JP2009034095A - Stabilizer for aqueous solution containing protein, method for stabilizing aqueous solution containing protein and aqueous solution containing protein - Google Patents
Stabilizer for aqueous solution containing protein, method for stabilizing aqueous solution containing protein and aqueous solution containing protein Download PDFInfo
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- JP2009034095A JP2009034095A JP2008133321A JP2008133321A JP2009034095A JP 2009034095 A JP2009034095 A JP 2009034095A JP 2008133321 A JP2008133321 A JP 2008133321A JP 2008133321 A JP2008133321 A JP 2008133321A JP 2009034095 A JP2009034095 A JP 2009034095A
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- Prior art keywords
- aqueous solution
- protein
- arginine
- containing aqueous
- acid
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- 239000001103 potassium chloride Substances 0.000 description 1
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- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
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- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
本発明は、タンパク質含有水溶液に含まれるタンパク質の安定化に関する。さらに詳しくは、酵素、組み換えタンパク質、抗体、ペプチドなどのタンパク質を含有する水溶液に含まれるタンパク質の安定化剤、この安定化剤を共存させるタンパク質含有水溶液の安定化方法、および安定化剤を共存させたタンパク質含有水溶液に関する。 The present invention relates to stabilization of a protein contained in a protein-containing aqueous solution. More specifically, a stabilizer for a protein contained in an aqueous solution containing a protein such as an enzyme, a recombinant protein, an antibody, or a peptide, a method for stabilizing an aqueous solution containing a protein containing the stabilizer, and a stabilizer. The present invention relates to a protein-containing aqueous solution.
酵素、抗体、ペプチドなどのタンパク質は、診断・検査薬、医薬品として広く利用されており、これらの製品においては、製造工程及び保存期間中に生理活性（力価）が損なわれないことが重要である。
製造工程及び保存期間中において、安定してタンパク質を取り出し精製するための一つの方法として凍結乾燥が一般的に行われている。タンパク質の多くは熱によって失活しやすい性質を有するが、凍結乾燥法では、熱をかけずにタンパク質を安定化することができる。
しかしながら、凍結乾燥法は、脱水により変性するタンパク質には使用できないこと、凍結乾燥工程中に吸湿や酸化による変質が起こりやすいこと等の難点がある。また、凍結乾燥製剤は使用時に溶媒（溶解液）に溶解して用いられるため、溶媒（溶解液）と組み合わせて供給される場合、試薬をその都度に必要量を調製しなければならないという煩雑さの問題がある。
このような理由からタンパク質を水溶液中で安定化させる技術が公開されている。たとえば、ウレアーゼパーオキシターゼの水溶液の安定化剤として、グリセリンなどの多価 アルコールを含有させたり（たとえば、特許文献１）、コレステロールオキシターゼを含む水溶液に、牛血清アルブミンやグルコース等の糖類あるいはリジン等のアミノ酸を添加する（たとえば、特許文献２）等が挙げられる。
しかし、これらはいずれも特定のタンパク質を安定化させるための方法であり汎用性があるとは言いがたく、タンパク質全般に適用して活性を長期間維持できる汎用的な安定化剤及び安定化方法はなかった。
Proteins such as enzymes, antibodies, and peptides are widely used as diagnostic / testing drugs and pharmaceuticals. In these products, it is important that physiological activity (titer) is not impaired during the manufacturing process and storage period. is there.
Lyophilization is generally performed as one method for stably extracting and purifying proteins during the production process and storage period. Many proteins have the property of being easily inactivated by heat, but the freeze-drying method can stabilize the protein without applying heat.
However, the lyophilization method has drawbacks such that it cannot be used for proteins that are denatured by dehydration and that alteration due to moisture absorption or oxidation tends to occur during the lyophilization process. In addition, since lyophilized preparations are used after being dissolved in a solvent (dissolution) at the time of use, when supplied in combination with a solvent (dissolution), the complexity of having to prepare the necessary amount of the reagent each time is required. There is a problem.
For these reasons, techniques for stabilizing proteins in aqueous solutions have been disclosed. For example, as a stabilizer of an aqueous solution of urease peroxidase, a polyhydric alcohol such as glycerin is contained (for example, Patent Document 1), or an aqueous solution containing cholesterol oxidase is added to a sugar such as bovine serum albumin or glucose or lysine. An amino acid is added (for example, patent document 2) etc. are mentioned.
However, it is difficult to say that these are methods for stabilizing a specific protein and are versatile, and general-purpose stabilizers and stabilization methods that can be applied to all proteins and maintain their activity for a long period of time. There was no.
そこで、水溶液中のタンパク質の変性、変質及び凝集を抑制し、タンパク質の水溶液を長期的に安定化させることができるタンパク質の安定化剤を提供することが課題である。 Therefore, it is an object to provide a protein stabilizer capable of suppressing protein denaturation, alteration and aggregation in an aqueous solution and stabilizing the aqueous solution of the protein for a long period of time.
本発明者は、上記の目的を達成するべく検討を行った結果、本発明に到達した。
すなわち、本発明は、酵素、組み換えタンパク質、抗体及びペプチドからなる群より選ばれる少なくとも１種のタンパク質を含有する水溶液の安定化剤であって、アルギニンと有機酸（ａ）との塩（Ａ）を含んでなるタンパク質含有水溶液の安定化剤；その安定化剤を含有させるタンパク質含有水溶液の安定化方法；安定化剤を含有するタンパク質含有水溶液である。
The inventor of the present invention has arrived at the present invention as a result of studies to achieve the above object.
That is, the present invention is an aqueous solution stabilizer containing at least one protein selected from the group consisting of an enzyme, a recombinant protein, an antibody and a peptide, and comprises a salt (A) of arginine and an organic acid (a). A method for stabilizing a protein-containing aqueous solution containing the stabilizer; a protein-containing aqueous solution containing the stabilizer.
本発明のタンパク質含有水溶液の安定化剤は、水溶液中のタンパク質を安定化し、水溶液中のタンパク質の生理活性が低下しないので、医薬品、および生化学の分野において有効に使用することができる。 Since the protein-containing aqueous solution stabilizer of the present invention stabilizes the protein in the aqueous solution and the physiological activity of the protein in the aqueous solution does not decrease, it can be effectively used in the fields of pharmaceuticals and biochemistry.
本発明は、酵素、組み換えタンパク質、抗体及びペプチドからなる群より選ばれる少なくとも１種のタンパク質を含有する水溶液の安定化剤であって、アルギニンと有機酸（ａ）との塩（Ａ）を含んでなるタンパク質含有水溶液の安定化剤である。
すなわち、塩基性アミノ酸であるアルギニンを有機酸で中和した塩を安定化剤として使用することを要旨とする。
The present invention is a stabilizer for an aqueous solution containing at least one protein selected from the group consisting of enzymes, recombinant proteins, antibodies and peptides, comprising a salt (A) of arginine and an organic acid (a). It is a stabilizer of protein-containing aqueous solution consisting of.
That is, the gist is to use a salt obtained by neutralizing arginine, which is a basic amino acid, with an organic acid as a stabilizer.
酵素、ペプチドなどのタンパク質を水溶液として保存する際にそのまま保存すると、これらが凝集や加水分解等を起こし力価が著しく低下するという問題点があるが、本発明では、特定の化学構造を有する上記の塩（Ａ）を水溶液の安定化剤として添加することにより解決することを見出した。 When proteins such as enzymes and peptides are stored as an aqueous solution as they are, they have the problem that they cause aggregation, hydrolysis and the like, and the titer is remarkably lowered. It has been found that this problem can be solved by adding the salt (A) as a stabilizer for an aqueous solution.
本発明のタンパク質水溶液安定化剤において、安定化剤として作用させるために存在させるアルギニンと有機酸（ａ）との塩（Ａ）は、 アルギニンを有機酸で中和したものである。 In the protein aqueous solution stabilizer of the present invention, the salt (A) of arginine and organic acid (a) that is present to act as a stabilizer is obtained by neutralizing arginine with an organic acid.
本発明における有機酸（ａ）としてはヒドロキシカルボン酸、カルボン酸、有機リン酸及び有機スルホン酸などが挙げられる。 Examples of the organic acid (a) in the present invention include hydroxycarboxylic acid, carboxylic acid, organic phosphoric acid, and organic sulfonic acid.
ヒドロキシカルボン酸としては、モノヒドロキシカルボン酸（乳酸、クエン酸、酒石酸、リンゴ酸及びサリチル酸等）、ジヒドロキシカルボン酸（オルセリン酸等）及びトリヒドロキシカルボン酸（没食子酸等）等が挙げられる。 Examples of the hydroxycarboxylic acid include monohydroxycarboxylic acid (such as lactic acid, citric acid, tartaric acid, malic acid, and salicylic acid), dihydroxycarboxylic acid (such as orthoric acid), and trihydroxycarboxylic acid (such as gallic acid).
カルボン酸としては、モノカルボン酸（蟻酸、酢酸、プロピオン酸、ブタン酸、オクタン酸、デカン酸及びオレイン酸等）、ジカルボン酸（フタル酸、シュウ酸及びアジピン酸等）、トリカルボン酸（トリメリット酸等）、テトラカルボン酸（エチレンジアミン４酢酸、ブタンテトラカルボン酸及びシクロペンタンテトラカルボン酸等）等が挙げられる。 Examples of carboxylic acids include monocarboxylic acids (such as formic acid, acetic acid, propionic acid, butanoic acid, octanoic acid, decanoic acid, and oleic acid), dicarboxylic acids (such as phthalic acid, oxalic acid, and adipic acid), and tricarboxylic acids (trimellitic acid). Etc.), tetracarboxylic acids (ethylenediaminetetraacetic acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, etc.) and the like.
有機リン酸としては、メチルリン酸、エチルリン酸等が挙げられる。 Examples of the organic phosphoric acid include methyl phosphoric acid and ethyl phosphoric acid.
有機スルホン酸としては、メタンスルホン酸、エタンスルホン酸、ベンゼンスルホン酸等が挙げられる。 Examples of the organic sulfonic acid include methanesulfonic acid, ethanesulfonic acid, and benzenesulfonic acid.
これらのうち、タンパク質の安定化の観点からヒドロキシカルボン酸及びカルボン酸が好ましく、さらに好ましくはヒドロキシカルボン酸であり、次にさらに好ましくは乳酸及びクエン酸であり、最も好ましくは乳酸である。 Of these, hydroxycarboxylic acids and carboxylic acids are preferable from the viewpoint of protein stabilization, more preferably hydroxycarboxylic acids, next more preferably lactic acid and citric acid, and most preferably lactic acid.
ヒドロキシカルボン酸は、得られた酵素、組み換えタンパク質、抗体、ペプチドなどが医薬品、食品分野で使用されることを考慮した場合、安全性の観点からも好ましい。 Hydroxycarboxylic acid is preferable from the viewpoint of safety when considering that the obtained enzyme, recombinant protein, antibody, peptide and the like are used in the pharmaceutical and food fields.
有機酸（ａ）による中和度は特に限定されず、部分中和塩であっても良い。例えば、２価の有機酸の場合、２価の完全中和塩であっても、１価分だけの部分中和塩であっても良い。
また、塩（Ａ）としては、１種だけを用いても複数種の塩（Ａ）の混合物を用いても良い。
The degree of neutralization with the organic acid (a) is not particularly limited, and may be a partially neutralized salt. For example, in the case of a divalent organic acid, it may be a divalent fully neutralized salt or a partially neutralized salt only for a monovalent component.
Moreover, as a salt (A), you may use only 1 type or a mixture of multiple types of salt (A).
本発明の安定化剤には緩衝剤及び多価アルコールを含んでもよい。
緩衝剤としては、公知（特開平０８−１８７０９５号公報等）に記載の緩衝剤を使用できる。多価アルコールとしては、グリセリン、ソルビトール及び公知（特開平０８−１８７０９５号公報等）に記載の糖類を使用できる。
The stabilizer of the present invention may contain a buffer and a polyhydric alcohol.
As the buffering agent, the buffering agents described in publicly known (Japanese Patent Laid-Open No. 08-187095 etc.) can be used. As the polyhydric alcohol, glycerin, sorbitol and saccharides described in publicly known (Japanese Patent Laid-Open No. 08-187095 etc.) can be used.
アルギニンと有機酸（ａ）との塩（Ａ）を、酵素、組み換えタンパク質、抗体及びペプチドなどのタンパク質を含有する水溶液に含有させることでタンパク質含有水溶液のタンパク質を安定化できる。 The protein in the protein-containing aqueous solution can be stabilized by containing the salt (A) of arginine and the organic acid (a) in an aqueous solution containing a protein such as an enzyme, a recombinant protein, an antibody, and a peptide.
塩（Ａ）の含有量は、長期間のタンパク質の安定化及びコストの観点から、これらのタンパク質を含有する水溶液の体積に基づいて０．００１〜０．５ｍｏｌ／Ｌの濃度で含有することが好ましく、さらに好ましくは０．０１〜０．４ｍｏｌ／Ｌ、特に好ましくは０．０４〜０．２５ｍｏｌ／Ｌである。 The content of the salt (A) may be contained at a concentration of 0.001 to 0.5 mol / L based on the volume of the aqueous solution containing these proteins from the viewpoint of long-term protein stabilization and cost. More preferably, it is 0.01-0.4 mol / L, Most preferably, it is 0.04-0.25 mol / L.
なお、アルギニン以外の塩基性アミノ酸であるヒスチジン、リシンでも、安定化の効果は認められるが、長期安定化の観点から、アルギニンに比べて劣る。 It should be noted that histidine and lysine, which are basic amino acids other than arginine, have a stabilizing effect, but are inferior to arginine from the viewpoint of long-term stabilization.
タンパク質含有水溶液におけるタンパク質の含有量（重量％）は、タンパク質の安定化の観点からタンパク質含有水溶液の重量に基づいて、０．００１〜１０が好ましく、０．０１〜２がさらに好ましい。 The protein content (% by weight) in the protein-containing aqueous solution is preferably 0.001 to 10, more preferably 0.01 to 2, based on the weight of the protein-containing aqueous solution from the viewpoint of protein stabilization.
タンパク質含有水溶液における塩（Ａ）の含有量（重量％）は、タンパク質の安定化の観点から、タンパク質の重量に基づいて８〜５，０００が好ましく、さらに好ましくは５０〜１，０００である。 The content (% by weight) of the salt (A) in the protein-containing aqueous solution is preferably 8 to 5,000, more preferably 50 to 1,000, based on the weight of the protein, from the viewpoint of protein stabilization.
本発明のタンパク質含有水溶液には、安定化効果を損なわない範囲で、緩衝剤、多価アルコール、金属塩、ゼラチン及び界面活性剤等を添加してもよい。 In the protein-containing aqueous solution of the present invention, a buffer, a polyhydric alcohol, a metal salt, gelatin, a surfactant, and the like may be added within a range that does not impair the stabilizing effect.
緩衝剤及び多価アルコールとしては前述のものが使用できる。金属塩としては、塩化ナトリウム、塩化カリウム等が使用できる。ゼラチンとしては変性コラーゲンであれば特に限定することなく使用することができる。界面活性剤としては公知（特開２００７−２０４４９８号公報等）に記載の非イオン性界面活性剤等が使用でき、市販のＴＷＥＥＮ（登録商標）８０等が容易に入手できる。 As the buffer and polyhydric alcohol, those described above can be used. As the metal salt, sodium chloride, potassium chloride and the like can be used. As the gelatin, any modified collagen can be used without particular limitation. As the surfactant, nonionic surfactants described in publicly known (Japanese Patent Application Laid-Open No. 2007-204498 etc.) can be used, and commercially available TWEEN (registered trademark) 80 can be easily obtained.
本発明のタンパク質含有水溶液の安定化剤の使用方法及び本発明の安定化方法の例を以下に説明するが、アルギニンと有機酸との塩（Ａ）又は本発明の安定化剤をそのまま、あるいは水に溶かしてタンパク質含有水溶液に加えてもよいし、タンパク質をアルギニンと有機酸との塩（Ａ）の水溶液又は本発明の安定化剤の水溶液に加えてもよいし、タンパク質と、アルギニンと有機酸との塩（Ａ）又は本発明の安定化剤と、水とを同時に混ぜてもよい。
分離精製工程で分離された酵素の安定化水溶液を作成する場合の一例を以下に挙げる。
１．アルギニンと有機酸との塩（Ａ）又は本発明の安定化剤を水に加え、水溶液を作製する。
２．分離精製後の酵素水溶液を上記水溶液に加える。
３．常温（１０〜２５℃）もしくは冷蔵（４℃〜１０℃程度）で密封保存する。
Examples of the method for using the stabilizer for the protein-containing aqueous solution of the present invention and the method for stabilizing the present invention will be described below. It may be dissolved in water and added to a protein-containing aqueous solution, or protein may be added to an aqueous solution of a salt (A) of arginine and an organic acid or an aqueous solution of the stabilizer of the present invention, or protein, arginine and organic You may mix the salt (A) with an acid or the stabilizer of this invention, and water simultaneously.
An example of preparing a stabilized aqueous solution of the enzyme separated in the separation and purification step is given below.
1. The salt (A) of arginine and organic acid or the stabilizer of the present invention is added to water to prepare an aqueous solution.
2. The separated aqueous enzyme solution is added to the aqueous solution.
3. Store sealed at room temperature (10-25 ° C) or refrigerated (about 4-10 ° C).
本発明のタンパク質水溶液安定化剤が適用できるタンパク質としては、酵素（Ｐ１）、組み換えタンパク質（Ｐ２）、抗体（Ｐ３）及びペプチド（Ｐ４）などが含まれる。 Proteins to which the aqueous protein stabilizer of the present invention can be applied include enzymes (P1), recombinant proteins (P2), antibodies (P3), peptides (P4) and the like.
酵素（Ｐ１）としては、加水分解酵素、異性化酵素、酸化還元酵素、転移酵素、合成酵素及び脱離酵素などが含まれる。
加水分解酵素としては、リゾチーム、プロテアーゼ、セリンプロテアーゼ、アミラーゼ、リパーゼ、セルラーゼ、グルコアミラーゼなどが挙げられる。
異性化酵素としては、グルコースイソメラーゼが挙げられる。
酸化還元酵素としては、ペルオキシダーゼなどが挙げられる。
転移酵素としては、アシルトランスフェラーゼ、スルホトランスフェラーゼなどが挙げられる。
合成酵素としては、脂肪酸シンターゼ、リン酸シンターゼ、クエン酸シンターゼなどが挙げられる。
脱離酵素としては、ペクチンリアーゼなどが挙げられる。
Examples of the enzyme (P1) include a hydrolase, an isomerase, an oxidoreductase, a transferase, a synthetic enzyme, and a desorbing enzyme.
Examples of hydrolases include lysozyme, protease, serine protease, amylase, lipase, cellulase and glucoamylase.
An example of the isomerase is glucose isomerase.
Examples of the oxidoreductase include peroxidase.
Examples of the transferase include acyltransferase and sulfotransferase.
Examples of the synthase include fatty acid synthase, phosphate synthase, and citrate synthase.
Examples of the desorption enzyme include pectin lyase.
組み換えタンパク質（Ｐ２）としては、タンパク製剤、ワクチン等が含まれる。
タンパク製剤としては、インターフェロンα、インターフェロンβ、インターロイキン１〜１２、成長ホルモン、エリスロポエチン、インスリン、顆粒状コロニー刺激因子（Ｇ−ＣＳＦ）、組織プラスミノーゲン活性化因子（ＴＰＡ）、ナトリウム利尿ペプチド、血液凝固第ＶＩＩＩ因子、ソマトメジン、グルカゴン、成長ホルモン放出因子、血清アルブミン、カルシトニン等が挙げられる。
ワクチンとしては、Ａ型肝炎ワクチン、Ｂ型肝炎ワクチン、Ｃ型肝炎ワクチン及びインフルエンザワクチン等が挙げられる。
The recombinant protein (P2) includes protein preparations, vaccines and the like.
Examples of protein preparations include interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), natriuretic peptide, Examples include blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like.
Examples of the vaccine include hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, and influenza vaccine.
抗体（Ｐ３）としては、モノクローナル抗体及びポリクローナル抗体等が挙げられる。モノクローナル抗体には、パーオキシターゼやアルカリ性フォスファターゼで標識した酵素標識抗体も含まれる。 Examples of the antibody (P3) include a monoclonal antibody and a polyclonal antibody. Monoclonal antibodies also include enzyme-labeled antibodies labeled with peroxidase or alkaline phosphatase.
ペプチド（Ｐ４）としては、アミノ酸の個数が２〜５０個の化合物であり、特にアミノ酸組成を限定するものではなく、ジペプチド、トリペプチドなどが含まれる。 The peptide (P4) is a compound having 2 to 50 amino acids, and is not particularly limited in amino acid composition, and includes dipeptides and tripeptides.
これらのうち、本発明は、タンパク質の安定化の観点から、酵素（Ｐ１）及び組み換えタンパク質（Ｐ２）に好適に適用され、特に（Ｐ１）に適している。 Among these, the present invention is preferably applied to the enzyme (P1) and the recombinant protein (P2) from the viewpoint of protein stabilization, and is particularly suitable for (P1).
以下の実施例により本発明を更に説明するが、本発明はこれらに限定されるものではない。 The following examples further illustrate the invention, but the invention is not limited thereto.
実施例１ （アルギニン・乳酸塩の合成）
２００ｍＬコルベンにアルギニン（和光純薬製）８．７ｇ（０．０５モル）を入れ、イオン交換水１００ｇに溶解させた。ここに９０％乳酸（和光純薬製）を５．０ｇ（０．０５モル）を少しずつ加え、６０℃で１時間加熱攪拌して反応させた。エバポレーターで濃縮後、水から再結晶して０．２Ｔｏｒｒで３時間減圧乾燥し、アルギニン・乳酸塩１１．９ｇ（収率９９％）を得た。核磁気共鳴スペクトル（ＮＭＲ）で測定した純度は９９％であった。
Example 1 (Synthesis of Arginine / Lactate)
In 200 mL Kolben, 8.7 g (0.05 mol) of arginine (manufactured by Wako Pure Chemical Industries, Ltd.) was added and dissolved in 100 g of ion-exchanged water. To this, 5.0 g (0.05 mol) of 90% lactic acid (manufactured by Wako Pure Chemical Industries) was added little by little, and the mixture was reacted by heating and stirring at 60 ° C. for 1 hour. After concentrating with an evaporator, it was recrystallized from water and dried under reduced pressure at 0.2 Torr for 3 hours to obtain 11.9 g (99% yield) of arginine / lactate. The purity measured by nuclear magnetic resonance spectrum (NMR) was 99%.
実施例２ （アルギニン・クエン酸塩の合成）
実施例１において、乳酸をクエン酸１水和物３．５ｇ（０．０１７モル）に変更する以外は実施１と同様におこない、アルギニン・クエン酸塩１１．０ｇ（収率９８％）を得た。純度は９９％であった。
Example 2 (Synthesis of Arginine / Citrate)
In Example 1, except that lactic acid was changed to citric acid monohydrate 3.5 g (0.017 mol), the same procedure as in Example 1 was carried out to obtain 11.0 g of arginine citrate (yield 98%). It was. The purity was 99%.
実施例３ （アルギニン・酢酸塩の合成）
実施例１において、乳酸を酢酸３．０ｇ（０．０５モル）に変更する以外は実施例１と同様におこない、アルギニン・酢酸塩１０．７ｇ（収率９８％）を得た。純度は９９％であった。
Example 3 (Synthesis of Arginine / Acetate)
Example 1 was carried out in the same manner as in Example 1 except that lactic acid was changed to 3.0 g (0.05 mol) of acetic acid to obtain 10.7 g of arginine / acetate (yield 98%). The purity was 99%.
実施例４ （アルギニン・蟻酸塩の合成）
実施例１において、乳酸を蟻酸２．３ｇ（０．０５モル）に変更する以外は実施例１と同様におこない、アルギニン・蟻酸塩１０．０ｇ（収率９７％）を得た。純度は９９％であった。
Example 4 (Synthesis of arginine / formate)
Example 1 was carried out in the same manner as in Example 1 except that lactic acid was changed to 2.3 g (0.05 mol) of formic acid to obtain 10.0 g (yield 97%) of arginine / formate. The purity was 99%.
実施例５
リゾチーム（市販品「リゾチーム」和光純薬製、力価２０，０００ｕｎｉｔｓ／ｍｇ）１０ｍｇを５０ｍＭリン酸緩衝液（ｐＨ＝７）０．９ｍＬに溶解させ、さらに、予め実施例１のアルギニン・乳酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・乳酸塩水溶液０．１ｍＬを加え、１分間かき混ぜ本発明のタンパク質含有水溶液（Ｒ−１）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４１ｍｏｌ／Ｌであった。
この（Ｒ−１）の酵素活性（力価）を後述の方法で測定した。
Example 5
10 mg of lysozyme (commercial product “Lysozyme” manufactured by Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg) was dissolved in 0.9 mL of 50 mM phosphate buffer (pH = 7), and the arginine / lactate salt of Example 1 was previously prepared. 0.1 g of 10 wt% arginine / lactate aqueous solution dissolved by stirring and mixing 1 g in 9 g of ion exchange water was added and stirred for 1 minute to obtain the protein-containing aqueous solution (R-1) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.041 mol / L based on the volume of the protein-containing aqueous solution.
The enzyme activity (titer) of this (R-1) was measured by the method described later.
実施例６
実施例５において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例２のアルギニンクエン酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・クエン酸塩水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、本発明のリゾチーム含有水溶液（Ｒ−２）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・クエン酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０１６ｍｏｌ／Ｌであった。
Example 6
In Example 5, instead of the 10 wt% arginine / lactate aqueous solution, 1 g of arginine citrate of Example 2 was previously stirred and mixed in 9 g of ion-exchanged water and dissolved in 10 g of arginine / citrate. Except having used 0.1 mL of aqueous solution, it carried out similarly to Example 5 and obtained the lysozyme containing aqueous solution (R-2) of this invention.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / citrate in the obtained protein-containing aqueous solution was 0.016 mol / L based on the volume of the protein-containing aqueous solution.
実施例７
実施例５において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例３のアルギニン・酢酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・酢酸塩水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、本発明のリゾチーム含有水溶液（Ｒ−３）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・酢酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４６ｍｏｌ／Ｌであった。
Example 7
In Example 5, instead of the 10 wt% arginine / lactate aqueous solution, 10 g of arginine / acetate aqueous solution in which 1 g of arginine / acetate of Example 3 was previously stirred and mixed in 9 g of ion-exchanged water was dissolved. Except having used 0.1 mL, it carried out similarly to Example 5 and obtained the lysozyme containing aqueous solution (R-3) of this invention.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / acetate in the obtained protein-containing aqueous solution was 0.046 mol / L based on the volume of the protein-containing aqueous solution.
実施例８
実施例５において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例４のアルギニン・蟻酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・蟻酸塩水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、本発明のリゾチーム含有水溶液（Ｒ−４）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・蟻酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０５０ｍｏｌ／Ｌであった。
Example 8
In Example 5, instead of the 10 wt% arginine / lactate aqueous solution, 1 g of arginine / formate of Example 4 was previously stirred and mixed in 9 g of ion-exchanged water and dissolved in 10 g of arginine / formate aqueous solution. Except having used 0.1 mL, it carried out similarly to Example 5 and obtained the lysozyme containing aqueous solution (R-4) of this invention.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / formate in the obtained protein-containing aqueous solution was 0.050 mol / L based on the volume of the protein-containing aqueous solution.
実施例９
実施例５において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例１のアルギニン・乳酸塩０．１ｇをイオン交換水４９．９ｇに撹拌混合して溶解させておいた０．２重量％アルギニン・乳酸塩水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、本発明のリゾチーム含有水溶液（Ｒ−５）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．００１ｍｏｌ／Ｌであった。
Example 9
In Example 5, in place of the 10 wt% arginine / lactate aqueous solution, 0.2 g of 0.1 g of arginine / lactate of Example 1 previously dissolved in 49.9 g of ion-exchanged water while being stirred and mixed. A lysozyme-containing aqueous solution (R-5) of the present invention was obtained in the same manner as in Example 5 except that 0.1 mL of a% arginine / lactate aqueous solution was used.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.001 mol / L based on the volume of the protein-containing aqueous solution.
実施例１０
実施例５において、１０重量％アルギニン・乳酸塩水溶液の代わりに、実施例１のアルギニン・乳酸塩０．１２３ｇをそのまま使用する以外は実施例５と同様に行い、本発明のリゾチーム含有水溶液（Ｒ−６）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に１．０重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．５０ｍｏｌ／Ｌであった。
Example 10
In Example 5, instead of the 10 wt% arginine / lactate aqueous solution, 0.123 g of arginine / lactate of Example 1 was used as it was, and the lysozyme-containing aqueous solution (R -6) was obtained.
The protein concentration in the obtained protein-containing aqueous solution was 1.0% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.50 mol / L based on the volume of the protein-containing aqueous solution.
比較例１
実施例５において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させた牛血清アルブミン（和光純薬製）の水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、リゾチーム含有水溶液（Ｒ’−１）を得た。
Comparative Example 1
In Example 5, instead of the arginine / lactate aqueous solution, an example was used except that 0.1 mL of an aqueous solution of bovine serum albumin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. 5 was performed to obtain a lysozyme-containing aqueous solution (R′-1).
比較例２
実施例５において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグルコース（和光純薬製）の水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、リゾチーム含有水溶液（Ｒ’−２）を得た。
Comparative Example 2
In Example 5, instead of the arginine / lactate aqueous solution, Example 5 was used except that 0.1 mL of an aqueous solution of glucose (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. It carried out similarly and obtained lysozyme containing aqueous solution (R'-2).
比較例３
実施例５において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグリセリン（和光純薬製）の水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、リゾチーム含有水溶液（Ｒ’−３）を得た。
Comparative Example 3
In Example 5, instead of the arginine / lactate aqueous solution, Example 5 was used except that 0.1 mL of an aqueous solution of glycerin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. It carried out similarly and obtained lysozyme containing aqueous solution (R'-3).
比較例４
実施例５において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたアルギニン・塩酸塩（和光純薬製）の水溶液０．１ｍＬを使用する以外は実施例５と同様に行い、リゾチーム含有水溶液（Ｒ’−４）を得た。
Comparative Example 4
In Example 5, in place of the arginine / lactate aqueous solution, 0.1 mL of an aqueous solution of arginine / hydrochloride (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. It carried out like Example 5 and obtained lysozyme containing aqueous solution (R'-4).
＜リゾチーム含有水溶液の酵素活性（力価）の測定＞
実施例５〜１０及び比較例１〜４で得られたリゾチーム含有水溶液（Ｒ−１）〜（Ｒ−６）及び（Ｒ’−１）〜（Ｒ’−４）が入った容積１．５ｍＬのエッペンドルフチューブを８０℃に温調した振とう器付き恒温槽で３０分インキュベートし、各溶液５０μＬを、２％枯草菌懸濁液（２０％枯草菌懸濁液２ｍＬに５０ｍＭリン酸緩衝液１８ｍＬを加えて前もって調製したもの）１ｍＬと５０ｍＭリン酸緩衝液２ｍＬとが入った試験管に加えた。加えた直後の４５０ｎｍにおける吸光度（Ａ0）を分光光度計（島津製作所製、ＵＶ−２５５０）で測定し、さらに測定開始から５分後にもう一度吸光度（Ａ5）を測定し、これらの４５０ｎｍにおける５分間の吸光度変化（ΔＡ）を以下の式で算出した。
ΔＡ＝Ａ5−Ａ0
また、実施例５〜１０及び比較例１〜４のリゾチーム水溶液のリゾチーム濃度と同濃度の市販品「リゾチーム」の水溶液で他に添加剤を加えないブランクの水溶液を調製し、これを用いて、インキュベートしないこと以外は上記と同様に測定し、４５０ｎｍにおける５分間の吸光度変化（ΔＡb）を測定し算出した。
ΔＡb＝Ａb5−Ａb0
<Measurement of enzyme activity (titer) of lysozyme-containing aqueous solution>
A volume of 1.5 mL containing the lysozyme-containing aqueous solutions (R-1) to (R-6) and (R′-1) to (R′-4) obtained in Examples 5 to 10 and Comparative Examples 1 to 4 The Eppendorf tube was incubated in a thermostatic chamber with a shaker at 80 ° C. for 30 minutes, and 50 μL of each solution was added to 2 mL of 2% Bacillus subtilis suspension (2 mL of 20% Bacillus subtilis suspension and 18 mL of 50 mM phosphate buffer). (Prepared in advance) and added to a test tube containing 1 mL and 2 mL of 50 mM phosphate buffer. The absorbance (A 0 ) at 450 nm immediately after the addition was measured with a spectrophotometer (manufactured by Shimadzu Corporation, UV-2550), and the absorbance (A 5 ) was measured again 5 minutes after the start of the measurement. The change in absorbance (ΔA) per minute was calculated by the following formula.
ΔA = A 5 −A 0
Moreover, the aqueous solution of the blank which does not add an additive with the aqueous solution of the commercial item "lysozyme" of the same density | concentration as the lysozyme density | concentration of the lysozyme aqueous solution of Examples 5-10 and Comparative Examples 1-4 was prepared, and using this, The measurement was performed in the same manner as described above except that the incubation was not performed, and the change in absorbance (ΔA b ) at 450 nm for 5 minutes was measured and calculated.
ΔA b = A b5 −A b0
８０℃で３０分間保管後の各リゾチーム含有水溶液の「力価の保持率」は以下の式を用いて算出した。 The “titer retention” of each lysozyme-containing aqueous solution after storage at 80 ° C. for 30 minutes was calculated using the following formula.
力価の保持率（％）＝（ΔＡ／ΔＡb） ×１００ Retention rate of titer (%) = (ΔA / ΔA b ) × 100
上記実施例及び比較例の力価の保持率を表１に示す。 Table 1 shows the retention ratios of the titers of the above examples and comparative examples.
従来から使用されている安定化剤を用いた比較例１〜３は、酵素含有水溶液の安定化効果がまだ不十分であることがわかる。また、アルギニンの無機酸塩を使用した比較例４では、安定化効果が高まり、比較例１〜３に比べて力価の保持率が改善されるものの、依然として十分な効果が得られなかった。一方、アルギニンを有機酸で中和した本発明のアルギニンと有機酸との塩を使用した実施例５〜１０の安定化剤の力価の保持率は非常に高いことがわかる。 It can be seen that Comparative Examples 1 to 3 using conventionally used stabilizers still have insufficient stabilizing effect on the enzyme-containing aqueous solution. Moreover, in Comparative Example 4 using the inorganic acid salt of arginine, the stabilizing effect was enhanced, and although the titer retention rate was improved as compared with Comparative Examples 1 to 3, a sufficient effect was still not obtained. On the other hand, the retention of the titer of the stabilizers of Examples 5 to 10 using the salt of arginine and organic acid of the present invention obtained by neutralizing arginine with organic acid is very high.
実施例１１
遺伝子組換えインターフェロンβ（和光純薬工業製、マウス由来）１００μｇをイオン水１００μＬに溶解させ、この水溶液１００μＬに、予め実施例１のアルギニン・乳酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・乳酸塩水溶液１０μＬを加えて軽く振り混ぜて均一化して本発明のインターフェロンβ水溶液（Ｉ−１）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０３７ｍｏｌ／Ｌであった。
この（Ｉ−１）の活性（力価）を後述の方法で測定した。
Example 11
100 μg of recombinant interferon β (manufactured by Wako Pure Chemical Industries, Ltd., derived from mouse) is dissolved in 100 μL of ionic water, and 1 g of arginine / lactate of Example 1 is previously mixed with 9 g of ion-exchanged water in 100 μL of this aqueous solution. 10 μL of the 10% by weight arginine / lactate aqueous solution was added, and the mixture was lightly mixed to homogenize to obtain an interferon β aqueous solution (I-1) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.037 mol / L based on the volume of the protein-containing aqueous solution.
The activity (titer) of this (I-1) was measured by the method described later.
実施例１２
実施例１１において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例２のアルギニン・クエン酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・クエン酸塩水溶液１０μＬを使用する以外は実施例１１と同様に行い、本発明のインターフェロンβ水溶液（Ｉ−２）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・クエン酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０１４ｍｏｌ／Ｌであった。
Example 12
In Example 11, instead of the 10% by weight arginine / lactate aqueous solution, 1 g of arginine / citrate of Example 2 was previously stirred and mixed in 9 g of ion-exchanged water, and dissolved therein. Except for using 10 μL of the aqueous salt solution, the same procedure as in Example 11 was performed to obtain an interferon β aqueous solution (I-2) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / citrate in the obtained protein-containing aqueous solution was 0.014 mol / L based on the volume of the protein-containing aqueous solution.
実施例１３
実施例１１において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例３のアルギニン・酢酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・酢酸塩水溶液１０μＬを使用する以外は実施例１１と同様に行い、本発明のインターフェロンβ水溶液（Ｉ−３）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・酢酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４２ｍｏｌ／Ｌであった。
Example 13
In Example 11, instead of the 10 wt% arginine / lactate aqueous solution, 10 g arginine / acetate aqueous solution in which 1 g of arginine / acetate of Example 3 was previously stirred and mixed in 9 g of ion-exchanged water was dissolved. Except using 10 microliters, it carried out like Example 11 and obtained interferon beta aqueous solution (I-3) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / acetate in the obtained protein-containing aqueous solution was 0.042 mol / L based on the volume of the protein-containing aqueous solution.
実施例１４
実施例１１において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例４のアルギニン・蟻酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・蟻酸塩水溶液１０μＬを使用する以外は実施例１１と同様に行い、本発明のインターフェロンβ水溶液（Ｉ−４）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・蟻酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４５ｍｏｌ／Ｌであった。
Example 14
In Example 11, instead of the 10 wt% arginine / lactate aqueous solution, 10 g% arginine / formate aqueous solution in which 1 g of arginine / formate of Example 4 was previously stirred and mixed in 9 g of ion-exchanged water was dissolved. Except using 10 microliters, it carried out like Example 11 and obtained interferon beta aqueous solution (I-4) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / formate in the obtained protein-containing aqueous solution was 0.045 mol / L based on the volume of the protein-containing aqueous solution.
実施例１５
実施例１１において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例１のアルギニン・乳酸塩０．２ｇをイオン交換水４９．８ｇに撹拌混合して溶解させておいた０．２重量％アルギニン・乳酸塩水溶液１０μＬを使用する以外は実施例１１と同様に行い、本発明のインターフェロンβ水溶液（Ｉ−５）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０１ｍｏｌ／Ｌであった。
Example 15
In Example 11, instead of the 10% by weight arginine / lactate aqueous solution, 0.2 g of 0.2 g of arginine / lactate of Example 1 previously stirred and mixed in 49.8 g of ion-exchanged water was dissolved. The same procedure was carried out as in Example 11 except that 10 μL of a% arginine / lactate aqueous solution was used to obtain an interferon β aqueous solution (I-5) of the present invention.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.01 mol / L based on the volume of the protein-containing aqueous solution.
実施例１６
実施例１１において、１０重量％アルギニン・乳酸塩水溶液の代わりに、実施例１のアルギニン・乳酸塩０．００１８ｇをそのまま使用する以外は実施例１１と同様に行い、本発明のインターフェロンβ水溶液（Ｉ−６）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・クエン酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．５０ｍｏｌ／Ｌであった。
Example 16
In Example 11, instead of the 10 wt% arginine / lactate aqueous solution, 0.0018 g of arginine / lactate of Example 1 was used as it was, and the interferon β aqueous solution (I -6) was obtained.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / citrate in the obtained protein-containing aqueous solution was 0.50 mol / L based on the volume of the protein-containing aqueous solution.
比較例５
実施例１１において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させた牛血清アルブミン（和光純薬製）の水溶液１０μＬを使用する以外は実施例１１と同様に行い、インターフェロンβ水溶液（Ｉ’−１）を得た。
Comparative Example 5
Example 11 is different from Example 11 except that 10 μL of an aqueous solution of bovine serum albumin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight is used instead of the arginine / lactate aqueous solution. In the same manner, an interferon β aqueous solution (I′-1) was obtained.
比較例６
実施例１１において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグルコース（和光純薬製）の水溶液１０μＬを使用する以外は実施例１１と同様に行い、インターフェロンβ水溶液（Ｉ’−２）を得た。
Comparative Example 6
In Example 11, instead of the arginine / lactate aqueous solution, 10 μL of an aqueous solution of glucose (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used in the same manner as in Example 11. This gave an interferon β aqueous solution (I′-2).
比較例７
実施例１１において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグリセリン（和光純薬製）の水溶液１０μＬを使用する以外は実施例１１と同様に行い、インターフェロンβ水溶液（Ｉ’−３）を得た。
Comparative Example 7
In Example 11, in place of the arginine / lactate aqueous solution, 10 μL of an aqueous solution of glycerin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used in the same manner as in Example 11. And an interferon β aqueous solution (I′-3) was obtained.
比較例８
実施例１１において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたアルギニン・塩酸塩（和光純薬製）の水溶液１０μＬを使用する以外は実施例１１と同様に行い、インターフェロンβ水溶液（Ｉ’−４）を得た。
Comparative Example 8
In Example 11, instead of the arginine / lactate aqueous solution, Example 11 was used except that 10 μL of an aqueous solution of arginine / hydrochloride (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. In the same manner as above, an interferon β aqueous solution (I′-4) was obtained.
＜インターフェロンβ水溶液の活性（力価）の測定＞
実施例１１〜１６及び比較例５〜８で得られたインターフェロン水溶液（Ｉ−１）〜（Ｉ−６）及び（Ｉ’−１）〜（Ｉ’−４）を恒温槽中で３７℃、３時間インキュベートし、この水溶液５０μＬを、インターフェロンβ ＥＬＩＳＡキット（「インターフェロンβ ＥＬＩＳＡキット」、鎌倉テクノサイエンス社製）を用いて、力価を測定した。力価はインターフェロンに作用する酵素標識抗インターフェロンモノクローナル抗体の酵素活性で評価した。酵素活性はキットに添付の説明書に従い、分光光度計で吸光度を読み取り検量線を作成し、検量線から各サンプルの力価を算出した。
また、インターフェロン水溶液（Ｉ−１）〜（Ｉ−６）及び（Ｉ’−１）〜（Ｉ’−４）を恒温槽でインキュベートする前に上記と同様に測定し、別途力価を算出した。
力価の保持率は、以下の式で計算した。
力価の保持率＝（インキュベート後の力価）／（インキュベート前の力価）×１００
<Measurement of activity (titer) of aqueous solution of interferon β>
The interferon aqueous solutions (I-1) to (I-6) and (I′-1) to (I′-4) obtained in Examples 11 to 16 and Comparative Examples 5 to 8 were maintained at 37 ° C. in a constant temperature bath. After incubating for 3 hours, the titer of 50 μL of this aqueous solution was measured using an interferon β ELISA kit (“Interferon β ELISA kit”, manufactured by Kamakura Technoscience). The titer was evaluated by the enzyme activity of an enzyme-labeled anti-interferon monoclonal antibody that acts on interferon. The enzyme activity was determined by reading the absorbance with a spectrophotometer according to the instructions attached to the kit, creating a calibration curve, and calculating the titer of each sample from the calibration curve.
Moreover, before incubating interferon aqueous solution (I-1)-(I-6) and (I'-1)-(I'-4) in a thermostat, it measured similarly to the above, and calculated the titer separately. .
The retention rate of titer was calculated by the following formula.
Retention rate of titer = (titer after incubation) / (titer before incubation) × 100
上記実施例及び比較例の力価の保持率を表２に示す。 Table 2 shows the retention ratios of the titers of the above examples and comparative examples.
従来から使用されている安定化剤を用いた比較例５〜７は、組換えタンパク質含有水溶液の安定化効果がまだ不十分であることがわかる。また、アルギニンの無機酸塩を使用した比較例８では、安定化効果が高まり、比較例５〜７に比べて力価の保持率が改善されるものの、依然として十分な効果が得られなかった。一方、アルギニンを有機酸で中和した本発明のアルギニンと有機酸との塩を使用した実施例１１〜１６の安定化剤の力価の保持率は非常に高いことがわかる。 It can be seen that Comparative Examples 5 to 7 using the conventionally used stabilizers still have insufficient stabilizing effect of the aqueous solution containing the recombinant protein. Moreover, in Comparative Example 8 using the inorganic acid salt of arginine, the stabilization effect was enhanced, and although the titer retention rate was improved as compared with Comparative Examples 5 to 7, a sufficient effect was still not obtained. On the other hand, it can be seen that the retention of the titers of the stabilizers of Examples 11 to 16 using the salt of arginine of the present invention obtained by neutralizing arginine with an organic acid and the organic acid is very high.
実施例１７
西洋ワサビ由来ペルオキシターゼ標識抗ヒトインターフェロンβマウスモノクローナル抗体溶液（鎌倉テクノサイエンス社製）１００μＬに、予め実施例１のアルギニン・乳酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・乳酸塩水溶液を１０μＬを加えて軽く振り混ぜて均一化して本発明のモノクローナル抗体水溶液（Ｍ−１）を得た。
この（Ｍ−１）の活性（力価）を後述の方法で測定した。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０３７ｍｏｌ／Ｌであった。
Example 17
Horseradish-derived peroxidase-labeled anti-human interferon β mouse monoclonal antibody solution (manufactured by Kamakura Techno Science Co., Ltd.) 100 μL, 10 g of arginine / lactate of Example 1 previously stirred and mixed in 9 g of ion-exchanged water A 10% arginine / lactate aqueous solution was added and lightly mixed to homogenize to obtain an aqueous monoclonal antibody solution (M-1) of the present invention.
The activity (titer) of this (M-1) was measured by the method described later.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.037 mol / L based on the volume of the protein-containing aqueous solution.
実施例１８
実施例１７において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例２のアルギニン・クエン酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・クエン酸塩水溶液１０μＬを使用する以外は実施例１７と同様に行い、本発明のモノクローナル抗体水溶液（Ｍ−２）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・クエン酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０１４ｍｏｌ／Ｌであった。
Example 18
In Example 17, instead of the 10% by weight arginine / lactate aqueous solution, 1 g of arginine / citrate of Example 2 was previously stirred and mixed in 9 g of ion-exchanged water, and dissolved in 10% by weight of arginine / citric acid. A monoclonal antibody aqueous solution (M-2) of the present invention was obtained in the same manner as in Example 17 except that 10 μL of the salt aqueous solution was used.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / citrate in the obtained protein-containing aqueous solution was 0.014 mol / L based on the volume of the protein-containing aqueous solution.
実施例１９
実施例１７において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例３のアルギニン・酢酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・酢酸塩水溶液１０μＬを使用する以外は実施例１７と同様に行い、本発明のモノクローナル抗体水溶液（Ｍ−３）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・酢酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４２ｍｏｌ／Ｌであった。
Example 19
In Example 17, instead of the 10 wt% arginine / lactate aqueous solution, 10 g of arginine / acetate aqueous solution in which 1 g of arginine / acetate of Example 3 was previously stirred and mixed in 9 g of ion-exchanged water was dissolved. A monoclonal antibody aqueous solution (M-3) of the present invention was obtained in the same manner as in Example 17 except that 10 μL was used.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / acetate in the obtained protein-containing aqueous solution was 0.042 mol / L based on the volume of the protein-containing aqueous solution.
実施例２０
実施例１７において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例４のアルギニン・蟻酸塩１ｇをイオン交換水９ｇに撹拌混合して溶解させておいた１０重量％アルギニン・蟻酸塩水溶液１０μＬを使用する以外は実施例１７と同様に行い、本発明のモノクローナル抗体水溶液（Ｍ−４）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・蟻酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０４５ｍｏｌ／Ｌであった。
Example 20
In Example 17, in place of the 10 wt% arginine / lactate aqueous solution, 1 g of arginine / formate of Example 4 was previously stirred and mixed in 9 g of ion-exchanged water and dissolved in 10 g of arginine / formate aqueous solution. A monoclonal antibody aqueous solution (M-4) of the present invention was obtained in the same manner as in Example 17 except that 10 μL was used.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / formate in the obtained protein-containing aqueous solution was 0.045 mol / L based on the volume of the protein-containing aqueous solution.
実施例２１
実施例１７において、１０重量％アルギニン・乳酸塩水溶液の代わりに、予め実施例１のアルギニン・乳酸塩０．１ｇをイオン交換水４９．９ｇに撹拌混合して溶解させておいた０．２重量％アルギニン・乳酸塩水溶液１０μＬを使用する以外は実施例１７と同様に行い、本発明のモノクローナル抗体水溶液（Ｍ−５）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・乳酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．０１ｍｏｌ／Ｌであった。
Example 21
In Example 17, in place of the 10 wt% arginine / lactate aqueous solution, 0.2 g of 0.1 g of arginine / lactate of Example 1 previously dissolved in 49.9 g of ion-exchanged water was mixed and dissolved. A monoclonal antibody aqueous solution (M-5) of the present invention was obtained in the same manner as in Example 17 except that 10 μL of a 10% arginine / lactate aqueous solution was used.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / lactate in the obtained protein-containing aqueous solution was 0.01 mol / L based on the volume of the protein-containing aqueous solution.
実施例２２
実施例１７において、１０重量％アルギニン・乳酸塩水溶液の代わりに、実施例１のアルギニン・乳酸塩０．００１４ｇをそのまま使用する以外は実施例１７と同様に行い、本発明のモノクローナル抗体水溶液（Ｍ−６）を得た。
得られたタンパク質含有水溶液中のタンパク質濃度は、タンパク質含有水溶液の重量を基準に０．０９１重量％であった。得られたタンパク質含有水溶液中のアルギニン・クエン酸塩の濃度は、タンパク質含有水溶液の体積を基準に０．５０ｍｏｌ／Ｌであった。
Example 22
In Example 17, in place of the 10% by weight arginine / lactate aqueous solution, 0.0014 g of arginine / lactate of Example 1 was used as it was, and the monoclonal antibody aqueous solution (M -6) was obtained.
The protein concentration in the obtained protein-containing aqueous solution was 0.091% by weight based on the weight of the protein-containing aqueous solution. The concentration of arginine / citrate in the obtained protein-containing aqueous solution was 0.50 mol / L based on the volume of the protein-containing aqueous solution.
比較例９
実施例１７において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させた牛血清アルブミン（和光純薬製）の水溶液１０μＬを使用する以外は実施例１７と同様に行い、モノクローナル抗体水溶液（Ｍ’−１）を得た。
Comparative Example 9
Example 17 is different from Example 17 except that 10 μL of an aqueous solution of bovine serum albumin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight is used instead of the arginine / lactate aqueous solution. In the same manner, a monoclonal antibody aqueous solution (M′-1) was obtained.
比較例１０
実施例１７において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグルコース（和光純薬製）の水溶液１０μＬを使用する以外は実施例１７と同様に行い、モノクローナル抗体水溶液（Ｍ’−２）を得た。
Comparative Example 10
In Example 17, in place of the arginine / lactate aqueous solution, 10 μL of an aqueous solution of glucose (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used in the same manner as in Example 17. And a monoclonal antibody aqueous solution (M′-2) was obtained.
比較例１１
実施例１７において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたグリセリン（和光純薬製）の水溶液１０μＬを使用する以外は実施例１７と同様に行い、モノクローナル抗体水溶液（Ｍ’−３）を得た。
Comparative Example 11
In Example 17, in place of the arginine / lactate aqueous solution, 10 μL of an aqueous solution of glycerin (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used in the same manner as in Example 17. Then, an aqueous monoclonal antibody solution (M′-3) was obtained.
比較例１２
実施例１７において、アルギニン・乳酸塩水溶液の代わりに、１０重量％となるようにイオン交換水で予め溶解させたアルギニン・塩酸塩（和光純薬製）の水溶液１０μＬを使用する以外は実施例１７と同様に行い、モノクローナル抗体水溶液（Ｍ’−４）を得た。
Comparative Example 12
In Example 17, instead of the arginine / lactate aqueous solution, Example 17 was used except that 10 μL of an aqueous solution of arginine / hydrochloride (manufactured by Wako Pure Chemical Industries) previously dissolved in ion-exchanged water so as to be 10% by weight was used. And an aqueous monoclonal antibody solution (M′-4) was obtained.
＜モノクローナル抗体水溶液の活性（力価）の測定＞
実施例１７〜２２及び比較例９〜１２で得られたモノクローナル抗体水溶液（Ｍ−１）〜（Ｍ−６）及び（Ｍ’−１）〜（Ｍ’−４）を恒温槽中で３７℃、３時間インキュベートし、この水溶液５０μＬを、インターフェロンβ ＥＬＩＳＡキット（「インターフェロンβ ＥＬＩＳＡキット」、鎌倉テクノサイエンス社製）を用いて、力価を測定した。力価はインターフェロン標準液（キットに付属）に作用する酵素標識抗インターフェロンモノクローナル抗体の酵素活性で評価した。酵素活性はキットに添付の説明書に従い、分光光度計で吸光度を読み取り検量線を作成し、検量線から各サンプルの力価を算出した。
また、モノクローナル抗体水溶液（Ｍ−１）〜（Ｍ−６）及び（Ｍ’−１）〜（Ｍ’−４）を恒温槽でインキュベートする前に上記と同様に測定し、別途力価を算出した。
力価の保持率は、以下の式で計算した。
力価の保持率＝（インキュベート後の力価）／（インキュベート前の力価）×１００
<Measurement of activity (titer) of monoclonal antibody aqueous solution>
The monoclonal antibody aqueous solutions (M-1) to (M-6) and (M′-1) to (M′-4) obtained in Examples 17 to 22 and Comparative Examples 9 to 12 were incubated at 37 ° C. in a thermostatic bath. After incubating for 3 hours, the titer of 50 μL of this aqueous solution was measured using an interferon β ELISA kit (“Interferon β ELISA kit”, manufactured by Kamakura Technoscience). The titer was evaluated by the enzyme activity of an enzyme-labeled anti-interferon monoclonal antibody that acts on an interferon standard solution (included in the kit). The enzyme activity was determined by reading the absorbance with a spectrophotometer according to the instructions attached to the kit, creating a calibration curve, and calculating the titer of each sample from the calibration curve.
Moreover, before incubating monoclonal antibody aqueous solution (M-1)-(M-6) and (M'-1)-(M'-4) in a thermostat, it measures similarly to the above and calculates titer separately. did.
The retention rate of titer was calculated by the following formula.
Retention rate of titer = (titer after incubation) / (titer before incubation) × 100
上記実施例及び比較例の力価の保持率を表３に示す。 Table 3 shows the retention ratios of the titers of the above examples and comparative examples.
従来から使用されている安定化剤を用いた比較例９〜１１は、抗体含有水溶液の安定化効果がまだ不十分であることがわかる。また、アルギニンの無機酸塩を使用した比較例１２では、安定化効果が高まり、比較例９〜１１に比べて力価の保持率が改善されるものの、依然として十分な効果が得られなかった。一方、アルギニンを有機酸で中和した本発明のアルギニンと有機酸との塩を使用した実施例１７〜２２の安定化剤の力価の保持率は非常に高いことがわかる。 It can be seen that Comparative Examples 9 to 11 using a conventionally used stabilizer are still insufficient in stabilizing effect of the antibody-containing aqueous solution. Moreover, in Comparative Example 12 using the inorganic acid salt of arginine, the stabilization effect was enhanced, and although the titer retention rate was improved as compared with Comparative Examples 9 to 11, a sufficient effect was still not obtained. On the other hand, it can be seen that the retention of the titers of the stabilizers of Examples 17 to 22 using the salt of arginine of the present invention obtained by neutralizing arginine with an organic acid and the organic acid is very high.
本発明のタンパク質含有水溶液のの安定化剤は、タンパク質を安定化し活性を長期間低下させないので、医薬品、食品、および生化学の分野において有効に使用することができる。たとえば、タンパク医薬品液体製剤、酵素液体製剤、工業用酵素水溶液、液体洗剤、飲料、診断薬用の測定試薬、タンパク質の標準液などに使用できる。 Since the stabilizer of the protein-containing aqueous solution of the present invention stabilizes the protein and does not reduce the activity for a long period of time, it can be effectively used in the fields of pharmaceuticals, foods, and biochemistry. For example, it can be used in protein pharmaceutical liquid preparations, enzyme liquid preparations, industrial enzyme aqueous solutions, liquid detergents, beverages, measuring reagents for diagnostic agents, protein standard solutions, and the like.
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| WO2011024862A1 (en) * | 2009-08-31 | 2011-03-03 | 三洋化成工業株式会社 | Stabilizer for aqueous protein solution, aqueous protein solution and liquid detergent composition each containing the stabilizer, and method for stabilization of protein using the stabilizer |
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| JP2011147440A (en) * | 2009-12-24 | 2011-08-04 | Sanyo Chem Ind Ltd | Method for preserving aqueous protein solution |
| CN110062620A (en) * | 2016-10-31 | 2019-07-26 | 德国费森尤斯卡比有限公司 | liquid pharmaceutical composition |
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| CN110062620B (en) * | 2016-10-31 | 2023-12-05 | 德国费森尤斯卡比有限公司 | liquid pharmaceutical composition |
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