Validated Biosystems, San Clemente, CA 92672, USA.
J Sep Sci. 2009 Nov;32(22):3857-65. doi: 10.1002/jssc.200900055.
Retention mapping of chimeric monoclonal IgG(1), Fc, Fab, F(ab')(2), and aggregated antibody was conducted on hydroxyapatite (HA) by systematically varying phosphate and chloride concentrations during gradient elution in order to characterize the interactions of each solute with calcium and phosphate residues on the solid phase. Lysozyme was used as a control to model cation exchange-dominant interactions. Bovine serum albumin was used as a control for calcium affinity-dominant interactions. Calcium affinity and phosphoryl cation exchange were positively cooperative for IgG-related species. Fc retention was dominated by calcium affinity, while retention of Fab was dominated by cation exchange. F(ab')(2) exhibited a curve shape similar to Fab, but stronger retention. The retention curve for intact IgG incorporated the distinctive elements of its fragments but stronger retention than that predicted by their addition to one another. Aggregate retention paralleled the curve for non-aggregated antibody, with stronger retention by both binding mechanisms. Experimental data revealed evidence of charge repulsion between IgG carboxyls and HA phosphate at low conductivity values. Electrostatic repulsion of amino residues and attraction of carboxyls by HA calcium appeared to be blocked by strong complexation of calcium with mobile phase phosphate.
采用梯度洗脱的方法，在羟磷灰石（HA）上进行嵌合单克隆 IgG(1)、Fc、Fab、F(ab')(2)和聚集抗体的保留映射，通过系统地改变磷酸根和氯离子浓度，以研究每种溶质与固相上钙和磷酸根残基的相互作用。溶菌酶被用作模型以模拟阳离子交换主导的相互作用。牛血清白蛋白被用作模型以模拟钙亲和力主导的相互作用。对于 IgG 相关物质，钙亲和力和磷酸阳离子交换呈正协同作用。Fc 的保留主要由钙亲和力决定，而 Fab 的保留则主要由阳离子交换决定。F(ab')(2)的保留曲线与 Fab 相似，但保留更强。完整 IgG 的保留曲线包含了其片段的独特元素，但保留强度大于它们相互叠加的预测值。聚集物的保留与非聚集抗体的保留曲线平行，两种结合机制的保留都更强。实验数据表明，在低电导率值下，IgG 羧基和 HA 磷酸根之间存在电荷排斥。HA 钙对氨基酸残基的静电排斥和对羧基的吸引似乎被钙与流动相磷酸根的强络合所阻断。
