Pan Jian, Tao Yunguo
Hisun Pharmaceutical(Hangzhou) Co. Ltd., Hangzhou 311404, China.
Se Pu. 2017 Aug 8;35(8):848-854. doi: 10.3724/SP.J.1123.2017.04014.
The development and optimization of reversed-phase preparative liquid chromatography method for insulin were performed. The chromatographic retention, sample loading and peak broadening were investigated. The mobile phase gradient conditions and stationary phases were optimized. The parameters of the method contained the peak broadening levels under different amounts of sample loading and the concentration distribution of the target compound in the elution curves. The parameters of peak broadening levels were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The most suitable chromatographic system (including stationary phases and mobile phase conditions) was selected. A gradient program with slow change of the strong elution solvent was used. The most suitable stationary phase should exhibit the narrower peak broadening and the peaks were best to broaden to both sides comparing to that under the analytical conditions. Besides, the concentration distribution of the target compound should be focused on the middle of the elution. The guide principles were validation by purification of crude insulin products. The preparative chromatographic system constructed through this method can remove impurities effectively. The method has good practical value. It can provide reference for the development of preparative chromatographic methods of other macromolecular compounds.
开展了胰岛素反相制备液相色谱方法的开发与优化。研究了色谱保留、进样量和峰展宽情况。对流动相梯度条件和固定相进行了优化。该方法的参数包括不同进样量下的峰展宽水平以及洗脱曲线中目标化合物的浓度分布。定义了峰展宽水平参数并表示为一个矩阵，该矩阵由进样量、前向展宽水平和后向展宽水平组成。选择了最合适的色谱系统（包括固定相和流动相条件）。使用了强洗脱溶剂变化缓慢的梯度程序。最合适的固定相应表现出较窄的峰展宽，并且与分析条件下相比，峰最好向两侧展宽。此外，目标化合物的浓度分布应集中在洗脱的中间部分。通过粗胰岛素产品的纯化验证了这些指导原则。通过该方法构建的制备色谱系统能够有效去除杂质。该方法具有良好的实用价值。可为其他大分子化合物制备色谱方法的开发提供参考。
