Bridonneau P, Lederer F
Centre National de Transfusion Sanguine, Les Ulis, France.
J Chromatogr. 1993 Jul 2;616(2):197-204. doi: 10.1016/0378-4347(93)80386-i.
We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not absorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for absorption on thiophilic gels at low and high salt concentration.
我们尝试使用嗜硫亲和色谱法和疏水相互作用色谱法从一种治疗性免疫球蛋白制剂中获得富集的人免疫球蛋白G（IgG）亚类。蛋白质在1M硫酸铵中吸附于嗜硫凝胶以及苯基、丁基或辛基琼脂糖上。用递减盐梯度洗脱未产生明显的亚类选择性，但辛基琼脂糖除外，它产生了一个吸附较差的级分，其中IgG2略有富集，约占初始总蛋白的20%。嗜硫亲和色谱法和疏水相互作用色谱法似乎都不适用于高效富集亚类，因为所有亚类对这些色谱柱的亲和力都表现出广泛的异质性。还研究了起始盐浓度的影响。对于嗜硫凝胶，在无硫酸铵的情况下，约30%的初始上样量未被吸附，且发现富含IgG2。在2.5%和5%的硫酸铵浓度下，几乎不发生吸附。在7.5%的硫酸铵浓度下，未吸附级分富含IgG3。对于苯基琼脂糖，吸附随盐浓度平稳增加。得出的结论是，在低盐浓度和高盐浓度下，嗜硫凝胶上的吸附涉及不同的作用力。
