Method and apparatus for analyzing human IgG4 antibodies

Abstract

To provide an analytic method of an affinity variant to an Fc receptor in which a result can be obtained with good reproducibility and highly accurately for a human IgG4 antibody whose analysis has conventionally been difficult.SOLUTION: A human IgG4 antibody is analyzed by an analytic method including: a process of passing buffer solution from pH5 to pH7 into a column filled with insoluble carrier in which Fc receptors are immobilized to bring the column into equilibrium; a process of injecting solution containing antibodies into the column which has been brought into equilibrium; and a gradient eluting process of gradually increasing the ratio of buffer solution from pH3 to pH5 while passing the buffer solution from pH5 to pH7 to elute the antibody, in which the buffer solution from pH3 to pH5 has higher buffer agent concentration than the buffer solution from pH5 to pH7.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and an analyzer for liquid chromatography analysis of a human IgG4 antibody. [Background technology]

[0002] In recent years, drugs containing antibodies (antibody drugs) have been used to treat cancer, immune diseases, and other conditions. Antibodies used in antibody drugs are produced by culturing cells capable of expressing the antibody (e.g., Chinese hamster ovary (CHO) cells, etc.) obtained by genetic engineering techniques, and then purifying them to a high degree using column chromatography, etc. In designing antibody molecules, the selection of the target molecule is of utmost importance, but it is also important to select an appropriate subclass as the basic structure. The majority of antibody drugs currently in practical use are designed to have the same structure as human antibodies, taking immunogenicity into consideration, and examples include chimeric antibodies, humanized antibodies, and fully human antibodies. In addition, subclasses are selected from the four subclasses of human IgG antibodies (IgG1, IgG2, IgG3, and IgG4) taking into consideration the strength of effector functions, stability, productivity, and other factors. Even with appropriate molecular design, it has been found that antibody molecules become diversified by modifications such as oxidation, reduction, isomerization, and glycosylation, and there are concerns about their impact on efficacy and safety. In particular, the structures of glycans bound to the Fc region of antibodies are diverse and are an important factor that greatly influences the efficacy and side effects of antibody drugs, but there are limited methods for controlling these structures (see non-patent document 1).

[0003] The development of an analytical column using human type 3 Fc receptor (FcγRIIIA) as a ligand (see Non-Patent Document 2) has made it possible to use pH gradient elution to perform detailed analysis based on the glycan structure and effector function of antibodies that strongly bind to Fc receptors, such as IgG1 and IgG3 antibodies (see Non-Patent Document 3).

[0004] However, IgG2 and IgG4 antibodies are known to have extremely low affinity for FcγRIIIA. In particular, IgG4 has a binding affinity that is 5 to 50 times lower than that of IgG1 and IgG3 (see Non-Patent Document 4), making its analysis difficult.

[0005] Under these circumstances, there has been no discussion of changes in effector function due to differences in glycan structure with respect to IgG4 antibodies. However, the possibility that glycan structure potentially affects the quality of pharmaceuticals cannot be denied, so there was a demand for the development of an effective analytical method. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Patrick Hossler et al., Glycobiology, 19, 9, 936-949(2009) [Non-Patent Document 2] Tosoh Research and Technology Report, Vol. 63 (2019), 77-82 [Non-Patent Document 3] Masato Kiyoshi et al., Scientific reports, 8:3955(2018) [Non-Patent Document 4] Pierre Bruhns et al., BLOOD, 113, 16, 3716-3725(2009) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a liquid chromatography analysis method and analyzer that can obtain highly reproducible and accurate results for human IgG4 antibodies, for which analysis of variants with different affinity for Fc receptors has traditionally been difficult. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors conducted extensive research and arrived at the present invention.

[0009] That is, one aspect of the present invention is a method for analyzing a human IgG4 antibody using a column packed with an insoluble carrier to which an Fc receptor has been immobilized, comprising the steps of: Equilibrating the column by passing a buffer solution having a pH of 5 to 7; injecting a solution containing an antibody into the equilibrated column; A gradient elution step of eluting the antibody by gradually increasing the ratio of a buffer solution having a pH of 3 to 5 while passing a buffer solution having a pH of 5 to 7, The method for analyzing a human IgG4 antibody is characterized in that a buffer solution of pH 3 to pH 5 has a higher buffer concentration than a buffer solution of pH 5 to pH 7.

[0010] The antibody can be detected using a UV absorption detector or a fluorescence detector, and more preferably, the absorbance of ultraviolet light at 280 nm can be used.

[0011] Fc receptors include human FcγRI, human FcγRII, human FcγRIII and complement protein C1q, and mutants in which the amino acids constituting each of these are partially substituted may also be used.

[0012] The insoluble carrier may be any substance that is insoluble in the solution or solvent used for antibody adsorption / elution and has a functional group (e.g., a hydroxyl group) for covalently immobilizing the Fc receptor, and may be a carrier derived from an inorganic substance such as zirconia, zeolite, silica, or coated silica; a carrier derived from a natural organic polymeric substance such as cellulose, agarose, or dextran; or a carrier derived from a synthetic organic polymeric substance such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, or vinyl polymer.

[0013] To equilibrate the column, it is sufficient to pass a liquid at a volume equal to or greater than the column volume, and it is more preferable to pass a liquid at a volume equal to or greater than the column volume.

[0014] The eluent preferably has a buffer concentration of 10 mM or more and 1000 mM or less, and more preferably 20 mM or more and 300 mM or less.

[0015] Examples of the buffer solution that can be used include citrate buffer, succinate buffer, tartrate buffer, GTA buffer, borate buffer, Tris buffer, sodium glycine buffer, phosphate buffer, acetate buffer, glycine hydrochloride buffer, Good's buffer such as MES buffer and HEPES buffer, etc. Inorganic salts and surfactants may be added to adjust affinity and suppress nonspecific adsorption.

[0016] A solution containing an antibody can be defined as a solution containing an antibody at a concentration of 10 μg / L or more. The amount of antibody added to the column is preferably 0.1 μg or more and 1 g or less, as long as it can be detected by the detector of the chromatography device. In addition, the solution may contain additives such as buffers, salts, preservatives, and surfactants.

[0017] The antibodies to be analyzed include human antibodies, humanized antibodies, chimeric antibodies, and their amino acid substitution products, and have the Fc region of human IgG4 as a basic structure. The origin of the antibody is not important, and it may be gamma globulin produced from human blood, or a recombinant protein obtained by animal cell culture or microbial culture. In addition, Fc fusion proteins and fragments of the Fc portion obtained by decomposing an antibody can also be used as antibodies in the broad sense.

[0018] Since the affinity of the column is temperature-dependent, it is desirable to keep the column at a constant temperature using a column oven or the like. The measurement temperature should be a temperature at which the inside of the column does not freeze and a temperature at which the Fc-binding protein immobilized as a ligand does not denature. Specifically, the temperature is preferably 4°C or higher and 50°C or lower, and more preferably 15°C or higher and 35°C or lower. Effect of the Invention

[0019] According to the present invention, it is possible to perform chromatographic separation of human IgG4 antibodies with good reproducibility and high accuracy, and to perform component analysis based on the binding affinity to Fc receptors. [Brief description of the drawings]

[0020] [Figure 1] This is a chromatograph obtained by separating human IgG4 antibody using 20 mM citrate buffer and 50 mM citrate buffer as eluents. [Diagram 2] This is a chromatograph obtained by separating human IgG1 and human IgG4 antibodies using 50 mM citrate buffer as an eluent. [Diagram 3] This is a chromatograph obtained by separating human IgG4 antibody using 25 mM citrate buffer as an eluent. [Figure 4] This is a chromatograph obtained by separating human IgG4 antibody using 20 mM citrate buffer as an eluent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The method of the present invention for analyzing human IgG4 antibodies comprises the steps of: adding a citrate buffer solution of pH 5 to pH 7 (hereinafter, eluent A) to a column packed with an insoluble carrier on which an Fc receptor is immobilized to equilibrate the column; adding a solution containing an antibody to the equilibrated column; and a gradient elution step of gradually increasing the proportion of a citrate buffer solution of pH 3 to pH 5 (hereinafter, eluent B) while passing the eluent A through the column to elute the antibody.

[0022] First, eluent A is added to a column packed with an insoluble carrier on which an Fc receptor is immobilized to equilibrate the column. EXAMPLES

[0023] The following examples are provided to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various aspects are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention.

[0024] The eluents and the like used in the examples were prepared as follows. (1) Eluents A and B 9.606 g of citric acid (manufactured by Fujifilm Wako Pure Chemical Industries) was taken and dissolved in 900 mL of Milli-Q water, and then the pH was adjusted to the specified level with sodium hydroxide solution (eluent A was pH 6.5, eluent B was pH 4.5), and then the solution was made up to 1000 mL in a graduated cylinder. The solution was filtered through a 0.2 μm filter and degassed by ultrasonic irradiation under reduced pressure to prepare a 50 mM citrate buffer. 20 mM and 25 mM citrate buffers were also prepared using the same procedure. (2) Antibody sample Two types of commercially available antibodies (human IgG1(κ): manufactured by Sigma-Aldrich, human IgG4(κ): manufactured by Sigma-Aldrich) were diluted with eluent A to a final concentration of 0.2 g / L. (3) Analytical column As the Fc receptor immobilized column, a commercially available TSKgel FcR-IIIA-NPR (manufactured by Tosoh Corporation) was used.

[0025] Example 1 Analysis using 20mM-50mM citrate buffer The column, eluent A (citric acid concentration 20 mM, pH 6.5), eluent B (citric acid concentration 50 mM, pH 4.5), and antibody sample (human IgG4) were placed in an HPLC device (manufactured by Tosoh). The column oven temperature was set to 15°C and 25°C, the flow rate was 1.0 mL / min, and the detector was set to measure absorbance at 280 nm. The gradient conditions were set to pass eluent A 100%-eluent B 0% for 2 minutes, then linearly switch from eluent A 100%-eluent B 0% to eluent A 0%-eluent B 100% over 18 minutes, pass eluent A 0%-eluent B 100% for 5 minutes, and then pass eluent A 100%-eluent B 0% for 5 minutes (hereinafter referred to as the standard gradient method). After equilibrating the column with eluent A, the antibody was loaded onto the column at a loading amount of 50 μL (10 μg), and then eluted using the standard gradient method to obtain a chromatogram, which is shown in Figure 1. A chromatogram with good retention times and peak separation was obtained. Furthermore, the good separation made it possible to clearly distinguish minor peaks, confirming the improvement in the reproducibility and accuracy of the analysis.

[0026] Comparative Example 1 Analysis under standard analytical conditions for human IgG1 The column, eluent A (citric acid concentration 50 mM, pH 6.5), eluent B (citric acid concentration 50 mM, pH 4.5), and antibody samples (human IgG1 and human IgG4) were placed in an HPLC system (manufactured by Tosoh). The column oven temperature was set to 25°C, the antibody load amount to 50 μL, the flow rate to 1.0 mL / min, and the detector to measure absorbance at 280 nm. The results of analysis using the standard gradient method described in Example 1 are shown in Figure 2. Although good analytical results were obtained for human IgG1, analysis of human IgG4 was difficult due to weak retention and close peaks.

[0027] Comparative Example 2 Analysis with 25 mM citrate buffer The column, eluent A (citric acid concentration 25 mM, pH 6.5), eluent B (citric acid concentration 25 mM, pH 4.5) and antibody sample (human IgG4) were placed in an HPLC device (manufactured by Tosoh). The column oven temperatures were set to 15°C and 25°C, the antibody load amount to 50 μL, the flow rate to 1.0 mL / min, and the detector to measure absorbance at 280 nm. The results of analysis using the standard gradient method described in Example 1 are shown in Figure 3. Compared to Comparative Example 1, it can be confirmed that the retention time was extended, but separation of some peaks was incomplete.

[0028] Comparative Example 3 Analysis with 20 mM citrate buffer The column, eluent A (citric acid concentration 20 mM, pH 6.5), eluent B (citric acid concentration 20 mM, pH 4.5) and antibody sample (human IgG4) were placed in an HPLC device (manufactured by Tosoh). The column oven temperatures were set to 15°C and 25°C, the antibody load amount to 50 μL, the flow rate to 1.0 mL / min, and the detector to measure absorbance at 280 nm. The results of analysis using the standard gradient method described in Example 1 are shown in Figure 4. Compared to Comparative Example 1, it was confirmed that the retention time was extended, but the peak width was larger and it was difficult to detect minor peaks.

Claims

1. 1. A method for analyzing a human IgG4 antibody using a column packed with an insoluble carrier having an Fc receptor immobilized thereon, comprising: Equilibrating the column by passing a buffer solution having a pH of 5 to 7; injecting a solution containing an antibody into the equilibrated column; A gradient elution step of eluting the antibody by gradually increasing the proportion of a buffer solution having a pH of 3 to 5 while passing a buffer solution having a pH of 5 to 7, Buffers from pH 3 to pH 5 have a higher buffering agent concentration than buffers from pH 5 to pH 7; The concentration of the buffer is in the range of 10 mM to 1000 mM; And the buffer is citric acid. Method for analyzing human IgG4 antibodies.

2. The method according to claim 1, wherein Fc receptors are FcγRI, FcγRII, FcγRIII, or amino acid variants thereof.

3. 3. The method according to claim 1, wherein the human IgG4 antibody is a human antibody, a humanized antibody, a chimeric antibody, an Fc fusion protein, or a minibody.

4. The analytical method according to any one of claims 1 to 3, characterized in that the measurement is carried out at a temperature in the range of 4°C to 50°C.

Images