In a recent study, ultrahigh molecular weight (Mw) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O-2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model. 1 In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G: Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O-2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G: Hb molar ratio. For low O-2 affinity PolybHbs, increasing the G: Hb molar ratio reduced the O-2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O-2 affinity PolybHbs, increasing the G: Hb molar ratio led to increased O-2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O-2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G: Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G: Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G: Hb molar ratio. To preliminarily evaluate the ability of low and high O-2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O-2 transport model was used to simulate O-2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O-2 affinity PolybHbs oxygenated the bioreactor better than high O-2 affinity PolybHbs. This result points to the suitability of low O-2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G: Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1172-1184, 2011