Purification and identification of PEGlated hemoglobin, a potential blood substitute, by chromatography and capillary electrophoresis

Bovine hemoglobin (Hb) has been chemically modified, by reaction of its lysine residues with the active ester of poly(ethylene glycol) (PEG, M-w = 5000), to produce a potential blood substitute for human therapy. Covalent attachment of PEG chains to the protein produced a heterogeneous mixture of PEGlated Hb (PEG-Hb). It is difficult but necessary to remove unmodified and poorly modified Hb from the mixture. This paper describes the use of cation-exchange chromatography (IEC), in flow-through mode, and size-exclusion chromatography (SEC) for purification of the PEG-Hb mixture. The highly modified Hb flowed through the IEC column in the loading buffer without adsorption by the chromatographic medium. SEC was then used for further purification. These two steps were suitable for pilot-scale preparation or for analytical chromatography. The purified product was assessed by high-performance capillary electrophoresis (HPCE), which was also used to optimize the chromatographic parameters.