Genentech Process Research and Development, 1 DNA Way MS 75A, South San Francisco, CA 94080, USA.
J Chromatogr A. 2010 Jan 29;1217(5):660-6. doi: 10.1016/j.chroma.2009.11.099. Epub 2009 Dec 4.
Large conductivity fluctuations were observed during a high pH wash step in a weak cation exchange chromatography process. These conductivity transitions resulted in a conductivity drop during pH increase and a conductivity rise during pH decrease. In some cases, the absolute conductivity change was greater than 6mS/cm which was sufficient to affect target protein retention on the column. Further investigation revealed that wash buffer concentration, resin ligand density, and resin ligand pK have a profound effect on the magnitude of the conductivity transitions and the shape of corresponding pH traces. A potentiometric electrode selective for sodium ions was used to measure effluent counterion concentrations from two preparative resins during high pH washes, and the number of exchangeable counterions was compared to predictions made using ion exchange equilibrium theory. Results from this analysis show that conductivity transitions can be effectively mitigated without compromising process performance by optimizing the trade-off between wash buffer concentration and wash phase duration.
在弱阳离子交换色谱过程的高 pH 洗脱步骤中观察到了大的电导率波动。这些电导率转变导致 pH 升高时电导率下降，pH 降低时电导率上升。在某些情况下，绝对电导率变化大于 6mS/cm，足以影响目标蛋白在柱上的保留。进一步的研究表明，洗脱缓冲液浓度、树脂配体密度和树脂配体 pK 对电导率转变的幅度和相应 pH 曲线的形状有深远的影响。使用对钠离子具有选择性的电位电极测量了两种制备性树脂在高 pH 洗脱过程中的流出物抗衡离子浓度，并将可交换抗衡离子的数量与使用离子交换平衡理论进行的预测进行了比较。该分析的结果表明，通过优化洗脱缓冲液浓度和洗脱阶段持续时间之间的权衡，可以有效地减轻电导率转变，而不会影响工艺性能。
