A hydrophobic interaction chromatography method was developed to analyze recombinant soluble Interleukin 1 receptor type II (sIL-1R type II) drug substance and assess the stability of the drug under accelerated degradation studies. HIC resolved the degraded molecules into three peaks. A combination of several analytical techniques, including cyanogen bromide cleavage, reversed-phase chromatography, mass spectrometry, and N-terminal sequencing, were used to identify the origins of these peaks. We found that accelerated degradation resulted from three different events, deamidation and isomerization at asparagine 317 (Asn317), C-terminal cleavage, and aggregation. The iso-aspartate 317 (iso-Asp317)-containing species were shown to elute in HIC peak I and the Asp317-containing species in HIC peak II, respectively. Deamidation-isomerization to iso-Asp317, but not deamidation to Asp317, resulted in altered retention time on HIC companied by loss of potency, presumably by introducing a significant conformational change. CNBr C-terminal analysis showed that the inactive HIC peak I consisted of sIL-1R type II with "large" C-terminal truncations of 13 or 14 amino acids, whereas the active HIC peak II contained C-terminally full length and "small" C-terminal clips of two amino acids. Molecular modeling indicates that the short loop D317-S320, in the third domain of IL-1R type II, has a crucial impact on the stability of the molecule.