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.