JP6111384B2 - Peptide column - Google Patents

Description

The present invention relates to a pair Puchidokara-time.

  TNF-α (Tumor necrosis factor alpha) is a cytokine produced by cells involved in the inflammatory immune response, and the anti-TNF-α antibody is used as a drug for rheumatism, Crohn's disease, ulcerative colitis and the like. An anti-TNF-α antibody is produced from an antibody molecule (IgG) produced by immunizing a different animal (usually a mouse) with TNF-α. The main mechanism of action of an anti-TNF-α antibody is to specifically bind to the target molecule TNF-α and inhibit its biological action. In addition, it also binds to membrane-type TNF-α molecules expressed on the surface of TNF-α producing cells such as monocytes, and damages these producing cells to suppress TNF-α production.

  This anti-TNF-α antibody is recovered by using affinity chromatography, as described in Patent Document 1, for example. Other methods for recovering the anti-TNF-α antibody include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydroxylatpatite chromatography, and the like.

Special table 2012-506383 gazette

  However, the above-described method for recovering anti-TNF-α antibody has a problem that the ease of operation or the recovery rate is insufficient.

The present invention has been made in view of such problems, and an object thereof is to provide a peptide column capable of recovering an anti- TNF-α antibody with a simple operation and a high recovery rate .

The peptide column according to the present invention is a peptide column in which a functional group that specifically binds to a peptide to be adsorbed is immobilized on the surface of a porous carrier, wherein the anti- TNF-α antibody affinity peptide IAVSYQTK is the functional group. It is characterized by being coupled to.

According to the present invention, a peptide column capable of recovering anti- TNF-α antibody with a simple operation and high recovery rate can be obtained .

FIG. 1 (a) is a total ion current chromatogram of an affinity peptide derived from adalimumab, FIG. 1 (b) is a total ion current chromatogram of an affinity peptide derived from infliximab, and FIG. 1 (c) is derived from golimumab. It is the total ion current chromatogram of the affinity peptide of. 2A is an MS spectrum of Peptide 1, and FIG. 2B is an MS / MS spectrum of Peptide 1. FIG. 3 is a schematic diagram of an anti-TNF-α antibody affinity peptide to which a linker sequence has been added. FIG. 4 is a schematic view of a production process of an anti-TNF-α antibody affinity peptide column. FIG. 5 (a) is an electrophoretic diagram of washing and elution fractions when human plasma added with adalimumab was used as a sample, and FIG. 5 (b) washes and washings when human plasma added with infliximab was used as a sample. Fig. 5 (c) is an electrophorogram of the eluted fraction, Fig. 5 (c) is an electrophoretic diagram of the washed and eluted fraction when human plasma added with golimumab is used as a sample, and Fig. 5 (d) is a sample of human plasma. FIG. 4 is an electrophoretogram of washing and elution fractions. FIG. 5 (e) is an electrophoretic diagram of washing and elution fractions when using a Protein A column with human plasma as a sample. FIG. 6 (a) is an electrophoretic diagram of the washing and elution fraction when the medium added with golimumab is used as a sample, and FIG. 6 (b) is the washing and elution image when the medium containing infliximab is used as a sample. Fig. 6 (c) is an electrophoretic diagram of washing and elution fractions using a culture supernatant to which golimumab is added as a sample, and Fig. 6 (d) is a culture to which infliximab is added. FIG. 3 is an electrophoretogram of washing and elution fractions when a supernatant is used as a sample. FIG. 7A is a deconvolution mass spectrum obtained by LC / MS of golimumab before recovery, and FIG. 7B is a deconvolution mass spectrum obtained by LC / MS of golimumab after recovery.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

(1) Anti-TNF-α antibody affinity peptide The anti-TNF-α antibody affinity peptide according to the present embodiment is obtained by the following method. That is, an antibody-immobilized gel preparation step for immobilizing an anti-TNF-α antibody on a gel to produce an anti-TNF-α antibody-immobilized gel, and a TNF-α-derived peptide by fragmenting TNF-α with an enzyme A peptide library preparation step for producing fragments, an anti-TNF-α antibody solid-phase gel obtained in the antibody-immobilized gel preparation step, a TNF-α-derived peptide fragment obtained in the peptide library preparation step, A step of removing a part of the TNF-α-derived peptide fragment that does not bind to the anti-TNF-α antibody and recovering the remainder of the TNF-α-derived peptide fragment bound to the anti-TNF-α antibody, It is a method to have. In the selection step, the TNF-α-derived peptide fragment bound to the anti-TNF-α antibody is an anti-TNF-α antibody affinity peptide having affinity for the anti-TNF-α antibody.

  In the antibody-immobilized gel preparation step, as the anti-TNF-α antibody immobilized on the gel, for example, commercially available antibodies such as adalimumab, infliximab, and golimumab can be used. The gel for immobilizing the anti-TNF-α antibody is not particularly limited, and for example, a silica-based gel can be used.

  In the peptide library production step, the enzyme that fragments TNF-α is not particularly limited, and for example, trypsin can be used.

  In the selection step, the anti-TNF-α antibody solid-phased gel obtained in the antibody-immobilized gel preparation step and the TNF-α-derived peptide fragment obtained in the peptide library preparation step are mixed to obtain anti-TNF -TNF-α-derived peptide fragments that do not bind to α antibody are removed. Removal of the TNF-α-derived peptide fragment that does not bind to the anti-TNF-α antibody is performed by washing the gel with, for example, ammonium acetate. After washing the gel, anti-TNF-α antibody affinity peptide having affinity for anti-TNF-α antibody can be recovered by eluting the TNF-α-derived peptide fragment bound to anti-TNF-α antibody with acid. .

  The recovered anti-TNF-α antibody affinity peptide can be identified by mass spectrometry. By performing identification, it can be confirmed that the obtained anti-TNF-α antibody affinity peptide is a peptide corresponding to a predetermined position of TNF-α.

(2) Peptide column The peptide column according to this embodiment is a peptide column in which a functional group is immobilized on the surface of a porous carrier, and the aforementioned anti-TNF-α antibody affinity peptide is bound to this functional group. Has the structure.

  The peptide column is based on a porous carrier such as a silica substrate. The functional group on the surface of the porous carrier is not particularly limited as long as it can specifically bind the anti-TNF-α antibody affinity peptide, and can be, for example, a tresyl group.

  More preferably, a linker is added to the N-terminus of the anti-TNF-α antibody affinity peptide, and the anti-TNF-α antibody affinity peptide is bound to the functional group via this linker. When the anti-TNF-α antibody affinity peptide is directly bound to the functional group on the surface of the porous carrier, the anti-TNF-α antibody is difficult to access near the binding site of the anti-TNF-α antibody affinity peptide due to steric hindrance. Because it is possible.

(3) Screening method The screening method according to the present embodiment comprises an antibody-immobilized gel preparation step for preparing an anti-TNF-α antibody-immobilized gel by immobilizing an anti-TNF-α antibody on a gel, and TNF-α. Peptide Library Preparation Process for Producing TNF-α Derived Peptide Fragments by Enzymatic Fragmentation, Anti-TNF-α Antibody Immobilized Gel Obtained in Antibody Immobilized Gel Preparation Process, and Peptide Library Preparation Process Mixed with the TNF-α-derived peptide fragment obtained in step 2 to remove a part of the TNF-α-derived peptide fragment that does not bind to the anti-TNF-α antibody, and to derive from TNF-α bound to the anti-TNF-α antibody And a screening step for recovering the remainder of the peptide fragment. In the selection step, the TNF-α-derived peptide fragment bound to the anti-TNF-α antibody is an anti-TNF-α antibody affinity peptide having affinity for the anti-TNF-α antibody.

Example 1
(1) In Example 1, an anti-TNF-α antibody affinity peptide was prepared.

(1-1) Preparation of antibody-immobilized gel Three types of anti-TNF-α antibodies (adalimumab (Abbott Japan, Tokyo, Japan), infliximab (Tanabe Seiyaku, Osaka, Japan), and golimumab (Jansen Pharma, Tokyo, Japan) ) Was dissolved in 0.5 M dipotassium hydrogen phosphate buffer (pH 7.5) (1.0 μg / μL). The anti-TNF-α antibody solution (100 μL) was added to a gel (TSKgel Tresyl-5PW, 10 mg) (Tosoh, Tokyo, Japan) and shaken overnight at room temperature. The functional group of the gel was a tresyl group, which reacted with the amino group or thiol group of the ligand. After washing the gel with 200 μL of 0.5 M NaCl, unreacted active groups were blocked with 100 μL of 0.1 M Tris-HCl buffer (pH 8.0) to prepare an anti-TNF-α antibody immobilized gel.

(1-2) Preparation of peptide library Trypsin Gold (Massspectrometry Grade) (Promega) to TNF-α (10 μL) (TNF-α (Miltenyi Biotech, Tokyo, Japan)) prepared to 1.0 μg / μL with purified water , Madison, WI, USA)) (1 μL), and incubated at 37 ° C. for 16 hours. The obtained TNF-α-derived peptide was used as a peptide library.

(1-3) Selection of peptide that binds to antibody After drying the peptide library, it was redissolved in purified water (100 μL), added to the anti-TNF-α antibody solid-phase gel, and shaken overnight at room temperature. . The gel was washed with 400 μL of 25 mM ammonium acetate (pH 7.0) to remove peptides that did not bind to the anti-TNF-α antibody. Anti-TNF-α antibody affinity that shows affinity for anti-TNF-α antibody by eluting and drying the peptide bound to anti-TNF-α antibody immobilized gel with 300 μL of 0.5 M acetic acid (pH 2.5) The peptide was obtained.

(1-4) Identification of Anti-TNF-α Antibody Affinity Peptide Anti-TNF-α antibody affinity peptide was dissolved in 40 μL of purified water to prepare a sample for mass spectrometry. After obtaining an MS / MS spectrum of the anti-TNF-α antibody affinity peptide by liquid chromatography / mass spectrometry (LC / MS) under the following conditions, the primary structure was determined by database search.

  The LC / MS conditions were as shown below.

(1-4-1) LC
Instrument: Paradigm MS4 (Michrom Bioresources, Auburn, CA, USA)
Trap column: L-column ODS (0.3 × 5 mm, 5μm, Agency for Chemical Evaluation)
Analytical column: L-column ODS (0.075 × 150 mm, 3 μm, Agency for Chemical Evaluation)
Mobile phase: Buffer A, 0.1% formic acid / 5% acetonitrile
Buffer B, 0.1% formic acid / 90% acetonitrile Gradient condition: 5-65% (B buffer) 45 minutes Flow rate: 300 nL / min
(1-4-2) MS
Equipment: LTQ-FT (Thermo Fisher Scientific, Waltham, MA, USA)
Spray voltage: 2.5 kV
Electrode: Positive ion mode
MS spectrum acquisition: full scan mode (measurement range: m / z 400-2,000)
MS / MS spectrum acquisition: data-dependent product ion scan mode
(1-4-3) Database Search Search Software: BioWorks 3.1 (Thermo Fisher Scientific)
Database: UniProt Database FIG. 1 (a) is a total ion current chromatogram of an affinity peptide derived from adalimumab, and FIG. 1 (b) is a total ion current chromatogram of an affinity peptide derived from infliximab. ) Is a total ion current chromatogram of an affinity peptide derived from golimumab. As a result, as shown in FIGS. 1A to 1C, peptides 1 to 5 were detected.

  Table 1 below shows the identification results of peptides 1 to 5. As shown in Table 1, peptides 1, 2, 3, 4 and 5 are amino acid sequences at positions 83-90, 7-32, 33-44, 16-31 and 91-98 of TNF-α, respectively. Was identified as the corresponding peptide.

  Among the identified peptides 1 to 5, peptide 1 derived from peak 1 which was found in all antibodies and had the highest recovery amount was used as an anti-TNF-α antibody affinity peptide used in the subsequent experiments. 2A is an MS spectrum of this peptide 1, and FIG. 2B is an MS / MS spectrum of peptide 1.

(Example 2)
(2) In Example 2, a peptide column having high affinity for the anti-TNF-α antibody was produced.

(2-1) Production of anti-TNF-α antibody affinity peptide using peptide 1 A linker sequence was added to the N-terminal side of the anti-TNF-α antibody affinity peptide (peptide 1). The linker sequence was CGSGSGGS, and as shown in FIG. 3, the linker sequence-peptide 1 was chemically synthesized as an affinity peptide column peptide (Toray Research Center (Kanagawa, Japan)).

(2-2) Production of Anti-TNF-α Antibody Affinity Peptide Column FIG. 4 is a schematic diagram of the production steps of an anti-TNF-α antibody affinity peptide column. As shown in FIG. 4, first, the TSKgelTresyl-5PW column (2 × 35 mm, Tosoh) was washed with 500 μL of 0.5 M dipotassium hydrogen phosphate solution (pH 7.5).

  Next, the above chemically synthesized peptide was dissolved in a 0.5 M dipotassium hydrogen phosphate solution (pH 7.5) (1.0 μg / μL), and 100 μL thereof was injected into the column and left at 4 ° C. overnight.

  Next, 200 μL of 0.2 MTris-HCl (pH 8.0) was injected to block unreacted active groups for 1 hour at room temperature.

  Next, the column was washed with 500 μL of 25 mM ammonium acetate (pH 7.0) to remove unreacted synthetic peptide. Thereby, an anti-TNF-α antibody affinity peptide column was prepared.

(Example 3)
(3) In Example 3, the recovery of anti-TNF-α antibody from human plasma was confirmed using the anti-TNF-α antibody affinity peptide column prepared in Example 2.

(3-1) Recovery of anti-TNF-α antibody from human plasma Anti-TNF-α antibodies (adalimumab, infliximab, and golimumab) were dissolved in purified water (1.0 μg / μL).

  Next, these anti-TNF-α antibody solutions (10 μL) were diluted with 90 μL of human plasma (KACcorporation (Kyoto, Japan)) and then injected into the anti-TNF-α antibody affinity peptide column prepared in Example 2. .

  Next, the column was washed with 1 mL of 25 mM ammonium acetate (pH 7.0).

  Next, the adsorbate was eluted from the column with 300 μL of 0.1 M Tris-NaOH (pH 11.5).

  Next, the washed and eluted fractions were dried and dissolved in 10 μL of purified water (1.0 μg / μL).

(3-2) SDS-PAGE
Using each fraction as a sample, SDS-PAGE was performed under the following conditions to verify the specificity of the peptide column of the present invention.

SDS-PAGE conditions Gradient gel: e-PAGEL (18-well, thickness 1.0mm, gradient 5-20%; ATTOCorporation, Tokyo, Japan)
Loading buffer: 0.5M Tris-HCl (pH6.8) 1.25 mL, SDS (Wako Osaka Japan) 0.2 g, Glycerol (Wako Osaka Japan) 2 mL, Bromophenol Blue (WakoOsaka Japan) small volume / 10 mL Purified water Electrophoresis buffer: Tris (Sigma Aldrich Tokyo Japan) 3g, Glycine (WakoOsaka Japan) 14.4g, SDS (Wako) 1g / 1L Purified water Sample preparation: Add 2μL of sample solution to 9μL of loading buffer and heat at 100 ℃ for 3 minutes The processed sample was used as a sample.

Gel staining: SYPRO Rudy Protein Gel Stain (Invitrogen)
Detection: Band confirmation with Typhoon 9400 (GE Healthcare) Fig. 5 (a) is an electrophoretogram of washing and elution fractions when human plasma added with adalimumab is used as a sample, and Fig. 5 (b) shows addition of infliximab. Fig. 5 (c) is an electrophoretic diagram of washing and elution fractions when human plasma added with golimumab is used as a sample. FIG. 5 (d) is an electrophoretic diagram of washing and elution fractions using human plasma as a sample, and FIG. 5 (e) is a Protein A column (manufactured by GE Healthcare Biosciences, HiTrap ProteinAHP) using human plasma as a sample. FIG. 2 is an electrophoretogram of washing and elution fractions when 1 mL) is used. In each figure, the control is electrophoresis when an anti-TNF-α antibody aqueous solution is used as a sample.

  As shown in FIGS. 5 (a) to 5 (c), as a result, the migration pattern of the control and elution fractions were similar regardless of which human plasma added with any antibody was used as a sample. When human plasma was used as a sample, no band was observed in the electrophoretic image of the eluted fraction. As shown in FIG. 5 (e), antibodies in plasma were detected in the elution fraction with the Protein A column, and could not be specifically recovered with the Protein A column. By using the anti-TNF-α antibody affinity peptide column according to the present invention, the anti-TNF-α antibody can be produced in a high yield without being affected by nonspecific binding or cross-reaction of endogenous antibodies in plasma. And it could be recovered specifically.

Example 4
(4) In Example 4, the anti-TNF-α antibody affinity peptide column prepared in Example 2 was used to confirm recovery of the anti-TNF-α antibody from the culture solution or culture supernatant.

(4-1) Recovery of anti-TNF-α antibody from culture medium or culture supernatant Anti-TNF-α antibodies (adalimumab, infliximab, and golimumab were dissolved in purified water (1.0 μg / μL).

  Next, these anti-TNF-α antibody solutions (10 μL) were diluted with 90 μL of a culture solution or culture supernatant and then injected into the anti-TNF-α antibody affinity peptide column prepared in Example 2. The medium was FreeStyleTM CHOExpression Medium (Invitrogen, Tokyo, Japan). The cell culture supernatant was a culture supernatant obtained by culturing CHO-S cells.

  Next, the column was washed with 500 μL of 25 mM ammonium acetate (pH 7.0).

  Next, the adsorbate was eluted from the column with 300 μL of 0.1 M Tris-NaOH (pH 11.5).

  Next, the washed and eluted fractions were dried and dissolved in 10 μL of purified water (1.0 μg / μL).

(4-2) SDS-PAGE
Next, using each fraction as a sample, SDS-PAGE was performed under the same conditions as in Example 3 to verify the specificity of this peptide column.

  FIG. 6 (a) is an electrophoretic diagram of the washing and elution fraction when the medium added with golimumab is used as a sample, and FIG. 6 (b) is the washing and elution image when the medium containing infliximab is used as a sample. Fig. 6 (c) is an electrophoretic diagram of washing and elution fractions using a culture supernatant to which golimumab is added as a sample, and Fig. 6 (d) is a culture to which infliximab is added. FIG. 3 is an electrophoretogram of washing and elution fractions when a supernatant is used as a sample. In each figure, the control is electrophoresis when an anti-TNF-α antibody aqueous solution is used as a sample.

  As shown in FIGS. 6 (a) to 6 (d), as a result, in any antibody, only the anti-TNF-α antibody is produced in a high yield and specific without being affected by the medium components or the host cell-derived protein. Could be recovered.

(Example 5)
(5) In Example 5, as the process analysis engineering (PAT), the sugar chain heterogeneity of the anti-TNF-α antibody changes before and after the recovery of the anti-TNF-α antibody from the anti-TNF-α antibody affinity peptide column. I verified not to.

(5-1) Process analysis engineering In recent years, the center of gravity of pharmaceutical quality control strategies has shifted from methods that guarantee quality in the final drug substance / product to methods that guarantee by controlling important intermediates or in-process management. However, the process analysis engineering (PAT) according to this example can measure the characteristics and processes of raw materials and intermediates of pharmaceuticals without delay.

  A column switching system capable of continuously performing desalting using an anti-TNF-α antibody affinity peptide column and a reverse phase system column prepared in Example 2 (see J Pharm Biomed Anal. 2012, 67-68, 1-9. ) Was built. The desalting column was a MassPREP ™ Micro desalting column (2.1 × 5.0 mm, 20 μm; Waters, Milford, Mass., USA). This system was connected to a liquid chromatography-mass spectrometer (LC-MS), and sample purification, desalting, and mass measurement were performed online. A solution obtained by adding golimumab to the culture supernatant of CHO-S cells at 1.0 μg / μL was used as a sample solution, and 6 μL was injected into column switching LC-MS. The anti-TNF-α antibody (golimumab) before and after being collected by the anti-TNF-α antibody affinity peptide column prepared in Example 2 was compared for sugar chain heterogeneity by LC / MS under the following conditions.

(5-2) Comparison of sugar chain heterogeneity First, an elution fraction was collected using a culture solution to which an anti-TNF-α antibody (golimumab) was added as a sample.

  Then, the deconvolution mass spectrum patterns obtained by LC / MS under the following conditions were compared using the antibody samples before and after recovery.

  The LC / MS conditions were as shown below.

(5-2-1) LC
Device: Paradigm MS4
Column: Peptide column, MassPREPTM Micro desalting column (2.1 × 5.0 mm, 20μm; Waters, Milford, MA, USA)
Mobile phase: Buffer A, 0.1% formic acid / 5% acetonitrile Buffer B, 0.1% formic acid / 90% acetonitrile Gradient condition: 10-80% (B buffer) 30 minutes Flow rate: 600 nL / min
(5-2-2) MS
Instrument: QSTAR Elite Qq-TOF mass spectrometer (AB Sciex, MA, USA)
Spray voltage: 4.0 kV
Electrode: Positive ion mode
MS spectrum acquisition: full scan mode (measurement range: m / z 800-4,000)
Software for deconvolution of mass spectra: Bioanalyst ™ software (AB Sciex)
FIG. 7A is a deconvolution mass spectrum obtained by LC / MS of golimumab before recovery, and FIG. 7B is a deconvolution mass spectrum obtained by LC / MS of golimumab after recovery.

  As a result, the same mass and sugar chain heterogeneity were confirmed in golimumab before and after the recovery, and it was demonstrated that this column can be used in a PAT system for monitoring sugar chain heterogeneity.

  It is useful for recovering antibodies with simple operation and high recovery rate.

  Sequence number 6: Linker

Claims (4)

A peptide column formed by fixing a functional group that specifically binds to a peptide to be adsorbed on the surface of a porous carrier,
A peptide column , wherein the anti- TNF-α antibody affinity peptide IAVSYQTK is bound to the functional group. A linker is added to the N-terminus of the anti-TNF-α antibody affinity peptide IAVSYQTK , and the anti-TNF-α antibody affinity peptide IAVSYQTK is bound to the functional group via the linker. The peptide column according to claim 1 . The peptide linker according to claim 2 , wherein the linker is CGSGSGS. The peptide column according to any one of claims 1 to 3 , wherein the functional group is a tresyl group.