title: Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation.
abstract: Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.
authors.affiliation: AntiCancer Inc., 7917 Ostrow Street, San Diego, CA 92111, USA.
authors.forename: Shukuan
authors.initials: S
authors.lastname: Li
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Yang
authors.forename: Xinghua
authors.initials: X
authors.lastname: Sun
authors.forename: Yuying
authors.initials: Y
authors.lastname: Tan
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yagi
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hoffman
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000627:therapeutic use; D011700:Putrescine / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
chemical_list: D000577:Amides; C117929:PEG-O-succinyl-O-Su; D011506:Proteins; D013388:Succinimides; D011092:Polyethylene Glycols; D011700:Putrescine