Hinz Steffen C, Elter Adrian, Rammo Oliver, Schwämmle Achim, Ali Ataurehman, Zielonka Stefan, Herget Thomas, Kolmar Harald
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
Merck Lab @ Technische Universität Darmstadt, Darmstadt, Germany.
Front Bioeng Biotechnol. 2020 Jun 23;8:688. doi: 10.3389/fbioe.2020.00688. eCollection 2020.
Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8-3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast screened FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.
亲和层析为蛋白质纯化提供了一个出色的平台，这是治疗性单克隆抗体（Mab）大规模下游加工中的关键步骤。蛋白A层析是Mab纯化的金标准。然而，从亲和基质上洗脱所需的酸性条件（2.8 - 3.5）限制了它们的适用性，特别是对于下一代抗体和抗体融合蛋白，因为酸性缓冲条件可能导致变性和不可逆聚集。在此，我们描述了一种生成具有定制洗脱条件的抗原特异性层析配体的通用方法。为此，我们基于用人Fc免疫的鸡的mRNA构建了一个单链抗体库（scFv）。抗体库展示在酵母上，通过荧光激活细胞分选术（FACS）筛选对pH和镁有反应的scFv，这些scFv能特异性识别人类IgG抗体。分离出的scFv被重新构建，在大肠杆菌中表达并固定在NHS - 琼脂糖柱上。鉴定出了几种scFv，它们在中性pH下介导抗体结合，并在pH值为4.5及更高时或甚至在暴露于MgCl₂时在中性pH下实现抗体回收。这里建立的迭代筛选方法通常适用于直接分离对刺激有反应的抗体，这些抗体可能成为各种应用中有价值的工具。
