WO1992008478A1

WO1992008478A1 - Method of enhancing long-term storage stability of hemoglobin products

Info

Publication number: WO1992008478A1
Application number: PCT/US1991/008622
Authority: WO
Inventors: Kwang Nho
Original assignee: Enzon, Inc.
Priority date: 1990-11-20
Filing date: 1991-11-19
Publication date: 1992-05-29
Also published as: AU9067791A

Abstract

The present invention relates to a method for enhancing the stability of hemoglobin products comprising deoxygenating hemoglobin by gas exchange through a permeable membrane. It is based, in part, on the observation that hemoglobin was rendered significantly more stable upon deoxygenation; methemoglobin formation from hemoglobin process by the methods of the invention was surprisingly low. In particular embodiments of the invention, the stable hemoglobin-based products are deoxygenated prior to storage, so as to prolong shelf-life. In alternative embodiments of the invention, hemoglobin may be deoxygenated prior to chemical treatment, including but not limited to conjugation to polyalkylene oxide. The present invention offers the advantages of decreasing the rate of conversion of hemoglobin to methemoglobin in solution, and diminishes the need for chemical reducing agents or stabilizing agents, such as sugars. Because the presence of chemical reducing agents or sugars may be clinically problematic, the present invention provides for hemoglobin pharmaceutical preparations of superior purity.

Description

METHOD OF ENHANCING LONG-TERM STORAGE STABILITY OF HEMOGLOBIN PRODUCTS

1. INTRODUCTION „ The present invention relates to a method for ^■ _{* s} enhancing the stability of hemoglobin products comprising

, deoxygenating hemoglobin via gas exchange through a gas- permeable membrane. It offers the advantages of increasing shelf-life as well as decreasing the need for chemical preservatives in hemoglobin-based products.

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2. BACKGROUND OF THE INVENTION 2.1. STROMA-FREE HEMOGLOBIN

In attempts to utilize free hemoglobin as a red blood cell substitute, erythrocyte hemolyzates have been

IS administered by infusion. However, the stromal components were found to be extremely toxic, resulting in coagulopathy and associated renal failure. In 1967, Rabiner used centrifugation and ultrafiltration procedures to prepare a stro a-free hemoglobin solution (Rabiner et al., 1967, J.

2_Q Exp. Med. 126;1127) ; by 1977, a crystalline form of stroma-free hemoglobin had been prepared (De Venuto et al., 1977, J. Lab. Clin. Med. 89:509) .

Stroma-free hemoglobin, taken out of the red blood cell microenvironment, was found to exhibit a propensity to

₉ bind oxygen too tightly (a low P₅₀) and also to have a short circulating half-life following transfusion. The low P₅₀, reflective of a leftward shift in the hemoglobin oxygen binding curve, was, in part, consequent to exposure of stroma-free hemoglobin to a higher pH in plasma (7.4)

30 than that experienced within the erythrocyte (7.2); furthermore, the natural association between hemoglobin and 2,3-diphosphoglycerate was destroyed when hemoglobin was * removed from the red cell. In terms of clearance, the..

^ stroma-free hemoglobin was observed to be rapidly eliminated by the kidneys, with a transfusion half-life of only about 100 minutes.

A number of chemical modifications have been introduced into stroma-free hemoglobin in attempts to increase the P₅_ and to render the hemoglobin more stable. Perhaps the most widely used chemical modification of stroma-free hemoglobin utilizes pyridoxal 5'-phosphate and sodium or potassium borohydride to increase the P_5Q (Benesch et al., 1972, Biochem. 11:3576) .

To extend the half-life of stroma-free hemoglobin, the hemoglobin has been linked to other macromolecules, such as dextran (Chang, J. E. et al., 1977, Can. J. Biochem. 55:398), hydroxyethyl starch (DE OS No. 2,616,085), gelatin (DE AS 2,449,885), albumin. (DE AS 2,449,885), and polyethyleneglycol (PEG) (DE 3026398; U. S. Patent No. 4,670,417; U. S. Patent No. 4,412,989; U. S. Patent No.

4,301,144). Technologies were also developed to cross-link stroma-free hemoglobin to form polyhemoglobin (U.S. Patent No. 4,001,200 and 4,001,401) or to internally cross-link hemoglobin molecules, for example, using 2-N-2-formyl- pyridoxal-5'-phosphate and borohydride (Benesch et al.,

1975, Biochem. Biophys. Res. Com un. 6_2^1123) or diaspirins (diesters of bis 3,5-dibromosalicylate; see U.S. Patent No. 4,529,719) .

Additional modifications of stroma-free hemoglobin included a method for decreasing the rate of methemoglobin formation using NADH and NADPH (Sehgal et al., 1981, J. Surg. Res. 3_1:13-17). Keipert and Chang (1985, Biomater. Med. Devices Artif. Organs 13_:156) tested the efficacy of pyridoxal phosphate treated polyhemoglobin in resuscitating rats acutely bled to 67 percent of total blood volume, and found it comparable to whole blood in providing for long- term survival.

The use of stroma-free hemoglobin from different - species as a human red blood cell substitute has been suggested (e.g., in U. S. Patent No. 4,670,417; U.S. Patent No. 4,584,130; U.S. Patent No. 4,529,719; U.S. Patent No. 4,412,989; U.S. Patent No. 4,377,512; U.S. Patent No. 4,301,144; U.S. Patent No. 4,061,736). However, Chang et al. (1987, Bio ater. Artif. Cells Artif. Organs 15:443-452) 5 performed immunologic studies which revealed that immunizing doses of heterologous (i.e. cross-species) hemoglobin was associated with antibody production by the recipient animal; furthermore, cross-linking the heterologous hemoglobin increased the immune response, 0 thereby teaching against the use of cross-species hemoglobins as human red cell substitutes.

2.2. CURRENT METHODS OF STABILIZING HEMOGLOBIN IN SOLUTION 5 In order to retard oxidation of cell-free hemoglobin in aqueous solution, additives which have been known to stabilize protein conformation and enzyme activity have been utilized to prolong the half-life of hemoglobin solutions. These additives include glycerol and sugars, 0 primarily mono- and di-saccharides. Thermodynamic studies (Gekko and Timasheff, 1981, Biochem. 2£:4677-4686) of several proteins, including ribonuclease A, chymotrypsinogen A, beta-lactoglobulin, alpha-chymotrypsin, lysozyme, insulin and bovine serum albumin have shown that ~ these proteins appear to be preferentially hydrated in glycerol-water mixtures, indicating that the chemical potential (activity coefficient) of a protein increases with increasing glycerol concentration. An increase in the activity coefficient of a solute corresponds to a decrease in its concentration at constant activity or a decrease in its solubility. Since glycerol interacts favorably with water, by entering into the water lattice and strengthening solvent structure, its presence in the aqueous medium could increase the hydrophobicity of the protein. The nonpolar groups on the protein surface may react unfavorably to contact with the mixed solvent. For example, surface hydrophobic groups may prefer to migrate into the interior of the protein in order to avoid contact with glycerol/water solvent, thereby distorting protein structure.

The alteration in protein-solvent interactions in the presence of sucrose also has been studied (Lee and Timasheff, 1981, J. Biol. 256:7193-78201) . Sucrose was not observed to induce a conformational change in the proteins studied. The enthalpy of thermal unfolding showed little dependence on the concentration of sucrose, while the apparent activation energy of the unfolding process was increased by the addition of sucrose. The study also indicated that sucrose was preferentially excluded from the protein domain, thereby increasing the free energy of the system. Thermodynamically, this may lead to protein stabilization since the unfolded state of the protein may become thermodynamically less favorable in the presence of sucrose. The exclusion of sucrose from the protein domain appeared to be related to higher cohesive force of the sucrose-water solvent system. A major factor in the stabilization is thought to have been the free energy required to form a cavity in the solvent needed for accommodating the protein molecule, the stabilization, perhaps, being conferred on the protein by the increase in the solvent cohesive force when sucrose was added. Glucose and maltose have also been added to aqueous hemoglobin solutions in attempts to minimize the rate of methemoglobin (MetHb) formation. According to a study (Iwasaki et al. U.S. Patent No. 4,670,417, filed February 21, 1986 and issued June 2 , 1987) moderate success has been achieved with 5% maltose in that 14 days of storage at 30^βC resulted in only 5.2% of metHb, when the starting value was 1.2%. Although the protective effect of these sugars appears to be beneficial, the amount of sugars which must be added in order to generate commercially viable storage life may be sufficient to pose clinical problems for patients such as diabetics who would be sensitive to a sugar overload, or patients with congestive heart failure who may be adversely affected by changes in serum osmolality.

Lyophilization of hemoglobin has also been utilized in efforts toward long-term preservation. However, the process of freeze-drying is harsh enough to generate unacceptable levels of oxidized hemoglobin. Additives, including cryoprotectors such as sugars that appear to protect the integrity of proteins during lyophilization, have been tested on hemoglobin solutions with some degree of success. Although the presence of cryoprotectors has appeared to significantly reduce the amount of MetHb generated during the lyophilization process, the MetHb in the final product has remained undesirably high.

3. SUMMARY OF THE INVENTION The present invention relates to a method for enhancing the stability of hemoglobin products comprising deoxygenating hemoglobin by gas exchange through a permeable membrane. It is based, in part, on the observation that hemoglobin was rendered significantly more stable upon deoxygenation; methemoglobin formation from hemoglobin processed by the methods of the invention was surprisingly low.

In particular embodiments of the invention, the stable hemoglobin-based products are deoxygenated prior to storage, so as to prolong shelf-life. In alternative embodiments of the invention, hemoglobin may be deoxygenated prior to chemical treatment, including but not limited to conjugation to polyalkylene oxide.

The present invention offers the advantages of decreasing the rate of conversion of hemoglobin to methemoglobin in solution, and diminishes the need for chemical reducing agents or stabilizing agents, such as sugars. Because the presence of chemical reducing agents or sugars may be clinically problematic, the present invention provides for hemoglobin pharmaceutical preparations of superior purity.

3.1. ABBREVIATIONS DeoxyHb deoxyhemoglobin Hb hemoglobin MetHb methemoglobin

PEG polyethyleneglycol

4. DESCRIPTION OF THE FIGURES Figure 1. Diagram of a hemoglobin solution-containing reactor connected to a gas exchange device for deoxygenation of hemoglobin.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods of deoxygenating hemoglobin products. The term hemoglobin products may be construed to refer to hemoglobin which is in solution and which may or may not be chemically modified, as well as to hemoglobin within living cells. Chemically modified hemoglobin may include but not be limited to hemoglobin which has been cross-linked, which has been treated with pyridoxal phosphate, or which has been conjugated to polyalkylene oxide. Hemoglobin products may be derived from a human or non-human source or by genetic engineering methods. According to the invention, the hemoglobin product is deoxygenated by exposing the hemoglobin to an inert gas via a gas permeable membrane, such that the inert gas must pass through the membrane in order to come in contact with the hemoglobin. For purposes of clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections:

(i) gas permeable membranes;

(ii) circulation of blood or hemoglobin over gas permeable membrane; and

(iii) pharmaceutical compositions of deoxygenated hemoglobin.

5.1. GAS PERMEABLE MEMBRANES According to the present invention, any membrane which is gas permeable but hemoglobin impermeable, and which does not chemically react with hemoglobin, may be utilized. Membranes used according to the invention may preferably be easily sterilized. Examples of gas permeable membranes which may be used according to the invention include, but are not limited to, polypropylene and cellulose acetate membranes.

In particular embodiments of the invention, membranes may be utilized such that a large surface area is available for gas exchange. Accordingly, it may be desirable to provide a large surface area of membrane for a relatively small volume of blood. In preferred specific embodiments of the invention, gas permeable membranes may be assembled into long cylindrical shapes, and groups of these cylinders may be assembled together; a hemoglobin solution may be passed through these cylinders while an inert gas is circulated outside the cylinders, or, alternatively, an inert gas may be passed through the cylinders while hemoglobin solution is circulated outside the cylinders [FIGURE l] . According to the invention, whole blood, red blood cells, or hemoglobin products, including chemically modified hemoglobin, may also be deoxygenated in this manner. If whole blood cells are to be deoxygenated, the membrane configuration should permit the passage of whole cells without lysis or accumulation of cells. In specific embodiments of the invention, gas-permeable membranes designed and manufactured so as to generate a large surface area of gas/liquid contact, such as those produced by Hoechst-Celanese, Celgard G-240/11, polypropylene fiber diameter 240 micron, surface area 11 square feet or CD Medical, Inc., Cell-Pharm Hollow Fiber Oxygenators, or any structurally and functionally equivalent apparatus, may be used.

5.2. CIRCULATION OF BLOOD OR HEMOGLOBIN

OVER GAS PERMEABLE MEMBRANE

Hemoglobin products may be circulated over the gas permeable membrane using any method known in the art so as to create a flow rate which permits effective gas exchange. - Appropriate circulation methods would include but not be limited to those associated with gravity, a peristaltic pump, capillary action, pressure differentials or centrifugal force.

In a preferred specific embodiment of the invention, 0 the Celgard G-240/11 gas exchange device may be used, and hemoglobin may be circulated through the device at a flow rate of about 500 ml/min or at such a flow rate that deoxygenation may be achieved after 10-15 minutes under constant inert gas flow at about 5-10 p.s.i. In this g specific embodiment, the exterior space of the fibers may be desirably under constant inert gas (e.g. nitrogen) flow at about 5-10 psi, in which case complete deoxygenation may be achieved after about 10-15 minutes.

The gas used to deoxygenate hemoglobin may be any gas 0 which does not react with the hemoglobin in solution, including, but not limited to, nitrogen, helium, argon, and carbon dioxide gas.

It may be desirable to monitor the extent of deoxygenation of hemoglobin during the deoxygenation process using for example a Radiometer OSM3 Hemoximeter. In preferred embodiments of the invention, at least about 90 percent of the hemoglobin in solution may be deoxygenated.

5.3. PHARMACEUTICAL COMPOSITIONS OF DEOXYGENATED HEMOGLOBIN

The present invention provides for pharmaceutical compositions comprising hemoglobin deoxygenated according to the invention. In particular embodiments of the invention, the hemoglobin is in aqueous solution at concentrations ranging from about 5-15%. The hemoglobin compositions of the invention differ from those produced by other known methods in that the rate of methemoglobin formation is extremely low in the absence of stabilizing compounds, such as sugars, as well as in the presence of extremely low concentrations of sugar, such as dextrose at a concentration of less than five percent g/dl, or at a concentration of one percent (see Example Sections 6 and 7, infra) .

6. EXAMPLE: IMPROVED STABILITY OF MEMBRANE- DEOXYGENATED BOVINE HEMOGLOBIN

Native bovine hemoglobin was purchased from Biopure,

Boston, MA. 500 ml of 6% (g/dl) Hb solution was prepared in 0.1 M NaCl, 0.01 M Na₂HP0₄ and 0.03 M NaHC0₃« This solution was put in a closed vessel whose gas phase was continuously replenished with a slow flow of nitrogen, and the temperature of the solution was maintained at 4-6°C.

The reactor was connected to a gas-exchange device, Celgard

(G-240/11) , polypropylene fiber diameter 20 micron, surface 2 area 11 ft from Hoechst-Celanese, so that the hemoglobin solution was in recirculation through the fibers of the gas-exchange device. The exterior space of the fibers was maintained under constant nitrogen flow at a pressure of 5-10 psi. The flow rate of the recirculating hemoglobin solution was set at 500 ml/min. Substantially complete deoxygenation was achieved after 10-15 in as measured by a Radiometer 0SM3 Hemoximeter. Hemoglobin was considered to be essentially deoxygenated when the percentage of deoxyhe oglobin was about 93%. Three separate samples were prepared to contain either 0% (g/dl) dextrose, 1% dextrose or 5% dextrose. Dextrose was added to the hemoglobin solution in order to determine its effect on the stability of deoxyHb. These samples were stored at 2-4^βC for ten months and MetHb levels were measured frequently on a Hemoximeter (Radiometer) (see Table I) . Methemoglobin formation from Hb not deoxygenated according to these methods (Hb(oxy)) was measured as a control.

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Table I. Methemoglobin formation from deoxyherooglobin and oxyhemoglobin over a ten month period

Hb(deoxy) Hb(deoxy) Hb(deoxy) Hb(oxy) in 0% in 1% in 5% in 1% Months Composition dextrose dextrose dextrose dextrose

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* The rest of the composition is consisted of 0-2 % HbCO.

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As shown in Table I, whereas oxyhemoglobin rapidly formed methemoglobin, resulting in 58.3% methemoglobin in ten months, hemoglobin deoxygenated according to the methods of the invention was significantly more stable, and formed only 5.9% methemoglobin after ten months of cold storage. While the addition of dextrose improved stability somewhat, its effect was relatively minor, decreasing methemoglobin formation by only about 1.0 percent.

7. EXAMPLE: IMPROVED STABILITY OF MEMBRANE- DEOXYGENATED PEG-BOVINE HEMOGLOBIN

200 ml of 6% bovine hemoglobin solution was prepared containing 0.5 M NaCl, -0.01 M Na_HP0₄, 0.03 M NaHCO . This hemoglobin solution was chemically modified with activated polyethylene glycol-succinimidyl carbonate (SC-PEG, M.W.

5141) . SC-PEG was prepared according to the method described in U. S. patent application serial number

07/440,553, filed November 22, 1989, which is incorporated in its entirety by reference herein. Reaction of hemoglobin with SC-PEG produced tetrameric hemoglobin with molecules of linear PEG attached to the surface lysines of hemoglobin. The molar ratio between SC-PEG and Hb in the reaction was 15:1. SC-PEG was rapidly added into the reactor and well agitated. Reaction was allowed to continue for two hours at pH 8.0 and 4^βC, followed by ultrafiltration to remove unreacted PEG and excess electrolytes. The product was found to be non-polymerized, tetrameric hemoglobin bearing 7-8 strands of PEG, and having a molecular weight of about 100,000-105,000 daltons.

The number of amino acids modified with PEG was determined by trinitrobenzene sulfonic acid (TNBS) assay (Habeeb,

1966, Analyt. Biochem. 14_:328-336) . The PEG-Hb was then membrane deoxygenated according to the method set forth in

Section 6, supra, and was then stored at 2-4^βC in solutions further comprising 0, 1, or 5% dextrose. Non-deoxygenated PEG-Hb was included in storage studies as a control. As shown in Table II, PEG-Hb deoxygenated by the methods of the invention was significantly more stable with respect to methemoglobin formation than non-deoxygenated PEG-Hb. Furthermore, the addition of dextrose resulted in a relatively minor improvement in stability.

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Table II .

Methemoglobin formation from deoxyhemoglobin over a ten month period

* The rest of the composition is consisted of 0-2% HbCO.

The present invention is not limited in scope by the cell lines deposited or the embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

Claims

WE CLAIM:

1. A method of deoxygenating a hemoglobin product comprising exposing the hemoglobin product to an inert gas via a gas permeable membrane such that the inert gas must pass through the membrane to come in contact with the hemoglobin.

2. The method according to claim 1 in which the hemoglobin is stroma-free hemoglobin.

3. The method according to claim 1 in which the hemoglobin is chemically modified.

4. The method according to claim 3 in which the hemoglobin is conjugated to a polyalkylene oxide.

5. The method according to claim 4 in which the polyalkylene oxide is polyethylene glycol.

6. The method according to claim 1 in which the gas permeable membrane is polypropylene.

7. The method according to claim 6 in which the inert gas is applied at a pressure of about 5-10 psi.

8. The method according to claim 2 in which the gas permeable membrane is polypropylene.

9. The method according to claim 8 in which the inert gas is applied at a pressure of about 5-10 psi.

10. The method according to claim 3 in which the gas permeable membrane is polypropylene.

11. The method according to claim 10 in which the inert gas is applied at a pressure of about 5-10 psi.

12. The method according to claim 4 in which the gas permeable membrane is polypropylene.

13. The method according to claim 12 in which the inert gas is applied.at a pressure of about 5-10 psi.

14. The method according to claim 5 in which the gas permeable membrane is polypropylene.

15. The method according to claim 14 in which the inert gas is applied at a pressure of about 5-10 psi.

16. A pharmaceutical composition comprising hemoglobin in a suitable pharmacologic carrier which is at least about 90% deoxygenated and which forms methemoglobin at a rate of less than 1.5 percent per month for the first four months of storage at 2-4°C.

17. The pharmaceutical composition of claim 16 which further comprises less than five percent (g/dl) sugar.

18. The pharmaceutical composition of claim 17 which further comprises less than one percent (g/dl) sugar.

19. The pharmaceutical composition of claim 16 in which the hemoglobin is stroma-free hemoglobin.

20. The pharmaceutical composition of claim 17 in which the hemoglobin is stroma-free hemoglobin.

21. The pharmaceutical composition of claim 18 in which the hemoglobin is stroma-free hemoglobin.

22. The pharmaceutical composition according to claim

16 in which the hemoglobin is chemically modified.

23. The pharmaceutical composition according to claim

17 in which the hemoglobin is chemically modified. 5

24. The pharmaceutical composition according to claim

18 in which the hemoglobin is chemically modified.

25. The pharmaceutical composition according to claim 10 22 in which the hemoglobin is conjugated to a polyalkylene oxide.

26. The pharmaceutical composition according to claim

23 in which the hemoglobin is conjugated to a polyalkylene 15 oxide.

27. The pharmaceutical composition according to claim

24 in which the hemoglobin is conjugated to a polyalkylene oxide.

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28. The pharmaceutical composition according to claim

25 in which the polyalkylene oxide is polyethylene glycol.

29. The pharmaceutical composition according to claim 25 26 in which the polyalkylene oxide is polyethylene glycol.

30. The pharmaceutical composition according to claim 27 in which the polyalkylene oxide is polyethylene glycol.

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