JP2020048524A - Method for producing antibody with modified carbohydrate chain - Google Patents

Abstract

To provide a method for producing conveniently and efficiently an antibody with a modified carbohydrate chain using an unstable carbohydrate chain oxazoline derivative as a carbohydrate chain donor.SOLUTION: The problem is solved by the following steps of: treating a carbohydrate chain donor with a dehydration condensation agent to obtain a solution containing a carbohydrate chain oxazoline derivative, and treating the solution containing the carbohydrate chain oxazoline derivative with an antibody with a hydrolysed asparagine bonding carbohydrate chain and a carbohydrate chain transferase.SELECTED DRAWING: None

Description

  The present invention relates to a simple method for producing an antibody having a modified asparagine-linked sugar chain, using a sugar chain hydrolase and a sugar chain transferase.

  Antibody drugs are biopharmaceuticals that utilize the properties of antibodies to bind to a specific antigen and subsequently remove foreign substances via the immune system, and are attracting attention as effective therapeutic drugs with few side effects. Antibodies are known to have two asparagine-linked sugar chains bound to the Fc region of their H chains, which strongly affects the activity and pharmacokinetics of the antibody drug. For example, Non-Patent Document 1 discloses removal of a fucose residue bonded to an N-acetylglucosamine residue on the reducing end side of a complex type sugar chain, which is one kind of asparagine-linked sugar chain, and bisecting a complex type sugar chain. It is disclosed that the antibody-dependent cytotoxic activity (ADCC activity) is enhanced by increasing the amount of binding of TingGlcNAc. Non-Patent Document 2 discloses that the greater the amount of galactose bound at the non-reducing end, the more the complement-dependent cytotoxicity is enhanced. Since the asparagine-linked sugar chain bound to the antibody drug has a great effect on the activity of the antibody drug, research and development of highly active antibody drugs in which the structure of the asparagine-linked sugar chain has been modified or controlled has been promoted. ing.

  As a method of modifying or controlling the asparagine-linked sugar chain bound to the antibody, for example, a method of modifying a biosynthesis gene of an asparagine-linked sugar chain of an antibody-producing cell such as a Chinese hamster ovary cell (CHO cell), There is known a method for preparing an antibody to which a desired asparagine-linked sugar chain is bound using a sugar chain donor and a sugar chain transferase. Among these, as a method for modifying a sugar chain biosynthesis gene, for example, Non-Patent Document 3 discloses the addition of bisecting GlcNAc in which ADCC activity is enhanced by co-expressing N-acetylglucosamine transferase III. Methods have been disclosed for increasing the proportion of antibodies made. Non-Patent Document 4 discloses a method in which α-fucosyltransferase such as FUT8 is knocked out to increase the proportion of an antibody from which core fucose that decreases ADCC activity has been removed. However, in these methods, since the biosynthetic pathway of glycoproteins to which asparagine-linked sugar chains are bound is complicated, for example, the asparagine-linked sugar chain structure of an antibody, such as an antibody having a uniform asparagine-linked sugar chain structure, is used. At present, it is difficult to prepare a completely controlled antibody.

  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 sugar chain transferase, for example, Patent Document 1 and Non-Patent Documents 5 to 7 disclose an asparagine-linked sugar chain of an antibody. An antibody obtained by cleaving an asparagine-linked sugar chain with endo-β-N-acetylglucosaminidase which is a hydrolyzing enzyme is added to a sugar chain oxazoline derivative as a sugar chain donor and a modified endo-β-N- sugar chain transferase. A method of transferring a desired sugar chain to an antibody whose sugar chain has been cleaved using acetylglucosaminidase S is disclosed. In these methods, for example, an antibody having a uniform sugar chain structure can be prepared by using a desired sugar chain donor such as a sugar chain having a uniform structure.

When a sugar chain oxazoline derivative is used as a sugar chain donor, there is an advantage that a sugar chain transfer reaction by a modified endo-β-N-acetylglucosaminidase S occurs with high efficiency, but as described in Non-Patent Document 8, Even when the sugar chain oxazoline derivative is at a low temperature of 4 ° C., it is very unstable in a neutral state and in a solution state of pH 7.0 or less. Therefore, when the sugar chain oxazoline derivative is purified, the yield is low. That is the challenge. Therefore, there is a need for a method for efficiently transferring a sugar chain to an antibody using an unstable sugar chain oxazoline derivative as a sugar chain donor. Patent Document 2 discloses a method for producing an oxazoline sugar chain derivative, which comprises treating a sugar chain with a haloformamidinium derivative which is a dehydration condensing agent. The crude product can be used for the next reaction, ie, a transglycosylation reaction to a sugar chain receptor, but can be isolated and purified by chromatography, crystallization, recrystallization, and the like. " However, in this patent document, there is no example in which a sugar chain oxazoline derivative was used for a sugar transfer reaction to a sugar chain receptor without purification. In addition, Patent Literature 1 and Non-Patent Literatures 5 to 7 also report examples of using a sugar chain oxazoline derivative as a sugar chain donor without purification and transferring the sugar chain to an antibody in which an asparagine-linked sugar chain has been cleaved. Never before.

JP-T-2015-507925A International Publication No. 2008/111526

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) Kurogoshi, M, et al., PLoS ONE. 10: e0132848 (2015) Small and Medium Enterprise Agency, 2015 Strategic Fundamental Technology Advancement Support Project “Development of Mass Production Method of Enzymes and Sialyl Glycan Derivatives for Production of Uniform Glycosylated Glycoproteins” R & D Report (March 2016)

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

  The present inventors have conducted intensive studies to solve the above-described problems, and found that an asparagine-linked sugar chain-cleaved antibody was added to a solution containing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydrating condensing agent. And that a sugar chain oxazoline derivative-transferred antibody can be easily and efficiently produced by the action of a sugar chain transferase, thereby completing the present invention.

That is, the present invention provides the inventions described in the following (1) to (11). (1) A solution containing an unpurified oxazoline derivative of a sugar chain obtained by treating a sugar chain with a dehydrating condensing agent is reacted with an antibody in which an asparagine-linked sugar chain has been cleaved and a sugar chain transferase. A method for producing an antibody having a modified sugar chain.
(2) The production method according to the above (1), which comprises the following steps (A) and (B): (A) treating the sugar chain with a dehydrating condensing agent to obtain an unpurified sugar; Step (B) of Obtaining a Solution Containing the Oxazoline Chain Derivative (B) The antibody containing the unpurified sugar chain oxazoline derivative obtained in the above (A) is reacted with an antibody having a cleaved asparagine-linked sugar chain and a glycosyltransferase Thereby obtaining an antibody having a sugar chain transferred thereto.
(3) The production method according to (2), wherein the steps (A) and (B) are performed in an aqueous solution containing a phosphate.
(4) The sugar chain treated with the dehydrating condensing 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 a saccharide). The production method according to any one of (1) to (3).
(5) The method according to any one of (1) to (4), wherein the dehydration condensing agent is 2-chloro-1,3-dimethylbenzimidazolinium chloride.
(6) The production according to any one of (1) to (5) above, wherein the sugar chain transferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into endo-β-N-acetylglucosaminidase. Method.
(7) any of (1) to (6) above, wherein the sugar chain transferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into endo-β-N-acetylglucosaminidase derived from Streptococcus pyogenes. The production method described in Crab.
(8) The antibody according to any one of (1) to (7) above, wherein the antibody in which the asparagine-linked sugar chain has been cleaved is an antibody obtained by allowing endo-β-N-acetylglucosaminidase to act on the antibody. Production method.
(9) The production method according to any one of (1) to (8), wherein the antibody is an IgG antibody.
(10) The method according to any one of (1) to (9), wherein the antibody is a non-human antibody, a chimeric antibody, a humanized antibody, or a human antibody.
(11) The method according to any one of (1) to (10), wherein the antibody having a modified sugar chain is separated using an adsorbent in which human FcγRIIIa is immobilized on an insoluble carrier. .

According to the method of the present invention, the target sugar chain oxazoline derivative is transferred to an antibody in which an asparagine-linked sugar chain has been cleaved by treatment with endo-β-N-acetylglucosaminidase using a sugar chain transferase with high efficiency. Antibody can be easily produced. The method of the present invention can transfer an unstable oxazoline derivative, which is a sugar chain donor, to an antibody with high efficiency without purifying it. It is suitable.
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 examining the effect of the sugar chain structure on the pharmacological activity of an antibody drug, but also as a standard product for quality control in antibody drug production and as an antibody drug itself. Can be used.

Fig. 4 is a comparison between the method for producing an antibody having a modified sugar chain of the present invention and a conventional technique. 5 is a result (photograph) of SDS-PAGE of the modified EndoS in Preparation Example 1. 5 is a result (photograph) of SDS-PAGE of the reaction solution a and the reaction solution b in the step (Example 1-1) of Example 1. FIG. 2 shows the results of HPLC analysis of the reaction solution a and the reaction solution b in the step (Example 1-1) of Example 1 using a column packed with a human FcγRIIIa-immobilized adsorbent. SDS- of the reaction solution a in the step (Example 1-1) of Example 1, the reaction solution c in the step (Example 1-3) of Example 1, the reaction solution d in Comparative Example 1, and the reaction solution e1 in Reference Example 1 It is a result (photograph) of PAGE. Results of HPLC analysis of the reaction solution a in the step (Example 1-1) of Example 1 and the reaction solution c in the step (Example 1-3) of Example 1 using a column packed with a human FcγRIIIa-immobilized adsorbent. It is. 5 shows the results of HPLC analysis of the reaction solution c in the step (Example 1-3) in Example 1 and the reaction solution d in Comparative Example 1 using a column packed with a human FcγRIIIa-immobilized adsorbent. 5 is a result (photograph) of SDS-PAGE of reaction solutions e1, e2, and e3 in Reference Example 1. 4 is a result of HPLC analysis of the reaction solutions e1, e2, and e3 in Reference Example 1 using a human FcγRIIIa-immobilized adsorbent.

Hereinafter, the present invention will be described in more detail.
The method for producing an antibody having a modified sugar chain of the present invention (hereinafter, referred to as the production method of the present invention) is a method for preparing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydrating condensing agent. , An asparagine-linked sugar chain-cleaved antibody, and a glycosyltransferase. In the production method of the present invention, the sugar chain is transferred to the antibody with high efficiency by subjecting the unstable oxazoline derivative obtained by treating the sugar chain to a dehydration condensing agent to a sugar chain transfer reaction without purification. This is what made it possible.
Here, “purification” means a known method for purifying a sugar chain oxazoline derivative such as gel filtration chromatography and ion exchange chromatography. Here, “purification” includes “isolation”.
Specifically, the production method of the present invention is characterized by including the following steps (A) and (B).
(A) a step of obtaining a solution containing the unpurified sugar chain oxazoline derivative by treating the sugar chain with a dehydrating condensing agent; and (B) a 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 with a cleaved asparagine-linked sugar chain and a glycosyltransferase.

Hereinafter, details of each of the steps (A) and (B) will be described.
Step (A) of the present invention is a step of reacting a sugar chain with a dehydrating condensing agent to obtain a solution containing an unpurified sugar chain oxazoline derivative in which the reducing end of the sugar chain has been oxazolined. The sugar chain that can be used in the step (A) is an antibody in which an asparagine-linked sugar chain is cleaved by the action of a glycosyltransferase in a step (B) described below (hereinafter referred to as a sugar chain-cleaved antibody). ), And specific examples include a sugar chain represented by the general formula (i), which is known as a basic structure of an asparagine-linked sugar chain. Here, R1 to R9 in the sugar chain represented by the general formula (i) will be described. Examples of the saccharides 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. Further, one or more monosaccharides of the same type or different types may be bonded to these monosaccharides. The sugar chain may be prepared according to a known method, and as an example, after hydrolyzing an asparagine-linked sugar chain bound to a glycoprotein such as an antibody or ribonuclease B with endo-β-N-acetylglucosaminidase, Can be mentioned. For example, a high mannose-type sugar chain can be obtained by a method described in a known document (J Biol Chem. 283: 4469-4479 (2008)). Further, a complex type sugar chain to which sialic acid is bound can be obtained by the method described in JP-A-2012-191932. In addition, a commercially available sugar chain having a uniform structure, for example, a sialyl sugar chain manufactured by Fushimi Pharmaceutical Co., Ltd. can be used as the sugar chain. Further, these sugar chains may be a sugar chain having a single structure or a mixture of sugar chains having a plurality of structures depending on the purpose.

  Examples of the sugar chains that can be used in step (A) of the production method of the present invention include sugar chains having the structures represented by the following formulas 1-1 to 1-46. In the 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. Α and β indicate the binding mode, and the numbers indicate the binding position of each monosaccharide.

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

The reaction of step (A), that is, the synthesis of a sugar chain oxazoline derivative using the dehydrating condensing agent can be performed in a solvent known in the art, as long as the reaction is not adversely affected. The solvent that can be used in the step (A) is not particularly limited as long as the solvent can dissolve the dehydrating condensing agent and the sugar chain. The reaction of obtaining a sugar chain oxazoline derivative by the action of glycan proceeds rapidly under basic conditions. In addition, as described in Non-Patent Document 8, the sugar chain oxazoline derivative is in a solution state of pH 7.0 or less. It is preferable to use a basic aqueous solution or a mixed solvent of a basic aqueous solution and an organic solvent because of being unstable. The pH of the solvent that can be used in the step (A) is not limited as long as the sugar chain oxazoline derivative is stable and does not adversely affect the dehydration / condensation reaction. For example, pH 7.5 to 12.5 is preferable. . In the step (B) described below, the sugar chain is transferred by reacting the sugar chain-cleaving type antibody and the sugar chain transferase with the solution containing the unpurified sugar chain oxazoline derivative obtained in the step (A). The solvent used in the step (A) is more preferably a basic aqueous solution not containing an organic solvent, from the viewpoint of suppressing the denaturation of the antibody and the glycosyltransferase, since the step of obtaining a purified antibody is preferred. More preferably, the aqueous solution is a basic aqueous solution having a buffering capacity near the optimum pH of the chain transferase. The compound having a buffering ability for preparing the basic aqueous solution is not particularly limited as long as the aqueous solution shows basicity and has a buffering ability near the optimum pH of the glycosyltransferase in the step (B). For example, trishydroxymethylaminomethane, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate And phosphates such as tripotassium phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and mixtures thereof. Among these, trisodium phosphate has a buffering capacity in a pH range of 11 or more and has a buffering capacity even in a pH range of 6.5 to pH 8.0, which is the optimum pH of a glycosyltransferase described later. Alternatively, a phosphate such as tripotassium phosphate is more preferred. Further, the concentration of the compound having a buffering ability in the basic aqueous solution is not particularly limited as long as it is within a range in which the base is dissolved, and examples thereof include 0.01 mol / L to 2.0 mol / L. Further, as long as the reaction in the step (A) is not inhibited, the basic aqueous solution may contain salts having no buffering ability such as sodium chloride in addition to these bases.
The temperature and time for carrying out the reaction in the step (A) may be appropriately set in consideration of the stability of the resulting oxazoline sugar chain derivative. For example, the temperature is preferably from 0 ° C to 30 ° C, more preferably from 0 ° C to 15 ° C. The time is preferably from 10 minutes to 24 hours, more preferably from 30 minutes to 12 hours.
The solution containing a sugar chain oxazoline derivative obtained by reacting a sugar chain with a dehydrating condensing agent in step (A) is used in the next step (B) without purifying the sugar chain oxazoline derivative.
The interval between the step (A) and the step (B) is preferably performed within 1 hour from the viewpoint of suppressing the decomposition of the obtained oxazoline sugar chain derivative, and more preferably performed continuously without an interval.

  In the step (B) of the present invention, the solution containing the unpurified oxazoline derivative obtained in the step (A) is reacted with a sugar chain-cleaved antibody and a sugar chain transferase, described below, in an aqueous solution having a buffering capacity. This is a step of obtaining an antibody to which a sugar chain has been transferred. In the step (B), a sugar chain-cleaving type antibody and a sugar chain transferase, which are likely to be denatured under basic conditions, are added to a solution containing a basic sugar chain oxazoline derivative. The reaction solution used in the step (B) is prepared by first adding an aqueous solution having a buffering capacity to be described later to a solution containing a basic sugar chain oxazoline derivative. It is preferable to adjust the pH of the solution so that the sugar chain-cleaving type antibody and the glycosyltransferase are not denatured, and then to add the sugar chain-cleaving type antibody and the glycosyltransferase.

The aqueous solution having a buffering ability used in the step (B), that is, the aqueous solution having a buffering ability to be added to the solution containing the sugar chain oxazoline derivative obtained in the step (A), has an optimum pH of the sugar chain transferase described later. There is no particular limitation as long as it has a buffering capacity in 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. 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) are included. In addition to the aqueous solution, commercially available buffers such as D-PBS (-) (manufactured by Wako Pure Chemical Industries) can be mentioned. Among these aqueous solutions having a buffer capacity, the oxazoline sugar chain of step (A) has a buffer capacity in the range of pH 6.5 to pH 7.5, which is the optimum pH for many glycosyltransferases. Phosphoric acid such as trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate can be used for the synthesis of derivatives. More preferably, it is an aqueous solution containing a salt or a mixture thereof. The concentration of the compound contained in the aqueous solution having such a buffer capacity is not particularly limited as long as the sugar chain transfer reaction by the sugar chain transferase is not inhibited, and is, for example, 5 mmol / L to 200 mmol / L.
Can be mentioned. In addition, for the purpose of improving the activity of glycosyltransferase, an inorganic salt such as sodium chloride, a hydrophilic polymer such as polyoxyethylene glycol, and a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) are added to the aqueous solution. May be. The amount of the aqueous solution having the buffering ability added to the solution containing the sugar chain oxazoline derivative obtained in the step (A) and the concentration of the compound contained in the aqueous solution depend on the amount of the glycosyltransferase used in the step (B). The pH may be adjusted appropriately in consideration of the pH and salt concentration that do not inhibit the activity. For example, the pH of the solution obtained after adding the aqueous solution having a buffer capacity to the solution obtained in step (A) is 6.5 to 7 5. The amount of the aqueous solution having the buffer capacity and the concentration of the compound contained in the aqueous solution may be adjusted so that the buffer concentration becomes 5 mmol / L to 200 mmol / L.

  The sugar chain transferase that can be used in the step (B) is a sugar chain-cleaved antibody, specifically, an antibody to which an asparagine-linked sugar chain is bound as described below, and endo-β-N-acetylglucosaminidase. By acting, the sugar chain oxazoline derivative prepared in the step (A) can be transferred to an antibody in which the asparagine-linked sugar chain is hydrolyzed and only GlcNAc on the reducing end 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 terminal side of the asparagine-linked sugar chain bound to the antibody, is converted into an asparagine-linked sugar by a reverse reaction of hydrolysis. It is known to have the ability to transfer an asparagine-linked sugar chain to a glycoprotein whose chain has been cleaved (for example, TIGG. 23: 33-52 (2011)). Further, it is known that by substituting the amino acid at the active center of endo-β-N-acetylglucosaminidase, the hydrolysis activity of asparagine-linked sugar chain is suppressed, and the transfer reaction of asparagine-linked sugar chain proceeds with high efficiency. (For example, J Biol Chem. 283: 4469-4479 (2008)). Furthermore, in the transfer reaction of the asparagine-linked sugar chain, the sugar chain itself is used as the sugar chain donor by using the sugar chain oxazoline derivative obtained by treating the reducing end of the sugar chain with a dehydrating condensing agent as the sugar chain donor. It is known that sugar chains can be transferred more efficiently than when they are used (for example, J Biol Chem. 285: 511-521 (2010)). Therefore, the sugar chain transferase that can be used in the step (B) may be a modified endo-β-N-acetylglucosaminidase in which an amino acid mutation has been introduced into the active center of endo-β-N-acetylglucosaminidase. Preferably, for example, EndoS-D233Q in which an amino acid mutation is introduced into the active center of EndoS (Non-patent Documents 5 and 7), EndoS2-D184M in which an amino acid mutation is introduced into the active center of EndoS2 (Non-patent Document 6), EndoM EndoM-N175Q (J Biol Chem. 285: 511-521 (2010)) having an amino acid mutation introduced into the active center thereof. 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 are high in glycosyltransferase activity to glycosylated antibodies and can transfer various types of sugar chains having different structures. Is more preferable. The method for adding the glycosyltransferase to the solution containing the sugar chain oxazoline derivative obtained in the step (A) is not particularly limited. For example, a method in which the purified glycosyltransferase is added as a powder by freeze-drying And a method of adding as a buffer solution containing a purified glycosyltransferase. When added as a buffer solution containing a glycosyltransferase, it is preferable that the buffer solution containing a phosphate contains a glycosyltransferase for the reasons described above.

The temperature and time for carrying out the step (B) are set at 10 ° C. or more and 50 ° C. or less in consideration of the fact that the sugar chain oxazoline derivative is suppressed from being decomposed and the sugar chain transfer reaction to the antibody is performed with high efficiency. Is preferably 0.5 to 48 hours, more preferably 25 to 40 ° C., and more preferably 1 to 24 hours. The mixing ratio of the sugar chain-cleaved antibody added in step (B) to the sugar chain used in step (B) is appropriately selected depending on the molar ratio of sugar chain-cleaved antibody to sugar chain and the activity of glycosyltransferase. For example, 0.1 mg to 10 mg of a sugar chain may be used for 1 mg of a sugar chain-cleaved type antibody. The amount of sugar chain transferase to be added is not particularly limited as long as the sugar chain is transferred to the sugar chain-cleaved antibody, and may be appropriately selected depending on the activity of the sugar chain transferase.

  The progress of the sugar chain transfer reaction to the sugar chain-cleaved antibody can be confirmed, for example, by analyzing the reaction solution for changes in the molecular weight of the antibody H chain by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Further, as a method other than the SDS-PAGE method, an improved Fc-binding protein described in JP-A-2006-169197 (an amino acid residue at a specific position in the extracellular region of human FcγRIIIa can be replaced with another specific amino acid) Can be obtained by analyzing the reaction solution using an adsorbent in which an Fc-binding protein having improved stability to heat and acid by replacement with an FcγRIIIa (hereinafter, referred to as human FcγRIIIa) is immobilized on an insoluble carrier. The progress of the chain transfer reaction can be confirmed. The adsorbent obtained by immobilizing human FcγRIIIa on an insoluble carrier (hereinafter referred to as “human FcγRIIIa-immobilized adsorbent”) can identify and separate the structure of an asparagine-linked sugar chain bound to an antibody. For example, when the reaction solution of step (B) is analyzed by HPLC connected to a column packed with a human FcγRIIIa-immobilized adsorbent (hereinafter referred to as an FcγRIIIa column), the antibody to which the sugar chain has been transferred is human FcγRIIIa. Antibody, which is eluted from the column with a delay, whereas the antibody to which the sugar chain was not transferred does not interact with human FcγRIIIa, and elutes in a flow-through fraction. The progress of the transfer reaction of the sugar chain to the antibody can be confirmed by the difference in the elution time of the antibody. 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 sugar chain transfer reaction can be measured by measuring the peak areas of the antibody with and without the sugar chain transferred. It can be evaluated quantitatively.

  By separating and purifying the reaction solution of the step (B) by liquid chromatography such as ion exchange chromatography or affinity chromatography, it is possible to obtain an antibody to which a sugar chain is transferred. As the liquid chromatography, affinity chromatography using a protein having an affinity for the antibody is preferable in that the separation and purification operation can be easily performed. For example, antibody purification in which protein A or protein G is immobilized on an insoluble carrier is preferable. A sugar chain-cleaved type antibody can be purified from the reaction solution by using the adsorbent for use or the adsorbent immobilized with human FcγRIIIa. When an antibody-purifying adsorbent on which protein A or protein G is immobilized is used, the antibody is adsorbed to the antibody-purifying adsorbent regardless of the presence or absence of asparagine-linked sugar chains. Is not suitable for the purpose of separating and purifying only the antibody to which the sugar chain has been transferred from the mixture of the antibody to which the asparagine-linked sugar chain is bound and the antibody to which the sugar chain is truncated, such as when the transfer of the sugar chain is incomplete. It is. On the other hand, the human FcγRIIIa-immobilized adsorbent is capable of discriminating the structure of the asparagine-linked sugar chain bound to the antibody. Therefore, by using the human FcγRIIIa-immobilized adsorbent, the asparagine-linked sugar chain is transferred. Among the mixed antibodies and the antibodies whose sugar chains have not been transferred, only the antibodies whose sugar chains have been transferred can be purified.

The sugar chain-cleaved type antibody used in the step (B) of the present invention is obtained by reacting the sugar chain oxidase with the sugar chain oxazoline derivative obtained in the step (A) to transfer the sugar chain to the antibody. By acting endo-β-N-acetylglucosaminidase on the antibody to which the asparagine-linked sugar chain is bound, the bond between the GlcNAcβ1-4GlcNAc present on the reducing end side of the asparagine-linked sugar chain becomes a chain acceptor. The antibody needs to be hydrolyzed and bound to one GlcNAc on the reducing end side of the asparagine-linked sugar chain. An endo-β-N-acetylglucosaminidase that can be used for preparing a glycosylated antibody hydrolyzes a bond between GlcNAcβ1-4GlcNAc present on the reducing terminal side of an 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, and Streptococcus.
pneumoniae-derived endo-β-N-acetylglucosaminidase D (EndoD), Flavobacterium meningosepticum-derived endo-β-N-acetylglucosaminidase F (EndoF), Streptomyces plicatus-derived endo-β-H-glucoseminidase, Endo-β-N-glucoseminidase Lactococcus lactis-derived endo-β-N-acetylglucosaminidase LL (EndoLL), Mucor HIemalis-derived endo-β-N-acetylglucosaminidase M (EndoM), and Streptococcus pyogenes-derived endo-β-N-acetyl-S-glucoseminidase. And Streptococcus pyogenes Can be mentioned end-beta-N-acetylglucosaminidase S2 (EndoS2) of come. 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. Although asparagine-linked glycans bound to antibodies have the diversity of being classified into high-mannose-type, complex-type, and hybrid-type glycans, most of the asparagine-linked glycans bound to the antibody are complex-type. It is already known to be a type sugar chain (for example, Proteomics, 8: 2858-2871 (2008)). Therefore, as an endo-β-N-acetylglucosaminidase used for preparing a sugar chain-cleaved antibody, one of the asparagine-linked sugar chains is a point that the asparagine-linked sugar chain bound to the antibody can be hydrolyzed with high efficiency. EndoS and / or EndoS2 capable of hydrolyzing the complex type sugar chain with high efficiency are used alone, or the EndoS and / or EndoS2 and a high mannose type which is one of asparagine-linked sugar chains It is more preferable to use a mixture of at least one selected from EndoCC, EndoD, EndoH and EndoLL, which can hydrolyze a sugar chain with high efficiency. As these endo-β-N-acetylglucosaminidases, commercially available products (for example, EndS from New England BioLabs, product name: Remove-iT EndoS) may be used, and known methods, such as Non-Patent Document 6 and Non-Patent Documents 7 or endo-β-N-acetylglucosaminidase prepared by the method described in JP-A-2017-158596 or the like. The mixing ratio of the antibody having the asparagine-linked sugar chain bound thereto and the endo-β-N-acetylglucosaminidase is not particularly limited as long as the asparagine-linked sugar chain of the antibody is hydrolyzed. What is necessary is just to select suitably according to the activity of acetylglucosaminidase.

  There is no particular limitation on the type of antibody that can be used for the preparation of a glycosylated antibody, and any of IgG, IgA, IgM, IgD and IgE may be used. It is preferably IgG. Examples of these antibodies include non-human, chimeric, humanized and human antibodies. Here, a non-human antibody is an antibody produced by immunizing a mammal other than a human, a chimeric antibody is an antibody obtained by linking a variable region and a constant region derived from a heterologous animal, and a humanized antibody is a human The complementarity-determining regions of antibodies derived from mammals other than the above were transplanted to the complementarity-determining regions of human-derived antibodies, and human antibodies indicate that all regions are human-derived antibodies. The antibody is not particularly limited, and may be any of a monoclonal antibody and a polyclonal antibody, and may be an Fc fragment constituting a part or all of the constant region of the antibody. Preferred examples of the antibody include commercially available immunoglobulin preparations and antibodies produced using mammalian cells such as CHO cells. Among them, antibody pharmaceuticals produced using mammalian cells such as CHO cells are particularly preferable, and examples thereof include rituximab, trastuzumab, infliximab, cetuximab and the like. When the immunoglobulin preparation or the antibody drug is used in the production method of the present invention, a commercially available solution may be used as it is, or may be used after being diluted with a buffer described later. Further, if necessary, it may be used after being concentrated by ultrafiltration or the like.

  The buffer used for the hydrolysis reaction of the asparagine-linked sugar chain bound to the antibody is not particularly limited as long as the hydrolysis reaction is not inhibited. For example, an acetate buffer, a phosphate buffer, 2-morpholinoethane sulfone Acid (MES) buffer, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer, tris (hydroxymethyl) aminomethane (Tris) buffer, and D-PBS (-) (sum) Commercially available buffers such as Kojundo Chemical Co., Ltd.) can be used. The type of these buffers may be appropriately selected depending on the stability of the antibody and the optimal pH of endo-β-N-acetylglucosaminidase. The concentration of these buffers is not particularly limited as long as they do 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, in order to improve the stability of the antibody and the sugar chain hydrolyzing activity of endo-β-N-acetylglucosaminidase, inorganic salts such as sodium chloride, hydrophilic polymers such as polyoxyethylene glycol, A surfactant such as ethylene sorbitan monolaurate (Tween 20) may be added.

  The temperature and time during which the antibody and endo-β-N-acetylglucosaminidase are reacted in a buffer solution are taken into consideration in that the sugar chain hydrolysis reaction is performed with high efficiency and endo-β-N-acetylglucosaminidase is not deactivated. The temperature is preferably from 10 ° C. to 50 ° C., and the time is preferably from 1 hour to 72 hours, more preferably from 25 ° C. to 40 ° C., and more preferably from 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 the analysis method using a human FcγRIIIa-immobilized adsorbent in the same manner as the analysis method described in the above step (B). You can check. As described above, since the human FcγRIIIa-immobilized adsorbent can identify and separate the structure of the asparagine-linked sugar chain bound to the antibody, for example, the hydrolysis reaction is performed by HPLC connected to an FcγRIIIa column. When the solution was analyzed, the antibody to which the asparagine-linked sugar chain was bound eluted later from the column because it interacted with human FcγRIIIa, whereas the antibody to which the asparagine-linked sugar chain was hydrolyzed Since it does not interact with FcγRIIIa, it elutes in the fraction without passing through, so that 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. In the analysis using the FcγRIIIa column, the analysis can be performed in a shorter time than the SDS-PAGE method, and the hydrolysis can be performed by measuring the peak areas of the antibody in which the asparagine-linked sugar chain is hydrolyzed and the antibody in which the asparagine-linked sugar chain is not hydrolyzed. 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, it is possible to obtain an antibody in which the target asparagine-linked sugar chain has been cleaved. As the liquid chromatography, affinity chromatography using a protein having an affinity for the antibody is preferable in that the separation and purification operation can be easily performed. For example, antibody purification in which protein A or protein G is immobilized on an insoluble carrier is preferable. A sugar chain-cleaved type antibody can be purified from the reaction solution by using the adsorbent for use or the adsorbent immobilized with human FcγRIIIa. In addition, when an adsorbent for antibody purification on which protein A or protein G is immobilized is used, the antibody is adsorbed to the adsorbent for antibody purification regardless of the presence or absence of asparagine-linked sugar chains, so that endo-β-N -When the asparagine-linked sugar chain bound to the antibody by acetylglucosaminidase is incompletely hydrolyzed, such as when the asparagine-linked sugar chain-bound antibody and the sugar chain-cleaved antibody are mixed, 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 can discriminate the structure of the asparagine-linked sugar chain bound to the antibody. It is possible to purify only a sugar chain-cleaved antibody from a mixture of a sugar chain-bound antibody and a sugar chain-cleaved antibody. The method of adding the sugar chain-cleaved antibody to the solution containing the sugar chain oxazoline derivative obtained in the step (A) is not particularly limited. For example, a purified sugar chain-cleaved antibody is added as a powder by freeze-drying. And a method of adding as a buffer containing a purified sugar chain-cleaved antibody. When added as a buffer solution containing a sugar chain-cleaved antibody, it is preferred that the buffer solution containing a phosphate contains the sugar chain-cleaved antibody for the above-mentioned reason.

  FIG. 1 shows a comparison between the method for preparing a sugar chain-modified antibody of the present invention and a conventional method for preparing a sugar chain-modified antibody. Since the production method of the present invention does not require the purification of unstable oxazoline sugar chains, the sugar chain can be prepared more easily and more efficiently than the methods described in Patent Documents 1 and 2 and Non-Patent Documents 5 to 7. Antibodies with altered chains can be produced.

  Hereinafter, the present invention will be described in more detail with reference to Production Examples, Examples, Comparative Examples, and Reference Examples, but the present invention is not limited thereto.

<Preparation 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. Was prepared by referring to the methods described in Non-Patent Document 5 and the literature (EMBO J. 20: 3046-55 (2001)), and using endo-β-derived from Streptococcus pyogenes MGAS315 strain (ATCC-BAA-595D-5). This was performed by introducing an amino acid mutation into N-acetylglucosaminidase S (hereinafter referred to as EndoS). Specifically, a modified GST-fused GST was prepared by preparing an EndoS expression vector in which Glutathione S-transferase (GST) was fused to the N-terminal side, and replacing 233rd aspartic acid in the amino acid sequence of EndoS with glutamine. After an EndoS expression vector (hereinafter referred to as an expression vector GST-EndoS.D233Q) is prepared, the modified EndoS is expressed by using Escherichia coli transformed with the expression vector under the conditions described in Non-Patent Document 6. Was prepared. Extraction of the protein soluble fraction from the Escherichia coli was performed using BugBuster 10X Protein Extraction Reagent (manufactured by Merck Millipore). In addition, purification of the modified EndoS was performed by 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. In accordance with the procedure attached to each product, an on-column digestion method using a disposable column with a lid (manufactured by Bio-Rad) was performed. As a purification buffer, a buffer consisting of 50 mM Tris-HCl, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, pH 8.0 was used, and a protease treatment for cleaving the GST tag using the same buffer. Was done.

  Escherichia coli expression of modified EndoS and GST tag were cleaved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a protein-soluble fraction solution from the obtained cells and a modified EndoS purified solution after the protease treatment. The progress of the protease treatment was performed.

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

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

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

  After allowing to stand in a thermostat at 37 ° C. for 20 hours, the antibody was recovered from each of the reaction solution a and the reaction solution b by the following method. Mini-bio spin column (manufactured by BIO-RAD) containing 50 μL of Protein A immobilized gel (product name: AF-rProtein A HC-650, manufactured by Tosoh) equilibrated with D-PBS (-) (manufactured by Wako Pure Chemical Industries, Ltd.) ) Was added thereto, and the antibody was adsorbed on the Protein A-immobilized gel by shaking for 60 minutes at 25 ° C and 1,000 rpm using a Deepwell maximizer manufactured by TAITEC. After completion of the adsorption treatment, the reaction solution is collected by a centrifugal separator, 150 μL of D-PBS (−) is added to the reaction vessel, and the operation of collecting the washing solution by the centrifuge is repeated three times, whereby a total of 650 μL of the washing solution is obtained. Was recovered. Next, 150 μL of 50 mM citrate buffer (pH 3.0) was added to the reaction vessel, and the mixture was shaken at 25 ° C. and 1,000 rpm for 5 minutes using a Deepwell maximizer, and then the washing solution was collected by a centrifuge. By repeating this operation four times, the antibody was desorbed from the Protein A-immobilized gel, and a total of 600 μL of the desorption washing solution containing the antibody was obtained. The antibody concentration in the washing solution for desorption was determined by concentrating the washing solution for desorption with an ultrafiltration device (Amicon Ultra-0.5 mL, 30K, manufactured by Merck Millipore), and then replacing the buffer solution with D-PBS (-) in the device. The measurement was performed using BCA Protein Assay Kit (manufactured by Thermo Scientific).

Next, SDS-PAGE and HPLC analysis using a column packed with a human FcγRIIIa-immobilized adsorbent (hereinafter, referred to as an FcγRIIIa column) are performed using the desorption washing solution of the reaction solution a and the reaction solution b, thereby obtaining an antibody. The progress of the hydrolysis reaction of the asparagine-linked sugar chain bound to was confirmed. The FcγRIIIa column was prepared according to the method described in Example 12 of JP-A-2006-169197. Specifically, JP-A-2016-169
An FcR5a-immobilized gel (1.2 mL) obtained by immobilizing human FcγRIIIa (FcR5aCys) prepared in Example 11 of Japanese Patent Publication No. 197 on a hydrophilic vinyl polymer for an adsorbent (manufactured by Tosoh: Toyopearl) was used as a φ4.6 mm × 75 mm. It was produced by packing in a stainless steel column. The HPLC analysis of the desorption washing solution of the reaction solution a and the reaction solution b using the column was performed by the following methods (a) to (c).

(A) The FcγRIIIa column was connected to an HPLC device (manufactured by Shimadzu Corporation), and equilibrated with buffer solution 1 (20 mM acetic acid containing 50 mM sodium chloride, pH 5.0).
(B) 0.1 mL of the desorption washing solution of the reaction solution a or the 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 solution 1 for 2 minutes while maintaining the flow rate of 0.6 mL / min, the pH gradient of the buffer solution 2 (10 mM glycine hydrochloride buffer, pH 3.0) (buffer solution 2 is 100% in 28 minutes) The elution pattern of the antibody contained in each solution was analyzed by the following linear gradient.

  FIG. 3 shows the results of SDS-PAGE. In FIG. 3, lane 1 shows the antibody (product name Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) used for preparing reaction solution a and reaction solution b, lane 2 shows the desorption washing solution for reaction solution b, and lane 3 shows the reaction solution a. The desorption cleaning liquid is shown. In addition, SDS-PAGE of each solution used 10% gel (manufactured by ATTO). As shown in FIG. 3, in the desorption washing solution of the reaction solution a to which endo-β-N-acetylglucosaminidase was added, a decrease in the molecular weight of the antibody H chain due to hydrolysis of the asparagine-linked sugar chain 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 the FcγRIIIa column. Since the antibody contained in the desorption washing solution of the reaction solution b interacted with human FcγRIIIa and was separated into a plurality of peaks, it was confirmed 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 the reaction solution a had no interaction with human FcγRIIIa, and was eluted in the fraction passing through. Thus, it was confirmed that the asparagine-linked sugar chain of the antibody was hydrolyzed. As described above, as is clear from the results of FIGS. 3 and 4, it was confirmed that the use of endo-β-N-acetylglucosaminidase could provide the desired sugar chain-cleaved antibody.

  By replacing the desorption washing solution of the reaction solution a with D-PBS (−) by the method using the ultrafiltration device described in Preparation Example 1, the sugar chain-cleaved antibody solution (concentration 3.42 mg / mL) was obtained. Obtained.

(Example 1-2) Preparation of a solution containing a sugar chain oxazoline derivative in an aqueous solution containing a phosphate (corresponding to step (A) of the present invention)
A sialyl sugar chain (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 freeze-dried. To this tube was added 5.0 μL of a 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.) as a dehydration condensing agent. . The tube was cooled to 4 ° C., and 5.0 μL of a 1.5 M aqueous solution of tripotassium phosphate (7.5 μmol) prepared from tripotassium phosphate (manufactured by Kanto Kagaku) was added and stirred (pH 12.0). The solution containing the oxazoline derivative of the sialyl sugar chain was prepared by allowing to stand at 2 ° C. for 2 hours.

(Example 1-3) Transfer of sugar chain to sugar chain-cleaved antibody in an aqueous solution containing phosphate (corresponding to step (B) of the present invention)
In a solution (10 μL) containing the sialyl sugar chain oxazoline derivative prepared in the above step (Ex. 1-2), 94 μL of 50 mM sodium phosphate buffer (pH 7.0) and the reaction solution a of (Ex. 1-1) were added. The prepared sugar chain-cleaved antibody solution (146 μL, concentration 3.42 mg / mL, antibody amount 500 μg) and the modified EndoS solution 2 prepared in Preparation Example 1 (50 μL, concentration 2.26 mg / mL, enzyme amount 113 μg) were used. A reaction solution c (pH 7.5) was prepared by successive addition, and the mixture was allowed to stand in a thermostat 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 (Example 1-1) of Example 1, and the antibody concentration of the obtained desorption washing solution was measured. Further, using the desorbed washing solution of the obtained reaction solution c, SDS-PAGE and HPLC analysis using an FcγRIIIa column are performed according to the method described in the above step (Ex. 1-1), whereby a sugar chain-cleaved antibody can be obtained. 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 shows the antibody (product name Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) used for preparing the reaction solution a, lane 2 shows the desorption washing solution of the reaction solution a in the aforementioned step (Ex. 1-1), Reference numeral 3 denotes a cleaning solution for desorbing the reaction solution e1 in Reference Example 1 described below, lane 4 denotes a cleaning solution for desorption of the reaction solution d in Comparative Example 1 described later, and lane 5 denotes a cleaning solution for desorption of the reaction solution c in this example. . In addition, SDS-PAGE of each solution used 10% gel (manufactured by ATTO). When lane 2 (reaction solution a) and lane 5 (reaction solution c) in FIG. 5 were compared, an increase in the molecular weight of the antibody H chain due to transfer of the sugar chain was confirmed in lane 5, indicating that the target asparagine-bound It was confirmed that an antibody with a sugar chain transferred was obtained.

  FIG. 6 shows the results of HPLC analysis of the reaction solution a and the reaction solution c using an FcγRIIIa column. Although the antibody contained in the reaction solution a eluted in the pass-through fraction, the antibody contained in the reaction solution c hardly eluted in the pass-through fraction, and interacted with human FcγRIIIa and eluted as a single peak. It was confirmed that an antibody to which the target asparagine-linked sugar chain was transferred was obtained. As described above, as is clear from the results of FIGS. 5 and 6, it was clarified that the method of Example 1 can efficiently obtain an antibody to which a target sugar chain has been transferred.

<Comparative Example 1> Oxazoline derivatization of sialyl sugar chain and transfer of sugar chain to sugar chain-cleaved antibody Comparative Example 1 includes the sugar chain oxazoline derivative obtained in step (Example 1-2) of Example 1. 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 sugar chain-cleaved type antibody obtained in Step (Example 1-1) of Example 1 By reacting the above, an attempt was made to produce an antibody having a sugar chain transferred thereto.

A sialyl sugar chain (Fushimi Pharmaceutical Co., Ltd., 1.0 mg, 500 nmol) was dissolved in a small amount of water, transferred to a PCR tube and freeze-dried. To this tube, 5.0 μL of a 0.5 M CDMBI aqueous solution (2.5 μmol) prepared from CDMBI (Fushimi Pharmaceutical) was added. The tube was cooled to 4 ° C., and 5.0 μL of a 1.5 M K ₃ PO ₄ aqueous solution (7.4 μmol) was added thereto. Was done. After the completion of the reaction, the reaction solution was desalted using a PD MiniTrap G-10 column (manufactured by GE Healthcare). By detecting the sugar chain by the phenol sulfate method, the fraction from which the sugar chain was eluted was collected from the column, and lyophilized in a 1.5 mL eppendorf tube to purify the oxazoline sialyl sugar chain.

Next, the purified oxazoline-modified sialyl sugar chain was dissolved in 100 μL of a 50 mM sodium phosphate buffer, pH 7.4, and 146 μL prepared from the reaction solution a obtained in the step (Example 1-1) of Example 1 described above. And the 50 μL modified EndoS solution (concentration 2.26 mg / mL) prepared in Preparation Example 1 and the sugar chain cleavage type antibody solution (concentration 3.42 mg / mL, antibody amount 500 μg)
, Modified EndoS amount 113 μg) to prepare a reaction solution d, which was allowed to stand in a thermostat at 37 ° C. for 3 hours.

  After allowing to stand at 37 ° C. for 3 hours, the antibody in the reaction solution d was recovered according to the method described in the step (Example 1-1) of Example 1, and the obtained desorption washing solution was used. By performing SDS-PAGE and HPLC analysis using an FcγRIIIa column according to the method described in the step (Exact 1-1), the progress of the transfer reaction of the sialyl sugar chain to the sugar chain-cleaved antibody was confirmed.

  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 the antibody H chain due to transfer of the sugar chain was confirmed. Thus, an antibody to which the target asparagine-linked sugar chain was transferred could not be obtained.

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

<Reference Example 1> Sugar chain transfer to sugar chain-cleaved antibody using modified EndoS and commercially available sugar chain oxazoline derivative In Reference Example 1, a commercially available sugar chain oxazoline derivative was used using the modified EndoS prepared in Preparation Example 1. And the sugar chain-cleaved antibody obtained in the step (Example 1-1) of Example 1 was reacted to prepare an antibody having a sugar chain transferred thereto.

  In a 1.5 mL eppendorf tube, an oxazoline sialyl glycan solution (100 μL) prepared from oxazoline sialyl glycan (manufactured by Fushimi Pharmaceutical Co., Ltd.), 100 μL of 100 mg of 50 mg sodium phosphate buffer, pH 7.4, was added. 146), a sugar chain-cleaved antibody solution (146 µL, concentration 3.42 mg / mL, antibody amount 500 µg) prepared from the reaction solution a of the step (Example 1-1) of Example 1; 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 are prepared, and one is 3 hours (reaction solution e1), the next one is 23 hours (reaction solution e2) in a thermostat set at 37 ° C. The last one was allowed to stand for 47 hours (referred to as reaction solution e3). The reaction solution e was prepared using the oxazoline sialyl sugar chain stored at -80 ° C. to prevent the decomposition of the oxazoline sialyl sugar chain, and from the preparation of the oxazoline sialyl sugar chain solution to the sugar chain cleavage type. The addition of the antibody solution and the modified EndoS solution was performed promptly.

  After allowing to stand 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 (Example 1-1) in Example 1 and the obtained desorption washing solution was used. By performing SDS-PAGE and HPLC analysis using an FcγRIIIa column in accordance with the method described in the step (Example 1-1) of Example 1, progress of the transfer reaction of the sialyl sugar chain to the sugar chain-cleaved antibody was confirmed. .

FIG. 8 shows the results of SDS-PAGE. In FIG. 8, Lane 1 shows the antibody (product name Rituxan, manufactured by Zenyaku Kogyo) used for preparing the reaction solution a described in the step (1-A) of Example 1, and Lane 2 shows the antibody of Example 1 described above. In the step (actual 1-1), the cleaning solution for desorption of the reaction solution a, the lane 3 shows the cleaning solution for desorption of the reaction solution e1, the lane 4 shows the cleaning solution for desorption of the reaction solution e2, and the lane 5 shows the cleaning solution for desorption of the reaction solution e3. In addition, SDS-PAGE of each solution used 10% gel (manufactured by ATTO). Comparing lane 3 (reaction solution e1) to lane 5 (reaction solution e3) in FIG. 8, as the standing time at 37 ° C. becomes longer, the ratio of the antibody H chain whose molecular weight increases due to sugar chain transfer gradually increases. , And the proportion of antibody H chains whose molecular weights did not increase gradually increased.

  FIG. 9 shows the results of HPLC analysis of the reaction solutions e1, e2, and e3 using an FcγRIIIa column. In the reaction solution e1 in which the standing time at 37 ° C. was 3 hours, it was revealed that almost all the antibodies interacted with human FcγRIIIa and eluted later. 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 fraction without passing through the interaction with human FcγRIIIa was reacted with the reaction solution e1. It was found that the number of antibodies that eluted later after interacting with human FcγRIIIa decreased compared to the reaction solution e1. Further, in the reaction solution e3 in which the standing time at 37 ° C. is 47 hours, the antibody eluted in the flow-through fraction further increases as compared with the reaction solution e2, and the antibody eluted later by interacting with human FcγRIIIa is eluted. It became clear that it was further reduced as compared with the reaction solution e2. As described above, from the results of FIGS. 8 and 9, the reaction time when a sugar chain-cleaved antibody is reacted with a commercially available sugar chain oxazoline derivative to transfer the sugar chain to the antibody using the modified EndoS is about 3 hours. It turned out to be favorable.

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

Claims (11)

  A solution containing an unpurified sugar chain oxazoline derivative obtained by treating a sugar chain with a dehydrating condensing agent, an asparagine-linked sugar chain-cleaved antibody, and a sugar chain transferase, wherein the sugar chain is reacted. A method for producing an antibody having a modified chain. The production method (A) according to claim 1, comprising the following steps (A) and (B): treating the sugar chain with a dehydrating condensing agent to convert the unpurified sugar chain oxazoline derivative. Step (B) of obtaining a solution containing the unpurified sugar chain oxazoline derivative obtained in (A) above, by reacting the antibody and the glycosyltransferase having the asparagine-linked sugar chain cleaved thereon with the solution containing the sugar chain oxazoline derivative. Obtaining an antibody to which the chain has been transferred.   The method according to claim 2, wherein the steps (A) and (B) are performed in an aqueous solution containing a phosphate. The sugar chain treated with the dehydrating condensing 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 a sugar chain selected from the group consisting of any saccharide). The method according to any one of claims 1 to 3.   The method according to any one of claims 1 to 4, wherein the dehydrating condensing agent is 2-chloro-1,3-dimethylbenzimidazolinium chloride.   The method according to any one of claims 1 to 5, wherein the glycosyltransferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into endo-β-N-acetylglucosaminidase.   The glycosyltransferase is a modified endo-β-N-acetylglucosaminidase obtained by introducing an amino acid mutation into endo-β-N-acetylglucosaminidase derived from Streptococcus pyogenes, according to any one of claims 1 to 6. Production method.   The production method according to any one of claims 1 to 7, wherein the antibody from which the asparagine-linked sugar chain has been cleaved is an antibody obtained by allowing endo-β-N-acetylglucosaminidase to act on the antibody.   The method according to any one of claims 1 to 8, wherein the antibody is an IgG antibody.   The production method according to any one of claims 1 to 9, wherein the antibody is a non-human antibody, a chimeric antibody, a humanized antibody, or a human antibody.   The method according to any one of claims 1 to 10, wherein the antibody having a modified sugar chain is separated using an adsorbent having human FcγRIIIa immobilized on an insoluble carrier.

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