Controlled Polymerization and Ultrafiltration Increase the Consistency of Polymerized Hemoglobin for Use as an Oxygen Carrier

Abstract

Polymerized human hemoglobins (PolyhHbs) are a promising class of red blood cell substitute for use in transfusion medicine. Unfortunately, the application of the commonly used glutaraldehyde cross-linking chemistry to synthesize these materials results in a complex mixture of PolyhHb molecules with highly varied batch-to-batch consistency. We implemented a controlled method of gas exchange and reagent addition that results in a homogeneous PolyhHb product. A fully coupled tangential flow filtration system was used to purify and concentrate the synthesized PolyhHb molecules. This improved method of PolyhHb production could be used to more precisely control the size and reduce the polydispersity of PolyhHb molecules, with minimal effects on the resulting oxygen-carrying capability. In addition to these factors, we assessed how the hemoglobin scavenging protein haptoglobin (Hp) would interact with PolyhHb molecules of varying sizes and quarternary states. Our results indicated that T-state PolyhHbs may be more efficiently detoxified by Hp compared with R-state PolyhHb and unmodified Hb.

Polymerized human hemoglobin facilitated modulation of tumor oxygenation is dependent on tumor oxygenation status and oxygen affinity of the hemoglobin-based oxygen carrier

“…Human Hb (hHb) used in these studies was first purified from human red blood cells (RBCs) as described previously 48 . PolyhHb was produced using methods described previously 17 . In brief, the resulting hHb solution was polymerized with glutaraldehyde while fully oxygenated or deoxygenated to form either R-state or T-state PolyhHb, respectively.…”

“…The hHb/ PolyhHb kinetics of O 2 offloading ( k off ,O 2 ) were measured with an Applied Photophysics SF-17 microvolume stopped-flow spectrophotometer (Applied Photophysics Ltd., Surrey, United Kingdom) using protocols previously described by Rameez and Palmer [51][52][53] . The MW distribution was estimated using an Acclaim SEC-1000 column (Thermo Scientific, Waltham, MA) on a Thermo Scientific Dionex Ultimate UHPLC system using previously described methods 17,54 . Dorsal chamber window model.…”

“…The elevated renal toxicity and hypertension associated with previous generations of commercial HBOCs have mainly been attributed to this delay in development. Current improvements in reactor design and product purification are now able to exclude the low molecular weight (MW) HBOC fractions (< 250 kDa ) that contributed to the toxicity of early generation HBOCs [15][16][17][18] .…”

“…The chemical modifications that are necessary to make HBOCs safe for infusion typically alter the HBOC O 2 affinity 16,17,[19][20][21] . For example, conjugating polyethylene glycol to the surface of hemoglobin (Hb) results in an increased O 2 affinity (P 50 : 5-6 mm Hg) compared to unmodified Hb 22,23 .…”

“…Alternatively, commercial polymerized Hbs (PolyHbs) prepared via glutaraldehyde or O-raffinose crosslinking in the tense (T) quaternary state all have significantly lower O 2 affinity (P 50 : 30-40 mm Hg) compared to unmodified Hb [24][25][26] . Recently, we demonstrated that the O 2 affinity of PolyHbs could be controlled by polymerizing the Hb under fully oxygenated or deoxygenated conditions 16,17,21 . By fully oxygenating the Hb during polymerization, the PolyHb is effectively locked into the high O 2 affinity, relaxed quaternary state (R-state).…”