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Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation
- Source :
-
Analytical Biochemistry . Jul2004, Vol. 330 Issue 2, p264-271. 8p. - Publication Year :
- 2004
-
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 #x03B5;-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 <f>α,γ</f>-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. [Copyright &y& Elsevier]
- Subjects :
- *PROTEINS
*POLYETHYLENE glycol
*BIOCONJUGATES
*THERAPEUTICS
Subjects
Details
- Language :
- English
- ISSN :
- 00032697
- Volume :
- 330
- Issue :
- 2
- Database :
- Academic Search Index
- Journal :
- Analytical Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 13474414
- Full Text :
- https://doi.org/10.1016/j.ab.2004.03.034