JP2011232098A - Measuring method for immunoglobulin amount in solution - Google Patents

Abstract

Provided is a simple and highly quantitative antibody assay method for antibodies.
An antibody sample solution is passed through a column having monolithic silica immobilized with antibody-binding protein as a carrier, and then the column is washed and regenerated, and then the protein recovered in the effluent by the regeneration process. A method for measuring the amount of immunoglobulin in a solution, characterized by measuring the amount of
[Selection figure] None

Description

  The present invention relates to a method for measuring the amount of immunoglobulin in a solution.

  Measuring the abundance of a specific protein in a mixed solution is important for estimating the properties of the solution. In particular, there are a wide range of fields in which the measurement technology can be used, such as measuring the abundance of specific substances in body fluids such as blood and pollutants in wastewater, and measuring nutrients and harmful substances in food.

  When measuring the amount of a specific substance present in a mixed solution, (1) a method (separation measurement) of measuring the amount of the target substance as a purified product by completely separating and purifying the target substance from other mixtures. In addition, (2) a method of labeling only the target substance in the mixed solution and estimating the amount of the target substance from the labeling signal, and measuring the function of the substance (eg, catalytic function in the case of an enzyme protein) There is non-separation measurement such as a method of estimating the amount of the target substance from the strength of. In the former, the operation of separating and purifying the target substance is necessary, and for that purpose, a large-scale purification apparatus is usually required. Furthermore, the separation measurement not only has the disadvantage that it takes time for the separation and purification operation, but also causes a problem such as a decrease in the recovery rate during the separation and purification. On the other hand, in the latter case, there are technical difficulties in labeling only the target substance, and there are problems such as interference with quantitative measurement due to contaminants when performing functional measurement. In addition, immunoassay using an antibody that specifically binds to a target substance is often used for quantifying a specific protein in a mixed solution. In an immunoassay method using an antibody, the amount of antibody bound to a target substance or unbound antibody in a mixed solution is measured. In that case, it is generally necessary to measure the amount of the antibody in the mixed solution, but there is a sufficient method for specifically and accurately measuring the amount of antibody (immunoglobulin) which is a protein. Since it has not been established, there are still many improvements in the quantitative measurement method of the target substance in the mixed solution using the immunoassay method.

  Thus, in any of the above methods for measuring the target substance in the mixed solution, it is complicated and requires time for measurement, or in the case of a simple measurement method, the reliability of the obtained value is low, Since various problems exist, the development of a simple and reliable measurement method is desired.

  Immunoglobulin IgG, which is an antibody molecule usually used in immunoassays, is widely used as a therapeutic antibody or a test antibody based on its excellent substance recognition mechanism and in vivo effector activity. As for therapeutic antibodies and test antibodies, a method of producing them by hybridoma cell culture is the mainstream.

  The present inventors have developed a monolith spin column for antibody purification (Patent Document 1), and have also developed an antibody-binding protein suitable for immobilizing the monolith spin column with a controlled orientation with respect to the monolith support. (Patent Documents 2 and 3). In antibody purification using this monolith spin column, an initialization solution is applied to the column and passed through centrifugation to stabilize the initial conditions (initialization step), and then a solution containing the antibody is applied and centrifuged. To allow only antibody molecules to be specifically adsorbed and bonded to the carrier (adsorption process), and then wash solution is applied and centrifuged to pass through the nonspecifically bound molecules from the carrier ( (Washing step), and then applying an eluate under mild conditions so as not to affect the function of the antibody molecule, passing the solution by centrifugation, eluting the antibody, and collecting the antibody molecule (elution step). Thereafter, the regeneration solution is applied to the column and passed through centrifugation to elute antibody molecules that could not be eluted, thereby regenerating the column (regeneration process step). However, even with this antibody purification method, a reduction in the antibody recovery rate in the elution step is inevitable.

Registered Utility Model No. 3149787 JP 2008-115151 A JP 2008-115152 A

  An object of the present invention is to provide a simple and highly quantitative antibody measurement method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have used an inorganic porous support column such as a monolithic silica column on which an antibody-binding protein is immobilized in a solution under appropriate conditions. Antibody can be adsorbed and separated, washed, and subjected to column regeneration without elution step, so that a very high antibody recovery rate can be obtained in the regeneration solution (regeneration effluent) that has flowed out of the column. The inventors have found that the target antibody in the solution applied to the column can be quantitatively measured by measuring the amount of protein in the effluent, and the present invention has been completed.

That is, the present invention includes the following.
[1] The amount of protein recovered in the effluent by regeneration after passing the antibody sample solution through a column having a monolithic silica carrier on which antibody-binding protein is immobilized, and then washing and regenerating the column A method for measuring the amount of immunoglobulin in a solution, characterized in that
[2] The method according to [1] above, wherein the column is a spin column.
[3] The method of [1] or [2] above, wherein the antibody-binding protein is an IgG-binding protein and the immunoglobulin is IgG.
[4] The method according to [1] to [3] above, wherein the antibody-binding protein is protein A or protein G or a variant thereof.
[5] The method of [1] to [4] above, wherein the antibody-binding protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2.
[6] The method according to [1] to [5] above, wherein the antibody sample solution is a mixed solution containing two or more proteins.
[7] The method of [1] to [6] above, wherein the antibody sample solution has a total protein concentration of 1 mg / mL or less.

  By using the method of the present invention, antibodies such as IgG in a solution can be measured easily and with high quantitativeness.

FIG. 1 is a diagram showing an outline of the configuration of a monolith spin column used in the present invention. FIG. 2 is a diagram illustrating a procedure for quantifying antibodies in a solution using a spin column. FIG. 3 shows the protein concentration at the time of addition to the column (applied) measured by applying a Rituxan solution with various protein concentrations to a monolith spin column in which a protein A variant is immobilized on a carrier, and regeneration treatment. It is the figure which showed the relationship with the density | concentration of the protein in the collect | recovered solution (regeneration effluent). In the figure, the horizontal axis represents the protein concentration of the added Rituxan, and the vertical axis represents the protein concentration in the recovered regeneration effluent. In the figure, a straight line indicates a straight line having an inclination of 1. FIG. 4 shows that the protein concentration at the time of addition to the column and the regeneration were collected from the column as measured by applying an Avastin solution of various protein concentrations to a monolith spin column in which the protein A mutant was immobilized on a carrier. It is the figure which showed the relationship with the density | concentration of the protein in a solution (regeneration effluent). In the figure, the horizontal axis represents the protein concentration of the added Avastin, and the vertical axis represents the protein concentration in the recovered regeneration effluent. In the figure, a straight line indicates a straight line having an inclination of 1. FIG. 5 shows that the protein concentration at the time of addition to the column and the regeneration were collected from the column as measured by applying a Herceptin solution of various protein concentrations to a monolith spin column in which the protein A mutant was immobilized on a carrier. It is the figure which showed the relationship with the density | concentration of the protein in a solution (regeneration effluent). In the figure, the horizontal axis represents the protein concentration of the added Herceptin, and the vertical axis represents the protein concentration in the recovered regenerated effluent. In the figure, a straight line indicates a straight line having an inclination of 1. FIG. 6 is measured by applying IgG1 human monoclonal antibody products prepared at various protein concentrations to a monolith spin column in which a protein A variant is immobilized on a carrier or a monolith spin column in which a protein G variant is immobilized on a carrier. It is a figure showing the relation between the protein concentration at the time of addition to the column and the concentration of the protein in the solution (regeneration effluent) recovered from the column by the regeneration treatment. In the figure, the horizontal axis represents the protein concentration of the added mixed solution sample, and the vertical axis represents the protein concentration in the recovered regeneration effluent. In the figure, white circles show the results when using a spin column with a protein A variant immobilized, and black circles show the results when using a spin column with a protein G variant immobilized. The solid line (protein G mutant) shows a straight line with a slope of 1, and the dotted line (protein A mutant) shows a straight line with a slope of 0.9. The regenerated solution has strong acidity and may affect protein quantification. To avoid this, neutralization is performed by adding 40 μl of 1M Tris.

Hereinafter, the present invention will be described in more detail.
1. Introduction In recent years, antibody proteins have great expectations as pharmaceuticals. When producing antibody proteins as pharmaceuticals, the process of secretory expression using CHO cells is mainly used, but in the culture management, development of a method for quickly and simply measuring the amount of secreted and produced antibody is required. Therefore, development of a technique for measuring the amount of antibody protein in a mixed solution is very useful in process management. When the antibody protein is used as a measurement target substance, the present inventors have used a monolith spin column developed by the present inventors in which the antibody-binding protein is immobilized and immobilized, whereby the antibody in the mixed solution is used. With the idea that the measurement of protein amount can be greatly simplified, we worked on the development of this measurement method.

  As a method considered optimal when measuring the amount of antibody in a mixed solution containing two or more proteins, 1. a method capable of quantitatively separating and recovering only the target antibody molecule; 2. the protein amount of the separated and recovered antibody can be measured easily and accurately; 3. The time required for measurement is short. 4. The apparatus required for measurement is simple and accurate. It was considered necessary to satisfy the following five conditions: it can be used for quantitative analysis of a wide range of antibodies rather than specific antibodies. However, a method that satisfies all of these conditions has not been developed so far.

  On the other hand, the present inventors have developed a spin column in which a modified antibody-binding protein is immobilized on a monolithic carrier such as monolithic silica while controlling the orientation (Registered Utility Model No. 3149787). (Actual application No. 2008-008179 filed on Nov. 21, 2008), JP 2008-115151 A, JP 2008-115152 A). By using this monolith spin column, it is possible to shorten the antibody separation / recovery, which has taken 10 minutes or more, within 2 minutes. Therefore, studies were made to construct a method for quantitatively measuring the amount of antibody protein in a mixed solution by using a spin column excellent in rapid separation and purification. As a result, by using a spin column equipped with a monolithic carrier such as monolithic silica in which antibody-binding protein is immobilized by controlling the orientation, it can be applied to various antibodies, has good reproducibility, and has high quantitativeness. Succeeded in building a measurement method.

  The present invention is based on such knowledge. The antibody sample solution is applied to a column having an inorganic porous carrier such as monolithic silica on which an antibody-binding protein is immobilized, and the column is allowed to pass (pass through) the column. After washing and regenerating the column, the amount of the protein recovered in the effluent from the column obtained by the regenerating process is measured to quantitatively determine the immunoglobulin (antibody) in the antibody sample solution. It relates to a measuring method.

2. Column for antibody adsorption separation The antibody measurement method according to the present invention uses a column having an inorganic porous carrier on which an antibody-binding protein is immobilized. As the inorganic porous carrier, a porous body mainly composed of silica (SiO ₂ ), for example, monolithic silica is preferably used. The column may be any column that can be used for column chromatography, but is more preferably a spin column. The spin column is a column that causes the carrier to pass through the solution applied to the column by centrifuging the column (that is, applying a centrifugal force) and adsorbs the target substance to the carrier. As such a column, a monolith spin column described as a chip for antibody purification in Registered Utility Model No. 3149787 (actual application No. 2008-008179 filed on Nov. 21, 2008), particularly a monolith silica spin column, Is preferred.

  The inorganic porous carrier attached to this monolith spin column has a co-continuous structure of macropores with a diameter of 100 nm to 10000 nm and a three-dimensional network skeleton, and the skeleton has mesopores with a diameter of 2 nm to 200 nm. To do. This inorganic porous carrier can be produced by causing a sol-gel transition with phase separation in the reaction solution. An inorganic porous carrier can also be produced by utilizing sol-gel transition by changing the pH of water glass or silicate aqueous solution.

  More specifically, the inorganic porous carrier is prepared by dissolving a water-soluble organic polymer and a thermally decomposable low-molecular compound in an acidic aqueous solution, and adding a metal compound having a hydrolyzable functional group to the hydrolysis reaction. Then, after the product solidifies, the wet gel is heated to thermally decompose the thermally decomposable low molecular weight compound, which can be dried and heated.

  The water-soluble organic polymer used here is a water-soluble organic polymer that can constitute an aqueous solution of an appropriate concentration, and is uniformly contained in a reaction system containing an alcohol generated by a metal compound having a hydrolyzable functional group. Any material that can be dissolved may be used. Examples of preferable water-soluble organic polymers include sodium salt or potassium salt of polystyrene sulfonic acid which is a polymer metal salt, polyacrylic acid which is a polymer acid and dissociates to form a polyanion, a polymer base which is an aqueous solution Examples thereof include polyallylamine and polyethyleneimine that generate a polycation, polyethylene oxide having a neutral polymer and having an ether bond in the main chain, and polyvinylpyrrolidone having a carbonyl group in the side chain. The addition amount of the water-soluble organic polymer is not limited, but is preferably 0.2 to 1.4 g per 10 g of the reaction solution.

  Instead of the water-soluble organic polymer, formamide, polyhydric alcohol, and / or surfactant may be used. In that case, glycerol is more preferable as the polyhydric alcohol, and polyoxyethylene alkyl ethers are more preferable as the surfactant.

  The thermally decomposable low molecular weight compound may be a compound that makes the solvent basic after thermal decomposition. Specific examples of thermally decomposable low molecular weight compounds include organic amides such as urea, hexamethylenetetramine, formamide, N-methylformamide, N, N-dimethylformamide, acetamide, N-methylacetamide, and N, N-dimethylacetamide. (Amide compounds) and the like. In the production of the inorganic porous carrier, the pH value of the solvent after heating is important, and it is necessary that the reaction solution becomes basic by this thermal decomposition. The amount of such a thermally decomposable low-molecular compound to be coexisted in the reaction solution depends on the kind of the compound. For example, in the case of urea, 0.05 to 0.8 g, preferably about 10 g of the reaction solution. 0.1 to 0.7 g is preferable.

  In addition, a compound that generates a compound having a property of dissolving silica, such as hydrofluoric acid, by thermal decomposition can be similarly used.

  As the metal compound having a hydrolyzable functional group, metal alkoxides, complexes, metal salts, organically modified metal alkoxides, organic cross-linked metal alkoxides, partial hydrolysis products thereof, and oligomers thereof can be used. The hydrolyzable functional group is preferably a group having a small number of carbon atoms such as a methoxy group, an ethoxy group, or a propoxy group. As the metal, for example, Si, Ti, Zr, Al or the like can be used. Any oligomer can be used as long as it can be uniformly dissolved and dispersed in alcohol, and specifically, for example, about 10-mer can be used. Specific examples of the metal compound having a hydrolyzable functional group include, but are not limited to, alkoxysilane compounds such as tetramethoxysilane. In order to produce an inorganic porous carrier mainly composed of silica, it is preferable to use a compound containing Si (silicon) as the metal compound having a hydrolyzable functional group. As such a compound containing Si (silicon), a precursor compound of silica that causes gel formation in a sol-gel reaction is preferable.

  As the acidic aqueous solution, any acid diluted aqueous solution, for example, a mineral acid such as hydrochloric acid or nitric acid (preferably an aqueous solution having a concentration of 0.001 molar or higher), or an organic acid such as acetic acid or formic acid (preferably having a molar concentration of 0.01 molar or higher) is used. Aqueous solution) can be preferably used.

  In the production of the inorganic porous carrier used in the present invention, for example, a water-soluble organic polymer and a heat-decomposable low-molecular compound are dissolved in an acidic aqueous solution, and a metal compound having a hydrolyzable functional group is added to add water. By performing the decomposition reaction, a gel separated into a solvent-rich phase and a skeleton phase can be generated. Phase separation and gelation can usually occur by storing the solution at room temperature 40-80 ° C. for 0.5-5 hours. In the phase separation and gelation step, the initially transparent solution becomes cloudy, undergoes phase separation between an inorganic phase such as a silica phase and an aqueous phase, and finally undergoes gelation. In this phase separation and gelation step, the water-soluble organic polymer is in a dispersed state and does not substantially precipitate. When the gel thus produced has solidified, the wet gel is heated after an appropriate aging time. By heating the gel, a thermally decomposable low molecular compound such as an amide compound dissolved in the reaction solution is thermally decomposed, and the pH of the solvent in contact with the inner wall surface of the skeleton phase is increased. The heating temperature may be, for example, 40 to 200 ° C. in the case of urea, and as a result, the pH value of the reaction solution after heating is preferably 6.0 to 12.0. As a result of the increase in pH value, the reaction solution erodes the inner wall surface, and the pore diameter gradually increases by changing the uneven state of the inner wall surface.

  In the case of gels composed mainly of silica, the degree of change is very small in the acidic or neutral region, but as the thermal decomposition becomes more active and the basicity of the aqueous solution increases, the portion constituting the pores dissolves. In addition, by reprecipitation in a flatter portion, a reaction that expands the average pore diameter significantly occurs.

  In gels that have only three-dimensionally confined pores without huge pores, the elution substance cannot be diffused to the external solution even if it can be dissolved as an equilibrium condition. Remains in a considerable proportion. On the other hand, in a gel having a solvent-rich phase that becomes giant pores, there are many pores that are restricted only two-dimensionally, and exchange of substances with an external aqueous solution occurs frequently enough. In parallel with the development, small pores disappear and the entire pore size distribution does not spread significantly.

In the heating process, it is preferable to place the gel in a hermetically sealed condition so that the vapor pressure of the thermal decomposition product is saturated and the pH of the solvent quickly takes a steady value.

  The above heat treatment time required for the dissolution / reprecipitation reaction to reach a steady state and generate a pore structure corresponding to the steady state varies depending on the size of the huge pores and the volume of the sample. Therefore, it is preferable to determine and use the shortest processing time at which the pore structure does not substantially change under each processing condition.

  The gel after the heat treatment becomes a dry gel by vaporizing (volatilizing) the solvent as the reaction solution by the drying treatment. In the dry gel, coexisting substances (such as water-soluble organic polymer and thermally decomposable low molecular weight compound) in the starting solution may remain. Therefore, the target inorganic porous carrier can be obtained with higher purity by further heat treatment at an appropriate temperature to thermally decompose the organic matter. In addition, what is necessary is just to perform a drying process by leaving at 30-80 degreeC for several hours-dozens of hours. Moreover, heat processing can be performed by heating a gel at about 200-800 degreeC.

  As described above, a rod-shaped inorganic porous carrier having continuous micrometer-sized pores and nanometer-sized mesopores can be produced. When the main component is silica, the inorganic porous carrier thus obtained is monolith type silica (monolith silica).

  Furthermore, using this inorganic porous carrier, it is possible to produce a column having a high resolution at a load pressure smaller than that of the particle packed column. For example, by mounting such an inorganic porous carrier or a modified surface thereof in a column container such as a centrifuge chip (centrifuge tube), the carrier is passed through the applied solution, preferably by centrifugation. A spin column can be manufactured.

  The inorganic porous carrier is usually processed into a size and shape suitable for mounting inside the column container. In one preferred embodiment, when the column container is cylindrical, the inorganic porous carrier can be processed into a disk shape. In order to process the inorganic porous carrier into a disk shape, a known method such as cutting or punching may be used.

  In the present invention, an antibody-binding protein or antibody-binding peptide is immobilized on the surface of an inorganic porous carrier, preferably an inorganic porous carrier that has been processed into a disk shape or the like, in order to adsorb the antibody in the solution to the carrier. . For this immobilization, it is preferable to first modify the surface of the inorganic porous carrier by introducing an amino group, and then bind the antibody-binding protein to the amino group by an amide bond. In the present invention, a peptide in which 2 to 10 amino acids are peptide-bonded is called “peptide”, and a peptide in which 11 or more amino acids are peptide-bonded is called “protein”.

  The antibody-binding peptide or protein has a binding ability to any of various immunoglobulins. The antibody-binding peptide or protein may bind to, for example, one or more of IgG, IgE, IgA, IgM, and IgD, but an IgG-binding protein is more preferable. Examples of antibody-binding peptides or proteins include, but are not limited to, protein A from Staphylococcus aureus (described in A. Forsgren and J. Sjoquist, J. Immunol. (1966) 97, 822-827.), Protein G derived from Streptococus sp. Group C / G (described in European Patent Application Publication No. 0131142A2 (1983)), Protein L derived from Peptostreptococcus magnus (described in US Pat. No. 5,965,390 (1992)), group A Streptococcus-derived protein H (described in US Pat. No. 5,180,810 (1993)), Haemophilus influenzae-derived protein D (described in US Pat. No. 6,025,484 (1990)), Streptococcus AP4-derived protein Arp (protein) Arp4) (described in US Pat. No. 5,210,183 (1987)), Streptococcal FcRc derived from group C Streptococcus (described in US Pat. No. 4,900,650 (1985)), group A streptococcus Type II strain A conventional protein (described in US Pat. No. 5,555,944 (1991)), a protein derived from human colonic mucosal epithelial cells (described in US Pat. No. 6,271,362 (1994)), derived from Staphylococcus aureu, 8325-4 strain Proteins (described in US Pat. No. 6,486,839 (1997)), proteins derived from Pseudomonas maltophilia (described in US Pat. No. 5,245,016 (1991)), and the like, or variants thereof can be used. For example, various mutant proteins in which protein A, protein G, protein L, etc. are appropriately changed in sequence so that the orientation control immobilization technology of the present inventors can be applied (JP 2008-115151, JP 2008-115152) Can be preferably used. These mutant proteins whose orientation is controlled and immobilized on the carrier have an amino acid sequence represented by the general formula PQR2 [in this formula, this sequence indicates a sequence from the amino terminal side to the carboxy terminal side. The sequence of the PQ portion is a sequence characterized by not containing lysine residues and cysteine residues; the sequence of the R2 portion may not be present, and if present, other than lysine and cysteine residues Is a spacer sequence composed of amino acid residues, and the sequence of the P portion may or may not exist, but if present, a sequence consisting of (Ser or Ala)-(Gly) n (n is The sequence of the Q moiety is 2 or more repeating units not containing lysine residues and cysteine residues (for example, 2 to 10, more preferably 2 to 5). Repetitive Represented by a is] array to return. In this mutant protein consisting of the amino acid sequence represented by the general formula PQR2, the PQ part is derived from an antibody-binding protein, and the Q part specifically interacts (binds) with the antibody molecule. This is an array part having a function. The sequence of the R2 portion may be a sequence consisting of 1 to 10 Gly (glycine). In one embodiment, a particularly suitable protein A variant includes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. Particularly preferred protein G variants include a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

  Binding of an antibody-binding peptide or protein to an inorganic porous carrier, for example, introduction of an amino group on the surface of an inorganic porous carrier and binding of an antibody-binding peptide or protein to the amino group are performed by conventional methods. Or it can implement by the method described in Unexamined-Japanese-Patent No. 2008-115152 and / or registration utility model 3149787. The antibody-binding peptide or protein is preferably bound to the amino group by an amide bond between the carboxy terminus of the antibody-binding peptide or protein and the amino group bound to the carrier. When the antibody-binding peptide or protein comprising the amino acid sequence represented by the general formula PQR2 represented by the general formula PQR2 to an amino group is bound to a carrier, for example, but not limited to, for example, the general formula R1-R2-R3- The amino acid sequence represented by R4-R5 [in this formula, this sequence represents a sequence from the amino terminal side to the carboxy terminal side, and the sequence of the R1 portion is the sequence of the PQ portion of the protein to be immobilized. Yes; the sequence of the R2 portion is the sequence of the R2 portion of the protein to be immobilized, that is, the sequence of the R2 portion may not be present, and if present, is composed of amino acid residues other than lysine and cysteine residues The sequence of the R3 moiety is composed of two amino acids represented by cysteine-X (X is an amino acid residue other than lysine and cysteine). The sequence of the R4 portion may not be present, and if present, is a sequence that does not include lysine residues and cysteine residues and is represented by the general formula R1-R2-R3-R4-R5 Comprising an acidic amino acid residue capable of bringing the isoelectric point of the entire protein consisting of the amino acid sequence to the acidic side; and the sequence of the R5 portion is an affinity tag sequence for purifying the protein] The portion represented by R1-R2 may be immobilized on a carrier using a protein comprising For specific experimental procedures for carrying out the binding of the antibody-binding peptide or protein to the carrier, reference can be made, for example, to the examples described later.

  In a column container such as a centrifuge chip equipped with an inorganic porous carrier on which an antibody-binding peptide or protein is immobilized, the load pressure applied to the inorganic porous carrier is determined by the thickness of the carrier. On the other hand, the volume of the inorganic porous carrier determines the amount of antibody adsorption. For example, the diameter of the disk-shaped inorganic porous carrier is determined depending on the size of the column container such as the centrifuge tip to be used. Therefore, when using a specific column container, the volume of the inorganic porous carrier is increased to increase the adsorption amount. In order to increase the thickness, it is necessary to increase the thickness of the inorganic porous carrier. However, if the thickness of the inorganic porous carrier is increased, the load pressure increases, and for example, there is a possibility that liquid passage by centrifugation becomes difficult. Therefore, the thickness of the inorganic porous carrier is preferably in the range of 0.1 to 1.0 with respect to the diameter 1.

  When mounting an inorganic porous carrier on which an antibody-binding peptide or protein is immobilized (preferably a discotic inorganic porous carrier) to a column container such as a centrifuge chip, the inorganic porous carrier is attached to the column container. It is preferable to fix it inside. In order to fix the inorganic porous carrier to the column container by an operation at room temperature or lower, it is also preferable to dispose a spacer inside the column container and fix the inorganic porous carrier through the spacer. The spacer is not limited, but is preferably made of a material such as elastic rubber.

  A column (preferably a spin column) equipped with such an inorganic porous carrier (preferably monolithic silica carrier) on which an antibody-binding peptide or protein is immobilized, adsorbs and separates antibodies from the solution with high efficiency. can do.

3. Antibody Measurement Method Using Antibody Adsorption Separation Column In the method of the present invention, the amount of antibody in a sample solution is determined using an antibody adsorption separation column such as the monolith spin column (for example, monolith silica spin column). It can be easily quantified.

  A preferable sample (antibody sample solution) for applying the antibody measuring method according to the present invention is a solution containing an antibody (immunoglobulin) or a solution containing an antibody, and is an object of antibody quantification. It is a solution. The antibody sample solution may be a solution containing or possibly containing one or more proteins, but a solution (mixed solution) containing two or more proteins is particularly suitable. The antibody sample solution may contain one type of antibody or two or more types of antibodies. The antibody sample solution may be an antibody solution prepared from an isolated antibody such as an antibody drug, a culture solution or culture supernatant of antibody-producing cells or hybridomas, or a solution prepared therefrom, A biological sample (for example, body fluid such as blood, serum, plasma, lymph, ascites, semen, etc.) or a solution prepared therefrom may be used, but is not limited thereto. The antibody sample solution is usually preferably prepared in an adsorption buffer. As the adsorption buffer, an adsorption buffer generally used in column chromatography including monolith column chromatography can be used. Although not limited thereto, for example, sodium phosphate buffer, preferably 0.1 M sodium phosphate buffer (pH 7.2) can be suitably used for preparing an antibody sample solution as an adsorption buffer. The antibody sample solution preferably has a total protein concentration of 0.0001 mg / mL or more and 10 mg / mL or less. Particularly preferred is an antibody sample solution having a total protein concentration of 1 mg / mL or less. If an antibody sample solution having such a concentration, particularly an antibody sample solution having a total protein concentration of 1 mg / mL or less is used, the antibody can be adsorbed to an inorganic porous carrier such as a monolithic silica carrier with high efficiency and from the carrier. Since it can elute efficiently, quantitative property can be improved significantly. Therefore, in the present invention, it is also preferable to apply the antibody sample solution to the column after adjusting it in these concentration ranges in advance.

  In the method for measuring an antibody in a solution according to the present invention, preferably, an initialization solution is applied (added) to the antibody adsorption separation column and passed therethrough (initialization step), and then the antibody sample solution is passed through the column. The solution is applied and allowed to pass through to adsorb and bind the target antibody molecule to the carrier (adsorption process). In this adsorption step, the antibody molecule that adsorbs and binds to the carrier is an antibody molecule that belongs to the antibody species to which the antibody-binding protein (or peptide) immobilized on the carrier binds. For example, if the antibody binding protein is an IgG binding protein, the IgG antibody adsorbs and binds to the carrier on which the antibody binding protein is immobilized.

  Next, a washing solution was applied to the column, and non-specifically bound molecules were washed out by passing through the column (washing step). Further, the regeneration solution was applied through the column and passed through the column to regenerate the column ( After the regeneration treatment step), the regeneration solution (regeneration effluent) that has passed through the column is recovered. In the antibody measurement method of the present invention, unlike the usual antibody purification method, the elution solution is applied between the column washing step and the regeneration treatment step under conditions that do not affect the function of the antibody molecule. A step (elution step) of eluting and recovering the antibody by passing the solution through is not performed. It is preferable that the solution is passed through the column by centrifugation (with a centrifugal force), usually using a centrifuge. Centrifugation may be usually performed using a centrifuge at an appropriate rotation speed and rotation time.

  As an initialization solution used in the initialization step, a phosphate buffer solution (for example, sodium phosphate buffer solution, potassium phosphate buffer solution), tris-hydrochloric acid buffer solution, sodium borate buffer solution or the like is preferably used. However, the present invention is not limited to these. The initialization solution may be, for example, a 0.001M to 0.8M buffer. The initialization solution is preferably a solution having a pH of 6.5 to 8.5. A particularly preferred example of the initialization solution is 0.1M sodium phosphate buffer (pH 7.2). The initialization solution is also preferably a solution having the same composition as the adsorption buffer used for preparing the antibody sample solution.

  The washing solution used in the washing step includes sodium chloride-containing phosphate buffer, sodium chloride-containing sodium phosphate buffer, sodium chloride-containing potassium phosphate buffer, sodium chloride-containing Tris-HCl buffer, sodium chloride-containing boric acid. A sodium chloride-containing buffer solution such as a sodium buffer solution (a buffer solution containing 0.5 M to 1.5 M NaCl in a preferred example) can be suitably used. For example, a phosphate salt containing 0.5 M to 1.5 M NaCl Although a buffer solution is mentioned, it is not limited to these. The washing solution may be, for example, a 0.001M to 0.8M buffer. As the cleaning solution, a solution having a pH of 6.5 to 8.5 is preferably used. A particularly preferred example of the washing solution is 0.1M sodium phosphate buffer (pH 7.2) containing 1M NaCl.

  As the regeneration solution used in the regeneration treatment step, a citric acid solution itself (pH about 2.25), a glycine-hydrochloric acid buffer solution (pH 2.5), or the like can be preferably used, but is not limited thereto. Absent. The regeneration solution may be, for example, a 0.05M to 1.0M solution. The regeneration solution is preferably a solution having a pH of 2.0 to 2.8. Particularly preferred examples of the regeneration solution include 0.02 M citric acid solution (pH 2.25) or 0.1 M glycine-hydrochloric acid buffer (pH 2.5).

  In a preferred embodiment, phosphate buffer (eg, sodium phosphate buffer) can be used as the initialization solution and wash solution, and glycine-hydrochloric acid buffer can be used as the regeneration solution.

  The cleaning and regeneration treatment may be performed once, or may be performed twice or more continuously. The washing can be performed, for example, 1 to 5 times, preferably 3 times. The regeneration process can be performed, for example, 1 to 5 times, preferably 2 or 3 times.

  Next, with respect to the regenerated effluent obtained as described above, the total amount of protein contained in the regenerated effluent is measured. Measurement of the total protein amount can be performed using any protein quantification technique. Examples of such protein quantification techniques include, but are not limited to, the Bradford protein quantification method, the bicinchoninic acid (BCA) quantification method, the Raleigh method, and the burette method. These protein quantification methods can also be more easily performed using a commercially available quantification kit.

For example, in the bicinchoninic acid (BCA) quantification method, a copper ion Cu ²⁺ is reduced to Cu ¹⁺ by a peptide bond in a protein under alkaline conditions, and then bicinchoninic acid (BCA) is added to form a complex of BCA2 molecule and Cu ^1+. Proteins are quantified by forming and measuring the red-violet visible absorbance (562 nm) resulting from the complex. This BCA method can be easily performed, for example, using a commercially available protein assay kit manufactured by Thermo Ficher Scientific.

  In the method of the present invention, the amount of protein in the regeneration effluent determined in this way is used to bind the target antibody molecule contained in the antibody sample solution, that is, the antibody binding protein (or peptide). It can be the amount of a species antibody molecule (immunoglobulin). By the antibody adsorption technique using the above-mentioned antibody adsorption separation column, the present inventors can adsorb the target antibody molecule very efficiently from the solution and can be eluted with high efficiency by the regeneration treatment. It was newly found that the yield was very high and the antibody molecule of interest could be almost completely separated from the solution. Moreover, in the present invention, surprisingly, similar results can be obtained even when a mixed solution containing two or more proteins is used as the antibody sample solution. In the present invention, based on this finding, the amount of the target antibody molecule contained in the antibody sample solution can be quantified as a value approximating the amount of protein in the regenerated effluent. Such quantification is possible because the measured value of the amount of the target antibody molecule (antibody molecule capable of binding to the antibody-binding protein) contained in the antibody sample solution is measured in the regeneration effluent. This is to show a proportional relationship with the measured amount of protein, for example, a linear proportional relationship with a slope of 0.9 to 1.1, preferably a linear proportional relationship with a slope of approximately 1.

  In the method of the present invention, when an antibody sample solution having a total protein concentration of 10 mg / mL or less, particularly 1 mg / mL or less is applied to the column, a very good proportional relationship can be realized, and the quantitative property can be achieved. Can be greatly increased.

  High quantitativeness in the method of the present invention is demonstrated in the examples described below. For example, the amount of protein in the mixed solution containing antibody molecules (antibody sample solution) is T (grams), and the measured amount of protein in the effluent (outflow fraction) in the adsorption step is A (grams). The measurement value of the amount of protein in the effluent (wash effluent) in the process is W (gram), and the amount of protein in the effluent (regeneration effluent) in the regeneration process step by passing the regeneration solution through the column The measured value is R (gram). Here, the initialization solution contains no protein. On the other hand, the antibody adsorbed and bound to the column carrier is eluted and recovered in the regeneration effluent. Since the protein in the mixed solution is either adsorbed to the column carrier in the adsorption step, the protein that is not adsorbed to the column carrier is recovered in the effluent fraction, while the protein adsorbed to the column carrier is washed. Or it will be recovered in the effluent in the regeneration process. Therefore, if measurement is performed correctly, T = A + W + R should hold. As shown in the examples described later, in the method of the present invention, the relationship of T = A + W + R is substantially established, and accurate measurement is possible. In addition, in the present invention, a protein in an amount equivalent to the content of the target antibody molecule in the antibody sample solution was recovered in the regeneration effluent. This means that in the method of the present invention, the target antibody molecule is adsorbed on the column carrier with high efficiency and hardly adsorbs in the adsorbed effluent or washing effluent while adsorbing firmly, but it is eluted efficiently in the regeneration process. It shows that That is, in the method of the present invention, as long as the measurement is appropriately performed, the value obtained as R is sufficiently reliable for the protein amount of the target antibody molecule (eg, IgG) in the antibody sample solution such as a mixed solution. It can be a measured value.

  In general, for protein measurement by immunoassay, it is necessary to use an antibody that specifically binds to the immunoglobulin to be measured (various antibody molecules such as IgG), and the concentration for preparing a calibration curve is known. Measurement using standard protein is also required. However, in the method of the present invention, it is only necessary to measure the amount of immunoglobulin protein recovered in the regenerated effluent, so that the application range of the measurement method can be further expanded, and the cost can be reduced. In the conventional measurement method including the purification separation step, the purification separation step generally takes time, and a large-scale apparatus such as chromatography is required. However, in the method of the present invention, the separation step can be performed in a few minutes. In addition, when a spin column is used, since it can be carried out only by using a simple desktop centrifuge, the working time and cost can be reduced.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

In the following production examples and examples, an initialization solution, a cleaning solution, and a regeneration solution having the following compositions were used in common.
-Initialization solution (initialization step): 0.1 M sodium phosphate buffer (pH 7.2)
Washing solution (washing step): 0.1 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl
・ Regeneration solution (regeneration treatment step): 0.1 M glycine-hydrochloric acid buffer (pH 2.5)

  The solution applied to the spin column was allowed to flow by centrifuging the spin column for 30 seconds (about 3000 rpm) with a table centrifuge.

  In the following Examples, the above-described initialization solution was also used as an adsorption buffer for preparation (dilution) of antibody sample solutions of various concentrations and a control sample (protein concentration 0 mg / mL).

(Production Example 1)
In the following examples, a spin column (FIG. 1) using a monolith disk on which a protein A or protein G variant capable of binding about 0.5 mg of IgG antibody as a binding capacity was immobilized was used. This spin column is manufactured by the method described in Japanese Utility Model Registration No. 3149787 (actual application No. 2008-8179).

  The protein A variant immobilized on the spin column carrier is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. This protein A variant is immobilized on the carrier by binding to the amino group of the silica monolith carrier with an amide bond at one carboxy terminus. The protein G variant immobilized on the spin column carrier is a protein having the amino acid sequence represented by SEQ ID NO: 2. This protein G variant is immobilized on a carrier by being bonded to the amino group of the silica monolith carrier by an amide bond at one position of the carboxy terminal. These protein A mutants and protein G mutants have high IgG adsorption ability.

  Specifically, this spin column was manufactured as follows. First, 0.90 g of polyethylene oxide (product number 85,645-2 made by Aldrich) and 0.90 g of urea, which are water-soluble polymers, are dissolved in 10 g of 0.01 N acetic acid aqueous solution, and 4 ml of tetramethoxysilane is stirred into this solution. In addition, a hydrolysis reaction was performed. After stirring for several minutes, the obtained clear solution was poured into a glass tube having an inner diameter of 6 millimeters and kept in a constant temperature bath at 40 ° C., and solidified after about 30 minutes.

  The solidified sample was further aged for several hours and kept at 140 ° C. for 1 hour under sealed conditions. After this treatment, the gel was dried at 40 ° C. for 3 days and heated to 800 ° C. at a temperature increase rate of 100 ° C./h to obtain a rod-shaped inorganic porous carrier having a diameter of 4.5 mm.

  In the obtained inorganic porous carrier, it was confirmed that through-holes having a central pore diameter of about 3 μm (= 3000 nm) were present in a structure entangled in a three-dimensional network (three-dimensional network). And it was confirmed by nitrogen adsorption measurement that many pores having a diameter of about 60 nm exist on the inner wall of the through hole. By cutting a monolith-type (integral type that is a continuous porous body) silica gel (also referred to as monolith silica or monolith-type silica) obtained as such a rod-like inorganic porous carrier into a thickness of 1.5 mm, a disc shape is obtained. An inorganic porous carrier (monolith disc) was obtained.

  A protein A mutant or protein G mutant was immobilized on the disc-shaped inorganic porous carrier by the following method. First, a disk-shaped inorganic porous carrier is immersed in a solution obtained by diluting aminopropyltriethoxysilane with a toluene solvent to a concentration of 20%, and heated and refluxed for 6 hours to cause a reaction. An amino group was introduced on the surface of Next, the amino group-modified disc-shaped inorganic porous carrier is immersed in a solution in which 20 mg of protein A mutant or protein G mutant is dissolved in 5 ml of 0.5M boric acid buffer, and reacted at room temperature for 20 hours. As a result, the protein A mutant or protein G mutant was bound to the surface of the disc-shaped inorganic porous carrier.

  Next, as shown in FIG. 1, a disc-shaped inorganic porous carrier 1 to which a protein A mutant or protein G mutant is bound is placed inside a centrifugal chip 3 having a capacity of 500 μL using a spacer 2 made of silicon rubber. Installed. The silicon rubber spacer 2 had an outer diameter of 8 mm, an inner diameter of 4.3 mm, and a thickness of 1.5 mm. The pore size of the inorganic porous carrier was about 3 μm for continuous pores and 60 nm for mesopores. The chip obtained as described above (FIG. 1) is a spin column using a monolith disc carrier on which a protein A mutant or protein G mutant is immobilized.

  A human IgG solution was added as an antibody sample solution to this spin column, and centrifuged for 30 seconds with a table centrifuge, and the liquid (outflow fraction) that passed through the monolith disk was collected. Subsequently, a washing solution was added and the mixture was centrifuged for 30 seconds with a desktop centrifuge, and the liquid (outflow fraction) that passed through the monolith disk was collected. A human IgG solution diluted with an adsorption buffer so as to be 1 mg / mL was used. The amounts of the antibody sample solution and the washing solution used are 500 μL, respectively. Based on the difference between the amount of IgG in the antibody sample solution and the amount of IgG in the effluent obtained by washing, the IgG binding capacity to the carrier of this spin column was calculated as about 0.5 mg.

Example 1 Measurement Using Purified IgG Protein As an antibody sample solution, rituximab (trade name Rituxan) sold as a monoclonal antibody was used, and various concentrations of monoclonal antibody solutions (5 types; 0.31 mg / mL, 0) .61 mg / mL, 0.92 mg / mL, 1.25 mg / mL, 1.48 mg / mL). An initialization solution (0.1 M sodium phosphate buffer (pH 7.2)) was used as a control sample (protein concentration 0 mg / mL). Further, 0.4 mL of the initialization solution was prepared in advance through the protein A mutant-immobilized spin column prepared in Production Example 1 prepared for the number of antibody sample solutions to be tested (initialization step). Each 0.4 mL of antibody solution is passed through the spin column (adsorption step), followed by 0.4 mL of washing solution three times (washing step), and then 0.4 mL of the regeneration solution is passed. Liquid (regeneration process step). And the density | concentration of the protein (monoclonal antibody rituximab) eluted in the regeneration solution (regeneration effluent) which flowed out from the column was measured by the bicinchoninic acid (BCA) method. An outline of the above procedure is shown in FIG. The protein concentration of each antibody sample solution prepared in advance was also measured by the BCA method.

  As a result, as shown in FIG. 3, the concentration of the applied monoclonal antibody is between the amount (concentration) of the monoclonal antibody applied to the spin column and the amount (concentration) of the monoclonal antibody recovered in the regeneration solution. When it was less than 1 mg / mL, it was shown that there was a proportional relationship of slope 1. That is, under conditions where an antibody sample solution of 1 mg / mL or less is applied at 0.4 mL or less (total amount of monoclonal antibody is 0.4 mg or less), the measurement of the amount of protein in the regenerated effluent enables highly quantitative monoclonal antibody measurement. Was possible. Similar results were obtained when the monoclonal antibody drugs bevacizumab (trade name Avastin) (FIG. 4) and trastuzumab (trade name Herceptin) (FIG. 5) were used, and an antibody sample solution of 1 mg / mL or less was applied to the spin column. When applying, adsorption of the antibody to the column was observed at a concentration (amount) approximate to the antibody concentration (antibody amount) in the antibody sample solution.

(Example 2) Measurement using human polyclonal antibody As an antibody sample solution, an antibody solution (0 IgG various human serum Technical grade, Sigma) sold as a human IgG protein (0) .35 mg / mL, 0.67 mg / mL, 1.08 mg / mL, 1.35 mg / mL, 1.70 mg / mL). A spin column prepared according to Production Example 1 and having a protein A variant immobilized on a carrier and a spin column having a protein G variant immobilized on a carrier were used.

  The amount of antibody bound to the spin column was measured in the same procedure as in Example 1. A spin column was prepared in the same manner as in Example 1 using an initialization solution. Apply the prepared antibody solution to the spin column after initialization, pass through the solution, then wash the column, regenerate, and recover the protein (human IgG polyclonal antibody) in the regenerated solution (regenerated effluent) that flowed out of the column ) Was measured by the BCA method.

  As a result, the result shown in FIG. 6 was obtained. The measurement using a spin column in which the protein G variant was immobilized on a carrier (results are indicated by black circles) showed that when the applied antibody concentration was less than 1 mg / mL, there was a proportional relationship of slope 1. .

  On the other hand, when a spin column in which a protein A variant was immobilized on a carrier was used (results are indicated by white circles), a proportional relationship with a slope of about 0.9 was shown. It is known that IgG-derived antibody products derived from humans contain 10% or more of IgG3 molecules that have a weak binding force to protein A. It was considered that the antibody recovery rate was slightly decreased.

Example 3 Measurement of Monoclonal Antibody Level in CHO Cell Culture Medium Expressing IgG1 Type Monoclonal Antibody A mixed solution sample as an antibody sample solution is a CHO cell line expressing an anti-IL8 human monoclonal antibody (IgG1 type) The culture supernatant obtained by culturing (ATCC catalog number CRL-12445) in a synthetic medium was directly used as a sample with unknown concentration. In the same manner as in Example 1, according to the procedure shown in FIG. 2, the mixed solution sample is applied and passed through a spin column in which the protein A variant prepared in Production Example 1 is immobilized on a carrier, and the column is washed and regenerated. did. Each effluent from the column (an effluent from three washes and an effluent from the regeneration process) was collected, and the protein concentration in each effluent was measured by the BCA method. The measurement was performed in parallel using two columns. The results are shown in Table 1.

  As shown in Table 1, the results of two measurements performed in the same experimental procedure showed almost equivalent values. In addition, the total amount of protein (concentration) in the sample and the total amount of protein in the effluent obtained in each step (total concentration) are almost equal, and the recovery rate of the protein itself is almost 100%. It was.

  The protein concentration in the culture solution was estimated to be 0.12 ± 0.02 mg / mL by taking the average of the protein concentrations in the regenerated effluent of Measurement 1 and Measurement 2. On the other hand, when the antibody concentration in the CHO cell culture supernatant was measured by an immunoassay using a rabbit antibody against a human antibody, a value of 0.13 ± 0.03 mg / mL was obtained. It was a consistent value. Since the IgG1 type monoclonal antibody, which is the antibody molecule to be measured, is adsorbed on the column and recovered in the regeneration effluent by the regeneration process, this result indicates that the target antibody molecule is recovered in the regeneration effluent by the method of the present invention. In addition, the total amount of protein in the regenerated effluent can be quantified as an approximation of the content of the antibody molecule of interest contained in the antibody sample solution applied to the column. It showed that it can be done.

  The method according to the present invention can be used for measuring the amount of antibody molecule immunoglobulin (eg, IgG) in a solution (such as a mixed solution) simply and with high quantitativeness. This measurement method is suitable for use in a wide range of fields such as antibody drug manufacturing field, antibody development, and manufacturing process analysis (PAT) field.

1. 1. Protein A or protein G binding inorganic porous carrier Spacer 3. Centrifugal tip

SEQ ID NO: 1: Protein A variant SEQ ID NO: 2: Protein G variant

Claims (7)

  The antibody sample solution is passed through a column having a monolithic silica carrier on which antibody-binding protein is immobilized, and then the column is washed and regenerated, and then the amount of protein recovered in the effluent by the regeneration is measured. A method for measuring the amount of immunoglobulin in a solution.   The method of claim 1, wherein the column is a spin column.   The method according to claim 1 or 2, wherein the antibody binding protein is an IgG binding protein and the immunoglobulin is IgG.   The method according to claim 3, wherein the antibody-binding protein is protein A or protein G or a variant thereof.   The method according to claim 4, wherein the antibody-binding protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2.   The method according to any one of claims 1 to 5, wherein the antibody sample solution is a mixed solution containing two or more kinds of proteins.   The method according to any one of claims 1 to 6, wherein the antibody sample solution has a total protein concentration of 1 mg / mL or less.

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