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.
Background and Objectives: Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. Materials and Methods:The TFF RBC washing system was run under hypothermic conditions at 4 C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre-and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis.Results: No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P 50 between the initial and final RBC sample Shuwei Lu and Megan Allyn contributed equally to the work.
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 O carrying capacity of PolyhHb makes it appealing as an O therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc., Evanston, IL) was never approved for clinical use due to the presence of large quantities of low molecular weight polymeric (LMW) 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 (TFF) 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, in order 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 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.
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