Isolation, physicochemical, and structure–function relationship of the hydrophobic variant of Fc-fusion proteins that bind to TNF-α receptor, HS002 and HS002A

Abstract

HS002 is the recombinant human tumor necrosis factor-α receptor Ⅱ: IgG Fc fusion protein licensed in China to treat rheumatism and psoriasis. The aim of this study was to isolate and characterize the hydrophobic freeze-dried powder injection (HS002) and ampoule injection (HS002A) variants derived from proteins of the same sequence and then to explore the structure–function relationship. Extensive physicochemical and structural testing was performed during a side-by-side comparison of the monomer peak and variant. Then the TNF-α-related binding activity, cell biological activity and affinity with FcRn were analyzed. Finally, a transformation study of the hydrophobic variant was performed under serum-like redox conditions. This research revealed that HS002A has similar physicochemical and structure–function relationship profiles to those of HS002. The hydrophobic variant exhibited the presence of new incorrect disulfide bridging. At the same time, this novel disulfide scrambled species structure–function relationship was found to be the molecular basis for reduced TNF-α binding and cell biological activities. In addition, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring TNF-α binding and cell biological activities to almost normal levels, all of which indicate that the variant is probably irrelevant to clinical efficacy once the drug enters the bloodstream.