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        Site-specific PEGylation at histidine tags

        Author
        Cong, Yuehua
        Pawlisz, Estera
        Bryant, Penny
        Balan, Sibu
        Laurine, Emmanuelle
        Tommasi, Rita
        Singh, Ruchi
        Dubey, Sitara
        Peciak, Karolina
        Bird, Matthew
        Sivasankar, Amrita
        Swierkosz, Julia
        Muroni, Maurizio
        Heidelberger, Sibylle
        Farys, Monika
        Khayrzad, Farzad
        Edwards, Jeff
        Badescu, George
        Hodgson, Ian
        Heise, Charles
        Somavarapu, Satyanarayana
        Liddell, John
        Powell, Keith
        Zloh, Mire
        Choi, Ji-won
        Godwin, Antony
        Brocchini, Steve
        Attention
        2299/9997
        Abstract
        The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.
        Publication date
        2012
        Published in
        Bioconjugate Chemistry
        Published version
        https://doi.org/10.1021/bc200530x
        Other links
        http://hdl.handle.net/2299/9997
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