Abstract
PEGylation of protein sulfhydryl residues is a common method used to create a stable drug conjugate to enhance vascular retention times. We recently created a putative haemoglobin-based oxygen carrier using maleimide-PEG to selectively modify a single engineered cysteine residue in the α subunit (αAla19Cys). However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions, with consequent cross-conjugation to endogenous plasma thiols such as those found on human serum albumin or glutathione. In previous studies mono-sulfone-PEG adducts have been shown to be less susceptible to deconjugation. We therefore compared the stability of our maleimide-PEG Hb adduct with one created using a mono-sulfone PEG. The corresponding mono-sulfone-PEG adduct was significantly more stable when incubated at 37 °C for 7 days in the presence of 1 mM reduced glutathione, 20 mg/mL human serum albumin, or human serum. In all cases haemoglobin treated with mono-sulfone-PEG retained >90% of its conjugation whereas maleimide-PEG showed significant deconjugation, especially in the presence of 1 mM reduced glutathione where <70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation of Hb seems adequate for an oxygen therapeutic intended for acute use, if longer vascular retention is required reagents such as mono-sulfone-PEG may be more appropriate.
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References
Bettati S, Mozzarelli A (eds) (2011) Chemistry and biochemistry of oxygen therapeutics: from transfusion to artificial blood. Wiley
Cooper CE, Silkstone GGA, Simons M et al (2020) Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality. Biomater Sci 8:3896–3906
Badescu G, Bryant P, Swierkosz J et al (2014) A new reagent for stable thiol-specific conjugation. Bioconjug Chem 25:460–469
Cooper CE, Bird M, Sheng X et al (2021) Stability of Maleimide-PEG and Mono-sulfone-PEG conjugation to a novel engineered cysteine in the human hemoglobin alpha subunit. Front Chem 9:707797
Acknowledgments
This research was funded in whole or in part by the UK research councils BBSRC (BB/L004232/1) and MRC (MR/L01310X/1). For the purpose of Open Access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.
Disclosure
Cooper and Reeder have patents relating to the use of recombinant haemoglobin as a component of an oxygen therapeutic. Bird and Sheng were employees of Abzena Ltd. at the time this work was undertaken.
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Cooper, C.E. et al. (2022). Stability of a Novel PEGylation Site on a Putative Haemoglobin-Based Oxygen Carrier. In: Scholkmann, F., LaManna, J., Wolf, U. (eds) Oxygen Transport to Tissue XLIII. Advances in Experimental Medicine and Biology, vol 1395. Springer, Cham. https://doi.org/10.1007/978-3-031-14190-4_48
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DOI: https://doi.org/10.1007/978-3-031-14190-4_48
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