KR20050020971A - Sterilization, stabilization and preservation of functional biologics - Google Patents

Abstract

Described herein are methods for sterilizing and preserving functional biological materials, biochemical entities and biologically active molecules for room temperature storage. Biological contaminants are markedly reduced or eliminated in titer while preserving the functional integrity of sterile and stabilized products derived from the methods of the present invention. These substances may be primarily functional constructs such as bone or may exhibit active molecular function as in the case of hemoglobin and antibodies. Treatments for sterilization and stabilization of biologics can be achieved by changing the temperature of the material, adjusting the radiation dose rate, optimizing the irradiation time, adjusting the oxygen content, using the stabilizer, specifying the pH, including the specific solvent and the type of radiation applied. Include a change. Such treated, sterile, stabilized biological products can be stored at room temperature, making them more available and easier to use than conventional lyophilized conventional frozen or frozen stored biologics.

Description

Sterilization, stabilization and preservation of functional biologics

1 shows a table showing the characteristics of the samples tested.

2 shows a graph of Hct vs Mrad in the presence / absence of methylene blue.

3 shows a graph of pO ₂ versus Mrad in the presence / absence of methylene blue.

4 shows a graph of O ₂ saturation versus Mrad in the presence / absence of methylene blue.

5 shows a graph of FO ₂ Hb vs Mrad in the presence / absence of methylene blue.

6 shows a graph of FMetHb vs. Mrad in the presence / absence of methylene blue.

7 shows a graph of FHHb vs. Mrad in the presence / absence of methylene blue.

8 shows a graph of Na versus Mrad in the presence / absence of methylene blue.

9 shows a graph of WBC vs. Mrad in the presence / absence of methylene blue.

10 shows a graph of RBC vs. Mrad in the presence / absence of methylene blue.

FIG. 11 shows a graph of HGB vs. Mrad in the presence / absence of methylene blue.

12 shows a graph of HCT vs. Mrad in the presence / absence of methylene blue.

FIG. 13 shows a graph of MCV vs. Mrad in the presence / absence of methylene blue.

FIG. 14 shows a graph of MCH vs Mrad in the presence / absence of methylene blue.

FIG. 15 shows a graph of PLT vs. Mrad in the presence / absence of methylene blue.

FIG. 16 shows a graph of MCHC vs. Mrad in the presence / absence of methylene blue.

FIG. 17 shows a graph of RDW vs Mrad in the presence / absence of methylene blue.

FIG. 18 shows a graph of MPV vs. Mrad in the presence / absence of methylene blue.

19 is a photograph of titer plates showing antibodies with biological activity preserved 4 weeks after irradiation in accordance with the present invention.

20 is a photograph of a titer plate showing the light staining of red blood cells after gamma radiation and storage for 4 weeks at room temperature in accordance with the present invention.

Figure 21 is a photograph of a portion of a titer plate that slaughtered the antibody whose biological activity was preserved 4 weeks after irradiation in accordance with the present invention.

The present invention sterilizes and preserves biological materials, biochemical entities, and biologically active molecules (biologics) to protect viruses, bacteria (intercellular and intracellular bacteria such as mycoplasma, ureaplasma, nanobacteria, chlamydia, Methods for reducing the level of one or more biological contaminants or pathogens, including rickettsia), yeast, fungi, fungi, unicellular or multicellular parasites and / or prions or similar factors that alone or together result in infectious cavernous encephalopathy (TSE). It is about. Current storage methods of such biologics typically involve lyophilization of the biologics prior to sterilization and / or refrigeration of the product after sterilization and prior to transfusion or use in humans. Although certain methods have already described the use of ionizing radiation, these methods use additional additives and solutions with radiation. For example, some studies have focused on removing leukocytes and have generally used low radiation doses that cannot effectively inactivate bacterial and plasma viral loads. In addition, the literature of the prior art does not mention the ability to store red blood cells at room temperature after irradiation. The process of the invention does not require this additional solution. Numerous studies suggest that red blood cells are not destroyed by doses that inactivate known pathogenic viruses. Accordingly, the present invention is a simpler method of preserving and sterilizing blood prior to transfusion and making the blood safer for the recipient.

The present invention makes it possible to more easily preserve and store red blood cells while improving the safety of the material after irradiation. The prior art does not mention the storage of red blood cells at ambient temperature (eg room temperature) after irradiation. The prior art also incorporates additional solutions during the irradiation process. It may be advantageous to add a solution that counteracts potassium leakage from erythrocytes, for example buffers with or without potassium binder. The solution may include additional glucose in the blood bag to provide an additional source of energy to the red blood cells. Simply irradiating red blood cells in an appropriate solution environment in a basic storage bag is suitable to remove many known pathogens and allow red blood cells to be stored at room temperature.

I. Pollution Sources

As used herein, the term "biological contaminant or pathogen" means a contaminant or pathogen (alone or in combination) that may have a detrimental effect on the biological material or its recipients upon direct or indirect contact with the biological material. do. Such biological contaminants or pathogens include a variety of viruses, bacteria (including intracellular and intracellular bacteria such as mycoplasma, ureaplasma, nanobacteria, chlamydia, rickettsia), yeast, fungi, fungi, unicellular or multicellular parasites, Prions, factors causing TSE and other factors found in or infecting biological materials, biochemical entities and biologically active molecules. Further examples of biological contaminants or pathogens include viruses (eg, human immunodeficiency virus and other retroviruses), herpes virus, filovirus, circovirus, paramyxovirus, cytomegalovirus, hepatitis virus (types A, B and C). Hepatitis virus and other variants thereof), pox virus, toga virus, Epstein-Barr virus and parvovirus; Bacteria such as Escherichia, Bacillus, Campylobacter, Streptococcus and Staphylococcus (including Mycoplasma, Ureaplasma, Nanobacteria, Chlamydia, Rickettsia); Parasites such as tripanosoma and malaria parasites including plasmodium species; leaven; mold; And alone or in combination with TSE, for example tremors, kuru's disease, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD), Gerstmann-Straeussler-Scheinkler Syndrome ) And prion or similar factors that cause fatal familial insomnia. As used herein, the term “active biological contaminant or pathogen” is used alone or in other factors in a biological material and / or recipient thereof, such as a second biological contaminant or pathogen or native protein (wild type or mutant). Or a biological contaminant or pathogen that can cause a deleterious effect with the antibody.

II. Biologics

A. Biological Substances

As used herein, the term "biological material" means any material derived or obtained from a living organism. Illustrative examples of biological substances include cells, tissues, blood, blood components, proteins (including recombinant proteins, transgenic proteins, and monoproteins), amino acids, peptides (including all natural and synthetic peptides), sugars, lipids, Enzymes, including degradable enzymes such as trypsin, chymotrypsin, alpha-glucosidase and iduronodate-2 sulfatase, immunoglobulins (including monoglobulins and polyimmunoglobulins), plant materials and food It is not limited to this. Preferred examples of biological materials include ligaments, tendons, nerves, bones (including demineralized bone matrix, grafts, joints, femurs and femoral heads), bone marrow (whole or processed bone marrow cell suspension), teeth, skin grafts, heart valves, Cartilage, cornea, artery, vein, organs (including transplant organs such as heart, liver, lungs, kidneys, intestines, pancreas), limbs, fingers, lipids, carbohydrates, collagen (natural, non-fibrous, endless, soluble and Insoluble, recombinant and transgenic collagen, including native sequences and modified collagen), enzymes, chitin and derivatives thereof (including NO-carboxy chitosan "NOCC"), stem cells, islets of Langerhans and other cells for transplantation Including, but not limited to, fully modified cells), red blood cells, white blood cells (including monocytes), and platelets.

1.blood

What was needed or has not been available so far is a rapid and safe means of preserving the vital function of the tangible components and proteins of blood transfusion while sterilizing the blood, blood components and blood products for transfusion. Radiation not only provides a means of making transfusion components with increased safety but also allows storage of already frozen or refrigerated blood components and whole blood at room temperature. Such products and methods for their preparation will prove very beneficial by making the blood supply and supply of blood components safer and more readily available due to room temperature storage. It is also expected that the shelf life of the irradiated blood and blood products will be longer than that of refrigerated blood. About 20% of current transfusion units have already expired before transfusion. Increased shelf life will enable the use of units that are past their expiration date. Thus, such extension of shelf life may have the effect of increasing blood supply, another beneficial effect of introducing this new technique.

The present invention relates to a method of treating tissue to remove biological contaminants, in particular for sterilization and storage of blood and blood components. The invention also relates to the storage of red blood cells at room temperature after irradiation. The present invention will make it possible to transport and store accumulated blood at room temperature. As used herein, the term "blood component" refers to one or more components that can be separated from whole blood, and includes cellular blood components (e.g. red blood cells, white blood cells and platelets), blood proteins (e.g. clotting factors, enzymes). , Albumin, plasminogen, fibrinogen and immunoglobulin) and liquid blood components such as plasma, plasma protein fraction "PPF", cryoprecipitate, plasma fraction and plasma containing compositions). As used herein, the term "liquid blood component" refers to plasma (fluid noncellular part of whole blood of human or animal found before coagulation) and serum (fluid noncellular part of whole blood of human or animal found after coagulation) By one or more of the flow non-cellular part of the whole blood, such as.

As used herein, the term "cellular blood component" refers to one or more of the components of whole blood, including cells such as red blood cells, white blood cells, stem cells, and platelets. Viable erythrocytes may be characterized by one or more of the following: the ability to synthesize ATP; Cell morphology; P ₅₀ value; Oxidized hemoglobin, methemoglobin and hemichrome values; MCV, MCH and MCHC values; Cellular enzyme activity; And in vivo viability. Thus, if a virus-activated cell, lyophilized and then reconstituted, is impaired to the extent that such cells cannot metabolize or synthesize ATP, or the circulation of such cells is weakened, its utility in transfusion medicine will be diminished. In contrast, irradiated erythrocytes can still perform biochemical functions without the need for nucleic acid synthesis activity. Unlike most other mammalian cells, red blood cells are unique in that they do not have a nucleus, so protein components are irradiated because the protein components are much smaller than the DNA of the cell and are less likely to be inactivated by the radiation of the sterile beam being projected. It represents a more resistant target in that it can still function later.

As used herein, the term "blood protein" refers to one or more of the proteins commonly found in whole blood. Illustrative examples of blood proteins found in animals include vitamin K-dependent (e.g. Factor VII and Factor IX) and vitamin K-independent (e.g. Factor VIII and von Willebrand Factor) coagulation protein, albumin, lipoprotein (HDL) , Low density lipoprotein (LDL), lowest density lipoprotein, and complement protein globulins (eg, immunoglobulins IgA, IgM, IgG and IgE). Preferred groups of blood proteins include factor I (fibrinogen), factor II (prothrombin), factor III (tissue factor), factor V (proaxlerin), factor VI (accelelin), factor VII (proconvertin, serum). Prothrombin conversion), factor VIII (anti-hemopathy factor A), factor IX (anti-hemopathy factor B), factor X (stuart-prowar factor), factor XI (plasma thromboplastin precursor), factor XII (hegemann factor ), Factor XIII (protransglutamidase), von Willebrand factor (vWF), factor Ia, factor IIa, factor IIIa, factor Va, factor VIa, factor VIIa, factor VIIIa, factor IXa, factor Xa, factor XIa , Factor XIIa and factor XIIIa. Other preferred groups of blood proteins include proteins found inside red blood cells (eg hemoglobin), various growth factors, and derivatives of such proteins.

Plasma and serum (fluid phase is formed after coagulation) are components of blood. Numerous proteins and other factors exist and are valuable in a wide range of medical applications. Antibodies are present in plasma and serum. It can be used in a number of applications and investigations when treating patients (gamma globulin, pooled antibodies). The method of preserving red blood cells described in detail herein also applies to the production of antibodies and other proteins so that such isolates can be irradiated for sterile purposes and stored at room temperature. This will be very useful in medical environment and laboratory investigations. The technique can be applied to antibodies prepared from human blood or other animal blood or from tissue cultures derived from cell cultures that produce antibodies. The technique can also be used to prepare sterile viral vaccines for storage at room temperature, which can simplify the distribution and administration of vaccines. In addition, erythrocytes can be processed and stored at room temperature for use in hemagglutination blood typing.

2. Blood bag

Materials suitable for making bags and other containers for sterile and stabilized biological materials, biochemical entities and biologically active molecules include, but are not limited to, silicones, plastics, and foils. For example, collapsible oxygen permeable silicone bags are suitable for storing irradiated biologics such as sterile, stabilized blood products. The use of such bags may be important for converting methemoglobin formed during irradiation to oxidized hemoglobin prior to transfusion. Oxygen-rich environments can also help irradiation be more effective at lower total radiation doses. Flexible collapsible bags made of poly (ethylene-vinyl acetate) (E.V.A.) plastic are manufactured by Fenwal Division of Baxter Travenol Laboratories, Inc. Deerfield, Illinois.

Red blood cells are often washed to reduce the large number of white blood cells in the blood. Leukocytes can induce an immune response from blood recipients. Because of the risk of bacterial contamination of the blood, this washing process reduces the shelf life of red blood cells to 24 hours. Resealing and irradiating bags used to store blood will significantly extend shelf life. The normal standard shelf life after irradiation is 28 days from the time of irradiation or the original expiration date, in units, whichever is earlier. This can significantly extend the shelf life of washed red blood cells.

B. Biochemical Entities

Irradiation of functional biochemical entities and biologically active molecules, including but not limited to hemoglobin (hemoglobin in erythrocytes and hemoglobin independent of erythrocytes), antibodies, peptides (both natural and synthetic peptides), vaccines and other antigens It is about preservation by. More particularly, the present invention relates to plasma protein fractions (eg albumin and coagulation factors) collected from antibodies, peripheral blood (eg erythrocytes and platelets), whole blood (eg blood from virus infected persons), body fluids (urine, spinal fluid, amniotic fluid). And lubricating fluids, potentials for compositions or products derived from such compositions, including, but not limited to, ex vivo media and cell culture media (eg, fetal calf serum and bovine serum) used in the manufacture of antiviral vaccines. It relates to the inactivation of biological contaminants such as viruses, bacteria, yeasts, fungi, mycoplasma and parasites. The invention further includes a method of preparing whole blood products for room temperature storage. The present invention further relates to blood-based proteins and biologically derived proteins, including but not limited to botulinum toxin and plant derived proteins.

In other embodiments of the present invention, antibodies, coagulation factors, growth factors, and other biologically derived proteins, including whole viruses or portions thereof, can be preserved by the irradiation techniques described above for blood and blood components. High dose gamma irradiation inactivates bacteria and viruses and allows the irradiated material to be stored at room temperature. This provides greater availability of very important biologicals, such as polio vaccines, which currently have to be refrigerated, which is an impediment to use in necessary areas, such as Central Africa, where few refrigeration facilities can store vaccines requiring frozen storage. can do.

C. Biologically Active Molecules

 As used herein, the term "proteinaceous substance" means any substance derived from or obtained from a living organism comprising one or more proteins or peptides. The proteinaceous material may be a natural material in its inherent state or a natural material after processing / purification and / or derivatization or artificially produced by chemical synthesis or recombinant / transformation techniques and optionally processed / purified and / or derivatized It may be a substance. Illustrative examples of proteinaceous materials include proteins and peptides produced from cell culture, milk and other dairy products; revenge; hormone; Growth factor; Substances extracted or separated from animal tissues or plant matter (including agents such as insulin); Plasma and plasma protein fractions (including fresh, frozen and lyophilized fractions); Fibrinogen and derivatives thereof such as fibrin, fibrin I, fibrin II, soluble fibrin, fibrin monomers and fibrin closure products; whole blood; Protein C; Protein S; Alpha-1 anti-trypsin (alpha-1 protease inhibitor); Butyl-cholinesterase; Anticoagulant; Streptokinase; Tissue plasminogen activator (tPA); Erythropoietin (EPO); Urokinase; NEUPOGEN (filgrastim, granulocyte stimulating factor); Anti-thrombin-3; Alpha-galactosidase; Iduraonate-2-sulfatase; (Fetal) bovine serum / horse serum; meat; Immunoglobulins (anti-serum, monoclonal antibodies, polyclonal antibodies and genetically engineered or prepared antibodies); albumin; Alpha-globulin; Beta-globulin; Gamma-globulin; Coagulation protein; Complement protein; And interferon, including but not limited to.

III. radiation

As used herein, the term “sterile” means reducing the level of one or more active or potentially active biological contaminants or pathogens found in the biological material treated in accordance with the present invention. As used herein, the term "radiation" means radiation of sufficient energy to sterilize at least some components of the irradiated biological material. Types of radiation include particulates (atomic particles—such as streams of neutrons, electrons and protons); Electromagnetic (caused by changing electromagnetic fields—for example radio waves, visible light—both monochromatic and polychromatic, invisible, infrared, ultraviolet radiation, x-radiation, gamma rays and mixtures thereof); Sound and pressure waves are included, but are not limited to these. Such radiation is often described as ionizing radiation (which can produce ions in the irradiated material)-eg gamma rays and non-ionizing radiation (eg visible light). These radiation sources can vary, and in general, the choice of a particular radiation source is not critical, as long as sufficient radiation is provided at the right rate at the right time to achieve sterilization. In practice, gamma radiation is generally produced by isotopes of cobalt or cesium, and UV and X-rays are produced by machines that emit UV and X-radiation, respectively. The former is often used to sterilize materials in a manner known as "e-beam" irradiation, including through mechanical generation. Both monochromatic and polychromatic visible light are produced by the machine and can actually be combined with invisible light such as infrared and UV generated by the same machine or different machines.

It is known in the art to irradiate, sterilize and protect biological materials from radiation at a rate effective to sterilize such biological materials for a time effective for sterilizing such biological materials on radiation-sensitive biological materials. Publication 2003/0012687 Al (Macphee et al.), 09 / 973,958, currently US Pat. No. 6, xxx, xxx; The contents of which are incorporated herein by reference]. However, the present invention is based on the contents of this prior art and describes the first known use of gamma irradiation for sterilizing and preparing biologically active molecules such as whole blood samples for use as biological agents that can be stored at ambient temperature. Doing. Due to the risk of infection of infectious diseases such as HIV, hepatitis and other viral diseases, the use of safe, effective and inexpensive methods has become evident. The only apparent factor limiting the utility of this technique is the availability of suitable biologically active molecules (eg blood, blood components, biological proteins, vaccines, viruses and other antigens) and cobalt-60 sources or other suitable radiation sources. The low cost of the methods of the present invention and the fact that the biologically active molecules do not contain viruses, in particular HIV, makes the method of the present invention the most interesting means for preparing such biologically active molecules for use in a variety of patients with various needs. something to do. Since the process can often be carried out at ambient temperature without the need to cool, freeze or chemically treat the product containing the biologically active molecule prior to the process according to the invention, some of the extra processing steps present in the process of the prior art Avoided.

By one method of the invention, gamma radiation is delivered over an extended period of time to substantially reduce damage to products containing biologically active molecules. Typically, irradiation is performed for a period of at least 10 hours, preferably from about 20 hours to about 40 hours, more preferably from about 20 hours to about 30 hours. Irradiation ranges from about 0.5 kGy / hour to about 3.0 kGy / hour depending on the product to be sterilized and the length of irradiation time. The total radiation dose is typically about 20 to about 32 kGy, which has been shown to be effective in reducing the levels of contaminants such as viruses. The radiation dose can be used for preservation of function and subsequent room temperature storage, with sterilization of biological products, with doses of radiation delivered at rates as high as 4.0 kGy / min for a time as short as 5 minutes.

Preservation by gamma irradiation of biologically active molecules (e.g. whole blood, blood components, biological proteins and viral entities) can have many benefits, and are currently used by such biologically active molecules, such as small hospitals, private hospitals and developing countries. It enables its use in areas that are not possible. The preparation of irradiated whole blood is inexpensive and only requires access to basic materials and cobalt-60 sources and is simple to perform. Irradiated whole blood can be stored at room temperature and does not require liquid nitrogen or cold freezer storage. Application of irradiated whole blood does not require thawing, washing or rehydration found in other whole blood preservation methods.

In one method of the invention, the product containing the biologically active molecule may be irradiated in a form containing preferably less than 20% solids. Thus, certain products may be diluted before irradiation. Treating the product in diluted form may serve to reduce degradation of the product during irradiation. The choice of diluent depends on the nature of the product to be investigated. For example, when irradiating blood cells, a physiologically acceptable diluent such as citrate phosphate dextrose can be selected.

The method of the present invention is useful for treating organic products that are sensitive to radiation. Such products may tend to degrade when irradiated with standard methods. However, irradiation of sensitive products by the method can be expected not to be harmful to the products. The method typically applies to, but is not limited to, biological products such as blood and blood components. When living cells (eg blood cells) are irradiated, scavenger can be added to bind to free radicals and other substances that are toxic to the cells. Suitable scavengers include, but are not limited to, antioxidants, free-radical scavengers and ligands that stabilize the molecule.

Another aspect of the invention may be practiced by irradiating the sample of biologically active molecule with radiation for a time sufficient to provide sterile amounts of radiation. Thus, the amount is calculated using conventional and routine dosimetry. Radiation doses sufficient to achieve sterilization are known in the art. Cleaning is not essential to practicing the present invention.

One example of a protocol that includes time to deliver gamma radiation and dose for irradiation of red blood cells includes:

(i) the dose delivered shall be 2500 cGy targeting the central portion of the vessel and the minimum dose should be 1500 cGy at any other point;

(ii) The time required to deliver the radiation dose should be based on the radiation intensity of the source. The collapse of the source should be calculated according to the manufacturer's instructions. FDA typically recommends recalibration of sources every year for cesium-137 and every half year for cobalt-60. The method of calculating decay, included in the operating manual of the irradiator, can be referred to in the standard operating method (SOP).

(iii) The SOP shall specify the maximum number of blood or blood component units to be irradiated at one time. This is a batch and can be determined by the device manufacturer's method and is based on the manufacturer's validation data.

(iv) The total radiation dose should not exceed 5000 cGy at any point in the container.

As an alternative, the blood may be exposed to a radiation dose of about 30.0 kGy to sterilize bacteria, viruses and other potential pathogens in the blood and then stored at room temperature.

IV. Example

1-21 summarize the test data performed on whole blood using one embodiment of the method of the present invention. In connection with FIG. 1, blood from the blood bank was irradiated with gamma rays during a total exposure of 30 kGy. Blood was recovered in USP anticoagulant citrate phosphate dextrose adenine solution (CPDA-1) blood-pack unit (Baxter Healthcare Corporation, Deerfield, IL). It was stored at 4 ° C. for one week after the expiry date before irradiation. The table lists some features of blood before and after irradiation. p0 ₂ , Hb0 ₂ and 0 ₂ saturation are significantly reduced after irradiation. MetHb (methhemoglobin), which reflects the oxidation of iron atoms in hemoglobin, is markedly increased. This finding suggests the need to seek ways to reduce the above changes so that the irradiated blood can carry oxygen immediately after transfusion.

Experimental results demonstrating the effect of irradiation of whole blood according to the invention can be found in FIG. 1. The protocol for this experiment resulting in this data is as follows: Newly harvested whole blood, 5 milliliters (ml) anticoagulated with EDTA in a suction tube, receives gamma radiation at room temperature in 0 to 50 kGy steps in 10 kGy steps. The change was investigated. One half of the number of tubes was used as a control for the paired tubes to which 0.01 ml (10 mg / ml) of methylene blue 1% solution was added. Prior to analysis, oxygen gas was simply bubbled through the tube to test if high concentrations of hemoglobin could be formed. After irradiation, blood samples were analyzed using the laboratory's standard machines in a hospital hematology laboratory. Many of the results are shown in FIGS.

With reference to FIG. 2, this experiment demonstrates that erythrocyte volume fraction is maintained after gamma irradiation of whole blood according to the teachings of the present invention. Methylene blue was added as 0.01 ml of a 1% (10 mg / ml) solution per 5 ml sample tube, corresponding to a pharmacological dose of 1 to 2 mg / kg used to treat patient's methemoglobinemia. As shown in FIG. 3, the pO ₂ of the sample tube is higher in the partial pressure curve of oxygen representing the partial pressure of oxygen after irradiation in the presence of methylene blue. In connection with FIG. 4, experimental data demonstrates uniformly high (about 100%) saturation of hemoglobin containing oxygen in methylene blue treated irradiated species. The saturation at 2 and 3 Mrads for tubes not containing methylene blue is believed to be due to the oxidation that occurs when the blood is stirred in the test formulation. This demonstrates that blood exposed to methylene blue treated, irradiated oxygen can bind to the gas. As shown in FIG. 5, the hemoglobin fraction bound to oxygen decreases with increasing radiation dose.

With reference to FIG. 6, this experiment demonstrates that the increase in radiation dose correlates with the increase in methemoglobin content. MetHb levels for tubes not containing methylene blue are unexpectedly low based on other observations including the results of FIG. 1 above. Similarly, as shown in FIG. 7, the fraction of hemolytic free hemoglobin was higher in the tube without methylene blue added. In addition, the sodium concentration in the blood sample tube demonstrates relative hyponatremia. Control and methylene blue containing samples show similar values (FIG. 8). Similarly, as shown in FIGS. 9 and 10, the white blood cell count and the red blood cell count are not significantly affected by irradiation or the presence of methylene blue.

In connection with FIG. 11, this experiment shows that the hemoglobin content (gm%) is affected by irradiation. This is believed to reflect artifacts caused by radiation induced changes. Increased erythrocyte volume ratios, which have also been shown in some samples herein (FIG. 12), with macrocyticemia after irradiation, have been reported. As shown in FIG. 13, the average erythrocyte volume appears to be increased by irradiation, as noted by others. 14 and 16 show mean erythrocyte hemoglobin and mean erythrocyte hemoglobin concentration measured by experiments performed in accordance with the present invention. The data also show that the red blood cell distribution width (FIG. 17) is not very different except for one that can represent some sort of systematic error. This data demonstrates an increase in platelet content, which may be the result of blood concentration (FIG. 15), while the average platelet volume does not change rapidly under the experimental conditions (FIG. 18).

Samples from the irradiated units of whole blood were examined under the microscope using appropriate blood staining and intact red blood cells were visualized after 30.0 kGy gamma radiation (FIG. 19). Biochemical analysis has shown that methemoglobinemia can be reversed using standard techniques used in medical practice. The addition of methylene blue and oxygen exposure appear to be beneficial for converting iron in hemoglobin from the oxidized Fe ⁺⁺⁺ state to the Fe ⁺⁺ state typically present in functional hemoglobin. In addition, silicone blood bags can assist in this conversion by allowing oxygen to diffuse into the blood and Fe ⁺⁺ atoms prior to transfusion.

In one embodiment of the invention, whole blood from HIV and hepatitis negative donors can be obtained at a time when the expiration date is near. The HIV infected blood can be stored frozen at 4 ° C. and irradiated with gamma radiation of 30 kGy. It can then be transported and stored at room temperature for extended periods of time.

20 shows evidence of the ability of anti-A antibodies to aggregate Group A erythrocytes. For this experiment, anti-A antiserum was divided into two aliquots. One was stored in the refrigerator and the other was irradiated with gamma radiation of 30 kGy and stored for 1 month at room temperature. In this experiment, the ability of two antisera to aggregate Group A cells was tested with a series of 1: 2 antibody dilutions in saline in a round bottom well containing a series of Group A cells. The titer of the refrigerated antibody was 1:80 and that of the irradiated and stored at room temperature was 1:40.

While certain forms of the invention have been described and described, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. More specifically, it will be apparent that the present invention is not only limited to the conservation of specifically mentioned biological materials, biochemical entities and biologically active molecules, but also applicable to the conservation of many suitable biologics not specifically mentioned. will be. Likewise, the invention is not limited to any particular medical device structure capable of carrying out the disclosed methods and products. Accordingly, the invention is not to be limited except as limited by the appended claims.

Cross Reference to Related Application

This application claims the benefit of US Provisional Patent Application No. 60 / 387,177, filed June 7, 2002, the content of which is incorporated herein by reference.

Background of the Invention

The present invention relates to the general biochemical and medical field, more particularly hemoglobin (hemoglobin in the red blood cell category and hemoglobin independent of erythrocytes), antibodies, cytokines, blood components, proteins and other cellular components, including both tangible and non-type components, intact And preservation by irradiation of animal tissues and functional biological materials, biochemical entities and biologically active molecules, including but not limited to antigens for sensitivity and desensitization testing and vaccines.

Biological materials have been used for many years in a wide range of applications, including human medicine and veterinary applications, diagnostic applications, and in experimental or chemical processes. Such materials are often biologically active, which can perform the same or similar structural, enzymatic or other molecular functions as those performed in the source organism or plant, making these materials available for use as diagnostics, preservatives or therapeutics. It means that there is. Despite the enormous benefits of biological materials, there are risks in using them. These substances can be contaminated with various biological contaminants, including viruses and bacteria. These pollutants can cause serious health problems when infected with humans or animals. In addition, these pollutants have the ability to reduce efficacy or even destroy material contaminated with such pollutants.

There are many techniques for testing and screening biological materials for biological contaminants or pathogens, but this screening process has disadvantages. The process is not always reliable, can be expensive and can only be tested for very specific contaminants. It is even more effective to reduce the risk of a broad group of sources. Numerous techniques, including heat treatment, chemical solvents and radiation, have been used to reduce the risk of pollutants. Heat treatment has often been shown to adversely affect the biological activity of the product, reducing the efficacy of the product. Chemical solvents, disinfectants and antibacterial agents have been used to reduce the potential for contamination, especially after packaging and before use. Such solvents and agents can be harmful when contacted with humans and have often been removed from the product before use in humans.

Radiation treatment is another way to sterilize the product. Radiation is particularly effective in reducing a wide range of biological contaminants, including bacteria and viruses. Literature published in the art is very easy to handle, and if a person skilled in the art is familiar with the radiation sterilization technique, specific criteria including the type of radiation used, oxygen content, pH, irradiation rate, temperature, solvents, stabilizers, etc. It is taught that manipulation can change the product's response to radiation. However, most of the research so far in developing radiation treatment protocols has focused on inactivating contaminants while rarely changing both the structural and biological functions of biological materials. Such sterile, stabilized products must be functional, which can perform the same or similar structural, enzymatic or other molecular functions as those performed in the source organism or plant and can be used as diagnostic, preservative or therapeutic agents. That means you can.

A. Blood and Blood Components

Deficiencies in the prior art are in the manufacture of medical blood and blood components. A wide variety of injuries and medical procedures require the administration of whole blood or various blood components. Not all patients need whole blood, and in fact the presence of all blood components can cause medical problems. Individual blood fractions can be stored under special conditions that are best suited to ensure their biological activity upon transfusion. For example, when donor blood is contained in a processing organ, red blood cells are isolated and stored in various ways. The cells are typically in units of packaged red blood cells having a volume of 200-300 ml and a red blood cell volume value (expressed as blood cell volume ratio) of about 70-90%, 5 at 4 ° C. in citrate-phosphate-dextrose. Can store for less than a week. Erythrocytes may also be treated with glycerol and then frozen at −30 ° C. to −196 ° C. and stored for up to 7 years in glycerol solution, but must be frozen at low temperature to survive sufficiently for transfusion. All of these methods require careful storage temperature maintenance to avoid disruption of the biological activity of the desired red blood cells and to provide a 24-hour survival time for at least 70% of transfused cells, which is the American Association of Blood Banks. It is considered an acceptable level for transfusion under the Regulation of Blood Bank.

One known method of storage of red blood cells has been to freeze (lyophilize) red blood cells, since red blood cells can be stored at room temperature for extended periods of time and can be easily reconstituted for use in mammals. When RBCs are lyophilized according to many previous methods, for example in aqueous or phosphate-buffered saline (PBS) solutions, reconstituted cells cannot metabolize these cells and cellular hemoglobin can carry oxygen. It is damaged enough. Lyophilized and reconstituted glutaraldehyde-fixed red blood cells have been used predominantly in aggregation assays.

Small amounts of gamma irradiation may be performed on the blood to prevent graft-versus-host disease associated with blood transfusions. Graft-versus-host disease (GVHD) occurs when donor lymphocytes are transplanted into sensitive recipients. The donor lymphocytes can proliferate and damage target organs, especially bone marrow, skin, liver and gastrointestinal tract, which can eventually be fatal. The disease was initially recognized as a complication of transfusion to allogeneic bone marrow graft recipients to patients undergoing intrauterine transfusion and systemic irradiation. GVHD has also been shown in other immunologically disabled patients who have been exposed to donor lymphocytes due to transfusion of cellular blood products or rarely due to transplanted organs. Finally, the most commonly reported environment for transfusion-related GVHD (TA-GVHD) is immunocompromised recipients who receive blood from biologically related or HLA-identical donors. Products implicated in the TA-GVHD case include non-irradiated whole blood, packaged red blood cells, platelets, granulocytes and fresh unfrozen plasma. Frozen deglycerolated red blood cells, fresh frozen plasma and cryoprecipitates were not involved.

It is understood that the term "blood product" as discussed herein may generally include whole blood (and fractions thereof), platelets and / or red blood cells. As used herein, the term “white blood cell” includes the general class of white blood cells, including mononuclear cells and neutrophils, lymphocytes and any other cells found in the blood, above and below red blood cells and platelets. In addition, substantially "cell-free" blood products may contain some leukocytes. At present, gamma irradiation of blood products is the only method known to prevent transfusion-related GVHD. The most common radiation sources are cobalt-60 and cesium-137. Most blood organs rely on face doses of 25 Gy, and in order to inactivate lymphocytes in transgenic cellular material, at least 15 Gy must be delivered to any area of the bag.

 One of the many problems that make the use of transfusions difficult is the infection of factors that cause infectious diseases. Because pathogenic organisms are found in different fractions of whole blood, the risk of post-transfusion disease depends on the blood product or component used. Some products prepared for human, veterinary or laboratory use may contain unwanted and potentially dangerous contaminants such as viruses, bacteria, yeasts, fungi, mycoplasma and parasites. Many of these infectious agents are of significant clinical importance, which can be dangerous to the receiving patient as well as to physicians and other hospital staff who handle blood and blood products. Therefore, before using such a product, it is most important to determine whether it contains no contaminant. This is particularly important when the product is administered directly to the patient, for example in transfusion, organ transplantation and other forms of human therapy.

B. Biological Molecules and Antibodies Acting as Antigens

Prior art methods are insufficient in the conservation of biological molecules and antibodies that act as antigens. Antibodies are currently widely used for diagnostic testing. In addition, many bacteriological and pathological diagnoses are made by tests that involve the reaction of antibodies with specific antigens. Such tests include simple hemagglutination assays used to determine the type of blood by ELISA (enzyme linked immunosorbent assay) tests. Antibody storage at room temperature allows for immediate use of blood in field hospitals such as battlefields, allowing precise and rapid diagnosis to be achieved on site without the need for established laboratory equipment. At present, the requirements and necessity for refrigeration make it difficult to treat patients with antibody preparations. Thus, the preparation, transport and storage of such antibody preparations is excessively uneconomical and often expensive.

C. Organizations and Institutions for Use in Medical Applications

Frozen storage is currently the primary means of preserving xenograft tissue and tissue-derived materials for transplantation. There have been numerous infections of bone and tendon that have accumulated until recently. Sterilization with radiation can prevent infection of both bacterial and viral pathogens. In addition, room temperature storage after irradiation will greatly simplify the manufacture, storage and use of xenograft tissue and its derivatives including bone, collagen and acellular dermis.

Already, most procedures have included methods for screening or testing biological materials, biochemical entities and biologically active molecules for specific contaminants rather than removing contaminants from the product. Products that proved positive for the contaminant are not used only. Examples of screening procedures include testing for specific viruses in human blood from blood donors. However, this process is not always reliable. This reduces the value or certainty of the test in terms of results associated with false negative results. False negative results can be life threatening in certain cases, for example in the case of acquired immunodeficiency syndrome (AIDS), for example when testing for human immunodeficiency virus (HIV). In addition, in some cases it can take weeks if not several months to determine if the product is contaminated.

More recent efforts have focused on removing or inactivating contaminants in biological materials, biochemical entities and biologically active molecules before use. Such methods include heat treatment, filtration, addition of chemical deactivators and gamma or other radiation treatments. It is well documented that gamma irradiation is effective in destroying viruses and bacteria. In fact, one author concluded that gamma irradiation was most effective in reducing or eliminating the level of virus. Although biological materials have been sterilized by irradiation, the materials have typically been stored or frozen after storage. Virus inactivation by stringent heat-based sterilization is not allowed because it can also destroy functional components of the blood, in particular red blood cells and platelets and unstable plasma proteins.

In view of the above, there is a need to provide a method for sterilizing products containing biological substances, biochemical entities and biologically active molecules that is effective at removing biological contaminants and at the same time does not adversely affect the product. Examples of unwanted biological contaminants include viruses, bacteria, yeasts, fungi, mycoplasma and parasites. Therefore, it is highly desirable to have a stable and economical method and apparatus that can eradicate pathogenic viruses, microorganisms or parasites present in human whole blood or blood products before such products are injected into recipients and the recipients are infected with these disease-producing agents. It is requested. At the same time, properly decontaminated blood will remove the daily threat of infection from hospital staff who need to manage these fluids. This need is even more urgent in blood banks that manage and process donor blood and blood products. By eradicating pathogenic viruses, microorganisms or parasites present in biological material or in human or animal tissues (e.g. skin, bone, fascia and tendon), the recipients will be prevented from infecting these diseases by introducing them into the recipients before they are introduced or transplanted. It is also desirable at the same time to have a stable and economical method and apparatus. Accordingly, the present invention addresses these and other needs.

Summary of the Invention

Briefly referred to in general terms, the present invention relates to the preservation by irradiation of biological materials, functional biochemical entities and biologically active molecules. The invention also relates to products derived from biological substances, biochemical entities, biologically active molecules for storage at ambient or room temperature, especially without the need for lyophilization (freeze-drying) prior to irradiation or for freezing or refrigeration after irradiation. It includes a method of producing. More particularly, the present invention relates to plasma protein fractions (e.g. albumin and coagulation factors) collected from antibodies, peripheral blood cells (e.g. red blood cells and platelets), whole blood (e.g. blood from virus infected persons), body fluids (urine, spinal fluid, Compositions, including but not limited to amniotic fluid and lubricating fluid, ex vivo media used in the manufacture of antiviral vaccines, and cell culture media (eg, fetal calf serum and bovine serum) or products from such compositions and veins It relates to the inactivation of potential biological contaminants (eg viruses, bacteria, yeasts, fungi, mycoplasma and parasites) in nutrient sugars, amino acids, peptides and lipid solutions. The invention further includes blood-based proteins and biologically, including but not limited to, monoclonal antibodies, botulinum toxins and plant-derived proteins, hemoglobin (hemoglobin in erythrocytes and hemoglobin independent of erythrocytes), antibodies and vaccines. To derived proteins. Pre-contamination or potential contamination is not a prerequisite to the value of the present invention.

Most studies of radiation treatment protocols to date have focused on inactivating contaminants while rarely altering both the structural and biological functions of biological materials. The present invention is based on known radiation treatment protocols and the radiation treatment is effective in preserving the function of biological materials, biochemical entities and biologically active molecules, as well as sterilization as well as without the need to strictly control the temperature of the sterilized product after irradiation. Teach that it can be. The unique method of preservation and treatment of the present invention allows the sterilized product to be stored at room temperature while maintaining the efficacy of the biological activity and mechanism of action of the product.

This is an important advance for a number of reasons. Most importantly, the ability to store the processed material at room temperature reduces the risk of impairing the efficacy of the material if the handling process is not strictly performed. In many parts of the world, even refrigeration is a luxury. This has a serious impact on public health in these areas because it is not possible to store drugs and vaccines refrigerated and the drugs and vaccines are usually inactivated. The ability to supply vaccines or biological materials that can be stored at room temperature in developing countries will have a significant impact on human health in this region. In addition, room temperature storage will have a significant impact, even in developed countries, by saving on storage and transportation costs, making the product convenient and easy to use in many cases, and making the production process cheaper.

One opportunity to use the present invention is in the manufacture of medical blood and blood components. The present invention sterilizes fractions of whole blood samples or biologically derived protein cells or structures comprising tangible components such as cells and tissues of plant or animal origin to substantially reduce or eliminate the risk of transmission of infectious diseases, particularly viral diseases. And how to save it. The present invention further includes a method of making a biological material, which is inexpensive and readily available to most medical organizations. The method allows for the preservation of biological materials without the need for refrigeration or other treatments that can result in significant additional costs. In addition, the present invention provides for the sterilization of biological proteins such as sterilization of antibodies and other chemical components of blood and for the stable storage of biological proteins at room temperature so that they can be used later with a greatly reduced risk of bacteria or certain viral contaminants. Teach stabilization.

Many functional biological materials can be prepared according to the methods of the present invention. Such sterile products can be used in methods of preventing or treating conditions or diseases such that biological materials can be stored at room temperature prior to administration of an effective amount of the biological material to a patient. Similarly, sterile, stabilized products formed from irradiated biological materials, biochemical entities and biologically active molecules can be incorporated into diagnostic test methods and kits and used as components in industrial and chemical processes. Likewise, nutrient solutions containing sugars, amino acids, peptides and lipids can be sterilized and prepared by this method for storage at room temperature (ambient temperature).

The method of the present invention includes a method of irradiating one or more biological materials, biochemical entities and biologically active molecules to preserve their function and to allow the obtained, sterile and stabilized product to be stored at or near ambient temperature. . Functional biological materials suitable for use in the present invention include, but are not limited to, blood or blood components, such as immunoglobulins including erythrocytes, leukocytes comprising monocytes, platelets, clotting factors, monoimmunoglobulins and polyimmunoglobulins. It is not limited. Likewise, suitable functional biological materials include cartilage, bone marrow (including bone marrow cell suspension), whole or processed ligaments, tendons, nerves, bone (including demineralized bone matrix), grafts, joints, femurs, femoral heads, teeth, skin grafts. , Heart valves, corneas, arteries, veins, lipids, carbohydrates, collagen (including natural, fibrous, non-terminal, soluble and insoluble, recombinant and transformed collagen, both native sequence collagen and modified collagen) and organs (heart, Animal tissues (including, but not limited to, tissues of mammals and other animal gates) such as liver, lungs, kidneys, intestines, pancreas, limbs and organs for transplantation such as fingers.

Similarly, the present invention relates to proteins (including recombinant and transgenic proteins), proteinaceous substances, amino acids, peptides, sugars, lipids, enzymes (trypsin, chymotrypsin, alpha-glucosidase and iduronodate-2-sulfa). It is applicable to non-cellular substances such as degrading enzymes such as other enzymes), antigens, bone marrow, chitin and derivatives thereof (including NO-carboxycytoic acid-NOCC). Sterilization and stabilization aspects of the methods of the invention and the products obtained calculate the measured amounts of biological material, biochemical entities and biologically active molecules to reduce the risk of viral and bacterial contaminants and to be effective in sterilizing and stabilizing running biological materials. Irradiating biological materials, biochemical entities, and biologically active molecules by introducing them into a predetermined amount of radiation.

In the present invention, the irradiation process can be carried out at a variety of temperatures, including but not limited to the usual range of room temperature to below cryogenic temperature, including the temperature of the liquid nitrogen. Rarely, it may also be advantageous to heat the target specimen while simultaneously irradiating the radiation.

Other features and advantages of the invention will be apparent from the accompanying drawings, which illustrate, by way of example, features of the invention in conjunction with the following detailed description.

Claims (22)

Providing a functional biological material; Irradiating the biological material to preserve its function; And Storing the biological material at a first temperature. The method of claim 1, wherein the step of providing a functional biological material comprises a blood component selected from the group consisting of red blood cells, white blood cells, monocytes, platelets, coagulation factors, immunoglobulins, monoglobulins and polyimmunoglobulins. A method comprising providing. The method of claim 1, wherein providing the functional biological material comprises cartilage, bone marrow, bone marrow cell suspension, ligaments, tendons, nerves, bones, demineralized bone matrix, grafts, joints, femurs, femoral heads, teeth, skin grafts, Supplying animal tissue selected from the group consisting of heart valves, corneas, arteries, veins, organs, carbohydrates and collagen. The method of claim 1, wherein providing a functional biological material comprises providing a non-cellular material selected from the group consisting of protein, proteinaceous material, enzyme, antigen, amino acid, peptide, sugar, lipid and bone marrow. The method of claim 1, wherein irradiating the biological material comprises introducing the biological material to an amount of radiation effective to sterilize the biological material. The method of claim 1, wherein irradiating the biological material comprises introducing the biological material into an amount of gamma radiation effective to sterilize the biological material. The method of claim 1, wherein irradiating the biological material comprises introducing the biological material to an amount of radiation effective to inactivate the virus. The method of claim 1, wherein irradiating the biological material comprises introducing the biological material into an amount of gamma radiation effective to kill the bacteria. The method of claim 1, wherein irradiating the biological material comprises introducing the biological material to gamma radiation for a total exposure of 30 kGy. The method of claim 1, wherein irradiating the biological material introduces the biological material to gamma radiation for a period of about 5 minutes to about 40 hours at a rate of about 0.5 kGy / hour to about 4.0 kGy / minute. How to include. The method of claim 1, wherein storing the biological material comprises allowing the biological material to be stored at room temperature before use. The method of claim 1, wherein storing the biological material comprises allowing the biological material to be stored at ambient temperature before use. The method of claim 1, wherein storing the biological material comprises maintaining the biological material at a first temperature of about 5 ° C. to 100 ° C. 7. The method of claim 1, wherein storing the biological material comprises maintaining the biological material at a first temperature of about 10 ° C. to about 30 ° C. 7. The method of claim 1, further comprising adjusting a second temperature of the biological material during irradiation of the biological material. The method of claim 1, further comprising maintaining a second temperature of the biological material at a temperature of about 10 ° C. to about 30 ° C. during irradiation of the biological material. Providing a functional biochemical entity; Irradiating the functional biochemical entity to preserve its function; And Storing the functional biochemical entity at ambient temperature. The method of claim 17, wherein irradiating the biochemical entity comprises introducing the biochemical entity into an amount of radiation effective to sterilize the biochemical entity. Providing a biologically active molecule; Irradiating the biologically active molecule to preserve its function; And Storing the biologically active molecule at room temperature. 20. The method of claim 19, wherein irradiating the biologically active molecule comprises introducing the biologically active molecule in an amount of radiation effective to sterilize the biologically active molecule. A functional biological material prepared according to the method according to any one of claims 1 to 16. Providing a biological material according to the method according to any one of claims 1 to 16; Storing the biological material at ambient temperature; And A method of preventing or treating a condition or disease, comprising administering to a patient an effective amount of the biological substance.