title: Site-specific PEGylation at histidine tags.
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.
authors.affiliation: PolyTherics Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, United Kingdom.
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Cong
authors.forename: Estera
authors.initials: E
authors.lastname: Pawlisz
authors.forename: Penny
authors.initials: P
authors.lastname: Bryant
authors.forename: Sibu
authors.initials: S
authors.lastname: Balan
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Laurine
authors.forename: Rita
authors.initials: R
authors.lastname: Tommasi
authors.forename: Ruchi
authors.initials: R
authors.lastname: Singh
authors.forename: Sitara
authors.initials: S
authors.lastname: Dubey
authors.forename: Karolina
authors.initials: K
authors.lastname: Peciak
authors.forename: Matthew
authors.initials: M
authors.lastname: Bird
authors.forename: Amrita
authors.initials: A
authors.lastname: Sivasankar
authors.forename: Julia
authors.initials: J
authors.lastname: Swierkosz
authors.forename: Maurizio
authors.initials: M
authors.lastname: Muroni
authors.forename: Sibylle
authors.initials: S
authors.lastname: Heidelberger
authors.forename: Monika
authors.initials: M
authors.lastname: Farys
authors.forename: Farzad
authors.initials: F
authors.lastname: Khayrzad
authors.forename: Jeff
authors.initials: J
authors.lastname: Edwards
authors.forename: George
authors.initials: G
authors.lastname: Badescu
authors.forename: Ian
authors.initials: I
authors.lastname: Hodgson
authors.forename: Charles
authors.initials: C
authors.lastname: Heise
authors.forename: Satyanarayana
authors.initials: S
authors.lastname: Somavarapu
authors.forename: John
authors.initials: J
authors.lastname: Liddell
authors.forename: Keith
authors.initials: K
authors.lastname: Powell
authors.forename: Mire
authors.initials: M
authors.lastname: Zloh
authors.forename: Ji-won
authors.initials: JW
authors.lastname: Choi
authors.forename: Antony
authors.initials: A
authors.lastname: Godwin
authors.forename: Steve
authors.initials: S
authors.lastname: Brocchini
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D000906:Antibodies; C033223:polyhistidine; D011092:Polyethylene Glycols; D006639:Histidine