Kangwa Martin, Yelemane Vikas, Ponnurangam Adilah, Fernández-Lahore Marcelo
Downstream Bioprocessing Laboratory, Department of Life Sciences & Chemistry, Jacobs University, Campus Ring 1, D-28759, Bremen, Germany.
Downstream Bioprocessing Laboratory, Department of Life Sciences & Chemistry, Jacobs University, Campus Ring 1, D-28759, Bremen, Germany.
Protein Expr Purif. 2019 Mar;155:27-34. doi: 10.1016/j.pep.2018.11.003. Epub 2018 Nov 13.
In antibody purification processes, affinity chromatography has been used with Staphylococcus aureus protein A (SpA) as the main ligand. In this work, we present a novel Staphylococcal Protein A (AviPure thereafter), a synthetic ligand analogue based on native SpA B domain, with a molecular weight of approximately 14 kDa. The binding affinity of mAbs to AviPure was evaluated using Surface Plasmon Resonance (SPR) and affinity chromatography methods. The equilibrium dissociation constant (K) between the AviPure and mAbs was systematically measured using 1:1 (Langmuir) model and found to be 4.7 × 10 M, with constant of dissociation at k ≤ 1.0 × 10 s and k being 3.1 × 10 M s. When immobilized on Sepharose, the AviPure ligand density was 429 nmol/g moist weight resin and was able to effectively bind immunoglobulin and Fc fragment samples with higher affinity and the most effective flow rate when using ligand - Sepharose beads was at 75 cm/h giving the dynamic binding capacity of 53 mg/mL and 91% recovery of IgG. Suitable ligands used in affinity purification should have a K ≤ 10 M and a dissociation rate (k) averaging 10 M s with the k ranging between 10 - 10 M. Therefore, the AviPure ligand can be used as an alternative to the standard protein A ligand in the purification of mAbs and Fc-fused proteins.
在抗体纯化过程中，亲和色谱法一直以金黄色葡萄球菌蛋白A（SpA）作为主要配体使用。在本研究中，我们展示了一种新型的葡萄球菌蛋白A（以下简称AviPure），它是一种基于天然SpA B结构域的合成配体类似物，分子量约为14 kDa。使用表面等离子体共振（SPR）和亲和色谱法评估了单克隆抗体与AviPure的结合亲和力。采用1:1（朗缪尔）模型系统地测量了AviPure与单克隆抗体之间的平衡解离常数（K），结果为4.7×10⁻⁸ M，解离常数k≤1.0×10⁻³ s⁻¹，k为3.1×10⁻³ M⁻¹ s⁻¹。当固定在琼脂糖凝胶上时，AviPure配体密度为429 nmol/g湿重树脂，能够以更高的亲和力有效结合免疫球蛋白和Fc片段样品，使用配体-琼脂糖凝胶珠时最有效的流速为75 cm/h，动态结合容量为53 mg/mL，IgG回收率为91%。亲和纯化中使用的合适配体应具有K≤10⁻⁸ M，解离速率（k）平均为10⁻³ M⁻¹ s⁻¹，k范围在10⁻⁴ - 10⁻² M⁻¹ s⁻¹之间。因此，AviPure配体可作为标准蛋白A配体的替代品，用于单克隆抗体和Fc融合蛋白的纯化。
