JP2022055933A - ANALYTIC METHOD AND ANALYZER OF HUMAN IgG4 ANTIBODY - Google Patents

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

The present invention relates to a liquid chromatography analysis method and an analyzer for human IgG4 antibody.

In recent years, drugs containing antibodies (antibody drugs) have been used for the treatment of cancer and immune diseases. Antibodies The antibodies used in pharmaceuticals are obtained by culturing cells capable of expressing the antibody (for example, Chinese hamster ovary (CHO) cells) obtained by genetic engineering techniques, and then purifying them to high purity using column chromatography or the like. Manufactured. Regarding the design of antibody molecules, the selection of target molecules is of utmost importance, but it is also important to select appropriate subclasses as the basic structure. The majority of antibody drugs currently in practical use are designed with molecules having the same structure as human antibodies in consideration of immunogenicity, and examples thereof include chimeric antibodies, humanized antibodies, and fully human antibodies. The subclass is selected from the four subclasses of human IgG antibody (IgG1, IgG2, IgG3 and IgG4) in consideration of the strength, stability, productivity and the like of the effector function. Even with appropriate molecular design, antibody molecules have been found to diversify by undergoing modifications such as oxidation, reduction, isomerization, and glycosylation, and there are concerns about their impact on drug efficacy and safety. ing. In particular, the structure of the sugar chain bound to the Fc region of the antibody is diverse and is an important factor greatly related to the efficacy and side effects of the antibody drug, but the method for controlling the structure is limited (Non-Patent Document 1). reference).

Due to the development of an analytical column using a human type 3 Fc receptor (FcγRIIIA) as a ligand (see Non-Patent Document 2), Fc receptors such as IgG1 antibody and IgG3 antibody can be used by using the pH gradient elution method. For antibodies with strong binding to, detailed analysis based on the sugar chain structure and effector function has become possible (see Non-Patent Document 3).

However, IgG2 antibody and IgG4 antibody are known to have extremely low affinity for FcγRIIIA, and in particular, IgG4 has a binding affinity that is 5 to 50 times lower than that of IgG1 and IgG3 (Non-Patent Document 4). See), difficult to analyze.

Against this background, the change in effector function due to the difference in sugar chain structure has not been discussed for IgG4 antibody, but the sugar chain structure may potentially affect the quality of pharmaceutical products. Since the sex cannot be denied, the development of an effective analysis method was required.

Patrick Hossler et al. , Glycobiology, 19, 9, 936-949 (2009) Tosoh Research and Technical Report, Vol. 63 (2019), 77-82 Masato Kiyoshi et al. , Scientific reports, 8: 3955 (2018) Pierre Bruhns et al. , BLOOD, 113, 16, 3716-3725 (2009)

An object of the present invention is to provide a liquid chromatography analysis method and an analyzer that can obtain highly reproducible and highly accurate results for a human IgG4 antibody, which has conventionally been difficult to analyze variants having different affinities for Fc receptors. To do.

In order to solve the above-mentioned problems, the present inventors have reached the present invention as a result of repeated diligent studies.

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 on which an Fc receptor is immobilized.
The step of equilibrating the column by passing a buffer solution of pH 5 to pH 7 and the process of equilibrating the column.
The step of injecting the solution containing the antibody into the equilibrated column, and
In an analytical method comprising a gradient elution step of elution of the antibody by gradually increasing the proportion of the buffer solution of pH 3 to pH 5 while passing a buffer solution of pH 5 to pH 7.
A method for analyzing a human IgG4 antibody, wherein the buffer solution having a pH of 3 to 5 has a higher buffer concentration than the buffer solution having a pH of 5 to 7.

A UV absorption detector or a fluorescence detector can be used for detecting the antibody, and more preferably, the absorbance of ultraviolet light of 280 nm can be used.

Examples of the Fc receptor include human FcγRI, human FcγRII, human FcγRIII and complement protein C1q, and variants in which the amino acids constituting each thereof are partially substituted may be used.

The insoluble carrier may be a substance that is insoluble in the solution or solvent used for adsorbing / elution of the antibody and has a functional group (for example, a hydroxy group) for immobilizing the Fc acceptor by a covalent bond. , A carrier derived from an inorganic substance such as zirconia, zeolite, silica, or film silica, a carrier derived from a natural organic polymer substance such as cellulose, agarose, or dextran, or a polyacrylic. The carrier may be derived from a synthetic organic polymer substance such as acid, polystyrene, polyacrylamide, polymethacrylicamide, polymethacrylate, and vinyl polymer.

In order to equilibrate the column, it is sufficient to pass the liquid 1 times or more with respect to the column volume based on the column volume, and it is more preferable to pass the liquid 3 times or more.

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.

The buffers include citric acid buffer, succinic acid buffer, tartrate buffer, GTA buffer, borate buffer, Tris buffer, sodium glycine buffer, phosphate buffer, acetate buffer, and glycine hydrochloride buffer. , Good buffers such as MES buffer and HEEPS buffer can be used. Inorganic salts and surfactants may be added for the purpose of adjusting affinity and suppressing non-specific adsorption.

The solution containing the antibody can be defined as a solution containing the antibody at a concentration of 10 μg / L or more. The amount of antibody added to the column may be as long as it can be detected by a detector of a chromatographic device, and is preferably 0.1 μg or more and 1 g or less. Further, as an additive of this solution, a buffering agent, salts, a preservative, and a surfactant may be contained.

Antibodies to be analyzed include human antibodies, humanized antibodies, chimeric antibodies and their amino acid substitutions, and have an Fc region of human IgG4 as a basic structure. Regardless of the origin of the antibody, gamma globulin produced from human blood may be used, or recombinant protein obtained by animal cell culture or microbial culture may be used. Further, an Fc fusion protein and a fragment of an Fc site obtained by degrading an antibody can also be used as an antibody in a broad sense.

Since the column has a temperature dependence in its affinity, it is desirable to keep it at a constant temperature by using a column oven or the like. The measured temperature may 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, it is preferably 4 ° C. or higher and 50 ° C. or lower, and more preferably 15 ° C. or higher and 35 ° C. or lower.

According to the present invention, it is possible to perform chromatographic separation of a human IgG4 antibody with good reproducibility and high accuracy, and perform component analysis based on the binding property to the Fc receptor.

6 is a chromatograph obtained by separating a human IgG4 antibody using 20 mM citrate buffer and 50 mM citrate buffer as eluents. 6 is a chromatograph obtained by separating human IgG1 and human IgG4 antibodies using 50 mM citrate buffer as an eluent. It is a chromatograph obtained by separating a human IgG4 antibody using 25 mM citrate buffer as an eluent. It is a chromatograph obtained by separating a human IgG4 antibody using 20 mM citrate buffer as an eluent.

In the method for analyzing a human IgG4 antibody according to the present invention, a citrate buffer solution having a pH of 5 to 7 (hereinafter referred to as eluent A) is added to a column packed with an insoluble carrier on which an Fc receptor is immobilized to equilibrate the column. Gradually the ratio of the citrate buffer solution (hereinafter, eluent B) of pH 3 to pH 5 while passing the eluent A and the step of adding the solution containing the antibody to the equilibrated column. It comprises a gradient elution step of increasing and eluting the antibody.

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

Examples are shown below, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible for details. Furthermore, the present invention is not limited to the above-described embodiments, and embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention.

The eluent and the like used in the examples were prepared as follows.
(1) Eluents A and B
Take 9.606 g of citric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dissolve it in 900 mL of Milli-Q water, and then adjust the pH to the specified pH with an aqueous solution of sodium hydroxide (pH 6.5 for eluent A and pH 4. for eluent B. 5) After that, the volume was increased to 1000 mL with a measuring cylinder. The solution was filtered through a 0.2 μm filter and degassed by ultrasonic irradiation while reducing the pressure to prepare a 50 mM citric acid buffer. 20 mM and 25 mM citric acid buffers were also prepared by the same procedure.
(2) Antibody sample Two commercially available antibodies (human IgG1 (κ): manufactured by Sigma-Aldrich, human IgG4 (κ): manufactured by Sigma-Aldrich) were diluted with eluent A so as to have a final concentration of 0.2 g / L. Prepared.
(3) Analytical column As an Fc receptor-immobilized column, a commercially available TSKgel FcR-IIIA-NPR (manufactured by Tosoh Corporation) was used.

(Example 1)
Analysis with 20 mM-50 mM citric acid buffer 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 used in an HPLC device (Tosoh). Made). The column oven temperature was set to 15 ° C. and 25 ° C., the flow rate was set to 1.0 mL / min, and the detector was set to measure the absorbance at 280 nm. The gradient conditions are as follows: after passing the eluent A100% -eluent B0% for 2 minutes, and then switching linearly from eluent A100% -eluent B0% to eluent A0% -eluent B100% in 18 minutes. It was set to pass the eluent A 0% -eluent B 100% for 5 minutes, and then eluate A 100% -eluent B 0% for 5 minutes (hereinafter referred to as the standard gradient method). FIG. 1 shows the results obtained by equilibrating the column with eluent A, adding the antibody to the column with a loading of 50 μL (10 μg), and then eluting by the standard gradient method to obtain a chromatogram. A chromatogram with good retention time and peak separation could be obtained. In addition, good separation made it possible to clearly discriminate minor peaks, confirming improvements in analysis reproducibility and accuracy.

(Comparative Example 1)
Analysis under standard analytical conditions for human IgG1 Column, eluent A (citric acid concentration 50 mM, pH 6.5), eluent B (citric acid concentration 50 mM, pH 4.5) and antibody sample (human IgG1 and human IgG4) HPLC. Installed in the device (manufactured by Tosoh). The column oven temperature was set to 25 ° C., the antibody loading was set to 50 μL, the flow rate was set to 1.0 mL / min, and the detector was set to measure the absorbance at 280 nm. The results of analysis by the standard gradient method described in Example 1 are shown in FIG. Although good analysis results were obtained for human IgG1, it was difficult to analyze human IgG4 because the peaks were close to each other due to its weak retention.

(Comparative Example 2)
Analysis with 25 mM citric acid buffer 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 used in an HPLC device (manufactured by Toso). Installed in. The column oven temperature was set to 15 ° C. and 25 ° C., the antibody loading was set to 50 μL, the flow rate was set to 1.0 mL / min, and the detector was set to measure the absorbance at 280 nm. The results of analysis by the standard gradient method described in Example 1 are shown in FIG. It can be confirmed that the retention time is extended as compared with Comparative Example 1, but the separation of some peaks was incomplete.

(Comparative Example 3)
Analysis with 20 mM citric acid buffer 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) are HPLC device (manufactured by Toso). Installed in. The column oven temperature was set to 15 ° C. and 25 ° C., the antibody loading was set to 50 μL, the flow rate was set to 1.0 mL / min, and the detector was set to measure the absorbance at 280 nm. The results of analysis by the standard gradient method described in Example 1 are shown in FIG. It can be confirmed that the holding time is extended as compared with Comparative Example 1, but the peak width is large and it is difficult to detect the minor peak.

Claims (7)

A method for analyzing a human IgG4 antibody using a column packed with an insoluble carrier on which an Fc receptor is immobilized.
The step of equilibrating the column by passing a buffer solution of pH 5 to pH 7 and the process of equilibrating the column.
The step of injecting the solution containing the antibody into the equilibrated column, and
In an analytical method comprising a gradient elution step of elution of the antibody by gradually increasing the proportion of the buffer solution of pH 3 to pH 5 while passing a buffer solution of pH 5 to pH 7.
The buffer solution of pH 3 to pH 5 is characterized by having a higher buffer concentration than the buffer solution of pH 5 to pH 7.
Analytical method for human IgG4 antibody. The analytical method according to claim 1, wherein FcγRI, FcγRII, FcγRIII and an amino acid variant thereof are used as the Fc receptor. The analysis method according to claim 1 or 2, wherein the human IgG4 antibody is a human antibody, a humanized antibody, a chimeric antibody, an Fc fusion protein, or a low molecular weight antibody. The analytical method according to any one of claims 1 to 3, wherein the buffer concentration is in the range of 10 mM to 1000 mM. The analytical method according to any one of claims 1 to 4, wherein the buffer is citric acid. The analysis method according to any one of claims 1 to 5, wherein the measurement is carried out in the range of 4 ° C to 50 ° C. The analyzer for human IgG4 antibody according to any one of claims 1 to 6.

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