As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z_Ca, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z_Ca to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z_CaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z_CaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

如此处报道，我们开发和优化了基于蛋白质A衍生结构域Z _Ca的纯化矩阵，该结构显示出钙依赖性抗体结合。它提供了常规蛋白A亲和色谱法酸性洗脱条件的替代方法，用于纯化敏感抗体和其他基于Fc的分子。我们描述Z _Ca的多聚化以产生具有更高结合能力的色谱树脂。最高阶多聚体变体Z _CaTetraCys展示了相当高的动态结合能力（35 mg IgG / ml树脂），同时保留了对IgG的特异性。获得了高回收率，纯化级分中的宿主细胞蛋白质和DNA含量被证明可与市售的MabSelect SuRe和MabSelect PrismA相提并论。研究了将该结构域用于抗体纯化的各种洗脱条件。此处提供的纯化数据揭示了人IgG不同亚类与Z _Ca相互作用的变化TetraCys。这导致了不同IgG的不同洗脱特性，其中在中性pH值条件下，所有捕获的IgG2和IgG4抗体的完全洗脱是可能的。这种优化的蛋白质配体和建议的纯化方法为有效，温和地纯化由于聚集形成或功能丧失而无法在常规酸性洗脱条件下纯化的抗体和Fc融合蛋白提供了独特的策略。