Bioconjugate Chem. 2001, 12, 163−169
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Drug Delivery Systems Employing 1,6-Elimination: Releasable Poly(ethylene glycol) Conjugates of Proteins Stanford Lee,* Richard B. Greenwald,* Jeffrey McGuire, Karen Yang, and Celine Shi Enzon, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854. Received June 3, 2000; Revised Manuscript Received October 21, 2000
Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the -amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.
INTRODUCTION
Conjugation of proteins with poly(ethylene glycol) (PEG),1 a process that has been termed PEGylation (1), imparts important pharmacological properties to the modified protein. The advantages of PEGylation include increased circulatory half-life, reduced immunogenicity, reduced loss of protein to protease cleavage, and increased water solubility. It is generally observed that protein functions, such as catalysis and receptor binding, are compromised to varying degrees following PEGylation, usually resulting in diminished activity; this may be due to the presence of the PEG substituent on or near the protein’s active or regulatory sites. Early attempts to improve this situation led to the use of differently activated PEG linkers, all producing conjugates with hydrolysis-resistant permanent bonds; no one particular linker has provided consistently superior results. Therefore, it seems likely that the most effective linker for maintaining activity will vary for different proteins and will have to be determined empirically for each case (2, 3). PEG mass and the degree of protein modification have also been explored as a means of preserving activity; the use of fewer PEG strands of higher molecular weight has been reported to produce conjugates with less loss of activity (4, 5). A novel approach for maintaining maximum activity in a conjugate would be to design a functional PEG linker that can predictably break down by enzymatic or pH directed hydrolysis. Such a releasable PEG (rPEG) would provide rPEGylated protein conjugates which are impermanent and could act as a depot or reservoir, continuously discharging native protein with full, albeit potentially short-acting, activity. Coupling of proteins to substituted maleic anhydrides at the -amino group of lysine residues results in the * To whom correspondence should be addressed. (S.L.) Phone: (732) 980-4909. E-mail:
[email protected]. (R.B.G.) Phone: (732) 980-4924. E-mail:
[email protected]. 1 References to PEG conjugates in this paper will be restricted to methoxy PEG with a molecular weight of 5000.
formation of maleic acid half amides which are labile at acidic pH (6, 7). By employing this property, it was demonstrated that conjugation of proteins through -amino groups with citraconic anhydride or 2,3-dimethylmaleic anhydride (8-11) led to conjugates that could be deacylated at pH
