A novel method for the preparation of haemoglobin-based oxygen carriers via near-complete polymerization under static conditions

The clinical development of haemoglobin-based oxygen carriers (HBOCs) is hindered by residual haemoglobin (Hb) from incomplete polymerization, which causes vasoconstriction and hypertension, while its removal requires complex and costly purification. To overcome this, we developed a static polymerization strategy for synthesizing near-completely polymerized haemoglobin (PolyHb) using Hb purified from porcine blood. Our innovative methodology integrates three critical process enhancements: (1) elevating solution viscosity to 13 cP to limit macromolecular mobility, (2) reducing reaction temperature to 4 °C to decelerate cross-linking kinetics and (3) eliminating mechanical agitation to eradicate forced convection. Combined with stepwise addition of low-concentration glutaraldehyde (0.04 mol/L), pH adjustment to 8.0, and optimized Hb concentration (5 mg/mL) and Hb:GA ratio (1:300), this strategy achieves >99% monomer conversion, a homogeneous particle size distribution (PDI = 0.163), and ∼96% product recovery after streamlined purification. In animal trials, PolyHb showed exceptional hemodynamic compatibility, inducing negligible blood pressure fluctuations. In haemorrhagic shock models, it outperformed 6% HES (hydroxyethyl starch) and matched whole blood in restoring hemodynamic stability and metabolic homeostasis. This approach markedly improves product yield and narrows molecular weight distribution for the prepared HBOCs compared to conventional processes, while eliminating size-based fractionation and requiring only diafiltration/ultrafiltration for buffer exchange and concentration, thereby reducing processing time and improving batch-to-batch consistency.