Klyushnichenko V E, Yakimov S A, Arutyunyan A M, Ivanov A E, Maltsev K V, Wulfson A N
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
J Chromatogr B Biomed Appl. 1994 Dec 9;662(2):363-9. doi: 10.1016/0378-4347(94)00306-8.
The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.
研究了反相高效液相色谱法（HPLC）在重组胰岛素产物分析中的适用性。考察了反相和离子对HPLC中几种流动相对选择性、分离度和灵敏度的影响。通过对选择性和分离度进行三维优化，建立了在市售和自制载体上分离胰岛素相关蛋白的最佳条件，该优化以pH值和离子强度（μ）为函数。提出了一种用含盐浓度高且pH值接近蛋白质等电点的流动相分离蛋白质的机制。考虑了放大问题。所提出的技术可用于分析主要杂质，并确保活性胰岛素生产的高质量。
