Anspach F B, Hilbeck O
GBF-National Research Centre for Biotechnology, Braunschweig, Germany.
J Chromatogr A. 1995 Sep 8;711(1):81-92. doi: 10.1016/0021-9673(95)00126-8.
Histidine, histamine and polymyxin B affinity sorbents were employed for the removal of Escherichia coli-derived endotoxins. Their effectiveness was compared with those of poly-L-lysine-Sepharose and DEAE-Sepharose. All sorbents reduced the concentration of endotoxins from an E. coli culture filtrate to tolerable levels. However, their effectiveness was not higher than that of the anion exchanger, which displayed clearance rates of up to 15,000. Endotoxin removal from protein solutions depended on the net charge of the desired protein. Lysozyme as a model for positively charged proteins enhanced endotoxin removal. In contrast, only low initial contamination levels (< 34 EU/ml) were reduced to tolerable levels from bovine serum albumin (BSA) as the negatively charged protein model owing to competition of BSA and endotoxins for adsorption sites. Hence also a low BSA recovery was observed after the treatment whereas the lysozyme recovery was almost 100%. At pH values below the isoelectric point of BSA, endotoxin removal was also more effective. The best conditions for the decontamination were found at neutral pH and low ionic strength (< or = 20 mM phosphate). Ionic forces between ligands and endotoxins are dominant at this ionic strength; hydrophobic interactions are not very effective. Hence the selectivities of all sorbents towards endotoxins are not exceptionally high. DEAE-anion exchangers are the most suitable sorbents for the removal of endotoxins from solutions accommodating positively charged proteins owing to their low cost and high capacity. Poly-L-lysine-Sepharose was most effective for the removal of small amounts of endotoxins from solutions of negatively charged proteins. The "affinity ligands" histamine, histidine and polymyxin B were effective for the removal of endotoxins from E. coli filtrate; however, their effectiveness decreased dramatically in the presence of BSA and it was lower than for poly-L-lysine- and DEAE-Sepharose in the presence of lysozyme.
使用组氨酸、组胺和多粘菌素B亲和吸附剂去除大肠杆菌衍生的内毒素。将它们的效果与聚-L-赖氨酸-琼脂糖和二乙氨基乙基-琼脂糖的效果进行了比较。所有吸附剂都将大肠杆菌培养滤液中的内毒素浓度降低到了可耐受水平。然而，它们的效果并不高于阴离子交换剂，阴离子交换剂的清除率高达15000。从蛋白质溶液中去除内毒素取决于所需蛋白质的净电荷。作为带正电荷蛋白质模型的溶菌酶增强了内毒素的去除。相反，由于牛血清白蛋白（BSA）作为带负电荷蛋白质模型与内毒素竞争吸附位点，只有低初始污染水平（<34 EU/ml）从BSA中降低到了可耐受水平。因此，处理后还观察到BSA回收率较低，而溶菌酶回收率几乎为100%。在低于BSA等电点的pH值下，内毒素去除也更有效。去污的最佳条件是在中性pH和低离子强度（≤20 mM磷酸盐）下。在这种离子强度下，配体和内毒素之间的离子力占主导；疏水相互作用不太有效。因此，所有吸附剂对内毒素的选择性都不是特别高。由于成本低且容量高，二乙氨基乙基阴离子交换剂是从容纳带正电荷蛋白质的溶液中去除内毒素的最合适吸附剂。聚-L-赖氨酸-琼脂糖对于从带负电荷蛋白质溶液中去除少量内毒素最有效。“亲和配体”组胺、组氨酸和多粘菌素B对于从大肠杆菌滤液中去除内毒素是有效的；然而，在存在BSA的情况下它们的效果急剧下降，并且在存在溶菌酶的情况下低于聚-L-赖氨酸-琼脂糖和二乙氨基乙基-琼脂糖。
