title: Chemical modifications of therapeutic proteins induced by residual ethylene oxide.
abstract: Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics.
authors.affiliation: Process and Product Development, Amgen Inc., Thousand Oaks, California.
authors.forename: Louise
authors.initials: L
authors.lastname: Chen
authors.forename: Christopher
authors.initials: C
authors.lastname: Sloey
authors.forename: Zhongqi
authors.initials: Z
authors.lastname: Zhang
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Bondarenko
authors.forename: Hyojin
authors.initials: H
authors.lastname: Kim
authors.forename: Da
authors.initials: D
authors.lastname: Ren
authors.forename: Sekhar
authors.initials: S
authors.lastname: Kanapuram
mesh_terms: D000595:Amino Acid Sequence; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics; D005027:Ethylene Oxide / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D004364:Pharmaceutical Preparations / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics
chemical_list: C554270:EPO protein, human; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; D005027:Ethylene Oxide
keywords: HPLC; analytical biochemistry; chromatography; drug delivery systems; formulation; injectables; mass spectrometry; peptides; protein delivery; proteins