title: 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 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.
authors.affiliation: Department of Bioisolation Process Development, Schering-Plough Research Institute, Union, New Jersey 07083, USA.
authors.forename: L L
authors.initials: LL
authors.lastname: Bondoc
authors.forename: J P
authors.initials: JP
authors.lastname: Varnerin
authors.forename: J C
authors.initials: JC
authors.lastname: Tang
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D016753:Interleukin-10 / Q000032:analysis*; D007525:Isoelectric Focusing / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D011993:Recombinant Fusion Proteins; D016753:Interleukin-10