Method for the preparation of a heat stable oxygen carrier-containing pharmaceutical composition and the use thereof

Abstract

A highly purified and heat stable cross-linked tetrameric hemoglobin suitable for use in mammals without causing renal injury and vasoconstriction is provided. A high temperature and short time (HTST) heat processing step is performed to remove undesired dimeric form of hemoglobin and the plasma proteins impurities effectively. Addition of N-acetyl cysteine to the heat stable cross-linked tetrameric hemoglobin maintains a low level of met-hemoglobin. The heat stable cross-linked tetrameric hemoglobin can improve and prolong oxygenation in normal and hypoxic tissue. In another aspect, the product is used in the treatment of various types of cancer such as leukemia, colorectal cancer, lung cancer, breast cancer, liver cancer, nasopharyngeal carcinoma and esophageal cancer. Another application is heart preservation in situations where there is a lack of oxygen supply in vivo, such as in heart transplant or oxygen-deprived heart. The presently claimed invention also includes a method for preparing a packaged cross-linked tetrameric hemoglobin composition.

What is claimed is: 
     
         1 . A method for the preparation of a highly purified and heat stable oxygen carrier-containing pharmaceutical composition packaged in a multilayer, flexible infusion package, the oxygen carrier-containing pharmaceutical composition including hemoglobin, the hemoglobin consisting essentially of nonpolymeric cross-linked tetrameric hemoglobin, the method comprising:
 a) providing mammalian whole blood including at least red blood cells and plasma;   b) separating the red blood cells from the plasma in the mammalian whole blood;   c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction;   d) washing the filtered red blood cell fraction to remove plasma protein impurities, resulting in washed red blood cells;   e) disrupting the washed red blood cells by a precise and controlled hypotonic lysis for 2 to 30 seconds or a time otherwise sufficient to lyse the red blood cells in an instant cytolysis apparatus to create a solution comprising a lysate of disrupted red blood cells at a flow rate of 50-1000 L/hr;   f) performing filtration to remove at least a portion of the waste retentate from the lysate;   g) extracting a first hemoglobin solution from the lysate;   h) performing a first ultrafiltration process, using an ultrafiltration filter configured to remove impurities having a higher molecular weight than hemoglobin to further remove any viruses and residual waste retentate from the first hemoglobin solution to obtain a second hemoglobin solution;   i) performing flowthrough column chromatography on the purified hemoglobin solution to remove protein impurities;   j) performing a second ultrafiltration process using an ultrafiltration filter configured to remove impurities and to concentrate the purified hemoglobin solution;   k) cross-linking the hemoglobin in a bis-3,5-dibromosalicyl fumarate cross-linking solution to form cross-linked hemoglobin in a deoxygenated environment wherein the cross-linked hemoglobin is nonpolymeric cross-linked tetrameric hemoglobin;   l) exchanging a suitable physiological buffer for the cross-linking solution;   m) removing any residual chemicals by tangential flow filtration;   n) heat treating the cross-linked hemoglobin in a deoxygenated environment to denature and precipitate any residual non-reacted hemoglobin, non-stabilized hemoglobin (dimer) and any other protein impurities such that the resulting heat stable cross-linked tetrameric hemoglobin has an undetectable concentration of dimer and consists essentially of nonpolymeric cross-linked tetrameric hemoglobin;   o) adding N-acetyl cysteine immediately following heat treating the cross-linked tetrameric hemoglobin to maintain a low level of met-hemoglobin;   p) removing precipitate by a centrifugation or a filtration apparatus to form a clear solution;   q) adding the purified and heat stable cross-linked tetrameric hemoglobin to a pharmaceutically acceptable carrier and   r) introducing the purified and heat stable cross-linked tetrameric hemoglobin and pharmaceutically acceptable carrier to a multilayer, flexible infusion package.   
     
     
         2 . The method of  claim 1  wherein said multilayer, flexible infusion package comprises a multilayer aluminum/polyethylene terephthalate (PET)/nylon/polyethylene (PE) overwrap with a thickness of 0.14 mm and an oxygen transmission rate of 0.006 cm 3  per 100 square inches per 24 hours per atmosphere at room temperature. 
     
     
         3 . The method of  claim 2  wherein said multilayer, flexible infusion package further comprises five layers of ethylene-vinyl-acetate (EVA)/ethylene-vinyl alcohol (EVOH) material with a thickness of 0.4 mm that has an oxygen permeability of 0.006-0.132 cm 3  per 100 square inches per 24 hours per atmosphere at room temperature. 
     
     
         4 . The method of  claim 1  further comprising storage at ambient conditions for a period of time up to two years. 
     
     
         5 . A packaged oxygen carrier-containing pharmaceutical composition formed according to the method of  claim 1 . 
     
     
         6 . A packaged oxygen carrier-containing pharmaceutical composition formed according to the method of  claim 2 . 
     
     
         7 . A packaged oxygen carrier-containing pharmaceutical composition formed according to the method of  claim 3 .