# US 11,504,417 B2 · Blood substitutes comprising hemoglobin and methods of making

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- Number: US 11,504,417 B2
- Document ID: US11504417B2
- Status: Granted
- Kind: B2
- Application number: 16/632,090
- Filed: 2018-07-17
- Published: 2020-07-16
- Granted: 2022-11-22
- Earliest claimed: 2017-07-18
- Assignees: VirTech Bio, Inc.
- Applicants: Virtech Bio, Inc.
- Inventors: William Richard Light; Joseph Tucker
- Web page: https://patentgrep.com/patent/US11504417B2

## CPC

- A61K 38/42: Haemoglobins; Myoglobins
- A61K 9/0026: {Blood substitute; Oxygen transporting formulations; Plasma extender}
- A61K 35/14: Blood; Artificial blood (perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42)
- A61K 35/18: Erythrocytes (haemoglobin A61K38/42)
- A61P 7/08: Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock (artificial tears A61P27/04)
- C07K 14/805: Haemoglobins; Myoglobins
- C12N 5/0641: {Erythrocytes}
- A61M 1/0281: {Apparatus for treatment of blood or blood constituents prior to transfusion, e.g. washing, filtering or thawing}
- A61M 2202/0429: Red blood cells; Erythrocytes
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)}

## 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.

## Claims

### Claim 1

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, the hemoglobin protein molecules intermolecularly cross-linked via cross-linkages, and the hemoglobin protein molecules intermolecularly cross-linked via cross-linkages to the endogenous non-hemoglobin protein complement, 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.

### Claim 2 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein about 90% (mole/mole) of the hemoglobin protein molecules in the erythrocyte protein fraction are cross-linked, wherein the average molecular mass of cross-linked proteins is at least 300 kDa.

### Claim 3 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein about 90% (mole/mole) of the hemoglobin protein molecules in the erythrocyte protein fraction are cross-linked, wherein the average molecular mass of cross-linked proteins is at least 1,000 kDa.

### Claim 4 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein the carbonic anhydrase, comprises about 0.2% (mol/mole) to about 15% (mole/mole) of the endogenous non-hemoglobin protein complement.

### Claim 5 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein the cross-linkages are reducible covalent cross-linkages.

### Claim 6 (depends on 5)

The finished blood substitute preparation according to claim 5, wherein the reducible covalent cross-linkages are Schiff bases.

### Claim 7 (depends on 6)

The finished blood substitute preparation according to claim 6, wherein the reduced covalent cross-linkages are secondary amines.

### Claim 8 (depends on 5)

The finished blood substitute preparation according to claim 5, wherein the cross-linkages are reduced Schiff bases.

### Claim 9 (depends on 8)

The finished blood substitute preparation according to claim 8, wherein the cross-linkages are reduced Schiff bases having been formed by reacting proteins in the low purity erythrocyte fraction with a polyaldehyde to form a Schiff base, and subsequent reduction of the Schiff base.

### Claim 10 (depends on 9)

The finished blood substitute preparation according to claim 9, wherein the polyaldehyde is glutaraldehyde.

### Claim 11 (depends on 1)

The finished blood substitute preparation according to claim 1, further comprising at least one additional ingredient suitable to form a finished blood substitute formulation.

### Claim 12 (depends on 11)

The finished blood substitute preparation according to claim 11, wherein the at least one additional ingredient is an excipient, diluent, or carrier.

### Claim 13 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein the blood substitute preparation is a formulation suitable for in-vivo use and/or for ex-vivo use.

### Claim 14 (depends on 13)

The finished blood substitute preparation according to claim 13, wherein the blood substitute preparation comprises at least one of a concentration of about 10 grams/deciliter to about 12 grams/deciliter of total hemoglobin, a pH of about 7.5, a partial pressure of oxygen (p50) of about 30 mmHg to about 50 mmHg, and a viscosity of about 12 cPs to about 18 cPs.

### Claim 15 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein the blood substitute preparation comprises at least one of a concentration of about 10 grams/deciliter of total hemoglobin, a pH of about 7.7, a partial pressure of oxygen (p50) of about 32 mmHg, and a viscosity of about 12 cPs.

### Claim 16 (depends on 1)

The finished blood substitute preparation according to claim 1, wherein the endogenous non-hemoglobin protein complement further comprises superoxide dismutase.