NZ617344B2 - Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with high temperature short time heat treatment apparatus - Google Patents

Abstract

Disclosed is a method for the preparation of a highly purified and heat stable oxygen carrier containing pharmaceutical composition, the oxygen carrier-containing pharmaceutical composition including haemoglobin, the haemoglobin including cross-linked polymeric haemoglobin, the method comprising: (a) providing whole blood including at least red blood cells and plasma; (b) separating the red blood cells from the plasma in the whole blood; (c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (d) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (e) extracting a first haemoglobin solution from the lysate; (f) performing one or more purification processes to produce a purified haemoglobin solution; (g) cross-linking haemoglobin tetramers to create a solution including cross-linked polymeric haemoglobin, the polymeric haemoglobin including two or more cross-linked haemoglobin tetramers; (h) heat treating the solution including the cross-linked polymeric haemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to approximately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non-cross-linked tetrameric haemoglobin, any dimeric haemoglobin, and any other impurities such to form a heat stable solution including a cross-linked polymeric haemoglobin; (i) cooling the heat-treated solution to a temperature approximately less than or equal to 25 °C in approximately two minutes or less to prevent formation of met-haemoglobin; (j) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat stable haemoglobin including cross-linked polymeric haemoglobin having an undetectable concentration of dimer. (a) providing whole blood including at least red blood cells and plasma; (b) separating the red blood cells from the plasma in the whole blood; (c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (d) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (e) extracting a first haemoglobin solution from the lysate; (f) performing one or more purification processes to produce a purified haemoglobin solution; (g) cross-linking haemoglobin tetramers to create a solution including cross-linked polymeric haemoglobin, the polymeric haemoglobin including two or more cross-linked haemoglobin tetramers; (h) heat treating the solution including the cross-linked polymeric haemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to approximately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non-cross-linked tetrameric haemoglobin, any dimeric haemoglobin, and any other impurities such to form a heat stable solution including a cross-linked polymeric haemoglobin; (i) cooling the heat-treated solution to a temperature approximately less than or equal to 25 °C in approximately two minutes or less to prevent formation of met-haemoglobin; (j) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat stable haemoglobin including cross-linked polymeric haemoglobin having an undetectable concentration of dimer.

Description

METHOD FOR REMOVING UNMODIFIED HEMOGLOBIN FROM CROSS— LINKED HEMOGLOBIN SOLUTIONS INCLUDING POLYMERIC HEMOGLOBIN WITH A HIGH TEIVIPERATURE SHORT TIME HEAT TREATMENT APPARATUS FIELD OF THE INVENTION The invention relates to the field of hemoglobin-based oxygen carriers and, more particularly, to heat treatment ques for purifying hemoglobin—based oxygen carriers including polymeric hemoglobin.

BACKGROUND OF THE INVENTION There exists a need for a blood-substitute to treat or prevent hypoxia resulting from blood loss (e. g., from acute hemorrhage or during surgical operations), resulting from anemia (e.g., pernicious anemia or sickle cell anemia) or resulting from shock (e.g., volume deficiency shock, anaphylactic shock, septic shock or allergic shock).

The use ofblood and blood fractions as in this capacity as a blood-substitute is fraught with disadvantages. For example, the use e blood often is accompanied by the risk of transmission of hepatitis—producing viruses and AIDS—producing Viruses which can complicate patient recovery or result in t fatalities. onally, the use of whole blood requires blood-typing and cross-matching to avoid immunohematological ms and inter donor incompatibility.

Hemoglobin, as a blood-substitute, possesses osmotic activity and the ability to transport and transfer oxygen. r, aqueous hemoglobin exists in brium between the eric (65 KDa) and dimeric (32 KDa) forms. Hemoglobin dimers are excreted by the kidney and result in rapid intravascular elimination of hemoglobin solutions with such solutions typically having a 2-4 hour plasma half—life.

Efforts have been directed to overcome the inherent limitations of hemoglobin solutions by molecularly modifying the hemoglobin. Intramolecularly and intermolecularly cross—linking hemoglobin has generally d renal elimination and increased intravascular retention time. r, solutions of cross-linked hemoglobin still typically contain a significant fraction ofunmodified tetrameric obin. This fied tetrameric hemoglobin can convert to dirneric hemoglobin and then be excreted from the body, thereby ng the average intravascular retention time for cross-linked obin blood-substitutes. Furthermore, current means for separation, such as standard filtration, do not adequately guish between unmodified tetrameric hemoglobin and modified tetramerie obin, Thus, in spite of the recent advances in the preparation of cross-linked hemoglobin blood-substitutes, the need continues to exist for a method to effectively separate unmodified hemoglobin from a solution of an intramolecularly and/or intermolecularly cross-linked hemoglobin blood—substitute to improve the e intravascular retention time of the blood- substitute and to prevent significant levels of renal excretion of hemoglobin.

Prior approaches to removal of various impurities from hemoglobin solutions has focused on relatively low temperature long term r than one hour) heat treatment processes.

US. Patent No. 5,281,579 describes heat treatment from 45 to 85 °C, and particularly 60-66 °C for 1 to 30 hours. US. Patent No. 5,741,894 describes a process for l of impurities from partially oxygenated hemoglobin solutions in a range of 45 to 85 °C, and particularly 76 °C for 90 minutes. r, such long term heat treatment conditions can lead to the formation of PCT/U82012/034608 met-hemoglobin, which cannot be used to oxygenate tissues. Further, such long term heat treatment processes are not compatible with commercial—scale production processes.

SUMMARY OF THE INVENTION The present invention relates to a method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution including ric hemoglobin. The method es contacting the hemoglobin solution with a high temperature short time apparatus wherein unmodified tetrameric hemoglobin is thermally denatured to form a precipitate. The denatured and precipitated hemoglobin dimers are then separated from the solution, while retaining the cross—linked hemoglobin in the solution.

The advantages of this invention include ing a substitute with an improved intrayascular retention time, a reduction or ation of significant renal elimination of hemoglobin and the side effects ated therewith, a suitable oncotic pressure, and reduced hypertensive effects.

BRIEF DESCRIPTION OF THE DRAWINGS represents a schematic flow m of a high temperature short time apparatus method for denaturing unmodified hemoglobin from d hemoglobin blood-substitute ing to the present invention. depicts high performance liquid chromatography analysis for non-heat treated polymeric hemoglobin.

PCT/U82012/034608 depicts high performance liquid chromatography analysis for (a) at treated polymeric hemoglobin with spiked hemoglobin dimer and (b) heat stable polymeric hemoglobin in which has undergone short term heat treatment at 80°C, 85°C, 90°C and 95°C respectively.

DETAILED DESCRIPTION OF THE INVENTION Hemoglobin (Hb) suitable for Hb solutions of this invention can be derived from new, old or outdated blood from humans and other vertebrates, such as cattle, pigs, sheep, ducks and chickens , The blood can be collected from live or freshly slaughtered donors. es of suitable methods for obtaining hemoglobin, derived from red blood cells, are described in US. Patent Nos. 5,084,558 and 5,296,465, issued to Rausch et al, US. Patent No. 6498141, issued to DeWoskin et al, and US. Patent No. 7291592, issued to Gould et al. The teachings of U.S.

Patent Nos. 5,084,558, 5,296,465, 6498141 and 2 are incorporated herein by reference in their entirety.

In a preferred ment, hemoglobin is derived from red blood cells as described in US. Patent No. 581, the teachings of which are incorporated herein by reference in their entirety.

Suitable hemoglobin solutions se s solutions of dissolved Hb wherein the dissolved Hb includes unmodified Hb in addition to modified tetrameric Hb and/or polymeric Unmodified hemoglobin, as defined , is hemoglobin in a non-dissociated and uncross-linked tetrameric form which can dissociate into Hb dimers in vitro or in vivo; WO 48832 unmodified hemoglobin may also include dissociated Hb dimers. Hb dimers can further dissociate into Hb subunits (monomers). Modified hemoglobin may be intramolecularly cross- linked into stable tetramers as well as intermolecularly cross-linked into a r chain within the Hb solution. A r-containing hemoglobin solution used as the starting solution of the present invention can e cross-linked tetrameric hemoglobin along with intermolecularly cross-linked polymeric hemoglobin, and also include undesirable unmodified hemoglobin in tetrameric or c form.

Polymeric hemoglobin may e only hemoglobin components as set forth in US.

Patent Nos. 5,753,616, 5,895,810 and 6,288,027, the disclosures h are incorporated by reference herein; it may include non-hemoglobin molecules conjugated with hemoglobin such as polyethylene glycol (PEG). Such materials are described in U.S. Patent Nos. 900, 7,501,499, and 7,795,401, the disclosures of which are incorporated by reference . All of the above materials can be used as starting hemoglobin-containing solutions in the dimer- removal processes described below. Commercially available hemoglobin-based oxygen carriers can also be used in the dimer-removal process of the present invention, including HEMOPURE®, OXYGLOBIN®, POLYHEME®, HEMOLINKTM and MP4.

In the process of the present invention, a polymeric obin-containing solution prepared according to any ofthe above ngs is subjected to a heat treatment process from approximately 80°C to approximately 95 °C, and, more preferably, greater than 85 °C to 95 °C to sfully remove uncross—linked tetrameric and dimeric forms of hemoglobin from the polymeric hemoglobin—containing solution. Any precipitates formed during the heat treatment are removed by centrifugation or a filtration apparatus to form a clear hemoglobin-containing solution. The high temperature short time heat treatment is preferably carried out using the PCT/U82012/034608 apparatus ed in and described in more detail in Example 1. In this temperature than one hour. range, all of the heat treatments can take place for durations of ntially less In the lower range of 80-85 °C, a time of about 20 to 40 minutes is sufficient. In a temperature minutes is sufficient. range of greater than 85 °C and less than 90 °C a period from 8 to about 30 However at higher temperatures, such as 90-95 °C, the heat treatment can be performed in an exemplary embodiment in 5 minutes or less, and more preferably in less than three minutes.

Cooling preferably takes less than two minutes, and more preferably less than one minute to minimize formation ofmet-hemoglobin.

The high temperature short time process for heat treating the hemoglobin solutions in the purification process also removes other impurities, for example immunoglobin—G and harmful microbial materials and viruses, so that renal injury, ar detrimental effects and other toxicity reactions can be avoided.

Heat treatment oftetrameric hemoglobin is described in US. Patent No. 7,932,356 and US. Patent Application Nos. 12/821,214 and 13/013,850 all of the sures h are incorporated herein by reference. The heat treated polymeric hemoglobin of the present invention can be packaged as described in US. 356 and can be used in various tissue ation techniques sed in the above patents and applications. The highly purified and heat stable oxygen carrier-containing pharmaceutical composition is used in methods of ating tissue in which the composition is provided to animal tissue either in vivo or ex vivo as described in the ‘356 patent.

EXAMPLE 1 HTST heat processing PCT/U82012/034608 A High Temperature Short Time (HTST) processing apparatus is shown in A heating process using the HTST sing apparatus is performed on the polymeric hemoglobin—containing ng material. In this e, the condition for heat treatment is 90°C for 30 seconds to 3 minutes, and preferably 45 to 60 seconds; although other conditions can be selected as discussed above and the tus modified ingly. A solution containing polymeric hemoglobin, that is commercially available Oxyglobin®, is optionally treated with 0.2% of N—acetyl cysteine and pumped into a HTST processing apparatus (first section of the HTST heat exchanger is pre—heated and ined at 90°C) at a flow rate of 1.0 liter per minute, the residence time of the first section of the apparatus is between 45 to 60 seconds, then the solution is passed through at the same flow rate into another section of the heat exchanger that is maintained at 25°C. The time required for cooling is between 15 to 30 s. After cooling down to approximately 25°C, N—acetyl cysteine is ally added immediately afterward at a concentration of 0.2% to 0.4%, preferably at 0.4%: This chemical addition after the HTST heating process maintains met-hemoglobin (inactive hemoglobin) at a low level. The set-up of the processing apparatus is easily controlled for industrial operation. If the hemoglobin is not linked, it is not heat stable and forms a precipitate after the heat treatment step. The precipitate is then removed by a centrifugation or a filtration apparatus to form a substantially clear solution thereafter. shows the molecular weight distribution of polymeric hemoglobin by size— ion High Performance Liquid Chromatography (HPLC). The molecular weight distribution for polymeric hemoglobin solution ranges from 32 KDa to >500 KDa. Following 2012/034608 the HTST heat process step (from 80°C to 95°C) in our invention, the spiked dimeric form of hemoglobin can be removed successfully from the polymeric hemoglobin solution (shown in . Any precipitates formed during the HTST heat process step are removed by centrifugation or a filtration apparatus to form a substantially clear cross-linked hemoglobin solution.

Hemox Analyzer for p50 (oxygen partial pressure, at which the hemoglobin solution is 50% saturated) measurement is used thereafter to analyze the (a) non-heat treated polymeric hemoglobin-containing solution, and (b) a heat treated polymeric hemoglobin-containing solution (undergo 80°C treatment). No significant change in hemoglobin content (as measured by Co-Oximetry) is detected between (1) before HTST heat process step, and (2) after HTST heat process step. However, the p50 value is shifted to a lower value (from 37.5 mmHg to 23.3 Hg) after HTST heat s step, This indicates that the hemoglobin-oxygen g affinity is higher. The lowering of p50 value is an advantage to upload oxygen in c tissues and hypoxic tumor cells. As a tumor grows, it rapidly outgrows its blood supply, leaving portions of the tumor with regions where the oxygen concentration is significantly lower than in y tissues. Denny (Denny W. A., Prodrug gies in cancer therapy, Eur. J. Med. Chem, 2001, 36, 5) reported that hypoxic tumor cells are usually resistant to radiotherapy and chemotherapy; r, they can be made more susceptible to treatment by increasing their oxygen t.

Table 1 p50 value tmmHg) Non-heat treated polymeric hemoglobin-containing 37.5 solution Heat treated lobin—containin_ solution 23.3 2012/034608 EXAMPLE 2 Oxyglobin® and/or Hemopure ® polymeric hemoglobin Synthesis of Stable Polymeric Hemoglobin Blood-Substitute based on the description of US Patent Nos. 5,895,810, 5,296,465, 5084558, 6 and 5955581, is also known as Oxyglobin® and/or Hemopure® product, the disclosures ofwhich are incorporated by reference herein.

The following example relates to a method for making polymeric—containing hemoglobin solutions which are suitable for treatment by the heat treatment apparatus and method ofthe present invention.

Bovine whole blood is collected, mixed with a sodium citrate anticoagulant to form a blood solution. The red blood cells (RBCs) are isolated from bovine whole blood. The RBCs are then washed to separate extracellular plasma proteins, such as BSA or IgG, from the RBCs.

Following separation of the RBCs, the RBCs are lysed to form a hemoglobin-containing solution.

The concentrated Hb solution is then directed from the ultrafiltrate tank onto the media contained in parallel chromatographic columns to te the Hb by high performance liquid chromatography. The chromatographic s n an anion exchange medium suitable to separate Hb from nonhemoglobin proteins. The anion exchange media is a quaternary ammonium anion exchange medium on silica gel. This method of ng silica gel is described in the Journal of Chromatography, 120:321-333 (1976).

The Hb solution is then deoxygenated to a level where oxyhemoglobin or HbOz t is about 101. During deoxygenation, temperature of the Hb on is maintained between PCT/U82012/034608 about 19°C and about 31°C. Also during deoxygenation, and uently throughout the process, the Hb is maintained in a low oxygen environment to minimize oxygen absorption by the Hb and to maintain an oxygenated Hb (HbOz) content of less than about 10% in the deoxy— Prior to the polymerization process, depyrogenated and oxygen-depleted “water for injection” (WFI) is added to the Hb solution to a concentration of about 40 g Hb/L. The polymerization is conducted in a 12 mM phosphate buffer with a pH of 7.8, having a chloride concentration less than or equal to about 35 mM.

The oxidation-stabilized deoxy-Hb and yl cysteine (NAC) are subsequently slowly mixed with the cross-linking agent glutaraldehyde, specifically 29.4 grams of glutaraldehyde for each am of Hb over a five hour period, While heating at 42°C and recirculating the Hb solution through a Kenics l-1/2 inch static mixer with 6 elements (Chemineer, Inc), to form a polymeric Hb solution (hereinafter Hb)"). After polymerization, the temperature of the poly(Hb) in the polymerization reactor is d to a temperature between about 18°C to about 22°C, The poly(Hb) is then concentrated by recirculating the poly(Hb) through the ultrafilter until the tration of the poly(Hb) is increased to about 85 g/L. A suitable ultrafilter is a 30 KDa ultra filter. Subsequently, the b) solution is then mixed with 66.75 g sodium dride, and then recirculated through the static mixer at a flow rate of 10-20 liters per minute.

After the pH and electrolytes are restored to physiologic levels, the stable polymeric Hb blood-substitute is then diluted to a concentration of 50 g/L by adding the filtered, deoxygenated low pH buffer to the polymerization reactor. The diluted blood-substitute is then diafiltered by recirculating from the polymerization reactor through the static mixer and a 100 KDa purification filter against a filtered deoxygenated buffer containing 27 mM sodium lactate, 12 mM NAC, 115 mM NaCl, 4 mM KCl and 1.36 mM CaClz in WFI, (pH 7.8). Diafiltration continues until the blood-substitute ns less than or equal to about 10% modified tetrameric and unmodified tetrameric species.

A polymeric Hb solution is formed according to the method described in this Example 2 (according to the description ofUS. Patent No. 5,084,558, issued to Rausch et al.). This Hb solution is analyzed by gel permeation chromatography and found to comprise about 45% Hb dimers, about 15% unmodified Hb tetramers, and about 40% polymeric Hb les which are larger than unmodified tetramers.

The polymeric hemoglobin-containing material may then be subjected to the heat treatment as discussed in Example 1 to remove dimer and unmodified Hb tetramer.

Polyheme® polymeric obin Synthesis of Stable Polymeric Hemoglobin Blood—Substitute based on the description of US Patent Nos. 6498141 and 7291592, is also known as Northfield laboratories Inc. product (Polyheme®), the sures of which are incorporated by reference .

The following example s to a method for making polymeric-containing hemoglobin solutions le for treatment by the heat treatment apparatus and method of the present invention. 2012/034608 (3a) Preparation of Red Blood Cells, Cell wash and Lysis Mix a blood solution with a 1% aqueous sodium chloride solution to form a 4% total hemoglobin solution; carbon monoxide is then introduced into the mixing tank so that the tank contains an here of carbon monoxide.

By coupling to a 0.65 um tangential flow filter, this 4% total hemoglobin solution is washed with about 8 volumes of the 1% sodium chloride solution to remove plasma protein contaminants. Subsequent to washing, the solution is concentrated to about 16% total hemoglobin, and "water for inj ection" (WFI) is added to bring the volume of the on up to about 2.5 times volume. With the addition of the WFI, the cells swell and rupture releasing obin into solution. The concentration of the resulting obin solution is about 7% total hemoglobin.

The resulting solution is clarified; red blood cells stroma contaminants and cell wall material is retained and removed by the filter. The remaining solution is about 3.3% total hemoglobin solution. (3b) Heat Treatment, Clarification and Viral Reduction The resulting solution of stroma-free hemoglobin is then heat treated at a ature of about 60-62°C over a period of about 10 hours. During this time, the on is moderately agitated. As the solution is heated and passes a temperature of about 55°C, a precipitate forms.

The resulting 3.3% total hemoglobin (w/v) stroma-free, heat treated hemoglobin solution is then filtered through a 0.2 um pre-filter ed by a 0.1 urn pre—filter and then pumped through a 100 KDa viral reduction ultra filter. 2012/034608 (3c) Ultra-filtration Concentration The filtered hemoglobin on is then concentrated to about 14% total Hb concentration and subsequently washed and diafilter with 4 volumes of WFI. The concentration and diafiltration is lished using a 10 KDa ultra filter. This hemoglobin in the solution is primarily carboxyhemoglobin. (3d) Gas exchange with Oxygen and Nitrogen The resulting yhemoglobin solution is sparged with a flow of oxygen for 18 hours at 10°C. The resulting solution contains less than 5% carboxyhemoglobin based on the weight of total hemoglobin. [005 0] After oxygenation, the solution is sparged with a similar flow of nitrogen for about 3 to 3.5 hours at 10°C. until less than 10% oxyhemoglobin based on the weight of total hemoglobin remains in the solution.

(Be) Chemical Modification The deoxyhemoglobin (at about 4°C) solution is then added an aqueous solution of pyridoxyl—S-phosphate (P5P) (93.75 g/L) at a 2:1 PSP to hemoglobin molar ratio. The pyridoxylation is conducted at a ature of about 4°C. The P5P solution is typically added over about 1 minute and mixed for approximately 15 minutes, after which a sodium borohydride/sodium hydroxide solution is added to the hemoglobin solution at a ratio of 0.8 g of sodium hydroxide and 90.8 g of sodium borohydride per 2 liters of hemoglobin on. The borohydride solution is added as rapidly as possible over a period of about 1 minute and then PCT/U82012/034608 stirred for one hour. The resulting solution of pyridoxylated obin is subsequently diafiltered using 10 KDa ultra filter to remove excess reactants with 4 volumes ofWFI.

(St) Polymeric Hemoglobin solution The pyridoxylated hemoglobin is added sufficient WFI to prepare a 4.5% total hemoglobin solution. A glutaraldehyde solution is added to the xylated hemoglobin solution at a molar ratio of glutaraldehyde to hemoglobin of about 24:1. The glutaraldehyde solution is typically added over a period of about 2.5 hours by metering pump to the hemoglobin solution. The polymerization reaction is allowed to proceed for about 15-18 hours.

The target molecular weight distribution is about 75% polymer and 25% er. The target polymers have molecular weights of less than about 600 KDa with a predominant fraction of the lar weights residing in the 100 KDa-350KDa range.

When the polymerization reaction reaches the target molecular weight distribution (after about 15-18 hours), aqueous glycine is added (as a quench) to the hemoglobin solution at a 140:1 molar ratio of glycine to hemoglobin. The solution pH at this point is 8.9. The resulting solution is then mixed for about 30-40 s after which a sodium borohydride sodium/hydroxide solution (having the concentration identified above) is added to the hemoglobin solution at a 28:1 molar ratio of sodium borohydride to hemoglobin. This resulting mixture is stirred for about 1 hour. The solution is then concentrated by ultrafiltration and washed with 4 volumes of WFI. An additional aliquot of sodium borohydride at the same molar ratio as indicated above is added to the concentrated solution and again mixed for 1 hour. The resulting solution is washed with 4 s ofWFI resulting in polymeric, pyridoxylated, -free obin that has been heat treated.

PCT/U82012/034608 The resulting solution is oxygenated by allowing the solution to stand under an oxygen atmosphere. The hemoglobin is then diluted to about 4% total hemoglobin. The 4% total hemoglobin solution is then diafiltered using 10 mM NaCl/20 mM NaOH and a 300 KDa ultra- filter. The filtration is continued until about 97% of the hemoglobin material passes through the filter and is continuously concentrated to 4-8% total hemoglobin using a 70 KDa ultrafilter.

(About 3% of the material, i.e., high molecular weight polymers is retained).

The resulting material is about 4-8% total hemoglobin and ns about 25% tetramer.

Subsequently, the polymeric hemoglobin-containing material may then be subjected to the heat treatment as sed in Example 1 to remove dimer and fied Hb tetramer.

Although various aspects of the ion are set out in the independent claims, other aspects of the invention comprise other combinations of features from the described embodiments and/or the dependent claims with the features of the independent claims, and not solely the combinations itly set out in the claims.

It is also noted herein that while the above describes exemplary embodiments of the invention, these descriptions should not be viewed in a ng sense. Rather, variations and modifications may be made without departing from the scope of the present invention as defined in the appended claims.

Claims (12)

CLAIMS 1.:

1. An ex Vivo method for the preparation of a highly purified and heat stable oxygen carrier-containing pharmaceutical composition, the oxygen r—containing pharmaceutical composition including hemoglobin, the hemoglobin including cross- linked ric hemoglobin, the method comprising: (a) separating red blood cells from plasma in whole blood; (b) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (0) lysing the red blood cells to create a solution sing a lysate of disrupted red blood cells; (d) extracting a first hemoglobin solution from the lysate; (e) performing one or more purification processes to produce a purified hemoglobin solution; (i) cross—linking hemoglobin tetramers to create a solution including cross-linked polymeric hemoglobin, the polymeric hemoglobin including two or more cross—linked hemoglobin tetramers; (g) heat ng the solution including the cross—linked polymeric hemoglobin in or equal to a deoxygenated environment at a temperature greater than 85°C and less than approximately 95°C for a period of less than imately 40 minutes to denature and precipitate any residual oss—linked tetrameric hemoglobin, any dimeric hemoglobin, and any other impurities such to form a heat stable solution including a linked polymeric hemoglobin; (h) cooling the heat—treated solution to a temperature approximately less than or equal to 25°C in approximately two minutes or less to prevent formation of met- hemoglobin; (i) removing precipitate by a centrifugation or a filtration apparatus to form a solution of purified, heat-stable hemoglobin including cross—linked polymeric hemoglobin having an undetectable concentration of dimer.

2. The method of claim 1 r comprising adding N—acetyl cysteine ately following heat treating.

3. The method of claim 1 wherein the g is performed in less than one minute.

4. The method of claim 1 wherein the heat treatment occurs at a temperature 8 minutes to about range of r than 85°C and less than 90°C for a period from about 30 minutes.

5. The method of claim 1 wherein the heat ent occurs at approximately 90 0C for a period from about 45 seconds to about 150 seconds.

6. The method of claim 1 wherein the heat treatment occurs at approximately 95 0C for a period from about 30 to about 100 seconds.

7. The method of claim 1 further sing adding N-acetyl cysteine immediately prior to heat treating.

8. The method of claim 2 fuither comprising adding yl cysteine immediately prior to heat treating.

9. The method of claim 1 n the performing one or more purification ultrafiltration. processes to produce a d hemoglobin solution includes

10. The method of claim 1 wherein the performing one or more ation processes to produce a purified hemoglobin solution includes purification by chromatography.

11. A highly purified and heat stable oxygen carrier—containing pharmaceutical composition including hemoglobin and N—acetyl cysteine, the hemoglobin including cross—linked polymeric hemoglobin, the linked polymeric hemoglobin prepared by: (a) separating red blood cells from plasma in whole blood; (b) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction; (c) lysing the red blood cells to create a solution comprising a lysate of disrupted red blood cells; (d) extracting a first hemoglobin solution from the lysate; (e) performing one or more purification processes on the first hemoglobin solution; (t) cross—linking hemoglobin tetramers in the first hemoglobin solution to create a second hemoglobin solution ing cross—linked ric hemoglobin, the polymeric hemoglobin including two or more cross—linked hemoglobin tetramers; (g) heat treating the second hemoglobin solution including the cross-linked ric hemoglobin in a deoxygenated environment at a temperature greater than 85°C and less than or equal to imately 95°C for a period of less than approximately 40 minutes to denature and precipitate any residual non—reacted hemoglobin, non—stabilized hemoglobin, dimer, and any other impurities such that the resulting hemoglobin solution has an undetectable amount of dimer when measured using high performance liquid chromatography; (h) cooling the heat—treated solution to a temperature approximately less than or equal to 25°C in approximately two minutes or less to prevent formation of met- hemoglobin; (i) adding N—acetyl cysteine either immediately prior to or after heat treating and cooling the second hemoglobin solution; (j) ng precipitate formed during the heat treating by a centrifugation or a filtration apparatus.

12. A method of ating tissue comprising providing the composition of claim ll to animal tissue ex vivo.