“Inside-Out” PEGylation of Bovine β-Cross-Linked Hemoglobin

Abstract

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to β-cross-linked (βXL-Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Study on Oxidation Stability and Oxygen Affinity of Hemoglobin During Storage

“…Hemoglobin‐based oxygen carriers (HBOCs) are blood substitutes derived from bovine, pig, or outdated human blood . The potential benefits of HBOCs include universal compatibility without the need for cross‐matching of donated blood, availability, and no infection risks . HBOCs in practice were expected to maintain stability in long production and storage processes .…”

Artificial Oxygen Carrier as Therapeutics Rather Than Blood Substitute for Transfusion

“…First of all, the imbalance between demands and resources prompted the hunt for sources of Hb other than human RBCs . Since bovine Hb is considered to be unlimited, it has been used as a source of Hb . While the risk of prion contamination needs to be minimized, its possibility cannot be completely ruled out as in the current practice of transfusion.…”

“…Synthetic AOCs have approached a level of experimental application following manufacturing renovations . More interesting are the characteristics of these new and innovative AOCs, which have a huge capacity to combine O 2 per single molecule, 156 O 2 molecules , and 3–8 O 2 molecules per single compound , suggesting that even a smaller dose is required to deliver the necessary amount of O 2 . In addition, control of O 2 affinity , and discrimination of the affinity to that of CO could become possible by advancements of the chemistry .…”

“…AOCs should be exposed to the rapidly developing field of artificial organs to support circulation. As opposed to the enthusiasm toward developing the “mechanical circulatory support,” AOCs and related compounds may be considered as “biological circulatory support.” In contrast to the closed circle of information and materials, this issue would share relevant information and welcome the offering of samples from developers to researchers to explore their utility or for researchers to suggest new ideas/utilities to manufacturers. As a result, new concepts and approaches are now being developed and tested with the use of the aforementioned AOCs and related compounds, which will be reported in the coming issue.…”

Artificial Oxygen Carriers: Exactly How Close Are We to an Ultimate Product?

“…This Special Issue of Artificial Organs , dedicated by Dr. Paul S. Malchesky, Editor‐in‐Chief, to Artificial Oxygen Carriers, contains some examples of work recently done in the field. It provides the information about clinical experience with Sanguinate (Prolong Pharmaceuticals, LLC, South Plainfield, NJ, USA) used in the treatment of SCA (Abuchowski, [ ]), use of solution containing marine worm molecule, HEMO2life and HEMOXCell (HEMARINA SA, Morlaix, France) in organ preservation and mesenchymal stem cell culture (Le Pape et al, [ ]), various therapeutic applications of liposome encapsulated Hb (Kawaguchi et al, [ ]), and novel modern Hb modification technologies (Webster et al, [ ]).…”