Stable hemoglobin protein compositions

Abstract

The present invention relates to a hemoglobin protein composition comprising biologically active hemoglobin and less than 2% metHb, wherein the metHb is maintained at less than 2% after reoxygenation of the hemoglobin. The present invention further provides a hemoglobin protein composition comprising biologically active hemoglobin and charge variants at less than 25% of total oxygenated hemoglobin. The present invention also provides an effective concentration of an antioxidant of 5 mM or more for use during heat treatment. The present invention also provides a pharmaceutically stable hemoglobin composition. The present invention also provides a process for reducing and / or controlling the formation of metHb, charge variants, and inactivating viruses and / or prions during the production of oxygenated hemoglobin. The present invention also provides therapeutic uses of oxygenated hemoglobin.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 416,434, filed October 14, 2022, the entire text of which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to a hemoglobin protein composition (e.g., a stable hemoglobin protein composition) comprising biologically active hemoglobin and less than 2% methemoglobin (metHb), wherein the metHb remains less than 2% even after reoxygenation of the hemoglobin. The present invention also relates to a hemoglobin protein composition comprising biologically active hemoglobin and charge variants that account for less than 25% of the total oxygenated hemoglobin. The present invention also provides an antioxidant at an effective concentration of greater than 5 mM for use during heat treatment. The present invention also provides a pharmaceutically stable hemoglobin composition. The present invention also provides a method for reducing and / or controlling methemoglobin, charge variants, and virus and / or prion inactivation during the production of oxygenated hemoglobin. The present invention also provides therapeutic uses for oxygenated hemoglobin. [Background technology]

[0003] The development of hemoglobin-based oxygen carriers (HBOCs) has focused on oxygen delivery in medical treatments such as blood transfusions. HBOCs have been shown to prevent or treat hypoxia resulting from blood loss (e.g., during acute hemorrhage or surgery), anemia (e.g., pernicious anemia or sickle cell anemia), or shock (e.g., hypovolemic, anaphylactic, septic, or allergic shock).

[0004] Existing hemoglobin-based oxygen carriers include synthetic hemoglobin analogues, liposome-encapsulated hemoglobin, chemically modified hemoglobin, and hemoglobin-based oxygen carriers in which the hemoglobin molecule is crosslinked.

[0005] Hemoglobin is highly susceptible to oxidation in the presence of oxygen, forming oxyhemoglobin called methemoglobin. Methemoglobin is a type of hemoglobin in which the iron in the heme group is replaced by the Fe in normal hemoglobin. 2+ (ferrous iron), but Fe 3+ Methemoglobin is hemoglobin in its ferric (ferric) state. It is sometimes called ferrihemoglobin. Methemoglobin cannot bind oxygen and therefore cannot transport oxygen to tissues. It is a bluish chocolate brown in color. Although trace amounts of MetHb are normally produced naturally in human blood, excessive MetHb causes blood to turn an abnormally dark blue-brown. The NADH-dependent enzyme methemoglobin reductase (a type of diaphorase) converts methemoglobin back to hemoglobin. Normally, 1–2% of a person's hemoglobin is methemoglobin. However, genetic factors and exposure to various chemicals and drugs can increase MetHb concentrations, resulting in the clinical condition known as methemoglobinemia. High MetHb levels can cause functional anemia and promote tissue hypoxia. Furthermore, extracellular MetHb has been shown to be a potent inflammatory risk factor (DAMP). The activity of these DAMPs includes activation of complement Toll-like receptor 4 (TLR4), which has been shown to promote macrophage activation, endothelial dysfunction, and other activities. Many acute illnesses, such as trauma, blood loss, and infection, can cause vascular injury, inflammation, and dysfunction, and transfusion of whole blood or early-generation HBOCs has been observed to increase morbidity and mortality rather than improve outcomes. These conditions are often exacerbated by elevated MetHb levels derived from endogenous red blood cells or transfusion products containing hemoglobin.

[0006] Typical manufacturing processes for purified hemoglobin include harsh purification and viral inactivation using high temperatures or chemicals. Unlike deoxygenated hemoglobin, which is stable at high temperatures, oxygenated (HbO2) hemoglobin denatures and precipitates at high temperatures, making the final HbO2 concentration (%) important. All of these processing steps promote hemoglobin autoxidation and the formation of MetHb. Importantly, this loss of hemoglobin purity is more pronounced during further processing steps, such as reoxygenation and cysteine ​​removal. Because hemoglobin is susceptible to oxidation in the presence of oxygen, suppression or control of MetHb concentration during the reoxygenation step (oxygenated hemoglobin) is important. Furthermore, control of charge variants during the reoxygenation step (oxygenated hemoglobin) has been shown to be important. Therefore, there is a need in the art to effectively control MetHb impurities and / or charge variants during manufacturing or purification processes and maintain hemoglobin purity.

[0007] The present invention effectively controls hemoglobin purity and MetHb formation in oxyhemoglobin by using a specific concentration or amount of L-cysteine ​​during the production / purification stage. The present invention surprisingly found that the use of an antioxidant concentration of more than 5 mM during the production / purification process effectively controls one or more of MetHb formation, charge variants, and hemoglobin purity in the oxyhemoglobin state (reoxygenated hemoglobin). Summary of the Invention

[0008] In some embodiments, the present invention provides a hemoglobin composition comprising: a) oxygenated hemoglobin; and b) MetHb less than 2% of total oxygenated hemoglobin.

[0009] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or greater; and b) one or more impurities selected from MeHb; an acidic variant, a basic variant; and optionally a viral particle and / or a prion.

[0010] In some embodiments, the oxygenated hemoglobin comprises no more than 2% MetHb of the total oxygenated hemoglobin.

[0011] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) containing one or more impurities selected from MeHb, acidic variants, basic variants, and optionally viral particles and / or prions; Here, the oxygenated hemoglobin includes MetHb, which comprises 2% or less of the total oxygenated hemoglobin.

[0012] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or greater and one or more impurities, including: b) Oxygenated hemoglobin containing 2% or less MetHb; c) Oxygenated hemoglobin containing less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin containing less than 11% of the main peak basic variant; e) oxygenated hemoglobin substantially free of viral particles; and f) Oxygenated hemoglobin that is virtually free of prions.

[0013] In some embodiments, MetHb is analyzed by co-oximetry and the main peak purity of oxygenated hemoglobin is analyzed by cIEF (capillary isoelectric focusing).

[0014] In some embodiments, the present invention provides a hemoglobin composition comprising the following components: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) acidic variants account for less than 12% of the main peak; and c) Basic variants account for less than 11% of the main peak.

[0015] In some embodiments, the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, wherein the heat treatment is carried out for at least about 4 hours.

[0016] In some embodiments, the present invention provides a hemoglobin composition comprising:

[0017] a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) the acidic variant is less than 12% of the main peak; and c) The basic variant is less than 11% of the main peak.

[0018] wherein the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, wherein the heat treatment is carried out for at least 4 hours.

[0019] In some embodiments, the present invention provides a hemoglobin composition comprising:

[0020] a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) the acidic variant is less than 12% of the main peak; and c) The basic variant is less than 9% of the main peak.

[0021] In some embodiments, the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, wherein the heat treatment is for at least 4 hours.

[0022] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) acidic variants account for less than 12% of the main peak; and c) Basic variants account for less than 9% of the main peak.

[0023] wherein the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, the heat treatment being for at least 4 hours.

[0024] In some embodiments, the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is performed for a suitable time period selected from 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, and 14 hours. In some embodiments, the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is performed for about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, or about 14 hours.

[0025] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin: d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin; f) heat inactivation of the deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature; g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the oxygenated hemoglobin composition; and i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0026] In some embodiments, the deoxygenated hemoglobin is inactivated by heat treatment. In some embodiments, the L-cysteine ​​concentration in the deoxygenated hemoglobin during step f) is maintained above 5 mM. In some embodiments, the oxygenated hemoglobin composition comprises a main peak purity of greater than 70% and one or more impurities selected from MetHb, acidic variants, basic variants, virus particles, and prions. In some embodiments, the oxygenated hemoglobin composition comprises 2% or less MetHb of the total oxygenated hemoglobin.

[0027] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of:

[0028] a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin; f) heat inactivation of the deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature; g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the oxygenated hemoglobin composition; and i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; wherein the virus inactivation of the deoxygenated hemoglobin is carried out by heat treatment, and the L-cysteine ​​concentration in the deoxygenated hemoglobin is maintained at 5 mM or more during step f), and wherein the oxygenated hemoglobin composition has a main peak purity of greater than 70% and one or more impurities selected from MetHb, acidic variants, basic variants, virus particles, and prions, and wherein the oxygenated hemoglobin composition contains 2% or less MetHb of the total oxygenated hemoglobin.

[0029] In some embodiments, the present invention provides a method for reducing viral and / or prion load in an oxygenated hemoglobin composition obtained from a mammalian source, the method comprising the steps of: a) heat-treating the deoxygenated hemoglobin at about 60°C for at least 4 hours; b) Maintaining an L-cysteine ​​concentration of 5.0 mM or higher during heat treatment; c) reoxygenating the heat-treated deoxygenated hemoglobin to form an oxygenated hemoglobin composition; d) optionally PEGylating the oxygenated hemoglobin composition; and e) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0030] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin; f) viral inactivation of the deoxygenated hemoglobin by heat treatment with an antioxidant at a suitable concentration of more than 5 mM at a suitable temperature to reduce viruses and / or prions; g) reoxygenating the heat-treated deoxygenated hemoglobin composition to produce an oxygenated hemoglobin composition; h) optionally PEGylating the heat-treated oxygenated hemoglobin composition; and i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0031] In some embodiments, the oxyhemoglobin compositions have a lower charge variant compared to the charge variant measured in an oxyhemoglobin composition prepared with 5 mM or less antioxidant.

[0032] In some embodiments, the present invention provides a method for preparing a stable hemoglobin composition, comprising the steps of:

[0033] a) washing fresh whole blood drawn from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells: c) filtering the extracted hemoglobin: d) Ultrafiltration and concentration step: e) ultrafiltration and deoxygenation of the concentrated hemoglobin: f) viral inactivation of the deoxygenated hemoglobin by heat treatment at a suitable temperature with an antioxidant at a suitable concentration of more than 5 mM to reduce viruses and / or prions; g) reoxygenating the heat-treated deoxygenated hemoglobin composition to produce an oxygenated hemoglobin composition: h) optionally PEGylating the heat-treated oxygenated hemoglobin composition; i) optionally performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; wherein the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the process using 5 mM or less antioxidant.

[0034] In some embodiments, the antioxidant is L-cysteine. In some embodiments, the present invention provides a process for controlling and / or reducing the formation of MetHb during the production of an oxygenated hemoglobin composition, the process comprising the steps of: a) performing deoxygenation of hemoglobin to form deoxygenated hemoglobin; b) heat-treating the deoxygenated hemoglobin with about 5.5 mM to about 15 mM L-cysteine; c) reoxygenating the deoxygenated hemoglobin to form an oxygenated hemoglobin composition; d) measuring the MetHb concentration in the oxygenated hemoglobin composition; wherein the oxygenated hemoglobin composition has a MetHb formation rate of less than 2% as analyzed by co-oximetry: e) optionally, PEGylating the oxygenated hemoglobin composition; and f) Optionally, a carboxylation process is performed to produce a carboxylated PEGylated hemoglobin composition.

[0035] In some embodiments, the PEGylated hemoglobin composition of step (e) or the PEGylated carboxylated hemoglobin composition of step (f) maintains MetHb at less than 4%, preferably less than 3%.

[0036] In some embodiments, the present invention provides a method for controlling and / or reducing the formation of MetHb during the production of an oxygenated hemoglobin composition, the method comprising the steps of: a) Deoxygenation of hemoglobin to form deoxyhemoglobin; b) heat-treating deoxygenated hemoglobin with about 5.5 mM to about 15 mM L-cysteine; c) reoxygenating the deoxygenated hemoglobin to form an oxygenated hemoglobin composition; d) measuring the MetHb concentration in the oxygenated hemoglobin composition; Here, the oxygenated hemoglobin composition has a MetHb formation rate of less than 2% as analyzed by co-oximetry.

[0037] g) Optionally, PEGylation of the oxygenated hemoglobin composition is performed.

[0038] h) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0039] wherein the PEGylated hemoglobin composition of step (e) or the PEGylated carboxylated hemoglobin composition of step (f) maintains MetHb at less than 4%, preferably less than 3%.

[0040] In some embodiments, the present invention provides a method for preparing a stable hemoglobin composition, the method comprising: a) Fresh whole blood drawn from an animal source is washed to generate washed red blood cells.

[0041] b) Extraction of hemoglobin from red blood cells.

[0042] c) Filter the extracted hemoglobin.

[0043] d) Ultrafiltration and concentration.

[0044] e) Ultrafiltration and deoxygenation of concentrated hemoglobin is performed.

[0045] f) Viral inactivation of deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature.

[0046] g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the heat-treated deoxygenated hemoglobin; and i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0047] In some embodiments, the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the method using 5 mM or less antioxidant.

[0048] In some embodiments, the present invention provides a method for preparing a stable hemoglobin composition, comprising the steps of: a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) deoxygenating the ultrafiltered and concentrated hemoglobin; f) Viral inactivation of deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature.

[0049] g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the heat-treated deoxygenated hemoglobin; i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Here, the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the present process using 5 mM or less antioxidant.

[0050] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin; and b) MetHb less than 9% of total oxygenated hemoglobin.

[0051] In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 12 hours. In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 11 hours. In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 10 hours. In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 9 hours.

[0052] In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 8 hours. In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 7 hours. In some embodiments, the oxygenated hemoglobin is subjected to heat treatment for about 6 hours.

[0053] In some embodiments, the hemoglobin composition comprises: c) oxygenated hemoglobin; and d) MetHb less than 9% of total oxygenated hemoglobin; Here, the heat treatment is carried out for about 10 hours.

[0054] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin; and b) MetHb less than 4% of total oxygenated hemoglobin; Here, the heat treatment is carried out for about 8 hours.

[0055] In some embodiments, the composition comprises oxygenated hemoglobin having a main peak hemoglobin purity of 70% or greater, less than 2% MetHb, and less than 25% charge variants, where the charge variants are acidic or basic variants of the main peak.

[0056] In some embodiments, the main peak purity of oxygenated hemoglobin is analyzed by cIEF (capillary isoelectric focusing) and MetHb is analyzed by co-oximetry.

[0057] In some embodiments, the acidic and basic variants of the oxygenated hemoglobin composition are analyzed by cIEF (capillary isoelectric focusing).

[0058] In some embodiments, the oxygenated hemoglobin composition has a total MetHb content of less than 2%. In some embodiments, the oxygenated hemoglobin composition comprises a total MetHb content ranging from about 0.5% to about 1.8% of total oxygenated hemoglobin.

[0059] In some embodiments, the stable hemoglobin composition is manufactured on a large scale.

[0060] In some embodiments, large scale is greater than 20 L. In certain embodiments, large scale is greater than 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 5000 L.

[0061] In some embodiments, the purity of the oxygenated hemoglobin composition is about 70% or greater, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, and about 85% or greater.

[0062] In some embodiments, the acidic variant is less than about 20%. In some embodiments, the acidic variant is less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less. In some embodiments, the basic variant is less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less.

[0063] In some embodiments, the heat-treated deoxygenated hemoglobin has a viral reduction of at least 1 log compared to treatment without heat treatment. 10 That's it, 2 log10 It has decreased by more than

[0064] In some embodiments, the heat-treated deoxygenated hemoglobin has a prion reduction of at least 1 log compared to a process performed without heat treatment. 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 or more decreased.

[0065] In some embodiments, a suitable concentration of L-cysteine ​​is 5 mM or greater, hi some embodiments, a suitable concentration of L-cysteine ​​is selected from about 5.5 mM to about 15 mM.

[0066] In some embodiments, the heat treatment process is carried out at a suitable temperature selected from about 55°C to about 75°C.

[0067] In some embodiments, the heat treatment step is carried out for at least 4 hours or more.

[0068] In some embodiments, the heat treatment step is carried out for a suitable time interval selected from about 4 hours to about 15 hours.

[0069] In some embodiments, the present invention provides methods of treating a condition that can be improved by oxygenating red blood cells in a patient in need thereof by administering to the patient a therapeutically effective amount of a stable hemoglobin composition comprising the following components: a) Oxygenated hemoglobin with a main peak purity of 70% or greater; and b) one or more impurities selected from MeHb, acidic variants, basic variants, viral particles and / or prions.

[0070] In some embodiments, the oxygenated hemoglobin comprises 2% or less MetHb of the total oxygenated hemoglobin.

[0071] In some embodiments, the present invention provides a method of treating a condition that can be ameliorated by oxygenating red blood cells in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a stable hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or greater; and b) containing one or more impurities selected from MeHb, acidic variants, basic variants, viral particles and / or prions; Here, the oxygenated hemoglobin contains MetHb in an amount of 2% or less of the total oxygenated hemoglobin.

[0072] In some embodiments, the present invention provides a method of treating a condition that can be improved by oxygenating red blood cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a stable hemoglobin composition comprising the following components: a) Oxygenated hemoglobin with MetHb ≤2%; b) Oxygenated hemoglobin with a main peak purity of 70% or more; c) Oxygenated hemoglobin containing less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin containing less than 11% of the main peak basic variant; and e) Oxygenated hemoglobin substantially free of viral particles and / or prions.

[0073] In some embodiments, the patient's condition is selected from the group comprising acute respiratory distress syndrome, bronchiectasis, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, lymphangiomatosis, primary ciliary dysfunction, cancer, tumors, lung cancer, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, pulmonary sarcoidosis, pneumonia and bronchitis, infections affecting the transport capacity of the lungs.

[0074] In some embodiments, the condition is anemia selected from the group consisting of anemia due to blood loss, anemia due to insufficient red blood cell production, anemia due to red blood cell destruction, and combinations thereof, wherein the cancer is selected from solid tumors, soft tissue cancer, lung cancer, bone cancer, metastatic cancer, adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, and breast cancer.

[0075] In some embodiments, the present invention provides a method of administering a stable hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or greater; and b) one or more impurities selected from MeHb, acidic variants, basic variants, viral particles and / or prions.

[0076] In some embodiments, the oxygenated hemoglobin comprises 2% or less of MetHb of total oxygenated hemoglobin.

[0077] In some embodiments, the present invention provides a method of administering a stable hemoglobin composition comprising:

[0078] c) Oxygenated hemoglobin with a main peak purity of 70% or greater; and d) one or more impurities selected from MetHb, acidic variants, basic variants, viral particles and / or prions.

[0079] Here, the oxygenated hemoglobin contains MetHb in an amount of 2% or less of the total oxygenated hemoglobin.

[0080] In some embodiments, the present invention provides a method of administering a stable hemoglobin composition comprising:

[0081] a) Oxygenated hemoglobin with MetHb ≤2%; b) Oxygenated hemoglobin with a main peak purity of 70% or more; c) Oxygenated hemoglobin with less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin with less than 11% of the main peak basic variant; e) oxygenated hemoglobin substantially free of viral particles and / or prions; Here, MetHb is analyzed by cooximetry and the purity of the oxygenated hemoglobin main peak is analyzed by cIEF (capillary isoelectric focusing).

[0082] In some embodiments, the dosing frequency is once daily or twice daily.

[0083] In some embodiments, the present invention provides a stable hemoglobin composition comprising: a) reoxygenated hemoglobin with a MetHb content of 2% or less; and b) The purity of the reoxygenated hemoglobin main peak is 60% or more; Here, MetHb is analyzed by co-oximetry and the purity of the reoxygenated hemoglobin main peak is analyzed by cIEF (capillary isoelectric focusing).

[0084] In some embodiments, the present invention provides a heat treatment process for removing or reducing potential endogenous adventitious substances in hemoglobin compositions by using an appropriate concentration of L-cysteine.

[0085] In some embodiments, a suitable concentration of L-cysteine ​​is greater than 5 mM. In some embodiments, a suitable concentration of L-cysteine ​​is selected from the range of about 5.5 mM to about 15 mM.

[0086] In some embodiments, the present invention provides a stable hemoglobin composition comprising the steps of:

[0087] a. heat treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time: b. Maintain an adequate concentration of antioxidants above 5mM: c. Reoxygenating hemoglobin compositions: d. Measuring the MetHb concentration in the reoxygenated hemoglobin composition: Here, the reoxygenated hemoglobin composition has a reduced MetHb concentration compared to the MetHb concentration measured in a reoxygenated hemoglobin composition treated with 5 mM or less of an antioxidant.

[0088] In some embodiments, the present invention provides for reoxygenation of the hemoglobin composition after heat treatment, such that the hemoglobin composition maintains a MetHb concentration of less than 2% as analyzed by co-oximetry.

[0089] In some embodiments, the heat treatment process is carried out at a suitable temperature selected from above 59°C to about 65°C.

[0090] In some embodiments, the heat treatment process is carried out for a suitable time period selected from about 5 hours to about 10 hours.

[0091] In some embodiments, the present invention can be applied to remove or reduce all forms of viruses, particularly DNA and RNA viruses, enveloped and non-enveloped viruses, as well as virions and prions or other similar biological systems, and bacteria and fungi. The method is preferably used to reduce bovine viral diarrhea virus (BVDV), retrovirus, parvovirus contamination, bovine spongiform encephalopathy (BSE), and transmissible spongiform encephalopathy (TSE) contamination from hemoglobin solutions.

[0092] In some embodiments, the present invention provides a stable hemoglobin composition comprising: a) Reoxygenated hemoglobin purity greater than 60%; b) reoxygenated hemoglobin that is substantially free of viral particles; and c) Reoxygenated hemoglobin that is substantially free of prions.

[0093] In some embodiments, the viral load of the heat-treated hemoglobin solution is reduced by 1 log compared to before heat treatment. 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 or more decreased.

[0094] In some embodiments, the present invention provides a method for preparing a stable hemoglobin composition, the method comprising the steps of: a. heat treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time; b. Maintaining adequate antioxidant concentrations above 5 mM; c. reoxygenating hemoglobin compositions; and d. measuring the MetHb concentration in the reoxygenated hemoglobin composition; Here, the reoxygenated hemoglobin composition has a lower MetHb concentration compared to the MetHb concentration measured in a reoxygenated hemoglobin composition prepared by the present method using 5 mM or less antioxidant.

[0095] In certain embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a. heat-treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time; b. maintaining an adequate antioxidant concentration above 5 mM; c. reoxygenating the hemoglobin composition; d. Measuring charge variants in the reoxygenated hemoglobin composition, Here, the reoxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in a reoxygenated hemoglobin composition prepared by the present method using 5 mM or less antioxidant. [Brief explanation of the drawings]

[0096] [Figure 1]FIG. 1 shows the relationship between L-cysteine ​​concentration and MetHb formation rate (%) during reoxygenation / oxygenation.

[0097] [Figure 2A] FIG. 2A shows the capillary isoelectric focusing (CIEF-Maurice) profile of a typical hemoglobin pre-heat treatment in the presence of 5 mM L-cysteine.

[0098] [Figure 2B] FIG. 2B shows the capillary isoelectric focusing (cIEF-Maurice) profile after treatment at 60° C. for 10 hours in the presence of 5 mM L-cysteine, showing an increase in acidic and basic charge variants.

[0099] [Figure 2C] FIG. 2C shows the capillary isoelectric focusing (cIEF-Maurice) profile after treatment at 60° C. for 10 hours in the presence of 15 mM L-cysteine, showing no increase in charge variants.

[0100] [Figure 3] FIG. 3 shows the effect of changing the L-cysteine ​​concentration during heat treatment on oxyhemoglobin purity / main peak purity, as analyzed by capillary isoelectric focusing (cIEF). DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention effectively controls MetHb formation during the production or purification process of oxygenated hemoglobin (e.g., reoxygenated hemoglobin). Furthermore, the present invention also provides effective control of charge variants and viruses and / or prions during the production or purification process of reoxygenated / oxygenated hemoglobin. According to the present invention, the percentage of MetHb in reoxygenated / oxygenated hemoglobin is less than about 2% (e.g., less than about 2%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.95%, less than about 0.9%, less than about Less than 0.85%, less than about 0.8%, less than about 0.75%, less than about 0.7%, less than about 0.65%, less than about 0.6%, less than about 0.55%, less than about 0.5%, less than about 0.45%, less than about 0.4%, less than about 0.35%, less than about 0.3%, less than about 0.25%, less than about 0.2%, less than about 0.15%, less than about 0.1%, or less than about 0.05%.

[0102] The present invention addresses the need to effectively control MetHb formation in reoxygenated / oxygenated hemoglobin compositions by using specific amounts or concentrations of antioxidants (e.g., cysteine, e.g., L-cysteine).

[0103] The present invention also provides hemoglobin stability at elevated temperatures during heat treatment.

[0104] As used herein, the term "comprises" or "comprising" does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is to be regarded as any embodiment of the term "comprising". Hereinafter, if a group is defined to include at least a certain number of embodiments, this is also to be understood as disclosing the group optionally consisting of only these embodiments.

[0105] As used throughout this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0106] As used herein, the term "about" refers to a range of about 10-20% more or less than a reference value. In certain circumstances, due to the nature of the reference value, one of ordinary skill in the art will recognize that the term "about" can mean a deviation of 10-20% or more or a deviation of less than 10-20% from the reference value.

[0107] As used herein, the term "substantially free" refers to a hemoglobin composition that is free of one or more impurities selected from MetHb, virions, prions, or such adventitious agents, charge variants, and has a purity of greater than about 60% (e.g., greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%). In some embodiments, the term "substantially free of viruses and / or prions" refers to a purified hemoglobin composition that is about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more free from adventitious agents, viruses, or prions. In some embodiments, the term "substantially free of viruses and / or prions" refers to a hemoglobin composition that is free of adventitious agents, viruses, or prions by 1 log 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 This refers to a decrease of more than

[0108] As used herein, the terms "hemoglobin" and "Hb" are used interchangeably and generally refer to the oxygen-transporting protein contained within red blood cells. The hemoglobin used in the present invention can be derived from virtually any mammal. Exemplary sources of hemoglobin include common livestock, such as cattle, pigs, and sheep. The present invention is not limited by the source of the hemoglobin. In various embodiments, the hemoglobin is bovine hemoglobin. The term "hemoglobin composition" refers to a hemoglobin composition containing a desired amount of MetHb, wherein the hemoglobin is reoxygenated hemoglobin. In some embodiments, a hemoglobin composition is considered stable when it contains 2% or less MetHb. In some embodiments, a hemoglobin composition contains 2% or less MetHb and has a main peak purity of 70% or greater for hemoglobin. In some embodiments, a hemoglobin composition contains charge variants that are less than 25% of the main peak purity of reoxygenated hemoglobin. Charge variants include acidic and basic variants with less than 12% of the main peak purity. In some embodiments, the hemoglobin composition is substantially free of viral particles and prions. In certain embodiments, the pH of the hemoglobin composition is 7.4 to 8.2. In some embodiments, the pH of the PEGylated and / or carboxylated hemoglobin composition is 7.4 to 8.0.

[0109] As used herein, the term "tHb" refers to the total hemoglobin content in the manufacturing process of purified hemoglobin.

[0110] The term "stable hemoglobin" composition refers to a hemoglobin composition that provides batch-to-batch stability, thereby helping to achieve batch-to-batch consistency. For further clarification, a stable hemoglobin composition maintains the desired level of hemoglobin / reoxygenated hemoglobin major purity peak at greater than 70% by cIEF analysis and maintains MetHb at less than 2% by co-oximetry analysis. This also aids in long-term storage of PEG-hemoglobin or PEG-Hb-CO compositions. For clarity, stable hemoglobin referred to herein refers to non-PEGylated oxygenated hemoglobin. PEGylated hemoglobin or carboxylated hemoglobin is produced by attaching PEG molecules to oxygenated hemoglobin. PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) maintains less than 5% MetHb during storage when MetHb is controlled at less than 2% during the production of oxygenated hemoglobin. In certain embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) maintains less than 4% MetHb during storage when MetHb is controlled to less than 2% during production of oxygenated hemoglobin. In some embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) maintains less than 3% MetHb during storage when MetHb is controlled to less than 2% during production of oxygenated hemoglobin.

[0111] In some embodiments, the oxygenated hemoglobin compositions maintain less than 2% MetHb during storage for 1 day, 3 days, 7 days, 1 month, and greater than 3 months at 2-8° C. In some embodiments, the oxygenated hemoglobin compositions maintain less than 2% MetHb when stored below −70° C. for 1 month, 3 months, 6 months, 9 months, and greater than 12 months. In some embodiments, the p50 value of reoxygenated / oxygenated hemoglobin is 7-16 mmHg when analyzed by a Hemox Analyzer.

[0112] In some embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) compositions maintain MetHb at less than 5%, preferably less than 4%, when stored for 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, and 48 months or longer at 2-8° C. In some embodiments, the p50 value for reoxygenated / oxygenated hemoglobin is 7-16 mmHg when analyzed by a Hemox Analyzer.

[0113] In some embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) compositions maintain MetHb at less than 5%, preferably less than 4%, when stored at 25° C. for 1 month or more, 3 months or more, 6 months or more, 9 months or more, 12 months or more, 15 months or more, 18 months or more, 21 months or more, 24 months or more, 27 months or more, 30 months or more, 33 months or more, 36 months or more, 39 months or more, 42 months or more, 45 months or more, and 48 months or more. In some embodiments, the p50 value for reoxygenated / oxygenated hemoglobin is 7-16 mmHg when analyzed by a Hemox Analyzer.

[0114] In some embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) compositions maintain MetHb concentrations below 5%, preferably below 4%, when stored at 40° C. for 1 month or more, 3 months or more, 6 months or more, 9 months or more, and 12 months or more. In some embodiments, the p50 value for reoxygenated / oxygenated hemoglobin is measured by a Hemox Analyzer between 7 and 16 mmHg. In some embodiments, PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) compositions maintain MetHb below 3% when stored at 2-8° C. for 1 month or more, 3 months or more, 6 months or more, 9 months or more, 12 months or more, 15 months or more, 18 months or more, 21 months or more, 24 months or more, 27 months or more, 30 months or more, 33 months or more, 36 months or more, 39 months or more, 42 months or more, 45 months or more, and 48 months or more. In some embodiments, the p50 value for reoxygenated / oxygenated hemoglobin is between 7 and 16 mmHg on a Hemox Analyzer. The term "reoxygenation" refers to the process of attaching oxygen (O2) molecules to deoxygenated hemoglobin by methods commonly known in the art, such as diafiltration.

[0115] As used herein, the terms "reoxygenated hemoglobin" or "reoxygenated hemoglobin" or "oxygenated hemoglobin" or "oxygenated hemoglobin" are used interchangeably and refer to an oxygenated hemoglobin molecule prepared after viral inactivation or heat treatment by attaching oxygen (O) that was deoxygenated before viral inactivation or heat treatment. In some embodiments, reoxygenation is performed by methods commonly known in the art.

[0116] The terms "heat-treated oxygenated hemoglobin" or "reoxygenated hemoglobin" or "oxygenated hemoglobin" are used interchangeably and refer to hemoglobin after the reoxygenation / oxygenation step.

[0117] The term "post-reoxygenation" refers to the hemoglobin composition after diafiltration or after the reoxygenation step. Reoxygenation of hemoglobin is accomplished with a buffer solution by diafiltration as commonly known in the art.

[0118] The terms "PEG-hemoglobin" and "PEG-Hb" are used interchangeably and refer to reoxygenated / oxygenated hemoglobin to which PEG molecules have been attached to improve the half-life of oxygenated hemoglobin. The PEGylation process involves attaching 8 to 10 chains of 5 kD activated polyethylene glycol (SC-PEG-5K) molecules to purified reoxygenated / oxygenated hemoglobin. PEG-Hb can deliver oxygen to tissues. Alternatively, PEG-Hb can be bound to carbon monoxide (CO) instead of oxygen, and is referred to as PEG-Hb-CO. In some embodiments, the p50 value of PEG-hemoglobin or PEG-Hb-CO is 7 to 16 mmHg when analyzed using a Hemox Analyzer.

[0119] In some embodiments, the PEG-hemoglobin or PEG-Hb-CO compositions contain less than 2% MetHb. In some embodiments, the PEG-hemoglobin or PEG-Hb-CO compositions contain about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, and about 1.8% MetHb of the total PEG-Hb. When MetHb is controlled to less than 2% during PEG-Hb manufacturing, it has been observed that MetHb remains below 3% even when the PEG-Hb is stored for long periods at 2-8°C. In certain embodiments, the PEG-hemoglobin or PEG-Hb-CO compositions maintain less than 2% MetHb when stored at 2-8°C for 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, and 48 months or longer.

[0120] As used herein, the terms "deoxygenated hemoglobin" or "deoxygenated Hb" are used interchangeably and refer to hemoglobin in which the Fe(II) atom is bound to a species other than oxygen (e.g., carbon monoxide) or is not bound to oxygen or other species. The hemoglobin is deoxygenated by passing it through a countercurrent membrane contactor (Liqui-Cel®). The hemoglobin is deoxygenated before heat treatment. The term "carboxylated" refers to gas-bound hemoglobin. In some embodiments, the gas-bound hemoglobin has CO bound to the hemoglobin.

[0121] As used herein, the term "antioxidant" refers to a molecule (e.g., an amino acid) that can inhibit or reverse oxidation reactions. In the present invention, antioxidants are used to prevent or reduce the formation of impurities associated with hemoglobin, where one or more impurities are selected from MetHb, acidic variants, and basic variants. In certain embodiments, antioxidants are useful during heat treatment of hemoglobin / deoxyhemoglobin to reduce viruses and / or prions. In some embodiments, the antioxidant is selected from N-acetylcysteine, glutathione, ascorbic acid, and preferably L-cysteine. In some embodiments, antioxidants are used at concentrations greater than 5 mM during heat treatment.

[0122] As used herein, the terms "cIEF" or "capillary isoelectric focusing" are used interchangeably and refer to an analytical method for determining the purity / impurity concentration in a hemoglobin solution. Capillary isoelectric focusing (cIEF) is a high-resolution analytical technique that allows for the separation of protein / peptide mixtures, protein glycoforms, and other charge variants based on their isoelectric points (pI). This method can be performed by any skilled technician.

[0123] As used herein, the term "reduced" means that the amount of impurity produced is reduced by about 60-80%.

[0124] As used herein, the term "impurities" refers to MetHb (inactive hemoglobin), charge variants, and adventitious agents from sources such as substances, viruses, and / or prions.

[0125] The term "MetHb" or "methemoglobin" refers to dysfunctional hemoglobin (an impurity) that cannot carry oxygen.

[0126] MetHb may be measured by any art-recognized analytical method. In the present invention, MetHb impurity is measured by co-oximetry.

[0127] As used herein, the term "co-oximetry" refers to the measurement of various forms of hemoglobin by a dedicated multi-wavelength spectrophotometer. This is a measure of the potential oxygen-carrying capacity of blood. A CO-oximeter is a multi-wavelength spectrophotometer that measures the absorbance of blood at different wavelengths and automatically calculates the fractional concentrations of the four major hemoglobin species (oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and MetHb) from the total hemoglobin concentration.

[0128] The terms "main peak purity" or "purity" can be used interchangeably and refer to the percentage of total hemoglobin excluding MetHb and other impurities (such as charge variants, viruses, prions, and other adventitious agents).

[0129] Purity is determined by art-recognized methods of analysis (eg, capillary isoelectric focusing, band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, HPLC, or similar methods).

[0130] The term "charge variants" includes product-related impurities. In certain embodiments, charge variants include acidic and / or basic variants.

[0131] As used herein, the terms "acidic variant" or "acidic species" and "AV" refer to a variant of a protein characterized by an overall acidic charge.

[0132] The term "basic variant" or "basic species" as used refers to variants that may arise from the presence of an additional positive charge on the C-terminal lysine or the removal of the negative charge.

[0133] The acidic and basic charge variants are analyzed by cIEF (capillary isoelectric focusing) methods commonly known in the art.

[0134] The term "heat treatment" refers to a method or process carried out at a specific temperature suitable for inactivating viruses and / or prions during the preparation of oxygenated hemoglobin, preferably at a temperature of about 55° C. to about 75° C., and particularly preferably at 60±1° C. Heat treatment has been observed to cause the formation of impurities in oxygenated hemoglobin.

[0135] In certain embodiments, the heat treatment is carried out using deoxygenated hemoglobin. As used herein, the term "high temperature" refers to a temperature of about 55°C to 75°C.

[0136] The term "large scale" refers to industrial scale of 10 L or greater. In embodiments, large scale is selected from 20 L to 1000 L. In certain embodiments, large scale is greater than 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 5000 L. In one aspect of such embodiments, the present invention provides batch-to-batch consistency at large scale. The present invention maintains impurities within tolerances required by regulatory agencies.

[0137] The meaning of other terms used herein will be readily apparent to those skilled in the art.

[0138] In some embodiments, the present invention provides a hemoglobin composition comprising: a) oxygenated hemoglobin; and b) MetHb less than 2% of total oxygenated hemoglobin.

[0139] In such embodiments, the oxygenated hemoglobin has a main peak purity of 70% or greater.

[0140] In some embodiments, the present invention provides a hemoglobin composition comprising:

[0141] a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) MetHb less than 2% of total oxygenated hemoglobin.

[0142] In some embodiments, the present invention provides a hemoglobin composition comprising:

[0143] a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) charge variants selected from MeHb, acidic variants, basic variants, and optionally one or more impurities selected from viral particles and / or prions; Here, oxygenated hemoglobin includes MetHb that accounts for 2% or less of the total oxygenated hemoglobin.

[0144] In some embodiments, the present invention provides the following hemoglobin compositions: a) Oxygenated hemoglobin with a main peak purity of 70% or greater and one or more of the following impurities: b) Oxygenated hemoglobin with a MetHb content of 2% or less; c) Oxygenated hemoglobin containing less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin containing less than 11% of the main peak basic variant; e) oxygenated hemoglobin substantially free of viral particles; and f) Oxygenated hemoglobin substantially free of prions: Here, MetHb is analyzed by cooximetry and the main peak purity of oxygenated hemoglobin is analyzed by cIEF (capillary isoelectric focusing).

[0145] In some embodiments, the present invention provides a hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) the acidic variant is less than 12% of the main peak; c) the basic variant is less than 11% of the main peak; wherein the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, the heat treatment being for at least 4 hours.

[0146] In some embodiments, the oxygenated hemoglobin is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is carried out for a suitable time period selected from 8 hours, 10 hours, 12 hours, and 14 hours.

[0147] In some embodiments, the present invention provides a hemoglobin composition comprising the following components: a) Oxyhemoglobin with a main peak purity of 70% or more; b) the acidic variant is less than 12% of the main peak; c) The basic variant is less than 9% of the main peak.

[0148] Here, the oxygenated hemoglobin composition is obtained from heat-treated deoxygenated hemoglobin, the heat treatment being for at least 4 hours.

[0149] In some embodiments, the oxygenated hemoglobin is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is carried out for a suitable time selected from 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, and 14 hours.

[0150] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin; f) heat inactivating the deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature; g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the oxygenated hemoglobin composition; i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; wherein the viral inactivation of the deoxygenated hemoglobin is carried out by heat treatment and during step f) an L-cysteine ​​concentration in the deoxygenated hemoglobin is maintained at 5 mM or greater; wherein the oxygenated hemoglobin composition has a main peak purity of greater than 70% and one or more impurities selected from MetHb, acidic variants, basic variants, virus particles, and prions; and wherein the oxygenated hemoglobin composition contains 2% or less MetHb of total oxygenated hemoglobin.

[0151] In some embodiments, the present invention provides a method for reducing viral and / or prion load in an oxygenated hemoglobin composition derived from a mammal, the method comprising the steps of: a) heat-treating the deoxygenated hemoglobin at about 60°C for at least 4 hours; b) Maintaining the L-cysteine ​​concentration at 5.0 mM or higher during heat treatment; c) reoxygenating the heat-treated deoxygenated hemoglobin to form an oxygenated hemoglobin composition; d) optionally PEGylating the oxygenated hemoglobin composition; e) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition.

[0152] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a) Washing fresh whole blood of animal origin to produce washed red blood cells; b) extracting hemoglobin from red blood cells: c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin; i) viral inactivation of the deoxygenated hemoglobin by heat treatment at a suitable temperature with an antioxidant at a suitable concentration of more than 5 mM to reduce viruses and / or prions; g) reoxygenating the heat-treated deoxygenated hemoglobin composition to produce an oxygenated hemoglobin composition; h) optionally PEGylating the heat-treated oxygenated hemoglobin composition; i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Here, the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the present process using 5 mM or less antioxidant.

[0153] In some embodiments, the antioxidant is selected from N-acetylcysteine, glutathione, ascorbic acid, and preferably L-cysteine.

[0154] In some embodiments, the present invention provides a method for controlling and / or reducing the formation of MetHb during the production of an oxygenated hemoglobin composition, the method comprising: a) deoxygenating hemoglobin to form deoxygenated hemoglobin; b) heat-treating the deoxygenated hemoglobin with about 5.5 mM to about 15 mM L-cysteine; c) reoxygenating the deoxygenated hemoglobin to form an oxygenated hemoglobin composition; d) measuring the concentration of MetHb in the oxygenated hemoglobin composition, wherein the oxygenated hemoglobin composition has a MetHb formation rate of less than 2% when analyzed by co-oximetry; e) optionally, PEGylating the oxygenated hemoglobin composition; f) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; wherein the PEGylated hemoglobin composition of step (e) or the PEGylated carboxylated hemoglobin composition of step (f) maintains MetHb at less than 4%, preferably less than 3%.

[0155] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a) washing fresh whole blood collected from an animal source to produce washed red blood cells; b) extracting hemoglobin from red blood cells; c) filtering the extracted hemoglobin; d) ultrafiltration and concentration; e) ultrafiltration and deoxygenation of the concentrated hemoglobin: f) viral inactivation of the deoxygenated hemoglobin by heat treatment at an appropriate temperature to reduce viruses and / or prions; g) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; h) optionally PEGylating the heat-treated deoxygenated hemoglobin; i) optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Here, the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the present method using 5 mM or less antioxidant.

[0156] In some embodiments, the hemoglobin composition comprises: a) Oxygenated hemoglobin: b) MetHb less than 9% of total oxygenated hemoglobin; where heat treatment is carried out for approximately 10 hours.

[0157] In some embodiments, the hemoglobin composition comprises the following components: a) Oxygenated hemoglobin; b) MetHb less than 4% of total oxygenated hemoglobin; Here, the heat treatment is carried out for about 8 hours.

[0158] In some embodiments, the composition comprises oxygenated hemoglobin having a main peak hemoglobin purity of greater than 70%, less than 2% MetHb, and less than 25% charge variants, wherein the charge variants are acidic or basic variants of the main peak.

[0159] In some embodiments, the oxygenated hemoglobin is obtained from heat-treated deoxygenated hemoglobin, wherein the heat treatment is for at least 4 hours.

[0160] In some embodiments, the main peak purity of oxygenated hemoglobin, acidic variants and basic variants is analyzed by cIEF (capillary isoelectric focusing).

[0161] In some embodiments, the main peak purity of oxygenated hemoglobin is analyzed by cIEF (capillary isoelectric focusing) and MetHb is analyzed by co-oximetry. In some embodiments, the main peak purity of oxygenated hemoglobin is greater than 70%. In some embodiments, the main peak purity of oxygenated hemoglobin is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, and about 85% or greater.

[0162] In some embodiments, the reoxygenated / oxygenated hemoglobin composition contains less than 25% charge variants. In some embodiments, the charge variants in the reoxygenated / oxygenated hemoglobin composition are acidic or basic charge variants. In some embodiments, the reoxygenated / oxygenated hemoglobin composition contains less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less charge variants, and has a main peak purity of greater than 60%. In some embodiments, the acidic charge variants in the reoxygenated / oxygenated hemoglobin composition are less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less.

[0163] In some embodiments, the basic charge variants in the reoxygenated / oxygenated hemoglobin composition are less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less.

[0164] In some embodiments, acidic and basic variants of oxygenated hemoglobin are analyzed by cIEF (capillary isoelectric focusing).

[0165] In some embodiments, the oxygenated hemoglobin is increased by 1 log10 have a reduced viral load.

[0166] In some embodiments, oxygenated hemoglobin is reduced by 1 log compared to a process performed without heat treatment. 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 A decrease in viral load, selected from ≥ 100 or ≥ 100, was observed.

[0167] In some embodiments, oxygenated hemoglobin is reduced by 1 log compared to a process performed without heat treatment. 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 In some embodiments, the suitable concentration of L-cysteine ​​is 5 mM or more. In some embodiments, the suitable concentration of L-cysteine ​​is selected from about 5.5 mM to about 20 mM.

[0168] In one aspect of such embodiment, a suitable concentration of L-cysteine ​​is selected from about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, mM, about 8.0mM, about 8.1mM, about 8.2mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9.0mM, about 9.1mM, about 9.2mM, about 9.3mM, about 9. 4mM, about 9.5mM, about 9.6mM, about 9.7mM, about 9.8mM, about 9.9mM, about 10mM, about 10.1mM, about 10.2mM, about 10.3mM, about 10.4mM, about 10.5mM, about 10.6mM, about 10.7mM, about 10 .8mM, about 10.9mM, about 11.0mM, about 11.1mM, about 11.2mM, about 11.3mM, about 11.4mM, about 11.5mM, about 11.6mM, about 11.7mM, about 11.8mM, about 11.9mM, about 12.0mM, about 12 .1mM, about 12.2mM, about 12.3mM, about 12.4mM, about 12.5mM, about 12.6mM, about 12.7mM, about 12.8mM, about 12.9mM, about 13.0mM, about 13.1mM, about 13.2mM, about 13.3mM, about 13 0.4 mM, about 13.5 mM, about 13.6 mM, about 13.7 mM, about 13.8 mM, about about 13.9 mM, about 14.0 mM, about 14.1 mM, about 14.2 mM, about 14.3 mM, about 14.4 mM, about 14.5 mM, about 14.6 mM, about 14.7 mM, about 14.8 mM, about 14.9 mM, about 15.0 mM, about 16.0 mM, about 16.5 mM, about 17.0 mM, about 17.5 mM, about 18.0 mM, about 18.5 mM, about 19.0 mM, about 19.5 mM, and about 20.0 mM.

[0169] In some embodiments, the heat treatment process is carried out at a suitable temperature selected from about 55°C to about 75°C.

[0170] In some embodiments, the heat treatment step is carried out at a suitable temperature selected from the following temperatures: about 57.0°C, about 57.1°C, about 57.2°C, about 57.3°C, about 57.4°C, about 57.5°C, about 57.6°C, about 57.7°C, about 57.8°C, about 57.9°C, about 58.0°C, about 58.1°C, about 58.2°C, about 58.3°C, about 58.4°C, about 58.5°C, About 58.6°C, about 58.7°C, about 58.8°C, about 58.9°C, about 59.0°C, about 59.1°C, about 59.2°C, about 59.3°C, about 59.4°C, about 59.5°C, about 59.6°C, about 59.7°C, about 59.8°C, about 59.9°C, about 60.0°C, about 60.1°C, about 60.2°C, about 60.3°C, about 60.4°C, about 60.5°C, about 60.6°C, about 60.7°C, About 60.8°C, about 60.9°C, about 61.0°C, about 61.1°C, about 61.2°C, about 61.3°C, about 61.4°C, about 61.5°C, about 61.6°C, about 61.7°C, about 61.8°C, about 61.9°C, about 62.0°C, about 62.1°C, about 62.2°C, about 62.3°C, about 62.4°C, about 62.5°C, about 62.6°C, about 62.7°C, about 62.8°C, about 62.9°C, about 63.0°C, about 63.1°C, about 63.2°C, about 63.3°C, about 63.4°C, about 63.5°C, about 63.6°C, about 63.7°C, about 63.8°C, about 63.9°C, about 64.0°C, about 64.1°C, about 64.2°C, about 64.3°C, about 64.4°C, about 64.5°C, about 64.6°C, about 64.7°C, about 64.8°C, about 64.9°C, and about 65.0°C.

[0171] In some embodiments, the heat treatment step is carried out for at least 4 hours or more.

[0172] In some embodiments, the heat treatment step is carried out for a suitable time period selected from about 4 hours to about 15 hours.

[0173] In some embodiments, the heat treatment step is carried out for a suitable time period selected from about 5 hours to about 10 hours.

[0174] In some embodiments, the heat treatment step is carried out for a suitable period of time selected from the following periods: about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5.0 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6. 0 hours, approximately 6.1 hours, approximately 6.2 hours, approximately 6.3 hours, approximately 6.4 hours, approximately 6.5 hours, approximately 6.6 hours, approximately 6.7 hours, approximately 6.8 hours, approximately 6.9 hours, approximately 7.0 hours, approximately 7.1 hours, approximately 7.2 hours, approximately 7.3 hours, approximately 7.4 hours, approximately 7.5 hours, approximately 7.6 hours, approximately 7.7 hours, approximately 7.8 hours, approximately 7.9 hours, approximately 8.0 hours, approximately 8.1 hours, approximately 8.2 Hours, approx. 8.3 hours, approx. 8.4 hours, approx. 8.5 hours, approx. 8.6 hours, approx. 8.7 hours, approx. 8.8 hours, approx. 8.9 hours, approx. 9.0 hours, approx. 9.1 hours, approx. 9.2 hours, approx. 9.3 hours, approx. 9.4 hours, approx. 9.5 hours, approx. 9.6 hours, approx. 9.7 hours, approx. 9.8 hours, approx. 9.9 hours, approx. 10.0 hours, approx. 10.1 hours, approx. 10.2 hours, approx. 10.3 hours, approx. 10.4 hours, about 10.5 hours, about 10.6 hours, about 10.7 hours, about 10.8 hours, about 10.9 hours, about about 11.0 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours, and about 15.0 hours.

[0175] In some embodiments, the reoxygenated hemoglobin comprises a total percentage of MetHb ranging from about 0.5% to about 1.8% of the total reoxygenated hemoglobin.

[0176] In some embodiments, the present invention provides reoxygenated hemoglobin comprising a total percentage of MetHb selected from about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, and about 1.8% of total reoxygenated hemoglobin.

[0177] In some embodiments, the stable hemoglobin composition is produced on a large scale. In some embodiments, the present invention provides a stable hemoglobin composition that is produced on a large scale of greater than 20 L.

[0178] In some embodiments, the stable hemoglobin composition is produced on a large scale selected from about 20 L to about 1000 L.

[0179] In some embodiments, large scale is greater than 20 L. In certain embodiments, large scale is greater than 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 5000 L.

[0180] In certain embodiments, the oxygenated hemoglobin composition maintains MetHb levels below 2% when stored for 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, and 48 months or longer at 2-8° C. In some embodiments, the p50 value of reoxygenated / oxygenated hemoglobin is 7-16 mmHg when analyzed by a Hemox Analyzer.

[0181] In some embodiments, the present invention provides methods for controlling MetHb formation in reoxygenated / oxygenated hemoglobin followed by viral inactivation of deoxygenated hemoglobin.

[0182] In some embodiments, the present invention provides a heat treatment process for removing or reducing viruses in hemoglobin compositions using L-cysteine ​​at a suitable concentration of greater than 5 mM, which has been observed to result in greater than 9% MetHb formation during about 9 to about 10 hours of heat treatment.

[0183] It has also been observed that 5 mM L-cysteine ​​results in greater than 4% MetHb formation during heat treatment for about 4 to about 5 hours.

[0184] In some embodiments, the present invention provides for reoxygenation of hemoglobin after heat treatment, and the oxygenated hemoglobin is maintained at a MetHb concentration of less than 2% as analyzed by co-oximetry.

[0185] In some embodiments, the present invention reoxygenates hemoglobin after heat treatment, where heat treatment with greater than 5 mM L-cysteine ​​for about 9 to about 10 hours maintains oxygenated hemoglobin at a MetHb concentration of less than 9% as analyzed by co-oximetry.

[0186] In some embodiments, the present invention reoxygenates hemoglobin after heat treatment, where heat treatment with greater than 5 mM L-cysteine ​​for about 4 to about 5 hours maintains oxygenated hemoglobin at a MetHb concentration of less than 4% as analyzed by co-oximetry.

[0187] In some embodiments, the reoxygenation of the deoxygenated hemoglobin composition is performed by diafiltration. In some embodiments, the diafiltration operating conditions (e.g., feed pressure, TMP, weight) are maintained by an automated recipe to consistently remove L-cysteine ​​from the viral inactivation process, reoxygenate intermediates, and perform buffer exchange.

[0188] In some embodiments, reoxygenation of the deoxygenated hemoglobin composition is achieved by maintaining the temperature of the buffer solution at about 5°C to about 10°C.

[0189] In some embodiments, the reoxygenated hemoglobin composition is further subjected to anion exchange purification by Sartobind Q filtration to produce purified hemoglobin.

[0190] In some embodiments, the purified hemoglobin of the Sartobind Q filtrate is subjected to a PEGylation process to produce PEGylated hemoglobin containing L-cysteine.

[0191] In some embodiments, the PEG-hemoglobin or PEG-Hb-CO compositions contain less than 2% MetHb. In some embodiments, the PEG-hemoglobin or PEG-Hb-CO compositions contain about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, and about 1.8% MetHb of the total PEG-Hb. When MetHb is controlled to less than 2% during the manufacture of the PEG-Hb hemoglobin, it has been observed that MetHb remains below 3% even during long-term storage of the PEG-Hb at 2-8°C. In certain embodiments, the PEGylated hemoglobin or PEG-Hb-CO compositions maintain less than 2% MetHb when stored at 2-8°C for 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, and 48 months or longer.

[0192] In some embodiments, the PEGylated hemoglobin or PEGylated carboxylated hemoglobin maintains MetHb at less than 4%, preferably less than 3%. In some embodiments, the PEGylated hemoglobin or PEGylated carboxylated hemoglobin maintains MetHb at less than 4%, preferably less than 3%, and is substantially free of viruses and prions.

[0193] In some embodiments, the PEGylated hemoglobin or PEGylated carboxylated hemoglobin maintains acidic charge variants at less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or below, and basic charge variants at less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or below.

[0194] In some embodiments, the present invention provides a method of treating a condition that can be improved by oxygenating red blood cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a stable hemoglobin composition comprising the following components: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) one or more impurities selected from MetHb, acidic variants, basic variants, viral particles and / or prions; Here, the oxygenated hemoglobin contains MetHb in an amount of 2% or less of the total oxygenated hemoglobin.

[0195] In some embodiments, the present invention provides a method of treating a condition that can be improved by oxygenating red blood cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a stable hemoglobin composition comprising the following components: a) Oxygenated hemoglobin with MetHb ≤2%; b) Oxygenated hemoglobin with a main peak purity of 70% or more; c) Oxygenated hemoglobin with less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin with less than 11% of the main peak basic variant; e) Oxygenated hemoglobin substantially free of viral particles and / or prions.

[0196] In some embodiments, the present invention provides a stable hemoglobin composition comprising the following components: a) reoxygenated hemoglobin containing 2% or less MetHb; and b) Reoxygenated hemoglobin main peak purity of 60% or more.

[0197] Here, MetHb is analyzed by co-oximetry and reoxygenated hemoglobin main peak purity is analyzed by cIEF (capillary isoelectric focusing).

[0198] In some embodiments, the present invention provides a heat treatment process for removing or reducing viruses in hemoglobin compositions using an appropriate concentration of L-cysteine.

[0199] In some embodiments, the suitable concentration of L-cysteine ​​is greater than 5 mM.

[0200] In one embodiment, the suitable concentration of L-cysteine ​​is selected from about 5.5 mM to about 15 mM.

[0201] In one aspect of such an embodiment, a suitable concentration of L-cysteine ​​is selected from the following concentrations: approx. 5.5mM, approx. 5.6mM, approx. 5.7mM, approx. 5.8mM, approx. 5.9mM, approx. 6.0mM, approx. 6.1mM, approx. 6.2mM, approx. 6.3mM , about 6.4mM, about 6.5mM, about 6.6mM, about 6.7mM, about 6.8mM, about 6.9mM, about 7.0mM, about 7.1mM, about 7.2m M, about 7.3mM, about 7.4mM, about 7.5mM, about 7.6mM, about 7.7mM, about 7.8mM, about 7.9mM, about 8.0mM, about 8.1 mM, about 8.2mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9.0 mM, about 9.1mM, about 9.2mM, about 9.3mM, about 9.4mM, about 9.5mM, about 9.6mM, about 9.7mM, about 9.8mM, about 9. 9mM, about 10mM, about 10.1mM, about 10.2mM, about 10.3mM, about 10.4mM, about 10.5mM, about 10.6mM, about 10. 7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.1 mM, about 11.2 mM, about 11.3 mM, about 11.4 mM, about 11.5 mM, about 11.6 mM, about 11.7 mM, about 11.8 mM, about 11.9 mM, about 12.0 mM, about 12.1 mM, about 12.2 mM, about 12.3mM, approx. 12.4mM, approx. 12.5mM, approx. 12.6mM, approx. 12.7mM, approx. 12.8mM, approx. 12.9mM, approx. 13.0mM , about 13.1mM, about 13.2mM, about 13.3mM, about 13.4mM, about 13.5mM, about 13.6mM, about 13.7mM, about 13.8 mM, approx. 13.9mM, approx. 14.0mM, approx. 14.1mM, approx. 14.2mM, approx. 14.3mM, approx. 14.4mM, approx. 14.5mM, approx. 14 .6mM, approx. 14.7mM, approx. 14.8mM, approx. 14.9mM, approx. 15.0mM, approx. 6.0mM, approx. 6.1mM, approx. 6.2mM, approx. 6.3m M, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM, about 8.1 mM mM, about 8.2mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9. 0mM, about 9.1mM, about 9.2mM, about 9.3mM, about 9.4mM, about 9.5mM, about 9.6mM, about 9.7mM, about 9.8mM, about 9.9 mM, about 10 mM, about 10.1 mM, about 10.2 mM, about 10.3 mM, about 10.4 mM, about 10.5 mM, about 10.6 mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, about 14.0 mM, about 14.0 mM, about 15.0 mM.

[0202] In some embodiments, the present invention provides a stable hemoglobin composition comprising the steps of: a. heat-treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time; b. maintaining an adequate antioxidant concentration above 5 mM; c. reoxygenating the hemoglobin composition; d. measuring the MetHb concentration in the reoxygenated hemoglobin composition, Here, the reoxygenated hemoglobin composition has a lower MetHb concentration compared to the MetHb concentration measured in a reoxygenated hemoglobin composition treated with 5 mM or less of an antioxidant.

[0203] In some embodiments, the present invention provides for reoxygenation of hemoglobin after heat treatment, such that the hemoglobin maintains a MetHb concentration of less than 2% as analyzed by co-oximetry.

[0204] In some embodiments, the percentage of MetHb available in the reoxygenated hemoglobin solution during or after heat treatment is selected from less than about 2%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, or less than about 1.0%.

[0205] In some embodiments, the heat treatment process is carried out at a suitable temperature selected from above 59° C. to about 65° C. In some embodiments, the heat treatment step is carried out at a suitable temperature selected from the following temperatures: about 60.0° C., about 60.1° C., about 60.2° C., about 60.3° C., about 60.4° C., about 60.5° C., about 60.6° C., about 60.7° C., about 60.8° C., about 60.9° C., about 60.0° C., about 61.1° C., about 61.2° C., about 61.3° C., about 61.4° C., about 61.5° C., about 61.5° C., about 61.7° C., about 61.8° C., about 61.9° C., about 62.0° C., about 62.1° C., about 62.2° C., about 62.3° C., about 62.4° C., about 62.5° C., about 62.6° C., about 62.7° C., about 62.8° C., about 62.9° C., about 62.10° C., about 62.11° C., about 62.12° C., about 62.13° C., about 62.14° C., about 62.15° C., about 62.16° C., about 62.17° C., about 62.18° C., about 62.19° C., about 62.16° C., about 62.19° C., about 62.16° C., about 62.19° C., about 62 .2℃, approximately 62.3℃, approximately 62.4℃, approximately 62.5℃, approximately 62.6℃, approximately 62.7℃, approximately 62.8℃, approximately 62.9℃, approximately 63.0℃, approximately 63.1℃, approximately 63.2℃, approximately 63.3℃, ​​approximately 63.4℃, approximately 63.5℃, approximately 63.6℃, ​​approximately 63.7℃, approximately 63.8℃, approximately 63.9℃, approximately 64.0℃, approximately 64.1℃, approximately 64.2℃, approximately 64.3℃, approximately 64.4℃, approximately 64.5℃, approximately 64.6℃, approximately 64.7℃, approximately 64.8℃, approximately 64.9℃, approximately 65.0℃.

[0206] In some embodiments, the heat treatment process is carried out for a suitable time interval selected from about 4 hours to about 15 hours. In some embodiments, the heat treatment step is carried out for a suitable time period selected from the following periods: about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6.0 hours, about 6.1 hours, about 6.2 hours, about 6.3 hours, about 6.4 hours, about 6.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours Time, approximately 7.9 hours, approximately 8.0 hours, approximately 8.1 hours, approximately 8.2 hours, approximately 8.3 hours, approximately 8.4 hours, approximately 8.5 hours, approximately 8.6 hours, approximately 8.7 hours, approximately 8.8 hours, approximately 8.9 hours, approximately 9.0 hours, approximately 9.1 hours, approximately 9.2 hours, approximately 9.3 hours, approximately 9.4 hours, approximately 9.5 hours, approximately 9.6 hours, approximately 9.7 hours, approximately 9.8 hours, approximately 9.9 hours, approximately 10.0 hours, approximately 10.1 hours, approximately 10.2 hours, approximately 10.3 hours, approximately 10.4 hours, approximately 10.5 hours, approximately 10.6 hours, approximately 10.7 hours, approximately 10.8 hours, approximately 10.9 hours, approximately 11.0 hours, approximately 11.1 hours, approximately 11.2 hours, approximately 11.3 hours, approximately 11.4 hours, approximately 11.5 hours, approximately 12.0 hours, approximately 12.5 hours, approximately 13.0 hours, approximately 13.5 hours, approximately 14.0 hours, approximately 14.5 hours, approximately 15.0 hours.

[0207] In some embodiments, the present invention can be applied to remove or reduce all forms of viruses, particularly DNA and RNA viruses, enveloped and non-enveloped viruses, as well as virions and prions or other similar biological systems, and even bacteria and fungi.

[0208] This method is preferably used to reduce contamination with bovine viral diarrhea virus (BVDV), retrovirus, parvovirus, bovine spongiform encephalopathy (BSE), and transmissible spongiform encephalopathy (TSE) from hemoglobin solutions.

[0209] In some embodiments, the present invention provides a stable hemoglobin composition comprising the following components: a) Reoxygenated hemoglobin purity greater than 60%; b) reoxygenated hemoglobin that is substantially free of viral particles; c) Reoxygenated hemoglobin that is substantially free of prions.

[0210] In some embodiments, the purity of the reoxygenated Hb is about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% or more.

[0211] In some embodiments, the heat-treated hemoglobin solution has a viral reduction of at least 1 log compared to before heat treatment. 10 That's it, 2 log 10 That's it, 3 logs 10 That's it, 4 logs 10 or more decreased.

[0212] In some embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a. heat-treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time; b. maintaining an appropriate concentration of antioxidant at 5 mM or above; c. reoxygenating the hemoglobin composition; d. measuring the MetHb concentration in the reoxygenated hemoglobin composition, wherein the reoxygenated hemoglobin composition has a lower MetHb concentration compared to the MetHb concentration measured in a reoxygenated hemoglobin composition prepared by the present method using 5 mM or less antioxidant.

[0213] In certain embodiments, the present invention provides a method for producing a stable hemoglobin composition, comprising the steps of: a. heat-treating the deoxygenated hemoglobin composition at a suitable temperature for a suitable period of time; b. maintaining an appropriate concentration of antioxidant at 5 mM or above; c. reoxygenating the hemoglobin composition; d. Measurement of charge variants in reoxygenated hemoglobin composition; Here, the reoxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in a reoxygenated hemoglobin composition produced with 5 mM or less antioxidant.

[0214] In some embodiments, the patient's condition is selected from the group comprising acute respiratory distress syndrome, bronchiectasis, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, lymphangiomatosis, primary ciliary dysfunction, cancer, tumors, lung cancer, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, pulmonary sarcoidosis, pneumonia and bronchitis, infections that affect the lung's ability to transport oxygen.

[0215] In some embodiments, the condition is selected from the group including acute respiratory distress syndrome, bronchiectasis, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, lymphangiomatosis, primary ciliary dysfunction, cancer, tumors, lung cancer, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, pulmonary sarcoidosis, pneumonia and bronchitis, and infections affecting the transport capacity of the lungs.

[0216] In some embodiments, the condition is anemia selected from the group consisting of anemia due to blood loss, anemia due to insufficient red blood cell production, anemia due to red blood cell destruction, and combinations thereof, wherein the cancer is selected from solid tumors, soft tissue cancer, lung cancer, bone cancer, metastatic cancer, adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, and breast cancer.

[0217] In some embodiments, the present invention provides a method of administering a stable hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or more; b) one or more impurities selected from MetHb, acidic variants, basic variants, viral particles and / or prions; Here, oxygenated hemoglobin includes MetHb that accounts for 2% or less of the total oxygenated hemoglobin.

[0218] In some embodiments, the present invention provides a method of administering a stable hemoglobin composition comprising: a) Oxygenated hemoglobin with a main peak purity of 70% or greater and one or more impurities including: b) Oxygenated hemoglobin with MetHb ≤2%; c) Oxygenated hemoglobin with less than 12% acidic variants of the main peak; d) Oxygenated hemoglobin with less than 11% of the main peak basic variant; e) oxygenated hemoglobin substantially free of viral particles and / or prions; Here, MetHb is analyzed by co-oximetry and the purity of the reoxygenated / oxygenated hemoglobin main peak is analyzed by cIEF (capillary isoelectric focusing).

[0219] In some embodiments, the dosing frequency is once daily or twice daily.

[0220] In some embodiments, the oxygenated hemoglobin is pharmaceutically stable.

[0221] The present inventors have surprisingly found that an L-cysteine ​​concentration of greater than 5 mM can effectively control one or more of MetHb formation, charge variants, and hemoglobin purity in the oxygenated hemoglobin state (reoxygenated hemoglobin) during production / purification.

[0222] The hemoglobin composition provides batch-to-batch stability, thereby helping to achieve batch-to-batch consistency. To further clarify, a stable hemoglobin composition maintains the main purity peak of hemoglobin / reoxygenated hemoglobin at a desired level (70% or greater) and MetHb below 2%. This also aids in long-term storage of the PEG-hemoglobin or PEG-Hb-CO composition.

[0223] In certain embodiments, the oxygenated hemoglobin composition maintains MetHb below 2% for greater than 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, and 48 months.

[0224] In some embodiments, oxygenated hemoglobin is obtained after post-thermal treatment of deoxygenated hemoglobin. In some embodiments, the present invention includes a method for synergistically treating inflammation, vasoconstriction, and hypoxia in a patient in need of such treatment by administering a therapeutically effective amount of an HBOC composition according to the present invention.

[0225] In some embodiments, the present invention also provides methods for treating trauma, shock, ischemia, and any other disease that can be ameliorated by increasing the oxygen or carbon monoxide content of tissues or organs. The compositions of the present invention rapidly restore tissue oxygenation and completely repay the oxygen debt in animal models of severe traumatic shock, in which at least 50% of subjects typically die from hemorrhagic shock. Using exemplary formulations, a single unit of the compositions of the present invention repays the oxygen debt to all major organs, opens the microvasculature, and restores mean arterial pressure.

[0226] In an exemplary embodiment, the method comprises administering to a subject a composition of the invention in an amount sufficient to achieve delivery of oxygen or carbon monoxide to one or more tissues and / or organs.

[0227] In various embodiments, the present invention provides methods for reversing oxygen debt in selected members of tissues and organs of a subject suffering from hemorrhagic shock. In exemplary embodiments, the method comprises administering to the subject a composition of the present invention in an amount sufficient to reverse the oxygen debt. Similar methods are provided for increasing carbon monoxide content in tissues in response to a decrease in carbon monoxide content due to disease, injury, etc., or as a means of achieving a therapeutic effect by increasing carbon monoxide content in tissues above normal levels in healthy or diseased states.

[0228] In various embodiments, the present invention provides a method for inducing angiogenesis in a tissue of a subject by administering to the subject an effective amount of a composition of the present invention to induce angiogenesis. In an exemplary embodiment, angiogenesis is induced in a tissue suffering from oxygen deficiency. In a further exemplary embodiment, the tissue or organ in which angiogenesis is induced is a tissue or organ of the subject suffering from oxygen deficiency. In an exemplary embodiment, the method comprises administering to the subject an amount of a composition of the present invention sufficient to restore the oxygen deficiency.

[0229] The following examples are intended to illustrate the purpose of the present invention, and the scope of the present invention should not be construed as being limited thereto.

[0230] Example 1: Hemoglobin extraction and purification This first step removes the plasma from the blood cells. At bench scale, this is accomplished by repeated washing, centrifugation, and decantation. This process appears to be more efficient than vertical continuous-flow centrifuges.

[0231] The red blood cells were washed four times with a buffer solution (1.2% NaCl, 10 mM phosphate, pH 7.8). The red blood cells were then lysed (hemoglobin extraction without true cell lysis) by slowly adding 1.5 volumes of WFI over a 2-hour period. The state of lysis (hemoglobin extraction) was monitored by sampling until the conductivity was in the range of 5.50–7.00 μS. The hemoglobin concentration (% Hb) was measured using a Radiometer OSM3 hemoximeter. Osmolality was also measured and ranged from 130–150 mOsmol.

[0232] Once hemoglobin extraction is complete, the contents are pressure filtered through a 1 μm cellulose depth filter, followed by 0.45 / 0.2 μm sterile polysulfone filters before being sent to a second tank, which serves as a holding tank for the next step. The hemoglobin solution is then passed through a 300 Kd ultrafiltration membrane and sent to a jacketed tank at 10°C. This tank serves as the first virus removal step, removing high molecular weight proteins.

[0233] Hemoglobin is concentrated using a 10 Kd MWCO system.

[0234] Deoxygenation process to produce deoxygenated hemoglobin: The concentrated hemoglobin is deoxygenated by passing it through a countercurrent membrane contactor. As the concentrated hemoglobin passes through the shell of the device, argon gas is pumped in the opposite direction into the core. The membrane within the device is permeable only to gas. The intermediate is circulated through the device from an intermediate tank until the oxygen content of the hemoglobin solution (HbO2) is less than 10.0%. Next, 7.5 mM to 15 mM L-cysteine ​​is added to the intermediate to produce deoxygenated hemoglobin.

[0235] The hemoglobin eluate can be deoxygenated by gas permeation through a phase separation membrane with an inert gas, such as argon. Other means known in the art for deoxygenating hemoglobin solutions can also be used to deoxygenate the hemoglobin eluate.

[0236] Deoxygenation is continued until the pO2 of the hemoglobin solution is reduced to a desired level, for example, until the oxygenated hemoglobin (oxyhemoglobin or HbO2) content in the hemoglobin solution is about 10% or less, 5% or less, 3% or less, or 1% or less.

[0237] Production of virus-inactivated hemoglobin by virus inactivation treatment: Viral inactivation begins after deoxygenation and involves heat treatment to inactivate adventitious substances. Deoxygenated hemoglobin containing L-cysteine ​​is required for the inactivation process. Residual oxygenated hemoglobin is denatured or precipitated. The deoxygenated hemoglobin is heated and held at approximately 60°C for 10 hours, then cooled again. After processing, the virally inactivated hemoglobin is filtered through a 0.45 μm / 0.2 μm capsule filter. In the case of deoxygenated hemoglobin, if the liquid temperature exceeds 65°C, MetHb (indicating hemoglobin decomposition) may form in the intermediate product, and at the same time, the temperature approaches the denaturation (thermal melting) temperature of bovine deoxygenated hemoglobin.

[0238] To achieve a significant adventitious log reduction, temperatures above 59°C must be maintained for a minimum of 2 hours.

[0239] Pre-PEG diafiltration process for producing diafiltered reoxygenated hemoglobin (reoxygenation of hemoglobin): The critical in-process quality attribute for this unit operation is % HbO2. % HbO2 is very important for controlling hemoglobin before PEGylation because structural changes in deoxygenated conditions affect the hemoglobin PEGylation reaction.

[0240] Additionally, operating conditions (e.g., feed pressure, TMP, weight) are maintained by automated recipes to consistently remove L-cysteine ​​from the virus inactivation process, reoxygenate intermediates, and perform buffer exchange. Co-oximetry (% HbO2), cIEF, pH, and osmolality measurements are performed to confirm that the buffer exchange has achieved the product quality required for hemoglobin PEGylation.

[0241] PEGylation process for the production of PEGylated hemoglobin using L-cysteine: Prior to PEGylation, the reoxygenated hemoglobin is subjected to extraneous material removal filtration. The PEGylation process involves the attachment of 8–10 chains of 5 kD activated polyethylene glycol (SC-PEG-5K) molecules to purified bovine hemoglobin molecules using the following controlled method. Purified hemoglobin is PEGylated by adding a specified molar ratio of PEG to hemoglobin at a specified rate. The reaction is allowed to proceed to completion (indicated by a stable pH for a specified time). During and after the PEG addition, 1 N NaOH is added directly to the reaction mixture to maintain the pH between 7.95 and 8.20. Once the intermediate pH has stabilized, 15 mM L-cysteine ​​is added to control the rate of MetHb production, and the PEGylation reaction is quenched overnight. The L-cysteine-added PEG-Hb is filtered into a stainless steel tank using a 0.45 μm / 0.2 μm filter membrane.

[0242] Production of purified PEGylated hemoglobin (DS) by post-PEG diafiltration process: The filtered PEGylated hemoglobin is diafiltered with 10 times the intermediate volume of FFB using a 50 kD MWCO diafiltration system. Impurities removed by diafiltration include N-hydroxysuccinimide (NHS), free PEG, unreacted bovine hemoglobin, and L-cysteine ​​due to the MWCO of the diafiltration membrane.

[0243] After diafiltration, the hemoglobin is concentrated to a final tHb (total hemoglobin concentration) of 3.6-4.6g% tHb. The diafiltered PEGylated hemoglobin is then filtered through a 0.45µm / 0.2µm capsule and transferred from a stainless steel skid into a final disposable bag. At this stage, the purified PEGylated hemoglobin bulk becomes the bulk drug substance.

[0244] Carboxylation process for producing carboxylated PEGylated hemoglobin (with additives): Using purified PEGylated hemoglobin bulk (drug substance bulk) as the raw material, sparge carbon monoxide into the bulk at a flow rate of 3 to 7 liters per minute until the HbCO concentration reaches 98% or higher.

[0245] The final excipients, L-cysteine ​​and dextrose, were dissolved in FFB, filtered through 0.45 and 0.2 μm filters, and dispensed into the carboxylated PEGylated hemoglobin at 0.61 g / kg and 5.0 g / kg, respectively. The pH of the L-cysteine ​​solution was adjusted to 8.1–8.2 before mixing with the excipient solution. The filtered excipient solution was added to the carboxylated PEGylated intermediate to prepare the excipient-loaded carboxylated PEGylated hemoglobin. Carboxylation was restarted to remove excess oxygen that may have been introduced into the carboxylated intermediate by the addition of the excipient solution. The excipient-loaded carboxylated PEGylated hemoglobin (drug substance) was blanketed with argon to minimize exposure to oxygen and maintained at 2–8°C before filling. The final product ranges for manufacturing the final product are osmolality (310-360 mOsm / kg), pH (7.8-8.2), g% tHb (3.8-4.2), and % HbCO (>98%).

[0246] Example 2: Process for producing purified reoxygenated hemoglobin: Hemoglobin extraction The red blood cells are washed four times with phosphate buffer (1.2% NaCl, 10 mM phosphate, pH 7.8). The red blood cells are then lysed by slowly adding WFI (hemoglobin is extracted without true cell lysis). Hb concentration is measured using a Radiometer OSM3 hemoximeter. Once hemoglobin extraction is complete, the contents are pressure filtered through a 1 μm cellulose depth filter, followed by a 0.45 / 0.2 μm sterile polysulfone filter, before being sent to a second tank, which serves as a holding tank for the next step. The hemoglobin solution is then sent to a jacketed tank at 10°C via a 300 Kd ultrafiltration membrane.

[0247] The hemoglobin is concentrated to a concentration of approximately 16g% using a 10KD MWCO system and then deoxygenated by recirculation through a hollow fiber membrane contactor until the HbO2 in the hemoglobin solution is less than 10%.

[0248] Heat treatment of hemoglobin The deoxygenated and concentrated hemoglobin is transferred to a jacketed vessel and an L-cysteine ​​solution is added to a concentration of 5 mM or higher. L-cysteine ​​acts as a reducing agent / oxygen scavenger, preventing hemoglobin from oxidizing to MetHb. Meanwhile, L-cysteine ​​itself is consumed during heat treatment. It is known that the virus removal rate is directly proportional to the heat treatment time and temperature.

[0249] Table 1 shows the removal rate of prions from hemoglobin solutions after heat treatment for different times. The removal rate increases linearly as the heat treatment time increases from 1 hour to 4 hours.

[0250] Table 1. Transmissible spongiform encephalopathy (TSE) / prion initial load, output load, and Log 10 Details of the reduction rate.

[0251] 1 PWBU = Prion Western Blot Units [Table 1] Therefore, for each type of therapeutic protein, the longest possible time is usually selected to prevent protein denaturation. In this example, heat inactivation of viruses and prions is achieved by raising the temperature to 60±1°C and gently stirring the solution for up to 10 hours. After heat treatment, the solution is rapidly cooled to refrigeration temperature. The heat-treated deoxygenated hemoglobin is then filtered through a 0.45 / 0.2 μm sterile polysulfone filter.

[0252] Purification and reoxygenation The heat-treated deoxygenated hemoglobin is filtered using a 10KD MWCO system to produce purified hemoglobin (containing no L-cysteine). During this process, the hemoglobin is exposed to an oxygenated buffer solution, and the purified hemoglobin becomes primarily (more than 90%) oxygenated hemoglobin. MetHb is controlled to approximately 2% or less.

[0253] Example 3 L-cysteine ​​concentration and hemoglobin purity In this example, after the deoxygenation step, the solution was divided into four aliquots, labeled A–D. Various concentrations of L-cysteine ​​(5 mM, 7.5 mM, 10 mM, and 15 mM) were added to each aliquot. The samples were then heat-treated at 60°C for 10 hours. Small test samples were taken from each reaction mixture at 0, 4, 6, 8, and 10 hours during the heat treatment. Before proceeding to the next step, all of these heat-treated samples were analyzed for MetHb by co-oximetry and hemoglobin purity by capillary isoelectric focusing (cIEF). Next, all of these samples were washed with at least 10 volumes of 1% buffered saline (10 kDa filter) to produce purified oxyhemoglobin. The resulting hemoglobin was analyzed for MetHb using co-oximetry. The table below shows the effect of L-cysteine ​​concentration on MetHb content in purified hemoglobin. At each L-cysteine ​​concentration, the MetHb level was highest (approximately 2%) after 10 hours of heat treatment, but when the heat treatment time exceeded 8 hours, 5 mM L-cysteine ​​was unable to control MetHb after the reoxygenation step, increasing up to 10%.

[0254] Table 2: Percentage of MetHb content in purified oxyhemoglobin samples produced using different L-cysteine ​​concentrations and heat treatment times. [Table 2]

[0255] Example 4 L-cysteine ​​concentration and hemoglobin purity Cooximetric analysis of tHB, percent MetHb, percent HbCO, percent deoxyhemoglobin, and percent HbO2 was performed on undiluted samples using a Radiometer OSM3 cooximeter.

[0256] To determine the ability of L-cysteine ​​to prevent hemoglobin oxidative damage, we measured hemoglobin charge purity by cIEF and examined the effect of L-cysteine ​​concentration. Briefly, samples were diluted to 1 mg / mL and analyzed on the Maurice platform. Figure 2 shows the charge profiles of hemoglobin samples spiked with 5 mM L-cysteine ​​at 0 hours (baseline) and 10 hours, compared to hemoglobin samples spiked with 15 mM L-cysteine ​​at 10 hours. Figure 2 shows the cIEF (Maurice) profiles of typical hemoglobin samples spiked with 5 mM L-cysteine ​​at 0 hours (baseline) and 10 hours (10 hours). As shown in Table 3, Figure 2 shows: a) cIEF (Maurice) profiles of typical hemoglobin samples spiked with 5 mM L-cysteine; b) cIEF (Maurice) profiles of typical hemoglobin samples spiked with 5 mM L-cysteine ​​at 10 hours; ... and cIEF (Maurice) profiles of typical hemoglobin samples spiked with 5 mM L-cysteine ​​at 10 hours. b) After 10 hours of treatment at 60°C in the presence of 5 mM L-cysteine, an increase in acidic and basic charge variants is observed. c) No increase in charge variants is observed after treatment at 60°C for 10 hours in the presence of 15 mM L-cysteine.

[0257] Across samples, the increase in acidic and basic variant levels closely matched the trend observed in MetHb levels after purification. That is, an increase in MetHb values ​​corresponded to a decrease in main peak purity. When 5 mM L-cysteine ​​samples were treated for up to 10 hours, the main peak purity decreased to less than 70%, while 10 mM and 15 mM L-cysteine ​​showed little decrease in hemoglobin main peak purity (above 75%). Figure 3 compares main peak purity at different L-cysteine ​​concentrations versus heating time.

[0258] Table 3: Percentage of acidic and basic charge variants in purified oxyhemoglobin samples produced using different L-cysteine ​​concentrations and heat treatment times. [Table 3]

[0259] equivalent While specific embodiments of the present disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will become apparent to those skilled in the art upon review of the specification. The full scope of the present disclosure should be determined by reference to the claims, their full scope of equivalents, and the specification, and such variations.

Claims

1. c) oxygenated hemoglobin; d) MetHb is less than 2% of total oxygenated hemoglobin; Hemoglobin composition.

2. c) Oxygenated hemoglobin with a main peak purity of 70% or more; d) comprising one or more impurities selected from MetHb; charge variants selected from acidic variants, basic variants; and optionally viral particles and / or prions; 2. The hemoglobin composition of claim 1, wherein the oxygenated hemoglobin comprises 2% or less of MetHb of total oxygenated hemoglobin.

3. 3. The hemoglobin composition of claim 2, wherein the oxygenated hemoglobin comprises a total percentage of MetHb selected from the range of about 0.5% to about 1.8% of total oxygenated hemoglobin.

4. 4. The hemoglobin composition of claim 3, wherein the oxygenated hemoglobin comprises a total percentage of MetHb selected from about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, and about 1.8% of total oxygenated hemoglobin.

5. The hemoglobin composition of claim 2 , wherein the stable hemoglobin composition is produced on a large scale.

6. 3. The hemoglobin composition of claim 2, wherein the main peak purity of oxygenated hemoglobin is greater than or equal to about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, or about 85%.

7. 3. The hemoglobin composition of claim 2, wherein the charge variants are less than 25%.

8. less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less acidic variants; 3. The hemoglobin composition of claim 2, wherein the basic variants are less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less.

9. 2. The hemoglobin composition of claim 1, wherein the oxygenated hemoglobin is obtained after post-heat treatment of deoxygenated hemoglobin.

10. The hemoglobin composition of claim 2 , wherein the oxygenated hemoglobin has a viral load and / or prion load reduced by more than 1 log 10.

11. The oxygenated hemoglobin is 1 log １０ That's it, 2 log １０ That's it, 3 logs １０ That's it, 4 log １０ The hemoglobin composition according to claim 10, wherein the amount of a virus and / or a prion selected from the above is reduced.

12. The composition of the oxygenated hemoglobin is as follows: a) Oxygenated hemoglobin with a main peak purity of 70% or greater and one or more impurities including: b) Oxygenated hemoglobin with a MetHb content of 2% or less; c) Oxygenated hemoglobin containing less than 12% of the acidic variant of the main peak; d) Oxygenated hemoglobin containing less than 11% of the main peak basic variant; e) oxygenated hemoglobin substantially free of viral particles; and f) oxygenated hemoglobin that is substantially free of prions; The hemoglobin composition of claim 2, comprising:

13. 1. A hemoglobin composition comprising: d) Oxygenated hemoglobin with a main peak purity of 70% or more; e) the acidic variant is less than 12% of the main peak; f) The basic variant is less than 11% of the main peak

14. 1. A hemoglobin composition comprising: d) Oxygenated hemoglobin with a main peak purity of 70% or more; e) the acidic variant is less than 12% of the main peak; f) The basic variant is less than 9% of the main peak

15. 15. The hemoglobin composition of claim 2, 13, or 14, wherein the oxygenated hemoglobin is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is for at least 4 hours.

16. 16. The hemoglobin composition of claim 15, wherein the oxygenated hemoglobin is obtained from heat-treated deoxygenated hemoglobin, and the heat treatment is carried out for a suitable time selected from the following:

17. 13. The hemoglobin composition of claim 12, wherein the acidic variants are less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less, and the basic variants are less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less.

18. 13. The hemoglobin composition of claim 12, wherein the acidic variants are less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less, and the basic variants are less than about 9%, less than about 8%, less than about 7%, or less.

19. 1. A hemoglobin composition comprising: e) oxygenated hemoglobin; f) MetHb less than 9% of total oxygenated hemoglobin; wherein the heat treatment is carried out for about 10 hours.

20. 1. A hemoglobin composition comprising: c) oxygenated hemoglobin; d) MetHb less than 4% of total oxygenated hemoglobin; wherein the heat treatment is carried out for about 8 hours.

21. 21. The hemoglobin composition of any of claims 13, 14, 19 and 20, wherein the main peak purity of oxygenated hemoglobin is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% or more.

22. 21. The hemoglobin composition of any one of claims 1, 13, 14, 19 and 20, wherein the oxygenated hemoglobin is further conjugated with PEG to form PEGylated hemoglobin, and the PEGylated hemoglobin maintains MetHb less than 5% during storage at 2-8°C for 1 month or more, 3 months or more, 6 months or more, 9 months or more, 12 months or more, 15 months or more, 18 months or more, 21 months or more, 24 months or more, 27 months or more, 30 months or more, 33 months or more, 36 months or more, 39 months or more, 42 months or more, 45 months or more, and 48 months or more.

23. 23. The hemoglobin composition of claim 22, wherein the PEGylated hemoglobin maintains MetHb below 7%, preferably below 3%.

24. the PEGylated hemoglobin is a) The main peak of oxygenated hemoglobin is 70% or more; b) the acidic variant is less than 12% of the main peak; c) the basic variant is less than 11% of the main peak; The hemoglobin composition of claim 22, wherein the

25. 21. The hemoglobin composition of any one of claims 1, 13, 14, 19, and 20, wherein the MetHb is maintained at less than 2% during storage at 2-8°C for 1 month or more, 3 months or more, 6 months or more, 9 months or more, 12 months or more, 15 months or more, 18 months or more, 21 months or more, 24 months or more, 27 months or more, 30 months or more, 33 months or more, 36 months or more, 39 months or more, 42 months or more, 45 months or more, and 48 months or more.

26. 21. The hemoglobin composition of any one of claims 2, 13, 14, 19 and 20, characterized in that the main peak purity of oxygenated hemoglobin, acidic variants and basic variants are analyzed by cIEF (capillary isoelectric focusing) and MetHb is analyzed by co-oximetry.

27. The following steps: j) washing fresh whole blood collected from an animal source to produce washed red blood cells; k) extracting hemoglobin from red blood cells; l) filtering the extracted hemoglobin; m) ultrafiltration and concentration; n) ultrafiltration and deoxygenation of the concentrated hemoglobin; o) heat inactivation of the deoxygenated hemoglobin to reduce viruses and / or prions by heat treatment at an appropriate temperature; p) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; q) optionally PEGylating the oxygenated hemoglobin composition; r) optionally performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Including, The virus inactivation of the deoxygenated hemoglobin is carried out by heat treatment, and the L-cysteine ​​concentration in the deoxygenated hemoglobin during step (f) is maintained at 5 mM or more; the oxygenated hemoglobin composition comprises a main peak purity of greater than 70% and one or more impurities selected from MetHb, acidic variants, basic variants, viral particles, and / or prions; and A method for preparing a stable hemoglobin composition, wherein the oxygenated hemoglobin comprises no more than 2% MetHb of the total oxygenated hemoglobin.

28. 28. The method of claim 27, wherein the acidic variant is less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7% or less, and the basic variant is less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less.

29. 28. The method of claim 27, wherein MetHb is analyzed by co-oximetry and the main peak purity of oxygenated hemoglobin, as well as acidic and basic variants of oxygenated hemoglobin, is analyzed by cIEF (capillary isoelectric focusing).

30. 28. The method of claim 27, wherein the oxygenated hemoglobin comprises a total percentage of MetHb selected from about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, and about 1.8% of the total oxygenated hemoglobin.

31. 28. The method of claim 27, wherein the main peak purity of oxygenated hemoglobin is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% or more.

32. Heat inactivation of deoxyhemoglobin reduces viral load and / or prions by 1 log compared to before heat treatment. １０ That's it, 2 log １０ 28. The method of claim 27, wherein the amount of β-glucan in the blood is reduced by more than or equal to 1000 mg / L.

33. 28. The method of claim 27, wherein the heat treatment step is carried out at a suitable temperature selected from about 55°C to about 75°C.

34. The heat treatment step may be performed at temperatures of about 57.0°C, about 57.1°C, about 57.2°C, about 57.3°C, about 57.4°C, about 57.5°C, about 57.6°C, about 57.7°C, about 57.8°C, about 57.9°C, about 58.0°C, about 58.1°C, about 58.2°C, about 58.3°C, about 58.4°C, about 58.5°C, about 58.6°C, about 58.7°C, about 58.8°C, about 58.9°C, about 59.0°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, about 101°C, about 102°C, about 103°C, °C, about 59.1°C, about 59.2°C, about 59.3°C, about 59.4°C, about 59.5°C, about 59.6°C, about 59.7°C, about 59.8°C, about 59.9°C, about 60.0°C, about 60.1°C, about 60.2°C, about 60.3°C, about 60.4°C, about 60.5°C, about 60.6°C, about 60.7°C, about 60.8°C, about 60.9°C, about 61.0°C, about 61.1°C, about 61. 2°C, about 61.3°C, about 61.4°C, about 61.5°C, about 61.6°C, about 61.7°C, about 61.8°C, about 61.9°C, about 62.0°C, about 62.1°C, about 62.2°C, about 62.3°C, about 62.4°C, about 62.5°C, about 62.6°C, about 62.7°C, about 62.8°C, about 62.9°C, about 63.0°C, about 63.1°C, about 63.2°C, about 63.3°C, about 63.4°C, about 63.5°C, about 63.6°C, about 63.7°C, about 63.8°C, about 63.9°C, about 63.10°C, about 63.11°C, about 63.12°C, about 63.13°C, about 63.14°C, about 63.15°C, about 63.16°C, about 63.17°C, about 63.18°C, about 63.19°C, about 63.19°C, about 63.16°C, about 63.19 ...

34. The method of claim 33, wherein the reaction is carried out at one or more suitable temperatures selected from about 63.4°C, about 63.5°C, about 63.6°C, about 63.7°C, about 63.8°C, about 63.9°C, about 64.0°C, about 64.1°C, about 64.2°C, about 64.3°C, about 64.4°C, about 64.5°C, about 64.6°C, about 64.7°C, about 64.8°C, about 64.9°C and about 65.0°C.

35. 28. The method of claim 27, wherein the heat treating step is carried out for at least 4 hours or more.

36. The heat treatment process lasts for about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6.0 hours, about 6.1 hours, about 6.2 hours, about 6.3 hours, about 6.4 hours, about 6. 5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.0 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours, about 8.6 hours , about 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.0 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 hours, about 10.0 hours, about 10.1 hours, about 10.2 hours, about 10.3 hours, about 10.4 hours, about 10.5 hours, about 10.6 hours, about 10.7 36. The method of claim 35, wherein the method is carried out for a suitable period of time selected from about 10.8 hours, about 10.9 hours, about 11.0 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours and about 15.0 hours.

37. The L-cysteine ​​concentrations maintained during heat treatment were about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM, about 8.1 mM, about 8.2 mM, about 8.3 mM, about 8.4 mM, about 8.5 mM, about 8.6 mM, about 8.7 mM, about 8.8 mM, about 8.9 mM, about 9.0 mM, about 9.1 mM, about 9.2 mM, about 9.3 mM, about 9.4 mM, about 9.5 mM, about 9.6 mM, about 9.7 mM, about 9.8 mM, about 9.9 ... .. 9mM, about 8.0mM, about 8.1mM, about 8.2mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9.0mM, about 9.1mM, about 9.2mM, About 9.3mM, about 9.4mM, about 9.5mM, about 9.6mM, about 9.7mM, about 9.8mM, about 9.9mM, about 10mM, about 10.1mM, about 10.2mM, about 10.3mM, about 10.4mM, about 10.5mM, About 10.6 mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.1 mM, about 11.2 mM, about 11.3 mM, about 11.4 mM, about 11.5 mM, about 11.6 mM, about 11.7 mM, about 11.8 mM, about 11.9 mM, about 12.0 mM, about 12.1 mM, about 12.2 mM, about 12.3 mM, about 12.4 mM, about 12.5 mM, about 12.6 mM, about 12.7 mM, about 12.8 mM, about 12.9 mM 28. The method of claim 27, wherein the ATP concentration is selected from about 13.0 mM, about 13.1 mM, about 13.2 mM, about 13.3 mM, about 13.4 mM, about 13.5 mM, about 13.6 mM, about 13.7 mM, about 13.8 mM, about 13.9 mM, about 14.0 mM, about 14.1 mM, about 14.2 mM, about 14.3 mM, about 14.4 mM, about 14.5 mM, about 14.6 mM, about 14.7 mM, about 14.8 mM, about 14.9 mM and about 15.0 mM.

38. 28. The method of claim 27, wherein the pegylated hemoglobin of step (h) or the pegylated carboxylated hemoglobin of step (i) maintains MetHb below 4%, preferably below 3%.

39. 1. A method for reducing viral and / or prion load in an oxygenated hemoglobin composition derived from a mammal, comprising the steps of: f) heat treating the deoxygenated hemoglobin at about 60°C for at least 4 hours; g) maintaining the L-cysteine ​​concentration at 5.0 mM or higher during the heat treatment; h) reoxygenating the heat-treated deoxygenated hemoglobin to form an oxygenated hemoglobin composition; i) optionally PEGylating the oxygenated hemoglobin composition; j) optionally performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Including, The method wherein the oxygenated hemoglobin composition has less than 2% MetHb as analyzed by cooximetry and provides at least a 1 log reduction in viral and / or prion reduction.

40. The heat treatment of deoxygenated hemoglobin was carried out at about 60° C. for about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6.0 hours, about 6.1 hours, about 6.2 hours, about 6.3 hours, about 6.4 hours, about 6.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours, about 8.6 hours, about 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 hours, about 9.9 hours, about 9.9 hours, about 10.1 hours, about 10.2 hours, about 10.3 hours, about 10.4 hours, about 10.5 hours, about 10.6 hours, about 10.7 hours, about 10.8 hours, about 10.9 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about .3 hours, about 6.4 hours, about 6.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.0 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours 0.5 hours, about 8.6 hours, about 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.0 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 hours, about 10.0 hours, about 10.1 hours, about 10.2 hours, about 10.3 hours, about 10.4 hours, about 10.5 hours, about 40. The method of claim 39, wherein the treatment is for 10.6 hours, about 10.7 hours, about 10.8 hours, about 10.9 hours, about 11.0 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours, or about 15.0 hours.

41. During the heat treatment, the L-cysteine ​​concentration was increased to about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM, about 8.1 mM, about 8.2 mM mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9.0mM, about 9.1mM, about 9.2mM, about 9.3mM, about 9.4mM, about 9.5mM, about 9.6mM, about 9. 7mM, about 9.8mM, about 9.9mM, about 10mM, about 10.1mM, about 10.2mM, about 10.3mM, about 10.4mM, about 10.5mM, about 10.6mM, about 10.7mM, about 10.8mM, about 10.9mM, about 11.0mM, about 11.1mM, about 11.2mM, about 11.3mM, about 11.4mM, about 11.5mM, about 11.6mM, about 11.7mM, about 11.8mM, about 11.9mM, about 12.0mM, about 12.1mM, about 12.2mM, about 12.3mM, about 12.4mM, about 12.5mM, about 12.6mM, about 12.7mM, about 12.8mM, about 12.9mM, about 13.0mM, about 13.1mM, about 13.2mM, about 13.3mM, about 13.4mM, about 13.5mM, about 13.6mM, about 1 40. The method of claim 39, wherein the ATP is maintained at a concentration selected from about 3.7 mM, about 13.8 mM, about 13.9 mM, about 14.0 mM, about 14.1 mM, about 14.2 mM, about 14.3 mM, about 14.4 mM, about 14.5 mM, about 14.6 mM, about 14.7 mM, about 14.8 mM, about 14.9 mM, about 15.0 mM, about 16.0 mM, about 16.5 mM, about 17.0 mM, about 17.5 mM, about 18.0 mM, about 18.5 mM, about 19.0 mM, about 19.5 mM, and about 20.0 mM.

42. The oxygenated hemoglobin composition after heat treatment reduced the number of viruses and prions by 1 log compared to before heat treatment. １０ That's it, 2 log １０ That's it, 3 logs １０ Above and 4 log １０ 40. The method of claim 39, wherein the density is substantially reduced by a factor selected from the above.

43. 40. The method of claim 39, wherein the oxygenated hemoglobin composition has a main peak purity of 70% or greater as analyzed by cIEF (capillary isoelectric focusing), and the oxygenated hemoglobin composition is substantially free of viruses and prions.

44. 40. The method of claim 39, wherein the PEGylated hemoglobin composition of step (d) or the PEGylated carboxylated hemoglobin composition of step (e) maintains MetHb at less than 4%, preferably less than 3%, and is substantially free of viruses and prions.

45. 1. A method for preparing a stable hemoglobin composition, comprising the steps of: j) washing fresh whole blood collected from an animal source to produce washed red blood cells; k) extracting hemoglobin from red blood cells; l) filtering the extracted hemoglobin; m) ultrafiltration and concentration; n) ultrafiltration and deoxygenation of concentrated hemoglobin: o) viral inactivation of the deoxygenated hemoglobin by heat treatment at a suitable temperature with an antioxidant at a suitable concentration of more than 5 mM to reduce viruses and / or prions; p) reoxygenating the heat-treated deoxygenated hemoglobin to produce an oxygenated hemoglobin composition; q) optionally PEGylating the heat-treated oxygenated hemoglobin composition; r) optionally performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition; Including, The method, wherein the oxygenated hemoglobin composition has a lower charge variant compared to the charge variant measured in an oxygenated hemoglobin composition prepared by the method using 5 mM or less antioxidant.

46. 46. ​​The method of claim 45, wherein the charge variants in the oxygenated hemoglobin composition are acidic or basic charge variants; the acidic charge variants in the oxygenated hemoglobin composition are less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less; and the basic charge variants in the oxygenated hemoglobin composition are less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less.

47. The heat treatment process is carried out at temperatures of about 57.0°C, about 57.1°C, about 57.2°C, about 57.3°C, about 57.4°C, about 57.5°C, about 57.6°C, about 57.7°C, about 57.8°C, about 57.9°C, about 58.0°C, about 58.1°C, about 58.2°C, about 58.3°C, about 58.4°C, about 58.5°C, about 58.6°C, about 58.7°C, about 58.8°C, about 58.9°C, and about 59. 0°C, about 59.1°C, about 59.2°C, about 59.3°C, about 59.4°C, about 59.5°C, about 59.6°C, about 59.7°C, about 59.8°C, about 59.9°C, about 60.0°C, about 60.1°C, about 60.2°C, about 60.3°C, about 60.4°C, about 60.5°C, about 60.6°C, about 60.7°C, about 60.8°C, about 60.9°C, about 61.0°C, about 61.1°C, about 6 1.2°C, about 61.3°C, about 61.4°C, about 61.5°C, about 61.6°C, about 61.7°C, about 61.8°C, about 61.9°C, about 62.0°C, about 62.1°C, about 62.2°C, about 62.3°C, about 62.4°C, about 62.5°C, about 62.6°C, about 62.7°C, about 62.8°C, about 62.9°C, about 63.0°C, about 63.1°C, about 63.2°C, about 63.3°C 46. ​​The method of claim 45, wherein the reaction is carried out at a suitable temperature selected from about 63.4°C, about 63.5°C, about 63.6°C, about 63.7°C, about 63.8°C, about 63.9°C, about 64.0°C, about 64.1°C, about 64.2°C, about 64.3°C, about 64.4°C, about 64.5°C, about 64.6°C, about 64.7°C, about 64.8°C, about 64.9°C and about 65.0°C.

48. The heat treatment step may be performed for about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6.0 hours, about 6.1 hours, about 6.2 hours, about 6.3 hours, about 6.4 hours, about 6.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.0 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours, about 8.6 hours, about 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.0 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 ... 0.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.0 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours, about 8.6 hours, About 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.0 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 hours, about 10.0 hours, about 10.1 hours, about 10.2 hours, about 10.3 hours, about 10.4 hours, about 10.5 hours, about 10.6 hours, about 10.7 46. ​​The method of claim 45, wherein the method is carried out for a suitable period of time selected from about 10.8 hours, about 10.9 hours, about 11.0 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours, and about 15.0 hours.

49. the antioxidant is selected from glutathione, ascorbic acid, and L-cysteine, preferably L-cysteine; Here, the preferred concentration of L-cysteine is about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM, about 8.1 mM, about 8.2 mM, about 8.3 mM, about 8.4 mM, about 8.5 mM, about 8.6 mM, about 8.7 mM, about 8.8 mM, about 8.9 mM, about 9.0 mM, about 9.1 mM, about 9.2 mM, about 9.3 mM, about 9.4 mM, about 9.5 mM, about 9.6 mM, about 9.7 mM, about 9.8 mM, about 9.9 mM, about 10 mM, about 10.1 mM, about 10.2 mM, about 10.3 mM, about 10.4 mM, about 10.5 mM, about 10.6 mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.1 mM, about 11.2 mM, about 11.3 mM, about 11.4 mM, about 11.5 mM, about 11.6 mM, about 11.7 mM, about 11.8 mM, about 11.9 mM, about 12.0 mM, about 12.1 mM, about 12.2 mM, about 12.3 mM, about 12.4 mM, about 12.5 mM, about 12.6 mM, about 12.7 mM, about 12.8 mM, about 12.9 mM, about 13.0 mM, about 13.1 mM, about 13.2 mM, about 13.3 mM, about 13.4 mM, about 13.5 mM, about 13.6 mM, about 13.7 mM, about 13.8 mM, about 13.9 mM, about 14.0 mM, about 14.1 mM, about 14.2 mM, about 14.3 mM, about 14.4 mM, about 14.5 mM, about 14.6 mM, about 14.7 mM, about 14.8 mM, about 14.9 mM, about 15.0 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM, about 8.1 mM, about 8.2 mM, about 8.3 mM, about 8.4 mM, about 8.5 mM, about 8.6 mM, about 8.7 mM, about 8.8 mM, about 8.9 mM, about 9.0 mM, about 9.1 mM, about 9.2 mM, about 9.3 mM, about 9.4 mM, about 9.5 mM, about 9.6 mM46. ​​The method of claim 45, wherein the concentration is selected from about 9.7 mM, about 9.8 mM, about 9.9 mM, about 10 mM, about 10.1 mM, about 10.2 mM, about 10.3 mM, about 10.4 mM, about 10.5 mM, about 10.6 mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, about 14.0 mM, about 14.5 mM and about 15.0 mM.

50. 46. ​​The method of claim 45, wherein the PEGylated hemoglobin composition of step (h) or the PEGylated carboxylated hemoglobin composition of step (i) maintains acidic charge variants at less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less, and maintains basic charge variants at less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less.

51. 1. A composition comprising oxygenated hemoglobin having a main peak purity of 70% or greater, less than 2% MetHb, and less than 25% charge variants, wherein the charge variants are acidic or basic variants of the main peak.

52. 1. A method for controlling and / or reducing the formation of MetHb during the manufacture of an oxygenated hemoglobin composition, comprising the steps of: e) deoxygenating hemoglobin to form deoxyhemoglobin; f) heat-treating the deoxygenated hemoglobin with about 5.5 mM to about 15 mM L-cysteine; g) reoxygenating the deoxygenated hemoglobin to form an oxygenated hemoglobin composition; h) measuring the MetHb concentration in the oxygenated hemoglobin composition; wherein the oxygenated hemoglobin composition has less than 2% MetHb formation as analyzed by co-oximetry; e. optionally, PEGylating the oxygenated hemoglobin composition; f. optionally, performing a carboxylation process to produce a carboxylated PEGylated hemoglobin composition, wherein the PEGylated hemoglobin composition of step (e) or the PEGylated carboxylated hemoglobin composition of step (f) maintains MetHb at less than 4%, preferably less than 3%; A method comprising:

53. 53. The method of claim 45 or 52, wherein the oxygenated hemoglobin composition is produced on a large scale selected from about 20 L to about 1000 L.

54. The heat treatment of deoxygenated hemoglobin at about 60°C was performed for about 4 hours, about 4.1 hours, about 4.2 hours, about 4.3 hours, about 4.4 hours, about 4.5 hours, about 4.6 hours, about 4.7 hours, about 4.8 hours, about 4.9 hours, about 5 hours, about 5.1 hours, about 5.2 hours, about 5.3 hours, about 5.4 hours, about 5.5 hours, about 6.0 hours, about 6.1 hours, about 6.2 hours, and about 6.3 hours. .3 hours, about 6.4 hours, about 6.5 hours, about 6.6 hours, about 6.7 hours, about 6.8 hours, about 6.9 hours, about 7.0 hours, about 7.1 hours, about 7.2 hours, about 7.3 hours, about 7.4 hours, about 7.5 hours, about 7.6 hours, about 7.7 hours, about 7.8 hours, about 7.9 hours, about 8.0 hours, about 8.1 hours, about 8.2 hours, about 8.3 hours, about 8.4 hours, about 8.5 hours 0.5 hours, about 8.6 hours, about 8.7 hours, about 8.8 hours, about 8.9 hours, about 9.0 hours, about 9.1 hours, about 9.2 hours, about 9.3 hours, about 9.4 hours, about 9.5 hours, about 9.6 hours, about 9.7 hours, about 9.8 hours, about 9.9 hours, about 10.0 hours, about 10.1 hours, about 10.2 hours, about 10.3 hours, about 10.4 hours, about 10.5 hours, about 53. The method of claim 52, wherein the treatment is for 10.6 hours, about 10.7 hours, about about 10.8 hours, about 10.9 hours, about 11.0 hours, about 11.1 hours, about 11.2 hours, about 11.3 hours, about 11.4 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours, or about 15.0 hours.

55. 53. The method of claim 52, wherein the L-cysteine ​​concentration during heat treatment is about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8.0 mM , about 8.1mM, about 8.2mM, about 8.3mM, about 8.4mM, about 8.5mM, about 8.6mM, about 8.7mM, about 8.8mM, about 8.9mM, about 9.0mM, about 9.1mM, about 9.2mM, about 9.3mM, about 9.4mM, about 9.5mM, About 9.6mM, about 9.7mM, about 9.8mM, about 9.9mM, about 10mM, about 10.1mM, about 10.2mM, about 10.3mM, about 10.4mM, about 10.5mM, about 10.6mM, about 10.7mM, about 10.8mM, about 10.9mM, about 11.0mM, about 11.1mM, about 11.2mM, about 11.3mM, about 11.4mM, about 11.5mM, about 11.6mM, about 11.7mM, about 11.8mM, about 11.9mM, about 12.0mM, about 12.1mM, about 12.2mM, about 12 .3mM, about 12.4mM, about 12.5mM, about 12.6mM, about 12.7mM, about 12.8mM, about 12.9mM, about 13.0mM, about 13.1mM, about 13.2mM, about 13.3mM, about 13.4mM, about 13.5mM, about 13.6m 53. The method of claim 52, wherein the ATP is maintained at a concentration selected from about 13.5 mM, about 13.7 mM, about 13.8 mM, about 13.9 mM, about 14.0 mM, about 14.1 mM, about 14.2 mM, about 14.3 mM, about 14.4 mM, about 14.5 mM, about 14.6 mM, about 14.7 mM, about 14.8 mM, about 14.9 mM, about 15.0 mM, about 16.0 mM, about 16.5 mM, about 17.0 mM, about 17.5 mM, about 18.0 mM, about 18.5 mM, about 19.0 mM, about 19.5 mM, and about 20.0 mM.

56. 1. A method of treating a condition that can be ameliorated by oxygenating red blood cells in a patient in need thereof, comprising administering to said patient: c) Oxygenated hemoglobin with a main peak purity of 70% or more; d) one or more impurities selected from MetHb, acidic variants, basic variants, viral particles, and / or prions; wherein said oxygenated hemoglobin comprises 2% or less of MetHb of total oxygenated hemoglobin; 20. A method comprising administering a therapeutically effective amount of a stable hemoglobin composition comprising:

57. 57. The method of claim 56, wherein: f) A process comprising oxygenated hemoglobin having a main peak purity of 70% or greater and one or more of the following impurities: g) Oxygenated hemoglobin with MetHb ≤2%; h) Oxygenated hemoglobin containing less than 12% of the acidic variant of the main peak; i) Oxygenated hemoglobin containing less than 11% of the main peak basic variant; j) Oxygenated hemoglobin substantially free of viral particles and / or prions

58. 57. The method of claim 56, wherein the condition is selected from the group comprising acute respiratory distress syndrome, bronchiectasis, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, lymphangiomatosis, primary ciliary dyskinesia, cancer, tumor, lung cancer, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, pulmonary sarcoidosis, pneumonia and bronchitis, infections affecting the transport capacity of the lungs.

59. 57. The method of claim 56, wherein the stable hemoglobin composition comprises pegylated oxygenated hemoglobin or pegylated carboxylated hemoglobin.

60. 1. A method of administering a stable hemoglobin composition, wherein the stable hemoglobin composition comprises: e) Oxygenated hemoglobin with a main peak purity of 70% or more; f) one or more impurities selected from MetHb, acidic variants, basic variants, viral particles and / or prions; wherein the oxygenated hemoglobin comprises 2% or less of MetHb of total oxygenated hemoglobin; Method of administration.

61. below: a) A method comprising producing oxygenated hemoglobin having a main peak purity of 70% or greater and one or more of the following impurities: b) MetHb ≤2% oxygenated hemoglobin; c) Oxygenated hemoglobin containing less than 12% of the acidic variant of the main peak: d) Oxygenated hemoglobin containing less than 11% of the main peak basic variant: e) Oxygenated hemoglobin substantially free of viral particles and / or prions The administration method according to claim 60, wherein

62. 61. The method of claim 60, wherein the administration frequency is once a day or twice a day.

63. 61. The method of claim 60, wherein the method is used to treat a condition selected from the group consisting of acute respiratory distress syndrome, anemia, bronchiectasis, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, lymphangiomatosis, primary ciliary dysfunction, cancer, tumors, lung cancer, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, pulmonary sarcoidosis, pneumonia and bronchitis, and infections that affect the oxygen-carrying capacity of the lungs.

64. The anemia is selected from the group consisting of blood loss anemia, anemia due to insufficient red blood cell production, anemia due to red blood cell destruction, and combinations thereof; or 61. The method of claim 60, wherein the cancer is selected from solid tumors, soft tissue cancer, lung cancer, bone cancer, metastatic cancer, adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, and breast cancer.

65. 61. The method of claim 60, wherein the stable hemoglobin composition comprises pegylated oxygenated hemoglobin or pegylated carbonated hemoglobin.

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