Extra pure semi-synthetic blood substitute
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 homoglobin 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.
Claims20
A hemoglobin solution for transporting and releasing oxygen to living cells, comprising bovine 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) 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 standary regression curve made from colormetric readings from said dilutions.
A blood-substitute comprising intramolecularly and intramolecularly crosslinked bovine hemoglobin in an aqueous pharmaceutically acceptable carrier medium, said hemoglobin being crosslinked to a sufficient degree to provide polyhemoglobin with at least about 50% of said polyhemoglobin of greater than tetrameric form, said blood-substitute being substantially free of cell stroma, non-hemoglobin proteins and pyrogens and having an endotoxin level of less than about 0.5 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.
A blood-substitute comprising an aqueous solution of hemoglobin derived from bovine red blood cells, said hemoglobin being crosslinked to a sufficient degree to provide polyhemoglobin with at least about 50% of said polyhemoglobin of greater than tetrameric form, said blood-substitute:
- a) having a P.sub.50 measured under human physiologic conditions in the range of from about 18 to about 36 mmHg;
- 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) 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.
A blood-substitute comprising an aqueous solution of crosslinked hemoglobin, said hemoglobin being derived from bovine red blood cells, 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 millimoles 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;
- h) an intravascular half-life of at least about four days;
- i) being substantially free of red blood cell stroma, non-hemoglobin proteins and pyrogen.
A mammalian blood-substitute comprising crosslinked hemoglobin suspended in an aqueous medium, said hemoglobin having been derived from mammalian blood cells, 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 180-230 milliosmoles per liter of solution;
- a final hemoglobin content of from about 9 to about 13 grams per deciliter;
- a methamoglobin 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;
- an intravascular half-life of at least about four days;
- and an endotoxin level of less than about 0.5 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.
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 bovine blood without prior washing to produce hemoglobin-containing fraction;
- 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 involving the steps of isolating hemoglobin from mammalian red blood cells, suspending said isolated hemoglobin in a pharmaceutically acceptable aqueous carrier medium to thereby form a hemoglobin solution, filtering said solution to remove bacteria, red blood cell stroma and phospholipids:
- the improvement comprising additionally chromatographically treating said hemoglobin solution 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 0.5 endotoxin units per milliliter (EU/ml) determined by a chromatographic 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.
A blood-substitute comprising intermolecularly and intramolecularly crosslinked bovine hemoglobin in an aqueous pharmaceutically acceptable carrier medium, said hemoglobin being crosslinked to a sufficient degree to provide polyhemoglobin with at least about 50% of said polyhemoglobin of greater than tetrameric form, 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, wherein the endotoxin level of said composition is less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromgenic Limulus Amebocytic Lystate (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 colormetric readings from said dilutions.
A blood-substitute comprising of from about 5 to about 25 grams of bovine hemoglobin per deciliter of an aqueous pharmaceutically acceptable carrier medium, said hemoglobin being intermolecularly and intramolecularly crosslinked, 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, wherein the endotoxin level of said composition is less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lystate (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 colormetric readings from said dilutions.
Dependencies are inferred from the claim text.