Zhang Shaojie, Iskra Tim, Daniels William, Salm Jeffrey, Gallo Christopher, Godavarti Ranga, Carta Giorgio
Dept. of Chemical Engineering, University of Virginia, Charlottesville, VA.
Pfizer, Andover, MA.
Biotechnol Prog. 2017 Mar;33(2):425-434. doi: 10.1002/btpr.2412. Epub 2017 Jan 28.
Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425-434, 2017.
有人提出采用弱分配色谱法（WPC），使用阴离子交换（AEX）树脂来纯化单克隆抗体，以同时去除酸性和碱性蛋白质杂质。尽管具有潜在优势，但尚未系统评估树脂结构与WPC性能之间的关系。在这项工作中，我们使用透射电子显微镜和反相尺寸排阻色谱法确定了代表性AEX树脂（Fractogel® EMD TMAE HiCap、Q Sepharose FF和POROS 50 HQ）的结构，并以两种单克隆抗体单体、一种单克隆抗体二聚体、单克隆抗体多聚体和牛血清白蛋白（BSA）作为模型产物和杂质，在WPC条件下操作这些树脂时对蛋白质相互作用进行了表征。我们通过共聚焦激光扫描显微镜确定了弱结合单体和二聚体物种的等度洗脱行为以及强结合多聚体和BSA的吸附和传质特性。结果表明，对于每种树脂，以产物K值为指导，可以找到同时去除单克隆抗体多聚体和BSA的盐和pH条件。然而，每种树脂以及不同组分的等度洗脱和吸附机制是不同的。在WPC条件下，Fractogel树脂的单克隆抗体单体和二聚体物种扩散非常缓慢，但对单克隆抗体多聚体和BSA的吸附迅速，对BSA的容量较高，而Sepharose树脂由于孔径较小，无法有效去除单克隆抗体多聚体。相反，POROS树脂能够有效结合多聚体和BSA，同时对单克隆抗体单体和二聚体物种具有更高的分辨率。© 2017美国化学工程师学会生物技术进展，33:425 - 434，2017。
