JP7236234B2 - Method for producing antibody with modified sugar chain - Google Patents

Description

TECHNICAL FIELD The present invention relates to a simple method for producing an antibody having modified asparagine-linked sugar chains using a glycohydrolase and a glycosyltransferase.

Antibody drugs are biopharmaceuticals that utilize the properties of antibodies, such as binding to specific antigens and subsequent removal of foreign substances via the immune system, and are attracting attention as effective therapeutic drugs with few side effects. An antibody has two asparagine-linked sugar chains bound to the Fc region of its H chain, and is known to strongly affect the activity and pharmacokinetics of antibody drugs. For example, Non-Patent Document 1 describes removal of a fucose residue bound to an N-acetylglucosamine residue on the reducing terminal side of a complex-type sugar chain, which is one type of asparagine-linked sugar chain, and bisecting of a bisecting residue bound to the complex-type sugar chain. It is disclosed that antibody-dependent cellular cytotoxicity (ADCC activity) is enhanced by increasing the amount of binding GlcNAc. In addition, Non-Patent Document 2 discloses that complement-dependent cytotoxicity activity is enhanced as the amount of galactose bound to the non-reducing end increases. Since the asparagine-linked sugar chains bound to antibody drugs have a significant effect on the activity of antibody drugs, research and development of highly active antibody drugs by modifying or controlling the structure of the asparagine-linked sugar chains has been promoted. ing.

Methods of modifying or controlling asparagine-linked sugar chains bound to antibodies include, for example, a method of modifying the biosynthetic genes of asparagine-linked sugar chains in antibody-producing cells such as Chinese hamster ovary cells (CHO cells); A method for preparing an antibody to which a desired asparagine-linked sugar chain is bound using a sugar chain donor and a glycosyltransferase is known. Among these, as a method of modifying a gene related to sugar chain biosynthesis, for example, Non-Patent Document 3 describes the addition of bisecting GlcNAc that enhances ADCC activity by co-expressing N-acetylglucosaminyltransferase III. Methods are disclosed for increasing the percentage of antibodies identified. In addition, Non-Patent Document 4 discloses a method of knocking out α-fucosyltransferase such as FUT8 to increase the ratio of antibodies from which core fucose, which reduces ADCC activity, has been removed. However, in these methods, the biosynthetic pathway of glycoproteins to which asparagine-linked sugar chains are bound is complicated. Currently, it is difficult to prepare completely controlled antibodies.

On the other hand, as a method for preparing an antibody to which a desired sugar chain is bound using a sugar chain donor and a glycosyltransferase, for example, Patent Document 1 and Non-Patent Documents 5 to 7 disclose asparagine-linked sugar chains of antibodies. Antibodies whose asparagine-linked sugar chains have been cleaved by a hydrolyzing enzyme, endo-β-N-acetylglucosaminidase, are combined with a sugar chain donor, a sugar chain oxazoline derivative, and a glycosyltransferase, a modified endo-β-N- A method using acetylglucosaminidase S to transfer a desired sugar chain to a sugar chain-cleaved antibody is disclosed. In these methods, for example, by using a desired sugar chain donor such as a sugar chain having a uniform structure, it is possible to prepare an antibody having a uniform sugar chain structure.

When a sugar chain oxazoline derivative is used as a sugar chain donor, there is an advantage that the transglycosylation reaction by modified endo-β-N-acetylglucosaminidase S occurs with high efficiency. Since sugar chain oxazoline derivatives are very unstable in a solution state of neutrality and pH 7.0 or less even at a low temperature of 4° C., the yield is low when the sugar chain oxazoline derivatives are purified. is the issue. Therefore, there is a demand for a method for efficiently transferring a sugar chain to an antibody using an oxazoline derivative with an unstable sugar chain as a sugar chain donor. Patent Document 2 discloses a method for producing a sugar chain oxazoline derivative, which comprises treating a sugar chain with a haloformamidinium derivative, which is a dehydration condensation agent. It can be used as a crude product for the next reaction, that is, transglycosylation reaction to a sugar chain acceptor, but it can also be isolated and purified by chromatography, crystallization, recrystallization, or the like." However, in this patent document, there are no examples in which a sugar chain oxazoline derivative is used for a transglycosylation reaction to a sugar chain acceptor without being purified. In addition, including Patent Document 1 and Non-Patent Documents 5 to 7, examples of reports in which a sugar chain oxazoline derivative was used as a sugar chain donor without purification to transfer a sugar chain to an antibody in which an asparagine-linked sugar chain has been cleaved. never before.

Japanese Patent Publication No. 2015-507925 WO2008/111526

Shinkawa, T. et al., J Biol Chem. 278:3466-3473 (2003) Hodoniczky, J et al., Biotechnol Prog. 21: 1644-1652 (2005) Davies, J et al., Biotechnol Bioeng. 74:288-294 (2001) Yamane-Ohnuki, N.; et al., Biotechnol Bioeng. 87:614-622 (2004) Huang, W. et al., J Am Chem Soc. 134: 12308-12318 (2012) Li, T et al., J Biol Chem. 291: 16508-16518 (2016) Kurogochi, M. et al., PLoS ONE. 10: e0132848 (2015) Small and Medium Enterprise Agency, Fiscal 2015 Strategic Fundamental Technology Advancement Support Project “Development of Enzymes for Manufacturing Uniform Glycoproteins and Mass Production of Sialyl Glycan Derivatives” Research and Development Results Report (March 2016)

An object of the present invention is to provide a method for simply and efficiently producing an antibody with modified sugar chains using an unstable sugar chain oxazoline derivative as a sugar chain donor.

As a result of intensive studies to solve the above problems, the present inventors found that an antibody obtained by cleaving an asparagine-linked sugar chain in a solution containing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydration-condensing agent. , and a glycosyltransferase, an antibody to which a sugar chain oxazoline derivative has been transferred can be produced simply and highly efficiently, leading to the completion of the present invention.

That is, the present invention provides the inventions described in (1) to (11) below. (1) A solution containing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydration-condensing agent is reacted with an antibody having a cleaved asparagine-linked sugar chain and a glycosyltransferase. A method for producing an antibody having a modified sugar chain.
(2) The production method according to (1) above, characterized by comprising the following steps (A) and (B): (A) treating the sugar chain with a dehydration-condensing agent to produce an unpurified sugar; Step (B) of obtaining a solution containing a chain oxazoline derivative: The solution containing the unpurified sugar chain oxazoline derivative obtained in (A) above is allowed to react with an antibody having cleaved asparagine-linked sugar chains and a glycosyltransferase. A step of obtaining an antibody to which a sugar chain has been transferred.
(3) The production method according to (2) above, wherein the steps (A) and (B) are performed in an aqueous solution containing a phosphate.
(4) The sugar chain to be treated with the dehydration-condensation agent is represented by the following general formula (i)

(Wherein, R1 to R9 may be the same or different, and each independently represents a hydroxyl group or one selected from the group consisting of any sugars.), which is a sugar chain represented by the above ( The production method according to any one of 1) to (3).
(5) The production method according to any one of (1) to (4) above, wherein the dehydration condensation agent is 2-chloro-1,3-dimethylbenzimidazolinium chloride.
(6) The production according to any one of (1) to (5) above, wherein the glycosyltransferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into endo-β-N-acetylglucosaminidase. Method.
(7) Any one of (1) to (6) above, wherein the glycosyltransferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into Streptococcus pyogenes-derived endo-β-N-acetylglucosaminidase. The manufacturing method according to
(8) The antibody having cleaved asparagine-linked sugar chains is an antibody obtained by treating the antibody with endo-β-N-acetylglucosaminidase, according to any one of (1) to (7) above. Production method.
(9) The production method according to any one of (1) to (8) above, wherein the antibody is an IgG antibody.
(10) The production method according to any one of (1) to (9) above, wherein the antibody is a non-human antibody, a chimeric antibody, a humanized antibody or a human antibody.
(11) The production method according to any one of (1) to (10) above, wherein the sugar chain-modified antibody is separated using an adsorbent in which human FcγRIIIa is immobilized on an insoluble carrier. .

According to the method of the present invention, an antibody whose asparagine-linked sugar chain has been cleaved by treatment with endo-β-N-acetylglucosaminidase is treated with a glycosyltransferase to efficiently transfer the desired sugar chain oxazoline derivative. Antibodies can be produced easily. The method of the present invention can transfer an unstable sugar chain oxazoline derivative, which is a sugar chain donor, to an antibody with high efficiency without purification. preferred.
Moreover, in the method of the present invention, by using a sugar chain having a uniform structure as a sugar chain donor, an antibody having a uniform sugar chain structure can be produced with high efficiency. The prepared antibody with a uniform sugar chain structure can be used not only for investigating the effect of the sugar chain structure on the pharmacological activity of antibody drugs, but also as a standard for quality control in the manufacture of antibody drugs and as an antibody drug itself. can be used.

The method for producing an antibody with modified sugar chains of the present invention is compared with conventional techniques. 1 is the result (photograph) of SDS-PAGE of modified EndoS in Preparation Example 1. FIG. 1 shows the results (photographs) of SDS-PAGE of reaction solution a and reaction solution b in the step of Example 1 (Ex. 1-1). 1 shows the results of HPLC analysis of reaction solution a and reaction solution b in the step (Ex. 1-1) of Example 1 using a column packed with human FcγRIIIa-immobilized adsorbent. SDS- It is the result (photograph) of PAGE. Results of HPLC analysis of reaction solution a in the step of Example 1 (Example 1-1) and reaction solution c in the step of Example 1 (Example 1-3) using a column packed with a human FcγRIIIa-immobilized adsorbent. is. 1 shows the results of HPLC analysis of reaction solution c in the step (Ex. 1-3) of Example 1 and reaction solution d in Comparative Example 1 using a column packed with a human FcγRIIIa-immobilized adsorbent. 1 shows the results (photographs) of SDS-PAGE of reaction solutions e1, e2 and e3 in Reference Example 1. FIG. 1 shows the results of HPLC analysis of reaction solutions e1, e2, and e3 in Reference Example 1 using a human FcγRIIIa-immobilized adsorbent.

The present invention will be described in more detail below.
In the method for producing an antibody having modified sugar chains of the present invention (hereinafter referred to as the production method of the present invention), a solution containing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydration condensation agent , an antibody with cleaved asparagine-linked sugar chains, and a glycosyltransferase. In the production method of the present invention, an unstable sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydration-condensing agent is subjected to a transglycosylation reaction without purification, thereby transferring a sugar chain to an antibody with high efficiency. This is what made it possible.
Here, the term “purification” means a known method for purifying sugar chain oxazoline derivatives such as gel filtration chromatography and ion exchange chromatography. In addition, "purification" also includes "isolation" here.
Specifically, the production method of the present invention is characterized by including the following steps (A) and (B).
(A) a step of treating a sugar chain with a dehydration-condensing agent to obtain a solution containing an unpurified sugar chain oxazoline derivative; (B) the solution containing the unpurified sugar chain oxazoline derivative obtained in (A); A step of obtaining an antibody to which a sugar chain has been transferred by reacting an antibody having a cleaved asparagine-linked sugar chain and a glycosyltransferase.

The details of each step (A) and (B) are described below.
The step (A) of the present invention is a step of reacting a sugar chain with a dehydration condensation agent to obtain a solution containing an unpurified sugar chain oxazoline derivative in which the reducing end of the sugar chain is oxazolated. The sugar chain that can be used in the step (A) is an antibody in which the asparagine-linked sugar chain has been cleaved by the action of a glycosyltransferase in the step (B) described later (hereinafter referred to as a sugar chain-cleaved antibody. ), and specific examples include the sugar chain represented by the general formula (i), which is known as the basic structure of asparagine-linked sugar chains. Here, R1 to R9 in the sugar chain represented by the general formula (i) will be explained. Examples of sugars represented by R1 to R9 include monosaccharides such as glucose, glucosamine, N-acetylglucosamine, mannose, mannosamine, N-acetylmannosamine, galactose, galactosamine, N-acetylgalactose, fucose, and sialic acids. can be mentioned. Moreover, one or more of the same or different monosaccharides may be bound to these monosaccharides. The sugar chain may be prepared according to a known method. For example, after hydrolyzing an asparagine-linked sugar chain bound to a glycoprotein such as an antibody or ribonuclease B with endo-β-N-acetylglucosaminidase, the sugar chain is can be mentioned. For example, a high mannose-type sugar chain can be obtained by the method described in a known document (J Biol Chem. 283:4469-4479 (2008)). In addition, a complex sugar chain to which sialic acid is bound can be obtained by the method described in JP-A-2012-191932. As the sugar chain, a commercially available sugar chain having a uniform structure, such as a sialyl sugar chain manufactured by Fushimi Pharmaceutical Co., Ltd., can also be used. Furthermore, these sugar chains may be sugar chains having a single structure or a mixture of sugar chains having multiple structures, depending on the purpose.

Sugar chains that can be used in step (A) of the production method of the present invention include, for example, sugar chains having structures represented by the following formulas 1-1 to 1-46. In formulas 1-1 to 1-46, Sia represents sialic acids (N-acetylneuraminic acid, N-glucolylneuraminic acid), Gal represents galactose, GlcNAc represents N-acetylglucosamine, and Man represents Indicates mannose. Also, α and β indicate the binding mode, and the numbers indicate the binding position of each monosaccharide.

The dehydration condensation agent that can be used in step (A) is not particularly limited as long as it can react with the sugar chain to produce a sugar chain oxazoline derivative. 2-chloro-1,3-dimethylimidazolinium chloride, 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate and 2-chloro-1,3-dimethylbenzo Imidazolinium chloride is preferred, and 2-chloro-1,3-dimethylbenzimidazolinium chloride is more preferred in terms of low deliquescence and easy handling. The amount of the dehydration condensation agent to be used is not particularly limited, but from the viewpoint of obtaining a high yield of the sugar chain oxazoline derivative, it is preferable to add 1 to 10 equivalents relative to the sugar chain used in step (A). It is more preferable to add 2 to 5 equivalents.

The reaction of step (A), ie, the synthesis of the sugar chain oxazoline derivative using the dehydration-condensation agent, can be carried out in a solvent known in the art as long as it does not adversely affect the reaction. The solvent that can be used in step (A) is not particularly limited as long as it can dissolve the dehydration condensation agent and the sugar chain. to obtain a sugar chain oxazoline derivative proceeds rapidly under basic conditions, and as described in Non-Patent Document 8, sugar chain oxazoline derivatives are Since it is unstable, it is preferably a basic aqueous solution or a mixed solvent of a basic aqueous solution and an organic solvent. The pH of the solvent that can be used in step (A) is not limited as long as the sugar chain oxazoline derivative is stable and the pH does not adversely affect the dehydration condensation reaction. For example, pH 7.5 to 12.5 is preferable. . In step (B) described later, the solution containing the unpurified sugar chain oxazoline derivative obtained in step (A) is allowed to act with a sugar chain-cleavage antibody and a glycosyltransferase to transfer a sugar chain. Since this is a step of obtaining an antibody prepared by the method, the solvent used in step (A) is more preferably a basic aqueous solution that does not contain an organic solvent, from the viewpoint of suppressing denaturation of the antibody and the glycosyltransferase. More preferably, it is a basic aqueous solution having a buffering capacity near the optimum pH of the chain transferase. The compound having a buffering capacity for preparing the basic aqueous solution is particularly limited if the aqueous solution exhibits basicity and has a buffering capacity near the optimum pH of the glycosyltransferase in step (B). such as trishydroxymethylaminomethane, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate , tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and the like, or mixtures thereof. Among these, trisodium phosphate has a buffering capacity in the range of pH 11 or higher and also has a buffering capacity in the range of pH 6.5 to pH 8.0, which is the optimum pH for glycosyltransferase described later. Alternatively, a phosphate such as tripotassium phosphate is more preferred. Also, the concentration of the compound having buffering ability in the basic aqueous solution is not particularly limited as long as the base is dissolved, and examples thereof include 0.01 mol/L to 2.0 mol/L. In addition to these bases, the basic aqueous solution may contain a salt having no buffering capacity, such as sodium chloride, as long as it does not inhibit the reaction in step (A).
The temperature and time for carrying out the reaction in step (A) may be appropriately set in consideration of the stability of the resulting sugar chain oxazoline derivative. For example, the temperature is preferably 0°C to 30°C, more preferably 0°C to 15°C. Also, the time is preferably 10 minutes to 24 hours, more preferably 30 minutes to 12 hours.
The solution containing the sugar chain oxazoline derivative obtained by reacting the sugar chain with the dehydration condensation agent in step (A) is used in the next step (B) without purifying the sugar chain oxazoline derivative.
The interval between step (A) and step (B) is preferably less than 1 hour from the viewpoint of suppressing decomposition of the obtained sugar chain oxazoline derivative, and more preferably continuous without an interval.

In step (B) of the present invention, the solution containing the unpurified sugar chain oxazoline derivative obtained in step (A) is treated with a glycosylation-cleaving antibody and a glycosyltransferase described later in an aqueous solution having a buffer capacity. This is a step of obtaining an antibody to which a sugar chain has been transferred by allowing it to react. In step (B), a sugar chain cleaving antibody, which is highly likely to be denatured under basic conditions, and a glycosyltransferase are added to a solution containing a basic sugar chain oxazoline derivative to obtain a sugar chain cleaving antibody. Since it is a step of transferring a sugar chain to the , the reaction solution used in step (B) is prepared by first adding an aqueous solution having a buffering capacity described later to a solution containing a basic sugar chain oxazoline derivative. It is preferable to adjust the pH of the solution to a pH range in which the sugar chain-cleaving antibody and the glycosyltransferase are not denatured by addition, and then add the sugar chain-cleaving antibody and the glycosyltransferase.

The aqueous solution having buffering capacity used in step (B), that is, the aqueous solution having buffering capacity to be added to the solution containing the sugar chain oxazoline derivative obtained in step (A) has the optimum pH for the glycosyltransferase described later. There is no particular limitation as long as it has a buffering capacity within the range of pH 6.5 to pH 8.0. Examples of the aqueous solution having a buffering capacity include phosphates such as trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate, or An aqueous solution containing a mixture thereof, an aqueous solution containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and tris(hydroxymethyl)aminomethane-hydrochloric acid (hereinafter referred to as Tris-HCl). In addition to aqueous solutions, commercially available buffer solutions such as D-PBS(-) (manufactured by Wako Pure Chemical Industries) can be used. These aqueous solutions with buffering ability have buffering ability in the range of pH 6.5 to pH 7.5, which is the optimum pH for many glycosyltransferases, and the sugar chain oxazoline in step (A) described above. Phosphoric acids such as trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, in that they can also be used in the synthesis of derivatives. More preferred are aqueous solutions containing salts or mixtures thereof. In addition, the concentration of the compound contained in the aqueous solution having these buffering capabilities is not particularly limited as long as it does not inhibit the glycosyltransferase reaction by the glycosyltransferase.
can be mentioned. In addition, inorganic salts such as sodium chloride, hydrophilic polymers such as polyoxyethylene glycol, and surfactants such as polyoxyethylene sorbitan monolaurate (Tween 20) are added to the aqueous solution for the purpose of improving the activity of the glycosyltransferase. You may The amount of the aqueous solution having buffering capacity added to the solution containing the sugar chain oxazoline derivative obtained in step (A) and the concentration of the compound contained in the aqueous solution are determined according to the glycosyltransferase used in step (B). The pH and salt concentration that do not inhibit the activity may be adjusted appropriately. 5. The amount of the buffering aqueous solution added and the concentration of the compound contained in the aqueous solution may be adjusted so that the concentration of the buffer solution ranges from 5 mmol/L to 200 mmol/L.

The glycosyltransferase that can be used in step (B) is a sugar chain-cleavage antibody, specifically, an antibody to which an asparagine-linked sugar chain is bound, as described later, with endo-β-N-acetylglucosaminidase. Asparagine-linked sugar chains are hydrolyzed by the action, and the sugar chain oxazoline derivative prepared in step (A) can be transferred to the antibody to which only the GlcNAc on the reducing terminal side of the asparagine-linked sugar chain is bound. There are no particular restrictions. Endo-β-N-acetylglucosaminidase, an enzyme that hydrolyzes the bond between GlcNAcβ1-4GlcNAc present on the non-reducing end side of the asparagine-linked sugar chain bound to the antibody, converts the asparagine-linked sugar chain to an asparagine-linked sugar chain through a reverse hydrolysis reaction. It is known to have the ability to transfer asparagine-linked sugar chains to glycoproteins with truncated chains (eg, TIGG.23:33-52 (2011)). It is also known that substitution of amino acids in the active center of endo-β-N-acetylglucosaminidase suppresses the hydrolysis activity of asparagine-linked sugar chains, and the transfer reaction of asparagine-linked sugar chains proceeds with high efficiency. (eg, J Biol Chem. 283:4469-4479 (2008)). Furthermore, in the transfer reaction of the asparagine-linked sugar chain, the sugar chain itself can be used as the sugar chain donor by using a sugar chain oxazoline derivative obtained by treating the reducing end of the sugar chain with a dehydration-condensing agent as the sugar chain donor. It is known that sugar chains can be transferred more efficiently than when using such a method (eg, J Biol Chem. 285:511-521 (2010)). Therefore, the glycosyltransferase that can be used in step (B) is a modified endo-β-N-acetylglucosaminidase in which an amino acid mutation is introduced into the active center of endo-β-N-acetylglucosaminidase. Preferably, for example, EndoS-D233Q (Non-Patent Document 5 and Non-Patent Document 7) in which an amino acid mutation is introduced into the active center of EndoS, EndoS2-D184M (Non-Patent Document 6) in which an amino acid mutation is introduced into the active center of EndoS2, EndoM EndoM-N175Q (J Biol Chem. 285: 511-521 (2010)) in which an amino acid mutation is introduced into the active center of These glycosyltransferases may be used alone or in combination, depending on the structure of the sugar chain to be transferred to the glycosylated antibody. Among these glycosyltransferases, EndoS-D233Q and EndoS2-D184M have high glycosyltransferase activity to glycosylation-cleavage antibodies and are capable of transferring various types of sugar chains having different structures. is more preferable. In addition, the method of adding the glycosyltransferase to the solution containing the sugar chain oxazoline derivative obtained in step (A) is not particularly limited. , a method of adding as a buffer solution containing purified glycosyltransferase. When added as a buffer containing a glycosyltransferase, it is preferable that the phosphate-containing buffer contains the glycosyltransferase for the reasons described above.

The temperature and time for performing the step (B) are determined in consideration of suppressing the decomposition of the sugar chain oxazoline derivative and performing the sugar chain transfer reaction to the antibody with high efficiency. is preferably 0.5 hours or more and 48 hours or less, more preferably 25° C. or more and 40° C. or less, and 1 hour or more and 24 hours or less. The mixing ratio of the glycolytic antibody added in step (B) and the sugar chain used in step (B) is appropriately selected depending on the molar ratio of the glycolytic antibody and the sugar chain and the activity of the glycosyltransferase. For example, 0.1 mg to 10 mg of sugar chain may be used for 1 mg of glycosylated antibody. The amount of glycosyltransferase to be added is not particularly limited as long as the sugar chain is transferred to the glycosylation-cleavage antibody, and may be appropriately selected depending on the activity of the glycosyltransferase.

The progress of the sugar chain transfer reaction to the glycosylated antibody can be confirmed, for example, by analyzing the change in the molecular weight of the antibody H chain of the reaction solution by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, as a method other than the SDS-PAGE method, the improved Fc-binding protein described in JP-A-2016-169197 (human FcγRIIIa, amino acid residue at a specific position in the extracellular region Fc-binding protein with improved stability against heat and acid by substituting with (hereinafter referred to as human FcγRIIIa.) by analyzing the reaction solution using an adsorbent immobilized on an insoluble carrier, sugar The progress of the strand transfer reaction can be confirmed. The adsorbent in which human FcγRIIIa is immobilized on an insoluble carrier (hereinafter referred to as human FcγRIIIa-immobilized adsorbent) is capable of identifying and separating the structures of asparagine-linked sugar chains bound to antibodies. For example, when the reaction solution in step (B) is analyzed by HPLC connected to a column (hereinafter referred to as an FcγRIIIa column) filled with a human FcγRIIIa-immobilized adsorbent, the antibody to which the sugar chain has been transferred is human FcγRIIIa. Because it interacts with the column, it is eluted from the column with a delay, whereas the antibody to which the sugar chain has not been transferred does not interact with human FcγRIIIa, and is eluted in the flow-through fraction. The progress of the sugar chain transfer reaction to the antibody can be confirmed by the difference in the elution time of . In the analysis using the FcγRIIIa column, the analysis can be performed in a shorter time than the SDS-PAGE method, and the degree of progress of the glycosylation reaction can be evaluated by measuring the peak areas of the antibody to which the sugar chain has been transferred and the antibody to which the sugar chain has not been transferred. It can be evaluated quantitatively.

By separating and purifying the reaction solution of step (B) by liquid chromatography such as ion exchange chromatography or affinity chromatography, the desired antibody to which the sugar chain has been transferred can be obtained. As the liquid chromatography, affinity chromatography using a protein having an affinity for the antibody is preferable because separation and purification operations can be easily performed. For example, antibody purification by immobilizing protein A or protein G on an insoluble carrier. The oligosaccharide chain-cleaved antibody can be purified from the reaction solution by using an adsorbent for human FcγRIIIa or the aforementioned human FcγRIIIa-immobilized adsorbent. When using an antibody purification adsorbent on which protein A or protein G is immobilized, the antibody is adsorbed to the antibody purification adsorbent regardless of the presence or absence of binding of asparagine-linked sugar chains. It is not suitable for the purpose of isolating and purifying only the antibody to which the sugar chain has been transferred from the mixture of antibodies bound with asparagine-linked sugar chains and sugar chain-cleaved antibodies, such as when the sugar chain transfer is incomplete. is. On the other hand, the human FcγRIIIa immobilized adsorbent is capable of identifying the structure of the asparagine-linked sugar chain bound to the antibody. Only the antibody to which the sugar chain has been transferred can be purified from a mixture of the antibody to which the sugar chain has been transferred and the antibody to which the sugar chain has not been transferred.

The glycosylated antibody to be used in step (B) of the present invention is the sugar chain oxazoline derivative obtained in step (A) that is reacted with the glycosyltransferase to transfer the sugar chain to the antibody. Since it acts as a chain acceptor, the binding between GlcNAcβ1-4GlcNAc present on the reducing terminal side of the asparagine-linked sugar chain is reduced by the action of endo-β-N-acetylglucosaminidase on the antibody to which the asparagine-linked sugar chain is bound. The antibody must be hydrolyzed and bound with one GlcNAc on the reducing terminal side of the asparagine-linked sugar chain. Endo-β-N-acetylglucosaminidase, which can be used for the preparation of glycosylated antibodies, hydrolyzes the bond between GlcNAcβ1-4GlcNAc present on the reducing terminal side of the asparagine-linked sugar chain bound to the antibody. is not particularly limited, for example, endo-β-N-acetylglucosaminidase A (EndoA) derived from Arthrobacter protophormiae, endo-β-N-acetylglucosaminidase CC (EndoCC) derived from Coprinopsis cinerus, Streptococcus
endo-β-N-acetylglucosaminidase D (EndoD) from pneumoniae, endo-β-N-acetylglucosaminidase F (EndoF) from Flavobacterium meningosepticum, endo-β-N-acetylglucosaminidase H (EndoH) from Streptomyces plicatus, Endo-β-N-acetylglucosaminidase LL (EndoLL) from Lactococcus lactis, Endo-β-N-acetylglucosaminidase M (EndoM) from Mucor hiemalis, Endo-β-N-acetylglucosaminidase S (EndoS) from Streptococcus pyogenes and endo-β-N-acetylglucosaminidase S2 (EndoS2) from Streptococcus pyogenes. These endo-β-N-acetylglucosaminidases may be used alone or in combination, depending on the structure of the asparagine-linked sugar chain bound to the antibody. Antibody-bound asparagine-linked glycans are diverse and can be classified into high-mannose-type, complex-type, and hybrid-type glycans. are already known to be polysaccharide chains (eg, Proteomics, 8: 2858-2871 (2008)). Therefore, as the endo-β-N-acetylglucosaminidase used for the preparation of a glycosylated antibody, it is one of the asparagine-linked sugar chains because it can efficiently hydrolyze the asparagine-linked sugar chain bound to the antibody. EndoS and/or EndoS2, which can hydrolyze the complex-type sugar chain with high efficiency, is used alone, or the EndoS and/or EndoS2 and the high-mannose-type sugar chain, which is one of the asparagine-linked sugar chains, are used. More preferably, it is used in combination with at least one selected from EndoCC, EndoD, EndoH and EndoLL, which can hydrolyze sugar chains with high efficiency. These endo-β-N-acetylglucosaminidases may be commercially available products (eg, EndoS manufactured by New England BioLabs, product name: Remove-iT EndoS), and known methods such as Non-Patent Document 6 and Non-Patent Document 7, and endo-β-N-acetylglucosaminidase produced by the method described in JP-A-2017-158596. In addition, the mixing ratio of the asparagine-linked sugar chain-bound antibody and the endo-β-N-acetylglucosaminidase is not particularly limited as long as the asparagine-linked sugar chain of the antibody is hydrolyzed. It may be appropriately selected depending on the activity of acetylglucosaminidase.

The type of antibody that can be used for the preparation of glycosylated antibodies is not particularly limited and may be any of IgG, IgA, IgM, IgD and IgE. IgG is preferred. Examples of these antibodies include non-human antibodies, chimeric antibodies, humanized antibodies and human antibodies. Here, a non-human antibody is an antibody prepared by immunizing a mammal other than a human, a chimeric antibody is an antibody in which a variable region and a constant region derived from a heterologous animal are linked, and a humanized antibody is a human It is obtained by grafting the complementarity-determining regions of an antibody derived from a mammal other than the human antibody to the complementarity-determining regions of a human-derived antibody, and a human antibody indicates that all regions are human-derived antibodies. The antibody is not particularly limited, and may be either a monoclonal antibody or a polyclonal antibody, and may be an Fc fragment that constitutes part or all of the antibody constant region. Preferred examples of the antibodies include commercially available immunoglobulin preparations and antibodies produced using mammalian cells such as CHO cells. Among these, antibody drugs produced using mammalian cells such as CHO cells are particularly preferred, and examples thereof include rituximab, trastuzumab, infliximab, and cetuximab. In addition, when the immunoglobulin preparation or antibody drug is used in the production method of the present invention, a commercially available product in a solution state may be used as it is, or it may be diluted with a buffer to be described later. Furthermore, it may be used after being concentrated by ultrafiltration or the like, if necessary.

The buffer used for the hydrolysis reaction of the asparagine-linked sugar chain bound to the antibody is not particularly limited as long as it does not inhibit the hydrolysis reaction. Examples include acetate buffer, phosphate buffer, and 2-morpholinoethanesulfone. Acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, D-PBS (-) (sum Commercially available buffers such as Kojunyaku Co., Ltd.) can be mentioned. The types of these buffers may be appropriately selected depending on the stability of the antibody and the optimum pH of endo-β-N-acetylglucosaminidase. Moreover, the concentration of these buffers is not particularly limited as long as it does not inhibit the hydrolysis of the asparagine-linked sugar chain bound to the antibody, and can be used, for example, in the range of 5 mM to 500 mM. Furthermore, for the purpose of improving antibody stability and glycosylation activity of endo-β-N-acetylglucosaminidase, inorganic salts such as sodium chloride, hydrophilic polymers such as polyoxyethylene glycol, polyoxyethylene A surfactant such as ethylene sorbitan monolaurate (Tween 20) may be added.

The temperature and time at which the antibody and endo-β-N-acetylglucosaminidase are allowed to react in the buffer should be determined by taking into consideration the fact that the sugar chain hydrolysis reaction is highly efficient and that endo-β-N-acetylglucosaminidase is not inactivated. The temperature is preferably 10° C. to 50° C., and the time is preferably 1 hour to 72 hours, more preferably 25° C. to 40° C., and 2 hours to 24 hours.

The progress of the hydrolysis reaction of the asparagine-linked sugar chain bound to the antibody can be determined by the SDS-PAGE method or an analysis method using a human FcγRIIIa-immobilized adsorbent, similar to the analysis method described in step (B) above. can be confirmed. As described above, the human FcγRIIIa immobilized adsorbent is capable of identifying and separating the structure of the asparagine-linked sugar chain bound to the antibody. When the solution was analyzed, the asparagine-linked sugar chain-bound antibody interacted with human FcγRIIIa and was eluted from the column with a delay, whereas the asparagine-linked sugar chain-hydrolyzed antibody was human. Since it does not interact with FcγRIIIa, it is eluted in the flow-through fraction, so the progress of the hydrolysis reaction of the asparagine-linked sugar chain of the antibody can be confirmed by the difference in elution time on the FcγRIIIa column. Analysis using the FcγRIIIa column can be performed in a shorter time than the SDS-PAGE method, and hydrolysis by measuring the peak areas of antibodies with and without hydrolysis of asparagine-linked sugar chains. The degree of progress of the reaction can be quantitatively evaluated.

By separating and purifying the hydrolysis reaction solution by liquid chromatography such as ion exchange chromatography or affinity chromatography, the desired antibody with cleaved asparagine-linked sugar chains can be obtained. As the liquid chromatography, affinity chromatography using a protein having an affinity for the antibody is preferable because separation and purification operations can be easily performed. For example, antibody purification by immobilizing protein A or protein G on an insoluble carrier. The oligosaccharide chain-cleaved antibody can be purified from the reaction solution by using an adsorbent for human FcγRIIIa or the aforementioned human FcγRIIIa-immobilized adsorbent. When using an antibody purification adsorbent on which protein A or protein G is immobilized, the antibody is adsorbed to the antibody purification adsorbent regardless of the presence or absence of binding of asparagine-linked sugar chains, so endo-β-N - When the hydrolysis of the asparagine-linked sugar chains bound to the antibody by acetylglucosaminidase is incomplete, among the mixture of antibodies with asparagine-linked sugar chains and glycosylated antibodies, It is not suitable for the purpose of separating and purifying only antibodies. On the other hand, as described above, the human FcγRIIIa immobilized adsorbent is capable of identifying the structure of the asparagine-linked sugar chain bound to the antibody. It is possible to purify only the glycosylated antibody from a mixture of the glycosylated antibody and the glycosylated antibody. In addition, the method of adding the glycosylated antibody to the solution containing the oligosaccharide oxazoline derivative obtained in step (A) is not particularly limited. and addition as a buffer solution containing purified glycosylated antibody. When added as a buffer containing a glycosylated antibody, the phosphate-containing buffer preferably contains the glycosylated antibody for the reasons described above.

FIG. 1 shows a comparison between the sugar chain-modified antibody preparation method of the present invention and a conventional sugar chain-modified antibody preparation method. Since the production method of the present invention does not require purification of an unstable sugar chain oxazoline derivative, compared with the methods described in Patent Documents 1 and 2 and Non-Patent Documents 5 to 7, it is possible to produce sugars easily and efficiently. Antibodies with altered chains can be produced.

EXAMPLES The present invention will be described in more detail below with reference to Production Examples, Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these.

<Production Example 1> Preparation of glycosyltransferase (modified endo-β-N-acetylglucosaminidase) Modified endo-β-N-acetylglucosaminidase S (hereinafter referred to as modified EndoS), which is a glycosyltransferase The preparation of Streptococcus pyogenes MGAS315 strain (ATCC-BAA-595D-5)-derived endo-β- This was carried out by introducing amino acid mutations into N-acetylglucosaminidase S (hereinafter referred to as EndoS). Specifically, an EndoS expression vector is prepared by fusing Glutathione S-transferase (GST) to the N-terminal side, and a modified form fused with GST is obtained by substituting glutamine for aspartic acid at position 233 in the amino acid sequence of EndoS. After constructing an EndoS expression vector (hereinafter referred to as the expression vector GST-EndoS D233Q), E. coli transformed with the expression vector is expressed under the conditions described in Non-Patent Document 6 to obtain modified EndoS. was prepared. Protein soluble fraction extraction from the E. coli was performed using BugBuster 10X Protein Extraction Reagent (manufactured by Merck Millipore). In addition, the modified EndoS was purified using Pierce Glutathione Agarose (manufactured by Thermo Scientific) and PreScission Protease (manufactured by GE Healthcare), which is a fusion protein of HRV 3C protease, which is a protease for cleaving the GST tag, and GST. Using a disposable column with a lid (manufactured by Bio-Rad), an on-column digestion method was performed according to the procedure attached to each product. A buffer consisting of 50 mM Tris-HCl, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, pH 8.0 was used as a purification buffer, and the same buffer was used for protease treatment to cleave the GST tag. did

E. coli expression of modified EndoS and GST tag are cleaved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using the protein-soluble fraction solution from the obtained bacterial cells and the modified EndoS purified solution after the protease treatment. The progress of protease treatment for

FIG. 2 shows the results of SDS-PAGE of modified EndoS. In FIG. 2, SF is the soluble fraction, P is the modified EndoS purified solution after protease treatment, and M is the molecular weight marker. For SDS-PAGE of each solution, 5-20% gradient gel (manufactured by ATTO) was used. From the results of FIG. 2, in the soluble fraction, the molecular weight of the modified EndoS fused with GST (13
A thick band was confirmed near 6 kDa). In addition, in the modified EndoS purified solution after protease treatment, a single band was confirmed near the molecular weight of the modified EndoS (109 kDa), confirming that the desired modified EndoS was obtained.

In order to replace the obtained purified EndoS solution after protease treatment with a buffer solution containing phosphate, the purified modified EndoS solution was passed through an ultrafiltration device (Amicon Ultra-0.5 mL, 30K, manufactured by Merck Millipore). was substituted with a 50 mM sodium phosphate buffer (pH 7.4), and used as a modified EndoS solution in Example 1 (Ex. 1-3) and Reference Example 1 described later. As a result of measuring the protein concentration contained in the modified EndoS solution using Micro BCA Protein Assay Kit (manufactured by Thermo Scientific), the protein concentration of the modified EndoS solution was 2.26 mg/mL.

<Example 1> Oxazoline derivatization of sialylglycans and transfer of sialylglycans by modified EndoS (Ex. 1-1) Preparation of antibodies with cleaved asparagine-linked sugar chains (antibodies with cleaved sugar chains) Reaction solution a As, 98 μL of 50 mM sodium phosphate buffer (pH 7.4), 100 μL of antibody solution (product name Rituxan (rituximab formulation), manufactured by Zenyaku Kogyo, concentration 10 mg / mL, amount of antibody 1,000 μg) and 2 μL of endo- A solution consisting of a β-N-acetylglucosaminidase solution (product name: Remove-iT EndoS, New England BioLabs, concentration: 200,000 units/mL) was added to a 1.5 mL Eppendorf tube (Eppendorf). In addition, as the reaction solution b, 100 μL of 50 mM sodium phosphate buffer (pH 7.4) and 100 μL of antibody solution (product name Rituxan (rituximab formulation), manufactured by Zenyaku Kogyo, concentration 10 mg/mL, amount of antibody 1,000 μg). A solution consisting of was added to a 1.5 mL Eppendorf tube. Both reaction solution a and reaction solution b were allowed to stand in a constant temperature bath set at 37° C. for 20 hours.

After standing in a constant temperature bath at 37° C. for 20 hours, the antibody was recovered from each solution of reaction solution a and reaction solution b by the following method. D-PBS (-) (manufactured by Wako Pure Chemical Industries) equilibrated with 50 μL of Protein A immobilized gel (product name: AF-rProtein A HC-650, manufactured by Tosoh) Mini Bio Spin Column (manufactured by BIO-RAD ) and shaken at 25° C. and 1,000 rpm for 60 minutes using a Deepwell maximizer manufactured by TAITEC to adsorb the antibody onto the Protein A-immobilized gel. After the end of the adsorption treatment, the reaction solution was collected with a centrifuge, then 150 μL of D-PBS(−) was added to the reaction vessel, and the operation of collecting the washing solution with a centrifuge was repeated three times to obtain a total of 650 μL of washing solution. recovered. Next, 150 μL of 50 mM citrate buffer (pH 3.0) was added to the reaction vessel, and after shaking for 5 minutes at 25° C. and 1,000 rpm using a Deepwell maximizer, the washing solution was recovered using a centrifuge. By repeating this operation four times, the antibody was detached from the Protein A-immobilized gel, and a total of 600 μL of detaching/washing solution containing the antibody was obtained. The antibody concentration in the desorption washing solution was determined by concentrating the desorption washing solution with an ultrafiltration device (Amicon Ultra-0.5 mL, 30K, manufactured by Merck Millipore), then buffer-exchanging to D-PBS (-) in the device, and filtering with Micro It was measured using BCA Protein Assay Kit (manufactured by Thermo Scientific).

Next, using the desorption/washing solution of reaction solution a and reaction solution b, HPLC analysis using SDS-PAGE and a column packed with human FcγRIIIa-immobilized adsorbent (hereinafter referred to as FcγRIIIa column) was performed to obtain antibodies. We confirmed the progress of the hydrolysis reaction of the asparagine-linked sugar chain bound to . The FcγRIIIa column was prepared according to the method described in Example 12 of JP-A-2016-169197. Specifically, JP 2016-169
FcR5a-immobilized gel (1.2 mL) in which human FcγRIIIa (FcR5aCys) prepared in Example 11 of JP-A-197 (FcR5aCys) was immobilized on a hydrophilic vinyl polymer for adsorbent (manufactured by Tosoh Corporation: Toyopearl) was placed in a φ4.6 mm × 75 mm It was prepared by filling a stainless steel column. The HPLC analysis of the desorption washing solutions of reaction solution a and reaction solution b using the column was performed by the following methods (a) to (c).

(a) The FcγRIIIa column was connected to an HPLC system (manufactured by Shimadzu Corporation) and equilibrated with buffer 1 (20 mM acetic acid containing 50 mM sodium chloride, pH 5.0).
(b) 0.1 mL of the desorption/washing solution of reaction solution a or reaction solution b diluted with the buffer solution 1 to an antibody concentration of 0.2 mg/mL was injected into the column at a flow rate of 0.6 mL/min.
(c) After washing with the buffer 1 for 2 minutes while maintaining a flow rate of 0.6 mL/min, a pH gradient of buffer 2 (10 mM glycine-HCl buffer, pH 3.0) (100% buffer 2 in 28 minutes) The elution pattern of the antibody contained in each solution was analyzed by a linear gradient of .

FIG. 3 shows the results of SDS-PAGE. In FIG. 3, lane 1 is the antibody (product name Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) used in the preparation of reaction solution a and reaction solution b, lane 2 is the desorption washing solution of reaction solution b, and lane 3 is reaction solution a. Desorption cleaning solution is shown. A 10% gel (manufactured by ATTO) was used for SDS-PAGE of each solution. As shown in FIG. 3, in the desorption/washing solution of reaction solution a to which endo-β-N-acetylglucosaminidase was added, a reduction in the molecular weight of antibody H chains due to hydrolysis of asparagine-linked sugar chains was confirmed. , no decrease in the molecular weight of the antibody H chain was observed in the desorption/washing solution of the reaction solution b to which endo-β-N-acetylglucosaminidase was not added.

FIG. 4 shows the results of HPLC analysis using an FcγRIIIa column. The antibody contained in the desorption/washing solution of reaction solution b interacted with human FcγRIIIa and was separated into multiple peaks, confirming that the asparagine-linked sugar chain of the antibody was not hydrolyzed. On the other hand, the antibody contained in the desorption/washing solution of reaction solution a did not interact with human FcγRIIIa and was eluted in the flow-through fraction, confirming that the asparagine-linked sugar chain of the antibody was hydrolyzed. As is clear from the results of FIGS. 3 and 4, it was confirmed that the desired glycosylated antibody can be obtained by using endo-β-N-acetylglucosaminidase.

The desorption washing solution of reaction solution a was replaced with D-PBS(−) by the method using the ultrafiltration device described in Preparation Example 1, to obtain a glycosylated antibody solution (concentration 3.42 mg/mL). Obtained.

(Ex. 1-2) Preparation of solution containing sugar chain oxazoline derivative in aqueous solution containing phosphate (corresponding to step (A) of the present invention)
A sialyl sugar chain (manufactured by Fushimi Pharmaceutical Co., Ltd., 1.0 mg, 500 nmol) was dissolved in a small amount of water, transferred to a 1.5 mL Eppendorf tube, and lyophilized. To this tube was added 5.0 μL of 0.5 M CDMBI aqueous solution (2.5 μmol) prepared from 2-chloro-1,3-dimethyl-benzimidazolium chloride (CDMBI, manufactured by Fushimi Pharmaceutical Co., Ltd.), which is a dehydration condensation agent. . The tube was cooled to 4° C., and 5.0 μL of 1.5 M tripotassium phosphate aqueous solution (7.5 μmol) prepared from tripotassium phosphate (manufactured by Kanto Kagaku) was added and stirred (pH 12.0). C. for 2 hours to prepare a solution containing an oxazoline derivative of a sialyl sugar chain.

(Ex. 1-3) Transfer of sugar chain to glycolytic antibody in aqueous solution containing phosphate (corresponding to step (B) of the present invention)
To the solution (10 μL) containing the sialyl sugar chain oxazoline derivative prepared in the step (Ex. 1-2), 94 μL of 50 mM sodium phosphate buffer (pH 7.0), from the reaction solution a in (Ex. 1-1) The prepared glycosylated antibody solution (146 μL, concentration 3.42 mg/mL, amount of antibody 500 μg) and modified EndoS solution 2 prepared in Preparation Example 1 (50 μL, concentration 2.26 mg/mL, amount of enzyme 113 μg) were added. A reaction solution c (pH 7.5) was prepared by adding them in order, and allowed to stand in a constant temperature bath at 37° C. for 3 hours.

After allowing to stand at 37° C. for 3 hours, the antibody in the reaction solution c was recovered according to the method described in the step (Ex. 1-1) of Example 1 above, and the concentration of the antibody in the resulting desorption/washing solution was measured. Furthermore, using the desorption/washing solution of the obtained reaction solution c, HPLC analysis using SDS-PAGE and an FcγRIIIa column was performed according to the method described in the above step (Ex. 1-1), whereby the oligosaccharide-cleavage antibody was analyzed. The progress of the transfer reaction of the sialyl sugar chain was confirmed.

FIG. 5 shows the results of SDS-PAGE. In FIG. 5, lane 1 is the antibody (product name: Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) used for the preparation of reaction solution a, lane 2 is the desorption washing solution of reaction solution a in the above-described step (Ex. 1-1), lane Lane 4 indicates the desorption/washing solution for reaction solution e1 in Reference Example 1 described later, Lane 4 indicates the desorption/washing solution for reaction solution d in Comparative Example 1 described later, and Lane 5 indicates the desorption/washing solution for reaction solution c in this example. . A 10% gel (manufactured by ATTO) was used for SDS-PAGE of each solution. Comparing lane 2 (reaction solution a) and lane 5 (reaction solution c) in FIG. It was confirmed that an antibody with transferred sugar chains could be obtained.

FIG. 6 shows the results of HPLC analysis of reaction solution a and reaction solution c using an FcγRIIIa column. The antibody contained in reaction solution a eluted in the flow-through fraction, but the antibody contained in reaction solution c hardly eluted in the flow-through fraction, interacting with human FcγRIIIa and eluting as a single peak. , it was confirmed that an antibody to which the desired asparagine-linked sugar chain was transferred was obtained. As is clear from the results of FIGS. 5 and 6, it was found that the method of Example 1 enables highly efficient production of the desired antibody to which the sugar chain has been transferred.

<Comparative Example 1> Oxazoline derivatization of sialyl sugar chains and transfer of sugar chains to glycolytic antibodies In Comparative Example 1, the sugar chain oxazoline derivative obtained in the step of Example 1 (Ex. 1-2) was included. After purifying the sugar chain oxazoline derivative from the solution, using the modified EndoS prepared in Preparation Example 1, the purified sugar chain oxazoline derivative and the glycosylated antibody obtained in the step of Example 1 (Ex. 1-1). An attempt was made to prepare an antibody to which a sugar chain was transferred by reacting with .

A sialyl sugar chain (manufactured by Fushimi Pharmaceutical Co., Ltd., 1.0 mg, 500 nmol) was dissolved in a small amount of water, transferred to a PCR tube, and lyophilized. To this tube was added 5.0 μL of 0.5 M CDMBI aqueous solution (2.5 μmol) prepared from CDMBI (manufactured by Fushimi Pharmaceutical Co., Ltd.). The tube was cooled to 4° C., 5.0 μL of 1.5 M K ₃ PO ₄ aqueous solution (7.4 μmol) was added, and after vortexing, allowed to stand at 4° C. for 2 hours to initiate the oxazoline derivatization reaction of the sialyl sugar chain. did After completion of the reaction, the reaction solution was desalted using a PD MiniTrap G-10 column (manufactured by GE Healthcare). Sugar chains were detected by the phenol-sulfuric acid method, and fractions in which sugar chains were eluted from the column were collected and lyophilized in 1.5 mL Eppendorf tubes to purify oxazoline sialyl sugar chains.

Next, the purified oxazolinated sialyl sugar chain was dissolved in 100 μL of 50 mM sodium phosphate buffer, pH 7.4, and 146 μL prepared from the reaction solution a obtained in the step of Example 1 (Ex. 1-1). of the glycosylated antibody solution (concentration 3.42 mg/mL, antibody amount 500 μg) and 50 μL of the modified EndoS solution prepared in Preparation Example 1 (concentration 2.26 mg/mL
, modified EndoS (113 μg) solution was sequentially added to prepare a reaction solution d, which was allowed to stand in a constant temperature bath at 37° C. for 3 hours.

After standing at 37 ° C. for 3 hours, the antibody in the reaction solution d was recovered according to the method described in the step (Ex. 1-1) of Example 1, and the obtained desorption/washing solution was used as described in Example 1. HPLC analysis using SDS-PAGE and an FcγRIIIa column was performed according to the method described in step (Ex. 1-1) to confirm the progress of the sialyl sugar chain transfer reaction to the glycosylated antibody.

FIG. 5 shows the results of SDS-PAGE. In FIG. 5, when lane 2 (reaction solution a) and lane 4 (reaction solution d) were compared, almost no increase in the molecular weight of antibody H chains due to sugar chain transfer was observed. However, it was not possible to obtain an antibody to which the desired asparagine-linked sugar chain was transferred.

7 shows the antibody (product name Rituxan, manufactured by Zenyaku Kogyo) used for the preparation of reaction solution a described in Example 1 (Ex. 1-1), and the antibody described in Example 1 (Ex. 1-3). HPLC analysis results of reaction solution c and reaction solution d using an FcγRIIIa column are shown. The antibody used in the preparation of reaction solution a and the antibody contained in reaction solution c interacted with human FcγRIIIa and eluted with a delay, whereas most of the antibody contained in reaction solution d was eluted in the flow-through fraction. . As described above, from the results of FIGS. 5 and 7, it was not possible to obtain an antibody to which the desired sugar chain was transferred by the method of this comparative example.

<Reference Example 1> Transferring a sugar chain to a sugar chain cleaving antibody using modified EndoS and a commercially available sugar chain oxazoline derivative was reacted with the glycosylated antibody obtained in the step of Example 1 (Example 1-1) to prepare an antibody to which a sugar chain had been transferred.

In a 1.5 mL Eppendorf tube, oxazoline sialyl sugar chain solution prepared from oxazoline sialyl sugar chain (manufactured by Fushimi Pharmaceutical Co., Ltd.) (100 μL, 1 mg of oxazoline sialyl sugar chain, 100 μL of 50 mM sodium phosphate buffer pH 7.4) ), a glycosylated antibody solution (146 μL, concentration 3.42 mg/mL, amount of antibody 500 μg) prepared from the reaction solution a in the step of Example 1 (Ex. 1-1), the above preparation example A reaction solution e was prepared by sequentially adding the modified EndoS solution prepared in 1 (50 μL, concentration 2.26 mg/mL, amount of enzyme 113 μg). A total of three reaction solutions e were prepared, and in a constant temperature bath set at 37 ° C., one was 3 hours (reaction solution e1), the next one was 23 hours (reaction solution e2), The last one was allowed to stand for 47 hours (referred to as reaction solution e3). In order to prevent decomposition of the oxazoline sialyl sugar chain, the reaction solution e was prepared by using the oxazoline sialyl sugar chain that had been stored at -80°C. Addition of antibody solution and modified EndoS solution was performed immediately.

After standing at 37° C. for a predetermined time, the antibodies in the reaction solutions e1, e2, and e3 were recovered according to the method described in the step (Ex. 1-1) of Example 1, and the obtained desorption/washing solution was used to HPLC analysis using SDS-PAGE and an FcγRIIIa column was performed according to the method described in the step of Example 1 (Ex. 1-1) to confirm the progress of the sialyl sugar chain transfer reaction to the glycosylated antibody. .

FIG. 8 shows the results of SDS-PAGE. In FIG. 8, lane 1 is the antibody (product name Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) used in the preparation of reaction solution a described in step (1-A) of Example 1, and lane 2 is the antibody of Example 1 described above. Lane 3 shows the desorption/washing solution of reaction solution e1, lane 4 shows the desorption/washing solution of reaction solution e2, and lane 5 shows the desorption/washing solution of reaction solution e3. A 10% gel (manufactured by ATTO) was used for SDS-PAGE of each solution. Comparing lanes 3 (reaction solution e1) to lane 5 (reaction solution e3) in FIG. It was revealed that the proportion of antibody H chains whose molecular weights did not increase gradually increased.

FIG. 9 shows the results of HPLC analysis of reaction solutions e1, e2 and e3 using an FcγRIIIa column. It was revealed that almost all antibodies interacted with human FcγRIIIa and were eluted with a delay in the reaction solution e1, which was allowed to stand at 37° C. for 3 hours. On the other hand, in the reaction solution e2 in which the standing time at 37° C. was 23 hours, the asparagine-linked sugar chain was not transferred, and the antibody eluted in the flow-through fraction without interacting with human FcγRIIIa was found in the reaction solution e1. It was found that the amount of antibody interacting with human FcγRIIIa and eluting with a delay was decreased compared to reaction solution e1. In addition, in the reaction solution e3 in which the stationary time at 37 ° C. is 47 hours, the antibody eluted in the flow-through fraction is further increased compared to the reaction solution e2, and the antibody that interacts with human FcγRIIIa and elutes later. It became clear that it was further reduced compared to the reaction solution e2. As described above, from the results of FIGS. 8 and 9, the reaction time for transferring a sugar chain to an antibody by reacting a sugar chain-cleavage antibody with a commercially available sugar chain oxazoline derivative using modified EndoS is around 3 hours. turned out to be favorable.

INDUSTRIAL APPLICABILITY The present invention can be applied to manufacture of reagents, pharmaceuticals, and the like.

Claims (7)

including the following steps (A) and (B),
The asparagine-linked sugar chain-cleaved antibody used in step (B) is an antibody to which N-acetylglucosamine on the reducing terminal side of the asparagine-linked sugar chain is linked,
The glycosyltransferase used in step (B) is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into Streptococcus pyogenes-derived endo-β-N-acetylglucosaminidase,
performing step (A) and step (B) in an aqueous solution containing a phosphate;
A method for producing an antibody with modified sugar chains.
(A) A step of obtaining a solution containing an unpurified sugar chain oxazoline derivative by treating the sugar chain with a dehydration-condensation agent
(B) After adjusting the pH of the solution containing the unpurified sugar chain oxazoline derivative obtained in (A) above to 6.5 to 7.5 with an aqueous solution containing phosphate, the asparagine-linked A step of obtaining an antibody to which a sugar chain has been transferred by reacting a sugar chain-cleaved antibody and a glycosyltransferase at a reaction temperature of 25° C. or higher and 40° C. or lower for a reaction time of 1 hour or longer and 3 hours or shorter. . The sugar chain to be treated with the dehydration-condensation agent has the following general formula (i)

(Wherein, R1 to R9 may be the same or different, and each independently represents a hydroxyl group or one selected from the group consisting of any sugars.) 1. The manufacturing method according to 1. 3. The production method according to claim 1 , wherein the dehydration condensation agent is 2-chloro-1,3-dimethylbenzimidazolinium chloride. 4. The production method according to any one of claims 1 to 3 , wherein the antibody in which the asparagine-linked sugar chain has been cleaved is an antibody obtained by reacting the antibody with endo-β-N-acetylglucosaminidase. 5. The production method according to any one of claims 1 to 4 , wherein the antibody is an IgG antibody. 6. The production method according to any one of claims 1 to 5 , wherein the antibody is a non-human antibody, chimeric antibody, humanized antibody or human antibody. 7. The production method according to any one of claims 1 to 6 , wherein the sugar chain-modified antibody is separated using an adsorbent in which human FcγRIIIa is immobilized on an insoluble carrier.

Images