Flow Diagram for Study Search and Inclusion.

<p>Flow Diagram for Study Search and Inclusion.</p

Summary of Key Characteristics of Included Studies.

<p>Summary of Key Characteristics of Included Studies.</p

Strength of Evidence and Generalizability/Transferability of Key Review Findings to High Prevalence Contexts.

<p>Strength of Evidence and Generalizability/Transferability of Key Review Findings to High Prevalence Contexts.</p

Antiretroviral medication regimens in included studies.

<p>*PMTCT prophylaxis refers to the use of ARV drugs solely for the purpose of reducing the risk of vertical transmission when a woman is not on standard ART for therapeutic reasons.</p><p>Antiretroviral medication regimens in included studies.</p

Site-Specific PEGylation at Histidine Tags

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-His₆) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His₈-IFN). The site of PEGylation at the His-tag for both dAb-His₆-PEG and PEG-His₈-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 His₈Met-IFN. Cyanogen bromide digestion studies of PEG-His₈Met-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 <i>in vitro</i> evaluation confirmed that these PEGylated proteins retained their biological activity. <i>In vivo</i> 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