title: Biophysical stability of hyFc fusion protein with regards to buffers and various excipients.
abstract: A novel non-cytolytic hybrid Fc (hyFc) with an intact Ig structure without any mutation in the hyFc region, was developed to construct a long-acting agonistic protein. The stability of interleukin-7 (IL-7) fused with the hyFc (GXN-04) was evaluated to develop early formulations. Various biophysical methods were utilized and three different buffer systems with various pH ranges were investigated including histidine-acetate, sodium citrate, and tris buffers. Various excipients were incorporated into the systems to obtain optimum protein stability. Two evident thermal transitions were observed with the unfolding of IL-7 and hyFc. The Tm and ΔH increased with pH, suggesting increased conformational stability. Increased Z-average size with PDI and decreased zeta potential with pH increase, with the exception of tris buffer, showed aggregation issues. Moreover, tris buffer at higher pH showed aggregation peaks from DLS. Non-ionic surfactants were effective against agitation by outcompeting protein molecules for hydrophobic surfaces. Sucrose and sorbitol accelerated protein aggregation during agitation, but exhibited a protective effect against oxidation, with preferential exclusion favoring the compact states of GXN-04. The stability of GXN-04 was varied by basal buffers and excipients, hence the buffers and excipients need to be evaluated carefully to achieve the maximum stability of proteins.
authors: College of Pharmacy, Dongguk University-Seoul, Donggukro 32, Ilsandonggu, Goyang, Gyeonggi 410-820, Republic of Korea.
authors: Jun Yeul
authors: JY
authors: Lim
authors: College of Pharmacy, Dongguk University-Seoul, Donggukro 32, Ilsandonggu, Goyang, Gyeonggi 410-820, Republic of Korea.
authors: Nam Ah
authors: NA
authors: Kim
authors: College of Pharmacy, Dongguk University-Seoul, Donggukro 32, Ilsandonggu, Goyang, Gyeonggi 410-820, Republic of Korea.
authors: Dae Gon
authors: DG
authors: Lim
authors: Research Institute, Genexine Co., Ltd., Gyeonggi 137-701, Republic of Korea.
authors: Chang-yong
authors: CY
authors: Eun
authors: Research Institute, Genexine Co., Ltd., Gyeonggi 137-701, Republic of Korea.
authors: Donghoon
authors: D
authors: Choi
authors: College of Pharmacy, Dongguk University-Seoul, Donggukro 32, Ilsandonggu, Goyang, Gyeonggi 410-820, Republic of Korea. Electronic address: shjeong@dongguk.edu.
authors: Seong Hoon
authors: SH
authors: Jeong
mesh_terms: D002021:Buffers; D005079:Excipients / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D055550:Protein Stability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D002021:Buffers; D005079:Excipients; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins
keywords: Biophysical methods; GXN-04; Stability