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Characterization of asparagine deamidation and aspartate isomerization in recombinant human interleukin-11
Wei Zhang1, J Marta J Czupryn, Philip T Boyle
1Wyeth BioPharma, Genetics Institute Campus, Andover, Massachusetts 01810, USA. wxzhang@wyeth.com
Unlabelled:
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 30 degrees C for 6 weeks in liquid resulted in significant isomerization of Asp45 and Asp47. Isomerization of Asp51 and deamidation of Asn49 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.