Methods are disclosed for separating hemoglobin from erythrocytes by contacting erythrocytes with a hypotonic buffer solution at a rate sufficient to render the release of hemoglobin from said erythrocytes without significant lysis. The hemoglobin is then separated from the erythrocytes. Methods are also disclosed for purifying hemoglobin solutions of DNA, endotoxins and phospholipids by contacting the hemoglobin solutions with an anion exchange medium.
Disclosed is an improved method for hemoglobin purification utilizing a novel two-step dual-aqueous-phase extraction technique to separate hemoglobin from red cell membrane stroma and other protein contaminants. In the first step, a first dual-aqueous-phase liquid system is prepared which comprises an upper aqueous phase containing polyethylene glycol in water and a lower aqueous phase containing a phosphate buffer at a pH of about 10. In the second step, a second dual-aqueous-phase liquid system is prepared which comprises an upper aqueous phase, which contains the polyethylene glycol phase containing the hemoglobin extracted in the first step, and a lower aqueous phase containing a new phosphate buffer at a pH of less than 7.5. After the second extraction step, purified hemoglobin solution can then be obtained by removing the phosphate salt and the minute amounts of polyethylene glycol contained in the lower phase.
Hemoglobin-containing solutions containing polyalkyene oxide-conjugated hemoglobin having a molecular weight greater than about 85,000 daltons and a degree of substitution of at least five polyalkylene oxide conjugates per hemoglobin molecule are described that are not associated with hemoglobinuria in mammals. A method of simultaneously fractionating and purifying polyalkylene oxide-conjugated hemoglobins is also disclosed.
Hemoglobin is purified from a crude solution thereof, to obtain an aqueous solution containing at least 99% of a preselected hemoglobin species, by a two stage displacement chromatography process. One of the stages is conducted under anionic exchange conditions, and the other under cationic exchange conditions. In both stages, 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. Normally, anionic exchange is conducted first, with contaminants more acidic than the hemoglobin displacing the hemoglobin from the column and themselves remaining attached to the column for separation. The cationic exchange process is conducted second, on the eluent from the first column, and in this stage, the more basic contaminants displace the hemoglobin from the column under overload conditions, to yield a substantially pure hemoglobin solution.
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 invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulent to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized. The polymerized hemoglobin solution is then diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavenges oxygen, to form a stable polymerized hemoglobin blood-substitute, which is then packaged and stored in an atmosphere substantially free of oxygen.
