title: Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency.
abstract: 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.
authors.affiliation: Department of Analytical and Formulation Sciences, Amgen Inc. 1201 Amgen Court West, Seattle, Washington 98119, USA. zhangy@amgen.com
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zhang
authors.forename: Theresa
authors.initials: T
authors.lastname: Martinez
authors.forename: Brian
authors.initials: B
authors.lastname: Woodruff
authors.forename: Andy
authors.initials: A
authors.lastname: Goetze
authors.forename: Robert
authors.initials: R
authors.lastname: Bailey
authors.forename: Dean
authors.initials: D
authors.lastname: Pettit
authors.forename: Alain
authors.initials: A
authors.lastname: Balland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D003488:Cyanogen Bromide / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D053573:Receptors, Interleukin-1 Type I / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013696:Temperature
chemical_list: D053573:Receptors, Interleukin-1 Type I; D011994:Recombinant Proteins; D003488:Cyanogen Bromide