position, wherein the collection parameters are as follows: 50mAU was collected initially and 50mAU was terminated. Peaks 1 to 4 were collected in order. The chromatogram is shown in FIG. 3.

Regenerating the used chromatographic column, washing with 0.5M sodium hydroxide at 40cm/h and 3CV, balancing with balance solution until pH is stable, and storing with 20% ethanol.

The components from peak 1 to peak 4 were collected, and the acid-base peaks of the respective components were detected using icIEF, and the results are shown in table 4.

TABLE 4

The results in table 4 show that the sample was able to bind effectively to the column, but the UV peak was not clearly characterized by separation according to the chromatogram of fig. 3, which is detrimental to the subsequent elution collection.

Comparative example 2: antibody samples were purified under pH 6.3 loading, 500mM NaCl linear elution conditions.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:50mM phosphate-citrate buffer, pH 6.3;

and (3) buffer solution B:50mM phosphate-citrate buffer, 500mM sodium chloride, pH 6.3.

The sample of cetuximab obtained in example 1 was adjusted to a conductance of < 6ms/cm with purified water while controlling the pH at 6.3, giving control sample 2.

A chromatography column HiTrap SP HP CV 4.2ml, column height 10cm was used. The column was equilibrated with buffer A, 5CV. Control sample 2 was loaded onto the column for a contact time of 6min, DBC < 7mg/ml. The column was re-equilibrated with buffer A, 3CV. Then, a mixed solution of buffer a and buffer B was used, and elution was performed with 15 CV. During elution, the volume of buffer B in the total volume increased linearly from 0 to 100%. Collecting a main peak at the ultraviolet 280nm position, wherein the collection parameters are as follows: 50mAU was collected initially and 50mAU was terminated. Peaks 1-4 were collected in order. The chromatogram is shown in FIG. 4.

Regenerating used chromatographic column, washing with 0.5M sodium hydroxide at 40cm/h for 3CV, balancing with balance solution until pH is stable, and storing with 20% ethanol.

The components from peak 1 to peak 4 were collected, and the acid-base peaks of the respective components were detected using icIEF, and the results are shown in table 5.

TABLE 5

The results in table 5 show that the sample was able to bind effectively to the column, but according to the chromatogram of fig. 4, the UV peak had no significant separation characteristics, which is detrimental to the subsequent elution collection.

Comparative example 3: antibody samples were loaded at pH 6.8 and purified using 300mM sodium chloride linear elution.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:20mM phosphate buffer, pH 6.8;

and (3) buffer solution B:20mM phosphate buffer, 300mM sodium chloride, pH 6.8.

The sample of cetuximab obtained in example 1 was subjected to a solution change using G25 packing (Cytiva, cat # 29048684) into buffer A at pH 6.8 to obtain control sample 3.

A chromatography column HiTrap SP HP CV 4.2ml, column height 10cm was used. The column was equilibrated with buffer A, 5CV. Control sample 3 was loaded onto the column for a contact time of 6min, DBC < 7mg/ml. The column was reequilibrated with buffer A, 3CV. Then, a mixed solution of buffer A and buffer B was used at 20CV to elute. During elution, the volume of buffer B in the total volume increased linearly from 0 to 100%. Collecting a main peak at the ultraviolet 280nm position, wherein the collection parameters are as follows: 50mAU starts to be collected, and 50mAU is stopped to be collected. Peaks 1 to 4 were collected in order. The chromatogram is shown in FIG. 5.

The used chromatographic column is regenerated, washed with 0.5M sodium hydroxide at 40cm/h and 3CV, equilibrated with an equilibration solution until pH is stable, and then stored with 20% ethanol.

The components from peak 1 to peak 4 were collected, and the acid-base peaks of the respective components were detected using icIEF, and the results are shown in table 6.

TABLE 6

The results in table 6 show that the sample was able to bind effectively to the column, but according to the chromatogram of fig. 5, the UV peak had no significant separation characteristics, which is detrimental to the subsequent elution collection.

Comparative example 4: antibody samples were purified under linear elution conditions of 300mM sodium chloride, loaded at pH 7.0.

Buffer a and buffer B were prepared separately.

And (3) buffer solution A:20mM phosphate buffer, pH 7.0;

and (3) buffer solution B:20mM phosphate buffer, 300mM sodium chloride, pH 7.0.

The sample of cetuximab obtained in example 1 was exchanged into buffer a, pH 7.0, using G25 filler to obtain control sample 4.

A chromatography column HiTrap SP HP CV 4.2ml, column height 10cm was used. The column was equilibrated with buffer A, 5CV. Control sample 4 was loaded onto the column with a contact time of 6min and DBC < 7mg/ml. The column was re-equilibrated with buffer A, 3CV. Then, a mixed solution of buffer A and buffer B was used at 20CV to elute. During elution, the volume of buffer B in the total volume increased linearly from 0 to 100%. Collecting a main peak at the ultraviolet 280nm position, wherein the collection parameters are as follows: 50mAU was collected initially and 50mAU was terminated. The chromatogram is shown in FIG. 6.

Regenerating used chromatographic column, washing with 0.5M sodium hydroxide at 40cm/h for 3CV, balancing with balance solution until pH is stable, and storing with 20% ethanol.

As can be seen from fig. 6, control sample 4 was loaded onto the column at pH 7.0, but the control sample failed to bind to the chromatography packing, so only one protein peak was detected at the upper sample on the chromatogram of fig. 6.

The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solutions of the present disclosure fall within the protection scope of the present disclosure.

Claims (10)

1. A method of protein purification, comprising:

adjusting the pH of the protein sample to be purified to a first pH;

loading the protein sample onto a cationic chromatography column; and

performing linear gradient elution with an eluent comprising a first buffer solution and a second buffer solution, wherein the pH value of the first buffer solution is the first pH value,

wherein the protein has an isoelectric point = a first pH + n, n being 1 to 2.

2. The method according to claim 1, wherein the first pH value is 5.0 to 6.8, preferably 5.5 to 6.5,

more preferably, the first buffer is selected from 10 to 100mM disodium hydrogen phosphate-citric acid buffer, 10 to 100mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, or 10 to 100mM citric acid-phosphoric acid-Tris buffer.

3. The method according to claim 1 or 2, wherein the pH of the second buffer is greater than the isoelectric point of the protein,

preferably, the second buffer is selected from 10 to 100mM disodium hydrogen phosphate-citric acid buffer, 10 to 100mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, or 10 to 100mM citric acid-phosphoric acid-Tris buffer.

4. The method according to any one of claims 1 to 3, characterized in that the protein has an isoelectric point of 6.5 to 8.0,

preferably, the protein is a fusion protein or a monoclonal antibody, more preferably a monospecific antibody or a bispecific antibody.

5. Method according to any of claims 1 to 4, characterized in that the packing of the cationic chromatography column comprises a cation exchanger containing sulfonic, sulfopropyl, phosphoric, carboxymethyl, carboxyl functional groups, preferably a sulfopropyl-containing cation exchanger.

6. The method according to any one of claims 1 to 5, wherein the method further comprises subjecting the protein sample to be purified to affinity chromatography prior to the adjusting the pH of the protein sample to be purified to a first pH,

preferably, the affinity medium of the affinity chromatography is protein a.

7. The method according to any one of claims 1 to 6, wherein the method further comprises subjecting the protein sample to be purified to anion chromatography prior to the adjusting the pH of the protein sample to be purified to a first pH,

preferably, the packing material for anion chromatography comprises Q-Sepharose, a resin containing quaternary aminoethyl or quaternary amine functional groups.

8. The method of any one of claims 1 to 7, wherein prior to loading the protein sample on the cationic chromatography column, the method further comprises equilibrating the cationic chromatography column with the first buffer.

9. The method of any one of claims 1 to 8, wherein prior to performing linear gradient elution, the method further comprises re-equilibrating the cationic chromatography column with the first buffer.

10. A purified protein obtained by the method of any one of claims 1 to 9.

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