US 3864478 A
Method for obtaining a storage-stable, infusionable and hepatitis-safe hemoglobin solution having a stabilized 2,3-diphospho-glycerate level which method comprises A. TREATING A STARTING MATERIAL CONTAINING HUMAN ERYTHROCYTE WITH A DILUTE SOLUTION OF beta -PROPIOLACTONE AT TEMPERATURES BETWEEN 5 DEG AND 15 DEG C. to result in a mixture containing beta -propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter B. TREATING THE MIXTURE WITH A NEUTRAL OR WEAKLY ALKALINE WASHING SOLUTION TO WASH OUT THE EXCESS beta -PROPIOLACTONE AND ITS REACTION PRODUCTS, C. THEN TREATING THE RESULTING HEMOLYSATE, AFTER MIXING THE ERYTHROCYTES IN DISTILLED WATER, WITH A CATION EXCHANGE RESIN IN H<+>-form, until the pH-value has dropped to 5.0 to 5.5, to result in precipitation of a stroma-lipid mass, D. SEPARATING THE HEMOLYSATE FROM THE RESIN AND THE PRECIPITATED STROMA MASS BY CENTRIFUGING, E. OPTIONALLY ADJUSTING THE DESIRED HEMOGLOBIN CONCENTRATION BY DILUTING WITH WATER, F. OPTIONALLY ADDING AGENTS FOR THE CONTROL OF THE ISOTONICITY AND FOR THE STABILIZATION OF PH-value and ferrous protein bound iron content and G. RECOVERING THE HEMOGLOBIN SOLUTION BY STERILE FILTRATION.
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United States Patent [191 Bonhard [451 Feb. 4, 1975 i 1 STORAGE-STABLE I-IEMOGLOBIN SOLUTIONS AND METHOD FOR THEIR PREPARATION Klaus Bonhard, Sandeldamm l6, Hanau, Germany 22 Filed: Oct.3, 1973 21 Appl. No.: 403,141
 Foreign Application Priority Data Primary ExaminerSam Rosen Attorney, Agent, or Firm-Burgess, Dinklage & Sprung  ABSTRACT Method for obtaining a storage-stable, infusionable and hepatitis-safe hemoglobin solution having a stabilized 2,3-diphospho-glycerate level which method comprises a. treating a starting material containing human erythrocyte with a dilute solution of B-propiolactone at temperatures between 5 and 15C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter b. treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
0. then treating the resulting hemolysate, after mixing the erythrocytes in distilled water, with a cation exchange resin in H form, until the pH-value has dropped to 5.0 to 5.5, to result in precipitation of a stroma-lipid mass,
(1. separating the hemolysate from the resin and the precipitated stroma mass by centrifuging,
e. optionally adjusting the desired hemoglobin concentration by diluting with water,
f. optionally adding agents for the control of the isotonicity and for the stabilization of pH-value and ferrous protein bound iron content and g. recovering the hemoglobin solution by sterile filtration.
15 Claims, N0 Drawings STORAGE'STABLE HEMOGLOBIN SOLUTIONS AND METHOD FOR THEIR PREPARATION The invention relates to a method for obtaining hemoglobin solutions, particularly hemoglobin solutions which are storage-stable and infusionable and which are free of potential hepatitis causing infectants or contaminants, and to such compositions per se.
The commercially available. so-called blood substitutes have the following advantages over conserved whole blood:
1. Their application does not require diagnosis of blood groups.
2. They are stable over longer periods of time.
3. They are free of heptatitis-causing contaminants and are thus hepatitis-safe.
The main disadvantage of such blood substitutes, however, is that they cannot replace many special functions of the blood, as for example the oxygen transport of the red blood cells. Particularly for this purpose, isotonic solutions of human hemoglobin have been used for some years in pre-clinical tests, as an alternative to whole blood. Since the decisive factor in causing shock is an insufficient oxygen supply, such preparations have been particularly useful in shock-therapy.
It has already been attempted to use aqueous emulsions of carbon fluoride compounds as oxygen carrier, especially for the perfusion of isolated organs. Since, however, there is no natural way of elimination for this kind of substance, an intravenous infusion thereof into the body is currently not practical, particularly since such foreign substances, not being metabolized, are necessarily stored in the tissue. Contrary thereto, free hemoglobin is capable of passing into the urine, or will be in part resorbed and subjected to natural metabolic processes. However, there is a potential hepatitis danger in connection with the known hemoglobin preparations and this is significant in clinical application, be cause in the case of donated blood as starting material, an infection potentional cannot be excluded.
Methods have already been described as to how hemoglobin solutions adjusted to the blood plasma physiology can be prepared from human erythrocytes with lowering of the potassium content and removal of the stroma-lipid also ocurring in the hemolysis. However, in testing these known hemoglobin solutions, disadvantages have been found which so far have prevented clinical application, particularly since kidney disturbances had been observed. Other known hemoglobin solutions are kidney-compatible, but for their preparation a dialysis method, among others, is being used, whereby the phosphate ester, the 2,3-di-phosphoglycerate, responsible for the physiologically important oxygen affinity, is quantitatively removed.
The present invention provides a method for obtaining hepatitis-safe, storage-stable and infusionable hemoglobin solutions with a stabilized 2,3-diphosphoglycerate level, which thus substantially overcame the disadvantages of known compositions.
Essentially, the inventive method comprises a. treating a starting material containing human erythrocyte with a dilute solution of B-propiolactone at temperatures between 5 and C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter b. treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
0. then treating the resulting hemolysate by mixing the erythrocytes in distilled water with a cation exchange resin in H* form, until the pH-value has dropped to 5.0 to 5.5, to result in precipitation of a stroma-lipid mass,
d. separating the hemolysate from the resin and the precipitated stroma mass by centrifuging,
e. adjusting the desired hemoglobin concentration by diluting with water,
f. adding agents for the control of the isotonicity and for the stabilization of pH-value and iron content and g. recovering the hemoglobin solution by sterile filtration.
By this combination of critical process steps, a product is obtained whose application to humans excludes the danger of hepatitis.
The treatment with the solution of the B-propiolactone (step (a), above) is performed preferably with 6 to 16 g of B-propiolactone in solutions of 1.2 tp 3.2 g/lOO ml per 1.5 liter erythrocyte sediment, and for no longer than 25 minutes. By washing four times on the centrifuge, the content of not yet completely reacted B-propiolactone in the erythrocyte supernatant, is decreased to 20 to 10 mg%, whereby preferably washing solutions are used which contain sodium chloride and- /or sodium bicarbonate. Other suitable washing agents are solutions of tert. sodium citrate, tert. sodium phosphate and sodium carbonate. In the subsequent hemolysis, the remaining B-propiolactone is further diluted to l/50. Only in such a concentration does the B-propiolactone come into direct contact with the hemoglobin and the other components of the cell nucleus, and it can then completely react.
Distilled water is used as a preferred diluent. However, also physiologically suitable electrolyte solutions can be added, for example sodium chloride solutions, glucose solutions or bicarbonate solutions.
It has surprisingly been found that with a method performed in such a manner, particularly the enzyme system responsible for the hydrolysis of the 2,3-diphospho glycerate is clearly inhibited. If in the method of the invention, the step of the B-propiolactone treatment is ommited, after 3 months of storage, the product has about double the content of free phosphate than has the product according to the invention The treatment according to the invention fi-propiolactone results in stabilization of the 2,3-diophospho-glycerate content in hemoglobin solutions at a value of about 0.4 mmole per 0.8 mmole of hemoglobin over a period of time of more than three month, as determined by direct measurements. Without this stabilization, the corresponding 2,3-diphospho-glcerate value fluctuates from charge to charge between 0 and 0.25 mmole.
Solutions of 3.8% of sodium bicarbonate or 1.6% of sodium chloride are particularly suitable solutions for the washing treatment according to the invention. When diluting, particularly the erythrocyte volume is diluted 4.5 times. Particularly suitable cation exchange resins are acid polystyrene-sulfonate cation exchangers, especially the resins commercially sold by the Dow Chemical Company, Midland, Michigan, under the designation Dowex 5O WX, which have an exchange capacity of 4 to 5 milliequivalents per each gram of dry resin. Other suitable exchangers are, for example, Amberlite lR-120 of the Rohm and Haas Company, Philadelphia "Zero-Karb 225" of the Permutit Company Ltd., London, and Lewatit" of Farbenfabriken Bayer, Leverkusen.
The lowering of the potassium content takes place in the method according to the invention preferably by the cation exchange 1t. against 11* and the subsequent centrifuge treatment. Advantageously, there should be present about 6.3 g of hemoglobin per 100 ml. The stabilized pH-value at the end of the process amounts advantageously to 7.4. A preferred agent for the control of isotonicity is glucose, however all other agents conventional for this purpose are also suitable.
In freezing the hemoglobin solution obtained according to the invention and partial re-thawing, a product can be withdrawn which is rich in hemoglobin, which product, by repetition of this process, can be brought up to a 15% hemoglobin content (this corresponds to the total hemoglobin content of blood). The relative viscosity of such a solution of about 1.3 at 37C. is still within the normal range of plasma. In this careful manner, by using sterile bottles and blood transfusion devices, there can be obtained in a closed system a variant with increased active material concentration. The final adjustment of the isotonicity is advantageously carried out only after the initial concentration, so that the obtained V hyperoncotic concentrate, i.e., concentrate whose colloidal osmotic pressure exceeds that of normal plasma is not also hypertonic.
The method according to the invention is illustrated by the following examples. The products obtained in accordance with these examples have already been tested on mammals such as mice, rats, rabbits, dogs, pigs, and humans, and have been found compatible as well as oxygen-transport effective.
The following examples are illustrative of, but not limitative of, the method of the invention.
EXAMPLE 1 Erythrocyte sediments from 6 blood donors, donating 500 ml of blood each, were separated from the plasma, combined, diluted with 1.6% sodium chloride solution to a volume of 2 liters and at C. mixed with 5 g of freshly distilled B-propiolactone for minutes at 10 to 12C. The mixture was subjected for minutes to a cooling centrifugation, the supernatant was sucked off and the erythrocyte sediments were washed with fresh, 1.6% sodium chloride solution four times in succession in proportions of about 1.1 liters, each, on the centrifuge and then placed into 5.2 liters of sterile, demineralized and distilled water. After stirring for 5 minutes, 500 ml of very pure (pA quality) cation exchanger Dowex 50 were added in H -form and the pl-l-value was stopped at 5.0 by addition of normal caustic soda, until stroma-lipid sedimentation became visible. The mixture was then adjusted to a pH value of 5.5 and after 10 minutes centrifuged at 2,000 g.
From about 1 liter of separated sediment (stroma and exchange resin) about 5 liters of 6-7% hemoglobin solution were removed by decantation. After addition of 40 g of glucose, the pH-value was adjusted to 7.4 with caustic soda, the solution was again centrifuged in the above described manner and sterilefiltered with a cellulose-asbestos filter (EKS I of the company Seitz- Werke, Bad Kreuznach, Germany). Also, the following types of filters can be used successfully: Filtrox sterile filters of the Filtrox-Werke AG, St., St. Gallen, Switzerland, filter candles of the company Pall, type Ultipore" (both are cellulose asbestos filters) or cellulose acetate membrane filters, such as Millipore type GSWP ofthe Millipore Corp., Bedford, Mass, Sartorius type SM 1 107 of the Sartorius-Werke Goettingen, Germany, and similar materials.
The oxygen binding curve of a product prepared in accordance with this example showed that after 2 months of storage at 10C., a P 50 value of 19 mm Hg and a pH-value of 7.4. Calculated on an intraerythrocytic pH-value of 7.2, the P 50 value of the hemoglobin of said preparation would be 23 mm Hg. The P 50 value is the oxygen partial pressure under which the hemoglobin preparation is saturated to 50% of its maximum oxygen binding capacity.
EXAMPLE 2 Example 1 was repeated but 16 g of ,B-propiolactone was added to 2 liters of erythrocyte suspension. After centrifugation and sucking off the supernatant, the erythrocyte sediment was washed with about 1.1 liters of each of the following solutions:
1. with a sodium bicarbonate solution with a content of 38 g per liter, 2. repeat of (l), 3. with a solution which contained 38 g of sodium bicarbonate and 16 g of NaCl per liter and mixed in the ratio 1:1,
4. with a sodium chloride solution having a content of 16 g of NaCl per liter.
The sediment was then further processed as in Example 1 through the stroma separation. Prior to the sterile filtration the pH-value was adjusted with n-NaOl-l to 7.0, 33 g per liter of glucose were added, then 2.5 g per liter sodium bicarbonate were added and adjusted with n-NaOH to a final pH-value of 7.4.
EXAMPLE 3 Example 1 was repeated through the stroma separation. Thereafter, the mixture was concentrated to blood-analogous hemoglobin content of 15 g/ ml by way of the following steps: the hemoglobin solution was frozen in portions of 500 ml each, in 1000 ml bottles lying on their side, and thereafter thawed at room temperature with the bottles placed in an upside down position in such a manner that by perforation of the stopper closure with a sterile blood-taking-device the deepred melt could be continuously removed from the remaining white ice stick. A repetition of the process resulted in a solution containing about g/liter of hemoglobin, which was then worked-up in accordance with Example 1 to an infusionable product.
It will be understood that the foregoing specification and examples are illustrative but not limitative of the present invention inasmuch as other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.
What is claimed is:
1. Method for obtaining a hemoglobin solution having a stabilized 2,3-diophospho-glycerate level which method comprises a. treating a starting material containing human erythrocytes with a diluted solution of B-propiolactone at temperatures between 5 and 15C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
c. then mixing the erythocytes with distilled water to form a hemolysate which is treated with a cation exchange resin in H*-form,-until the pH-value has dropped to 5.0 to 5.5, to result in precipitation of a stroma-lipid mass,
d. separating the hemolysate from the resin and the precipitated stroma mass by centrifuging, and e. recovering the hemoglobin solution by sterile filtration.
2. Method as claimed in claim 1, wherein, in step (a) of the method, from 6 to 16 g. of ,B-propiolactone are used in solutions of 1.2 to 3.2 g/lOO ml per 1.5 liter erythrocyte sediment.
3. Method as claimed in claim 1, wherein prior to the sterile filtration of step (e) of the method, the solution present after the stroma separation of step (d) is frozen in stick-shaped form and then thawed in vertical position and the melt rich in hemo-globin is seperated from the ice block.
4. Method as claimed in claim 1, wherein the B-propiolactone treatment of step (a) is performed at 10 to 12C.
5. Method as claimed in claim 1, wherein the washing step (b) for the removal of excess B-propiolactone is repeated at least four times.
6. Method as claimed in claim 1, wherein the washing step (b) is effected with a solution of sodium bicarbonate.
7. Method as claimed in claim 6, wherein said solution is 3.8% sodium bicarbonate.
8. Method as claimed in claim 1, wherein the washing step (b) is effected with a solution of sodium chloride.
9. Method as claimed in claim 8, wherein said solution is l.6sodium chloride.
10. Method as claimed in claim I, wherein the cation exchange resin used in step (c) is a polystyrenesulfonate resin in H-form.
11. Method as claimed in claim 1, including the additional step between steps (d) and (e) of adjusting the isotonicity by adding glucose, sodium acetate or sodium bicarbonate.
12. Method as claimed in claim 1, including the additional step between steps (d) and (e) of adjusting the pl-l-value to about 7.4 with caustic soda.
13. Method as claimed in claim 1, wherein sterile filtration of step (e) is effected by use of celluloseasbestos filters or cellulose acetate discs.
14. A hemoglobin solution having a stabilized 2,3-diphospho-glycerate level, prepared by the method claimed in claim 1.
15. A hemoglobin solution having a stabilized 2,3-diphospho-glycerate level, prepared by diphosphoglycerate method claimed in claim 3.