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 (MW), 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 MW (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 MW (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.