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Received October 14, 2002; accepted January 22, 3003
Purpose. N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw 2000 and 5000) through a reactive terminal aldehyde group.
Methods. The site-specific PEG conjugation was conducted at a slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an -amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF.
Results. The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such as cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability when incubated with tissue homogenate.
Conclusion. N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.
KEY WORDS: epidermal growth factor (EGF); poly(ethylene glycol) (PEG); site-specific PEGylation; biologic activity.
INTRODUCTION
Many therapeutic proteins have been conjugated with a water-soluble synthetic polymer, poly(ethylene glycol) (PEG), to enhance their pharmacological activities (1). Conjugation of PEG (PEGylation) to various proteins not only increases their half-life in the blood stream but also significantly reduces their immunogenicities (2,3). In particular, the prolonged circulation of PEGylated proteins reduces the necessity of multiple injections to patients. A wide range of therapeutic proteins have been PEGylated; for example, human growth hormone, interferon, insulin, granulocyte-colony stimulating factor (G-CSF), and interleukin II (1). Generally speaking, whereas PEGylation of proteins beneficially results in a longer circulation time in blood, it also causes the problem of reducing their intrinsic biologic activities. The steric hindrance effect of conjugated PEG significantly suppresses specific binding of PEGylated proteins to counterpart cellular receptors or substrates (4,5). However, the prolonged in vivo half-life of PEGylated proteins often compensates for the reduction of biologic activity, leading to an overall increase in their therapeutic effects in vivo. This is the so-called counteracting effect (6). Therefore, PEGylation of proteins at specific residues is necessary to exert minimal effect...