Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross‐link densities, and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand‐binding kinetics, auto‐oxidation, and haptoglobin [Hp]‐binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta‐data analysis to evaluate correlations between procedural parameters (i.e., cross‐linker:bovine hemoglobin (bHb) molar ratio, gas‐liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T‐ and R‐state PolybHbs. Our results showed that, the duration of the fast‐step auto‐oxidation phase of R‐state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T‐state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O2 offloading rate constants compared to R‐state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as artificial red blood cell (RBC) substitutes for use in transfusion medicine. Unfortunately, prior generations of HBOCs were not able to successfully minimize key side effects, including vasoconstriction, systemic hypertension, and oxidative tissue injury, which is primarily due to the extravasation of cell-free Hb from the vascular space into the tissue space. Therefore, to potentially reduce these side effects, we successfully encapsulated Hb within a zeolitic imidazolate framework (ZIF-8) to form ZIF-8-Hb nanoparticles (ZIF-8P-Hb). Both ZIF-8 and ZIF-8P-Hb nanoparticles were synthesized at a relatively high molar ratio of 2-methylimidazole:zinc, which resulted in a monodisperse nanoparticle size distribution. In addition, the flow conditions for tangential flow filtration-facilitated purification of the nanoparticles did not exert a strong effect on the nanoparticle size distribution. ZIF-8P-Hb nanoparticles exhibited high stability, ultrahigh Hb encapsulation efficiency, and a monodisperse size distribution. Additionally, ZIF-8P-Hb nanoparticles exhibited a ζ-potential of −11.2 ± 0.9 mV, demonstrating its potentially enhanced biocompatibility in comparison to bare ZIF-8 nanoparticles (40.7 ± 2.0 mV). More significantly, ZIF-8P-Hb nanoparticles exhibited significantly enhanced hydrothermal stability with negligible release of cell-free Hb. Furthermore, ZIF-8P-Hb displayed a significantly lower haptoglobin binding rate constant compared to cell-free Hb, indicating its potentially slower in vivo clearance in comparison to cell-free Hb. Moreover, we observed a relatively low level of hemolysis when ZIF-8P-Hb nanoparticles were incubated with RBCs (<5%), which demonstrates a suitable safety profile. To further optimize the ZIF-8P-Hb nanoparticle synthesis protocol, various procedural parameters were systematically investigated to evaluate their impact on the size distribution of ZIF-8 and ZIF-8P-Hb nanoparticles. Taken together, this work provides a comprehensive approach for synthesizing a monodisperse HBOC as a potential artificial RBC substitute.

Red blood cell (RBC) substitutes tested in latephase clinical trials contained low-molecular-weight hemoglobin species (<500 kDa), resulting in vasoconstriction, hypertension, and oxidative tissue injury; therefore, contributing to poor clinical outcomes. This work aims to improve the safety profile of the RBC substitute, polymerized human hemoglobin (PolyhHb), via in vitro and in vivo screening of PolyhHb fractionated into four molecular weight brackets (50−300 kDa [PolyhHb-B1]; 100−500 kDa [PolyhHb-B2]; 500−750 kDa [PolyhHb-B3]; and 750 kDa to 0.2 μm [PolyhHb-B4]) using a two-stage tangential flow filtration purification process. Analysis showed that PolyhHb's oxygen affinity, and haptoglobin binding kinetics decreased with increasing bracket size. A 25% blood-for-PolyhHb exchange transfusion guinea pig model suggests that hypertension and tissue extravasation decreased with increasing bracket size. PolyhHb-B3 demonstrated extended circulatory pharmacokinetics, no renal tissue distribution, no aberrant blood pressure, or cardiac conduction effects, and may therefore be appropriate material for further evaluation.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (O2) carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench‐top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two‐stage tangential flow filtration purification process to remove almost all undesirable Hb species (>0.2 µm and <500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto‐oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

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