MXPA98002059A - Preparation of pharmaceutical grade hemoglobins by means of heating treatment, in partially oxygen unaform - Google Patents

Abstract

During the purification of chemically modified hemoglobin mixtures of pharmaceutical grade, cross-linked hemoglobin and hemoglobin without crosslinking, they are heated in the presence of non-stoichiometric amounts of oxygen, highlighting in the selective precipitation of unbound hemoglobin. After separation of the tetramers without binding precipitates, the bound hemoglobin remaining in the supernatant is purified, that an additional chromatography procedure is no longer necessary. This hemoglobin is highly crosslinked, absolutely free of chromatography residues and has a low content of metemoglobi

Description

PREPARATION OF PHARMACEUTICAL GRADE HEMOGLOBINS BY MEANS OF HEATING TREATMENT, IN A PARTIALLY OXYGENATED FORM BACKGROUND OF THE INVENTION The use of free hemoglobin in the treatment of a large number of clinical conditions, rather than the requirement of transfusion of all blood, has been proposed for several years. For a historical review, see VJinslow, Hemoglobin-based Red Cell Substitutes, Johns Hopkins U. Press (1992). Many of the obstacles in the use of free hemoglobin have included the toxicity of hemoglobin, up to its dissociation into subunits and a high affinity of clustering of free hemoglobin with oxygen, thus limiting the oxygen release capacity of tissues . Additional obstacles have achieved a preparation of hemoglobin, free of cellular stroma, viral and bacterial pathogens, and endotoxins. The first of these obstacles, referring to unfavorable dissociation, has been largely eliminated, using several methods of tetrameric hemoglobin reticular bonding. This serves for the purpose of physically avoiding, that the tetrameric subunits are dissociated into alpha-beta dimers. U.S. Patent Number 4,061,736 (Bonson, et al.) Describes bound hemoglobin REF: 26931 intramolecularly, in which the crosslinking agent is either a heterocyclic or a aromatic triazine, cycloalkane, halogenated dialdehyde, etc. In U.S. Patent No. 4,826,811 (Sehgal, et al.), Hemoglobin is first pyridoxylated and then c-linked intramolecularly and intermolecularly with glutaraldehyde.

Other reticular bonding strategies combine the objectives of conformational immobilization and molecular stability, without antigenicity, by using binders that have low immunogenicity. For example, U.S. Patent 4,377,512 (Ajisaki, et al.), Discloses the reticular bond, through a polyalkaline oxide linker group. U.S. Patent Number 5,234,903 similarly discovers the conjugation of hemoglobin with a polyalkaline oxide through the urethane linkage. Finally, a strategy in which all the above objectives are developed, in addition to obtaining high yields, using reagents of an effective cost, involves bonding diaspirin, in accordance with US Patents Nos. 4, 598, 064, and 4,600,531. Another obstacle to achieving a therapeutically acceptable hemoglobin is purity. The concept of product purity refers in part to the removal of endogenous contaminants, such as the red blood cell stroma and the non-hemoglobin proteins, which are removed from the hemoglobin solution. Purity also refers to the absence of introduced foreign contaminants, such as viruses, bacteria and endotoxins. A large number of schemes to purify hemoglobin have been developed. The larger cellular components, resulting from cell lysis, are typically removed by filtration. The filter may be diatomaceous earth (See US Patent Number 4,001,200, Bonson) or, more typically, a membrane filter with a small pore size (for example, in US Patent Nos. 4,001,200, 3,991,181 and 4,473,494). Generally, the raw filtration process can be followed by ultrafiltration, that is, through a hemodialysis filter cartridge as set forth in U.S. Patent Nos. 4,598,064 and 4,401,652. Alternatively, large debris and particulate matter can be removed by means of continuous fluid centrifugation. The purification of early stage, has been generally improved and has overcome historical difficulties, in direct proportion to the improvements in the art of filtration, in such a way that the current and conventionally available filtration technology, are suitable for removing particulate matter in most pharmaceutical applications. Further purification to remove non-hemoglobin soluble proteins and other materials is carried out, typically by means of some form of gel filtration or ion exchange chromatography. By selecting an appropriate gel exclusion chromatography procedure, the removal of soluble particles from non-hemoglobin can be affected. For example, U.S. Patent No. 4,136,093 (Bonhard) discloses a method for purifying filtered hemoglobin by passing through a Sephadex G-150 gel filtration column. A higher level of purity, say to reduce endotoxin levels, to pharmaceutically acceptable levels, below 0.5 EU / mL, using a double rechromatography procedure on a Sephadex G-200, optionally followed by an affinity chromatography procedure of haptoglobin (see U.S. Patent Nos. 5,084,558 and 5,296,465). Another solution for purification involves the differential precipitation of proteins, reflecting the observation that in a complex mixture, certain proteins are more stable to stress conditions than others. In heating protein mixtures, it has been found that the individual proteins will denature and will precipitate out of the solution at characteristic temperatures. The removal of the resulting precipitate, comprised of the denatured proteins, results in a partial purification. U.S. Patent No. 4,861,867 discloses the differential inactivation of viruses in a hemoglobin solution, by heating it in the form of deoxyheglobin, at a temperature between 145 and 85 degrees Celsius. The viruses are the labile ones to the heating that the hemoglobin, and the reduction in the volume of concentration of the virus, by means of several registries, is immediately obtained by means of a heating treatment, without the appreciable loss of biologically active hemoglobin. U.S. Patent No. 4,861,867 discloses a heating treatment process in which deoxyhemoglobin is purified from nonhemoglobin proteins, by heating the solution at a temperature between 45 and 86 SC, for varying times of up to ten hours. Since it is important to limit the conversion of hemoglobin to ethemoglobin, deoxygenation is carried out both in the presence of reducing agents, such as ascorbate, and gas release procedures at the electrode, using gas exchange devices. with membrane, to exert an essentially complete deoxygenation. In a typical practice with a heating treatment of 5 hours at 602C, it resulted in a 93% recovery of the total hemoglobin. Another object of the production of blood substitutes based on hemoglobin is the manufacture of material, which has low levels of this protein, in the form of inactive oxidized methemoglobin. This is often elaborated by performing manufacturing operations at low temperatures, since the formation of metemoglobin is substantially accelerated as the temperature is increased. When it is exposed to high temperatures is required, this is during the reaction of hemoglobin with certain modifying agents or during the treatment of heating to inactive viruses, the deoxygenation of the protein can be used, to inhibit the formation of metemoglobin, as it was discovered by U.S. Patent Number 4,861,867. On the basis of the literature pertaining to the oxidation of hemoglobin, a condition that must be avoided is the exposure of hemoglobin to partially oxygenated conditions, since the range of methemoglobin formation is greater, when the hemoglobin is partially saturated of hemoglobin. See Brooks., Proc. Roy. Soc. Lond. Ser. B. 118: 560-51, 1935, which shows that the oxidation range of hemoglobin is the maximum at an oxygen pressure of 20 mm Hg.

BRIEF COMPENDIUM OF THE INVENTION It is an object of the present invention to provide a hemoglobin composition of pharmaceutical quality, which is linked cross-linked, to maintain the optimal oxygen pool affinity and which contains less than 0. 25 EU / mL of endotoxin, in such a way that it does not produce an adverse physiological reaction, that it is substantially free of non-hemoglobin proteins and the unbound hemoglobin is absolutely free of residues of chromatographies or other contaminating polymer species, derived from a matrix of chromatography, which is substantially free of contaminating viruses and has a methemoglobin content of less than 5 percent, at the time of delivery of the product for distribution. It is a further object to provide a method of producing said hemoglobin, comprising the dosing procedures, which do not involve an expensive and troublesome solid phase chromatography system. In the present invention, solutions containing a mixture of bound and unbound hemoglobin are heated between approximately 45 ° C and 85 ° C, for a period ranging from thirty minutes to ten hours in the presence of non-stoichiometric amounts of oxygen, with a pH of 7.25-7.55. Until the non-hemoglobin proteins precipitated, and the bulk of the hemoglobin unbound, the resulting bound hemoglobin, contains less than one percent unbound material. The composition of the present invention is a highly purified, pharmaceutically acceptable bound hemoglobin solution having less than one percent unbound hemoglobin, residual amounts of residual non-hemoglobin proteins (less than 0.01% w / w), less than 0.25 EU / mL of endotoxin, being absolutely free of residues of chromatography, or other residues of procedures by purification systems containing matrices and having a content of metemoglobin of less than 5 percent, at the time of delivery of the product for distribution. In the method of the invention, a mixture of estrous-free, deoxygenated, bound and unbound hemoglobin is placed in an oxygen-impermeable reactor apparatus, partially reacted with oxygen, to obtain from 11 to 28 percent oxyhemoglobin, heated at a temperature between about 45 ° C and 85 ° C, differential or preferably precipitating unbound hemoglobin. Equivalently, the level of partial oxygenation, which can achieve the purification objective, of the present invention can be conveniently measured as parts of a million (ppm) of dissolved oxygen. Accordingly, a pharmaceutical grade hemoglobin can be obtained without the purification process by chromatography, by placing the mixture of hemoglobin bound, stromal-free and deoxygenated, in a waterproof, oxygen reactor apparatus, introducing the oxygen into a content of dissolved oxygen from 0.7 to 1.7 ppm, heating the hemoglobin to a temperature of approximately 45 to 85sec, and removing the non-hemoglobin precipitated and the hemoglobin unbound.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a flow diagram of heating treatment and reticular bonding procedures to produce pharmaceutical grade bound hemoglobin. FIGURE 2 is a rectilinear plot of obtaining bound hemoglobin, as a percentage function of oxyhemoglobin, present during the heating treatment step, of the manufacturing process.

DETAILED DESCRIPTION OF THE PREFERRED MODALITY It has been widely known of free human hemoglobin and some other hemoglobins, detached from the disrupted red cells of the blood, have an affinity to group significantly higher, than their natural counterpart in the red cell. This high affinity of grouping, makes hemoglobin less useful in its function as an oxygen carrier molecule, because of its poor detachment properties in tissues. It was subsequently discovered that the lattice binding, made with certain agents, forces the tetramer of hemoglobin to a conformation in which the affinity of oxygen grouping approaches that of intact red cells. Acceptable levels of P50 (the partial pressure of oxygen, in which it is saturated by half) of the bound hemoglobins of the present invention, are even within 20 and 45 mm Hg. The linked also stabilizes tetrameric hemoglobin, which would otherwise tend to dissociate into dimers. Also, within the scope of this invention, there are crosslinked hemoglobins, which have been further polymerized to produce micromolecules ranging from 120,000 to 600,000 Daltons in molecular weight. The acellular hemoglobin used in the present invention can be of any type, which is stromal free and chemically modified to prevent dissociation into subunits and to increase the affinity to oxygen grouping, in a range of P50 with values of between 20 and 20. and 45 mm Hg, as long as the chemical groups formed are stable upon heating, under the conditions denoted by the following. Modified hemoglobin can be conjugated hemoglobin, cross-linked hemoglobin, polymerized hemoglobin. Several examples of technology in the modification of hemoglobin have been described in the scientific literature, which can be used to overcome the practice of the present invention. For example, see the bulletin contained in Winslow, R.M., Hemoalobin-based Red Cell Substitutes. The John Hopkins U. Press (1992). More specifically, the chemically modified hemoglobin manufacturing methods will be established here, below.

A conjugated hemoglobin is one in which a non-protein macromolecule is covalently linked to hemoglobin. An example is a hemoglobin chemically modified with poly-alkylene glycol, which is described together with a process for its preparation and provided in WO 9107190 (Enzon). An example of poly (alkylene oxide) conjugated hemoglobin and a process for its preparation are described in U.S. Patent Nos. 4,301,144 4,412,989 and 4,670,417, and in Japanese Patent Nos. 59-104,323 and 61-053,223 (from Ajinomoto). Hemoglobin can be conjugated to insulin, in a process discovered by US Pat. No. 4,377,512 (Ajinomoto). The patents WO 91/07190, Patents US Nos. 4,301,144, 4,670,412, 4,377,512 and Japanese Patent Nos. 59-104,323 and 61-053,233, are incorporated herein by reference, a bound hemoglobin contains an intramolecular chemical bond. Examples of linked hemoglobin and methods for their preparation are described in U.S. Patent Nos. 4,001,401 and 4,053,590, which disclose an intramolecular network link between an alpha and beta subunit of a hemoglobin tetramer, using compounds such as halogenated cycloalkanes, diepoxides and diazobenzidines. In the present purification method with heating treatment, a preferred modified hemoglobin is linked with a bis (3, 5-dibromosalicyl) fumarate, to create a cross linkage of fumarate between the two alpha subunits. This bound hemoglobin is mostly described, together with its methods of preparation, in US Patent Nos. 4,598,064, 4,600.31, RE 34,271, omitting the chromatography step. It is preferably manufactured under the conditions found in U.S. Patent No. 5,128,452 (Hai), to avoid linkage between the β chains. US Patent Numbers 4,598,064, 4,600,531, RE 34,271 and 5,128,452 are incorporated herein by reference. WO 90/13309 (Staat Der Nederlanden De Minister Van Defeuric), discovers a method to bind hemoglobin through a ß-ß bond. A polymerized hemoglobin is one in which the crosslinked, intermolecular binding of the hemoglobin tetramers has been used to increase the molecular weight of the modified hemoglobin. The hemoglobins modified by pyridoxal-5'-phosphate to adjust the affinity of oxygen and by the conjugation of polyethylene glycol and the processes for their preparation are described in Japanese Patent Numbers 59-089,629, 59-103,322 and 59-104,323 (Ajinimoto ). U.S. Patent No. 5,248,766 discloses a lattice bond polymerization strategy and a process for covalently interconnecting tetrameric units with oxiranes to form polyhemoglobins with molecular weights of up to 120,000 Daltons. These following Patents, disclose polymerized hemoglobins, U.S. Patent Nos. 5,194,590, 5, 248,766, Japanese Patent Nos. 59-103,322, 59-089,629 and 59-104,323 are incorporated herein by reference. hemoglobin can be modified by in situ directed mutagenesis and expressed in micro organisms or transgenic animals. The Recombinant Mutation, the artificial hemoglobin and its production in cell cultures or fluids, are described in U.S. Patent Number 5,028,588 (Soma t agen). Globinei di-alpha and di-beta type polypeptide (s) used for the production of hemoglobin in bacteria and yeast are described in WO 90 * 13645 (Soma togen). A unnatural multimeric hemoglobin protein is described in WO 93 * 09143 (Somatogen). In general, any method of binding, polymerization, encapsulation or genetic modification, or a combination of these that produce a tetramer, having a P50 in the operating range of 20 to 45 mm Hg, will be effective in the present method. The conditions can be adjusted for each linked tetramer and polymer derived from these, without the need for experimentation. FIGURE 1 is a flow chart for the manufacturing process, involved in the production of pharmaceutical grade Diaspirin linked hemoglobin, and hereafter referred to as "DCLHb ™." Where other hemoglobins in loops can be purified in a similar process, the manufacture of the DCLHb is described herein in detail, in the form of a preferred embodiment. Its state of preference reflects its ease of synthesis and purification in large-scale, commercial quantities and its usefulness as a therapeutic agent in many indications. The red blood cells are deposited, washed, to reduce the level of plasma protein residues, by means of methods such as constant volume diafiltration and then concentrated. The washed cells are disintegrated by lysis in three volumes of a hypotonic buffer. The resulting hemolysate is filtered using a fiber membrane with a pore size of 500 K. to produce a stromal-free hemoglobin solution. Stromal-free hemoglobin is concentrated by ultrafiltration. This solution is filtered through a filter with a pore size of 0.2 microns. The stromal-free hemoglobin is then deoxygenated in the presence of sodium tripolyphosphate (0.01 M.). The oxyhemoglobin content of the solution is reduced to less than three percent. It is important to remove oxygen at a low level, to establish a baseline, because of re-adding oxygen, which is critical for the present process, requires a precise determination. After deoxygenation, hemoglobin is crosslinked with a stoichiometric excess of bis (3,5-dibromosalicyl) fumarate (DBBF).

Up to this point, the oxygen is introduced into the reactor and the solution at a concentration of about 5 to 6 g / dL and thus heated to about 76 for 90 minutes with a pH of 7.4, in a normal practice. The range of temperatures and the duration of the heating treatment, which can be used, is 45se to 85se, for 30 minutes to 10 hours, respectively. Within this range, 65SC to 80SC are preferred and 74SC to 78SC are even more preferred. The hemoglobin concentration can vary from 3g / dL to 20 g / dL. The pH can be varied, from 7.25 to 7.55. In the selection of conditions, within the established ranges, some experimentation will be required to optimize production, while obtaining a high level of purity. For example, if a shorter process is desired in time, the heating treatment should be carried out at a temperature higher than 76se; In spite of this, certain adjustments of concentration or pH will be required. They should not be wrapped in more than a few production practices to obtain the specifications for optimum production, within the established ranges. The oxygen content can be measured according to two parameters. Oxygen is added until the total content of oxyhemoglobin is between about 11 and 28 percent, preferably 13 to 18 percent. Alternatively, the percentage of oxygen dissolved in the hemoglobin solution will be measured. The content of dissolved oxygen should be maintained between approximately 0.7 and 1.7 ppm. FIGURE 1 shows a typical sequence of the steps for linking and applying the heating treatment to hemoglobin. The diagram provides the parameters of the preferred modality. Despite this, the times and temperatures of the heating treatment may vary. In general, the higher the treatment temperature, the less time will be required to complete the process. In spite of this, for any particular modified hemoglobin, the temperature must be maintained below the denaturation temperature, with the desired protein being treated with heat, under these conditions. With respect to this, 85SC can be very high for DCLHb, under these conditions, but acceptable for other derivatives. The control of the process in the step of the heating treatment is critical. Although the construction of specialized equipment, it is not necessary, the integrity of the system with respect to the atmosphere and the control of dissolved gas, should not be compromised. It is essential that the reaction vessels are impervious to gas and precautions should be taken so that no leakage occurs. Precision valves should be used to prevent leakage through the gaskets and to ensure accurate oxygen measurement. The level of dissolved oxygen must be meticulously controlled in the range of 0.7 to 1.7 ppm (11 to 28% oxyhemoglobin) during the heating treatment step. Under these conditions, the level of methemoglobin will be less than one percent, after the heating treatment, and will remain low (<5%) until its packaging and delivery. By maintaining these low levels of methemoglobin during the manufacturing process, it is not statistically probable that any dosage exceeds the 5% limit on the delivery of the product for distribution. It has been determined empirically that unbound hemoglobin is preferentially precipitated, particularly when partially oxygenated. Since the unbound hemoglobin groups oxygen more closely than the bound molecules, it can be expected that the unbound material will preferably be oxygenated. Despite this, the Applicants have no explanation for this, since the unbound hemoglobin comprises more than 30% of the total hemoglobin, the operating range of the oxyhemoglobin content is 11 to 28 percent. The oxygen content, it is so, not stoichiometric and the addition of more oxygen loads to a drastic reduction in production. It was surprising to find that a more highly purified bound hemoglobin preparation can be obtained by partial reoxygenation of the reaction mixtures, before the heating treatment at elevated temperatures, in view of the teachings of Brooks supra. The solution obtained after the heating treatment, after the partial reoxygenation contained less than two percent unbound unbound hemoglobin, but the soluble bound protein was not highly oxidized. This represents a significant improvement in the chemical purity, on the materials resulting from a heating treatment of reaction mixtures, which were completely deoxygenated, while the latter contained enough percentage or higher bound hemoglobin. The result is even more surprising in view of the fact that the optimum amount of oxygen present in the solution, before starting the heating treatment, was insufficient to saturate the unsaturated hemoglobin, which was present. The invention with all this, enables an improved purification of bound hemoglobin, from a reaction mixtures containing substantial amounts of unbound hemoglobin, without increasing the level of methemoglobin in the final bound product. FIGURE 2 shows the effect of varying the oxyhemoglobin content in obtaining the product. As the oxygen content increases, the percentage of binding increases, but if the specified range is exceeded, the production begins to fall. The operable range is from about 11 to 28 percent, preferably from 13 to 18 percent oxyhemoglobin, corresponding to 0.7 to 1.7 ppm of dissolved oxygen.

The heating treatment can be carried out in the temperature range of 45 to 85 e and the temperature can be varied within this range, in the total treatment time, from 30 minutes to 10 hours. There is usually a reciprocal relationship between time and temperature, requiring less time for treatment, at higher temperatures. Heating for 90 minutes to 76 has been determined empirically, since it has been specially adapted for large-scale manufacturing purposes. The heating treatment should be continued for a sufficient time to obtain an optimal precipitation of the unbound hemoglobin, for the particular temperature selected in the range of between 45 and 85SC. Following the heating treatment, the precipitate is removed through a series of conventional filtration step, or by centrifugation. The concentration of DCLHb ™, is facilitated by means of diafiltration (as, for example, against a Mill ipore 30K spiral ultrafilter). The bound hemoglobin produced by the process described above is compositionally distinct and unique. The purity levels achieved make the chromatography procedure unnecessary. This has a profound effect on the cost, since preparation chromatography in the context of manufacturing is quite expensive and produces waste from resins or gels, since the matrix beds are not immediately restructured. Also, the chromatography residues will appear in the final product and if they are not present in the quantities poured into the adulterated product, it can cause the quality control tests to be false or ambiguous, as for example in the LAL test for endotoxin . In addition to the extremely low percentage of unbound hemoglobin and non-hemoglobin proteins, and the absolute absence of chromatography residues, the bound hemoglobin of the present invention has an endotoxin level of less than 0.25 EU / mL, as measured by the test described in the ÜSP, chapter 85, and a content of methemoglobin of hands of 5 percent at the time of delivery of the final product. In the manufacturing process, endotoxin sources are strictly excluded, since ultrapure water and reagents are used. The equipment is pharmaceutically energetic in its design and is designed to allow internal cleaning. The design specifications for such facility will be known to those skilled in the art of pharmaceutical engineering. Further advantages of the present invention will be visible from the following example.

EXAMPLE The heating treatment process will be carried out on previously obtained reaction mixtures, by means of reacting DBBF with stroma-free human hemoglobin, under varying conditions. Table 1 shows the effect in obtaining and percentage of bound hemoglobin in the final product of varying amounts of oxygen, present during the heating treatment. In all situations, the stromal-free hemoglobin was vigorously deoxygenated before being bound and then reoxygenated to different degrees before the heating treatment. The results showed that at oxyhemoglobin levels greater than 28 percent, production falls from the 77 to 83% range to only 59%, with only a marginal increase in the purity of the final product, with respect to hemoglobin without binding residual (99.6% to 99.9%). Taking away from these data, it is clear that the introduction of oxygen to achieve between about 11 and 18 percent oxyhemoglobin, it is desirable to reach the maximum levels of production with a high purity and keep the levels of methemoglobin low.

TABLE I Effects of the Warming Treatment on the Presence of Certain Oxygen Before Heating Treatment Oxygen ppm (O2) * 0.08 0.2 0.8 101 1.6 -% Hemoglobin (g / dL) 1.6 4.0 7.3 11.1 18.2 28.9 Total Hemoglobin 6.3 6.4 6.3 6.3 6.4 4.6 (g / dL)% of Metemoglobin 0 0 0 or 0.4 0.5 pH at 372C 7.35 7.40 7.38 7.47 7.35 7.48 % Linkage 72 72 72 72 72 73 During the Warming Treatment Time Curve 64 66 64 62 66 65 Temperature (mi n) pH at 762C 6.81 6.89 6.85 6. 89 6.85 Cooling Time 64 67 64 60 65 70 (min.) After the Warming Treatment% of Recovered Volume 76 74 75 72 77 82% of Metemoglobin 2.2 3.1 3.5 2.4 0.2 2.0% Obtaining (DCLHb 83 80 83 82 77 59 recovered) Link% 95.4 96.4 98.8 99.4 99.6 99.9 Total hemoglobin 5.2 5.2 5.1 5.2 4.6 2.4 (g / dL) 'Accurate reading not available A. The "in-process" method for determining the percentage of binding in the unheated reaction mixtures was determined using a B10-S1I TSK 250 column and a 1 M MgCl2 buffer in BisTris, pH 7.2, in the form of a mobile phase.

B. The percentage of obtaining was calculated as follows:% of Obtained = THb Final x% Link x% Recovered Volume Initial THb x 72 100 C. The percentage of binding in the heat-treated solution was determined using a Superóse ™ 12 column and a 0.75 M MgCl 2 buffer in BisTris, pH 6.5, in the form of a mobile phase.

Table 2A and 2B compare the values of methemoglobin, at the time of delivery of the product before the passage of micro oxygenation was implemented in the process (2A) with the corresponding values, after the implementation of micro oxygenation (2B) The average value before micro oxygenation was 4.9 percent compared to only 1.6 percent, after the implementation of micro oxygenation.

TABLE 2A METEMOGLOBIN VALUES FROM PRODUCTION PRACTICES, BEFORE OF THE INTRODUCTION OF MICRO OXYGENATION d = not detected For the latter, note that the consistency of the values below 1 percent, for the ste coarse dosages and less than 5 percent for all dosages delivered. Statistically there is a 95% reliability, that 99% of all manufactured dosages according to the present process, will have delivery values of metemoglobin less than the target of the percent. a level of less than one percent methemoglobin immediately after the heating treatment is a strong indicator (though not necessarily a causal one) that the level of methemoglobin will make up less than 5% of the standard product delivery. TABLE 2B VALUES OF METEMOGLOBIN FROM PRODUCTION PRACTICES, AFTER THE INTRODUCTION OF MICRO OXYGENATION It is noted that, with regard to this date, the best method known by the requested, to carry out the present invention, is that which is clear from the present, discovg the invention. Having described the invention as above, the content of the following is claimed as property.

Claims (21)

1. A method for preparing a pharmaceutical-grade cross-linked hemoglobin is characterized in that it comprises: reacting oxygen with a hemoglobin mixture, comprising chemically modified hemoglobin and hemoglobin, without chemically modifying, to form a reaction mixture, comprising from about 11 to 28 % oxyhemoglobin; heating the reaction mixture, to precipitate chemically modified hemoglobin; and remove the hemoglobin without chemical modification, precipitated.

2. A method for preparing a cross-linked hemoglobin of pharmaceutical grade, is characterized in that it comprises: adding oxygen to a hemoglobin mixture, comprising chemically modified hemoglobin and chemically modified hemoglobin, to form a reaction mixture, having a content of dissolved oxygen from 0.7 to 1.7 ppm; heating the reaction mixture, to precipitate chemically modified hemoglobin; and remove the hemoglobin without chemical modification, precipitated.

3. A method according to claim 1 or 2 is characterized in that the chemically modified hemoglobin is predominantly deoxygenated hemoglobin.

4. A method according to claim 3 is characterized in that the hemoglobin mixture has less than 3% oxyhemoglobin.

5. A method according to claim 1, 2, 3 or 4 is characterized in that the chemically modified hemoglobin is stromal free.

6. A method according to claim 5 is characterized in that the chemically modified, stromal-free hemoglobin is bound, polymerized or conjugated and is stable upon heating of the reaction mixture.

7. A method according to claim 6 is characterized in that chemically modified hemoglobin, stromal free, is hemoglobin crosslinked by diaspirin.

8. A method according to claim 1, 2, 3, 4, 5, 6 or 7, is characterized in that the chemically modified hemoglobin is differentially precipitated.

9. A method according to claim 1, 2, 3, 4, 5, 6 or 7, is characterized in that the chemically modified hemoglobin is preferably precipitated.

10. A method according to claim 1, 2, 3, 4, 5, 6, 7, 8 or 9, is characterized in that the reaction mixture is heated to a temperature between about 45 e to about 85se.

11. A method according to claim 10 is characterized in that the reaction mixture is heated to a temperature of between 65SC and 80SC.

12. The method according to claim 11 is characterized in that the reaction mixture is heated to a temperature of between 74SC and 78SC.

13. A method according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, is characterized in that the reaction mixture is heated for a time of 30 minutes to 10 minutes. hours.

14. A method according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, is characterized in that the reaction mixture has a pH of 7.25 to 7.55 and a hemoglobin concentration of 3 to 20 g / dL.

15. A chemically modified hemoglobin solution is characterized in that it contains less than 5% methemoglobin, less than 0.25 endotoxin units, per ml. of endotoxin, and being free of chromatography residues.

16. A hemoglobin solution according to claim 15 is characterized in that the chemically modified hemoglobin solution is comprised of cross-linked, polymerized or conjugated hemoglobin.

17. A hemoglobin solution according to claim 16, characterized in that the chemically modified hemoglobin is bound hemoglobin.

18. A hemoglobin solution according to claim 17, characterized in that the bound hemoglobin, is hemoglobin linked by diaspirin.

19. A hemoglobin solution according to claim 17, characterized in that it has an unbonded tetrameric content of less than two percent.

20. A hemoglobin solution according to claim 15, 16, 17, 18 or 19, is characterized by being stromal-free and substantially free of non-hemoglobin proteins and viral contamination.

21. A hemoglobin solution according to claim 15, 16, 17, 18, 19 or 20, is characterized by having a p50 of from about 20 to about 45 m Hg.