JP6387640B2 - Method for preserving Fc receptor - Google Patents

Description

  The present invention relates to a method capable of storing an Fc receptor in an aqueous solution for a long time.

  Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Fc receptors are classified according to the type of immunoglobulin to which they bind, and include Fcγ receptors that bind to the Fc region of IgG, Fcε receptors that bind to the Fc region of IgE, Fcα receptors that bind to the Fc region of IgA, etc. Patent Document 1). Each receptor is further classified according to the difference in structure. In the case of an Fcγ receptor, the presence of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb has been reported (Non-patent Document 1).

FcγRI, which is one of the Fcγ receptors, is expressed in monocytes and macrophages, and is inducibly expressed in neutrophils by γ interferon (Non-patent Document 1). FcγRI has high binding affinity for IgG, and its equilibrium dissociation constant (Kd) is 10 ⁻⁸ M or less (Non-patent Document 2). FcγRI is divided into an extracellular region, a transmembrane region, and an intracellular region, and binding to IgG occurs in the Fc region of IgG and the extracellular region of FcγRI, and then a signal is transmitted to the cytoplasm. FcγRI is composed of two types of subunits, an α chain with a molecular weight of about 42000 that is directly involved in binding to IgG, and a γ chain. The γ chain covalently binds at the boundary between the cell membrane and the extracellular region, thereby (Non-Patent Document 3).

  The amino acid sequence (SEQ ID NO: 1) and gene sequence of human FcγRI are published in public databases such as ExPASy (Primary accession number: P12314). Similarly, the functional domain in the structure of FcγRI, the signal peptide sequence for penetrating the cell membrane, and the position of the cell membrane penetrating region are also published. The α chain of human FcγRI has the structure shown in FIG. 1, and is a signal peptide region consisting of 15 amino acids from the N-terminal side (SS: region from the first to the 15th of the amino acid sequence described in SEQ ID NO: 1), 277 amino acids An extracellular region (EC: the region from the 16th to the 292th of the amino acid sequence described in SEQ ID NO: 1), a transmembrane region consisting of 21 amino acids (TM: the 293rd of the amino acid sequence described in SEQ ID NO: 1) To 313th region), an intracellular region consisting of 61 amino acids (C: the region from 314th to 374th of the amino acid sequence described in SEQ ID NO: 1).

  As described above, the protein constituting the extracellular region of FcγRI has an excellent discrimination function for human antibodies with high affinity. Based on this high affinity, a method of utilizing as a diagnostic reagent, a research tool for antibody drugs, or a ligand for affinity chromatography used in the production process of antibody drugs such as IgG has been reported (Patent Document 1). . In recent years, it has been discovered that it has unexpected immunosuppressive biological properties, and has attracted attention as a pharmaceutical in the areas of autoimmune disease or autoimmune syndrome, transplant rejection and malignant lymphoproliferation. (Non-Patent Document 2).

  Regarding expression of Fcγ receptor using gene recombination technology, E. coli (Patent Document 1), animal cells (Non-Patent Document 3), Bacillus bacteria (Patent Document 2), yeast (Patent Document 3), Neisseria gonorrhoeae Expression using (Patent Document 4) as a host has been reported. In particular, in systems using Escherichia coli as a host, methods for producing Fcγ receptors by high-density culture (Patent Document 5), purification methods for Fcγ receptors using hydrophobic chromatography (Patent Document 6) and cation exchange chromatography have also been reported. ing. Furthermore, interest in industrial use of Fcγ receptors is increasing, such as the modification of Fcγ receptor structural genes improves the stability and productivity of Fcγ receptors (Patent Document 7).

  On the other hand, the Fcγ receptor has a problem that it easily aggregates when stored in a solution state after purification, which has been a serious drawback in industrial use. That is, if the purified Fcγ receptor is allowed to stand until it is used, there is a problem of aggregation and precipitation. Furthermore, there is a problem that if the aggregated Fcγ receptor is left as it is, it is denatured and cannot be redissolved.

JP 2008-245580 A JP 2009-201403 A JP 2011-072246 A JP 2011-200203 A JP 2012-034591 A JP 2011-126825 A JP 2011-206046 A

J. et al. V. Ravetch et al., Annu. Rev. Immunol. , 9, 457, 1991 Toshiyuki Takai, Jpn. J. et al. Clin. Immunol. , 28, 318, 2005 A. Paetz et al., Biochem. Biophys. Res. Commun. , 338, 1811, 2005

  An object of the present invention is to provide a solution that can be stably stored for a long period of time without aggregating the Fcγ receptor, and a method for stably storing the Fcγ receptor for a long period of time without aggregating.

  As a result of intensive studies to solve the above problems, it was found that the aggregation of Fcγ receptor is caused by halide ions, and the present invention has been completed.

That is, the present invention includes the following inventions:
(I) An aqueous solution containing an Fc-binding protein, wherein the concentration of halide ions contained in the aqueous solution is less than 200 mM.

  (Ii) A method for storing an Fc binding protein, wherein the Fc binding protein is stored in an aqueous solution having a halide ion concentration of less than 200 mM.

  Hereinafter, the present invention will be described in detail.

In the present invention, the Fc-binding protein refers to a protein constituting the extracellular region of the Fcγ receptor. As an example, when the Fcγ receptor is a natural human FcγRI, the region from the 16th glutamine to the 292nd histidine in the amino acid sequence shown in SEQ ID NO: 1 corresponds to the Fc-binding protein. However, it does not necessarily have to be the entire extracellular region of the Fcγ receptor. Of the proteins (polypeptides) constituting the extracellular region of the Fcγ receptor, at least the original function of binding to the Fc region of an antibody (immunoglobulin) is required. It only needs to contain a polypeptide in a region that can be expressed. As an example of the Fc binding protein,
(I) a protein comprising an amino acid residue from at least the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 1,
(II) contains at least amino acid residues from the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 1, and one or more of the amino acid residues are substituted with other amino acid residues Inserted or deleted protein,
Can be given.

  Specific examples of the (II) include Fc-binding proteins disclosed in JP2011-206046A and JP2014-027916A.

  The present invention is characterized in that, when an aqueous solution containing an Fc-binding protein is stored, the concentration of halide ions contained in the aqueous solution is less than 200 mM. As used herein, halide ions refer to fluoride ions, chloride ions, bromide ions, and iodide ions. There is no particular limitation on the method for removing halide ions from an aqueous solution containing an Fc-binding protein until the concentration is lower than the above-mentioned concentration, a method by dialysis, a method by ultrafiltration, a method in which halide ions are diluted to less than 200 mM, Does not contain halide ions such as ammonium sulfate, a method that adsorbs and removes halide ions by passing through ion-exchange membranes, ion-exchange resins and ion-exchange chromatography carriers, a method that removes precipitates formed by adding silver nitrate, etc. Examples thereof include a method of adding an organic solvent miscible with water such as salt, acetone or ethanol in an arbitrary volume ratio and recovering it as a precipitate, and then re-dissolving it in water or a buffer containing less than 200 mM halide ions. Among these, from the viewpoint of simplicity of treatment, a method using dialysis or ultrafiltration is preferably used industrially. The concentration of halide ions contained in the Fc-binding protein aqueous solution is preferably as low as possible, preferably less than 150 mM, more preferably less than 80 mM, and even more preferably less than 50 mM.

  The present invention is characterized in that, when an aqueous solution containing an Fc-binding protein is stored, the concentration of halide ions contained in the aqueous solution is less than 200 mM. This prevents loss of the Fc-binding protein caused by aggregation before the purified Fc-binding protein is used for other applications (for example, ligands for affinity chromatography), and improves the utilization efficiency of the Fc-binding protein. Can be greatly improved.

The figure which shows the structure of human type | mold FcγRI. The figure which shows the influence on the storage stability of Fc binding protein by the sodium chloride concentration contained in Fc binding protein aqueous solution. The figure which shows the influence on the storage stability of Fc binding protein by the sodium chloride concentration contained in Fc binding protein aqueous solution in presence of 200 mM sodium sulfate. The figure which shows the influence on the storage stability of Fc binding protein by the anion seed | species contained in Fc binding protein aqueous solution. The figure which shows the long-term storage stability of the Fc binding protein aqueous solution (protein concentration: 10 mg / mL) of this invention. The figure which shows the long-term storage stability of the Fc binding protein aqueous solution (protein concentration: 30 mg / mL) of this invention.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the said example.

Example 1 Preparation of Fc-binding protein aqueous solution (1) Recombinant Escherichia coli obtained by transforming Escherichia coli with an expression vector containing a polynucleotide encoding an Fc-binding protein is described in JP2012-034591A. The protein was expressed by culturing according to the above method.
(2) After recovering bacterial cells from the recombinant Escherichia coli culture solution, the extract (650 mM sodium chloride, 1 mM EDTA, 6 mM magnesium sulfate, 250 U / L Benzonase (trade name), 0.05 g / L lysozyme, 0.4%) (V / v) Triton X-100 (trade name) and 50 mM Tris-HCl (pH 8.0) containing 50 mM calcium chloride) were added to suspend the cells, and then cetyltrimethylammonium bromide (CTAB) Was added at a final concentration of 0.5% (w / v) and stirred to extract the protein expressed in the cells.
(3) Acid treatment is performed by adding dropwise acetic acid to the bacterial cell extract obtained in (2) while stirring so that the pH of the solution becomes 3.5, and the supernatant (acid treatment) is centrifuged. Liquid).
(4) Cation exchange chromatography after equilibration with equilibration buffer A (20 mM phosphate buffer (pH 6.0) containing 1M urea) after adding urea to the acid treatment solution to a final concentration of 1M. The gel was added to a column packed with a chromatography gel (TOYOPEARL CM-650, manufactured by Tosoh Corporation), washed thoroughly with equilibration buffer A, and then Fc-binding protein was eluted with equilibration buffer A containing 1 M sodium chloride.
(5) The human Fc-binding protein was purified from the eluate of (4) by the method using hydrophobic chromatography described in JP2011-126927A.
(6) A column packed with a gel for cation exchange chromatography (TOYOPEARL CM-650, manufactured by Tosoh) equilibrated with equilibration buffer B (20 mM phosphate buffer (pH 6.0)) in advance (5 Fc-binding protein aqueous solution purified in (1) was added.
(7) After washing a sufficient amount with equilibration buffer B, elution was performed with equilibration buffer B containing 1 M sodium chloride to obtain an aqueous solution containing highly purified Fc-binding protein.
(8) 10 mL of the aqueous solution obtained in (7) is desalted by dialysis three times against 1 L of 20 mM MES (2-morpholinoethanesulfonic acid) buffer (pH 5.0) containing 1 mM EDTA. The sodium chloride contained in the aqueous solution was completely removed. The Fc binding protein concentration after dialysis was 6 mg / mL.
(9) The solution after desalting obtained in (8) was stored at 4 ° C. for 20 days.

  The results are shown in FIG. Precipitation was not observed after storage for 20 days, and no change was observed in the protein concentration in the supernatant (result of NaCl 0 mM).

Example 2 Effect of Sodium Chloride Contained in Fc Binding Protein Aqueous Solution (Part 1)
After adding 4 M sodium chloride aqueous solution to the aqueous solution after desalting obtained in Example 1 (8), each prepared Fc binding protein aqueous solution containing 78 mM, 154 mM, 262 mM, 364 mM or 525 mM sodium chloride, The solution was stored at 4 ° C. for 20 days, and the protein concentration in the solution was measured.

  The results are shown in FIG. The Fc-binding protein aqueous solution containing 78 mM sodium chloride did not show precipitation or change in protein concentration in the solution after storage for up to 24 hours. The protein concentration also decreased to 4.8 mg / mL. The Fc-binding protein aqueous solution containing 154 mM NaCl contained a precipitate after storage for 24 hours, and the protein concentration in the solution also decreased to 1.2 mg / mL. The Fc-binding protein aqueous solution containing 262 mM or more of sodium chloride decreased the protein concentration in the solution to almost 0 after storage for 24 hours.

  From the above results, when sodium chloride (chloride ion) is contained in the Fc binding protein aqueous solution, the aggregation of the Fc binding protein is promoted, and when the sodium chloride (chloride ion) concentration is 200 mM or more, the Fc binding protein. Is quickly aggregated (within 24 hours).

Example 3 Effect of sodium sulfate contained in an aqueous Fc-binding protein solution (Part 2)
(1) Dialyze 10 mL of the Fc-binding protein aqueous solution obtained in Example 1 (7) three times against 20 mM MES buffer (pH 5.0) containing 1 mM EDTA and 0.2 M sodium sulfate. The sodium chloride contained in the solution was completely removed. The Fc binding protein concentration after dialysis was 6 mg / mL.
(2) The solution obtained in (1) was stored at 4 ° C. for 20 days.

  The results are shown in FIG. No precipitate was observed during storage, and no change was observed in the Fc-binding protein concentration in the supernatant (result of NaCl 0 mM). From this result, it can be seen that even when sodium sulfate (sulfate ion) is added, the storage stability of the aqueous Fc-binding protein solution is not affected.

Example 4 Effect of Sodium Chloride Contained in an Fc Binding Protein Aqueous Solution (Part 3)
After adding 4 M sodium chloride aqueous solution to the aqueous solution after desalting obtained in Example 3 (1), Fc-binding protein aqueous solutions containing NaCl of 78 mM, 154 mM, 262 mM, 364 mM, or 525 mM were prepared. The plate was stored at 20 ° C. for 20 days, and the protein concentration in the solution was measured.

  The results are shown in FIG. In the case of an Fc-binding protein aqueous solution containing 78 mM sodium chloride, no precipitate was observed during storage, and no change was observed in the Fc-binding protein concentration in the supernatant, as in the case of no sodium chloride. It was. The Fc-binding protein aqueous solution containing 154 mM sodium chloride did not show precipitation or changes in protein concentration in the solution after storage for up to 5 days, but precipitates were generated in the solution after storage for 20 days. The protein concentration dropped to 4.2 mg / mL. The Fc-binding protein aqueous solution containing 262 mM or more of sodium chloride showed almost no change in precipitation or protein concentration in the solution when stored for up to 24 hours, but the protein in the supernatant when stored for 5 days. Concentrations decreased to 4.7 mg / mL (262 mM), 1.4 mg / mL (364 mM), and 0.7 mg / mL (525 mM), respectively, and the protein concentration in the supernatant was nearly zero after 20 days of storage. .

  That is, when sodium sulfate (sulfate ions, that is, ions other than halides) is contained in the Fc-binding protein aqueous solution, aggregation promotion by sodium chloride (chloride ions) is suppressed, but sodium chloride (chloride ions) is 200 mM or more. Then, it can be seen that the aggregation of Fc-binding protein is promoted and the storage stability is lowered.

Example 5 Influence of anions (1) To the aqueous solution after desalting obtained in Example 1 (8), pure water, 2M sodium chloride aqueous solution, 2M sodium bromide aqueous solution, 2M sodium iodide aqueous solution, 2M phosphoric acid An aqueous solution containing 200 mM of each anion (anion) by adding monosodium dihydrogen buffer (pH 5.3), sodium oxalate powder, 2M sodium citrate buffer (pH 5.4), or 2M aqueous sodium sulfate solution. It prepared so that it might become. At this time, the protein concentration was adjusted to 5 mg / mL with pure water so that the protein concentration was constant.
(2) The solution prepared in (1) was stored at 4 ° C. for 22 days.

  The results are shown in FIG. In aqueous solutions to which pure water (none), monosodium dihydrogen phosphate (PO4), sodium oxalate (oxalic acid), sodium citrate (citric acid), and sodium sulfate (SO4) were added, No precipitation was observed, and no change was observed in the Fc-binding protein concentration in the supernatant. On the other hand, in an aqueous solution to which sodium chloride (Cl), sodium bromide (Br), and sodium iodide (I) were added, precipitation of the precipitate progressed during storage, and the protein concentration in the supernatant was almost constant after storage for 22 days. 0. In particular, in the solution to which sodium bromide (Br) and sodium iodide (I) were added, a precipitate was deposited immediately after the addition, and the protein concentration in the supernatant was also reduced.

  From the above results, it was found that aggregation precipitation is promoted by halide ions in Fc-binding proteins, while other anions (anions) have no aggregation promoting effect.

Example 6 Long-term storage stability test (1) Fc-binding protein was prepared in the same manner as in Examples 1 (1) to (7), concentrated to about 10 mg / mL with an ultrafiltration membrane, and then pure water was added. While performing ultrafiltration, desalting was performed until the electric conductivity reached 2 mS / cm. Note that the weight of the mother liquor is measured with an electronic balance so that the addition rate of pure water and the filtration rate by ultrafiltration are matched, and the weight is controlled to be constant.
(2) After desalting, the Fc-binding protein was concentrated to 30 mg / mL.
(3) A part of the concentrated solution was collected, and pure water was added to prepare a 10 mg / mL Fc-binding protein aqueous solution.
(4) The 30 mg / mL Fc-binding protein aqueous solution obtained in (2) and the 10 mg / mL Fc-binding protein aqueous solution prepared in (3) were filtered through a 0.2 μm sterilization filter and sterilized. And then stored at −20 ° C., 4 ° C., 25 ° C. or 37 ° C.
(5) Observation of precipitate formation in each aqueous solution and measurement of protein concentration in the aqueous solution were conducted every month.

  Changes in protein concentration are shown in FIG. 5 (protein concentration: 10 mg / mL) and FIG. 6 (protein concentration: 30 mg / mL). Under either condition, no precipitation occurred during the storage period of 5 months, and no decrease in protein concentration in the supernatant was observed.

  From the above, it was confirmed that the Fc-binding protein can be stably stored in a solution state for a long period of time by the storage method of the present invention.

Claims (2)

An aqueous solution comprising an Fc binding protein,
Ri concentration 80 mM less than Der halide ions contained in the aqueous solution,
pH is 5.0,
Ru FcγRI Der comprising amino acid residues from at least the 16th glutamine to 289 valine of the amino acid sequence of the Fc binding protein is described in SEQ ID NO: 1,
The aqueous solution. A method for storing an Fc binding protein, comprising storing the Fc binding protein in an aqueous solution having a halide ion concentration of less than 80 mM and a pH of 5.0 ,
The method, wherein the Fc-binding protein is FcγRI containing at least the 16th glutamine to 289th valine amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1 .

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