Knödler Matthias, Rühl Clemens, Opdensteinen Patrick, Buyel Johannes F
Institute for Molecular Biotechnology, RWTH Aachen University; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
Sanofi Deutschland GmbH.
J Vis Exp. 2019 Aug 16(150). doi: 10.3791/59933.
The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.
单克隆抗体（mAb）的纯化通常通过蛋白A亲和色谱法实现，该方法可占整个工艺成本的25%。因此，具有成本效益的替代捕获步骤对于工业规模生产大量单一mAb而言很有价值。在此，我们展示了一种将基于DsRed的表位配体固定到交联琼脂糖树脂上的方法，该方法能够从粗植物提取物中选择性捕获HIV中和抗体2F5，而无需使用蛋白A。线性表位ELDKWA首先与荧光蛋白DsRed进行基因融合，融合蛋白在转基因烟草（Nicotiana tabacum）植株中表达，然后通过固定金属离子亲和色谱法进行纯化。此外，针对高配体密度、高效偶联和低成本对基于活化交联琼脂糖的方法进行了优化。pH值、缓冲液组成和可溶性配体浓度是偶联过程中最重要的参数，采用实验设计方法对其进行了改进。对所得的亲和树脂在粗植物提取物中选择性结合目标mAb的能力进行了测试，并对洗脱缓冲液进行了优化，以实现高mAb回收率、产品活性和亲和树脂稳定性。该方法可轻松适用于其他具有线性表位的抗体。新型树脂允许比蛋白A更温和的洗脱条件，还可降低mAb生产初始捕获步骤的成本。
