1 Introduction

Hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) are a class of O2 therapeutics that are currently in development (A. F. Palmer & Intaglietta, 2014). Among the myriad of methods used to produce HBOCs, glutaraldehyde-based protein crosslinking is the most frequently employed due to its low cost. Glutaraldehyde has been used to synthesize commercial polymerized bovine Hb (PolybHb) based HBOCs (Oxyglobin® (Cabrales, Tsai, & Intaglietta, 2008) and Hemopure® (Rice et al., 2008); Biopure Corporation, Cambridge, MA) and polymerized human Hb (PolyhHb) based HBOCs (PolyHeme® (Day, 2003; Sehgal, Gould, Rosen, Sehgal, & Moss, 1984); Northfield Laboratories, Evanston, IL). O-raffinose, another common cross-linking agent, has also been used to synthesize PolyhHb (Hemolink™ (Cheng et al., 2004; Leytin, Mazer, Mody, Garvey, & Freedman, 2003); Hemosol Ltd, Toronto, Canada). Due to the observation of deleterious side-effects in phase III clinical trials, which included vasoconstriction, systemic hypertension and oxidative tissue injury, none of these commercial products are FDA approved for clinical applications in the United States (Moradi, Jahanian-Najafabadi, & Roudkenar, 2016).
Vasoactivity via nitric oxide (NO) scavenging and oxidative tissue injury via tissue deposition of iron was determined to result from extravasation of low molecular weight (MW) polymerized Hb (PolyHb) and tetrameric Hb (α2β2) from the blood vessel lumen into the tissue space (Marret et al., 2004). For example, the aforementioned commercial PolyHbs with an average MW of 150-250 kDa and 1-5% unmodified Hb induced hypertension, stroke, myocardial infarction, renal toxicity, and even death in clinical trials (Gould & Moss, 1996; Levy et al., 2002; Marret et al., 2004; Napolitano, 2009b). The harmful side-effects of these small molecular diameter commercial PolyHbs in clinical trials underscore the need to eliminate these low MW fractions in future generations of PolyHbs. (Meng et al., 2018a). Moving forward, revived interest in PolyHb-based HBOCs as oxygen therapeutics must incorporate the lessons learned from these failed trials. Zhanget al. integrated these lessons into their method of synthesis and purification of PolyHb by polymerizing hHb at higher glutaraldehyde:hHb molar ratios and removing low MW PolyHb species with a high cutoff MW filter (Zhang, Jia, Chen, Cabrales, & Palmer, 2011). The PolyhHb synthesized in that study had an average MW of 1.1 - 18 Mda. Similar to Zhang et al.’s work, Zhou et al. synthesized PolybHb at various glutaradehyde:bHb molar ratios which had an averaged MW of 0.1 – 6.3 MDa (Zhou et al., 2011). The small library of glutaraldehyde polymerized bHb (PolybHb) evaluated by Baek et al. demonstrated a direct correlation between the cross-link density (i.e. glutaraldehyde:Hb molar ratio) and PolybHb MW (Baek et al., 2012). Administration of these PolybHbs in vivo confirmed the vasoactive effects of low MW PolybHbs. Low MW PolybHb also displayed reduced circulatory lifetime, and increased renal tissue deposition (Baek et al., 2012). Therefore, PolybHbs with MW greater than 500 kDa are expected to be less vasoactive and exihibit less tissue toxicity compared to PolybHbs with MW under 500 kDa. Unfortunately, not all PolybHb fractions greater than 500 kDa are equally safe. For PolybHb fractions greater than 500 kDa, there is an inverse relationship between PolybHb MW and vasoactivity at the same concentration (Baek et al., 2012). This inverse ratio also suggests that the polymer distribution has an impact on the toxiciological properties of HBOCs (Baek et al., 2012; Cabrales et al., 2009; Rice et al., 2008).
In this work, we synthesized a library of low O2-affinity tense quarternary state (T-state) PolybHbs and high O2-affinity relaxed quarternary state (R-state) PolybHbs of varying sizes with low batch-to-batch variability. These materials had very low levels of small MW PolybHbs and improved batch-to-batch consistency of biophysical properties via clarification with a 0.2 µm hollow fiber (HF) filter and diafiltration with a 500 kDa HF filter (Cabrales, Zhou, Harris, & Palmer, 2010; A. F. Palmer, Sun, & Harris, 2009). To investigate the nano-structure of PolybHb, transmission electron microscopy (TEM) was performed for the first-time on our material. Furthermore, to optimize the PolybHb synthesis protocol, we conducted a meta-data analysis of the procedural data during both polymerization and TFF purification to evaluate the correlation between procedural parameters and PolybHb biophysical properties. For example, increasing the number of diafiltration cycles resulted in higher final product purity, but produced materials with high methemoglobin (metHb) levels since the longer processing time resulted in increased PolybHb oxidation. In this study, we found the optimal number of diafiltrations (i.e. 14 diacycles), that led to the lowest metHb level, while not significantly affecting overall product yield and purity. Such findings could be used as future guidance to minimize batch-to-batch variances when synthesizing PolyHbs.