Resolution of Recombinant Human Interleukin 10 from Variants by Recycling Free Flow Focusing

Abstract 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 inclusion 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 (p I ) 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 p I '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-10 from variant C by successive RFFF treatments.