Beckman Coulter, Inc., Fullerton, CA 92834-3100, USA.
Electrophoresis. 2009 Dec;30(23):4049-58. doi: 10.1002/elps.200800690.
A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10<pI) sacrificial ampholyte to the CIEF sample solution. Optimization of the concentrations of the sacrificial ampholytes, iminodiacetic acid and arginine to 1.7 and 40 mM, respectively, yielded a stable focused CA train that spanned the 4<pH<10 range without sacrificing either mAb isoform resolution or sample throughput. Intermediate precision studies performed on the CIEF method with three basic mAbs yielded 0.04 to 0.09% CVs for the estimated pI values and 0.6-3% CVs for isoform group percent composition, indicating that the two-step CIEF method developed meets the rigorous demands of therapeutic mAb analysis.
完成了两步 CIEF 分析的系统研究，以确定关键组件，可以对其进行优化以增强 CIEF 分析 mAb 的性能。通过选择窄的检测器孔径、利用化学而非压力迁移、以及通过在载体两性电解质（CA）溶液中加入脲来提高蛋白质溶解度，增加了 mAb 异构体之间的分辨率。通过将磷酸阳极电解液的浓度设置为 200 mM 和氢氧化钠阴极电解液的浓度设置为 300 mM 并向 CIEF 样品溶液中加入足够量的酸性（pI<3）和碱性（10<pI）牺牲两性电解质，避免了IEF 固有的双向等电聚焦（ITP）中极端 pI CA 和样品成分的损失。优化牺牲两性电解质、亚氨基二乙酸和精氨酸的浓度分别为 1.7 和 40 mM，得到了稳定的聚焦 CA 馏分，覆盖了 4<pH<10 范围，既不牺牲 mAb 异构体分辨率，也不牺牲样品通量。对三种基本 mAb 的 CIEF 方法进行中间精密度研究，得到的估计 pI 值的 CV 值为 0.04 至 0.09%，异构体组百分比组成的 CV 值为 0.6-3%，表明两步 CIEF 方法满足治疗性 mAb 分析的严格要求。
