Blood substitutes comprising hemoglobin and methods of making
Inventors
Light, William Richard • Tucker, Joseph
Assignees
Publication Number
US-11504417-B2
Publication Date
2022-11-22
Expiration Date
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Abstract
Methods for making hemoglobin based blood substitute preparations and hemoglobin based blood substitute preparations. The methods involve preparing a low purity erythrocyte protein fraction comprising hemoglobin protein and endogenous non-hemoglobin protein complement, and chemically modifying the proteins in the protein fraction to form a cross-linked hemoglobin containing blood substitute preparation. The low purity erythrocyte protein preparation can contain from at least about 0.2% (mole/mole) up to about 20% (mole/mole) endogenous non-hemoglobin protein complement. At least about 90% (mole/mole) of the hemoglobin proteins can be cross-linked, so that the average molecular mass of cross-linked proteins comprising hemoglobin protein molecules in the preparation is at least about 300 kDa. The preparations can be used to prepare finished blood substitute formulations for in-vivo and ex-vivo use.
Core Innovation
Methods for making hemoglobin based blood substitute preparations and hemoglobin based blood substitute preparations that involve preparing a low purity erythrocyte protein fraction comprising hemoglobin protein molecules and an endogenous non-hemoglobin protein complement, and chemically modifying the proteins in the protein fraction to form cross-linked hemoglobin-containing blood substitute preparations. The low purity erythrocyte protein fraction can comprise from at least about 0.2% (mole/mole) up to about 20% (mole/mole) endogenous non-hemoglobin protein complement, and at least about 90% (mole/mole) of the hemoglobin protein molecules can be cross-linked so that the average molecular mass of cross-linked proteins comprising hemoglobin protein molecules in the preparation is at least about 300 kDa (and in some embodiments about 1,000 kDa). The resulting preparations can be used to prepare finished blood substitute formulations for in-vivo and ex-vivo use. [procedural detail omitted for safety]
The disclosure addresses limitations in existing blood substitute approaches, including blood donor shortages and safety concerns associated with blood-borne pathogenic agents, drawbacks of erythrocyte preparations, nephrotoxicity and suboptimal oxygen delivery characteristics observed with early hemoglobin preparations, the manufacturing complexity and cost associated with producing highly purified hemoglobin for hemoglobin-based oxygen carriers, hazards associated with certain polymerization reactants (notably sodium borohydride releasing hydrogen gas), and the formation of methemoglobin which is unable to bind oxygen.
Claims Coverage
Independent claim identified: one independent claim. The main inventive features recited in the independent claim and its dependent aspects include features directed to a finished blood substitute formulation comprising a blood substitute preparation made from a low purity erythrocyte protein fraction, the nature and amount of endogenous non-hemoglobin protein complement, cross-linking characteristics, cross-link chemistry, optional additives, intended uses, and selected physicochemical attributes.
Finished blood substitute formulation comprising low purity erythrocyte protein fraction
A finished blood substitute formulation comprising a blood substitute preparation comprising a low purity erythrocyte protein fraction comprising chemically modified cross-linked proteins comprising hemoglobin protein molecules and endogenous non-hemoglobin protein complement, with the low purity protein fraction comprising about 0.2% (mole/mole) to about 20% (mole/mole) of the endogenous non-hemoglobin protein complement, wherein the endogenous non-hemoglobin protein complement comprises carbonic anhydrase.
High degree of hemoglobin cross-linking and molecular mass
About 90% (mole/mole) of the hemoglobin protein molecules in the erythrocyte protein fraction are cross-linked so that the average molecular mass of cross-linked proteins is at least 300 kDa (and in another recited embodiment is at least 1,000 kDa).
Reducible covalent cross-link chemistry
Cross-linkages are reducible covalent cross-linkages, specifically reciting Schiff bases and reduced Schiff bases, and reciting polyaldehydes (for example glutaraldehyde) in association with formation of Schiff bases and subsequent reduction to reduced covalent cross-linkages (secondary amines).
Inclusion of additional formulation ingredients
The finished blood substitute preparation may further comprise at least one additional ingredient suitable to form a finished blood substitute formulation, wherein the at least one additional ingredient is an excipient, diluent, or carrier.
Intended in-vivo and ex-vivo uses and administration
The blood substitute preparation and finished blood substitute formulation are recited as suitable for in-vivo use and/or for ex-vivo use, and the disclosure recites administration into the circulatory system including injection (for example intravenous or intra-arterial injection) for in-vivo administration.
Selected physicochemical attributes
Selected recited attributes for the finished blood substitute preparation include, in various recited embodiments, total hemoglobin concentration (about 10 g/dL to about 12 g/dL), a pH of about 7.5 (or about 7.7 in another recited embodiment), a partial pressure of oxygen (p50) of about 30 mmHg to about 50 mmHg (or about 32 mmHg in another recited embodiment), and a viscosity of about 12 cPs to about 18 cPs.
The independent claim covers a finished blood substitute formulation made from a low purity erythrocyte protein fraction containing 0.2%–20% endogenous non-hemoglobin protein complement (including carbonic anhydrase), wherein hemoglobin is chemically cross-linked at high conversion to form high molecular mass cross-linked proteins, employing reducible covalent cross-link chemistries (e.g., Schiff bases/reduced Schiff bases using polyaldehydes such as glutaraldehyde), optionally formulated with excipients/diluents/carriers, and recited for in-vivo and ex-vivo use with specified physicochemical attributes.
Stated Advantages
Enables manufacture of blood substitute preparations from lower purity hemoglobin-containing erythrocyte protein fractions, avoiding the need for highly purified hemoglobin and reducing manufacturing complexity and cost.
Avoids the use of borohydride (BH4−) and the associated safety and operational challenges of hydrogen gas release in manufacturing.
Provides preparations that can deliver tissue oxygenation and thereby mediate tissue survival.
Exhibits a low colloid osmotic pressure and a high viscosity that are associated with limited or no vasoconstriction and limited or no extravasation of hemoglobin upon administration.
Limits formation of reactive oxygen species in the preparations, thereby minimizing damage to the endothelial glycocalyx of blood vessels.
Exhibits limited quantities of methemoglobin in the preparations.
Documented Applications
Preparation of finished blood substitute formulations for in-vivo administration to subjects in need thereof, including administration into the circulatory system by injection (for example intravenous or intra-arterial injection).
Preparation of finished blood substitute formulations for ex-vivo use to transport and deliver oxygen to organs, tissues or cells outside a living subject, including for organ preservation for transplantation.
Ex-vivo preservation of organs or tissue (e.g., liver, kidney, heart, lung, intestine, pancreas) in static or dynamic mode using the finished blood substitute formulation to maintain oxygen content.
Use during surgical procedures, resuscitation, or to treat hypoxic tissue resulting from myocardial infarction, stroke, anemia, trauma or shock (including anaphylactic, septic or allergic shock), and for acute hemorrhage replacement.
Use to maintain organs for research and development purposes, for example for biomarker discovery or use of organ tissue as a bioreactor.
Whole body preservation of living donors, brain-dead individuals, or cadavers.
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