Ultra pure hemoglobin solutions and blood-substitutes
AssigneeBiopure Corporation
InventorsCarl W. Rausch, Mario Feola
Life of this patent
Estimating the term…
Abstract
A blood substitute and plasma expander comprising a cross-linked, substantially endotoxin-free hemoglobin solution and process for preparing same. The process comprises fractionating whole blood, separating out a stromal-free, sterile hemoglobin solution, chromatographically separating endotoxins from said hemoglobin solution and crosslinking the resulting endotoxin-free hemoglobin solution.
Claims48
A blood-substitute comprising of from about 5 to about 25 grams of crosslinked hemoglobin per deciliter of an aqueous pharmaceutically acceptable carrier medium, said blood-substitute being substantially free of cell stroma, non-hemoglobin proteins and pyrogens and said blood-substitute being sufficiently purified with respect to endotoxin so that the blood-substitute does not, upon in vivo administration to a mammal in a significant dosage, cause complement activation.
A blood-substitute comprising an aqueous solution of crosslinked hemoglobin, said blood-substitute characterized by:
- a) an endotoxin concentration of less than about 0.02 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilutions of a reference endotoxin solution and thereafter interpolating from a standard regression curve made from colorometric readings from said dilutions;
- b) a phospholipid concentration of less than about 1 nanogram per milliliter;
- c) a hemoglobin molecular weight distribution of greater than about 90% in the range of 68,000-500,000 daltons;
- d) an osmolarity as measured by freezing point depression in the range of 180-320 milliosmoles per liter;
- e) a hemoglobin content of from about 5 to about 25 grams per deciliter;
- f) a methemoglobin content of less than about 20%, by weight;
- g) a P.sub.50 in the range of about 18-36 mm Hg;
- and
- h) being substantially free of red blood cell stroma, non-hemoglobin proteins and pyrogens.
A mammalian blood-substitute comprising crosslinked hemoglobin suspended in an aqueous medium, said blood-substitute having:
- crosslinked hemoglobin having a molecular weight distribution of greater than about 90% in the range of about 68,000 to 500,000 daltons;
- an osmolarity as measured by freezing point depression in the range of about 180-320 milliosmoles per liter of solution;
- a final hemoglobin content of from about 9 to about 13 grams per deciliter;
- a methemoglobin content of less than about 10%, by weight;
- physiologic levels of sodium chloride and potassium chloride;
- less than about one nanomole of phospholipid per milliliter;
- less than about one part per million of crosslinking agent;
- a P.sub.50 in the range of about 18-36 mm Hg;
- and an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A blood-substitute comprising intramolecularly and/or intermolecularly crosslinked hemoglobin in an aqueous pharmaceutically acceptable carrier medium, said hemoglobin being crosslinked and said blood-substitute being substantially free of cell stroma, non-hemoglobin proteins and pyrogens and said blood-substitute, upon in vivo administration to a mammal in a therapeutically significant dosage, having an endotoxin level which does not cause complement activation.
A blood-substitute comprising an aqueous solution of crosslinked hemoglobin, said blood-substitute:
- a) having a P.sub.50 measured under human physiologic conditions in the range of from about 18 to about 36 mm Hg;
- b) being substantially free of red blood cell stroma and non-hemoglobin proteins;
- and
- c) having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A hemoglobin solution for transporting and releasing oxygen to living cells comprising hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A method for producing a hemoglobin solution substantially free of endotoxins by performing the following sequence of steps under substantially endotoxin free conditions:
- a) disrupting the red blood cell fraction of blood without prior washing to produce a hemoglobin-containing solution;
- b) clarifying said hemoglobin-containing solution to produce a substantially stromal-free hemoglobin-containing solution;
- c) microporously filtering said substantially stromal-free hemoglobin-containing solution to produce a partially sterilized, stromal-free, hemoglobin-containing solution;
- d) ultrafiltering said partially sterilized, stromal-free hemoglobin-containing solution to produce a size-separated, partially sterilized, stromal-free, hemoglobin-containing solution;
- and
- e) chromatographically treating said size-separated, partially-sterilized, stromal-free, hemoglobin-containing solution in a high performance liquid chromatographic column containing a packing for the ion-exchange affinity separation of endotoxin from hemoglobin under conditions sufficient to produce a substantially phospholipid-free, substantially endotoxin-free hemoglobin solution.
In the preparation of an ultra pure hemoglobin solution, from mammalian blood with an endotoxin level of less than 7 endotoxin units per milliliter, involving the steps of separating the red blood cells from said blood by centrifugation, mechanically degrading said red blood cells to release hemoglobin, clarifying said hemoglobin to remove cell debris, suspending said hemoglobin in a pharmaceutically acceptable aqueous carrier medium to thereby form a hemoglobin solution, microporously filtering said solution to remove bacteria and red blood cell stroma, and ultrafiltering said solution to remove phospholipids and to reduce the endotoxin level to less than about 0.5 endotoxin units per millimeter:
- the improvement comprising additionally chromatographically treating said hemoglobin solution in a high performance liquid chromatographic column by passing said solution over a chromatographic medium for the ion-exchange affinity separation of endotoxin under conditions sufficient to produce a hemoglobin solution sufficiently purified with respect to endotoxin so that the hemoglobin solution does not, upon in vivo administration to a mammal in a significant dosage, cause complement activation.
In the preparation of an ultra pure hemoglobin solution, from mammalian blood with an endotoxin level of less than 7 endotoxin units per milliliter, involving the steps of separating the red blood cells from said blood by centrifugation, mechanically degrading said red blood cells to release hemoglobin, clarifying said hemoglobin to remove cell debris, suspending said hemoglobin in a pharmaceutically acceptable aqueous carrier medium to thereby form a hemoglobin solution, microporously filtering said solution to remove bacteria and red blood cell stroma, and ultrafiltering said solution to remove phospholipids and to reduce the endotoxin level to less than about 0.5 endotoxin units per millimeter:
- the improvement comprising additionally chromatographically treating said hemoglobin solution in a high performance liquid chromotographic column by passing said solution over a chromatographic medium for the ion-exchange affinity separation of endotoxin under conditions sufficient to produce a hemoglobin solution having an endotoxin level of less than about 0.02 endotoxin units per milliliter (EU/ml).
A pharmaceutical composition for transporting and releasing oxygen to living cells comprising a suspension of hemoglobin in a pharmaceutically acceptable carrier, said suspension having an endotoxin level of less than about 0.02 EU/ml.
A method of treating a mammal which has sustained a loss of blood by administering to said mammal a therapeutically efficacious amount of a suspension of hemoglobin in a pharmaceutically acceptable aqueous carrier, said suspension of hemoglobin having an endotoxin level of less than about 0.5 EU/ml.
A hemoglobin solution comprising hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A blood-substitute for transporting and releasing oxygen to living cells comprising cross-linked hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A plasma-expander for transporting and releasing oxygen to living cells comprising cross-linked hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
A resuscitation fluid for transporting and releasing oxygen to living cells comprising cross-linked hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml).
Dependencies are inferred from the claim text.