PEGylation of native disulfide bonds in proteins

Abstract

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

“…The next step uses MS to confirm the PEGylated protein's mass and the polydispersity of the reactive PEG. Finally, CD is used to confirm the secondary structure of the protein [38]. Current technological advances are enabling the use of these techniques for optimization of different manufacturing processes, for example product yields and heterogeneity, and have great advantages over time-consuming chromatographic steps.…”

“…When the conjugates have been separated and analyzed a series of assays should be performed to confirm the biological activity of the protein [38]. Selection of these assays depends entirely on the activity of the protein in its native form and the characteristics PEGylation is intended to improve for the desired application.…”

Chemical and Genetic Modification

“…Another approach is to use conjugation reagents capable of bis-alkylation that can conjugate to both cysteine thiols from an existing disulfide in the protein [147,148]. Conjugation results in a 3-carbon bridge spanning the two cysteine thiols of the original disulfide with PEG conjugated site-specifically on the bridge.…”

“…Conjugation of the drug to the unpaired cysteine residues using maleimide chemistry yields ADCs with homogeneous DAR [147,346,348]. Genentech's THIOMABs is the most advanced technology using engineered unpaired cysteine residues, and this approach is currently being investigated by several other companies, including MedImmune, Seattle Genetics, and Pfizer.…”

Site-selective in situ growth of fluorescent polymer–antibody conjugates with enhanced antigen detection by signal amplification

“…Traditionally, inter-chain disulfide bonds are selectively reduced to reactive thiols for alkylation with drugs [16,17], which may have negative effects on the stability [14] and effectorfunctions of antibodies [18]. A promising solution to these problems is to restore a covalent linkage between the reduced cysteine residues by the utilization of bis-reactive bridging reagents such as monosulfone [19,20] and 3, 4-substituted maleimide [21e24]. Here, we envisage that it would be possible to selectively attach polymerization initiator molecules to an antibody at its inter-chain disulfide bridging sites by disulfide re-bridging to yield a macroinitiator, followed by in situ growth of dye or drug-labelled polymer conjugates.…”