Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

Abstract

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.

Hemoglobin-Based Oxygen Carriers: Where Are We Now in 2023?

“…When extremely low O 2 tensions exist in ischemic tissues, the HBOC may need high affinity to optimize O 2 delivery [ 32 ]. Sanguinate’s structural characteristics and altered hemoglobin-oxygen binding affinity allow it to bypass obstructions in the microcirculation and effectively deliver oxygen to ischemic tissues [ 33 ]. Sanguinate is also able to endogenously deliver carbon monoxide, which has been shown to reduce inflammation and oxidative stress, mitigate ischemia-reperfusion injury, and promote vasodilation.…”

“…The additional therapeutic effects (anti-inflammatory, anti-vasoconstrictive, plasma expansion) may make Sanguinate useful in the treatment of disorders in which blood is ineffective, such as stroke, inflammatory diseases, and sepsis. As such, Sanguinate is more effectively termed a resuscitation fluid [ 33 ]. Cooper et al recently engineered a hemoglobin molecule with a single reactive cysteine residue on the surface of the α-subunit, creating a single PEGylation site (βCys93Ala/αAla19Cys).…”

Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity

“…Recently, Zuma and colleagues (2022) recently demonstrated that site-specific PEGylation enhances the biological activity and stability of recombinant DNA ligase proteins [ 23 ]. Cooper and colleagues showed that proteins treated with mono-sulfone-PEG retained higher significantly conjugation [ 24 ].…”

“…This method was used in PEGylation of HSA molecule-free Cy34 with PEG-maleimide for the protein sulfhydryl (-SH) groups (highly specific) ( Figure 3 ) [ 16 , 27 ]. Cooper and colleagues showed that proteins treated with mono-sulfone-PEG retained higher significantly conjugation [ 24 ]. Dozier and Distefano used this method by PEGylating L-lacate oxidase which retained activity after PEGylation.…”