Purification of hemoglobin by displacement chromatography

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A preselected hemoglobin species is separated from contaminants having a different acidity from that of the preselected hemoglobin species, by an overload displacement chromatography process. To remove more acidic contaminants, the process is conducted under anion exchange conditions. To remove more basic contaminants, the process is conducted under cation exchange conditions. In either case, the exchange column is overloaded to displace the hemoglobin species therefrom with contaminants having greater affinity for the column, and using the impure hemoglobin solution as the displacer.

The development of hemoglobin based blood substitutes continues to command commercial attention, and recent developments have shown that hemoglobin from mammalian blood cells, after suitable modification such as intramolecular crosslinking and in some instances polymerization, shows great promise as the basis of a blood substitute. As development has proceeded, however, the requirements for purity of the hemoglobin have steadily increased. At one time it was believed that hemoglobin simply needed to be stroma free, a condition achieved by washing and gentle lysing of the red blood cells, followed by filtration of the lysate. Subsequently, it was found that the presence of trace residues of impurities such as phospholipids led to more specific reactions, e.g. vasoconstriction, to the product in animal trials. Even after the product has been subjected to several diafiltration steps, it still contains unacceptably high traces of potentially harmful impurities such as erythrocyte enzymes, modified and variant forms of hemoglobin, phospholipids and surface antigens.

A hemoglobin-based blood substitute needs to be based either on a single hemoglobin species, or, if more than one species is present, a carefully controlled composition of known hemoglobin species. Accordingly, a successful hemoglobin protein purification process needs to be capable of separating one hemoglobin form from another, as well as separating the desired hemoglobin form from contaminating red blood cell such components such as erythrocyte enzymes, proteins, phospholipids and antigens.

Chemical crosslinking of hemoglobin for the preparation of the basis of a blood substitute commonly produces a mixture of hemoglobin species. These should subsequently be separated. Since they are in many cases of almost identical molecular weight and chemical composition, their separation presents difficulties.

Art example of a crosslinking reagent which produces a mixture of hemoglobin species, some crosslinked between certain pairs of positions on globin chains and others between other such positions, along with modified, uncrosslinked material and unchanged starting material, is the trig acyl (methyl phosphate) ester of 1,3,5-benzenetricarboxylic acid (TMMP), as disclosed in U.S. Pat. No. 5,250,665 Kluger et. al., issued Oct. 5, 1993.

Chromatographic methods have been applied to the protein purification of hemoglobin solutions. U.S. Pat. No. 4,925,474 Hsia et. al. describes the application of the techniques of affinity chromatography to hemoglobin purification, using columns in which a ligand showing preferential chemical binding affinity to the DPG site of hemoglobin was bound to the stationary phase of the column.

Ion exchange chromatographic techniques have also been applied to hemoglobin purification. The basic principles of the techniques of ion exchange chromatography are well known. A mixture of different species in a solution is applied to a suitably prepared ion exchange column. Each of the species in the mixture has a different affinity for the chemical reactant groups on the column. By varying the conditions on the column, e.g. the pH of the solution, the individual species can be arranged to bind or to elute from the column selectively, so as to separate one species individually from the mixture. The application of the technique to the protein purification of proteins such as hemoglobin is economically unattractive, except when used for small scale operations and analytical work. When hemoglobin is to be purified on a manufacturing scale, for use for example as an oxygen carrying resuscitative fluid (blood substitute), the technique, as conventionally applied, is impractical. The amounts of hemoglobin to be absorbed on and subsequently eluted from a chromatography column are so large that the column size requirements become impractically large and expensive.