Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Shaunak, Sunil; Godwin, Antony; Choi, Ji-Won; Balan, Sibu; Pedone, Elisa; Vijayarangam, Damotharan; Heidelberger, Sibylle; Teo, Ian; Zloh, Mire; Brocchini, Steve

doi:10.1038/nchembio786

Cited by 242 publications

(245 citation statements)

References 10 publications

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“…5a), Silver staining and Western blotting (Fig. 5b) to confirm the identity of the PEGylated protein 22,23 .…”

Section: Timingmentioning

confidence: 85%

“…For modeling studies, we recommend that, in the first instance, only one disulfide in a monomeric protein is modified. In the case of multimeric proteins, our studies suggest that a single PEG can be attached to each of the monomers that make up the protein without disrupting its structure or biological function 22 . For example, L-asparaginase is active as a tetramer.…”

Section: Thiol-specific Bis-alkylationmentioning

confidence: 92%

“…As a result, only a three-carbon disulfide single bridge was inserted into the antibody fragment. These observations suggest that when proteins have multiple disulfides with varying degrees of accessibility for reduction, only the accessible disulfide that has been reduced will be PEGylated 22,23 . .…”

Section: Thiol-specific Bis-alkylationmentioning

confidence: 99%

“…1). Our approach enables the covalent re-bridging of the two thiols that are derived from the original disulfide bond [21][22][23] with a three-carbon methylene bridge to which PEG has been covalently attached.…”

Section: Existing Pegylation Technologiesmentioning

confidence: 99%

“…(b) MALDI-TOF spectrometry to confirm the PEGylated protein's mass and polydispersity of the PEG 22,23 .…”

Section: Timingmentioning

confidence: 99%

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PEGylation of native disulfide bonds in proteins

et al. 2006

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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

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“…5a), Silver staining and Western blotting (Fig. 5b) to confirm the identity of the PEGylated protein 22,23 .…”

Section: Timingmentioning

confidence: 85%

Section: Thiol-specific Bis-alkylationmentioning

confidence: 92%

Section: Thiol-specific Bis-alkylationmentioning

confidence: 99%

Section: Existing Pegylation Technologiesmentioning

confidence: 99%

“…(b) MALDI-TOF spectrometry to confirm the PEGylated protein's mass and polydispersity of the PEG 22,23 .…”

Section: Timingmentioning

confidence: 99%

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PEGylation of native disulfide bonds in proteins

et al. 2006

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Proteins: Chemistry and Chemical Reactivity

Tilley

Joshi

Francis

2008

Wiley Encyclopedia of Chemical Biology

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Protein bioconjugation is a critically important tool for the elucidation of enzyme mechanisms, the tracking of biomolecules in living systems, the improvement of pharmacokinetic properties, and the construction of new materials. All these applications rely on a continually expanding set of chemical reactions that can modify native protein functionality in aqueous solution under mild pH and temperature conditions. To survey these techniques, this article provides an introduction to the chemical reactivity of the amino acid side chains, with an emphasis on the selectivity that can be achieved using a particular reactive strategy. Site‐selective techniques that target the unique reactive properties of N‐terminal residues are also reviewed, as are native chemical ligation methods for the modification of the C‐terminus. Whenever possible, the mechanistic aspects of the reactions are discussed, as these considerations provide the foundation for future reaction development. The article concludes with a brief description of labeling reactions that selectively target unnatural functional groups in the presence of native protein functionality.

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Chemical and Genetic Modification

Farys¹,

Ginn²,

Badescu³

et al. 2013

Pharmaceutical Sciences Encyclopedia

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There are many natural post‐translational modifications of proteins (e.g., glycosylation, ubiquitination, acylation,and amino acid derivatization).Glycosylation is common for therapeutic proteins. Classes of proteins modified by glycosylation include monoclonal antibodies, blood factors, hematopoietic proteins, and cytokines. Excluding monoclonal antibodies, many proteins were, at least when they were first introduced to the clinic, designed to be used as a replacement protein for the corresponding endogenous protein. Maintaining control of the protein structure to be as close as possible to the structure of the endogeneous protein is therefore a key goal. In this review, we focus on (i) optimization of protein pharmacokinetics, (ii) improvement of protein properties to be used as medicines (this primarily includes reduction of immunogenic sequences and improvement of protein stability), (iii) addition of a therapeutic effect, and (iv) use of proteins as diagnostics.

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Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Cited by 242 publications

References 10 publications

PEGylation of native disulfide bonds in proteins

PEGylation of native disulfide bonds in proteins

Proteins: Chemistry and Chemical Reactivity

Chemical and Genetic Modification

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