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.