Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, SC, USA.
J Sep Sci. 2014 Mar;37(5):495-504. doi: 10.1002/jssc.201301205. Epub 2014 Jan 25.
A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary-channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary-channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.
一种新型的蛋白 A 亲和层析固定相已经从经过重组蛋白 A 配体修饰的聚丙烯毛细管通道聚合物纤维中开发出来，用于高特异性和高收率地捕获和回收免疫球蛋白 G（IgG）。采用 SPE 微量离心管管尖形式，从而最小化溶剂、蛋白质和抗体的消耗。评估了纤维表面与蛋白 A 的吸附修饰作为进料浓度和体积的函数。蛋白 A 的纤维表面的最佳修饰产生了 5.7mg/mL（床体积）的配体容量，并且修饰后的纤维在多种溶剂环境下表现出稳定性。通过单独和混合暴露于人 IgG 和肌红蛋白来评估性能。肌红蛋白被用作生长培养基中常见的宿主细胞蛋白的替代物。通过与配体的选择性结合的 2.9:1（IgG/蛋白 A）结合比例证明了结合的功效。根据收集的洗脱液的吸收测量，用 0.1 M 乙酸洗脱可得到 89%的捕获 IgG 的回收率。用 10 mM NaOH 可以实现再生。蛋白 A 修饰的聚丙烯毛细管通道聚合物纤维作为亲和相，用于有效捕获和纯化 IgG，具有良好的初步效果。
