Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-10-14
2001-08-07
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C514S006900, C604S403000, C604S408000
Reexamination Certificate
active
06271351
ABSTRACT:
BACKGROUND OF THE INVENTION
There exists a need for a blood-substitute to treat or prevent hypoxia resulting from blood loss (e.g, from acute hemorrhage or during surgical operations), resulting from anemia (e.g., pernicious anemia or sickle cell anemia), or resulting from shock (e.g, volume deficiency shock, anaphylactic shock, septic shock or allergic shock). The use of blood and blood fractions as in these capacities as a blood-substitute is fraught with disadvantages. For example, the use of whole blood often is accompanied by the risk of transmission of hepatitis-producing viruses and AIDS-producing viruses which can complicate patient recovery or result in patient fatalities. Additionally, the use of whole blood requires blood-typing and cross-matching to avoid immunohematological problems and interdonor incompatibility.
Human hemoglobin, as a blood-substitute, possesses osmotic activity and the ability to transport and transfer oxygen, but it has the disadvantage of rapid elimination from circulation by the renal route and through vascular walls, resulting in a very short, and therefore, a typically unsatisfactory half-life. Further, human hemoglobin is also frequently contaminated with toxic levels of endotoxins, bacteria and/or viruses.
Non-human hemoglobin suffers from the same deficiencies as human hemoglobin. In addition, hemoglobin from non-human sources is also typically contaminated with proteins, such as antibodies, which could cause an immune system response in the recipient.
Previously, at least four other types of blood-substitutes have been utilized, including perfluorochemicals, synthesized hemoglobin analogues, liposome-encapsulated hemoglobin, and chemically-modified hemoglobin. However, many of these blood-substitutes have typically had short intravascular retention times, being removed by the circulatory system as foreign substances or lodging in the liver, spleen, and other tissues. Also, many of these blood-substitutes have been biologically incompatible with living systems.
Thus, in spite of the recent advances in the preparation of hemoglobin-based blood-substitutes, the need has continued to exist for a blood-substitute which has levels of contaminants, such as endotoxins, bacteria, viruses, phospholipids and non-hemoglobin proteins, which are sufficiently low to generally prevent an immune system response and any toxicological effects resulting from an infusion of the blood-substitute. In addition, the blood-substitute must also be capable of transporting and transferring adequate amounts of oxygen to tissues under ambient conditions and must have a good intravascular retention time.
Further, it is preferred that the blood-substitute 1) has an oncotic activity generally equivalent to that of whole blood, 2) can be transfused to most recipients without cross-matching or sensitivity testing, and 3) can be stored with minimum amounts of refrigeration for long periods.
SUMMARY OF THE INVENTION
The present invention relates to a method for producing a stable polymerized hemoglobin blood-substitute from whole blood.
In one embodiment, the invention relates to a method for preserving the stability of a hemoglobin blood substitute by maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen.
The invention also involves a method for preparing the hemoglobin blood substitute comprising mixing blood with an anticoagulant to form a blood solution and then washing the red blood cells in the blood solution to separate small plasma proteins from the red blood cells. The method also includes the steps of separating the washed red blood cells from the white blood cells and then disrupting the red blood cells to release hemoglobin and form a hemoglobin solution. Non-hemoglobin components in the hemoglobin solution are subsequently separated from the hemoglobin solution by molecular weight fractionation on 100 kD and 30 kD nominal molecular weight cut-off ultrafilters and high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated and subsequently contacted with a sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, which is subsequently mixed with a cross-linking agent to form a polymerization reaction mixture. The polymerization reaction mixture is then stabilized and diafiltered with a physiologic solution and with a sulfhydryl compound, whereby the polymerized hemoglobin solution is made physiologically acceptable, and whereby the sulfhydryl compound scavenges trace levels of oxygen, thereby forming said stable polymerized hemoglobin blood-substitute.
The advantages of this invention are numerous. One advantage is that the hemoglobin produced and stored according to the methods of this invention has a greater degree of purity and longer shelf-life. A primary package having a high oxygen barrier allows the primary package to protect product stability before a high barrier overwrap is applied or after the overwrap is removed. In addition, transparent primary packaging allows the visual inspection of the production condition.
The present invention is drawn to a method for preserving a deoxygenated hemoglobin blood substitute comprising maintaining the deoxygenated hemoglobin blood substitute in a transparent primary package that includes a polymer material having at least one layer, said polymer material having an oxygen penneability of less than about 0.6 cc per 100 square inches per 24 hours per atmosphere at about 25° C. and an external relative humidity of about 50%.
The present invention is also drawn to a preserved deoxygenated hemoglobin blood substitute. Said preserved blood substitute comprises a deoxygenated hemoglobin blood substitute and a transparent primary package. Said transparent primary package includes a polymer material having at least one layer, and an oxygen permeability of less than about 0.6 cc per 100 square inches per 24 hours per atmosphere at about 25° C. and an external relative humidity of about 50%, within which the deoxygenated hemoglobin blood substitute is sealed, thereby preserving the deoxygenated hemoglobin blood substitute in an environment that is substantially free of oxygen.
The blood-substitute can remain stable at room temperature for periods up to two years or more, a significant improvement over previous methods. Furthermore, with the purified hemoglobin of the present invention one species of hemoglobin can be successfully used as a blood-substitute in a different species without the recipient species suffering significant side effects.
DETAILED DESCRIPTION OF THE INVENTION
The features and other details of the process of the invention will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the present invention.
The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. This method can be accomplished by maintaining the blood substitute in an oxygen-impermeable container, such as, an oxygen barrier primary package, an oxygen barrier film overwrap (e.g., a bag), glass container (e.g., a vial) or a steel container. Where the primary package is an oxygen barrier film, the container can be manufactured from a variety of materials, including polymer films, (e.g., an essentially oxygen-impermeable polyester, polyvinylidene chloride, ethylene vinyl alcohol (EVOH), silicon oxide (SiO
x
) or nylon, and laminates thereof).
The laminate material can be transparent. In one embodiment, the transparent material can be made to prevent photodegradation, using methods known in the art. In one embodiment, the laminate comprises a medium density polymer layer (such as pol
Gawryl Maria S.
Houtchens Robert A.
Light William R.
Biopure Corporation
Gupta Anish
Hamilton Brook Smith & Reynolds P.C.
Low Christopher S. F.
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