Biophysical properties of tense quaternary state polymerized human hemoglobins bracketed between 500 kDa and 0.2 μm in size

AbstractPolymerized hemoglobin (Hb)-based oxygen carriers (HBOCs) are a scalable and cost-effective red blood cell (RBC) substitute. However, previous generations of commercial polymerized HBOCs elicited oxidative tissue injuryin vivo due to the presence of low molecular weight polymeric Hb species (<  500 kDa) and cell-free Hb (64 kDa). Polymerized human Hb (PolyhHb) locked in the tense quaternary state (T-state) exhibits great promise to meet clinical needs where past polymerized HBOCs failed. This work shows that purifying T-state PolyhHb via a two-stage tangential flow filtration (TFF) puri fication train such that the Hb polymers are bracketed between 500 kDa and 0.2 μm creates a uniform polymer size and largely eliminates the Hb species which elicit deleterious side effectsin vivo. Biophysical characterization of these materials demonstrates their potential effectiveness as an RBC substitute and verifies their low percentage of low molecular weight Hb polymers and cell-free Hb. Size exclusion chromatography confirms that T-state PolyhHb can be consistently produced in a size range between 500 kDa and 0.2 μm. Furthermore, the average molecular weight of all PolyhHb species produced are one or two orders of magnitude larger than that of the commercial polymerized HBOCs Hemolink and Oxyglobin, respectively. Haptoglobin binding kinetics confirms that two-stage TFF processing of PolyhHb reliably removes cell-free Hb and low molecular weight polymeric Hb species. T-sta...

Source: Biotechnology Progress - October 9, 2021 Category: Biotechnology Authors: Clayton T. Cuddington, Savannah R. Wolfe, Andre F. Palmer Tags: RESEARCH ARTICLE Source Type: research