Characterization of asparagine deamidation and aspartate isomerization in recombinant human interleukin-11

Purpose. The aim of this study was to investigate asparagine (Asn) deamidation and aspartate (Asp) isomerization and to measure the content of isoaspartate (isoAsp) in recombinant human interleukin-11 (rhIL-11). Methods. The rhIL-11 control and heat stressed samples were characterized with trypsin and endoproteinase Asp-N peptide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI MS) and capillary electrophoresis (CE). The total isoAsp content and bioactivity were also assessed. Results. Stress of rhIL11 at 30degreesC for 6 weeks in liquid resulted in significant isomerization of Asp(4)5 and Asp(4)7. Isomerization of Asp(5)1 and deamidation of Asn(4)9 were also detected at low levels. The stressed rhIL-11 molecule contained 0.3 mol of isoAsp per mol of protein, compared to only 0.007 mol/mol of protein in the control. Conclusions. Asp and Asn residues, located in a loop structure of rhIL-11, undergo isoAsp formation under stressed conditions.