Department of Chemical and Biomolecular Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Raleigh, NC 27695-7006, USA.
J Chromatogr A. 2011 Apr 1;1218(13):1691-700. doi: 10.1016/j.chroma.2010.11.071. Epub 2010 Dec 4.
Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.
先前的工作已经报道了六肽配体 HWRGWV、HYFKFD 和 HFRRHL 的鉴定和特性，这些配体通过与 IgG 的 Fc 片段特异性结合来实现 IgG 的亲和捕获。本文解决了在商业甲基丙烯酸盐色谱树脂上成功应用这些配体，从哺乳动物细胞培养物中纯化 IgG 的相关问题。优化了结合缓冲液中氯化钠和辛酸钠的浓度，以在洗脱时最大限度地提高 IgG 的纯度和产率。筛选了几种再生条件，发现 2M 盐酸胍或 0.85%磷酸随后 2M 尿素的组合均可完全恢复 IgG 吸附能力，且该柱可在多个循环中重复使用。六肽配体用于从两种商业 CHO 细胞培养物中纯化人源化和嵌合单克隆抗体。IgG1 亚类的嵌合 MAb 用 HWRGWV 树脂纯化，而 IgG4 亚类的人源化 MAb 则用 HWRGWV、HYFKFD 和 HFRRHL 树脂纯化。两种 MAb 的纯度和产率均高于 94%和 85%。这些结果与使用 Protein G 柱获得的产率和纯度相当。HWRGWV 树脂获得的残余 DNA 和宿主细胞蛋白减少值分别为 4 对数减少值 (LRV) 和 2LRV，与 Protein A 树脂报道的值相当。三种肽树脂对人源化单克隆抗体的动态结合能力均在 20mg/mL 范围内。
