Biophysical and Biochemical Characterization of High Molecular Weight Co-Polymerized Human Hemoglobin and Albumin as a Potential Hemoglobin-Based Oxygen Carrier

Human hemoglobin (hHb) in the tense (T) quaternary state was copolymerized with human serum albumin (HSA) at various hHb:HSA mass fractions to form polymerized hHb-HSA Poly(hHb:HSA) conjugates as a potential next-generation hemoglobin-based oxygen carrier (HBOC). These conjugates were evaluated for molecular weight (M_W), hydrodynamic size, oxygen transport characteristics, heme and oxidative stability, as well as hemorheological and colloid osmotic pressure (COP) properties. Among the variants, Poly(hHb₇₅:HSA₂₅) achieved a high M_W (2024 ± 262 kDa), hydrodynamic diameter (29.6 ± 2.3 nm), yield (39 ± 1%), and batch mass (11.8 ± 0.2 g), closely matching PolyhHb₁₀₀. In comparison, Poly(hHb₅₀:HSA₅₀) exhibited a lower M_W (875 ± 84 kDa) and diameter (21.5 ± 1.9 nm), with a reduced yield (26 ± 4%) and batch mass (7.7 ± 1.3 g). Both formulations demonstrated rapid oxygen offloading (63.1 ± 0.5 and 59.0 ± 1.4 s^-1) and low oxygen affinity (P₅₀ = 49.04 ± 0.95 and 41.79 ± 0.81 mmHg), indicating effective oxygen delivery under moderate oxygen tensions. Although polymerization modestly elevated the auto-oxidation rate compared to the precursor hHb, the oxidative stability remained comparable between Poly(hHb₇₅:HSA₂₅) and Poly(hHb₅₀:HSA₅₀), suggesting that HSA incorporation does not significantly impact the rate of auto-oxidation. Both Poly(hHb₇₅:HSA₂₅) and Poly(hHb₅₀:HSA₅₀) reduced haptoglobin binding (0.005 and 0.004 μM^-1 s^-1) and heme release rates, reflecting enhanced heme retention and reduced oxidative risk. Both Poly(hHb₇₅:HSA₂₅) and Poly(hHb₅₀:HSA₅₀) exhibited similar zeta potentials (-23.2 ± 1.3 mV and -27.0 ± 1.7 mV respectively). Structural analyses confirmed the preserved α-helical content, thermal stability (69.5-70.9 °C), and retained intrinsic catalase activity of the two variants. Hemorheological and COP analyses further revealed that both Poly(hHb₇₅:HSA₂₅) and Poly(hHb₅₀:HSA₅₀) exhibited low COP, were hyperviscous solutions with shear-thinning behavior, and exhibited reversible red blood cell (RBC) aggregation at low shear rates. Therefore, both T-state Poly(hHb₇₅:HSA₂₅) and Poly(hHb₅₀:HSA₅₀) offer an optimal balance of oxygen delivery, oxidative resilience, manufacturability, shear-thinning behavior, making them strong candidates for further HBOC development.