JP6889265B2 - 微小球が結合したクロマトグラフィー媒体、及びその製造方法 - Google Patents
微小球が結合したクロマトグラフィー媒体、及びその製造方法 Download PDFInfo
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Description
多孔質マトリックスと、
非多孔質微小球と、
を含むクロマトグラフィー媒体であって、
非多孔質微小球は、多孔質マトリックスの内表面及び外表面上に結合されており、かつ、
微小球の平均半径は、多孔質マトリックスの平均細孔径の30%以下である、クロマトグラフィー媒体が提供される。
多孔質出発マトリックスを準備することと、
少なくとも1種のモノマー、二官能性、三官能性、又は多官能性の架橋剤、重合開始剤、及び溶剤又は溶剤混合物を含み、少なくとも1種のモノマー、二官能性、三官能性、又は多官能性の架橋剤、及び重合開始剤が、溶剤又は溶剤混合物中に完全に可溶性である重合溶液を準備することと、
重合溶液における重合を多孔質出発マトリックスの存在下で開始させることで、溶剤又は溶剤混合物中に不溶性であり、かつ多孔質出発マトリックスの内表面及び外表面へと結合されている非多孔質微小球を形成することと、
を含み、微小球の平均半径が、多孔質出発マトリックスの平均細孔径の30%以下である、方法に関する。
本発明による方法において出発マトリックスとして使用される0.01μm〜20μm、好ましくは0.1μm〜10μm、より好ましくは0.2μm〜4μmの細孔サイズを有する多孔質マトリックスを、当業者に既知の慣例的な製造方法により製造した。細孔サイズは、キャピラリーフローポロメトリー試験の実施により測定した。その測定のための詳細は、対応する作業説明書(Capillary Flow Porometer 6.0、CAPWIN Software System、Porous Materials Inc.社)から集めることができる。
典型的な重合溶液のために、例えば1.7mL(1.82g、12.79mmol、0.10当量)のグリシジルメタクリレート(モノマー)、0.16g(0.71mmol、7×10−3当量)の2−ヒドロキシ−4−(2−ヒドロキシエトキシ)−2−メチルプロピオフェノン(重合開始剤)、0.33mL(0.347g、1.75mmol、0.013当量)のエチレングリコールジメタクリレート(架橋剤)、0.34mL(0.28g、1.78mmol、0.014当量)の1−デカノール、及び13.36mL(12.85g、128.32mmol、1.00当量)のシクロヘキサノール(溶剤混合物)を秤量又は量り、そして完全に溶解するまで30分間にわたり超音波浴中で処理した。その後に、重合溶液で濡らした試料に、不活性条件(N2雰囲気)下でUV光(紫外光、254nm、40W)(5分間)を照射した。ここで行われるUVで開始される慣用のフリーラジカル重合のために、製造業者LLG社製のBLX−E254 BIO−Link UV架橋剤を使用した。次いで重合されたメンブレンを、流れているRO水のもとで10分間すすぎ、その後にアセトン中で20分間にわたり振盪し、風乾させた。
丸形パンチを使用して、47mmの円形のブランクを実施例2によるクロマトグラフィー媒体から打ち抜いた。得られた47mmの打ち抜き物を逆浸透水で濡らし、流れている逆浸透水のもと5分間すすいだ。各々の打ち抜き物を、Sartorius社のモデル16249のトップフィード型濾過ハウジング内に取り付けた。
タンパク質(リガンド)を、本発明によるクロマトグラフィー媒体の微小球のエポキシ官能基(epoxide functionalities)をアルデヒド官能基へと変換することにより固定化した。タンパク質(リガンド)を、直接的にそこに固定化することが可能であった。このために、本発明により変性されたクロマトグラフィー媒体を、RO水中の3%(重量%)の過ヨウ素酸ナトリウム溶液中に入れて、引き続き、流れているRO水のもと10分間すすぎ、空気中でアセトンから乾燥させた。その後に、こうして製造されたクロマトグラフィー媒体を、リン酸水素カリウム緩衝液(pH8)中の2.5%(重量%)のシアノ水素化ホウ素ナトリウム溶液中に2時間入れて、そこに4.958μL/cm2の組み換えタンパク質溶液(1mLは50.4mgのタンパク質と定義する)を添加することで、7.2mg/mLの濃度を達成した。その後に、該クロマトグラフィー媒体を再びRO水ですすぎ、RO水中の0.01%(重量%)の水素化ホウ素ナトリウム溶液中に10分間入れた。その後に、該クロマトグラフィー媒体を、1×PBS緩衝液(リン酸緩衝食塩溶液)で洗浄し、引き続き20%のエタノール/PBS溶液中で貯蔵した。
免疫グロブリンG(IgG)の結合を、PBS緩衝液(pH=7.5)中の適切なタンパク質溶液(1mg/mL)を使用することにより測定した。実施例2により本発明に従って変性されて、実施例4によりプロテインAで官能化された3層のメンブレンを、メンブレンホルダー中に張着させた。その積層メンブレンは、該メンブレンホルダー中で15cm2のメンブレン面積、5cm2の流入面積、及び500μmのベッド高さ(積層メンブレンの厚さ)を有していた。メンブレンホルダー中のメンブレンに1×PBS緩衝液(pH=7.5)を注入して、空気を排除し、その後にGeneral Electric Health Care社製のAekta Explorer 100 FPLCシステムに接続した。
1. メンブレンを25mLの1×PBS緩衝液(pH=7.5)で平衡化する工程、
2. メンブレンに50mLのIgG溶液をロードする工程、
3. 20mLの1×PBS緩衝液(pH=7.5)で洗浄する工程、及び、
4. 0.1%のグリシン溶液(pH=3.5)で溶出させる工程。
行われる表面積測定は、micromeritics社製のGemini Vを用いて実施された。測定されるべき試料を事前に秤量し、試料チャンバー中に入れた。それを引き続き70℃及び2mbar(micromeritics社製のVacPrep 061により発生された負圧)で2時間にわたり全体的に加熱した。その後に、試料チャンバーを窒素でフラッシングし、再び70℃及び2mbarで1時間にわたり全体的に加熱した。実際の測定のために、窒素を、分析されるべき材料に導通させた。液体窒素による冷却(−196℃)に基づいて、標準的な圧力測定機器を使用して窒素の飽和蒸気圧未満で、吸着された量を測定することが可能であった(吸着)。引き続いての該装置内の圧力の減少により、吸着されたガス量の一部が表面から引き離された(脱着)。結果として、吸脱着等温線を求めることが可能であった。0.05mbarから0.3mbarまでの相対圧力範囲内では、この関連において測定される吸着されたガスの量又は放出されたガスの量は、表面積に比例している。それらの結果は、直接的にm2/gで出力された。
細孔サイズは、キャピラリーフローポロメトリー試験の実施により測定した。詳細は、対応する作業説明書(Capillary Flow Porometer 6.0、CAPWIN Software System、Porous Materials Inc.社)から集めることができる。
膨潤度は、溶剤中での膨潤に際してのポリマーメンブレンの相対的な体積増加を表す。実用的な理由から、膨潤度を、PBS緩衝液中でプロテインAアフィニティー吸着剤について測定した。このために、まずは乾燥状態でのクロマトグラフィー媒体の体積(V0)を測定し、次に湿潤状態での体積(VQ)を測定した。含水量による測定誤差をできる限り排除するために、乾燥体積の測定は、乾燥棚において80℃(30分)で乾燥させた後に実施した。膨潤度Qは、以下の式を介して測定した:
メンブレン面積当たりのクロマトグラフィー活性中心(リガンド)としての固定化されたタンパク質の測定のために、BCA(ビシンコニン酸)アッセイを使用した。このために、本発明によるクロマトグラフィー媒体から打ち抜き物(d=13mm)を打ち抜き、それらを最初にペトリ皿(3mL容)中に入れて、BCA試薬(2mL)で濡らした。BCA試薬は、比率50:1でBCA Protein Assay Reagent A(Pierce社の製品番号23221)とBCA Protein Assay Reagent B(Pierce社の製品番号23224)とからなっていた。その後に、こうして製造された試料を、振盪プラットフォーム(80rpm)上で60分間にわたり揺り動かした。
Claims (15)
- 多孔質マトリックスと、
非多孔質微小球と、
を含むクロマトグラフィー媒体であって、
前記非多孔質微小球は、前記多孔質マトリックスの内表面及び外表面上に共有結合的手段により結合されており、かつ、
前記微小球の平均半径は、前記多孔質マトリックスの平均細孔径の30%以下である、クロマトグラフィー媒体。 - 前記クロマトグラフィー媒体の透過性は、微小球が結合していない多孔質マトリックスの透過性の少なくとも40%である、請求項1に記載のクロマトグラフィー媒体。
- 前記非多孔質微小球は、グリシジル(メタ)アクリレート、置換又は非置換のアルキル(メタ)アクリレート及びそれらの誘導体、スチレン及びその誘導体、2−ビニル−4,4−ジメチルアズラクトン、置換又は非置換のN−アルキル(メタ)アクリルアミド及びそれらの誘導体、並びに置換又は非置換のN,N’−ジアルキル(メタ)アクリルアミド及びそれらの誘導体からなる群より選択される少なくとも1種のモノマーから構成される実質的に球形のオリゴマー及び/又はポリマーである、請求項1又は2に記載のクロマトグラフィー媒体。
- 前記非多孔質微小球は、その微小球に結合されているか、又はその微小球上に固定化されている追加のクロマトグラフィー活性中心又はリガンドを含む、請求項1〜5のいずれか一項に記載のクロマトグラフィー媒体。
- 請求項1〜6のいずれか一項に記載のクロマトグラフィー媒体の製造方法であって、
多孔質出発マトリックスを準備することと、
少なくとも1種のモノマー、二官能性、三官能性、又は多官能性の架橋剤、重合開始剤、及び溶剤又は溶剤混合物を含み、前記少なくとも1種のモノマー、前記二官能性、三官能性、又は多官能性の架橋剤、及び前記重合開始剤が、前記溶剤又は溶剤混合物中に完全に可溶性である重合溶液を準備することと、
前記重合溶液における重合を前記多孔質出発マトリックスの存在下で開始させることで、前記溶剤又は溶剤混合物中に不溶性であり、かつ前記多孔質出発マトリックスの内表面及び外表面へと結合されている非多孔質微小球を形成することと、
を含み、前記微小球の平均半径が、前記多孔質出発マトリックスの平均細孔径の30%以下である、方法。 - 前記少なくとも1種のモノマーは、グリシジル(メタ)アクリレート、置換又は非置換のアルキル(メタ)アクリレート及びそれらの誘導体、スチレン及びその誘導体、2−ビニル−4,4−ジメチルアズラクトン、置換又は非置換のN−アルキル(メタ)アクリルアミド及びそれらの誘導体、並びに置換又は非置換のN,N’−ジアルキル(メタ)アクリルアミド及びそれらの誘導体からなる群より選択される、請求項7に記載のクロマトグラフィー媒体の製造方法。
- 前記二官能性、三官能性、又は多官能性の架橋剤は、エチレングリコールジメタクリレート、トリメチルプロパントリメタクリレート、ジビニルベンゼン、及びN,N−メチレンビスアクリルアミドからなる群より選択される、請求項7又は8に記載のクロマトグラフィー媒体の製造方法。
- 前記重合開始剤は、2−ヒドロキシ−4−(2−ヒドロキシエトキシ)−2−メチルプロピオフェノン、アゾビス(イソブチロニトリル)、4,4−アゾビス(4−シアノ吉草酸)、1,1’−アゾビス(シクロヘキサンカルボニトリル)、ベンゾイルペルオキシド、2,2−ビス(tert−ブチルペルオキシ)ブタン、1,1−ビス(tert−ブチルペルオキシ)シクロヘキサン、tert−ブチルヒドロペルオキシド、tert−ブチルペルオキシイソプロピルカーボネート、シクロヘキサノンペルオキシド、及び2,4−ペンタンジオンペルオキシドからなる群より選択される、請求項7〜9のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
- 前記溶剤又は溶剤混合物は、シクロヘキサノール/ドデカン−1−オール、オクタン−2−オン、n−ブチルアセテート、p−キシレン、トルエン、エチルアセテート、ベンゾニトリル、シクロヘキサノン、ドデカン−1−オール、アセトニトリル/エタノール/水、デカン−1−オール、及びイソプロパノール/デカン−1−オールからなる群より選択される、請求項7〜10のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
- 前記重合溶液の全容量に対するモノマー及び架橋剤の全容量が、20容量%以下である、請求項7〜11のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
- 前記重合溶液の全容量に対する架橋剤の全容量が、6容量%以下である、請求項7〜12のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
- 前記重合溶液中の重合開始剤の濃度が、1重量%〜3重量%である、請求項7〜13のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
- 巨視的に確認可能な相分離が、最大30秒後に起こる、請求項7〜14のいずれか一項に記載のクロマトグラフィー媒体の製造方法。
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