Antibody population uniformly containing antibodies having asymmetric glycans and method for producing same

Abstract

Provided are: a method for producing an antibody population uniformly including antibodies having a left-right asymmetric sugar chain; and a uniform antibody population of antibodies having a left-right asymmetric sugar chain, which is obtained by said method.　This method for producing an antibody population, which uniformly includes antibodies in which N-type complex sugar chains bound to asparagines (Asn) at the 297th positions in CH domains of Fc regions of two heavy chains on the left and right sides of an antibody are sugar chains having different structures, comprises: (i) a step for cleaving, by endo-β-N-acetylglucosaminidase (ENGase), sugar chains bound to two heavy chains on the left and right sides of the antibody in an antibody composition, and purifying and isolating antibodies in which sugar chains of both heavy chains are cleaved; (ii) a step for mixing a population of the antibodies in which sugar chains of both heavy chains are cleaved and which are obtained in step (i), any oxazolined sugar chain X or a sugar derivative which generates oxazoline and of which the sugar chain is a sugar chain X, and ENGase which is modified so as to suppress sugar chain cleavage activity and improve sugar chain transfer activity, to produce antibodies in which the sugar chain X is bound to only one heavy chain among two heavy chains on the left and right sides, and purifying and isolating the antibodies; and (iii) a step for mixing the antibodies in which the sugar chain X is bound to only one heavy chain among two heavy chains on the left and right sides and which are obtained in step (ii), an oxazolined sugar chain Y having a structure different from that of the sugar chain X or a sugar derivative which generates oxazoline and of which the sugar chain is a sugar chain Y, and ENGase which is modified so as to suppress sugar chain cleavage activity and improve sugar chain transfer activity, to produce antibodies in which the sugar chain Y is bound to the other heavy chain among the two heavy chains on the left and right sides and the sugar chains bound to the two heavy chains on the left and right sides are sugar chains having different structures, and purifying and isolating the antibodies.

Description

[Technical field]

[0001] The present invention relates to an antibody population of antibodies in which the left and right sugar chain structures are different but the sugar chain structures are uniform, and to a method for producing the same. [Background technology]

[0002] The glycan of an antibody is heterogeneous, and is a mixture of slightly different glycan structures. It is known that the glycan of an antibody affects antibody functions such as effector functions such as ADCC (antibody dependent cellular cytotoxicity) activity and immunogenicity. In order to produce antibodies with higher functions, attention has been paid to modifying the glycan structure. It is currently impossible to isolate an antibody with a uniform glycan structure from antibodies with different glycan structures produced in CHO (Chinese Hamster Ovary) cells, because the antibodies are an aggregate of glycan structures with different fine structures and have very similar physicochemical properties. The glycan was modified and homogenized using endo-β-N-acetylglucosaminidase (ENGase) / modified ENGase, and an antibody with a glycan of a symmetrical uniform structure was produced. That is, an antibody with a uniform glycan structure was produced by cleaving a glycan of a heterogeneous structure with ENGase, and then adding a glycan to a synthesized or isolated glycan and modified ENGase (Patent Document 1). With the previous method, only antibodies with symmetrical glycan structures could be synthesized. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. WO2013 / 120066 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for preparing an antibody population homogeneously containing antibodies having asymmetric sugar chains, and a homogeneous antibody population of antibodies having asymmetric sugar chains obtained by the method. [Means for solving the problem]

[0005] The present inventors discovered that antibodies with different and uniform left and right glycan structures can be obtained by appropriately adjusting the amount of glycan-linked oxazoline X (oxazolinated glycan X) in the glycan-linking reaction, isolating an antibody with only one glycan linked using a column carrying FcγRIIIa, and then reacting the antibody with structurally different glycan-linked oxazoline Y (oxazolinated glycan Y), thereby completing the present invention. No technology for producing an antibody population with different and uniform left and right glycan structures has been known until now.

[0006] That is, the present invention is as follows. [1] An antibody population that homogeneously contains antibodies in which the N-linked complex glycan attached to asparagine (Asn) at position 297 in the CH domain of the Fc region of the two heavy chains on the left and right of the antibody has a glycan structure that is different from that of the other heavy chains. [2] An antibody group classified as [1], in which 90% or more of the antibodies have N-linked complex glycans that are different from each other at asparagine (Asn) 297 in the CH domain of the Fc region of the two heavy chains on the left and right of the antibody. [3] An antibody group according to [1] or [2], in which fucose is bound to the N-acetylglucosamine (GlcNAc) at the reducing end of the N-linked complex glycan that is bound to asparagine (Asn) at position 297 located in the CH domain of the antibody's Fc region. [4] A method for producing an antibody population that uniformly contains antibodies in which the N-linked complex glycans bound to asparagine (Asn) at position 297 in the CH domain of the Fc region of the two left and right heavy chains of the antibody have glycans with different structures, comprising: (i) cleaving sugar chains attached to the two heavy chains, left and right, of an antibody in the antibody composition with endo-β-N-acetylglucosaminidase (ENGase), and purifying and isolating the antibody from which the sugar chains of both heavy chains have been cleaved; (ii) mixing the antibody population obtained in step (i) in which the glycans of both heavy chains have been cleaved, an oxazolinated glycan X or a glycan derivative that generates an oxazoline and in which the glycan of the glycan derivative is glycan X, and ENGase modified to suppress the glycan cleavage activity and improve the glycosylation activity, to produce an antibody in which glycan X is bound to only one of the two heavy chains, and purifying and isolating the antibody; (iii) a step of mixing the antibody obtained in step (ii) having glycan X bound to only one of the two heavy chains, an oxazolinated glycan Y having a structure different from that of glycan X, or a glycan derivative which generates oxazoline and has glycan Y as the glycan of the glycan derivative, and ENGase modified to suppress the glycosylation activity and improve the glycosylation activity, to produce an antibody having glycan Y bound to the other heavy chain and having glycan structures different from each other bound to the two heavy chains, and purifying and isolating the antibody; The method includes: [5] The method of [4], in which the antibody is purified and isolated by affinity chromatography using a carrier carrying an Fcγ receptor as a ligand. [6] The method of [4] or [5], wherein the ENGase is EndoS. [7] Any of the methods according to [4] to [6], wherein the ENGase modified to suppress glycosylation activity and retain glycosylation activity is selected from the group consisting of EndoS D233Q, Endo S2 D184M, Endo S2 D184Q, EndoS D233Q / Q303L, D233Q / A303L / E350Q, and Endo M N175Q. [8] Any of the methods [4] to [7], wherein the molecular weight of the glycan X to be bound in step (ii) is greater than the molecular weight of the glycan Y to be bound in step (iii). [9] Any of the methods [4] to [8] for producing an antibody population containing 90% or more of antibodies in which the N-linked complex glycan bound to asparagine (Asn) at position 297 in the CH domain of the Fc region of the two left and right heavy chains of the antibody are different from each other.

[10] Any of the methods [4] to [9] for producing an antibody population in which fucose is bound to N-acetylglucosamine (GlcNAc) at the reducing end of an N-linked complex glycan that is bound to asparagine (Asn) at position 297 located in the CH domain of the Fc region of the antibody.

[11] A method of fractionating antibodies in an antibody population into antibodies with no glycosylation, antibodies with one glycosylation on one heavy chain, and antibodies with two glycosylation on two heavy chains by affinity chromatography using a carrier that supports Fcγ receptors as ligands.

[12] The method of

[11] , in which an antibody population is treated with endo-β-N-acetylglucosaminidase (ENGase), and then the antibodies in the antibody population are fractionated using affinity chromatography using a carrier carrying an Fcγ receptor as a ligand into antibodies without glycosylation, antibodies with one glycosylation on one heavy chain, and antibodies with glycosylation on two heavy chains. This specification includes the disclosures of Japanese Patent Application No. 2020-002968, which is the priority basis of this application. Effect of the Invention

[0007] By cleaving the glycan with a heterogeneous structure with ENGase and then adding a glycan using a synthetic or isolated sugar oxazoline and modified ENGase, an antibody population that uniformly contains antibodies with asymmetric glycan structures can be produced. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of analysis of commercially available Herceptin on Tosoh TSKgel® FcR-IIIA-NPR. [Diagram 2] This is a diagram showing the glycan structure of commercially available Herceptin. The "heterogeneous portion" in the diagram means that commercially available Herceptin contains antibody molecules with different structures in this portion. [Diagram 3]FIG. 1 shows the results of analysis of deglycosylated antibody: Herceptin [GlcNAc-Fucose / GlcNAc-Fucose] on Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. [Figure 4] FIG. 1 shows deglycosylated antibody: Herceptin [GlcNAc-Fucose / GlcNAc-Fucose]. [Diagram 5] FIG. 1 shows the results of Protein A purification of a deglycosylation reaction solution. [Figure 6] FIG. 1 shows the structure of SG-Oxazoline used in glycosyltransferase reaction. [Figure 7] FIG. 1 shows the results of Protein A purification of the glycosylation reaction solution using SG-Ox. [Figure 8] FIG. 1 shows the results of analysis of a glycosyltransfer reaction solution using SG-Ox on Tosoh TSKgel® FcR-IIIA-NPR. [Figure 9] FIG. 1 shows the antibody of Fr. 1: Herceptin [GlcNAc-Fucose / GlcNAc-Fucose]. [Figure 10] FIG. 1 shows the results of intact mass analysis of Fr. 1; Herceptin [GlcNAc-Fucose / GlcNAc-Fucose]. [Figure 11] FIG. 1 shows the antibody in Fr. 2: Herceptin [SG-F / GlcNAc-Fucose]. [Figure 12] FIG. 1 shows the results of intact mass analysis of Fr. 2; Herceptin [SG-F / GlcNAc-Fucose]. [Figure 13] FIG. 3 shows the antibody in Fr. 3: Herceptin [SG-F / SG-F]. [Figure 14] FIG. 1 shows the results of intact mass analysis of antibody Fr. 3: Herceptin [SG-F / SG-F]. [Figure 15] FIG. 1 shows the structure of G2-Oxazoline used in the glycosyltransferase reaction. [Figure 16] FIG. 1 shows the results of analysis of a glycosyltransfer reaction solution using G2-Ox on Tosoh TSKgel® FcR-IIIA-NPR. [Figure 17] FIG. 1 shows the structure of the isolated antibody: Herceptin [G2-F / GlcNAc-Fucose]. [Figure 18] FIG. 1 shows the results of intact mass analysis of the separated antibody: Herceptin [G2-F / GlcNAc-Fucose]. [Figure 19] FIG. 1 shows the structure of G0-Oxazoline used in the glycosyltransferase reaction. [Figure 20] FIG. 1 shows the results of analysis of a glycosyltransfer reaction solution using G0-Ox on Tosoh TSKgel® FcR-IIIA-NPR. [Figure 21] FIG. 1 shows the structure of the isolated antibody: Herceptin [G0-F / GlcNAc-Fucose]. [Figure 22] FIG. 1 shows the results of intact mass analysis of the isolated antibody: Herceptin [G0-F / GlcNAc-Fucose]. [Figure 23] FIG. 1 shows the structure of SG-Oxazoline used in glycosyltransferase reaction. [Figure 24] FIG. 1 shows the results of analysis of a glycosyltransfer reaction solution using M3-Ox on Tosoh TSKgel® FcR-IIIA-NPR. [Diagram 25] FIG. 1 shows the structure of the isolated antibody: Herceptin [M3-F / GlcNAc-Fucose]. [Figure 26] FIG. 1 shows the results of intact mass analysis of the isolated antibody: Herceptin [M3-F / GlcNAc-Fucose]. [Figure 27] FIG. 1 shows the results of Protein A purification of the glycosylation reaction solution using G2-Ox on SG single-chain antibody. [Figure 28]FIG. 13 shows the results of analysis of the G2 transfer reaction solution to SG single-chain antibody using Tosoh TSKgel® FcR-IIIA-NPR. [Figure 29] FIG. 1 shows the structure of the isolated antibody: Herceptin [SG-F / G2-F]. [Diagram 30] FIG. 1 shows the results of intact mass analysis of the isolated antibody: Herceptin [SG-F / G2-F]. [Diagram 31] FIG. 13 shows the results of analysis of the SG transfer reaction solution to G0 single-chain antibody using Tosoh TSKgel® FcR-IIIA-NPR. [Diagram 32] FIG. 1 shows the structure of the isolated antibody: Herceptin [SG-F / G0-F]. [Diagram 33] FIG. 1 shows the results of intact mass analysis of the isolated antibody: Herceptin [SG-F / G0-F]. [Diagram 34] FIG. 13 shows the results of analysis of the G2 transfer reaction solution to M3 single-chain antibody using Tosoh TSKgel® FcR-IIIA-NPR. [Diagram 35] FIG. 1 shows the structure of the isolated antibody: Herceptin [G2-F / M3-F]. [Diagram 36] FIG. 1 shows the results of intact mass analysis of the isolated antibody: Herceptin [G2-F / M3-F]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below.

[0010] Currently, monoclonal antibodies produced in CHO (Chinese Hamster Ovary) cells are used as antibody drugs. The protein structure consisting of the heavy and light chains of the monoclonal antibodies produced in this way is uniform, but the sugar chain structure is heterogeneous among antibodies. Here, the sugar chain bound to the antibody refers to an N-type complex sugar chain bound to the 297th asparagine (Asn) located in the CH domain of the Fc region of the antibody. In nature, there are antibodies with sugar chains having different structures, that is, asymmetric structures, bound to the two heavy chains of the antibody. However, when viewed as an antibody population, there are also antibodies with symmetric sugar chains, in which the sugar chains bound to the two heavy chains of the antibody have the same structure. In addition, the type of sugar chain varies depending on the antibody, even for antibodies with asymmetric sugar chains. Therefore, there has been no antibody population that uniformly contains antibodies with different structures of sugar chains bound to the two heavy chains of the antibody. On the other hand, by replacing the glycans of antibodies in an antibody population containing antibodies with heterogeneous glycans with other glycans using endo-β-N-acetylglucosaminidase and its modified forms, it was possible to obtain an antibody population consisting of antibodies with symmetrical and uniform glycans.

[0011] The present invention is a method for producing an antibody population that uniformly contains antibodies with asymmetric glycans, which was not possible with conventional methods. The present invention also relates to an antibody population that uniformly contains antibodies with asymmetric glycans, and the population is an isolated population. An antibody population refers to an antibody group that contains a large number of antibody molecules. The antibody population refers to a population that contains antibodies with asymmetric glycans at a certain ratio, or a population that contains 100% of antibodies with asymmetric glycans, that is, a population consisting only of antibodies with asymmetric glycans. An antibody with asymmetric glycans refers to an antibody in which the glycans that bind to asparagine (Asn) at position 297 in the CH domain of the Fc region of the two heavy chains on the left and right of the antibody have different structures. Alternatively, the antibody refers to an antibody in which the glycans that bind to each monomer portion of a dimeric antibody are different.

[0012] An antibody population that uniformly contains antibodies with asymmetric glycan chains refers to a population in which the types of glycan bound to the CH domains of the two heavy chains on the left and right sides of the antibody are uniform in each antibody molecule in the antibody population. In other words, an antibody population that uniformly contains antibodies with asymmetric glycan chains refers to an antibody population in which the glycan bound to each of the two heavy chains is different in each antibody in the antibody population, but there is no difference between antibodies and the glycan bound to each antibody is uniform.

[0013] Here, homogeneity means that the antibody population contains 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 98% or more, more preferably 99% or more, and particularly preferably 100% of antibody molecules having the same asymmetric glycan bound to the left and right heavy chains. The antibody population can also be called an antibody composition. When called an antibody composition, the composition may contain water, a buffer component, a stabilizing component, and the like. The antibody population may be a liquid antibody solution, or a lyophilized or frozen product. Furthermore, an antibody population that uniformly contains antibodies having asymmetric glycans is isolated by chromatography, and therefore can also be called an antibody fraction that uniformly contains antibodies having asymmetric glycans.

[0014] In the method of the present invention, the sugar chains of the antibody population can be replaced with different sugar chains on each of the two heavy chains, left and right, using modified endo-β-N-acetylglucosaminidase (ENGase).

[0015] Specifically, for example, sugar chains are replaced by the following method. The following is an example in which sugar chain X and sugar chain Y, which is a sugar chain different from sugar chain X, are replaced on the two left and right heavy chains of an antibody.

[0016] In the following method, the antibody itself can be purified using Protein A or the like.

[0017] (1) Cleavage of the glycans originally attached to the antibody Endo-β-N-acetylglucosaminidase (ENGase) is used to cleave the glycan originally bound to the antibody. In this process, both glycans bound to the two heavy chains, left and right, of the antibody are cleaved. ENGase is an enzyme that hydrolyzes N,N'-diacetylchitobiose present on the reducing end side of the N-linked glycan of glycoproteins such as antibodies, and releases the glycan into an endo-type. ENGase cleaves the glycan, leaving one N-acetylglucosamine (GlcNAc) residue at the reducing end of the N-linked complex glycan bound to asparagine (Asn) 297 located in the CH domain of the Fc region of the antibody. The N-acetylglucosamine at the reducing end may or may not have fucose bound to it. Cleaving the glycan is called deglycosylation. As a result, an antibody is produced that has no glycan bound to it and only GlcNAc bound to it.

[0018] This enzyme reaction may be carried out for 5 to 30 hours, preferably 10 to 25 hours, by mixing the antibody and ENGase at pH 6 to 9, preferably pH 6 to 8, and at 15 to 40° C., preferably 25 to 35° C. The amounts of antibody and ENGase added may be appropriately determined, but for example, 5 to 100 μL of 0.2 to 20 mg / mL ENGase may be added to 0.1 to 10 mL of a 5 to 100 mg / mL antibody solution.

[0019] As the endo-β-N-acetylglucosaminidase (ENGase), EndoS derived from Streptococcus pyogenes is preferred. EndoS is described in Collin, M., Olsen, A. (2001), EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. EMBO J. 20, 3046-3055., and its amino acid sequence is also disclosed.

[0020] Thereafter, the antibody with the cleaved sugar chain may be purified and isolated. Purification may be performed using chromatography. In this case, purification may be performed by affinity chromatography using a carrier such as a column carrying the Fcγ receptor FcγRIIIa as a ligand. The Fcγ receptor is a receptor protein for the Fc site of immunoglobulin, and is a receptor protein that can identify structural changes in the Fc region caused by the N-linked sugar chain of an antibody. An example of an Fcγ receptor is FcγRIIIa. In affinity chromatography using a column carrying an Fcγ receptor, antibodies with a sugar chain structure that does not contain fucose or antibodies with galactose at the end of the sugar chain structure have a higher binding affinity and a slower elution time.

[0021] An example of a column carrying FcγRIIIa is TSKgel (registered trademark) FcR-IIIA-NPR (Tosoh Corporation).

[0022] This process involves cleaving the glycans attached to the two heavy chains, left and right, of an antibody in the antibody composition using endo-β-N-acetylglucosaminidase (ENGase), and purifying and isolating the antibody from which the glycans of both heavy chains have been cleaved.

[0023] (2) Preparation of antibodies with glycans on only one heavy chain Next, the antibody in which both glycans bound to the two heavy chains have been cleaved is mixed with an oxazolinated glycan X or a glycan derivative that generates oxazoline, the glycan of which is glycan X, and the glycan is bound to the antibody using an ENGase modified to suppress glycan cleavage activity and improve glycosyltransferase activity, i.e., an ENGase that has suppressed hydrolysis ability but has glycosyltransferase activity. Examples of glycan derivatives that generate oxazoline include glycopeptides and paranitrophenyl oligosaccharides. Examples of ENGase modified to suppress glycan cleavage activity and improve glycosyltransferase activity include all ENGas that have suppressed glycan cleavage ability and retained glycosyltransferase activity. For example, EndoS with a D233Q mutation (substitution of aspartic acid (D) at position 233 with glutamine (Q)) is exemplified. The mutated antibody is called EndoS D233Q. In addition, ENGase modified to suppress glycosylation activity and improve glycosylation activity includes Endo S2 D184M and Endo S2 D184Q (Li, T. et al., J. Biol. Chem. 291, 16508-16518, (2016)), and Endo M N175Q (Katoh, T. et al., The Journal of Biological Chemistry, 291, 23305-23317). In addition, EndoS D233Q / Q303L, D233Q / A303L / E350Q (International Publication No. WO2017 / 010559) are also included.

[0024] Oxazolination of a sugar chain is carried out by oxazolinating a sugar chain having GlcNAc at the reducing end with an oxazolinating agent. The oxazolinated sugar chain is called an oxazolinated sugar chain. Using the oxazolinated sugar chain X as a substrate, the sugar chain is transferred to a GlcNAc-antibody by the modified ENGase of the present invention, thereby binding the sugar chain X to the antibody. That is, using an oxazoline sugar chain (azidooxazoline sugar chain) as a donor, the sugar chain is bound to the N-acetylglucosamine (GlcNAc) residue of the antibody whose acceptor sugar chain has been cleaved. In this reaction, the oxazoline ring of the donor reacts with the 4-position of the N-acetylglucosamine (GlcNAc) residue of the antibody, forming a chitobiose structure and binding. Examples of oxazolination agents include CDMBI (2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride, Fushimi Pharmaceutical Co., Ltd.) and DMC (2-chloro-1,3-dimethyl-imidazolinium chloride). Oxazolination can be carried out by the method described in J. Org. Chem. 74,5,2210 (2009). Oxazolinated sugar chains can be obtained from Fushimi Pharmaceutical Co., Ltd.

[0025] A sugar derivative that generates oxazoline when the sugar chain is cleaved by modified ENGase can also be used. An oxazolinated sugar chain is generated from a sugar derivative, and in situ, the sugar chain is bound to an N-acetylglucosamine (GlcNAc) residue of an antibody whose sugar chain has been cleaved as an acceptor, using the oxazolinated sugar chain as a donor. A method using a sugar derivative that generates oxazoline is described in Manabe, S. et al., R. Soc. Open Sci. 5, 171521 (2018) and Iwamoto, M. et al., PLoS One 13:e0193534 (2018).

[0026] In both cases, when an oxazolinated sugar chain is used, and when a sugar derivative that generates oxazoline is used, the oxazolinated sugar chain is used as an intermediate, so it can be said that "sugar is transferred using oxazoline as an intermediate."

[0027] This enzyme reaction may be carried out for 5 to 30 hours, preferably 10 to 25 hours, by mixing an antibody in which both the left and right glycans have been cleaved, a glycan derivative which generates an oxazolinated glycan or oxazoline, the glycan of the glycan derivative being glycan X, and modified ENGase at pH 6 to 9, preferably pH 6 to 8, at 15 to 40° C., preferably 25 to 35° C. The amounts of antibody and ENGase added may be appropriately set, but for example, 0.1 to 100 μL of 0.2 to 20 mg / mL modified ENGase may be added to 0.1 to 10 mL of a 5 to 100 mg / mL antibody solution in which both the left and right glycans have been cleaved.

[0028] In this case, an antibody having a sugar chain X on only one of the two left and right heavy chains can be obtained as a main product by adjusting the amount of a sugar derivative that generates an oxazolinated sugar chain X or oxazoline and has a sugar chain X. For example, a sugar derivative that generates an oxazolinated sugar chain X or oxazoline and has a sugar chain X can be added to a concentration of 25 μM to 5.0 mM.

[0029] This reaction can yield both antibodies with no sugar chain attached to either of the two chains, and biantennary sugar chain antibodies with sugar chain X attached to both of the two chains. The elution times are shorter for antibodies with no sugar chain attached, antibodies with sugar chain attached to one heavy chain, and antibodies with sugar chains attached to two heavy chains. In order to produce the antibody population of the present invention having sugar chains with an asymmetric uniform structure, it is necessary to purify and isolate only monoantennary sugar chain antibodies having sugar chain X on only one of the two heavy chains. To purify monoantennary sugar chain antibodies, they may be purified by affinity chromatography using a column carrying the above-mentioned Fcγ receptor FcγRIIIa.

[0030] This step is a step of mixing the antibody population obtained in step (1) above in which the glycans of both heavy chains have been cleaved, an arbitrary oxazolinated glycan X or a glycan derivative that generates an oxazoline and in which the glycan of the glycan derivative is glycan X, and ENGase modified to suppress the glycan cleavage activity and improve the glycosylation activity, to produce an antibody in which glycan X is bound to only one of the two heavy chains on the left and right, and purifying and isolating the antibody.

[0031] (3) Preparation of antibodies with asymmetric glycans After purifying a single-chain glycan antibody having glycan X on only one of the two heavy chains on the left and right sides by the method (2), glycan Y is bound to the other heavy chain to which no glycan is bound. Glycan Y may be bound by the same method as glycan X. That is, the antibody and modified ENGase are mixed at pH 6-9, preferably pH 6-8, and at 15-40°C, preferably 25-35°C, and the enzyme reaction is carried out for 5-30 hours, preferably 10-25 hours. The amounts of antibody and ENGase to be added can be appropriately set, but for example, 5-100 μL of 0.2-20 mg / mL ENGase may be added to 0.1-10 mL of 5-100 mg / mL antibody solution.

[0032] As a result, an antibody having asymmetric sugar chains in which the types of sugar chains bound to the two left and right heavy chains are different can be produced. By purifying an antibody having asymmetric sugar chains in which the types of sugar chains bound to the two left and right heavy chains are different by affinity chromatography using a column carrying FcγRIIIa, an antibody population homogeneously containing antibodies having asymmetric sugar chains in which the types of sugar chains bound to the two left and right heavy chains are different can be isolated and produced.

[0033] This step is a step of mixing the antibody having glycan X bound to only one of the two heavy chains obtained in step (2) above, oxazolinated glycan Y which is structurally different from glycan X, or a glycan derivative which generates oxazoline and whose glycan is glycan Y, and ENGase modified to suppress glycosylation activity and improve glycosylation activity, to produce an antibody having glycan Y bound to the other heavy chain and having glycan structures different from each other bound to the two heavy chains, and purifying and isolating the antibody.

[0034] The sugar chain to be bound to the antibody is appropriately selected depending on the purpose, and examples thereof include high-mannose sugar chains having a structure in which a mannose (Man) oligomer is bound to diacetylchitobiose (GlcNAc-GlcNAc) including N-acetylglucosamine (GlcNAc) at the reducing end, complex sugar chains in which Man and at least one of GlcNAc, galactose (Gal), and sialic acid (Neu5Ac) are bound to diacetylchitobiose, and hybrid sugar chains having a sugar chain structure in which high-mannose and complex types are mixed to diacetylchitobiose. Examples of high-mannose sugar chains include sugar chains called Man3 type, Man5 type, Man6 type, Man8 type, and Man9 type, in which 3, 5, 6, 8, and 9 mannoses are bound, respectively. Complex glycans include, for example, glycans called triple-antennary, quadruple-antennary, Asialo2-antennary, Agalacto2-antennary, bisecting2-antennary, and Sialo2-antennary. There are glycans with various structures depending on the presence or absence of sialic acid, core fucose, and branched chains. Glycans are represented by abbreviations such as SG and M3, glycans with sialic acid (sialyl glycans) are represented as SG, high mannose glycans are represented as M3, M5, etc., glycans without galactose are represented as G0, those with one galactose are represented as G1, and those with two galactose are represented as G2. When fucose is further present, they are represented as M3-F, G0-F, and G2-F. When bisecting N-acetylglucosamine is bound, they are represented as G0B, G0B, etc. with the addition of B. The glycan structure of an antibody (IgG) molecule as a whole can be expressed using the glycans bound to each of the left and right heavy chains, such as [SG-F / SG-F], [SG-F / G2-F], [SG-F / G0-F], and [G2-F / M3-F]. In these examples, [SG-F / SG-F] is an antibody with symmetric glycans, and [SG-F / G2-F], [SG-F / G0-F], and [G2-F / M3-F] are antibodies with asymmetric glycans. In addition, the name of the antibody is added before these notations, such as Herceptin[SG-F / G2-F]. This example is Herceptin, which has asymmetric glycans in which SG-F is bound to one heavy chain and G2-F is bound to the other heavy chain.

[0035] Moreover, an antibody in which both the sugar chains of the left and right heavy chains have been cleaved is represented as [GlcNAc / GlcNAc] or [GlcNAc-Fucose / GlcNAc-Fucose]. The former represents an antibody in which the sugar chains of both the left and right heavy chains have been cleaved, leaving one N-acetylglucosamine (GlcNAc) residue, and the latter represents an antibody in which fucose is bound to the remaining GlcNAc. Moreover, an antibody in which a sugar chain is bound to only one of the left and right heavy chains is represented as [SG-F / GlcNAc-Fucose]. This antibody represents an antibody in which SG-F is bound to only one heavy chain, no sugar chain is bound to the other heavy chain, and GlcNAc bound to fucose remains.

[0036] The sugar chain may contain an azide group, an alkyne group, etc. Sugar chains having these groups can be used in the synthesis of antibody-drug conjugates (ADCs).

[0037] Furthermore, chemically synthesized non-natural sugar chains can also be introduced. For example, non-natural sugar chains include non-human sugar chains that contain galactose at the non-reducing end, such as N-glycolylneuraminic acid (NeuGc) instead of N-acetylneuraminic acid, or galactose α1-3 galactose (Galα1-3Gal). These sugar chains are immunogenic and need to be removed from antibody pharmaceutical compositions, but the method of the present invention allows the synthesis of antibodies having these sugar chains as standard products.

[0038] In (2), it is desirable that the molecular weight of the glycan X that is attached first is smaller than that of the glycan Y that is attached second. If a glycan with a smaller molecular weight is attached first, the glycan attached first is more likely to be cleaved by hydrolysis when the glycan to be attached second is attached.

[0039] The present invention also encompasses a method for fractionating antibodies in an antibody population into non-glycosylated antibodies, antibodies having one glycosylated heavy chain, and antibodies having two glycosylated heavy chains using affinity chromatography with a carrier carrying an Fcγ receptor as a ligand, and the method can be carried out, for example, after treating the antibody population with endo-β-N-acetylglucosaminidase (ENGase) to partially cleave the glycosylation.

[0040] The antibody produced by the method of the present invention is not limited, and may be an antibody having core fucose in which fucose is bound to GlcNAc at the base of the N-glycan, or an antibody not having core fucose. The antibody may be derived from humans or non-human animals including rodents such as mice and rats. The antibody class may be any of IgG, IgM, IgA, IgD, and IgE.

[0041] The method of the present invention makes it possible to produce antibodies with glycans having a uniform, asymmetric structure for use as antibody drugs, antibody-drug conjugates (ADCs), and antibody standards. It is also possible to produce biosimilars and biobetters by modifying the glycans of antibody drugs. It is also possible to obtain antibody standards with different glycan structures.

[0042] Examples of antibody drugs include, but are not limited to, the following antibodies:

[0043] Trastuzumab (Herceptin®), Rituximab (Rituxan®), Mogamulizumab (Potelizio®), Adalimumab, Alilocumab, Alemtuzumab, Ixekizumab, Idarucizumab, Ipilimumab, Infliximab, Ustekinumab, Eculizumab, Evolocumab, Elotuzumab, Ofatumumab, Omalizumab, Canakinumab, Gemtuzumab Ozogamicin, Golimumab, Secukinumab, Cetuximab, Certolizumab Pegol, Denosumab, Tocilizumab, Trastuzumab, Trastuzumab Emtansine, Natalizumab, Nivolumab, Basiliximab, Panitumumab, Palivizumab, Brentuximab Vedotin, brodalumab, bevacizumab, pembrolizumab, pertuzumab, mepolizumab, ranibizumab, ramucirumab, etc. EXAMPLES

[0044] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0045] Preparation of antibodies with asymmetric glycan structures The antibody used was the anti-Her2 antibody Herceptin (registered trademark) (Trastuzumab). Trastuzumab is an antibody drug for breast cancer and stomach cancer. The sugar chain portion was synthesized as follows.

[0046] [Example 1] Preparation of an antibody having only reducing end GlcNAc (Fucose bond): Herceptin [GlcNAc-Fucose / GlcNAc-Fucose] 1-1. Commercially available Herceptin (Chugai Pharmaceutical, 150 and 177 mg for injection) was dissolved in 15 mL of 100 mM phosphate buffer (pH 6.5). The solution was then concentrated to approximately 5 mL using a Sartorius Vivaspin Turbo 15 (50 K) (3,000×g, 4°C). 10 mL of 100 mM phosphate buffer (pH 6.5) was added to the concentrated solution, and the process of concentrating the solution to 5 mL was repeated three times.

[0047] The results of analyzing the commercially available Herceptin using a Tosoh TSKgel® FcR-IIIA-NPR column (analysis condition 1) are shown in Figure 1. An image of the antibody structure at that time is shown in Figure 2.

[0048] 1-2. 10 μL of EndoS (2.0 mg / mL) was added to the Herceptin solution and reacted for 18 hours at 30° C. After the reaction, the disappearance of the raw material was confirmed by HPLC. The analysis results are shown in Figure 3 (analysis condition 1). An image of the structure of the antibody after deglycosylation is shown in Figure 4.

[0049] Analysis conditions 1 Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm ID x 10 cm, 5 m Column temperature: 25℃ Flow rate: 1 ml / min Detection: Ex. 280 nm, Em. 348 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0050] Gradient conditions 1. 0-2 min A 100% 2. 2-45 min A 100-0% 3. 45-55 min A 0% 4. 55-60 min A 0-100% 5. 60-80 min A 100%

[0051] 1-3. The antibody was purified from the reaction solution using a Protein A column. The reaction solution was divided into 10 fractions. The chromatogram during antibody purification is shown in Figure 5. The peak at a retention time of approximately 25 minutes was collected as the antibody.

[0052] Purification conditions Equipment: Shimadzu HPLC system Column: Tosoh ToyoScreen AF-ProteinA HC-650F 1ml Column temperature: 25°C Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 0.1 M phosphate buffer (pH 6.5) Mobile phase B: 0.1 M citrate buffer (pH 3.5)

[0053] Gradient conditions 1. 0-15 min A 100% 2. 15-15.01 min A 100-0% 3. 15.01-25 min A 0% 4. 25-25.01 min A 0-100% 5. 25.01-50 min A 100%

[0054] 1-4. The collected fraction was concentrated by ultrafiltration using a Sartorius Vivaspin Turbo 15 (50 K), and then replaced with 50 mM citrate buffer (pH 6.5) (2,460×g, 4°C).

[0055] 1-5. The protein concentration of the obtained Herceptin solution was measured using NanoDrop (trademark) 2000C (supplied software, mode; Protein A280, Type; IgG measurement) manufactured by Thermo Scientific, and was found to be 165 mg (30 mL, 5.5 mg / mL).

[0056] [Example 2] Preparation of an antibody having only one glycan 2-1. Preparation of Herceptin [SG-F / GlcNAc-Fucose] 2-1-1. Herceptin (20 mg; 2 mL) deglycosylated with ENGase, SG-Oxazoline (Fushimi Pharmaceutical; 1 mM; 1000 μL, FIG. 6, hereafter referred to as SG-Ox), and EndoS D233Q (4.35 μg; 1 μL) were added to 995 μL of 100 mM phosphate buffer (pH 6.5), and the reaction was carried out at 30°C for 12 hours.

[0057] 2-1-2. The antibody was purified from the reaction solution using a Protein A column. The chromatogram obtained during fractionation is shown in Figure 7. The peak at a retention time of approximately 25 minutes (the antibody fraction in Figure 7) was collected as the antibody.

[0058] Purification conditions Equipment: Shimadzu HPLC system Column: Tosoh ToyoScreen AF-ProteinA HC-650F 1ml Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 0.1 M phosphate buffer (pH 6.5) Mobile phase B: 0.1 M citrate buffer (pH 3.5)

[0059] Gradient conditions 1. 0-15 min A 100% 2. 15-15.01 min A 100-0% 3. 15.01-25 min A 0% 4. 25-25.01 min A 0-100% 5. 25.01-50 min A 100%

[0060] 2-1-3. The collected fraction was concentrated by ultrafiltration using a Sartorius Vivaspin Turbo 15 (50 K), and then replaced with 50 mM citrate buffer (pH 6.5) (2,460×g, 4°C).

[0061] 2-1-4. The protein concentration of the obtained Herceptin solution was measured using NanoDrop (trademark) 2000C (supplied software, mode; Protein A280, Type; IgG measurement) and was found to be 19 mg (6 mL, 3.3 mg / mL).

[0062] 2-1-5. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 8. The peaks shown in Figure 8 (Fr. 1, Fr. 2, and Fr. 3 in Figure 8) were each fractionated.

[0063] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0064] Gradient conditions 1. 0-2 min A 100% 2. 2-45 min A100-0% 3. 45-55 min A 0% 4. 55-60 min A 0-100% 5. 60-80 min A 100%

[0065] 2-1-6. The obtained fractions were classified as Fr.1 (2-10 min), Fr.2 (28-37 min), and Fr.3 (48-55 min) in ascending order of retention time, and each was subjected to intact mass analysis by LC-MS / MS.

[0066] Intact Mass Analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 1. [Table 1] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0067] Fr. 1 has a mass of 145,865 and is found to have a structure in which a pair of GlcNAc-Fucose is attached to Asn297 (Figure 9) (Figure 10).

[0068] Fr.2 has a mass of 147,868 and is found to have a structure in which one SG is attached to the GlcNAc-Fucose at Asn297 (Figure 11) (Figure 12).

[0069] Fr.3 has a mass of 149,872 and is found to have a structure in which GlcNAc-Fucose at Asn297 has two SGs attached (Figure 13) (Figure 14).

[0070] In this way, by using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR, it is possible to separate those with only GlcNAc-Fucose glycans, those with only one site, and those with two sites.

[0071] 2-2. Preparation of Herceptin [G2-F / GlcNAc-Fucose] 2-2-1. Herceptin (2 mg; 200 μL) deglycosylated with ENGase, G2-Oxazoline (Fushimi Pharmaceutical; 1 mM; 1000 μL, FIG. 15, hereafter referred to as G2-Ox), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the mixture was reacted at 30° C. for 12 hours. After the reaction, the antibody in the solution was purified in the same manner as in 2-1-2.

[0072] 2-2-2. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 16. The peak at a retention time of approximately 36 minutes (G2F / GlcNAc-Fucose in Figure 16) was fractionated.

[0073] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0074] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0075] 2-2-3. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 2. [Table 2] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0076] The detected mass was 147285, which indicates that the structure (Figure 17) has one G2-F attached to the GlcNAc-Fucose at Asn297 (Figure 18).

[0077] 2-3. Preparation of Herceptin [G0-F / GlcNAc-Fucose] 2-3-1. Herceptin (2 mg; 200 μL) deglycosylated with ENGase, G0-Oxazoline (Fushimi Pharmaceutical; 1 mM; 1000 μL, FIG. 19, hereafter referred to as G0-Ox), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the mixture was reacted at 30° C. for 12 hours. After the reaction, the antibody in the solution was purified in the same manner as in 2-1-2.

[0078] 2-3-2. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 20. The peak at a retention time of about 30 minutes (G0-F / GlcNAc-Fucose in Figure 20) was fractionated.

[0079] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0080] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0081] 2-3-3. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 3. [Table 3] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0082] The detected mass was 146961, which indicates that the structure (Figure 21) has one G0-F attached to the GlcNAc-Fucose at Asn297 (Figure 22).

[0083] 2-4. Preparation of Herceptin [M3-F / GlcNAc-Fucose] 2-4-1. Herceptin (2 mg; 200 μL) deglycosylated with ENGase, M3-Oxazoline (Fushimi Pharmaceutical; 1 mM; 1000 μL, FIG. 23, hereafter referred to as M3-Ox), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the reaction was carried out at 30° C. for 12 hours. After the reaction, the antibody in the solution was purified in the same manner as in 2-1-2.

[0084] 2-4-2. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 24. The peak at a retention time of approximately 26 minutes (M3-F / GlcNAc-Fucose in Figure 24) was fractionated.

[0085] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0086] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0087] 2-4-3. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80 °C Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 4. [Table 4] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0088] The detected mass was 146554, which indicates that the structure (Figure 25) has one M3-F attached to the GlcNAc-Fucose at Asn297 (Figure 26).

[0089] [Example 3] Preparation of an antibody with asymmetric glycan Using Herceptin [SG-F / GlcNAc-Fucose], which has SG attached at only one site, and G2-G2-Ox, which is different from SG, we prepared an antibody with different glycans on the left and right sides of the antibody (asymmetric).

[0090] 3-1. Preparation of Herceptin [SG-F / G2-F] 3-1-1. Herceptin [SG-F / GlcNAc-Fucose] (2 mg; 200 μL), which had a single SG attached by ENGase, G2-Ox (Fushimi Pharmaceutical; 1 mM; 100 μL), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the reaction was carried out at 30°C for 12 hours.

[0091] 3-1-2. The antibody was purified from the reaction solution using a Protein A column. The chromatogram obtained during fractionation is shown in Figure 27. The peak at a retention time of approximately 25 minutes (the antibody fraction in Figure 27) was collected as the antibody.

[0092] Purification conditions Equipment: Shimadzu HPLC system Column: Tosoh ToyoScreen AF-ProteinA HC-650F 1ml Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 0.1 M phosphate buffer (pH 6.5) Mobile phase B: 0.1 M citrate buffer (pH 3.5)

[0093] Gradient conditions 1. 0-15 min A 100% 2. 15-15.01 min A 100-0% 3. 15.01-25 min A 0% 4. 25-25.01 min A 0-100% 5. 25.01-50 min A 100%

[0094] The collected fraction was concentrated by ultrafiltration using a Sartorius Vivaspin Turbo 15 (50K), and then replaced with 50 mM citrate buffer (pH 6.5) (2,460×g, 4°C).

[0095] 3-1-3. The protein concentration of the obtained Herceptin solution was measured. Thermo Scientific NanoDrop(TM) 2000C (attached software, mode: Protein A280, Type: IgG). Recovery amount: 2 mg (1.8 mL, 1.1 mg / mL).

[0096] 3-1-4. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 28. The peak at a retention time of about 49 minutes (SG-F / G2-F in Figure 28) was fractionated.

[0097] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0098] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0099] 3-1-5. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 5. [Table 5] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0100] The detected mass was 149288, indicating that the structure (Figure 29) consists of one SG-F and one G2-F attached to each of the GlcNAc-Fucose residues at Asn297 (Figure 30).

[0101] 3-2. Preparation of Herceptin [G0-F / SG-F] 3-2-1. Herceptin [G0-F / GlcNAc-Fucose] (2 mg; 200 μL) with G0 attached at only one site by ENGase, SG-Ox (Fushimi Pharmaceutical; 1 mM; 100 μL), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the reaction was carried out at 30°C for 12 hours. After the reaction, the antibody in the solution was purified by the same procedure as in 3-1-2.

[0102] 3-2-2. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 31. The peak at a retention time of approximately 46 minutes (SG-F / G0-F in Figure 31) was fractionated.

[0103] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0104] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0105] 3-2-3. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 6. [Table 6] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0106] The detected mass was 148964, indicating that the structure (Figure 32) consists of one SG-F and one G0-F attached to each of the GlcNAc-Fucose residues at Asn297 (Figure 33).

[0107] 3-3. Preparation of Herceptin [G2-F / M3-F] 3-3-1. Herceptin [G2-F / GlcNAc-Fucose] (2 mg; 200 μL) with G2 attached at only one site by ENGase, M3-Ox (Fushimi Pharmaceutical; 1 mM; 100 μL), and EndoS D233Q (4.35 μg; 2 μL) were added to 98 μL of 100 mM phosphate buffer (pH 6.5), and the reaction was carried out at 30°C for 12 hours. After the reaction, the antibody in the solution was purified by the same procedure as in 3-1-2.

[0108] 3-3-2. The purified Herceptin was fractionated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR. The fractionation results are shown in Figure 34. The peak at a retention time of approximately 45 minutes (G2-F / M3-F in Figure 34) was fractionated.

[0109] Fractionation conditions Equipment: Shimadzu HPLC system Prominence Column: Tosoh TSKgel® FcR-IIIA-NPR 4.6 mm I.D. x 10 cm, 5 μm Column temperature: 25℃ Flow rate: 1 ml / min Detection: UV 280 nm Mobile phase A: 50 mM citrate buffer (pH 6.5) Mobile phase B: 50 mM citrate buffer (pH 4.5)

[0110] Gradient conditions 1. 0-2min A 100% 2. 2-45min A100-0% 3. 45-55min A 0% 4. 55-60min A 0-100% 5. 60-80min A 100%

[0111] 3-3-3. The isolated peaks were subjected to intact mass analysis by LC-MS / MS. Intact mass analysis Equipment: Waters Acquity H-Class Bio UHPLC System with Vion IMS Qtof Column: Waters MassPREP Desalting Column Column temperature: 80℃ Mobile phase: Solution A 0.1% formic acid B liquid Acetonitrile The gradient conditions are shown in Table 7. [Table 7] m / z range: 400-4000 Capillary voltage: 3.00 kV Cone voltage: 150 V Source temperature: 150℃ Desolvation temperature: 600℃ Deconvolution Software:Waters UNIFI software v1.8.2.

[0112] The detected mass was 147975, indicating that the structure (Figure 35) consists of one G0-F and one M3-F attached to the GlcNAc-Fucose at two Asn297 positions (Figure 36).

[0113] In this way, by using an antibody having one glycan separated using Tosoh TSKgel (registered trademark) FcR-IIIA-NPR, it is possible to produce an antibody having an asymmetric glycan by introducing a different glycan into the second glycan. [Industrial Applicability]

[0114] An antibody population that uniformly contains antibodies with asymmetric glycans can be used as an antibody drug, etc. It can also be used as a standard for quality control of antibody drugs. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An IgG antibody population that uniformly contains IgG antibodies in which the N-type complex glycan bound to the 297th asparagine (Asn) in the CH domain of the Fc region of the two left and right heavy chains of the IgG antibody has a glycan structure that differs from one another, and the IgG antibody population contains 95% or more IgG antibodies in which the N-type complex glycan structures differ from one another.

2. The IgG antibody population described in claim 1, in which fucose is bound to N-acetylglucosamine (GlcNAc) at the reducing end of an N-linked complex glycan that is bound to asparagine (Asn) at position 297 located in the CH domain of the Fc region of an IgG antibody.

3. A method for producing an IgG antibody population that uniformly contains IgG antibodies in which the N-linked complex glycan bound to asparagine (Asn) at position 297 in the CH domain of the Fc region of two left and right heavy chains of the IgG antibody has a glycan structure that is different from each other, comprising: (i) cleaving sugar chains bound to the two heavy chains, left and right, of an IgG antibody in the IgG antibody composition with endo-β-N-acetylglucosaminidase (ENGase), and purifying and isolating the IgG antibody from which the sugar chains of both heavy chains have been cleaved; (ii) mixing the IgG antibody population in which the sugar chains of both heavy chains obtained in step (i) have been cleaved, a sugar derivative which generates an oxazolinated sugar chain X or an oxazoline and in which the sugar chain of the sugar derivative is sugar chain X, and ENGase modified to suppress sugar chain cleavage activity and improve glycosylation activity, adding a sugar derivative which generates an oxazolinated sugar chain X or an oxazoline and in which the sugar chain of the sugar derivative is sugar chain X to a concentration of 25 μM to 5.0 mM to prepare an IgG antibody in which sugar chain X is bound to only one of the two heavy chains on the left and right, and purifying and isolating the IgG antibody by affinity chromatography using a carrier carrying an Fcγ receptor as a ligand; (iii) mixing the IgG antibody having glycan X bound to only one of the two heavy chains obtained in step (ii), an oxazolinated glycan Y having a structure different from that of glycan X, or a glycan derivative generating oxazoline, the glycan of the glycan derivative being glycan Y, and ENGase modified to suppress glycosylation activity and maintain or improve glycosylation activity, to produce an IgG antibody having glycan Y bound to the other heavy chain of the two heavy chains, and the glycans bound to the two heavy chains have different structures, and purifying and isolating the IgG antibody by affinity chromatography using a carrier carrying an Fcγ receptor as a ligand; The method includes:

4. The method according to claim 3, wherein the ENGase is EndoS or EndoS2.

5. The method according to claim 4, wherein the ENGase modified to suppress glycosylation activity and improve glycosylation activity is selected from the group consisting of EndoS D233Q, Endo S2 D184M, Endo S2 D184Q, EndoS D233Q / Q303L, D233Q / A303L / E350Q, and Endo M N175Q.

6. The method according to claim 4 or 5, wherein the molecular weight of the sugar chain X to be bound in step (ii) is greater than the molecular weight of the sugar chain Y to be bound in step (iii).

7. A method according to any one of claims 4 to 6, which produces an IgG antibody population containing 95% or more IgG antibodies in which the N-linked complex glycan bound to the 297th asparagine (Asn) of the CH domain of the Fc region of the two left and right heavy chains of the IgG antibody is different from each other.

8. A method according to any one of claims 4 to 7, which produces an IgG antibody population in which fucose is bound to N-acetylglucosamine (GlcNAc) at the reducing end of an N-type complex glycan that is bound to asparagine (Asn) at position 297 located in the CH domain of the Fc region of an IgG antibody.

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