Site-Specific PEGylation at Histidine Tags

Cong, Y., Pawlisz, E., Bryant, P., Balan, S., Laurine, E., Tommasi, R., Singh, R., Dubey, S., Peciak, K., Bird, M., Sivasankar, A., Swierkosz, J., Muroni, M., Heidelberger, S., Farys, M., Khayrzad, F., Edwards, J., Badescu, G., Hodgson, I., Heise, C., Somavarapu, S., Liddell, J., Powell, K., Zloh, M., Choi, J-W., Godwin, A. and Brocchini, S. (2012) Site-Specific PEGylation at Histidine Tags. Bioconjugate Chemistry, 23 (2). pp. 248-263. 10.1021/bc200530x.

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DOI: 10.1021/bc200530x


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 8Met-IFN. Cyanogen bromide digestion studies of PEG-His 8Met-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.

Item Type:Article
Departments, units and centres:Department of Pharmaceutical and Biological Chemistry > Department of Pharmaceutical and Biological Chemistry
ID Code:3042
Journal or Publication Title:Bioconjugate Chemistry
Deposited By:Library Staff
Deposited On:23 Mar 2012 09:01
Last Modified:23 Mar 2012 09:01


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