Fan Jinxin, Luo Jianquan, Chen Xiangrong, Wan Yinhua
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100190, PR China.
J Chromatogr A. 2017 Mar 24;1490:54-62. doi: 10.1016/j.chroma.2017.02.016. Epub 2017 Feb 14.
In this study, a polyvinylidene fluoride (PVDF) hydrophobic membrane with high mechanical property was used as substrate to prepare salt-tolerant anion-exchange (STAE) membrane adsorber. Effective hydrophilization and functionalization of PVDF membrane was realized via polydopamine (PDA) deposition, thus overcoming the drawbacks of hydrophobic substrates including poor water permeability, inert property as well as severe non-specific adsorption. The following polyallylamine (PAH) coupling was carried out at pH 10.0, where unprotonated amine groups on PAH chains were more prone to couple with PDA. This membrane adsorber could remain 75% of protein binding capacity when NaCl concentration increased from 0 to 150mM, while its protein binding capacity was independent of flow rate from 10 to 100 membrane volume (MV)/min due to its high mechanical strength (tensile strength: 43.58±2.30MPa). With 200mM NaCl addition at pH 7.5, high purity (above 99%) and high recovery (almost 100%) of Immunoglobulin G (IgG) were obtained when using the STAE membrane adsorber to separate IgG/human serum albumin (HSA) mixture, being similar to that without NaCl at pH 6.0 (both under the flow rate of 10-100MV/min). Finally, the reliable reusability was confirmed by five reuse cycles of protein binding and elution operations. In comparison with commercial membrane adsorber, the new membrane adsorber exhibited a better mechanical property, higher IgG polishing efficiency and reusability, while the protein binding capacity was lower due to less NH density on the membrane. The outcome of this work not only offers a facile and effective approach to prepare membrane adsorbers based on hydrophobic membranes, but also demonstrates great potential of this new designed STAE membrane adsorbers for efficient monoclonal antibody (mAb) polishing.
在本研究中,使用具有高机械性能的聚偏氟乙烯(PVDF)疏水膜作为基材来制备耐盐阴离子交换(STAE)膜吸附器。通过聚多巴胺(PDA)沉积实现了PVDF膜的有效亲水化和功能化,从而克服了疏水基材的缺点,包括透水性差、惰性以及严重的非特异性吸附。随后在pH 10.0下进行聚烯丙胺(PAH)偶联,此时PAH链上未质子化的胺基更容易与PDA偶联。当NaCl浓度从0增加到150mM时,这种膜吸附器可以保留75%的蛋白质结合能力,同时由于其高机械强度(拉伸强度:43.58±2.30MPa),其蛋白质结合能力在10至100膜体积(MV)/分钟的流速范围内不受流速影响。在pH 7.5下添加200mM NaCl,使用STAE膜吸附器分离IgG/人血清白蛋白(HSA)混合物时,可获得高纯度(高于99%)和高回收率(几乎100%)的免疫球蛋白G(IgG),这与在pH 6.0下不添加NaCl时的情况相似(两者流速均为10 - 100MV/分钟)。最后,通过蛋白质结合和洗脱操作的五个重复循环证实了其可靠的可重复使用性。与商业膜吸附器相比,新型膜吸附器表现出更好的机械性能、更高的IgG纯化效率和可重复使用性,而由于膜上NH密度较低,其蛋白质结合能力较低。这项工作的成果不仅提供了一种基于疏水膜制备膜吸附器的简便有效方法,还展示了这种新设计的STAE膜吸附器在高效单克隆抗体(mAb)纯化方面的巨大潜力。