High-efficiency affinity precipitation of multiple industrial mAbs and Fc-fusion proteins from cell culture harvests using Z-ELP-E2 nanocages: SWARTZ et al.
High-efficiency affinity precipitation of multiple industrial mAbs and Fc-fusion proteins from cell culture harvests using Z-ELP-E2 nanocages: SWARTZ et al.
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使用 Z-ELP-E2 纳米笼对细胞培养收获物中的多种工业 mAb 和 Fc 融合蛋白进行高效亲和沉淀：SWARTZ 等人。
DOI：
10.1002/bit.26717
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发表时间：
2018
影响因子：
3.8
通讯作者：
Chen, Wilfred
中科院分区：
文献类型：
--
作者：
Swartz, Andrew R.;Xu, Xuankuo;Traylor, Steven J.;Li, Zheng Jian;Chen, Wilfred
使用Z-弹性蛋白样多肽官能化的E2蛋白纳米笼的亲和沉淀已被证明是用于单克隆抗体（mAb）纯化的蛋白A色谱的有前景的替代方案。我们之前已经描述了一种高产的亲和沉淀方法，该方法能够通过自发的多价交联从细胞培养物中快速捕获mAb，形成大的聚集体。为了挑战该技术的能力，使用具有不同分子性质的四种工业mAb（mAb A-D）和一种Fc融合蛋白（Fc A）研究纳米笼亲和沉淀。3：1的Z：mAb的摩尔结合比足以在溶液中沉淀>95%的mAb，对于在环境温度下评价的所有分子，不添加盐。使用简化的两步模型研究溶液pH对聚集动力学的影响，以研究mAb-纳米笼交联期间发生的蛋白质相互作用，并确定沉淀的最佳溶液pH。离心后，沉淀的mAb-纳米笼复合物保持不溶，并且能够在pH ≥ 5下洗涤，并在pH下洗脱，< 4 with >所有分子的mAb回收率为90%。使用最佳工艺条件从细胞培养物中纯化四种mAb和一种Fc融合物，并且获得>94%产率和>97%单体含量。mAb A-D纯化导致来自细胞培养液的宿主细胞蛋白质减少99.9%和DNA减少>99.99%。纳米笼亲和沉淀相当于或超过预期的蛋白A色谱性能。这项研究强调了纳米颗粒交联增强亲和捕获的好处，并提出了一个强大的平台，可以应用于任何目标mAb或含Fc的蛋白质，最小限度地优化工艺参数。
Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.