WO1997033914A1

WO1997033914A1 - Methods for producing hemoglobin preparations and preparations obtainable thereby

Info

Publication number: WO1997033914A1
Application number: PCT/NL1997/000134
Authority: WO
Inventors: Eric Jeroen La Hey; Joachim Cornelis Bakker; Willem Karel Bleeker
Original assignee: Staat Der Nederlanden, De Minister Van Defensie
Priority date: 1996-03-15
Filing date: 1997-03-17
Publication date: 1997-09-18
Also published as: AU1946397A

Abstract

This invention relates to a glutaraldehyde cross-linked and polymerized hemoglobin (polyHbXl), which possesses the property of reversibly binding gaseous ligands such as oxygen and is useful for transporting and supplying oxygen to vital tissues and organs. This product is produced in such a way that administration of the modified hemoglobin solution prevents toxic hemorrhagic disorders which normally occur with a glutaraldehyde polymerized hemoglobin solution.

Description

Title: Methods for producing hemoglobin preparations and preparations obtainable thereby

Summary of the invention

This invention relates to a cross-linked and polymerized hemoglobin, which possesses the property of reversibly binding gaseous ligands such as oxygen and is useful for transporting and supplying oxygen to vital tissues and organs. This product should be produced in such a way that administration of the modified hemoglobin solution prevents toxic side effects which may occur with a polymerized hemoglobin solution. In routine preclinical safety studies these toxic side effects can easily remain undetected.

Background of the invention

For many years research has been carried out on modified hemoglobin solutions, which can serve as oxygen-carrying plasma expanders. The advantages over erythrocytes or blood is that the solutions are universally applicable without typing blood groups and that the hemoglobin solutions have much longer storage lives. However, the hemoglobin solutions must be modified in such a way that the three main problems of such solutions are minimized: the presence of residues of membrane fragments (originating from erythrocytes), the high intrinsic oxygen affinity outside the environment of the erythrocyte and the short retention time in the circulation because dissociated hemoglobin disappears rapidly from the vascular system because of leakage through the kidneys (2-4 hour plasma half-life) .

By intramolecular cross-linking stroma-free hemoglobin, the dissociation into 1/2- monomers (32 kD) can be avoided and the vascular retention time can further be prolonged by intermolecularly cross-linking, i.e. polymerization. Intra- molecularly cross-linking results into monomers (64 kD), inter-molecularly cross-linking results into dimers (128 kD) , oligo ers (128 - 500 kD) and polymers (> 500 kD) . A problem of these modifications of hemoglobin is that oxygen affinity substantially increases so that the oxygen release in vivo to the tissues can not be optimal. To solve this problem, many researchers coupled bis-pyridoxal phosphates (PLP, Bonhard et al., 1979, PLP, Seghal et al., 1986, NFPLP, Berbers et al. , 1991) to the hemoglobin because of the similarity of these compounds with 2,3 diphosphoglycerate. Many different cross-linkers have been investigated in the past. Some of them, e.g. bis(3,5-dibromosalicyl)fumarate (Walder et al. , 1979) and diisothiocyanatobenzenesulfonate (Manning et al., 1991) cross-link the α-chains, some, e.g. 2- nor-2-formylpyridoxal-5-phosphate (NFPLP, van der Plas, 1986), o-raffinose (Hsia, 1989) the β-chains, some both the α- and β- chains, like glycoaldehyde (Manning and Manning, 1988). Most of these compounds react significantly with the lysines 82 or 99 and the terminal valine groups. After intramolecularly cross-linking with one of the above mentioned cross-linkers, glutaraldehyde is widely used as the inter- molecular/polymerizing agent (Keipert and Chang, 1983, Seghal et al., 1983, Marini et al. , 1990, Berbers et al. , 1991, Nelson et al. , 1992) .

All the cross-linkers contain aldehyde groups which mainly react with the primary -NH2 groups of lysines. In all cases described in literature the intra-molecular cross- linking is reduced with sodium(cyano)borohydride or dimethylamineborane. The inter-molecular cross-linking is reduced either with sodium(c ano)borohydride or dimethylamineborane (Hsia, 1987, Berbers et al., 1991) or quenched with an excess of primary amino acids, i.e. glycine, lysine, serine, threonine, alanine etc. (Bonsen et al., 1977, Seghal et al., 1983, Keipert and Chang, 1984). It has been observed that these latter mentioned products are not stable regarding the polymer content. The product may continue to polymerize (Marini et al. , 1990, Berbers et al. , 1991) or just depolymerize (Nelson et al., 1992) depending upon the concentration of the quenching amino acid.

The invention thus provides a method for producing a polymerized hemoglobin preparation essentially free of the risk of causing hemorrhagic disorders, comprising intra¬ molecular cross-linking and intermolecular cross-linking of a deoxyhemoglobin using glutaraldehyde or a functional equivalent thereof, whereby the cross-linked material is treated with a blocking agent and a fixating agent. Functional equivalents of glutaraldehyde are molecules such as

0 0 w // C-(CH₂)n-C

/ \ H H

whereby n ⁼ 1~6 (possibly including side-chains) (including of course glutaraldehyde) .

For the purposes of this invention hemoglobin may include any mammalian hemoglobin or derivatives thereof.

The reaction between an aldehyde and a blocking agent such as an amine may form reversible bond, such as a Schiff- base, thus an aldehyde can be formed again. Therefore all products described in literature by this procedure are instable and physiologically not acceptable. Addition of a fixating agent such as sodiu (cyano)borohydride or functional equivalents thereof such as dimethylamineborane reduces the Schiff-base to a stable secondary amine and therefore a stable product can be obtained.

A major advantage of glutaraldehyde as a cross-linker is that it is a rather simple and small molecule and that it is easy to obtain and therefore cheap. In contrast to what is sometimes written in literature about glutaraldehyde

(MacDonald, 1994), it is possible to cross-link hemoglobin intra- and intermolecularly with only glutaraldehyde, which will be described herein. The resulting product is called polyHbXl. Large amounts (> 95%) of intra-molecularly cross- linked hemoglobin are obtained quite easily and reproducibly with varying polymer content, depending on the final product specifications (viscosity, osmolality etc.). The oxygen affinity of such a product can be manipulated by the presence of oxygen during polymerization. This was firstly described by Bonssen et al. (1977). Polymerization of deoxy-hemoglobin produces a product with lowered oxygen affinity. Compared to other procedures to make modified hemoglobin solutions, where separate special intra-molecular cross-linkers are needed, the procedure to make polyHbXl is clearly the cheapest.

In literature toxic effects of glutaraldehyde are only described about skin contact, inhalation and oral intake. However, hardly anything is known about the toxic effect after intravenous administration of pure glutaraldehyde and almost nothing about glutaraldehyde coupled to a protein. Discrepancy in literature existed about toxic effects of glutaraldehyde polymerized hemoglobin (Feola et al., 1989, Nelson et al., 1992) .

However, Bleeker et al. (1996) clearly observed in three animal models (rats, rabbits and rhesus monkeys) transient hemorrhagic disorders after administration of glutaraldehyde polymerized hemoglobin. This side effect was further analyzed in a histopathological study with rats. Hemorrhagic lesions were observed in several tissues (with predilection for the intestinal wall), two days after administration of glutaraldehyde polymerized hemoglobin and the lesions were resolved after one week. Microscopically, the bleedings were characterized as small vessel vasculitis" with a neutrophil infiltration. Platelet numbers, bleeding time and (A)PTT values were normal, which lead to the hypothesis that endothelial cell injury played a central role in the pathophysiology. By comparing different modified hemoglobin preparations they concluded that the toxic factor was the result of the cross-linking procedure with glutaraldehyde. Moreover, Murray et al. (1995) showed that side effects of recombinant hemoglobin preferably manifest themselves in the esophagus, indicating that the study of Bleeker et al. was extremely important for in vivo characteristics of modified hemoglobin solutions.

First Bleeker et al. tested pasteurized stroma-free hemoglobin (SFHb) . Stroma-free hemoglobin was obtained from red blood cells, which undergo leukocyte filtration, centrifugation (to remove the plasma), washing with 0.9% NaCl, lysis in a 100 mM PBS buffer at 2-8 °C, deoxygenation, pasteurization (10 hours at 60.5 °C) and filtration (in succession 20, 0.65, 0.45 and 0.22 mm filter). The SFHb caused abnormalities in the kidneys (severe black discoloration) but no hemorrhagic disorders. Thus disorders were not caused by impurities of the basic intermediate. The cross-linked SFHb with glutaraldehyde, however, caused severe hemorrhagic disorders. This SFHb was cross-linked was a 30-fold molar excess glutaraldehyde and reduced with a 3-fold molar excess sodiumborohydride. After stabilizing the product was dialyzed against Ringer lactate to remove all the excess glutaraldehyde and sodiumborohydride. The product contained an average of 30% polymers (> 500 kD), 30% oligomers (128-500 kD), 16% dimers (128 kD), 24% monomers (64 kD + 32 kD) . The total percentage dissociable monomers was only ± 2%. This polyHbXl-like product was fractionated into a product (HMW-polyHb) containing a polymer fraction of > 70% (> 500 kD) and a product (LMW- polyHb) containing a polymer fraction of < 1% (> 500 kD) . Both fractions induced hemorrhagic lesions, but there was a remarkable difference in localization. After administration of HMW-polyHb the lesions were mainly localized in the small intestine and mesenterial lymph nodes, whereas after administration of the LMW-polyHb no intestinal hemorrhage was observed. Thus the polymer size seemed to have some influence on the toxic effect, but it was not the decisive factor. Also a directly made polymerized product with ± 1% polymers (> 500 kD), 21% oligomers (128 - 500 kD) , 26% dimers (128 kD) and 52% monomers (32 + 64 kD) showed the same result as the LMW-polyHb product. The amount of dissociable monomers of this product was ± 5%. The product was polymerized with a 18-fold excess glutaraldehyde. Also a polymerized σyanomethemoglobin was tested. This product was made by incubation of polyHb with a 20% excess of sodiumferricyanide and sodiumcyanide. This reaction gives an almost complete conversion of hemoglobin to methemoglobi . The product will both prevent oxygen transport but also for example binding of NO. Also this cyanopolymethemoglobin showed hemorrhagic lesions. Finally a human albumin product, polymerized according to the procedure for polyHb, was tested. This product contained 33% polymers, 19% oligomers, 24% dimers and 25% monomers (69 kD), thus having the same composition as polyHb. But poly-albumin also induced hemorrhagic lesions in the skin and the thy us, indicating that the hemorrhagic lesions were not inherent to polymerized hemoglobin solutions. From these experiments Bleeker et al. concluded the following: - glutaraldehyde-polymerized hemoglobin may cause transient hemorrhagic disorders, since the observed disorders were usually mild and since these lesions became manifest after more than one day and were resolved in about one week they might easily remain undetected in routine preclinical safety studies.

Especially this latter statement indicates that other glutaraldehyde modified hemoglobin solutions in the market might have these toxic side effects, but can easily be remained undetected. In practice, no known modified hemoglobin solution has been successful in totally avoiding toxicity problems. It is an object of this invention to provide a glutaraldehyde modified hemoglobin (polyHbXl) which can serve as an oxygen- carrying plasma expander and prevents toxic side effects after administration. Detailed description of the invention

The present invention concerns a glutaraldehyde polymerized hemoglobin solution which prevents toxic side effects such as hemorrhagic disorders after administration. It was found that toxic side effects only can be prevented if hemoglobin is polymerized with glutaraldehyde under special conditions.

For purposes of this invention, the term intra- molecularly cross-linking means the chemical covalent binding of molecular bridges between two 1/2-monomers (32 kD) . The term inter-molecularly cross-linking means the covalent binding of a polymerizing agent, such as glutaraldehyde, between two hemoglobin molecules (64 kD) . Intra-vascular half- life is the period of time in which the initial amount of modified hemoglobin in and in vivo environment falls to half of its initial value. The term P-50 represents the partial pressure of oxygen (pθ2) at a 50% saturation of hemoglobin. The interaction between oxygen and hemoglobin is frequently represented as an oxygen dissociation curve with the percent saturation of hemoglobin plotted on the ordinate axis and the partial pressure of oxygen in Pa (or mm Hg) plotted on the abcissa.

The product if this invention is free from stroma and virally inactivated and is physiologically acceptable as well as therapeutically and clinically useful. The product is free from microbial and viral antigens and pathogens. Most importantly, it is free from viruses that can cause hepatitis or AIDS. The product has reversible oxygen binding capacities which are necessary for oxygen transport properties, i.e. the oxygen dissociation curve is quite similar to that of whole blood, i.e. a P-50 in the range of 25 - 35 mm Hg, see figure 1. The product has a viscosity between 1 and 1.8 cP, see figure 2, less than 10% methemoglobin, physiologic levels of sodium chloride and potassium chloride, less than 1 nanomole of phopholipid per milliliter and is free of endotoxine, see table 1. 8

The product of this invention is unique since it is both intra- and intermolecularly cross-linked with only glutaraldehyde under deoxy-conditions. The product of this invention is further unique since it prevents toxic side effects such as hemorrhagic, which normally may occur after administration of glutaraldehyde polymerized hemoglobin solution. In a preferred embodiment the glutaraldehyde polymerization and thus the free aldehyde groups are quenched with an excess glycine and reduced with sodiumborohydride or dimethylamineborane. This combination and sequence is very efficient for preventing toxic side effects. All prior art procedures to make a modified glutaraldehyde-polymerized hemoglobin will lead to toxic effects or to unstable products. The costs for making a product according to the invention are obviously the lowest in comparison with all other modified hemoglobin solutions in this field.

The preferred starting material is stroma-free hemoglobin obtained from fresh human red blood cells. First leukocyte filtration is applied, next the red blood cells are centrifuged, the supernatant is discarded and the cells are mixed with 0.9% NaCl. Then, the solution is again centrifuged and the supernatant and "buffy coat" are discarded. Again 0.9% NaCl is added. The cells are pooled and swelled for two hours at 2-8 °C in 100 mM PBS. After swelling diafiltration is performed with a 0.3 mm tangential microfiltration unit.

Finally this stroma-free hemoglobin is pasteurized for ten hours at 61 °C under deoxy-conditions.

The next step is the intra- and inter-molecular cross- linking of hemoglobin with glutaraldehyde. Before cross- linking the solution must completely be deoxygenated for instance by use of a hollow fibre module. Through this hollow fibre module flows nitrogen, helium or any other inert gas. Tubing must be impermeable or minimal impermeable to oxygen. The preferred temperature lies between 15 and 25 °C. The hemoglobin concentration at the start of the process must preferably be adjusted to 15 or 20 g 1 depending on the desired composition of the final product. The hemoglobin must preferably be solved in 50 mM phosphated buffered saline (pH = 7.4). On one hand a high molecular weight polymerized hemoglobin can be made. This polyHbXl HMW contains ± 30% polymers (> 500 kD) , 30% oligomers (128 - 500 kD) , 15% dimers (128 kD) and 25% monomers (32 kD) according to gel permeation chromatography, see figure 3. The total amount of 1/2-monomers is less than 5%. This product has a intravascular half life of 48 hours. On the other hand a low molecular weight product can be made. This polyHbXl LMW contains ± 1% polymers (> 500 kD), 20% oligomers (128 - 500 kD) , 25% dimers (128 kD) and 54% monomers (32 + 64 kD), see figure 4. The total amount of 1/2-monomers is also less than 5%. An advantage of this product is the smaller molecular weight distribution and another advantage is that less glutaraldehyde is needed for cross-linking.

Two methods are preferred to make this polyHbXl-HMW or -LMW. The first option makes it possible to carry out the whole polymerization process within only 270 minutes. The second option makes it possible to carry out the polymerization process within twenty-six hours. The cross-linking process may be carried out between 2 and 8 °C, preferably at 5 °C, If the short process is chosen, then a 10-fold molar excess glutaraldehyde must be added in three steps to the hemoglobin solution. In succession at t = 0, 30 and 60 minutes. Thus a total 30 : 1 molar ratio to hemoglobin is needed. If a high molecular (HMW) modified hemoglobin (25 - 35% > 500 kD) is wanted, then the hemoglobin concentration during cross-linking must preferably be between 90 and 110 g 1 If a low molecular (LMW) modified hemoglobin (0 - 5% > 500 kD) is needed, then the hemoglobin concentration during cross-

-1 linking must preferably be between 60 and 80 g 1 The polymerization process is monitored by use of gel permeation chromatography. The polymerization time for the high molecular hemoglobin polyHbXl-HMW is ± 150 minutes, for the low molecular hemoglobin polyHbXl-LMW ± 120 minutes. The sequence of addition of glutaraldehyde in three steps is very important in order to obtain a highly intra-molecularly cross- linked fraction (> 95%) in such a short reaction time. If the desired polymer composition is reached, an excess primary amino acid, preferably glycine, solved in phosphate buffered saline, should be added. This excess amino acid must be added between a 1 : 20 to 1 : 40 molar ratio to the total amount glutaraldehyde. This glycine shouls react between 60 and 120 minutes before sodiumborohydride or dimethylamineborane, solved in phosphated buffered saline, is added. This excess must be between a 1 : 1 to 1 : 5 molar ratio to the total amount glutaraldehyde. Since sodiumborohydride is very reactive, only 60 minutes is needed for the reaction.

A disadvantage is the forming of hydrogen gas and thus foam forming. Dimethylamineborane is less reactive and the reaction time must lie between 10 and 20 hours. This sequence and these amounts of quenching agents are essential for the composition of the final product in order to prevent hemorrhagic disorders after administration of the glutaraldehyde-polymerized hemoglobin solution.

If the longer process is chosen, then a 25 to 35-fold molar excess glutaraldehyde must be added at one time for a high molecular hemoglobin product (polyHbXl-HMW) ; a 15 to 20- fold molar excess for a low molecular hemoglobin (polyHbXl- LMW) . The quenching procedure is the same as described above. After this quenching and reducing procedure the modified hemoglobin solution is concentrated to 10 - 20 g 1 and dialysed against Ringer lactate or a Hartmann solutio .

Brief description of the drawings

For a better understanding of the nature and objects of the invention, there are provided 5 figures and tables to be hereinafter described in detail.

FIG.l shows an oxygen dissociation curve of polyHbXl with a low molecular weight. P-50 = 33.5 mm Hg and the Hill coefficient is 0.94.

FIG. 2 shows the dynamic viscosity of polyHbXl HMW and LMW as a function of the hemoglobin concentration.

FIG. 3 shows an elution pattern on a FPLC column of polyHbXl with a high molecular weight (HMW) . Peak 1 refers to polymers (> 500 kD) , peak 2 to oligomers (128 - 500 kD), peak 3 to dimers (128 kD) and peak 4 to monomers (32 + 64 kD) . Hemoglobin is cross-linked with a 30-fold molar excess glutaraldehyde.

FIG.4 shows an elution pattern on a FPLC column of polyHbXl with a low molecular weight (LMW) . Peak 1 refers to polymers (> 500 kD), peak 2 to oligomers (128 - 500 kD), peak 3 to dimers (128 kD) and peak 4 to monomers (32 + 64 kD) . Hemoglobin is cross-linked with a 20-fold molar excess glutaraldehyde.

TABLE 1. shows the properties of polyHbXl (HMW and LMW).

Examples

Example 1

Preparation of a pasteurized stroma-free hemoglobin solution

Fresh human blood is released from plasma by centrifugation, from leukocytes through leukocyte filter and from thrombocytes by removal of the buffy-coat. The units of red blood cells are washed three times in sterile plastic bags with 0.5 1 0.9% NaCl and centrifuged for seven minutes at 4500 g. The washed cells are pooled with 0.9% NaCl and swelled for two hours at 2 - 8 °C in a 100 mM phosphate buffered saline solution. After swelling diafiltration is performed with a 0.3 μ tangential microfiltration unit and the solution is concentrated to ± 100 - 200 g 1 ^"1 by use of a 10 kD cut-off filter and is sterilized by filtration. The yield of the stroma-free hemoglobin solution is 80 - 90%. Then the solution is deoxygenated by use of a hollow fibre module through which pure nitrogen flushes. When the saturation of the solution is lower than 5%, the pasteurization is started. The stroma-free hemoglobin is pasteurized for ten hours at 61 °C.

Example 2 Polymerization of stroma-free hemoglobin - PolyHbXl with a high molecular weight (HMW)

Stroma-free hemoglobin was obtained as described in the previous example. Adjust the concentration of the hemoglobin solution to 20 g 1 with 50 mM phosphated buffered saline

(pH = 7.4) till a solution of 500 ml at 20 °C. Deoxygenate the hemoglobin solution with the use of a hollow fibre module. Adjust the hemoglobin flow to 500 ml min . Flush the hollow fibre module with pure nitrogen, 5 - 10 L min . Wait until the saturation < 5%, cool the reactor to 5 °C and then start the polymerization. 1. Dilute 0.625 ml glutaraldehyde (25%, grade I) in a 400 ml deoxygenated 50 mM phosphated buffered saline solution. Add this solution to the hemoglobin solution preferably through the deoxygenator. Let the hemoglobin solution flow through the hollow fibre module during polymerization. Mix the solution slowly,

2. Dilute 0.625 ml glutaraldehyde (25%, grade I, 10-fold molar excess with respect to the hemoglobin concentration) in a 100 ml deoxygenated 50 mM phosphate buffered saline solution. Add this solution thirty minutes after the first addition preferably through the deoxygenator,

3. Dilute 0.625 ml glutaraldehyde (25%, grade I) in a 50 ml deoxygenated 50 mM phosphate buffered saline solution. Add this solution thirty minutes (total time = 60 minutes) after the second addition preferably through the deoxygenator.

4. Allow this reaction to proceed until the total time of 150 minutes. Dissolve 10.6 gram glycine (30-fold molar excess with respect to the total concentration glutaraldehyde) in a 100 ml deoxygenated 50 mM phosphate buffered saline and add this solution preferably through the deoxygenator. Allow this quenching reaction to proceed for 60 minutes (total time 210 minutes) ,

5. Dissolve 0.82 gram dimethylamineborane (3-fold molar excess with respect to the total concentration glutaraldehyde) in 10 ml 2 mM NaOH and add this to the solution,

6. Allow dimethylamineborane to react for twenty hours (at

5 °C),

-1

7. Concentrate this hemoglobin solution to 100 g 1 with ultrafiltration equipment (10 kD filter) after the reaction with dimethylamineborane is completed and dialyse against two litres Ringer lactate to remove excess glutaraldehyde, glycine and dimethylamineborane. Adjust the inlet flow to 500 ml min and set the transmembrane pressure at 200 kPa. This product contains ± 25% polymers (> 500 kD) and less than 5% 1/2-monomers (32 kD) . Example 3

Polymerization of stroma- free hemoglobin - PolyHbXl with a low molecular weight ( LMW)

Procedure similar as described in example 2 with the exceptions that the starting hemoglobin concentration is 15 g

-1

1 and that glycine is added after 120 minutes.

Example 4 Polymerization of stroma-free hemoglobin - PolyHbXl with a high molecular weight (HMW) . Alternative method.

Procedure similar as described in example 2, with the exception that in step five sodiumborohydride is used as a reducing agent. Dissolve 0.53 gram sodiumborohydride in 10 ml 4 mM NaOH and add this to the hemoglobin solution. Allow this reducing to reaction proceed for 60 minutes (total reaction time 270 minutes) .

Example 5

Polymerization of stroma-free hemoglobin - PolyHbXl with a high molecular weight (HMW). Alternative method.

Procedure similar as described in example 3, with the exception that in step five sodiumborohydride is used as a reducing agent. Dissolve 0.53 gram sodiumborohydride in 10 ml 4 mM NaOH and add this to the hemoglobin solution. Allow this reducing reaction to proceed for 60 minutes (total reaction time 240 minutes) .

Example 6

Stroma-free hemoglobin was obtained as described in the previous section. Adjust the concentration of the hemoglobin solution to 15 g 1 with 50 mM phosphate buffered saline (pH = 7.4) till a solution of 500 ml at 20 °C. Deoxygenate the hemoglobin solution with the use of a hollow fibre module. Adjust the hemoglobin flow to 500 ml min . Flush the hollow fibre module with pure nitrogen, 5 - 10 L min . Wait until the saturation < 5%, cool the reactor to 5 °C and then start the polymerization.

1. Dilute 1.4 ml glutaraldehyde (25%, grade I, 30-fold molar excess with respect to the total concentration glutaraldehyde) in a 500 ml deoxygenated 50 mM phosphate buffered saline solution. Add this solution to the hemoglobin solution preferably through the deoxygenator. Let the hemoglobin solution flow through the hollow fibre module during polymerization. Mix the solution slowly,

2. Allow this reaction proceed for twenty-four hours. Dissolve 7.9 gram glycine (30-fold molar excess with respect to the total concentration glutaraldehyde) in a 500 ml deoxygenated 50 mM phosphate buffered saline and add this solution preferably through the deoxygenator. Allow this quenching reaction to proceed for 60 minutes (total time 25 hours) ,

3. Dissolve 0.60 dimethylamineborane (3-fold molar excess with respect to the total concentration glutaraldehyde) in 10 ml 2 mM NaOH and add this to the solution,

4. Allow dimethylamineborane to react for twenty hours (at 5 °C) ,

5. Concentrate this hemoglobin solution to 100 g 1 with ultrafiltration equipment (10 kD filter) after the reaction with dimethylamineborane is completed and dialyse against two litres Ringer lactate to remove excess glutaraldehyde, glycine and dimethylamineborane. Adjust the inlet flow to 500 ml min and set the transmembrane pressure at 200 kPa. Example 7

Polymerization of stroma-free hemoglobin - PolyHbXl with a high molecular weight (HMW). Alternative method,

Procedure similar as described in example 6, with the exception that in step five sodiumborohydride is used as a reducing agent. Dissolve 0.53 gram sodiumborohydride in 10 ml 4 mM NaOH and add this to the hemoglobin solution. Allow this reducing reaction to proceed for 60 minutes (total reaction time 26 hours).

Example 8

Polymerization of stroma-free hemoglobin - PolyHbXl with a low molecular weight (LMW). Alternative method.

Stroma-free hemoglobin was obtained as described in the previous section. Adjust the concentration of the hemoglobin solution to 15 g 1 with 50 mM phosphate buffered saline (pH = 7.4) till a solution of 500 ml at 20 °C. Deoxygenate the hemoglobin solution with the use of a hollow fibre module.

Adjust the hemoglobin flow to 500 ml min . Flush the hollow fibre module with pure nitrogen, 5 - 10 L min . Wait until the saturation < 5%, cool the reactor to 5 °C and then start the polymerization. 1. Dilute 0.84 ml glutaraldehyde (25%, grade I) in a 500 ml deoxygenated 50 mM phosphated buffered saline solution. Add this solution to the hemoglobin solution preferably through the deoxygenator. Let the hemoglobin solution flow through the hollow fibre module during polymerization. Mix the solution slowly,

2. Allow this reaction to proceed for twenty-four hours. Dissolve 4.7 gram glycine (30-fold molar excess with respect to the total concentration glutaraldehyde) in a 500 ml deoxygenated 50 mM phosphate buffered saline and add this solution preferably through the deoxygenator. Allow this quenching reaction proceed for 60 minutes (total time 25 hours) , 3. Dissolve 0.36 dimethylamineborane (3-fold molar excess with respect to the total concentration glutaraldehyde) in 10 ml 2 mM NaOH and add this to the solution,

4. Allow dimethylamineborane to react for twenty hours (at 4 °C),

5. Concentrate this hemoglobin solution to 100 g 1 with ultrafiltration equipment (10 kD filter) after the reaction with dimethylamineborane is completed and dialyse against two litres Ringer lactate to remove excess glutaraldehyde, glycine and dimethylamineborane. Adjust the inlet flow to 500 ml min and set the transmembrane pressure at 200 kPa.

Example 9

Polymerization of stroma-free hemoglobin - PolyHbXl with a low molecular weight (LMW) . Alternative method.

Procedure similar as described in example 8, with the exception that in step five sodiumborohydride is used as a reducing agent. Dissolve 0.24 gram sodiumborohydride in 10 ml 4 mM NaOH and add this to the hemoglobin solution. Allow this reducing reaction to proceed for 60 minutes (total reaction time 26 hours) .

Literature

Beauchamp jr. R.O. , M.G. St. Clair, T.R. Fenell, D.O. Clarke, K.T. Morgan, T.W. Kari, Critical reviews in toxicology of glutaraldehyde, 22, p.p. 143-174, 1992

Berbers W.A.M., P.T.M. Biessels, W.K. Bleeker, J.C. Bakker, A hemoglobin composition and its use, patent, int. no. WO 90/13309, publication date: 15-11-1990

Bleeker W. et al., Hemostatic disorders after administration of glutaraldehyde polymerized hemoglobin, to be published 1996

Bonssen, P., M.B. Laver, K.C. Morris, Compositions of matter comprising macromolecular hemoglobin, US-patent, patent- number: 4,053,590, 1977.

Feola, M. , J. Simoni, R. Tran, P.C. Canizaro, Mechanisms of toxicity of hemoglobin solutions . In: Blood substitutes. Ed. by TMS Chang and RP Geyer. Marcel Dekker Inc. NY and Basel, p.p. 217-226, 1989.

Hsia, J.C, Pasteur izable, freeze -driable hemoglobin based blood substitute, US Patent, patent-number: 4,857,636, 1989

Keipert, P.E., T.M.S. Chang, Preparation and in vitro characteristics of a blood substitute based on pyridoxilated poly hemoglobin . Appl. Biochem. and Biotechnol. 10, p.p. 133- 141, 1984.

Manning, L.R., J.M. Manning, Influence of ligation state concentration of hemoglobin A on its cross-linking by glycolaldehyde : functional properties of cross-linked, carhoxymethylated hemoglobin . Biochemistry 27, p.p. 6640-6644 1988. MacDonald, S.L. and D.S. Pepper, Hemoglobin polymerization, Method. Enzymol., 231, p.p. 287-308, 1994.

Marini, M.A., G.L. Moore, S.M. Christensen, R.M. Fishman, R.G. Jessee, F. Medina, S.M. Snell, A.I. Zegna, Reexamination of the polymerization of pyridoxialted hemoglobin with glutaraldehyde . Biopolymers 29, p.p. 871-882, 1990.

Murray, J.A., A. Ledlow, J. Launspach, D.Evans, M. Loveday, J.L. Conklin, The effects of recombinant human hemoglobin on Esophageal motor functions in humans . Gastroentrology, vol. 109, p.p. 1241 - 1248, 1995.

Nelson, D.J., A. Srnak, Hemoglobin -oligomer based composition and method to make same, patent, int. no. WO 92/03153, 1992

Seghal, L.R., R.E. Woskin De, G.S. Moss, S.A. Gould, A.L.

Rosen, H. Seghal, Acellular red blood cell substitute, LTS- patent, patent-number: 5,464,814, 1995.

Walder, J.A., R.H. Zaugg, R.Y. Walder, J.M. Steele, I.M.

Klotz, Diaspirins that cross-link b chains of hemoglobin : bis

(3, 5-dibromosalicyl )succinate and (3, 5- dibromosalicyl ) fumarate . Biochemistry 18, p.p. 4265-4270, 1979.

Table 1

Claims

CLA IMS

1. The invention thus provides a method for producing a polymerized hemoglobin preparation essentially free of the risk of causing hemorrhagic disorders, comprising intra¬ molecular cross-linking and intermolecular cross-linking of a deoxyhemoglobin using glutaraldehyde or a functional equivalent thereof, whereby the cross-linked material is treated with a blocking agent and a fixating agent.

2. A method according to claim 1 whereby the blocking agent is provided in excess of the remaining free aldehyde groups.

3. A method according to claim 1 or 2, whereby the fixating agent is provided in excess of the remaining free aldehyde groups.

4. A method according to anyone of claims 1-3, whereby the blocking agent comprises a quenching agent.

5. A method according to anyone of claims 1-4, whereby the fixating agent comprises a reducing agent.

6. A method according to anyone of claims 4-5, whereby the quenching agent is an amino acid.

7. A method according to claim 6, whereby the amino acid is glycine.

8. A method according to anyone of the claims 6-7 whereby the reducing agent is sodiumborohydride or a functional equivalent thereof.

9. A method according to anyone of the aforegoing claims whereby the hemoglobin preparation is derived from mammalian hemoglobin.

10. An intra-molecularly cross-linked, polymerized hemoglobin preparation obtainable by anyone of claims 1-9.

11. A hemoglobin preparation according to claim 10 having an oxygen affinity (P-50) in physiological conditions of 25 to 35 mm Hg.

12. A hemoglobin preparation according to claim 10 or 11 having a methemoglobin concentration of 10% or less.

13. A hemoglobin preparation according to anyone of claims of 10-12 which is free of red blood cell stroma.

14. A hemoglobin preparation according to anyone of claims 10-13 comprising 30% or less hemoglobin polymers having a molecular weight above 500 kD.

15. A hemoglobin preparation according to anyone of claims 10-14 comprising 5% or less hemoglobin 1/2 monomers (32 kD) .

16. A hemoglobin preparation according to anyone of claims of 10-15 which is a solutions having an iso-oncotic hemoglobin concentration of 90 g 1 and a dynamic viscosity below 2 cP, preferably below 1.5 cP