非天然型立体構造抗体検出技術の開発

摘要

凝集体形成の要因となる非天然型立体構造抗体に特異的に結合するべプチドAF.2A1を利用した抗体医薬品分析用のELISA法を開発した。AF.2A1を用いたELISA法により、酸や加熱などのストレス処理を施した抗体に含まれる非天然型立体構造杭休を特異的に検出可能であることを確認した。SEC法またはSLS法との比校により、開発したELISA法が既存の凝集体検出技術、抗体安定性評価技術と相関があり、さらに既存分析技術では検出限界以下の凝集体やその核となる非天然ゆ立体構造抗体を高感度に検出できる点において優位であることを確認した。また、コールター力ゥンター法との比敉試験の結果から、ELISA法により凝集体形成前の立体構造変化を検出することで、凝集体形成のV.期段階をモニタリングでき、凝集予測に有用であることを示した。%Therapeutic immunoglobulin G (IgG) is unstable under various stresses in the manufacturing process, delivery and storage. When it is exposed to such stress factors, it becomes non-native IgG. If the molecules are present in the therapeutic IgG productions, they tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk for immunogenicity, therapeutic IgG should contain as small amount of them as possible and rapid analysis of aggregation is required for quality control.We have developed a novel ELISA system for detection of non-native IgG using AF.2A1 peptide. AF.2A1 is a 25-residue artificial peptide which binds specifically to non-native IgG. We also established the same assay system using automated assay equipment. This assay is performed in a short period of time. Our results show that AF.2A1 ELISA can detect only non-native IgG generated under acid, heat and freeze/thaw conditions but not native IgG. Because AF.2A1 recognizes the non-native structure in the fragment crystallizable region (Fc region) of IgG, this assay can be used to evaluate the various types of IgG. We also found that AF.2A1 ELISA is available for the method. In addition, this technique is effective in monitoring of the early stages of the IgG aggregation process. These results indicate that AF.2A1 ELISA can be useful for various types of applications such as screening for high performance cell, process analytical technology, process monitoring and quality control of therapeutic IgG.