Bondoc L L, Varnerin J P, Tang J C
Department of Bioisolation Process Development, Schering-Plough Research Institute, Union, New Jersey 07083, USA.
Anal Biochem. 1997 Mar 15;246(2):234-8. doi: 10.1006/abio.1997.2023.
Recombinant human interleukin 10 (rhIL-10) is a potential human therapeutic agent for treating inflammatory bowel diseases and rheumatoid arthritis. The rhIL-10 molecule derived from Escherichia coli including bodies consists of two identical subunits forming a noncovalent dimer. Since the ability to separate rhIL-10 from closely related impurities was highly desirable, recycling free flow focusing (RFFF) was utilized for the purification process development of rhIL-10. Under nondenaturing conditions, RFFF was able to separate rhIL-10 from fractions enriched in rhIL-10 variants. Three major monomeric variants (A, B, and C) can be identified and quantitated by reversed phase HPLC. The isoelectric point (pI) of rhIL-10 was empirically determined to be 8.2 while that for the three variant populations were in the range 7.3-7.5. Knowledge of these pI's would potentially facilitate the optimization process for ion-exchange chromatography. Furthermore, the technique provided a mild and fast preparation procedure for obtaining the recombinant protein and its variants for further characterization, as evidenced in the separation of rhIL-1- from variant C by successive RFFF treatments.
重组人白细胞介素10（rhIL-10）是一种用于治疗炎症性肠病和类风湿性关节炎的潜在人类治疗剂。源自大肠杆菌包含包涵体的rhIL-10分子由两个相同的亚基组成，形成一个非共价二聚体。由于非常需要将rhIL-10与密切相关的杂质分离的能力，因此采用循环自由流聚焦（RFFF）来开发rhIL-10的纯化工艺。在非变性条件下，RFFF能够从富含rhIL-10变体的级分中分离出rhIL-10。三种主要的单体变体（A、B和C）可以通过反相高效液相色谱法进行鉴定和定量。rhIL-10的等电点（pI）经实验测定为8.2，而三种变体群体的等电点在7.3-7.5范围内。了解这些pI值可能有助于优化离子交换色谱法的工艺。此外，该技术为获得重组蛋白及其变体以进行进一步表征提供了一种温和且快速的制备方法，连续的RFFF处理从变体C中分离出rhIL-10就证明了这一点。
