Sluzky V, Shahrokh Z, Stratton P, Eberlein G, Wang Y J
Pharmaceutical R & D, Scios Nova Inc., Mountain View, California.
Pharm Res. 1994 Apr;11(4):485-90. doi: 10.1023/a:1018946011652.
High-performance liquid chromatography (HPLC) methods were developed for evaluating stability of human recombinant basic fibroblast growth factor (bFGF) against denaturation and aggregation in solution formulations. Reversed-phase chromatography (RP-HPLC)-insensitive to bFGF tertiary structure--was used to measure total soluble protein; heparin affinity chromatography (HepTSK) provided quantitative analysis of native bFGF species. The folding state of bFGF was determined by fluorescence spectroscopy: Tryptophan emission, which was quenched in native protein, increased upon unfolding. Slow unfolding/refolding kinetics of bFGF in 2 M guanidine hydrochloride made possible the separation of native from denatured species by size exclusion chromatography (SEC). Although the tertiary structure affected bFGF retention times, it did not change the sample recovery by SEC. These chromatographic techniques, which quantitatively measure physical and chemical changes taking place in solution formulations, can be used in future investigations of bFGF stability.
开发了高效液相色谱（HPLC）方法，用于评估重组人碱性成纤维细胞生长因子（bFGF）在溶液制剂中抗变性和聚集的稳定性。反相色谱（RP-HPLC）对bFGF三级结构不敏感，用于测定总可溶性蛋白；肝素亲和色谱（HepTSK）提供天然bFGF种类的定量分析。bFGF的折叠状态通过荧光光谱法测定：色氨酸发射在天然蛋白质中被淬灭，在展开时增加。bFGF在2 M盐酸胍中的缓慢展开/重折叠动力学使得通过尺寸排阻色谱（SEC）分离天然和变性种类成为可能。尽管三级结构影响bFGF的保留时间，但它不会改变SEC的样品回收率。这些色谱技术能够定量测量溶液制剂中发生的物理和化学变化，可用于未来bFGF稳定性的研究。
