Chunyang Zheng

Solid Phase Pegylation of Hemoglobin

A solid phase conjugation process was developed for attachment of polyethylene glycol to hemoglobin molecule. Bovine hemoglobin was loaded onto an ion exchange chromatography column and adsorbed by the solid medium. Succinimidyl carbonate mPEG was introduced in the mobile phase after the adsorption. Pegylation took place between the hemoglobin on the solid phase, and the pegylation reagent in the liquid phase. A further elution was carried out to separate the pegylated and the unpegylated protein. Analysis by HPSEC, SDS-PAGE, and MALLS demonstrated that the fractions eluted from the solid phase contained well-defined components. Pegylated hemoglobin with one PEG chain was obtained with the yield of 75%, in comparison to the yield of 30% in the liquid phase pegylation. The P50 values of the mono-pegylated hemoglobin, prepared with SC-mPEG 5kDa, 10kDa and 20kDa, were 19.97, 20.23 and 20.54 mmHg, which were much closer to the value of red blood cells than that of pegylated hemoglobin prepared with the conventional method.

Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute: Characteristics and effects on rats

Abstract: Conjugate of bovine hemoglobin (bHb) and human serum albumin (HSA) was prepared. The product was simply composed of 89.7% one-to-one Hb-HSA conjugate, 6.0% oligomer of Hb and HSA, 3.5% unconjugated HSA and 0.8% unconjugated Hb, with an average molecular weight of 157 kD. The physicochemical characteristics were determined. Effects of single replacement on blood pressure and long-term survival of rats with 30% and 60% acute blood loss were studied, in comparison with Ringer-lactate solution, stroma-free hemoglobin (SFHb), 5% HSA in Ringer-lactate, whole blood and no resuscitation fluid. Results showed that Hb-HSA conjugate maintained the mean arterial pressure of rats to initial level with no pressor effect. Long-term effects of the replacement fluids on 30% bleeding rats showed that, for the group infused with Hb-HSA conjugate, histology of five major organs, heart, kidney, liver, spleen and lung, were essentially normal, similar to that of whole blood, while obviously renal side-effects appeared in other groups. The efficacy of the conjugate was further demonstrated by the resuscitation of lethal hemorrhagic shock rats (60% acute blood loss) with 100% survival rate (followed for 14 days), the same result as whole blood. The Hb-HSA conjugate can thus be another candidate for blood substitute in emergency.

Polyethylene glycol improves conjugation of bovine hemoglobin and human serum albumin in a controlled ratio.

Direct conjugation of bovine hemoglobin (Hb) and human serum albumin (HSA) with glutaraldehyde would result in a complex mixture of dimers Hb-Hb, Hb-HSA, HSA-HSA and other oligomers. To obtain a high yield of target Hb-HSA, modulation of the reaction environment was carried out. It was found that polyethylene glycol (PEG), a hydrophilic polymer, could improve the yield of Hb-HSA conjugate. The degree of improvement depended on the molecular weight and concentration of PEG. Under optimum condition of 9% (w/v) of PEG 4000, the reaction proceeded in a controlled mode with conversion yield of starting proteins to Hb-HSA increasing from 6% to 30%. The purity was about 88% of the total conjugates. Furthermore, the impurities were mainly tetrameric molecules of two Hb-HSA conjugates. The improvement could be attributed to the “micro-compartment” created by addition of polyethylene glycol, which brings HSA and Hb close together, thus increasing the chance of conjugation between the two molecules.