A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
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DOI：
10.3389/fbioe.2020.00688
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发表时间：
2020-06-23
影响因子：
5.7
通讯作者：
Kolmar, Harald
中科院分区：
文献类型：
--
作者：
Hinz, Steffen C.;Elter, Adrian;Kolmar, Harald
亲和层析为蛋白质纯化提供了一个极好的平台，蛋白质纯化是治疗性单克隆抗体（Mab）大规模下游加工的关键步骤。蛋白A层析构成Mab纯化的金标准。然而，从亲和基质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，因为由于酸性缓冲液条件可能发生变性和不可逆聚集。在这里，我们描述了一个通用的程序，用于生成抗原特异性层析配体与定制的洗脱条件。为此，我们基于来自用人Fc免疫的鸡的mRNA产生了scFv文库。抗体库通过FACS展示在酵母酵母菌筛选的pH和镁响应性scFv上，这些scFv特异性识别人IgG抗体。将分离的scFv重组，在大肠杆菌中产生并固定在NHS-琼脂糖柱上。鉴定了几种scFv，其在中性pH下介导抗体结合，并且在MgCl（2）暴露后在4.5和更高的pH值下或甚至在中性pH下介导抗体恢复。这里建立的迭代筛选方法通常适合于直接分离刺激响应性抗体，其可能成为用于各种应用的有价值的工具。
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 yeastSaccharomyces cerevisiaescreenedviaFACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced inEscherichia coliand 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(2)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.