Department of Protein Science, KTH-Royal Institute of Technology, SE-10691, Stockholm, Sweden.
Department of Protein Science, KTH-Royal Institute of Technology, SE-10691, Stockholm, Sweden.
J Chromatogr A. 2021 Jan 25;1637:461843. doi: 10.1016/j.chroma.2020.461843. Epub 2020 Dec 24.
The manufacturability of therapeutic monoclonal antibodies is limited by the harsh conditions that antibodies are subjected to during the purification procedure, which in turn restricts the development of novel acid-sensitive antibodies. The gold standard for antibody purification, Protein A affinity chromatography, offers the selective capture of antibodies with great yields, but also poses a threat to the quality of the antibodies. Antibodies and Fc-fusion proteins risk forming aggregates as a consequence of the acidic elution from the Protein A ligands, compromising the potency and safety of the drug. Here, we present a novel, mild purification strategy based on a calcium-dependent ligand derived from Protein A, called Z. Antibodies captured on a high-capacity tetrameric Z resin in the presence of calcium can be eluted by removing the calcium ions through the addition of a chelator, and we describe the strive to find a sustainable alternative to the previously applied chelator EDTA. The naturally occurring chelator citrate is shown to seamlessly replace EDTA. Further buffer optimization reveals that the elution can be considerably improved by increasing the conductivity through the addition of 300 mM sodium chloride, leading to a very concentrated eluate. Remarkably, merely sodium chloride at a concentration of 50 mM is proven to be sufficient for calcium-dependent antibody release in a cost-efficient manner. Antibodies of subclasses IgG2 and IgG4 are eluted with sodium chloride at neutral pH and IgG1 at pH 6, due to varying affinities for the tetrameric Z, ranging between 90-780 nM. The mild elution of an IgG4 antibody eliminated the formation of aggregates, which constituted as much as 34% of all eluted antibody from MabSelect SuRe at pH 3. This novel purification strategy thus combines the valuable qualities of a Protein A resin, by providing high selectivity and a recovery of 88-99%, with an exceptionally mild elution step similar to ion-exchange chromatography, rendering considerably more functional antibody.
治疗性单克隆抗体的可制造性受到抗体在纯化过程中所经历的苛刻条件的限制，这反过来又限制了新型酸敏感抗体的发展。抗体纯化的金标准，Protein A 亲和层析，提供了对抗体的高选择性捕获和高产量，但也对抗体的质量构成威胁。由于从 Protein A 配体中酸性洗脱，抗体和 Fc 融合蛋白有形成聚集体的风险，从而影响药物的效力和安全性。在这里，我们提出了一种基于 Protein A 的新型温和的纯化策略，该策略基于一种称为 Z 的钙依赖性配体。在存在钙的情况下，抗体可以被捕获在高容量的四聚体 Z 树脂上，通过添加螯合剂去除钙离子，可以被洗脱，我们还描述了寻找一种可持续替代以前应用的 EDTA 螯合剂的努力。天然存在的螯合剂柠檬酸盐被证明可以无缝替代 EDTA。进一步的缓冲优化表明，通过添加 300 mM 氯化钠来增加电导率，可以显著改善洗脱效果，从而得到非常浓缩的洗脱液。值得注意的是，仅仅 50 mM 的氯化钠浓度就足以以经济高效的方式实现钙依赖性抗体的释放。亚类 IgG2 和 IgG4 的抗体在中性 pH 和 pH 6 下用氯化钠洗脱，而 IgG1 在 pH 6 下用氯化钠洗脱，这是由于它们与四聚体 Z 的亲和力不同，范围在 90-780 nM 之间。IgG4 抗体的温和洗脱消除了聚集体的形成，而 MabSelect SuRe 在 pH 3 下洗脱的抗体中有高达 34%的抗体形成了聚集体。因此，这种新型的纯化策略结合了 Protein A 树脂的宝贵特性，提供了高选择性和 88-99%的回收率，以及类似于离子交换层析的非常温和的洗脱步骤，从而产生了更多具有功能性的抗体。
