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.

Pegylation and Its Impact on the Design of New Protein-Based Medicines

“…IFNα-2b, IFNβ-1b Preclinical [135] Cysteine Thiol alkylation Affibody Preclinical [106,136] Disulfide bonds Bis-alkylation Leptin, IFNα-2, Fab, somatostatin Preclinical [137][138][139] Bis-alkylation Somatostatin, salmon calcitonin Preclinical [109,140] Glutamine Enzyme-mediated transglutaminases G-CSF, hGH Preclinical [120,141] Glycosylation site Enzymatic transfer glycosyltransferases G-CSF, IFNα-2, blood factors Clinical [123] Histidine tags Bis-alkylation IFNα-2 and a domain antibody Preclinical [142] Non-native amino acids Cycloaddition SOD, HGH Preclinical [143] Key term Site-specific conjugation: Conjugation at a specific amino acid residue in the protein of interest.…”

Chemical and Genetic Modification

“…The defined incorporation of non-native amino acids for site-specific conjugation in an optimal location on the protein may provide the option to achieve good potency for a polymer conjugated protein [117]. It has also been hypothesized that selective PEGylation at either termini of a protein might offer the potential to achieve good potency [118].…”

“…Conjugation results in a 3-carbon bridge spanning the two cysteine thiols of the original disulfide with PEG conjugated site-specifically on the bridge. An alternative approach for site-specific conjugation using bis-alkylating reagents is that they can conjugate a protein His-tag site-specifically [118]. Because it is rare to have multiple histidines next to each other in a protein [149,150], it makes the His tag a good target for selective conjugation of the PEG.…”

“…staphylokinase, growth hormone) [170,174] as a means to maximize N-terminal selectivity and conjugation efficiency. The His tag-selective PEGylation mentioned earlier [118] is a potential alternative to C-and N-terminal PEGylation.…”

Genetic PEGylation

“…To overcome this difficulty, a more specific modification was achieved by exploiting the difference in the pKa value of the amine in the lysine side chain or the replacement of lysine residues with other amino acids [15]–[18]. The thiol groups in cysteine residues, the phenol groups of tyrosines, the amide groups of glutamines, or the incorporated His tag have also been used for this specific modification [19]–[29]. The site-specific polymer attachment has also been achieved with the complete synthetic construction of an erythropoietic protein [30], [31], consisting of an α-amino acid polypeptide chain of 166 residues by using native chemical ligation.…”