4 Conclusion

In this study, high MW R-state and T-state PolybHb were synthesized with negligible amounts of free Hb and low MW polymer order species (i.e. 1st order polymer, 128 kDa) in the final product, which is a significant improvement compared to previously failed commercial PolyHbs. Although the 35:1 T-state PolybHb and 30:1 R-state PolybHb had similar MW, the polymer order composition was found to be different. 35:1 T-state PolybHb was found to contain a higher fraction of 2nd and 5th order polymers than 30:1 R-state PolybHb. By using a 0.2 µm HF module for product clarification and sterile filtration, we were able to sterile filter the PolybHb solutions and remove ultrahigh MW polymer order species which might induce macrophage uptake in the reticuloendothelial system. For ligand-binding kinetics, T-state PolybHb exhibited significantly higher Hp-PolybHb binding rate constant and O2 offloading rate constant compared to R-state PolybHb. MALDI analysis indicated that R-state PolybHbs contain more β-β crosslinked polymers compared to T-state PolybHb.
Furthermore, for synthesis and TFF optimization, our results show that the metHb level in the final product of T-state PolybHb was sensitive to the temperature during sodium dithionite addition. During TFF of the final product, 14 difiltration cycles was identified as being optimal, since it resulted in the lowest PolybHb metHb level. Taken together, these results provide comprehensive guidance for synthesis and purification of PolybHbs as a HBOC.