title: Analysis and characterization of aggregation of a therapeutic Fc-fusion protein.
abstract: Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at -30 °C, even though the protein was stable at 4 and -80 °C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC-MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein.
authors.affiliation: Analytical Sciences, Process and Product Development, Amgen Inc., Thousand Oaks, CA, 91320, United States. tianw@amgen.com
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
authors.forename: Szilan
authors.initials: S
authors.lastname: Fodor
authors.forename: Suminda
authors.initials: S
authors.lastname: Hapuarachchi
authors.forename: Xinzhao Grace
authors.initials: XG
authors.lastname: Jiang
authors.forename: Ken
authors.initials: K
authors.lastname: Chen
authors.forename: Izydor
authors.initials: I
authors.lastname: Apostol
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D002479:Inclusion Bodies / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012492:Salts; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
chemical_list: D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D012492:Salts; D013438:Sulfhydryl Compounds