Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
J Chromatogr A. 2011 Oct 28;1218(43):7821-7. doi: 10.1016/j.chroma.2011.08.084. Epub 2011 Sep 3.
In this work, we systematically evaluated the potential of using boronic acid functionalized magnetic particles in the capturing of human immunoglobulin G under typical mammalian cell culture conditions. For comparison, Protein A coated magnetic particles were also used. The binding pH was found to significantly influence the adsorption isotherms of boronic acid particles with the higher capacities (0.216 g IgG/g support) being observed at pH 7.4. Comparatively, this value was 0.109 g IgG/g support, for Protein A particles under the same conditions. Both particles revealed very fast adsorption kinetics with more than 70% of the maximum binding capacity being achieved in a few seconds. The effect of glucose and lactate, which are known to interact with boronic acid, was evaluated. For glucose, the binding capacity was significantly influenced by the pH and decreased as pH increased. At pH 9.5, a 70% lower binding capacity was observed for glucose concentrations as low as 0.5 g/l. The effect of lactate was less pronounced and almost pH independent reaching at most 20% decrease in binding capacity. Nevertheless, the effect of both molecules was always lower at pH 7.4. The optimization of the elution conditions enabled complete recovery of bound IgG from boronic acid particles using 50mM Tris-HCl, 200 mM sorbitol, 200 mM NaCl at pH 8.5.
在这项工作中，我们系统地评估了硼酸功能化磁性颗粒在典型的哺乳动物细胞培养条件下捕获人免疫球蛋白 G 的潜力。为了进行比较，我们还使用了 Protein A 涂层的磁性颗粒。研究发现，结合 pH 值显著影响硼酸颗粒的吸附等温线，在 pH 值为 7.4 时观察到更高的容量（0.216 g IgG/g 载体）。相比之下，在相同条件下，Protein A 颗粒的这个值为 0.109 g IgG/g 载体。两种颗粒都表现出非常快速的吸附动力学，在几秒钟内就达到了超过 70%的最大结合容量。我们还评估了已知与硼酸相互作用的葡萄糖和乳酸的影响。对于葡萄糖，其结合容量受 pH 值的显著影响，并随着 pH 值的升高而降低。在 pH 值为 9.5 时，对于低至 0.5 g/l 的葡萄糖浓度，观察到结合容量降低了 70%。乳酸的影响则不那么明显，几乎与 pH 值无关，结合容量最多降低 20%。然而，在 pH 值为 7.4 时，这两种分子的影响总是更低。通过优化洗脱条件，可以使用 50mM Tris-HCl、200mM 山梨醇、200mM NaCl 在 pH 值 8.5 时从硼酸颗粒上完全回收结合的 IgG。
