JP6977536B2 - Method for evaluating the degree of antibody denaturation using gel filtration chromatography - Google Patents
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Description
本発明は、抗体の変性度合をゲル濾過クロマトグラフィを用いて評価する方法に関する。 The present invention relates to a method for evaluating the degree of denaturation of an antibody using gel filtration chromatography.
抗体医薬品市場の成長に伴い、安定性試験や品質評価などの規制への迅速な技術対応が求められている。抗体医薬品をはじめとする生物薬品は生体による生合成過程を生産に利用していることから、原理的に分子構造上不均一なものが産生される可能性が存在する。故に生物薬品の規格（非特許文献１）には、開発段階での広範かつ詳細な特性解析が必要であることが記載されており、前記特性解析の一つとして特定の生物学的効果を発揮するための特異的な機能やその程度を表す生物活性をあげている。 With the growth of the antibody drug market, prompt technical response to regulations such as stability tests and quality evaluations is required. Since biopharmaceuticals such as antibody drugs utilize the biosynthetic process by living organisms for production, there is a possibility that non-uniform molecular structures will be produced in principle. Therefore, the biopharmaceutical standard (Non-Patent Document 1) states that extensive and detailed characteristic analysis is required at the development stage, and exhibits a specific biological effect as one of the characteristic analyzes. It raises the specific function to do so and the biological activity that expresses its degree.
抗体医薬品の生物活性、すなわち抗体（免疫グロブリン）の結合活性の評価方法として、従来よりＥＬＩＳＡ（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）法や表面プラズモン共鳴法を用いた方法が知られている。しかしながら、これらの方法は、酵素やセンサーチップなどの高価な試薬や備品が必要なこと、および複数の工程を必要とし操作が煩雑なことが問題であった。 As a method for evaluating the biological activity of an antibody drug, that is, the binding activity of an antibody (immunoglobulin), a method using an ELISA (Enzyme-Linked ImmunoSorbent Assay) method or a surface plasmon resonance method has been conventionally known. However, these methods have problems that they require expensive reagents and equipment such as enzymes and sensor chips, and that they require a plurality of steps and are complicated to operate.
本発明の課題は、抗体の結合活性より変性度合を従来よりも短時間かつ簡便に評価可能な方法を提供することにある。 An object of the present invention is to provide a method capable of evaluating the degree of denaturation based on the binding activity of an antibody in a shorter time and more easily than before.
上記課題を解決するために本発明者らは、抗体を含む溶液に当該抗体のＦｃ領域と結合可能なタンパク質を添加し、前記抗体と前記タンパク質との複合体を形成させることで、ゲル濾過クロマトグラフィを用いた短時間かつ簡便な方法で抗体の結合活性および変性度合を評価できることを見出し、本発明を完成させるに至った。すなわち本発明は以下の［１］〜［３］の態様を包含する。
［１］
（１）一定量の未変性抗体Ａを含む溶液に、抗体ＡのＦｃ領域と結合可能なタンパク質を一定量添加し、抗体Ａと前記タンパク質との複合体を形成させる工程、
（２）（１）の工程で得られた溶液をゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程、
（３）（２）の工程で得られたクロマトグラムのうち、前記複合体または前記複合体を形成しない前記タンパク質に相当するピークの高さまたは面積を算出する工程、
（４）一定量の被検抗体Ａを含む溶液に、抗体ＡのＦｃ領域と結合可能なタンパク質を一定量添加し、抗体Ａと前記タンパク質との複合体を形成させる工程、
（５）（４）の工程で得られた溶液をゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程、
（６）（５）の工程で得られたクロマトグラムのうち、前記複合体または前記複合体を形成しない前記タンパク質に相当するピークの高さまたは面積を算出する工程、および
（７）（３）の工程での算出結果と（６）の工程での算出結果とを比較する工程、
を含む、被検抗体Ａの変性度合を評価する方法。
［２］
抗体のＦｃ領域と結合可能なタンパク質が、以下の（ｉ）〜（ｉｉｉ）から選択される何れかである、［１］に記載の評価方法：
（ｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を少なくとも含むポリペプチド
（ｉｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基において１〜１０個のアミノ酸残基の置換、欠失、挿入、または付加を含むアミノ酸配列を含み、かつ、抗体結合活性を有するタンパク質
（ｉｉｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基からなるアミノ酸配列に対して、９０％以上の相同性を有するアミノ酸配列を含み、かつ、抗体結合活性を有するタンパク質。
［３］
様々な濃度の未変性抗体Ａについて前記工程（１）〜（３）を行い、前記複合体量または前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質量と前記抗体量とで検量線Ｉを作成する工程、
様々な濃度の被検抗体Ａについて前記工程（４）〜（７）を行い、前記複合体量または前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質量と前記抗体量とで検量線ＩＩを作成する工程、並びに
前記検量線ＩおよびＩＩを比較する工程
を含む、［１］又は［２］に記載の評価方法。
In order to solve the above problems, the present inventors add a protein capable of binding to the Fc region of the antibody to a solution containing the antibody to form a complex of the antibody and the protein, thereby performing gel filtration chromatography. We have found that the binding activity and the degree of denaturation of an antibody can be evaluated by a simple method in a short time using the above, and have completed the present invention. That is, the present invention includes the following aspects [1] to [3].
[1]
(1) A step of adding a certain amount of a protein capable of binding to the Fc region of antibody A to a solution containing a certain amount of undenatured antibody A to form a complex of antibody A and the protein.
(2) A step of subjecting the solution obtained in the step (1) to gel filtration chromatography to obtain a chromatogram.
(3) Of the chromatograms obtained in the step (2), the step of calculating the height or area of the peak corresponding to the complex or the protein that does not form the complex.
(4) A step of adding a certain amount of a protein capable of binding to the Fc region of antibody A to a solution containing a certain amount of test antibody A to form a complex of antibody A and the protein.
(5) A step of subjecting the solution obtained in the step (4) to gel filtration chromatography to obtain a chromatogram.
(6) Of the chromatograms obtained in the steps (5), the steps of calculating the height or area of the peak corresponding to the complex or the protein that does not form the complex, and (7) (3). A step of comparing the calculation result in the step (6) with the calculation result in the step (6),
A method for evaluating the degree of denaturation of the test antibody A, which comprises.
[2]
The evaluation method according to [1], wherein the protein capable of binding to the Fc region of the antibody is selected from the following (i) to (iii).
(I) Polypeptide containing at least the amino acid residues from the 33rd glycine to the 208th glutamine in the amino acid sequence shown in SEQ ID NO: 2 (ii) From the 33rd glycine in the amino acid sequence shown in SEQ ID NO: 2. To protein (iii) SEQ ID NO: 2, which contains an amino acid sequence containing substitutions, deletions, insertions, or additions of 1 to 10 amino acid residues in the amino acid residues up to the 208th glutamine and has antibody-binding activity. A protein containing an amino acid sequence having 90% or more homology with respect to an amino acid sequence consisting of amino acid residues from the 33rd glycine to the 208th glutamine among the described amino acid sequences, and having antibody binding activity.
[3]
The steps (1) to (3) are performed on the unmodified antibody A having various concentrations, and the amount of the complex or the amount of protein that can bind to the Fc region of the antibody that does not form the complex and the amount of the antibody are used for calibration. The process of creating line I,
The steps (4) to (7) are performed on the test antibody A having various concentrations, and the amount of the complex or the amount of protein that can bind to the Fc region of the antibody that does not form the complex and the amount of the antibody are used for calibration. The evaluation method according to [1] or [2], which comprises a step of producing a line II and a step of comparing the calibration curves I and II.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
抗体を含む溶液に前記抗体のＦｃ領域と結合可能なタンパク質を添加すると、前記抗体と前記タンパク質との複合体が形成される。前記複合体と前記タンパク質とは分子量が異なるため、ゲル濾過クロマトグラフィに供することで前記複合体と前記タンパク質とを分離できる。ここで用いるゲル濾過クロマトグラフィ用担体は抗体や前記抗体のＦｃ領域と結合可能なタンパク質および前記抗体と前記タンパク質との複合体に対し反応性や吸着性をもたないものであればよく、個々の試料成分を分離するために十分な細孔径を有していればよい。例えば、担体の平均粒子径は２μｍ〜１０μｍ、より好ましくは４μｍ〜８μｍであり、担体の平均細孔径は２０ｎｍ〜５０ｎｍ、より好ましくは２５ｎｍ〜３０ｎｍであってよいが、これらに限定されない。本発明は、当該分離で得られた前記複合体または（前記複合体を形成しない）前記抗体もしくは前記タンパク質を定量することで、抗体の結合活性を評価する。なお本抗体結合活性評価を、前記複合体を形成しない前記抗体または前記タンパク質を定量することで評価する場合、抗体が前記抗体のＦｃ領域と結合可能なタンパク質に対し過剰量の場合は前記複合体を形成しない前記抗体を定量すればよく、抗体に対し前記抗体のＦｃ領域と結合可能なタンパク質が過剰量の場合は前記複合体を形成しない前記タンパク質を定量すればよい。 When a protein capable of binding to the Fc region of the antibody is added to the solution containing the antibody, a complex of the antibody and the protein is formed. Since the complex and the protein have different molecular weights, the complex and the protein can be separated by subjecting them to gel filtration chromatography. The carrier for gel filtration chromatography used here may be an antibody, a protein capable of binding to the Fc region of the antibody, or a complex of the antibody and the protein, as long as it does not have reactivity or adsorptivity. It suffices to have a pore diameter sufficient to separate the sample components. For example, the average particle size of the carrier may be 2 μm to 10 μm, more preferably 4 μm to 8 μm, and the average pore size of the carrier may be 20 nm to 50 nm, more preferably 25 nm to 30 nm, but is not limited thereto. The present invention evaluates the binding activity of an antibody by quantifying the complex or the antibody (which does not form the complex) or the protein obtained by the separation. When the antibody binding activity is evaluated by quantifying the antibody or the protein that does not form the complex, the complex is used when the antibody is in excess of the protein capable of binding to the Fc region of the antibody. The antibody that does not form the complex may be quantified, and if the amount of the protein that can bind to the Fc region of the antibody is excessive with respect to the antibody, the protein that does not form the complex may be quantified.
抗体のＦｃ領域と結合可能なタンパク質としては、抗体精製用アフィニティリガンドとして広く用いられている黄色ブドウ球菌由来のＰｒｏｔｅｉｎ Ａ、連鎖球菌由来のＰｒｏｔｅｉｎ Ｇ、Ｐｅｐｔｏｓｔｒｅｐｔｏｃｏｃｃｕｓ ｍａｇｎｕｓ由来のＰｒｏｔｅｉｎ Ｌ、Ｆｃ受容体が例示できる。Ｆｃ受容体は、抗体が単に結合するための器としての役割だけでなく生体内の免疫機構に関与するタンパク質であり、ＩｇＧに対する受容体であるＦｃγ受容体、ＩｇＥに対する受容体であるＦｃε受容体、ＩｇＡに対する受容体であるＦｃα受容体、等いくつかのサブタイプに分類される。また各サブタイプの受容体は更に細かく分類されており、例えばＦｃγ受容体は、ＦｃγＲＩ、ＦｃγＲＩＩａ、ＦｃγＲＩＩｂ、ＦｃγＲＩＩＩａ、ＦｃγＲＩＩＩｂの存在が報告されている（Ｔａｋａｉ．Ｔ．，Ｊｐｎ．Ｊ．Ｃｌｉｎ．Ｉｍｍｕｎｏｌ．，２８，３１８−３２６，２００５）。 Examples of proteins that can bind to the Fc region of an antibody include Protein A derived from yellow staphylococcus, Protein L derived from Peptostreptococcus magnus, and Fc receptor, which are widely used as affinity ligands for antibody purification. can. Fc receptors are proteins that are involved not only as a vessel for antibodies to bind but also in the immune system in the body, and are Fcγ receptors that are receptors for IgG and Fcε receptors that are receptors for IgE. , Fcα receptor, which is a receptor for IgA, etc., are classified into several subtypes. Receptors of each subtype are further subdivided, for example, Fcγ receptors have been reported to have the presence of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb (Takai.T., Jpn.J.Clin.Immunol). ., 28, 318-326, 2005).
抗体（免疫グロブリン）がＩｇＧであり、抗体のＦｃ領域と結合可能なタンパク質がＦｃγ受容体の場合、当該Ｆｃγ受容体として、ＦｃγＲＩ、ＦｃγＲＩＩａ、ＦｃγＲＩＩｂ、ＦｃγＲＩＩＩａ、ＦｃγＲＩＩＩｂのいずれも用いることができる。前記Ｆｃγ受容体の好ましい態様として、
（Ａ）配列番号１に記載のヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）のアミノ酸配列のうち１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を少なくとも含む、Ｆｃ結合性タンパク質、および
（Ｂ）配列番号１に記載のヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）のアミノ酸配列のうち１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を少なくとも含み、ただし当該１７番目から１９２番目までのアミノ酸残基において、１以上のアミノ酸残基が欠失、他のアミノ酸残基に置換、または付加されたポリペプチドを含む、Ｆｃ結合性タンパク質、
があげられる。また前記（Ｂ）の好ましい態様として、
配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を少なくとも含むポリペプチドを含む、Ｆｃ結合性タンパク質、
配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を少なくとも含み、ただし当該３３番目から２０８番目までのアミノ酸残基において、１９２番目のバリンがフェニルアラニンに置換されたポリペプチドを含む、Ｆｃ結合性タンパク質、
特開２０１５−０８６２１６号公報で開示のＦｃ結合性タンパク質、
特開２０１６−１６９１９７号公報で開示のＦｃ結合性タンパク質、
特開２０１７−１１８８７１号公報で開示のＦｃ結合性タンパク質、
配列番号１に記載のアミノ酸配列の１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を少なくとも含み、但し当該１７番目から１９２番目までのアミノ酸残基において、少なくとも１７６番目のフェニルアラニンがイソロイシン、アラニン、チロシンのいずれかにアミノ酸置換された、Ｆｃ結合性タンパク質、
があげられる。上記列挙したＦｃ結合性タンパク質は、抗体結合活性を有する限り、さらに１以上のアミノ酸残基が欠失、他のアミノ酸残基に置換、または付加されていてもよい。１以上のアミノ酸残基とは、アミノ酸残基のタンパク質の立体構造における位置やアミノ酸残基の種類によっても異なるが、具体的には、例えば１〜５０個、１〜４０個、１〜３０個、１〜２０個、１〜１０個を意味する。上記のアミノ酸の置換、欠失、挿入、または付加等には、遺伝子が由来する微生物の個体差、種の違いに基づく場合などの天然に生じる変異（ｍｕｔａｎｔ又はｖａｒｉａｎｔ）によって生じるものも含まれる。また、本発明のＦｃ結合性タンパク質は、抗体結合活性を有する限り、配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基からなるアミノ酸配列に対し、８０％以上、８５％以上、９０％以上、９５％以上の相同性を有するアミノ酸配列を含むものであってもよい。
When the antibody (immunoglobulin) is IgG and the protein capable of binding to the Fc region of the antibody is an Fcγ receptor, any of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb can be used as the Fcγ receptor. As a preferred embodiment of the Fcγ receptor,
(A) An Fc-binding protein containing at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence of human FcγRIIIa (UniProt No. P08637) shown in SEQ ID NO: 1, and the sequence (B). In the amino acid sequence of human FcγRIIIa (UniProt No. P08637) according to No. 1, at least the amino acid residues from the 17th glycine to the 192nd glutamine are contained, but in the 17th to 192nd amino acid residues. An Fc-binding protein, which comprises a polypeptide in which one or more amino acid residues have been deleted, replaced or added to another amino acid residue.
Can be given. Further, as a preferred embodiment of the above (B),
An Fc-binding protein comprising a polypeptide containing at least the amino acid residues from the 33rd glycine to the 208th glutamine in the amino acid sequence set forth in SEQ ID NO: 2.
Of the amino acid sequence set forth in SEQ ID NO: 2, at least the amino acid residues from the 33rd glycine to the 208th glutamine are contained, but in the 33rd to 208th amino acid residues, the 192nd valine is replaced with phenylalanine. Fc-binding protein, including the polypeptide
Fc-binding protein disclosed in Japanese Patent Application Laid-Open No. 2015-08626,
Fc-binding protein disclosed in JP-A-2016-169197,
Fc-binding protein disclosed in JP-A-2017-118871
It contains at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, but in the 17th to 192nd amino acid residues, at least the 176th phenylalanine is isoleucine, alanine. , Fc-binding protein, amino acid substituted for any of tyrosine,
Can be given. The Fc-binding proteins listed above may have one or more amino acid residues deleted, replaced with other amino acid residues, or added as long as they have antibody-binding activity. The amino acid residue of 1 or more varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of the amino acid residue, but specifically, for example, 1 to 50, 1 to 40, and 1 to 30. , 1 to 20 pieces, 1 to 10 pieces. The above-mentioned amino acid substitutions, deletions, insertions, additions, etc. include those caused by naturally occurring mutations (mutants or variants) such as those based on individual differences and species differences of the microorganism from which the gene is derived. Further, the Fc-binding protein of the present invention has 80 as long as it has antibody-binding activity, with respect to the amino acid sequence consisting of amino acid residues from the 33rd glycine to the 208th glutamine in the amino acid sequence shown in SEQ ID NO: 2. It may contain an amino acid sequence having a homology of% or more, 85% or more, 90% or more, and 95% or more.
なお配列番号２に記載のアミノ酸配列からなるポリペプチドは、配列番号１に記載のヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）の１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を含み、ただし当該１７番目から１９２番目までのアミノ酸残基において、Ｖａｌ２７Ｇｌｕ（この表記は配列番号１の２７番目（配列番号２では４３番目）のバリンがグルタミンに置換していることを表す、以下同じ）、Ｐｈｅ２９Ｉｌｅ、Ｔｙｒ３５Ａｓｎ、Ｇｌｎ４８Ａｒｇ、Ｐｈｅ７５Ｌｅｕ、Ａｓｎ９２Ｓｅｒ、Ｖａｌ１１７Ｇｌｕ、Ｇｌｕ１２１ＧｌｙおよびＰｈｅ１７１Ｓｅｒのアミノ酸置換が生じたポリペプチドである。 The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 contains amino acid residues from the 17th glycine to the 192nd glutamine of the human FcγRIIIa (UniProt No. P08637) set forth in SEQ ID NO: 1, but said 17 At amino acid residues from the th to the 192nd, Val27Glu (this notation indicates that the 27th valine of SEQ ID NO: 1 (43rd in SEQ ID NO: 2) is replaced with glutamine, the same applies hereinafter), Phe29Ile, Tyr35Asn. , Gln48Arg, Phe75Leu, Asn92Ser, Val117Glu, Glu121Gly and Phe171Ser with amino acid substitutions.
抗体と前記抗体のＦｃ領域と結合可能なタンパク質との複合体形成工程で用いる緩衝液としては、通常当業者が緩衝液として用いる、酢酸緩衝液、リン酸緩衝液、ＭＥＳ（２−Ｍｏｒｐｈｏｌｉｎｏｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃｉｄ）緩衝液、ＨＥＰＥＳ（４−（２−ｈｙｄｒｏｘｙｅｔｈｙｌ）−１−ｐｉｐｅｒａｚｉｎｅｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃｉｄ）緩衝液、トリス緩衝液、ホウ酸緩衝液を用いてもよいが、ＮａＨ２ＰＯ４・２Ｈ２ＯとＮａ２ＳＯ４とを含む緩衝液が好ましい。前記緩衝液成分の濃度は３０ｍＭから２００ｍＭまでの間とすると好ましく、５０ｍＭから１５０ｍＭまでの間とするとより好ましい。緩衝液のｐＨまたは反応温度は抗体の抗原への結合活性を損なわない範囲で設定すればよい。
As the buffer solution used in the complex formation step of the antibody and the protein capable of binding to the Fc region of the antibody, an acetate buffer solution, a phosphate buffer solution, or MES (2-Morphosphoethanesulphonic acid), which is usually used as a buffer solution by those skilled in the art, is used. buffer, HEPES (4- (2-hydroxyethyl ) -1-piperazineethanesulfonic acid) buffer, tris buffer, may be used boric acid buffer, but with NaH 2 PO 4 ·
本抗体結合活性評価法は、例えば以下の（Ｉ−１）から（Ｉ−６）に示す工程または（ＩＩ−１）から（ＩＩ−６）に示す工程に基づき行なえばよい。 The present antibody binding activity evaluation method may be carried out, for example, based on the following steps (I-1) to (I-6) or (II-1) to (II-6).
（Ｉ−１）種々の量、好ましくは０〜１５ｍｇ／ｍＬ、より好ましくは０〜１０ｍｇ／ｍＬ、更に好ましくは０〜５ｍｇ／ｍＬの抗体を含む溶液に、一定量の前記抗体のＦｃ領域と結合可能なタンパク質をそれぞれ添加し、前記抗体と前記タンパク質との複合体を形成させる工程
（Ｉ−２）（Ｉ−１）の工程で得られた溶液をそれぞれゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程
（Ｉ−３）（Ｉ−２）の工程で得られたクロマトグラムのうち、前記複合体（または前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質）に相当するピークの高さまたは面積を算出する工程
（Ｉ−４）（Ｉ−３）の算出結果に基づき、前記複合体量（または前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質量）と、前記抗体量とで検量線を作成する工程
（Ｉ−５）（Ｉ−４）で作成した検量線に基づき、前記一定量の抗体のＦｃ領域と結合可能なタンパク質全てを前記複合体（以下、完全複合体化とも表記）にするために必要な抗体量を算出する工程
（Ｉ−６）（Ｉ−５）で算出した抗体量に基づき、抗体の結合活性を評価する工程
（ＩＩ−１）一定量の抗体を含む溶液に、種々の量の前記抗体のＦｃ領域と結合可能なタンパク質をそれぞれ添加し、前記抗体と前記タンパク質との複合体を形成させる工程
（ＩＩ−２）（ＩＩ−１）の工程で得られた溶液をそれぞれゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程
（ＩＩ−３）（ＩＩ−２）の工程で得られたクロマトグラムのうち、前記複合体（または前記複合体を形成しない前記抗体）に相当するピークの高さまたは面積を算出する工程
（ＩＩ−４）（ＩＩ−３）の算出結果に基づき、前記複合体量（または前記複合体を形成しない前記抗体）と、前記タンパク質量とで検量線を作成する工程
（ＩＩ−５）（ＩＩ−４）で作成した検量線に基づき、前記一定量の抗体を完全複合体化するために必要な前記タンパク質量を算出する工程
（ＩＩ−６）（ＩＩ−５）で算出した前記タンパク質量に基づき、抗体の結合活性を評価する工程
本抗体結合活性評価法を利用して、抗体の変性度合も評価できる。抗体が、熱、酸、アルカリ、撹拌、凍結融解などのストレスにさらされると、二次構造などが変化し、変性することがある。当該変性した抗体は、未変性の抗体と比較し、結合活性が低下している。そこで変性した抗体を含む抗体溶液に対し、本抗体結合活性評価法を適用した。その結果、未変性の抗体のみを含む抗体溶液と比較し、抗体と当該抗体のＦｃ領域と結合可能なタンパク質との複合体の量が低下（前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質の量が増加）していた（後述の実施例２参照）。このことから、本抗体結合活性評価法により、抗体の変性度合の評価もできることがわかった。
(I-1) In a solution containing various amounts, preferably 0 to 15 mg / mL, more preferably 0 to 10 mg / mL, even more preferably 0 to 5 mg / mL, with a constant amount of the Fc region of the antibody. The solutions obtained in the steps (I-2) and (I-1) of adding the bindable proteins to form a complex of the antibody and the protein are subjected to gel filtration chromatography, respectively, and chromatograms are obtained. In the chromatograms obtained in the steps (I-3) and (I-2), the peak corresponding to the complex (or a protein capable of binding to the Fc region of the antibody that does not form the complex). Based on the calculation results of the steps (I-4) and (I-3) for calculating the height or area, the amount of the complex (or the amount of protein that can bind to the Fc region of the antibody that does not form the complex) and Based on the calibration line prepared in the steps (I-5) and (I-4) for preparing a calibration line with the amount of the antibody, all the proteins that can bind to the Fc region of the fixed amount of the antibody are combined with the complex (hereinafter referred to as the complex). Step of evaluating the binding activity of an antibody based on the amount of protein calculated in steps (I-6) and (I-5) of calculating the amount of antibody required to obtain a complete complex (also referred to as complete complexation) (II-1). Steps (II-2) (II-1) of adding various amounts of proteins capable of binding to the Fc region of the antibody to a solution containing a certain amount of the antibody to form a complex of the antibody and the protein. ) Is subjected to gel filtration chromatography, respectively, and among the chromatograms obtained in the steps (II-3) and (II-2) to obtain a chromatogram, the complex (or the complex) is obtained. The amount of the complex (or the antibody that does not form the complex) based on the calculation results of the steps (II-4) and (II-3) for calculating the height or area of the peak corresponding to the antibody that does not form the complex. And, based on the calibration line prepared in the steps (II-5) and (II-4) for creating a calibration line with the protein amount, the amount of the protein required for completely complexing the fixed amount of the antibody is determined. Step to evaluate the binding activity of the antibody based on the amount of the protein calculated in the steps (II-6) and (II-5) to be calculated Using this antibody binding activity evaluation method, the degree of denaturation of the antibody can also be evaluated. When an antibody is exposed to stress such as heat, acid, alkali, stirring, freeze-thaw, etc., the secondary structure and the like may change and denature. The denatured antibody has a lower binding activity than the undenatured antibody. Therefore, this antibody binding activity evaluation method was applied to an antibody solution containing a denatured antibody. As a result, the amount of the complex of the antibody and the protein that can bind to the Fc region of the antibody is reduced as compared with the antibody solution containing only the undenatured antibody (binding to the Fc region of the antibody that does not form the complex). The amount of possible protein was increased) (see Example 2 below). From this, it was found that the degree of denaturation of the antibody can also be evaluated by this antibody binding activity evaluation method.
本抗体結合活性評価法を利用した抗体の変性度合評価法（以下、単に本発明の抗体変性度合評価法とも表記する）は、例えば以下に示す工程に基づき行なえばよい。 The antibody denaturation degree evaluation method using the antibody binding activity evaluation method (hereinafter, also simply referred to as the antibody denaturation degree evaluation method of the present invention) may be carried out based on, for example, the following steps.
（ｉ−１）一定量の未変性抗体Ａを含む溶液および前記と同量の被検抗体Ａを含む溶液に対し、抗体ＡのＦｃ領域と結合可能なタンパク質をそれぞれ一定量添加し、抗体Ａと前記タンパク質との複合体を形成させる工程
（ｉ−２）（ｉ−１）の工程で得られた溶液をそれぞれゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程
（ｉ−３）（ｉ−２）の工程で得られたクロマトグラムのうち、前記複合体（または前記複合体を形成しない抗体ＡのＦｃ領域と結合可能なタンパク質）に相当するピークの高さまたは面積を算出する工程
（ｉ−４）未変性抗体Ａを含む溶液を用いたときの（ｉ−３）の算出結果と、被検抗体Ａを含む溶液を用いたときの（ｉ−３）の算出結果との差に基づき、抗体Ａの変性度合を評価する工程
前述した本発明の抗体変性度合評価法では、ある一点の（抗体Ａ量および抗体ＡのＦｃ領域と結合可能なタンパク質量を固定した）条件における、抗体Ａと前記タンパク質との複合体（または前記複合体を形成しない抗体ＡのＦｃ領域と結合可能なタンパク質）の量の変化に基づき評価しているが、前記（Ｉ−１）から（Ｉ−４）の工程により作成した検量線の変化に基づき、抗体の変性度合を評価してもよい。前記検量線の変化に基づく、本発明の抗体変性度合評価法は、例えば以下の（ｉｉ−１）から（ｉｉ−３）に示す工程または（ｉｉｉ−１）または（ｉｉｉ−３）に示す工程に基づき行なえばよい。
(I-1) To a solution containing a certain amount of the unmodified antibody A and a solution containing the same amount of the test antibody A as described above, a certain amount of a protein capable of binding to the Fc region of the antibody A is added to the solution, and the antibody A is added. The solutions obtained in the steps (i-2) and (i-1) for forming a complex between the protein and the protein are subjected to gel filtration chromatography, respectively, to obtain chromatograms (i-3) (i-2). ), The height or area of the peak corresponding to the complex (or a protein that can bind to the Fc region of antibody A that does not form the complex) is calculated from the chromatogram obtained in step (i-). 4) Based on the difference between the calculation result of (i-3) when the solution containing the unmodified antibody A is used and the calculation result of (i-3) when the solution containing the test antibody A is used. Step for evaluating the degree of denaturation of antibody A In the above-mentioned method for evaluating the degree of denaturation of antibody of the present invention, the antibody A and the antibody A are subjected to a certain condition (the amount of antibody A and the amount of protein that can bind to the Fc region of antibody A are fixed). The evaluation is based on the change in the amount of the complex with the protein (or the protein capable of binding to the Fc region of antibody A that does not form the complex), and the above (I-1) to (I-4). The degree of denaturation of the antibody may be evaluated based on the change in the calibration line prepared by the step. The antibody denaturation degree evaluation method of the present invention based on the change in the calibration curve is, for example, the steps shown in (ii-1) to (ii-3) below or the steps shown in (iii-1) or (iii-3) below. It should be done based on.
（ｉｉ−１）種々の量の未変性抗体Ａを含む溶液に対し、前記（Ｉ−１）から（Ｉ−４）の工程を実施し、検量線を作成する工程
（ｉｉ−２）（ｉｉ−１）と同じ種々の量の被検抗体Ａを含む溶液に対し、前記（Ｉ−１）から（Ｉ−４）の工程を実施し、検量線を作成する工程
（ｉｉ−３）（ｉｉ−１）で作成した検量線の傾きと、（ｉｉ−２）で作成した検量線の傾きとを比較し、抗体Ａの変性度合を評価する工程
（ｉｉｉ−１）種々の量の未変性抗体Ａを含む溶液に対し、前記（Ｉ−１）から（Ｉ−４）の工程を実施し、検量線を作成する工程
（ｉｉｉ−２）（ｉｉｉ−１）と同じ種々の量の被検抗体Ａを含む溶液に対し、前記（Ｉ−１）から（Ｉ−４）の工程を実施し、検量線を作成する工程
（ｉｉｉ−３）（ｉｉｉ−１）および（ｉｉｉ−２）で作成した検量線をそれぞれ外挿して求められる、抗体のＦｃ領域と結合可能なタンパク質を完全複合体化するために必要な抗体量の差に基づき、抗体Ａの変性度合を評価する工程
本発明の抗体変性度合評価法に基づく、被検抗体Ａが変性しているか否かの判断は、前記（ｉ−１）から（ｉ−４）の工程に基づき実施する場合は、例えば未変性抗体Ａを含む溶液を用いたときの結果から一定値以上乖離しているとき、変性していると判断すればよい。また前記（ｉｉ−１）から（ｉｉ−３）の工程に基づき実施する場合は、例えば未変性抗体Ａを含む溶液を用いたときの検量線の傾きから一定値以上または一定の割合以上乖離しているとき、変性していると判断すればよい。また前記（ｉｉｉ−１）から（ｉｉｉ−３）の工程に基づき実施する場合は、例えば未変性抗体Ａを含む溶液を用いたときの、抗体のＦｃ領域と結合可能なタンパク質を完全複合体化するのに必要な抗体量から一定値以上または一定の割合乖離しているとき、変性していると判断すればよい。
(Ii-1) Steps (ii-2) (ii-2) in which the steps (I-1) to (I-4) are carried out on a solution containing various amounts of the unmodified antibody A to prepare a calibration line. Steps (ii-3) (ii) for preparing calibration lines by carrying out the steps (I-1) to (I-4) above for a solution containing the same various amounts of the test antibody A as in -1). Step of comparing the inclination of the calibration line prepared in -1) with the inclination of the calibration line prepared in (ii-2) to evaluate the degree of denaturation of antibody A (iii-1) Various amounts of unmodified antibody. For the solution containing A, the same various amounts of the test antibody as in the steps (iii-2) and (iii-1) in which the steps (I-1) to (I-4) are carried out to prepare a calibration line are performed. The steps (I-1) to (I-4) were carried out on the solution containing A, and the steps (iii-3), (iii-1) and (iii-2) for creating a calibration line were prepared. A step of evaluating the degree of denaturation of antibody A based on the difference in the amount of antibody required to completely complex the Fc region of the antibody and the protein that can bind to the Fc region of the antibody, which is obtained by extrapolating the calibration lines. When the determination of whether or not the test antibody A is denatured based on the degree evaluation method is carried out based on the steps (i-1) to (i-4) above, for example, a solution containing the unmodified antibody A. When it deviates from the result when using the above by a certain value or more, it may be judged that the antibody is degenerated. Further, in the case of carrying out based on the steps (ii-1) to (ii-3), for example, the slope of the calibration curve when a solution containing the undenatured antibody A is used deviates from a certain value or more or a certain percentage or more. When it is, it should be judged that it is denatured. In the case of carrying out based on the steps (iii-1) to (iii-3), for example, when a solution containing the undenatured antibody A is used, a protein that can bind to the Fc region of the antibody is completely complexed. When the amount of antibody deviated from the amount of antibody required for the protein is more than a certain value or a certain percentage, it may be judged that the antibody is denatured.
本抗体結合活性評価法は、一定量の抗体を含む溶液に前記抗体のＦｃ領域と結合可能なタンパク質を一定量添加し前記抗体と前記タンパク質との複合体を形成させる第一の工程、前記第一の工程で得られた溶液をゲル濾過クロマトグラフィに供し、クロマトグラムを得る第二の工程、および前記第二の工程で得られたクロマトグラムのうち、前記複合体、前記抗体または前記タンパク質に相当するピークの高さまたは面積を算出する第三の工程を含むことを特徴としている。本抗体結合活性評価法は、溶液中に含まれる抗体のＦｃ領域と結合可能なタンパク質を添加後、ゲル濾過クロマトグラフィに供し、得られた画分中に含まれる前記複合体または前記タンパク質を定量するのみで抗体の結合活性を評価でき、抗体の結合活性を従来の方法（ＥＬＩＳＡ法や表面プラズモン共鳴法など）よりも短時間かつ煩雑な実験操作なしに評価できる。したがって、抗体の開発コストの削減に寄与できる。 The present antibody binding activity evaluation method is a first step of adding a certain amount of a protein capable of binding to the Fc region of the antibody to a solution containing a certain amount of the antibody to form a complex of the antibody and the protein, the first step. The solution obtained in one step is subjected to gel filtration chromatography to obtain a chromatogram, and the chromatogram obtained in the second step corresponds to the complex, the antibody or the protein. It is characterized by including a third step of calculating the height or area of the peak. In this antibody binding activity evaluation method, a protein capable of binding to the Fc region of an antibody contained in a solution is added and then subjected to gel filtration chromatography to quantify the complex or the protein contained in the obtained fraction. The antibody binding activity can be evaluated only by the conventional method (ELISA method, surface plasmon resonance method, etc.) in a shorter time and without complicated experimental operations. Therefore, it can contribute to the reduction of antibody development cost.
また本抗体結合活性評価法を利用して、溶液中に含まれる抗体の変性度合も評価できる。したがって、本発明は変性しにくい抗体の開発や抗体製造における工程分析に有用である。 In addition, the degree of denaturation of the antibody contained in the solution can be evaluated by using this antibody binding activity evaluation method. Therefore, the present invention is useful for the development of antibodies that are difficult to denature and for process analysis in antibody production.
以下、ヒトＦｃγＲＩＩＩａの細胞外領域のアミノ酸置換体（以下、ＦｃＲ９＿Ｆとも表記）を含むポリペプチドである、配列番号２に記載のアミノ酸配列からなるポリペプチドを抗体のＦｃ領域と結合可能なタンパク質としたときの実施例を用いて、本発明をさらに詳細に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, which is a polypeptide containing an amino acid substitution product in the extracellular region of human FcγRIIIa (hereinafter, also referred to as FcR9_F), was designated as a protein capable of binding to the Fc region of an antibody. The present invention will be described in more detail with reference to the examples of the above, but the present invention is not limited to these examples.
実施例１ 抗体結合活性評価法
以下に示す方法で、抗体結合活性評価法を検証した。
Example 1 Antibody-binding activity evaluation method The antibody-binding activity evaluation method was verified by the method shown below.
（１）下記（Ａ−１）から（Ａ−６）に示す、配列番号２に記載のアミノ酸配列からなるポリペプチドまたは前記ポリペプチドと抗体との混合物を、リン酸緩衝生理食塩水（ＰＢＳ）を用いて１０００μＬにメスアップし、３０℃で３０分間静置し、測定試料を調製した。なお配列番号２に記載のアミノ酸配列からなるポリペプチドのうち、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ９＿Ｆ（配列番号１の１７番目から１９２番目までの領域に相当）であり、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。またＦｃＲ９＿ＦはヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）の１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基において、Ｖａｌ２７Ｇｌｕ、Ｐｈｅ２９Ｉｌｅ、Ｔｙｒ３５Ａｓｎ、Ｇｌｎ４８Ａｒｇ、Ｐｈｅ７５Ｌｅｕ、Ａｓｎ９２Ｓｅｒ、Ｖａｌ１１７Ｇｌｕ、Ｇｌｕ１２１ＧｌｙおよびＰｈｅ１７１Ｓｅｒのアミノ酸置換が生じたポリペプチドである。
（Ａ−１）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ
（Ａ−２）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン（全薬工業製）１，０００μｇ
（Ａ−３）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン２，０００μｇ
（Ａ−４）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン３，０００μｇ
（Ａ−５）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン４，０００μｇ
（Ａ−６）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン５，０００μｇ
（２）高速液体クロマトグラフィー装置（ＨＬＣ−８０２０、東ソー製）にゲル濾過クロマトグラフィ用カラムであるＴＳＫｇｅｌ Ｇ３０００ＳＷＸＬカラム（東ソー製）を接続し、ｐＨ７．４のリン酸緩衝生理食塩水（ＰＢＳ）を流速１ｍＬ／分で流すことにより平衡化した後、（１）の試料５０μＬ（下記（Ａ−７）から（Ａ−１２））を注入し、３０分間分析した。
（Ａ−７）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ
（Ａ−８）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン（全薬工業製）５０μｇ
（Ａ−９）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン１００μｇ
（Ａ−１０）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン１５０μｇ
（Ａ−１１）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン２００μｇ
（Ａ−１２）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン２５０μｇ
得られたクロマトグラム中、配列番号２に記載のアミノ酸配列からなるポリペプチド（すなわち抗体（リツキサン）との複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質）に相当するピーク部分（保持時間９．５分から１２分の位置）を拡大した図を図１に示す。抗体（リツキサン）を添加すると、抗体未添加時と比較し、配列番号２に記載のアミノ酸配列からなるポリペプチドに相当するピークの高さおよび面積が減少すること、および抗体（リツキサン）の添加量が増加すると、前記ピークの高さおよび面積がより減少することがわかる。この結果から、種々の量の抗体を含む溶液に、前記抗体のＦｃ領域と結合可能なタンパク質を一定量添加し、前記抗体と前記タンパク質との複合体を形成させた後、ゲル濾過クロマトグラフィにより得られた、前記抗体との複合体を形成しない前記タンパク質を定量することで、前記抗体の結合活性を評価できることがわかる。
(1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 shown in (A-1) to (A-6) below or a mixture of the polypeptide and an antibody is added to phosphate buffered saline (PBS). Was used to make up to 1000 μL, and the mixture was allowed to stand at 30 ° C. for 30 minutes to prepare a measurement sample. Among the polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 2, the 1st methionine (Met) to the 26th alanin (Ala) are MalE signal peptides, and the 27th lys to the 32nd polypeptide. Up to methionine (Met) is the linker sequence, and from the 33rd glycine (Gly) to the 208th glutamine (Gln) is FcR9_F (corresponding to the 17th to 192nd regions of SEQ ID NO: 1), and the 209th. The 210th to 210th glycine (Gly) is a linker sequence, and the 211th to 216th histidine (His) is a tag sequence. In addition, FcR9_F produced Val27Glu, Phe29Ile, Tyr35Asn, Gln48Arg, Phe75Leu, Asn92Ser, Val117Ser, Val117Sr, Val117Glu in the amino acid residues from the 17th glycine to the 192nd glutamine of human FcγRIIIa (UniProt No. P08637). It is a polypeptide.
(A-1) 620 μg of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(A-2) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 1,000 μg of Rituxan (manufactured by Zenyaku Kogyo)
(A-3) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 2,000 μg of Rituxan
(A-4) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 3,000 μg of Rituxan
(A-5) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 4,000 μg of Rituxan
(A-6) Peptide 620 μg + Rituxan 5,000 μg consisting of the amino acid sequence shown in SEQ ID NO: 2.
(2) A TSKgel G3000SWXL column (manufactured by Tosoh), which is a column for gel filtration chromatography, is connected to a high performance liquid chromatography device (HLC-8020, manufactured by Tosoh), and a phosphate buffered saline solution (PBS) having a pH of 7.4 is passed through a flow rate. After equilibration by flowing at 1 mL / min, 50 μL of the sample (1) ((A-7) to (A-12) below) was injected and analyzed for 30 minutes.
(A-7) 31 μg of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(A-8) Peptide 31 μg consisting of the amino acid sequence shown in SEQ ID NO: 2 + Rituxan (manufactured by Zenyaku Kogyo) 50 μg
(A-9) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 100 μg of Rituxan
(A-10) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 150 μg of Rituxan
(A-11) Peptide 31 μg +
(A-12) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 250 μg of Rituxan
In the obtained chromatogram, the peak portion (retention) corresponding to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 (that is, a protein capable of binding to the Fc region of the antibody that does not form a complex with the antibody (Rituxan)). An enlarged view of the time (position from 9.5 minutes to 12 minutes) is shown in FIG. When the antibody (Rituxan) is added, the height and area of the peak corresponding to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is reduced as compared with the case where the antibody is not added, and the amount of the antibody (Rituxan) added. It can be seen that as the amount increases, the height and area of the peak decrease. From this result, a certain amount of a protein capable of binding to the Fc region of the antibody was added to a solution containing various amounts of the antibody to form a complex of the antibody and the protein, and then gel filtration chromatography was performed. It can be seen that the binding activity of the antibody can be evaluated by quantifying the protein that does not form a complex with the antibody.
また抗体（リツキサン）添加量（横軸）に対する、配列番号２に記載のアミノ酸配列からなるポリペプチドに相当するピーク面積（縦軸）を前記クロマトグラフィー装置の自動計算により算出してプロットした結果を図２に示す。前記（Ａ−７）から前記（Ａ−１１）の結果では、抗体添加量と前記ピーク面積との間に良好な直線性が得られ（Ｒ２＝０．９９９７）、前記ピーク面積の減少が添加した抗体量に依存していることがわかる。またこの結果から、抗体の結合活性を図２で得られた検量線の傾きから評価できることもわかる。さらに前記検量線を外挿して求められる、抗体のＦｃ領域と結合可能なタンパク質全てが複合体を形成（完全複合体化）するのに必要な抗体量からも、抗体の結合活性を評価できる。
Further, the peak area (vertical axis) corresponding to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 with respect to the amount of antibody (Ritzan) added (horizontal axis) was calculated and plotted by automatic calculation of the chromatographic apparatus. It is shown in FIG. The results of the (A-11) from (A-7), good linearity is obtained between the peak area and
なお前記（Ａ−１２）の結果が前記検量線から外れた結果となっているが、前記検量線を外挿して求められる、完全複合体化するのに必要な、抗体量が２４３μｇであることから、前記（Ａ−１２）の結果（抗体添加量：２５０μｇ）は抗体を過剰量添加したときの結果といえる。 Although the result of (A-12) is a result deviating from the calibration curve, the amount of antibody required for complete complex formation, which is obtained by extrapolating the calibration curve, is 243 μg. Therefore, it can be said that the result of (A-12) (antibody addition amount: 250 μg) is the result when an excessive amount of antibody is added.
実施例２ 本発明の抗体変性度合評価法
（１）配列番号２に記載のアミノ酸配列からなるポリペプチドまたは下記（Ｂ−１）から（Ｂ−５）および（Ｃ−１）から（Ｃ−５）に示す前記ポリペプチドと抗体との混合物を、０．１Ｍ Ｎａ２ＳＯ４を含むｐＨ６．７の０．１Ｍ ＮａＨ２ＰＯ４・２Ｈ２Ｏを用いて１０００μＬにメスアップし、３０℃で３０分間静置し、測定試料を調製した。なお熱変性処理したリツキサンは、リツキサンを５０℃で１８時間処理して調製した。
（Ｂ−１）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン２６０μｇ
（Ｂ−２）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン１，３２０μｇ
（Ｂ−３）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン３，３２０μｇ
（Ｂ−４）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン５，５２０μｇ
（Ｂ−５）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋リツキサン６，９００μｇ
（Ｃ−１）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋熱変性処理したリツキサン２６０μｇ
（Ｃ−２）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋熱変性処理したリツキサン１，３２０μｇ
（Ｃ−３）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋熱変性処理したリツキサン３，３２０μｇ
（Ｃ−４）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋熱変性処理したリツキサン５，５２０μｇ
（Ｃ−５）配列番号２に記載のアミノ酸配列からなるポリペプチド６２０μｇ＋熱変性処理したリツキサン６，９００μｇ
（２）高速液体クロマトグラフィー装置（Ｐｒｏｍｉｎｅｎｃｅ、島津製作所製）にゲル濾過クロマトグラフィ用カラムであるＴＳＫｇｅｌ Ｇ３０００ＳＷＸＬカラムを接続し、０．１Ｍ Ｎａ２ＳＯ４を含むｐＨ６．７の０．１Ｍ ＮａＨ２ＰＯ４・２Ｈ２Ｏを流速１ｍＬ／分で流すことにより平衡化した後、（１）の試料５０μＬ（下記（Ｂ−６）から（Ｂ−１０）および（Ｃ−６）から（Ｃ−１０））を注入し、３０分間分析した。
（Ｂ−６）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン１３μｇ
（Ｂ−７）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン６６μｇ
（Ｂ−８）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン１６６μｇ
（Ｂ−９）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン２７６μｇ
（Ｂ−１０）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋リツキサン３４５μｇ
（Ｃ−６）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋熱変性処理したリツキサン１３μｇ
（Ｃ−７）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋熱変性処理したリツキサン６６μｇ
（Ｃ−８）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋熱変性処理したリツキサン１６６μｇ
（Ｃ−９）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋熱変性処理したリツキサン２７６μｇ
（Ｃ−１０）配列番号２に記載のアミノ酸配列からなるポリペプチド３１μｇ＋熱変性処理したリツキサン３４５μｇ
実施例１と同様に配列番号２に記載のアミノ酸配列からなるポリペプチドに相当するピーク面積（縦軸）を前記クロマトグラフィー装置の自動計算により算出し、得られた検量線を図３に示す。熱変性処理したリツキサンを用いたとき（前記（Ｃ−６）から前記（Ｃ−１０）の結果）に得られた検量線の傾きは、熱変性処理しない（熱変性処理前の）リツキサンを用いたとき（前記（Ｂ−６）から前記（Ｂ−１０）の結果）と比較し、小さく（緩やかに）なった。さらに、熱変性処理したリツキサンを用いたときの完全複合体化に必要な抗体量は３３２μＬ、熱変性処理しないリツキサンを用いたときの完全複合体化に必要な抗体量は３０７μＬであり、完全複合体化に必要な抗体量は熱変性処理により７％増加した。つまり、リツキサンを熱変性処理することにより、抗体と当該抗体のＦｃ領域と結合可能なタンパク質との結合活性が低下していることがわかる。以上の結果から、本抗体結合活性評価法を用いて、検量線の傾きや完全複合体化に必要な抗体量を比較することで、被検抗体の変性度合を評価できることがわかる。
Example 2 Evaluation method for antibody denaturation degree of the present invention (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the following (B-1) to (B-5) and (C-1) to (C-5). the mixture of the polypeptide and the antibody shown in), measured up to 1000μL using 0.1M NaH 2 PO 4 · 2H 2 O of pH6.7 containing 0.1
(B-1) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 260 μg of Rituxan
(B-2) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 1,320 μg of Rituxan
(B-3) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 3,320 μg of Rituxan
(B-4) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 5,520 μg of Rituxan
(B-5) Polypeptide 620 μg consisting of the amino acid sequence shown in SEQ ID NO: 2 + Rituxan 6,900 μg
(C-1) 620 μg of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 260 μg of heat-denatured Rituxan
(C-2) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 1,320 μg of heat-denatured Rituxan
(C-3) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 3,320 μg of heat-denatured Rituxan
(C-4) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 5,520 μg of heat-denatured Rituxan
(C-5) 620 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 6,900 μg of heat-denatured Rituxan
(2) A TSKgel G3000SWXL column, which is a column for gel filtration chromatography, is connected to a high performance liquid chromatography device (Prominence, manufactured by Shimadzu Corporation), and a pH 6.7 0.1M NaH 2 PO 4 containing 0.1M Na 2 SO 4 is connected. After equilibration by flowing 2H 2 O at a flow rate of 1 mL / min, 50 μL of the sample of (1) ((B-6) to (B-10) and (C-6) to (C-10) below). Was injected and analyzed for 30 minutes.
(B-6) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 13 μg of Rituxan
(B-7) Peptide 31 μg + Rituxan 66 μg consisting of the amino acid sequence shown in SEQ ID NO: 2.
(B-8) Peptide 31 μg + Rituxan 166 μg consisting of the amino acid sequence shown in SEQ ID NO: 2.
(B-9) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 276 μg of Rituxan
(B-10) Peptide 31 μg + Rituxan 345 μg consisting of the amino acid sequence shown in SEQ ID NO: 2.
(C-6) 31 μg of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 13 μg of heat-denatured Rituxan
(C-7) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 66 μg of heat-denatured Rituxan
(C-8) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 166 μg of heat-denatured Rituxan
(C-9) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 276 μg of heat-denatured Rituxan
(C-10) 31 μg of polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 + 345 μg of heat-denatured Rituxan
Similar to Example 1, the peak area (vertical axis) corresponding to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 was calculated by automatic calculation of the chromatography apparatus, and the obtained calibration curve is shown in FIG. The slope of the calibration curve obtained when heat-denatured Rituxan was used (results from (C-6) to (C-10) above) used Rituxan not heat-denatured (before heat denaturation). Compared with the time when it was (result of (B-6) to (B-10) above), it became smaller (slowly). Further, the amount of antibody required for complete complexing when heat-modified Rituxan was used was 332 μL, and the amount of antibody required for complete complexation when heat-modified Rituxan was used was 307 μL. The amount of antibody required for embodying was increased by 7% by the heat denaturation treatment. That is, it can be seen that the heat denaturation treatment of Rituxan reduces the binding activity between the antibody and the protein that can bind to the Fc region of the antibody. From the above results, it can be seen that the degree of denaturation of the test antibody can be evaluated by comparing the slope of the calibration curve and the amount of antibody required for complete complexation using this antibody binding activity evaluation method.
Claims (3)
（２）（１）の工程で得られた溶液をゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程、
（３）（２）の工程で得られたクロマトグラムのうち、前記複合体または前記複合体を形成しない前記タンパク質に相当するピークの高さまたは面積を算出する工程、
（４）一定量の被検抗体Ａを含む溶液に、抗体ＡのＦｃ領域と結合可能なタンパク質を一定量添加し、抗体Ａと前記タンパク質との複合体を形成させる工程、
（５）（４）の工程で得られた溶液をゲル濾過クロマトグラフィに供し、クロマトグラムを得る工程、
（６）（５）の工程で得られたクロマトグラムのうち、前記複合体または前記複合体を形成しない前記タンパク質に相当するピークの高さまたは面積を算出する工程、および
（７）（３）の工程での算出結果と（６）の工程での算出結果とを比較する工程、
を含む、被検抗体Ａの変性度合を評価する方法。 (1) A step of adding a certain amount of a protein capable of binding to the Fc region of antibody A to a solution containing a certain amount of undenatured antibody A to form a complex of antibody A and the protein.
(2) A step of subjecting the solution obtained in the step (1) to gel filtration chromatography to obtain a chromatogram.
(3) Of the chromatograms obtained in the step (2), the step of calculating the height or area of the peak corresponding to the complex or the protein that does not form the complex.
(4) A step of adding a certain amount of a protein capable of binding to the Fc region of antibody A to a solution containing a certain amount of test antibody A to form a complex of antibody A and the protein.
(5) A step of subjecting the solution obtained in the step (4) to gel filtration chromatography to obtain a chromatogram.
(6) Of the chromatograms obtained in the steps (5), the steps of calculating the height or area of the peak corresponding to the complex or the protein that does not form the complex, and (7) (3). A step of comparing the calculation result in the step (6) with the calculation result in the step (6),
A method for evaluating the degree of denaturation of the test antibody A, which comprises.
（ｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を少なくとも含むポリペプチド
（ｉｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基において１〜１０個のアミノ酸残基の置換、欠失、挿入、または付加を含むアミノ酸配列を含み、かつ、抗体結合活性を有するタンパク質
（ｉｉｉ）配列番号２に記載のアミノ酸配列のうち３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基からなるアミノ酸配列に対して、９０％以上の相同性を有するアミノ酸配列を含み、かつ、抗体結合活性を有するタンパク質。 The evaluation method according to claim 1, wherein the protein capable of binding to the Fc region of the antibody is selected from the following (i) to (iii).
(I) Polypeptide containing at least the amino acid residues from the 33rd glycine to the 208th glutamine in the amino acid sequence shown in SEQ ID NO: 2 (ii) From the 33rd glycine in the amino acid sequence shown in SEQ ID NO: 2. To protein (iii) SEQ ID NO: 2, which contains an amino acid sequence containing substitutions, deletions, insertions, or additions of 1 to 10 amino acid residues in the amino acid residues up to the 208th glutamine and has antibody-binding activity. A protein containing an amino acid sequence having 90% or more homology with respect to an amino acid sequence consisting of amino acid residues from the 33rd glycine to the 208th glutamine among the described amino acid sequences, and having antibody binding activity.
様々な濃度の被検抗体Ａについて前記工程（４）〜（７）を行い、前記複合体量または前記複合体を形成しない前記抗体のＦｃ領域と結合可能なタンパク質量と前記抗体量とで検量線ＩＩを作成する工程、並びに
前記検量線ＩおよびＩＩを比較する工程
を含む、請求項１又は２に記載の評価方法。 The steps (1) to (3) are performed on the unmodified antibody A having various concentrations, and the amount of the complex or the amount of protein that can bind to the Fc region of the antibody that does not form the complex and the amount of the antibody are used for calibration. The process of creating line I,
The steps (4) to (7) are performed on the test antibody A having various concentrations, and the amount of the complex or the amount of protein that can bind to the Fc region of the antibody that does not form the complex and the amount of the antibody are used for calibration. The evaluation method according to claim 1 or 2, comprising a step of creating a line II and a step of comparing the calibration curves I and II.
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