JP7540346B2 - How to analyze intact antibodies - Google Patents

Description

The present invention relates to a method for analyzing an intact antibody. In particular, the present invention relates to a method for analyzing an intact antibody by combining chromatography and mass spectrometry.

In recent years, pharmaceuticals containing antibodies (antibody drugs) have been used to treat cancer, immune diseases, and the like. Antibodies used in antibody drugs are produced by culturing cells capable of expressing the antibody (e.g., Chinese hamster ovary cells, etc.) obtained by genetic engineering techniques, and then purifying them to a high degree using column chromatography or other methods. In recent years, it has been discovered that antibody drugs become aggregates of various molecules through modifications such as oxidation, reduction, isomerization, and glycosylation, raising concerns about the impact on their efficacy and safety. In particular, it has been reported that differences in the glycan structure bound to antibodies have a significant impact on the activity, kinetics, and safety of antibody drugs, and detailed analysis of the glycan structure is necessary (Non-Patent Document 1).

Methods for analyzing the glycan structure of antibodies used in antibody drugs include LC-MS analysis involving the excision of glycans (Patent Document 1 and Patent Document 2), and a method for directly analyzing intact antibodies by LC-MS without excision of glycans (Patent Document 3). However, these analytical methods involve very complicated operations and require a great deal of time.

Chromatographic analysis is a simpler method for analyzing the molecular structure of antibodies. Specifically, gel filtration chromatography can be used to separate antibodies based on their molecular weight, allowing for the separation and quantification of aggregates and degradation products. Ion exchange chromatography can also be used to separate antibody molecules based on the differences in their electric charges. However, these chromatographic analyses cannot distinguish minute structural changes in antibody molecules, such as their glycan structures, and the analytical results obtained are limited.

Analysis using affinity chromatography allows separation based on the affinity between affinity ligands and antibodies, and may therefore be able to observe minute structural changes. For example, affinity chromatography using Fc-binding proteins as ligands allows separation of antibodies based on differences in glycan structures, making it possible to analyse differences in the glycan structures bound to antibodies (Patent Documents 4 and 5). However, it is difficult to determine the detailed content and composition of glycan structures using this method alone.

In Patent Documents 4 and 5, detailed content and composition of glycan structures are analyzed by combining affinity chromatography using the aforementioned Fc-binding protein as a ligand with mass spectrometry. However, the methods disclosed in these patent documents involve complicated procedures such as glycan excision during mass spectrometry, and are time-consuming.

JP 2015-034712 A JP 2016-099304 A JP 2015-121539 A JP 2016-169197 A JP 2018-197224 A

CHROMATOGRAPHHY, 34(2), 83-88 (2013)

The objective of the present invention is to provide a method for analyzing antibodies contained in a sample that combines chromatography and mass spectrometry, and that can directly, easily, and quickly analyze intact antibodies contained in a sample.

As a result of intensive research conducted by the inventors to solve the above problems, they have completed the present invention by using affinity chromatography as the chromatography method and optimizing the components of the elution buffer in said chromatography.

That is, the present invention includes the following aspects [1] to [9].

[1]
A method for eluting an intact antibody adsorbed to an adsorbent with an elution buffer, comprising:
A method, wherein said elution buffer contains only volatile components.

[2]
A method for analyzing an intact antibody, comprising the step of subjecting an elution buffer containing the intact antibody eluted by the method according to [1] to mass spectrometry.

[3]
The method according to [1] or [2], wherein the adsorbent is a carrier having an Fc-binding protein immobilized thereon.

[4]
The method according to [3], wherein the intact antibody is an intact IgG comprising a human Fc region, and the Fc binding protein is human FcγRIIIa.

[5]
Human FcγRIIIa,
(1) A polypeptide comprising at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, or (2) a polypeptide comprising at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, further comprising one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions among the amino acid residues, and having IgG binding activity.
The method according to [4].

[6]
The method according to any one of [1] to [5], wherein the elution buffer is an aqueous ammonium acetate solution having a pH of 2.5 to 6.5.

[7]
A method for analyzing an intact antibody, comprising the step of directly subjecting a buffer solution containing the intact antibody eluted by the method according to [1] to a mass spectrometer.

[8]
A method for analyzing an intact antibody, comprising the step of adding 40 to 60% acetonitrile as a make-up solvent for directly subjecting the intact antibody eluted by the method according to [7] to mass spectrometry.

[9]
[8] An antibody analysis method in which chromatograms of spectra of antibodies containing each glycan structure are collected over time, and differences in antibody separation due to differences in the glycan structures bound to the antibodies are clarified.

The present invention is described in detail below.

The present invention is a method for analyzing intact antibodies by adsorbing intact antibodies contained in a sample to a chromatographic adsorbent, eluting the adsorbed intact antibodies using an elution buffer, and then subjecting a fraction containing the eluted intact antibodies to mass spectrometry. The method is characterized in that an affinity chromatographic adsorbent is used as the chromatographic adsorbent, and a buffer containing only volatile salts is used as the elution buffer.

In the present invention, an intact antibody refers to an antibody that contains a light chain variable region (VL) and a heavy chain variable region (VH), as well as a complete light chain constant region (CL) and a complete heavy chain constant region (CH), and that has N-linked glycans bound to two sites in the Fc region of a full-length antibody that is bound via at least one non-covalent interaction, thereby retaining a pair of glycan structures.

The affinity chromatography adsorbent used in the present invention for adsorbing intact antibodies is a carrier on which a substance capable of specifically binding to the intact antibody is immobilized, and the intact antibody can be specifically adsorbed based on the affinity between the intact antibody and the adsorbent. Examples of substances capable of specifically binding to intact antibodies include substances capable of specifically binding to the Fc region, Fab region, and glycans of antibodies. Among these, Fc-binding proteins, which are substances capable of specifically binding to the Fc region, are preferred.

Examples of Fc-binding proteins include Protein A and Protein G derived from the genus Staphylococcus, Fc receptors, and partial regions of the Fc receptors that have Fc-binding ability. Among these, when the intact antibody is an intact IgG containing a human Fc region, it is preferable to use human FcγRIIIa, one of the human Fc receptors, as the Fc-binding protein, since the intact antibody is separated by affinity chromatography based on differences in glycan structure, which makes it easy to analyze the detailed content and composition of the glycan structure bound to the intact antibody by subsequent mass spectrometry.

In the present specification, intact IgG containing a human Fc region is sufficient if it has at least a human-derived Fc region that retains a glycan structure as a partial structure, and examples of such antibodies include chimeric antibodies, humanized antibodies, and human antibodies, which are generally used as antibody pharmaceuticals, as well as their amino acid substitution products. In addition, it also includes artificially structurally modified antibodies, such as bispecific antibodies, antibody-drug conjugates (ADCs), and fusion proteins of an antibody Fc region with another protein.

A preferred embodiment of human FcγRIIIa is
(1) A polypeptide comprising at least the extracellular region of native human FcγRIIIa (amino acid residues from glycine at position 17 to methionine at position 192 of the amino acid sequence set forth in SEQ ID NO:1), and (2) a polypeptide comprising at least the extracellular region of native human FcγRIIIa, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions among the amino acid residues constituting the extracellular region, and having antibody binding activity.
Examples include:

In the above (2), "one or several" means, for example, 1 to 30, 1 to 20, 1 to 10, or 1 to 5, although this varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of the amino acid residue.
As a further preferred embodiment of the above (2),
Fc-binding proteins disclosed in JP2015-086216A;
Fc-binding proteins disclosed in JP2016-169197A;
Fc binding proteins disclosed in JP2017-118871A;
Fc-binding proteins disclosed in JP2018-197224A;
Fc binding proteins as disclosed in WO2019/083048;
Examples include:

In the present invention, the carrier for immobilizing a substance capable of specifically binding to an intact antibody may be any substance that is insoluble in the solution or solvent used for adsorption/elution of the antibody and has a functional group (e.g., a hydroxyl group) for immobilizing the substance by covalent bond. The carrier may be derived from an inorganic substance such as zirconia, zeolite, silica, or coated silica, or may be derived from a natural organic polymeric substance such as cellulose, agarose, or dextran, or may be derived from a synthetic organic polymeric substance such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, or vinyl polymer.

When the functional group on the carrier surface is a hydroxy group, an activating agent can be used to form from the hydroxy group an activated group capable of covalently bonding with a substance capable of specifically binding to an intact antibody. Specific examples of such activating agents include epichlorohydrin (which forms an epoxy group as the activated group), 1,4-butanediol diglycidyl ether (which forms an epoxy group as the activated group), tresyl chloride (which forms a tresyl group as the activated group), and vinyl bromide (which forms a vinyl group as the activated group).

In addition, the hydroxyl group may be converted to an amino group or a carboxyl group, and then activated by the action of an activator. Specific examples of such activators include N-succinimidyl 3-maleimidopropionate (which forms a maleimide group as the activating group), 1,1'-carbonyldiimidazole (which forms a carbonylimidazole group as the activating group), and halogenated acetic acid (which forms a halogenated acetyl group as the activating group).

In the present invention, intact antibodies contained in a sample adsorbed to an affinity chromatography adsorbent are eluted with a buffer solution containing only volatile components. As a result of this operation, the fraction containing the eluted antibodies contains only volatiles as a solvent, and the fraction is directly subjected to mass spectrometry. In other words, while recovery of antibodies from the obtained fraction, cleavage of glycans from the recovered antibodies, purification of the cleaved glycans, and fluorescent labeling and purification for mass spectrometry are normally required prior to mass spectrometry, the fraction can be subjected to mass spectrometry without undergoing these steps.

Examples of volatile components include formic acid, acetic acid, carbonic acid, bicarbonate, pyridine, trimethylamine, triethylamine, ammonia, N-methylmorpholine, ethanolamine, and salts thereof. Among them, ammonium acetate, which is an acetate salt, is preferred as the volatile component of the present invention. The pH of the elution buffer may be appropriately determined based on the affinity between the intact antibody and a substance capable of specifically binding to the antibody, and when ammonium acetate is used as the volatile component, the pH is preferably in the range of pH 2.5 to pH 6.5. The concentration of the volatile component contained in the elution buffer may be appropriately determined based on the pKa of the component, within the range of 1 mM to 1000 mM, preferably 10 mM to 500 mM, and more preferably 20 mM to 200 mM.

In the present invention, an organic solvent may be added as an auxiliary solvent when the intact antibody contained in the sample adsorbed to the affinity chromatography adsorbent is eluted with a buffer containing only volatile components and then the sample is subjected to a mass spectrometer. The organic solvent is not particularly limited as long as it is a solvent normally used in mass spectrometry, and examples of the organic solvent include methanol, ethanol, 2-propanol, and acetonitrile, but preferably methanol and acetonitrile, and more preferably acetonitrile. The ratio of the organic solvent added can be changed as appropriate within a range that does not affect the mass spectrometry, but for example, the organic solvent may be added to the elution solution at a ratio of 1 to 99 v/v%, preferably 20 to 80 v/v%, and more preferably 40 to 60 v/v%.

The present invention is a method for analyzing intact antibodies by adsorbing intact antibodies contained in a sample to a chromatographic adsorbent, eluting the adsorbed intact antibodies using an elution buffer, and then subjecting a fraction containing the eluted intact antibodies to mass spectrometry. The method is characterized in that an affinity chromatographic adsorbent is used as the chromatographic adsorbent, and a buffer containing only volatile salts is used as the elution buffer.

The present invention allows fractions containing eluted intact antibodies to be directly introduced into a mass spectrometer, so that, for example, the glycan structures bound to the antibodies can be directly analyzed simply, quickly, and with high resolution. Therefore, manufacturing process management and quality control of antibody pharmaceuticals can be carried out more simply, quickly, and accurately.

Separation pattern (chromatogram) of the antibody obtained in Example 2 Separation pattern (chromatogram) of the antibody obtained in Example 3 Separation pattern (chromatogram) of the antibody obtained in Example 4 Mass spectrogram of the antibody obtained in Example 5 A spectrogram obtained by deconvoluting the mass spectrogram of the antibody obtained in Example 5. Mass spectrogram of the antibody obtained in Example 6 A spectrogram obtained by deconvoluting the mass spectrogram of the antibody obtained in Example 6. List of glycan structure names and corresponding glycan structures Chromatograms of antibodies bound to each glycan structure extracted from the spectrogram obtained in Example 7

The present invention will be described in detail below using examples, but the present invention is not limited to these examples.

Example 1 Preparation of Fc-binding protein immobilized carrier 2 mL of hydrophilic vinyl polymer for separation agent (manufactured by Tosoh: packing material for liquid chromatography) was activated with iodoacetyl groups on the surface, and then 4 mg of Fc-binding protein FcR9_F_Cys (SEQ ID NO: 2) disclosed in JP-A-2018-197224 was reacted to obtain an FcR9_F immobilized gel. In addition, in FcR9_F_Cys (SEQ ID NO: 2), the first methionine (Met) to the 22nd alanine (Ala) are PelB signal peptides, and the 24th glycine (Gly) to the 199th glutamine (Gln) are Fc-binding protein FcR9_F (amino acid residues from the 17th to the 192nd of SEQ ID NO: 1, except that Val27Glu [this notation means that the 27th valine in SEQ ID NO: 1 is replaced with glutamic acid]. the same below], Phe29Ile, Tyr35Asn, Gln48Arg, Phe75Leu, Asn92Ser, Val117Glu, Glu121Gly, Phe171Ser, and Val176Phe have been substituted; JP 2018-197224 A), and the sequence from the 200th glycine (Gly) to the 207th glycine (Gly) is a cysteine tag sequence.

Example 2 Separation of antibodies using a column packed with an Fc-binding protein-immobilized carrier (part 1)
(1) 1.2 mL of the FcR9_F immobilized gel prepared in Example 1 was packed into a φ4.6 mm×75 mm stainless steel column to prepare an FcR9_F column.

(2) The FcR9_F column prepared in (1) was connected to a high-performance liquid chromatography device (manufactured by Tosoh Corporation) and equilibrated with 50 mM ammonium acetate buffer (pH 6.0) (hereinafter also referred to as equilibration buffer) at a flow rate of 1.0 mL/min.

(3) 25 μL of intact antibody (Rituxan, Zenyaku Kogyo Co., Ltd., mouse-human chimeric antibody) diluted to 1.0 mg/mL with equilibration buffer was added at a flow rate of 1.0 mL/min.

(4) After washing with the equilibration buffer for 2 minutes at a flow rate of 1.0 mL/min, the adsorbed intact antibody was eluted with a pH gradient of 50 mM ammonium acetate buffer (pH 3.0) (a gradient in which 50 mM ammonium acetate buffer (pH 3.0) reached 100% in 18 minutes).

The separation pattern of the obtained intact antibody is shown in Figure 1.

Example 3 Separation of antibodies using a column packed with an Fc-binding protein-immobilized carrier (part 2)
The intact antibody was separated in the same manner as in Example 2, except that the equilibration buffer used in Examples 2(2) and 2(3) was 50 mM ammonium acetate buffer (pH 5.0).

The separation pattern of the obtained intact antibody is shown in Figure 2.

Example 4 Separation of antibodies using a column packed with an Fc-binding protein-immobilized carrier (part 3) Intact antibodies were separated in the same manner as in Example 2, except that the equilibration buffer used in Examples 2 (2) and (3) was 50 mM citrate buffer (pH 6.5), and elution in Example 2 (4) was performed with a pH gradient of 50 mM citrate buffer (pH 4.5) (a gradient in which 50 mM citrate buffer (pH 4.5) becomes 100% in 18 minutes).

The separation pattern of the obtained intact antibody is shown in Figure 3.

From the results of Examples 2 to 4, it was confirmed that even when a buffer containing only a volatile component (ammonium acetate) was used as the elution buffer (Examples 2 and 3), antibody separation could be performed to the same extent as with a conventional buffer containing a non-volatile component (citric acid) (Example 4). Furthermore, since the fractions containing the eluted intact antibodies obtained in Examples 2 and 3 contain only volatile components as a solvent, the fractions can be directly subjected to mass spectrometry.

Example 5 Mass Analysis of Intact Antibody Using the volatile buffer used in Example 2, analysis of the intact antibody was carried out using a mass spectrometer as follows.
Measurement conditions: Apparatus: Waters Xevo G2 XS Q TOF
Infusion rate: 0.1 mL/min
Ionization: ESI+
Measurement mode: Sensitivity MS Mode
Capillary voltage: 4 kV
Cone voltage: 165V
Source offset: 50V
Source temperature: 150° C.
Desolvation temperature: 600°C
Cone gas: 50L/Hr
Desolvation gas: 1200 L/Hr
Data acquisition: m/z 500 4000, 0.5 sec
The results of mass spectrum analysis of the intact antibody measured using a liquid chromatography device are shown in Figure 4. In addition, from the spectrum in Figure 4, a deconvolution operation was performed using analysis software UNIFI attached to a mass spectrometer manufactured by Waters Corporation, to obtain the spectrum in Figure 5.

Example 6
The same procedure as in Example 5 was carried out, except that acetonitrile was added to the volatile buffer solution at a ratio of 50 v/v % as a make-up solvent, which is an auxiliary solvent when introduced into the mass spectrometer, in order to improve the sensitivity of the analysis by the mass spectrometer. The mass spectrum analysis results obtained are shown in Figure 6. Similarly, the spectrum in Figure 7 was obtained by a deconvolution operation. By identifying the molecular weights of the peaks in Figures 5 and 7, it is possible to assign the compositions of the two N-linked glycan structures bound to the intact antibody, and it is clear that the glycan structures contained in the intact antibody can be directly and simply analyzed.

The types of glycan structures (G0F, G1F, G2F) in Figures 5 and 7 correspond to the glycan structures shown in Figure 8.

Example 7
9 shows chromatograms extracted over time from mass spectra due to differences in N-linked glycan structures bound to intact antibodies obtained in Example 6. From this figure, it can be seen that in antibody separation using a column packed with an Fc-binding protein immobilized carrier, the combination G0F/G0F not containing galactose at the end is eluted first, followed by the combination G0F/G1F in which galactose is bound to one end, then the combination G1F/G1F in which galactose is bound to both ends, then the combination G1F/G2F, and finally the combination G2F/G2F. Similarly, it can be seen that antibodies containing the combination G0/G0F not containing fucose and the combination G0/G0 are eluted in this order. These make it possible to clarify the differences in separation patterns due to differences in combinations of glycan structures.

The present invention makes it possible to more accurately and quickly evaluate the separation and analysis of glycan structures of antibody drugs and antibody-related drugs based on differences in their glycan structures, which was previously difficult. The present invention improves the quality of glycan structures, which has been an issue when manufacturing antibody drugs, i.e., reduces the variation in the proportion and type of glycan structures between lots.

Claims (8)

A method for eluting an intact antibody adsorbed to an adsorbent with an elution buffer, comprising:
said elution buffer containing only volatile components ;
A method wherein the adsorbent is a carrier having an Fc binding protein immobilized thereon. A method for analyzing an intact antibody, comprising a step of subjecting an elution buffer containing an intact antibody eluted by the method of claim 1 to mass spectrometry. The method of claim 1 or 2 , wherein the intact antibody is an intact IgG comprising a human Fc region and the Fc binding protein is human FcγRIIIa. Human FcγRIIIa,
(1) A polypeptide comprising at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, or (2) a polypeptide comprising at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that among the amino acid residues , there is further one or more of substitutions, deletions, insertions and additions of 1 to 10 amino acid residues, and having IgG binding activity.
The method according to claim 3 . 5. The method according to claim 1, wherein the elution buffer is an aqueous solution of ammonium acetate at a pH of from 2.5 to 6.5. A method for analyzing an intact antibody, comprising the step of directly subjecting a buffer solution containing an intact antibody eluted by the method of claim 1 to a mass spectrometer. A method for analyzing an intact antibody, comprising the step of adding acetonitrile to a buffer solution containing the intact antibody eluted by the method of claim 1 so as to have a concentration of 40 to 60 v/v % as a make-up solvent for direct supply to a mass spectrometer, and then subjecting the solution to mass spectrometry. The intact antibody analysis method according to claim 7 , further comprising collecting chromatograms of spectra of antibodies containing each glycan structure over time, and clarifying differences in antibody separation due to differences in the glycan structures bound to the antibodies.