Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin

Abstract

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.

Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs)

“…Some liposome based HBOCs have been able to nearly match the level of methemoglobin initially after formulation when freshly isolated hemoglobin from expired blood units are used. 21,59 We hypothesize that improved hemoglobin sourcing and timelines can further improve the level of methemoglobin we observe in our system. However, for applications not replacing more than 30% of the blood volume methemoglobin levels of ~20% in HBOCs can be helpful in supporting native red blood cells.…”

“…18 To address these challenges, strategies to increase the molecular diameter of the HBOC and limit free hemoglobin in circulation have become requirements in the design of modern HBOCs. These strategies include chemical crosslinking of hemoglobin, 19,20 polymerization of hemoglobin and crosslinked hemoglobins, [21][22][23] and encapsulation of hemoglobin in liposome and nano-and microparticle systems. 24,25 Liposome encapsulated hemoglobins have demonstrated many of the desired design criteria for an alternative oxygen carrier, including absence of blood group antigens and blood-borne pathogens and a prolonged storage capacity.…”

Research Progress in Oxygen Carrier Design and Application

“…Palmer et al further purified and isolated T-state PolyhHbs by removing cell-free Hb and low-molecular-weight aggregated Hb, which achieved a lower autoxidation rate than previous generations of commercially polymerized HBOCs . This research has been scaled-up on a pilot scale as shown in Figure , which provides a new idea for further designing cross-linked HBOCs based on the allosteric properties of Hb.…”

“…Stage 2 facilitates the exchange of PolyhHb solution with modified Ringer’s lactate solution excipients, removing LMW Hb aggregates (<500 kDa), unmodified cell-free hHb, and free reactants from the final product. Reprinted with permission from ref . Copyright (2022) Wiley.…”

Silk Fibroin Particles as Carriers in the Development of Hemoglobin‐Based Oxygen Carriers

“…[21] To address these challenges, strategies to increase the molecular diameter of the HBOC and limit free hemoglobin in circulation have become requirements in the design of modern HBOCs. These strategies include chemical cross-linking of hemoglobin, [22,23] polymerization of hemoglobins, [24][25][26] and encapsulation of hemoglobin in liposome and nano-and microparticle systems. [27,28] Liposome encapsulated hemoglobin's have demonstrated many of the desired design criteria for an alternative oxygen carrier, including an absence of blood group antigens and blood-borne pathogens, and a prolonged storage capacity.…”