JP2015083558A - Antibody sorbent, and antibody purifying method and antibody identifying method using same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an identifiable method of the presence or absence of the addition of a sugar chain to an antibody and to provide antibodies obtained by the method concerned.SOLUTION: The invention provides a purification method of antibodies having a sugar chain using a sorbent which may specifically adsorb antibodies having a sugar chain, and is obtained by immobilizing, to an insoluble carrier, human FcγRIIIa protein produced by gene recombination technology (a polypeptide comprising at least amino acid residues from glycine in position 17th to glutamine in position 192nd among a human FcγRIIIa amino acid sequence, and having one or more amino acid residues substituted or inserted by other amino acid residues or deleted); a method for identifying the presence or absence of the addition of a sugar chain; and a separation method of a sugar chain.

Description

  The present invention relates to an antibody adsorbent and an antibody purification method and identification method using the adsorbent. In particular, the present invention relates to an adsorbent capable of specifically adsorbing an antibody having a sugar chain among antibodies, a method for purifying an antibody having a sugar chain using the adsorbent, and whether or not a sugar chain is added to the antibody. It relates to a method for identifying.

  In recent years, the development of a medicine (antibody medicine) utilizing the specificity of a monoclonal antibody has been promoted. Among human IgGs used in antibody drugs, ADCC (antibody-dependent cytotoxicity) activity is known to change depending on the difference in the N-type sugar chain added to the 297th asparagine residue in the Fc region. It has been reported that ADCC activity is improved by an antibody from which fucose, which is a kind of the above, is removed (Non-patent Document 1). That is, in antibody medicine, the sugar chain structure possessed by an antibody has an important meaning. However, antibody drugs are usually produced using gene recombination techniques using animal cells as hosts, and it is difficult to control the sugar chains added to the antibodies in the host. In addition, it takes a lot of time and effort to analyze the sugar chain of the produced antibody.

Shinkawa. T. T. et al. , J .; Biol. Chem. , 278, 3466-3473, 2003

  An object of the present invention is to provide a method capable of discriminating the presence or absence of sugar chain addition to an antibody and a material used in the method.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have used an adsorbent obtained by immobilizing FcγRIIIa, which is one of the receptors for IgG (Fcγ receptor), on an insoluble carrier, so that a sugar chain to the antibody can be obtained. The inventors have found that the presence or absence of addition can be identified, and have completed the present invention. That is, the present invention includes the following aspects:
(1) An adsorbent for an antibody having a sugar chain, obtained by immobilizing human FcγRIIIa on an insoluble carrier.

  (2) The adsorbent according to (1), wherein human FcγRIIIa is a polypeptide comprising an amino acid residue from at least the 17th glycine to the 192nd glutamine in the amino acid sequence described in SEQ ID NO: 1.

  (3) The human FcγRIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 1, and at least one of the amino acid residues is a residue of another amino acid. The adsorbent according to (1), which is a polypeptide substituted, inserted or deleted in a group.

(4) human FcγRIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, and the following amino acid residues from the 17th to the 192nd ( The adsorbent according to (3), which is a polypeptide in which at least one amino acid substitution has occurred among A) to (D).
(A) The 66th leucine of SEQ ID NO: 1 is replaced with histidine or arginine (B) The 147th glycine of SEQ ID NO: 1 is replaced with aspartic acid (C) The 158th tyrosine of SEQ ID NO: 1 is replaced with histidine (D ) The 176th valine of SEQ ID NO: 1 is substituted with phenylalanine. (5) A solution containing an antibody having a sugar chain is added to the adsorbent according to any one of (1) to (4) and adsorbed on the adsorbent. And a method for purifying an antibody having a sugar chain, comprising a step of eluting an antibody having a sugar chain adsorbed on the adsorbent with an eluent.

  (6) An antibody obtained by the purification method according to (5).

(7) A method for identifying the presence or absence of sugar chain addition to an antibody using the adsorbent according to any one of (1) to (4).

  (8) A method for separating sugar chains using the adsorbent according to any one of (1) to (4).

  (9) A sugar chain obtained by the separation method according to (8).

  Hereinafter, the present invention will be described in detail.

  The amino acid sequence (SEQ ID NO: 1) of human FcγRIIIa used as a ligand in the adsorbent of the present invention is published in public databases such as UniProt (Accession number: P08637). Similarly, the functional domain in the structure of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the position of the cell membrane penetrating region are also published. FIG. 1 shows a schematic diagram of the structure of human FcγRIIIa. The amino acid numbers in FIG. 1 correspond to the amino acid numbers described in SEQ ID NO: 1. That is, the signal sequence (S) is from the first methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1, and the extracellular region is from the 17th glycine (Gly) to the 208th glutamine (Gln). (EC), from the 209th valine (Val) to the 229th valine (Val) is the transmembrane region (TM) and from the 230th lysine (Lys) to the 254th lysine (Lys) is the intracellular region (C ). Note that the human FcγRIIIa used as a ligand in the adsorbent of the present invention does not necessarily need to use the full length of human FcγRIIIa (SEQ ID NO: 1). Any polypeptide containing amino acid residues up to glutamine may be used. Furthermore, among the amino acid sequence set forth in SEQ ID NO: 1, it contains at least amino acid residues from the 17th glycine to the 192nd glutamine, and one or more of the amino acid residues are substituted with other amino acid residues, Even an inserted or deleted polypeptide is included in human FcγRIIIa used as a ligand in the adsorbent of the present invention. In the natural human FcγRIIIa, the 66th leucine (Leu) is histidine (His) or arginine (Arg), the 147th glycine (Gly) is aspartic acid (Asp), and the 158th tyrosine (Tyr). Is known in which histidine (His) or 176th valine (Val) is substituted with phenylalanine (Phe). Is also included in human FcγRIIIa used as a ligand for the adsorbent of the present invention.

  Human FcγRIIIa, which is a ligand of the adsorbent of the present invention, may be further added with an oligopeptide useful for separation from a solution in the presence of contaminants on the N-terminal side or C-terminal side. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid and the like. In addition, an oligopeptide containing cysteine useful for immobilizing human FcγRIIIa, which is a ligand of the adsorbent of the present invention, on a solid phase such as a support for chromatography is used as a human FcγRIIIa, which is a ligand of the adsorbent of the present invention. It may be further added to the N-terminal side or the C-terminal side of. The length of the oligopeptide added to the N-terminal side or C-terminal side of human FcγRIIIa is not particularly limited as long as it does not impair the IgG binding and stability of human FcγRIIIa, which is the ligand of the adsorbent of the present invention. When the oligopeptide is added to human FcγRIIIa, which is a ligand of the adsorbent of the present invention, a polynucleotide encoding the oligopeptide is prepared and then genetically engineered using N methods of human FcγRIIIa using a method well known to those skilled in the art. The oligopeptide may be added to the terminal side or the C-terminal side, or the chemically synthesized oligopeptide may be added by chemically binding to the N-terminal side or the C-terminal side of human FcγRIIIa. Furthermore, a signal peptide for promoting efficient expression in the host may be added to the N-terminal side of human FcγRIIIa which is the ligand of the adsorbent of the present invention. Examples of the signal peptide in the case where the host is Escherichia coli include PelB, DsbA, MalE (uniprot No. P0AEX9 in the amino acid sequence described in UniProt No. 1 to 26th region), TorT, and other proteins that secrete proteins. A signal peptide can be exemplified (Japanese Patent Laid-Open No. 2011-097898).

As an example of a method for producing a polynucleotide encoding human FcγRIIIa which is a ligand of the adsorbent of the present invention,
(I) a method of artificially synthesizing a polynucleotide containing the nucleotide sequence by converting the amino acid sequence of human FcγRIIIa to a nucleotide sequence,
(II) A polynucleotide containing the entire or partial sequence of human FcγRIIIa is prepared directly or artificially or from a human FcγRIIIa cDNA or the like using a DNA amplification method such as a PCR method, and the prepared polynucleotide is linked by an appropriate method. Can be exemplified.
In the method (I), when converting from an amino acid sequence to a nucleotide sequence, the conversion is preferably performed in consideration of the frequency of codon usage in the host to be transformed. For example, when the host is Escherichia coli, AGA / AGG / CGG / CGA is used for arginine (Arg), ATA is used for isoleucine (Ile), CTA is used for leucine (Leu), and GGA is used for glycine (Gly). However, since CCC is less frequently used in proline (Pro) (because it is a so-called rare codon), it may be converted so as to avoid those codons. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute).

  There are no particular limitations on the host that expresses human FcγRIIIa, which is the ligand of the adsorbent of the present invention. Examples include animal cells (CHO cells, HEK cells, Hela cells, COS cells, etc.), yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe etc., Insect cells (Sf9, Sf21 etc.), E. coli (JW109, etc.) Use of animal cells or Escherichia coli as a host is preferable in terms of productivity, and more preferable when Escherichia coli is used as a host.

  In preparing the adsorbent of the present invention, the insoluble carrier used for immobilizing human FcγRIIIa is not particularly limited, and polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, and starch. And a carrier made of a synthetic polymer such as polyvinyl alcohol, polymethacrylate, poly (2-hydroxyethyl methacrylate) and polyurethane, and a carrier made of ceramics such as silica. Of these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carrier include polymethacrylate gels introduced with hydroxyl groups such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC). . The shape of the insoluble carrier is not particularly limited, and may be granular or non-particulate, porous or non-porous.

  In order to immobilize human FcγRIIIa on an insoluble carrier, N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group haloacetamide, etc. What is necessary is just to fix | immobilize by providing an active group and covalently bonding human FcγRIIIa and an insoluble carrier through the active group. The carrier provided with the active group may be a commercially available carrier as it is, or may be prepared by introducing an active group on the surface of the carrier under appropriate reaction conditions. Commercially available carriers to which an active group has been added include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4F, Examples thereof include 6B (both manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Scientific).

  On the other hand, examples of the method for introducing an active group on the surface of the carrier include a method in which one of compounds having two or more active sites reacts with a hydroxyl group, an epoxy group, a carboxyl group, an amino group, etc. present on the surface of the carrier. it can. Among examples of the compound, examples of the compound that introduces an epoxy group into the hydroxyl group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of the compound that introduces an epoxy group on the carrier surface with the compound and then introduces a carboxyl group on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3- Examples thereof include aminopropionic acid, 4-aminobutyric acid and 6-aminohexanoic acid.

  Examples of the compound that introduces a maleimide group into a hydroxyl group, epoxy group, carboxyl group, or amino group present on the surface of the carrier include N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidopropionic acid) hydrazide, 4- [ 4-N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [α-maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N-hydroxysuccinimide ester, Succinimidyl-4- [maleimidomethyl] cyclohexane-1-carbonyl- [6-aminohexanoic acid]), (succinimidyl-4- [maleimidomethyl] cyclohexane-1-carboxylic acid), (p-maleimidobenzoyl) N-hydroxysuccinimide Examples of the ester include (m-maleimidobenzoyl) N-hydroxysuccinimide ester.

  Compounds that introduce a haloacetyl group into the hydroxyl group or amino group present on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated. An example is a method in which a ω-alkenyl alkanglycidyl ether is reacted with a hydroxyl group or amino group present on the surface of the carrier, and then the ω-alkenyl moiety is halogenated with a halogenating agent to activate. Examples of ω-alkenyl alkanglycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.

  As another example of the method for introducing an active group on the surface of the carrier, there is a method for introducing an activating group into the carboxyl group present on the surface of the carrier using a condensing agent and an additive. Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

  Buffers used when immobilizing human FcγRIIIa on an insoluble carrier include acetate buffer, phosphate buffer, MES (2-Morpholineethanesulfide acid) buffer, HEPES (2- [4- (2-Hydroxyethyl) -1- piperazinyl] ethanesulfonic acid) buffer, Tris buffer, borate buffer. The reaction temperature at the time of immobilization may be appropriately set in the temperature range from 5 ° C. to 50 ° C. in consideration of the reactivity of the active group and the stability of human FcγRIIIa, preferably 10 ° C. to 35 ° C. Range.

  In order to purify an antibody having a sugar chain using the adsorbent of the present invention obtained by immobilizing human FcγRIIIa on an insoluble carrier, for example, an antibody having a sugar chain in a column packed with the adsorbent of the present invention After adding a buffer solution containing a solution using a liquid delivery means such as a pump to specifically adsorb an antibody having a sugar chain to the adsorbent of the present invention, an appropriate eluate is added to the column. Thus, the antibody having a sugar chain may be eluted. The antibody having a sugar chain that can be purified by the adsorbent of the present invention may be any antibody that has at least the Fc region of an antibody having a sugar chain that has affinity for an Fc receptor such as FcγRIIIa. Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substitutions thereof that are generally used as antibodies used in antibody pharmaceuticals. In addition, bispecific antibodies (bispecific antibodies), Fc regions of antibodies with sugar chains and fusion proteins of other proteins, Fc regions of antibodies with sugar chains and drugs (ADC), etc. Even an antibody whose structure has been artificially modified can be purified with the adsorbent of the present invention. In addition, it is preferable to equilibrate the column with an appropriate buffer before adding a buffer containing an antibody having a sugar chain to the column, because the antibody having a sugar chain can be purified with higher purity. Examples of the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution, and the pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8.

  In order to elute the antibody having a sugar chain adsorbed on the adsorbent of the present invention, the interaction between the antibody having a sugar chain and the ligand (human FcγRIIIa) may be weakened. Can be exemplified by pH change, counter peptide, temperature change, and salt concentration change. As a specific example of the eluate for eluting the antibody having a sugar chain adsorbed to the adsorbent of the present invention, it is more acidic than the solution used for adsorbing the antibody having a sugar chain to the adsorbent of the present invention. Side buffer. Examples of the buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side. The pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.

  When the antibody is purified from a solution containing an antibody having a sugar chain using the adsorbent of the present invention, the elution position (elution fraction) of the antibody varies depending on the sugar chain structure of the antibody. Therefore, by separating the antibody using the adsorbent of the present invention, the difference in the sugar chain structure of the antibody can be identified. There are no particular limitations on the structure of the glycans that can be identified. For example, sugar chains added when antibodies are expressed using animal-derived cells such as CHO cells, yeasts such as Pichia yeast and Saccharomyces yeast, and humans. Examples include sugar chains possessed by antibodies and sugar chains added to antibodies by chemical synthesis methods. Moreover, since the adsorbent of the present invention can be separated based on the difference in the sugar chain structure of the antibody, it can also be used for separation of the sugar chain itself.

  As described above, the adsorbent of the present invention can identify the difference in the sugar chain structure of the antibody. As a ligand protein used in the adsorbent, an Fc receptor other than FcγRIIIa (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb, FcRn) was used. Even in this case, the difference in the sugar chain structure can be similarly identified.

  The present invention relates to an adsorbent obtained by immobilizing human FcγRIIIa on an insoluble carrier, and the adsorbent specifically adsorbs an antibody having a sugar chain among antibodies, and thus has been difficult so far. The presence / absence of sugar chain addition to the antibody can be easily identified. In addition, since the antibody having a sugar chain can be specifically purified by using the adsorbent of the present invention, it is possible to efficiently produce an antibody having a sugar chain.

1 is a schematic diagram of human FcγRIIIa. The numbers in the figure indicate the amino acid sequence numbers described in SEQ ID NO: 1. In the figure, S represents a signal sequence, EC represents an extracellular region, TM represents a transmembrane region, and C represents an intracellular region. It is the figure which compared molecular weight by SDS-PAGE with human IgG1 (sugar chain-equipped human IgG1) having a sugar chain and human IgG1 (sugar chain-removed human IgG1) from which a sugar chain was removed. Lane (1) in the figure is a human IgG1 with a sugar chain, and lane (2) is a human IgG1 with a sugar chain removed. It is the figure which compared the binding property of human FcγRIIIa and protein A to human IgG1 with sugar chain and human IgG1 with sugar chain removed. (A) shows the results for human FcγRIIIa, and (B) shows the results for protein A. It is the result of separating the human IgG1 with sugar chain and the human IgG1 with sugar chain removed using the adsorbent of the present invention. (1) is the result for human IgG1 with a sugar chain, and (2) is the result for human IgG1 with a sugar chain removed.

Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to the examples.

Example 1 Preparation of Human FcγRIIIa Expression Vector (1) Among the human FcγRIIIa amino acid sequences described in SEQ ID NO: 1, based on the amino acid sequence from the 17th glycine (Gly) to the 192nd glutamine (Gln), the DNAworks method (Nucleic Acids Res., 30, e43, 2002) was used to design a nucleotide sequence in which the codon was converted from human type to E. coli type. The designed nucleotide sequence is shown in SEQ ID NO: 2.
(2) In order to prepare a polynucleotide containing the sequence shown in SEQ ID NO: 2, an oligonucleotide consisting of the sequences shown in SEQ ID NO: 3 to 20 was synthesized, and the two-step PCR shown below using the oligonucleotide Was done.
(2-1) In the first stage PCR, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, and 5 ° C. at 62 ° C. A polynucleotide was synthesized by repeating a reaction in which the second step for 2 seconds and the third step for 90 seconds at 72 ° C. were repeated 10 cycles, and this was designated as FcRp1. The DNA mix in Table 1 means a solution obtained by sampling a predetermined amount of each of 18 types of oligonucleotides having the sequences described in SEQ ID NOs: 3 to 20 and mixing them.

（２−２）二段階目のＰＣＲは、（２−１）で合成したＦｃＲｐ１を鋳型とし、配列番号２１（５’−ＴＡＧＣＣＡＴＧＧＧＣＡＴＧＣＧＴＡＣＣＧＡＡＧＡＴＣＴＧＣＣＧＡＡＡＧＣ−３’）および配列番号２２（５’−ＣＣＣＡＡＧＣＴＴＡＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＧＣＣＣＣＣＴＴＧＧＧＴＡＡＴＧＧＴＡＡＴＡＴＴＣＡＣＧＧＴＣＴＣＧＣＴＧＣ−３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして実施した。具体的には、表２に示す組成の反応液を調製し、当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、６２℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。

(2-2) The second-stage PCR uses FcRp1 synthesized in (2-1) as a template, SEQ ID NO: 21 (5′-TAGCCCATGGGCATGCCGTACGAGAATCTGCCCGAAAGCTGCTATGGATCCTGCTGGGTATCG An oligonucleotide consisting of the sequence described in 1 was used as a PCR primer. Specifically, a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was heat treated at 98 ° C. for 5 minutes, followed by a first step at 98 ° C. for 10 seconds, a second step at 62 ° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at ° C. for 30 cycles.

（３）（２）で得られたポリヌクレオチドを精製し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１−２０６０４６号公報）にライゲーションし、当該ライゲーション産物を用いて大腸菌ＢＬ２１株（ＤＥ３）を形質転換した。
（４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンンを含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いて、発現ベクターｐＥＴ−ｅＦｃＲ３を抽出した。
（５）（４）で作製した発現ベクターｐＥＴ−ｅＦｃＲ３のうち、ＦｃγＲＩＩＩａをコードするポリヌクレオチドおよびその周辺の領域について、チェーンターミネータ法に基づくＢｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ＦＳ ｒｅａｄｙ Ｒｅａｃｔｉｏｎ ｋｉｔ（ライフサイエンス社製）を用いてサイクルシークエンス反応に供し、全自動ＤＮＡシークエンサーＡＢＩ Ｐｒｉｓｍ ３７００ ＤＮＡ ａｎａｌｙｚｅｒ（ライフサイエンス社製）にてヌクレオチド配列を解析した。なお当該解析の際、配列番号２３（５’−ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧ−３’）または配列番号２４（５’−ＴＡＴＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧ−３’）に記載のオリゴヌクレオチドをシークエンス用プライマーとして使用した。

(3) The polynucleotide obtained in (2) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Application Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. Escherichia coli BL21 strain (DE3) was transformed with the ligation product.
(4) After culturing the obtained transformant in an LB medium containing 50 μg / mL kanamycin, the expression vector pET-eFcR3 was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) In the expression vector pET-eFcR3 prepared in (4), the Big Dye Terminator Cycle Sequencing Reaction Kit based on the chain terminator method for the polynucleotide encoding FcγRIIIa and its surrounding region (manufactured by Life Sciences) The nucleotide sequence was analyzed using a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Sciences). In the analysis, the oligonucleotide described in SEQ ID NO: 23 (5′-TAATACGACTCACTATAGGGG-3 ′) or SEQ ID NO: 24 (5′-TATGCTAGTTATTGCTCAG-3 ′) was used as a sequencing primer.

  The amino acid sequence of the polypeptide expressed by the expression vector pET-eFcR3 is shown in SEQ ID NO: 25, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26, respectively. In SEQ ID NO: 25, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. , From the 33rd glycine (Gly) to the 208th glutamine (Gln) is the extracellular region of human FcγRIIIa (the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine ( Gly) is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.

Example 2 Preparation of sugar chain-removed human IgG1 N-type sugar chains were removed from human IgG1 by the method shown below.
(1) It was diluted with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of human IgG1 (Fitzgerald: 31-AI17) was 3 mg / mL.
(2) 100 μL of 1M Tris-HCl buffer (pH 8.6) was added to 100 μL of the diluted solution of (1), 10 μL of N-glycosidase F (500 mU / μL (Takara Bio Inc .: 4450) was added, and then 37 ° C. The N-type sugar chain of IgG1 was removed by allowing to stand for 24 hours.
(3) Open column (manufactured by Bio-Rad) filled with 100 μL of Toyopearl AF-r Protein A-650F (manufactured by Tosoh Corporation: 22803) equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride ) Was applied with the treatment solution of (2).
(4) 20 mM Tris-HCl buffer (pH) containing 500 μL of 150 mM sodium chloride
7.4) was washed 3 times, and then eluted with 100 μL of 0.1 M glycine hydrochloride buffer (pH 3.0) 7 times to remove human IgG1 from which N-type sugar chains were removed (hereinafter simply referred to as humans from which sugar chains had been removed). IgG
1) was purified. The eluate was neutralized by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0).
(5) The same amount of sample buffer (2 (w / v)% sodium dodecyl sulfate, 6 (w / v)% β-mercaptoethanol, (W / v)% glycerin and 0.005 (w / v)% bromophenol blue-containing 50 mM Tris-HCl buffer (pH 6.8)) and heat-treated, thereby removing human IgG1 from which sugar chains were removed. Reduced.
(6) Human IgG1 was separated by electrophoresis using a 5 to 20% gradient SDS-PAGE gel (manufactured by Ato). For comparison, an aqueous solution (concentration: 0.5 mg / mL) of human IgG1 that has not been subjected to sugar chain treatment (hereinafter referred to as human IgG1 with sugar chain) was subjected to the same reduction treatment as described in (5), and SDS- Separated by PAGE.

  The results are shown in FIG. Since the N-type sugar chain is removed from the heavy chain of human IgG1 having a sugar chain removed, the molecular weight is lower than that of the heavy chain of human IgG1 having a sugar chain, but this was confirmed by SDS-PAGE (FIG. 2). Lane (2)). That is, it was confirmed that human IgG1 from which the N-type sugar chain was removed could be prepared by the method of this example.

Example 3 Large-scale preparation of human FcγRIIIa (1) 400 mL of 2YT liquid medium containing 50 μg / mL kanamycin in a 2 L baffle flask containing the transformant capable of expressing human FcγRIIIa obtained in Example 1 (3) (Peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) was inoculated, and precultured by aerobic shaking culture at 37 ° C. overnight.
(2) Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and kanamycin sulfate 50 mg 180 L of the culture solution of (1) was inoculated into 1.8 L of a liquid medium containing / L, and main culture was performed using a 3 L fermentor. The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation. The pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali. The dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. . When the glucose concentration could not be measured after the start of culture, fed-batch medium (glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L) was dissolved in dissolved oxygen (DO ) Was added while controlling.
(3) When the absorbance at 600 nm (OD _{600 nm} ) reaches approximately 150 as a measure of the amount of bacterial cells, the culture temperature is lowered to 25 ° C., and after confirming that the set temperature has been reached, the final concentration becomes 0.5 mM. IPTG (isopropyl-β-thiogalactopyranoside) was added, and the culture was continued at 25 ° C.
(4) About 48 hours after the start of culture, the culture was stopped, and the cells were collected by centrifugation at 8000 rpm for 20 minutes.
(5) The cells recovered in (4) are suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration is 5 mL / 1 g (cells). The cells were crushed with a power of about 150 W at 4 ° C. for about 10 minutes using a netter 201M (trade name, manufactured by Kubota Corporation). The cell disruption solution was centrifuged twice at 10,000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
(6) After adding imidazole to the disrupted solution obtained in (5) to a final concentration of 20 mM, equilibrate with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole in advance. Applied to an XK 26/20 column (GE Healthcare) packed with 50 mL of Ni Sepharose 6 Fast Flow (GE Healthcare).
(7) After washing with the buffer used for equilibration, human FcγRIIIa was eluted using 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 0.5 M imidazole.
(8) HR filled with 10 mL of IgG Sepharose (GE Healthcare) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride in advance. This was applied to a 16/10 column (manufactured by GE Healthcare). After washing with the buffer used for equilibration, human FcγRIIIa was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was returned to near neutrality by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0).

Example 4 Measurement of binding of human FcγRIIIa to antibody (1) Human FcγRIIIa prepared in Example 3 was treated with phosphate buffer (137 mM NaCl, 8.1 M Na ₂ HPO ₄ , 2.68 mM KCl and 1. The buffer was exchanged by dialysis with 47 mM KH ₂ PO ₄ (pH 7.4 buffer), and the concentration of human FcγRIIIa was measured from the absorbance at 280 nm.
(2) The human FcγRIIIa whose concentration was measured in (1) was diluted to 10 μg / mL with 20 mM acetate buffer (pH 5.5), and then sensor chip CM5 (by GE Healthcare) was used using an amine coupling kit (manufactured by GE Healthcare). The amount of human FcγRIIIa immobilized was measured using Biacore T-100 (GE Healthcare). As a result, the immobilized amount of human FcγRIIIa was 488.2 RU (1RU = 1 pg / mm ² ). Similarly, protein A (manufactured by Protenova) was diluted to 10 μg / mL with 20 mM acetate buffer (pH 5.5) and immobilized on CM5 (manufactured by GE Healthcare). As a result of measuring the amount of immobilization with Biacore T-100, the amount of immobilization was 290.0 RU.
(3) Sugar chain-containing human IgG1 and sugar chain-removed human IgG1 prepared in Example 2 were mixed with HBS-EP (+) (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.005 (v / v)% Surfactant P20 (manufactured by GE Healthcare, pH 7.4 solution) 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL Dilute to mL.
(4) Among the protein-immobilized chips prepared in (2), in the case of the human FcγRIIIa-immobilized chip, 4 μg / mL to 128 μg / mL sugar chain-containing human IgG1 or sugar chain-removed human IgG1 is flowed at a flow rate of 30 μL / min. In the case of a protein A-immobilized chip, human IgG1 with a sugar chain of 1 μg / mL to 16 μg / mL or human IgG1 with a sugar chain removed is flowed at a flow rate of 30 μL / min to bind human IgG1 and the protein immobilized on the chip. Thereafter, the binding between human IgG1 and the protein immobilized on the chip was measured by measuring with Biacore T-100 under conditions of a contact time of 210 seconds and a dissociation time of 400 seconds.

  The measurement results are shown in FIG. It can be seen that human FcγRIIIa binds to human IgG1 with a sugar chain, but is extremely difficult to bind to human IgG1 with a sugar chain removed (see FIG. 3 (A)). That is, it can be seen from this result that human FcγRIIIa recognizes the difference due to the addition of a sugar chain to the antibody. On the other hand, it can be seen that protein A has the same binding ability to human IgG1 with or without sugar chain (see FIG. 3B).

Example 5 Preparation of human FcγRIIIa-immobilized gel (1) Human FcγRIIIa prepared in Example 3 was concentrated and buffer exchanged using an ultrafiltration membrane (Millipore: Amicon Ultra-15). The solution was concentrated to a concentration of 2.6 mg / mL in 20 mM Tris-HCl buffer (pH 7.4) containing sodium chloride.
(2) 1,6-hexanediol diglycidyl ether was reacted with the hydroxyl group of a hydrophilic vinyl polymer (Tosoh Corporation: Toyopearl) as a carrier to prepare an epoxy toyopearl gel.
(3) Put 70 μL of the epoxy toyopearl gel prepared in (2) into a spin column (Bio-Rad) and add 0.5 mL of 0.1 M borate buffer (pH 9.0) containing 0.5 M sodium chloride. Washed 3 times.
(4) A solution prepared by mixing 0.4 mL of the human FcγRIIIa solution prepared in (1) with 0.6 mL of 0.1 M borate buffer (pH 9.0) containing 0.5 M sodium chloride (2 ) And shaken at 35 ° C. for 3 hours.
(5) The gel prepared in (4) was washed three times with 0.2 mL of 0.1 M glycine hydrochloride buffer (pH 3.0), and then 20 mM Tris hydrochloride buffer (pH 7.5) containing 150 mM sodium chloride. 4) The pH was returned to near neutrality by washing with 0.5 mL three times to prepare 0.2 mL of human FcγRIIIa-immobilized gel.
(6) The protein concentration contained in the solution and washing solution added to the gel of (2) was measured, and the amount of human FcγRIIIa immobilized on the gel was calculated to calculate the immobilization rate. 84% of this was immobilized on the gel.

Example 6 Antibody Separation Using Human FcγRIIIa Immobilized Gel (1) The human FcγRIIIa immobilized gel prepared in Example 5 was packed into an HR 16/5 column (manufactured by GE Healthcare), and the column was subjected to liquid chromatography. It connected to apparatus AKTAprime (made by GE Health).
(2) The column prepared in (1) was equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and prepared with human IgG1 (Fitzgerald: 31-AI17) or Example 2. 0.1 mL of the sugar chain-removed human IgG1 (the solution concentration was 1 mg / mL) was added at a flow rate of 0.1 mL / min. After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.5).

  The results are shown in FIG. Despite the flow of the same amount of IgG1, sugar chain-removed human IgG1 (see FIG. 4 (2)) does not adsorb to the gel as compared to human IgG1 with sugar chain (see FIG. 4 (1)). It can be confirmed that the amount of antibody passing through is large. In human IgG1 with sugar chain, when 0.1 M glycine-hydrochloric acid buffer (pH 3.5) was added, the antibody was eluted (see FIG. 4 (1)), but in the case of human IgG1 with sugar chain removed, almost no gel was present. The antibody was not eluted because it was not adsorbed (see FIG. 4 (2)). In other words, the adsorbent obtained by immobilizing human FcγRIIIa on an insoluble carrier has the ability to specifically adsorb an antibody having a sugar chain, and this ability can be used to add a sugar chain to the antibody. It can be seen that the presence or absence can be identified.

  Since the adsorbent of the present invention specifically adsorbs an antibody having a sugar chain, the antibody having a sugar chain can be separated and purified with high purity. Therefore, the adsorbent of the present invention can be used for antibody drug production and quality control.

Claims (9)

An adsorbent for an antibody having a sugar chain, obtained by immobilizing human FcγRIIIa on an insoluble carrier. The adsorbent according to claim 1, wherein the human FcγRIIIa is a polypeptide comprising an amino acid residue from at least the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1. Human FcγRIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine of the amino acid sequence shown in SEQ ID NO: 1, and one or more of the amino acid residues are substituted with other amino acid residues The adsorbent according to claim 1, which is an inserted or deleted polypeptide. Human FcγRIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid residues from the 17th to the 192nd are as follows (A) The adsorbent according to claim 3, wherein the adsorbent is a polypeptide in which at least one amino acid substitution in (D) occurs.
(A) The 66th leucine of SEQ ID NO: 1 is replaced with histidine or arginine (B) The 147th glycine of SEQ ID NO: 1 is replaced with aspartic acid (C) The 158th tyrosine of SEQ ID NO: 1 is replaced with histidine (D ) Substitution of 176th valine of SEQ ID NO: 1 with phenylalanine A step of adding a solution containing an antibody having a sugar chain to the adsorbent according to any one of claims 1 to 4 and adsorbing the adsorbent to the adsorbent, and eluting the antibody having a sugar chain adsorbed on the adsorbent A method for purifying an antibody having a sugar chain, comprising a step of eluting with a solution. An antibody obtained by the purification method according to claim 5. A method for identifying the presence or absence of sugar chain addition to an antibody using the adsorbent according to claim 1. A method for separating sugar chains using the adsorbent according to claim 1. A sugar chain obtained by the separation method according to claim 8.

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