Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA.
Biologics Process Development, Bristol-Myers Squibb, Hopewell, NJ, USA.
J Chromatogr A. 2018 Nov 30;1578:28-34. doi: 10.1016/j.chroma.2018.10.003. Epub 2018 Oct 7.
The gradient elution hydrophobic interaction chromatography of a monoclonal antibody that exhibits U-shaped retention as a function of the ammonium sulfate concentration is investigated for overloaded conditions at protein loads up to 30% of the column equilibrium binding capacity. Protein load and gradient slope affect both elution peak shape and protein recovery during the gradient. Higher protein loads result in tailing peaks with near 100% recovery that transition to fronting peaks and incomplete recovery as the protein load is reduced. The gradient slope also affects peak shape and recovery. Tailing peaks with lower recovery are obtained with sharper gradients and the most tailing peak and lowest recovery are obtained when step elution rather than gradient is implemented. Modeling the chromatographic elution based on independently determined adsorption isotherms as a function of protein and ammonium sulfate concentration predicts results in agreement with the experimental trends confirming that the unusual chromatographic behavior observed is due to the U-shaped protein binding as a function of the ammonium sulfate concentration. Although less pronounced than in the dilute limit, the U-shaped binding still produces peak shapes and recovery losses as a function of gradient slope that differ from those seen for systems where retention is a monotonic function of salt concentration.
研究了在蛋白质负载高达柱平衡结合容量的 30%的情况下，对表现出 U 形保留行为的单克隆抗体进行梯度洗脱疏水性相互作用色谱。蛋白质负载和梯度斜率都会影响梯度洗脱过程中的洗脱峰形状和蛋白质回收率。较高的蛋白质负载会导致峰尾几乎 100%回收，然后逐渐变为峰前和不完全回收，随着蛋白质负载的减少。梯度斜率也会影响峰形状和回收率。采用较陡的梯度可得到回收率较低的拖尾峰，而采用阶跃洗脱而不是梯度洗脱则会得到峰拖尾最严重和回收率最低的情况。基于独立确定的吸附等温线作为蛋白质和硫酸铵浓度的函数来模拟色谱洗脱，预测结果与实验趋势一致，证实观察到的异常色谱行为是由于蛋白质与硫酸铵浓度的 U 形结合所致。尽管不如在稀溶液极限下明显，但 U 形结合仍会导致峰形状和回收率损失，这与那些保留是盐浓度单调函数的系统中看到的情况不同。
