KR20010099700A - Method for production of stroma-free hemoglobin - Google Patents

Abstract

The method utilizes a commercially available blood cell separator consisting of a computer-controlled centrifuge 2 having a rotor 24 of a blood processing bag 6 containing donated blood. Once the blood has been collected, this treatment is carried out completely in a closed centrifuge bowl, preferably at the collecting sugars of the donating blood. In the first step, blood is centrifuged to separate plasma from cellular components. Erythrocyte separation from other blood components is followed by washing of the red blood cells with standard saline or other solution. The erythrocytes are then lysed by hypotonic shock to separate the erythrocyte membrane (epilepsy) and the lysate is collected in a sterile container 44, leaving only the epilepsy in the centrifuge bowl. The final product can be used as a raw material for hemoglobin-based oxygen carriers currently under development as red blood cell substitutes. All steps are performed in a blood bag 6 in a treatment container or bowl centrifuge to minimize direct manipulation and maintain sterilization. The process for preparing the modified hemoglobin solution is combined with the preparation of interstitial hemoglobin, the predetermined agent is reacted with the solution and the solution is filtered.

Description

Method for producing interstitial hemoglobin-free {METHOD FOR PRODUCTION OF STROMA-FREE HEMOGLOBIN}

Related Applications

This application claims the priority of US Patent 60 / 104,319, filed October 15, 1998, and US Patent 60 / 122,180, filed March 1, 1999. The technical content of the same application is hereby incorporated by reference in its entirety.

Transfusion of stored human blood in medical practice is an alternative to whales. However, its efficacy is not precise and the method also has significant deficiencies. For example, when the need for a blood transfusion is recognized, even in the best medical centers, treatment is delayed because the blood is transported to the patient or to the operating room or emergency room after cross testing of the blood type and the patient's blood. In addition, some evidence exists that transfusions are immunosuppressive and autologous lymphocytes can become chimeras at the receptor. The risk of infection of viral diseases by blood transfusion is well known.

Despite these problems, the need for blood transfusions is increasing worldwide as medical practice becomes more complex and the population ages. Extending the transfusion rate in the US to 6 billion people worldwide, a total of about 300 million units of red blood cells are needed annually. While we cannot reliably estimate the number of units that can actually transfuse blood, that number would be as much as 90 million units. If these assumptions are correct, there will be more than 200 million units annually worldwide. Developing countries will not be able to maintain effective elaborate blood banking practices as developed countries, and most developing countries will not be able to get enough blood transfusions to meet their needs because public health is generally inadequate.

In an effort to address this potential shortage of blood supply, therapeutic oxygen carriers, or "blood substitutes," have been developed intensively in laboratories and even laboratories at commercial colleges since the mid-1980s. Overcoming serious problems, including purification of hemoglobin used as raw material, characterization of solutions and hemoglobin modification chemistry.

The success of blood substitutes in the market depends on several important factors. First, it must be effective. However, no clear test of efficacy has been established. One possible test is whether the product can effectively reduce the patient's contact with allogenic blood. Second, it must be a safe product. The safety discussions so far center on the known properties of vasoconstrictive hemoglobin. At least part of this property is that nitric oxide (NO) binds very strongly with hemoglobin at the sulfhydryl site as a heme ligand. Third, the red blood cell surrogate must successfully compete with the blood for clinical use. Blood substitutes currently under development have a plasma retention time of 12 to 58 hours (half-time). Thus, they will be used only in temporary conditions or in situations where repeated administrations can be made.

Human blood is extremely safe by the intense scrutiny of blood banks, with the discovery that HIV can be transmitted by blood transfusion. To survive as a product, the red blood cell substitute must be safe and relatively economical. Higher prices than blood are acceptable only if they have obvious advantages in safety, efficacy, ease of use or patient acceptance. Therefore, blood substitutes that are close to the clinical use, source, price and supply of raw materials are more important. For clinical trial products, raw hemoglobin was obtained from stored human blood, bovine or recombinant ( E. coli, E. coli ) sources over time. The blood available for the manufacture of blood substitutes was only about 120,000 units per year, with 1% or less of the stored blood estimated to be out of date. Thus, this period of blood has a high competitive price. Bovine blood has the advantage of being obtained in large quantities. However, for this purpose, the health of cattle should be kept strictly standard and the management of veterinary animals, frequent experiments, and the large amounts of land and diet provided to them. In addition, bovine blood must be collected by a specific device designed for this purpose. Recombinant sources may be the ideal solution because the risk of contamination from human pathogens is reduced. However, recombinant hemoglobin requires large scale purification to separate proteins from other components of the fermentation, and large amounts of water are used and face significant problems in treating waste. For such processing reasons, the cost of blood substitutes made with recombinant hemoglobin is many times higher than that of conventional-bank blood.

Current methods for preparing hemoglobin solutions from human blood include washing, pooling, or diafiltration erythrocytes pooled with saline on a large scale, gently dissolving them in storage buffer, and strictly removing the red blood cell membrane. These methods require huge sterilization containers and expensive filters and take a long time to complete. Many of the components and solutions used in this process must be stored in a cold and / or clean room. The reported pilot device required four individual or connected spaces, occupying about 1000 square feet of laboratory space, as long as 5 liters of interstitial erythrocyte solution was treated per week. The production cost was about $ 1000 / liter (Winslow and Chapmanm "Pilot-scale preparation of hemoglobin solution", Meth. Enzymol ., 231: 3-16, 1994, hereby incorporated by reference). In addition to the significant disadvantages of high production costs, the large scale and complexity of the pilot system required several auxiliary personnel and a high risk of contamination.

In order to provide a blood substitute in an amount that adequately meets the lack of blood supply worldwide, the current treatment of raw materials for the production of blood substitutes should be improved. Such needs focus on the production of interstitial hemoglobin for use in the production of blood substitutes, while lowering cost, complexity, and risk of contamination of the product.

Summary of the Invention

The present invention has the advantage of providing a process for preparing interstitial hemoglobin in a self-contained, automated device.

Another advantage of the present invention is that the blood is collected directly from the recipient, so hemoglobin solution as a raw material for the preparation of blood substitutes, using blood which does not need to determine or store the blood type and reduces the risk of contamination. It is to provide a method for producing.

In an embodiment, the method utilizes a commercially available blood cell separator consisting of a computer-controlled centrifuge of a rotor with a blood treatment bag containing donor blood. Once the blood is collected, the treatment is carried out completely in a closed centrifuge bowl, preferably at the donor blood collection site in its original position. The rotor of the centrifuge includes one or more treatment chambers for receiving the treatment bag (s). In the first step, blood is centrifuged to separate plasma from cellular components. The supernatant, ie, white blood cells, platelets and plasma, is removed with hydraulic fluid through a flexible diaphragm present in the blood treatment bag and the solenoid-controlled pinch valve and leaves packed cells. The pinch valve works by clamping the tube containing the solution while avoiding direct contact to maintain sterility of the fluid passage. The rotating sealing device allows fluid to enter, pass through and exit the blood treatment bag while the centrifuge rotates. After red blood cells are separated from other blood components, the red blood cells are washed with standard saline or other solution. The washing solution is hydraulically removed and transported through a tube to a supernatant collection container. There is an optical sensor that monitors the tubes leading to the supernatant collection container to detect the presence of red blood cells. If red blood cells are detected, a stop or hold function is triggered by the system regulator. The erythrocytes are then dissolved in a hypotonic shock and centrifugal force is used to separate the erythrocyte membrane (epilepsy) from the lysate, leaving only the epilepsy in the centrifuge bowl and collecting it in a sterile container. The final product can be used as raw material for all hemoglobin-based oxygen carriers currently being developed as red blood cell substitutes. All these steps are performed in a harvester to maintain the bowl or sterile of the cell separator.

The blood can be treated at the donating sugar, allowing faster and less expensive collection of red blood cells to prepare blood substitutes. More specifically, the method does not need to determine or store the blood type of the collected blood units, and the collected units can be mixed for this purpose, thus reducing the cost of testing for infectious agents such as HIV or hepatitis C. have.

The present invention relates to a system and a method for producing a high-quality hemoglobin solution as a raw material for preparing a hemoglobin-based therapeutic oxygen carrier ("blood substitute") using an automated cell separator.

Together with the description and the preferred embodiments of the present invention, the accompanying drawings will be considered to facilitate an understanding of the present invention. Numbers refer to parts,

1 is a schematic of a red blood cell processor for use in the production of hemoglobin free of epilepsy;

2 is a flow diagram of producing interstitial hemoglobin from packed red blood cells,

3 is a block diagram of a processing flow according to the present invention,

4 is a schematic diagram of one possible configuration of the present invention for providing a "blood substitute".

The process for producing interstitial hemoglobin ("SFH") uses the commercially available blood cell processing apparatus illustrated in FIG. Such a system is used to separate complete blood from a patient or donor into its components. After collecting the red blood cells, the remaining components can be returned to the donor or discarded. The device comprises a centrifuge 2 holding a blood bag 6 and a plurality of reservoirs 8 and 10 containing the treatment solution, a reservoir 14 for receiving the supernatant, a number of valves and aseptic valves connecting the various reservoirs and the blood bag 6. Tube device and regulator 20 for controlling the centrifuge and valve. A disposable rotary seal device 34 is included in the tube device that allows fluid to enter and exit the blood treatment bag while the centrifuge rotor is rotating. The sealing device prevents fluid from leaking out of the fluid path and air from leaking into the fluid. Blood collected from the donor (denoted 22) is delivered directly to the blood bag 6 via tube 30 and valve 32, and is preferably processed immediately. In a preferred embodiment the blood bag 6 and the tube device are a partially pre-sterilized, disposable treatment set. The valve functions to clamp the tube so that fluid does not come into direct contact with the tube.

Centrifuge rotor 24 is used to separate erythrocytes from plasma. Centrifugal force provides pumping (or return to the patient or presenter if desired) to compress the plasma into the supernatant reservoir 14. More specifically, substitution chambers are included in centrifuge rotor 24 consisting of hydraulically-operated diaphragms 4. The hydraulic fluid flow into and out of the region of the flexible diaphragm 4 is controlled by a system regulator 20 which controls the rotation of the centrifuge driver 36 and the direction of the reverse hydraulic pump 38. Regulator 20 opens valve 16 to allow plasma to exit the blood bag through tube 18, remove plasma and close valve 16. Complete removal of plasma can be determined using optical detector 28 and recognizes the presence of red blood cells in a transparent tube. Drain the live solution through tube 26 from reservoir 18 and open valve 12 to enter blood bag 6. Optionally, an anti-bacterial, cleaning or other suitable purifying agent may be used as a washing solution or in addition to a saline solution to remove any bacterial contaminants that may be present in the blood. The centrifuge rotor rotates at a relatively low speed providing agitation to wash red blood cells. After washing, the speed of rotation is increased and regulator 20 opens valve 16 so that the centrifugal force and flexible diaphragm compress the wash solution into the supernatant reservoir 14.

Once the wash solution is removed from the blood bag 6, erythrocytes are dissolved by introducing distilled water to cause a hypotonic shock to separate the epilepsy from the hemoglobin. Distilled water stored in reservoir 10 is opened via valve 13 and conveyed through tube 11. When red blood cells first come into contact with distilled water, some of them dissolve and free hemoglobin comes into solution. Here cell-free components can be recovered and distilled water injection is continued to free more hemoglobin, which reduces the ionic strength of the suspension medium. Interstitial hemoglobin will be distributed throughout the bowl while red blood cells and membranes accumulate outside the bowl edge, using a slow-down centrifuge rotor to minimize damage to hemoglobin. Since a large amount of distilled water is required, several iterations are necessary to liberate most hemoglobin. Removal of hemoglobin is via outlet 40 sterilized in blood bag 6 and passes through tube 42 into sterile container 44. It is desirable to include filter 46 in the delivery path (tube) to remove trace amounts of membrane particles. Given sufficient water and time all hemoglobin should be removed, but post-treatment may be required to concentrate the hemoglobin depending on the intended use. Completion of extraction of hemoglobin can be determined by measuring the concentration of ions in the hemoglobin solution. A conductivity meter can be connected to system regulator 20 to terminate the process when the ion concentration drops below a predetermined level. The predetermined level is selected based on the efficiency of time versus hemoglobin extraction, ie the balance of cost-benefit analysis.

Other methods of dissolving erythrocytes to cause hypotonic shock are known and can be used instead of, or in combination with, distilled water washing. An optional dissolution method is described in more detail below.

Sterile containers in which the hemoglobin solution is carried include pre-evaluated reagents such as buffer salts, Traut's reagent and activated polyethylene glycol (PEG) for the preparation of blood substitutes. Specific examples of methods for preparing blood substitutes are described in Patents 5,814,601 and 5,296,465, which are hereby incorporated by reference.

The treatment method is entirely in a single use, pre-sterilized treatment set, which preferably includes all tubes, reservoirs, centrifuge bowls and in-line filters. This sealed system is free from contamination.

The following embodiments are merely illustrative and are not intended to limit the scope of the invention.

Example 1

Experimental apparatus for producing interstitial hemoglobin from blood includes branching tubes for emptying bags of expired blood; Cross-flow filtration units for washing, dissolving and purification; Bioreactors for cross-linking hemoglobin; Pre-scaled HPLC apparatus; And a hood that finally fills the product with a sterile bag. The flow loop containing the PFW distribution loop was sealed. All connections were hygienic triangular and manufactured in a laminar flow environment. The whole process requires about 1000 ft ² of experimental space, except for the office. The pilot unit had a capacity of about 5 liters per week and a production cost of about $ 1000 / liter.

The cross-flow filtration system consists of three pumps, four tanks and four distinct filter packages. All components were made of 316L stainless steel with 180 grit interior finishes. The tube is flexible and made of reinforced silicone. Filter cartridges for hollow fibers are non-blood, nonpyrogenic polysulfone membranes.

All solutions were prepared and cooled in a coated stainless-steel 500 liter tank. The erythrocyte washers and lysis tanks were glycol-coated for temperature control. 500- and 10-kDa ultrafiltration loops used a double-tube heat exchanger. The PHW was cooled before dialysis of the concentrated hemoglobin. The rotary plate pump guarantees low shear stress. The pressure profile was monitored throughout the system and adjusted to below 20 psi. The pH of all solutions was kept in physiological range.

Sterility was maintained throughout the process. The cross-flow filtration system was fitted to a class 100 environment. All tanks, pipes and filters of the process were steamed with pure steam and chemically sterilized with 0.1N NaOH. The sterile solution was flushed from the system using PFW. The sterilization of the system was assured prior to treatment and validated through the process, with exothermic material taken using the Limulus amebocye lysate (LAL) test.

A flow diagram showing the progress of typical FSH production is shown in FIG. 2. Oversealed human packed red blood cells (PRBC's) were used up to 4 weeks after expiration. All units were tested for HIV and hepatitis B antigens for negative. In a typical batch, about 20 liters of PRBC's were mixed using a blood collection set with 80 liters of standard saline in a coated 100-liter stainless-steel tank. RBC's were washed to 7 standard volumes of cooled standard saline by dialysis through three membrane cartridges (total area 69 ft ² ) of three 0.65 μm hollow fibers at a constant volume (80 liters). To confirm that plasma was removed, the filtrate for albumin was analyzed as a marker of plasma protein. During a typical wash, albumin concentrations consistently decreased from above 2 mg / ml to undetectable levels (<2 μg / ml). The RBC's were slowly dissolved and 5 volumes of cooled pH 7.6, 10 mM NaHPO ₄ were used. Epilepsy was removed by dialysis through a membrane cartridge of 0.1 μm hollow fibers at a constant volume (80 liters). The filtrate of the 0.1 μm cartridge was placed in a 100-liter stainless-steel tank. It was then dialyzed through a 500-kDa membrane cartridge at a constant volume (80 liters) to remove all interstitial particles. The filtrate (SFH) obtained from the 500-kDa cartridge was concentrated to 10 g / dl by circulation through a 10-kDa hollow fiber membrane cartridge.

The resulting SFH solution was dialyzed with 6 volumes of Ringer's acetate, pH 7.4, and transferred from a stainless-steel concentration tank to a 40 liter stainless-steel holding tank through a 0.2 μm, 10-inch filter. Finally, the SFH product was transferred to a plastic bag under sterile conditions and frozen at -80 ° C. Optionally, SFH was transferred to a 70-liter bioreactor for cross-linking.

The interstitial hemoglobin solution prepared using the apparatus and method was combined with water or phosphate buffer. The methemoglobin concentration is typically less than 1% and the solution can be stored indefinitely at -80 ° C. The pyrogenic test of the rabbits was negative and the solution was not contaminated by bacteria. Purity was determined by HPLC analysis, indicating that this solution was essentially free of non-hemoglobin protein. Other quality control tests include oxygen binding (P50, Hill's variable, n), pH, endotoxin (LAL test) and SDS gel electrophoresis. The results of these tests for representative batches of SFH provided are shown in Table 1.

Specific Characteristics of SFH Solution Hemoglobin (g / dl) 7.5 Metemoglobin (%) <1 P50 (Torr, pH 7.2, 37 ° C) 12.0 n (Hill's variable) 3.0 Pi (μm) 42 pH 7.45 Bactericidal Pass Heating material voice Endotoxin (EU / ml) 0.02-0.10 Free iron (μγ / μλ) 2.8

Perhaps the biggest concern in the preparation of interstitial hemoglobin, excluding contamination, is the precise removal of the phospholipid component of the membrane. Phospholipids used a simpler total phosphate assay because it is customarily very difficult to measure. Although fairly delicate, it does not identify the source of phosphate. It is not clear whether intracellular enzymes constitute the second group of erythrocyte contaminants, and in view of their antioxidant activity, it is desirable to remove all erythrocyte enzymes from the final product.

The "units" of the SFH thus collected can be frozen and stored indefinitely, sent to a central processing facility (although plasma treatment is performed at some locations) or locally mixed for quality control testing. Another advantage of the proposed system is that it can drastically reduce costs. At present, all collected blood has to undergo expensive viral testing and this is the purest cost of blood collection. When using the new system, the unit can be mixed before the test, which greatly reduces the overall cost. The cost of collecting a blood unit is $ 18, of which a virus test is estimated to be $ 15. This cost can be reduced, for example, by a factor of 10 for testing performed on a 10-unit pool.

The blood thus collected can be prepared again as a "blood substitute", as described, for example, in Patent 5,814,601. However, this does not meet current FDA standards for donated blood. For example, current FDA inhibits hemochromatosis patients from donating blood for transfusion. This was a significant expense to the patient, causing them to see a doctor personally for a phlebotomy. The Iron Overload Disease Society estimates that 1.5 million individuals in the United States alone have significant iron overload. The association has a record of about 5,000 patients in the usual phlebotomy program, which eliminates about 6 units / year, or 30,000 units. The dose-equivalent of the erythrocyte substitute was determined so that 1 g / dl of plasma hemoglobin was effective for the cerebral hemorrhage model as 7 g / dl of red blood hemoglobin. Thus, patients in this group alone could supply as much as 150,000 units of artificial blood per year.

Example 2

In an embodiment of the invention, a self-contained, disposable unit designed to collect donor / patient blood in the clinic was used. Suitable systems for this application include the Haemonetics MCS + 8150 Blood Collection System (Haemonetics Corporation, Braintree, Mass.) And COBE 2991 Cell Processor (COBE Laboratories, Inc., Lakewood, Co.). Blood was collected at a 1:16 ratio of anticoagulant to anticoagulant blood in the CP2D / AS-3 anticoagulant / additive system. The machine is configured to collect at least 2 units of red blood cells for about 25 minutes from a single donor. Each unit collected produces about 180 ml of packed red blood cells (“RBCs”) and 400 ml of plasma. The following relationship can be used to determine the efficiency of separation.

Post-treatment RBC count × Post-treatment weight × 100 =% Recovery RBC

RBC coefficient before treatment × weight before treatment

The white blood cell count remaining in the packed red blood cells should be less than 5 × 10 ⁸ . Pure white blood cell ("WBC") counts are:

WBC factor in milliliters × product volume in milliliters

And the remaining percentage is:

Post-treatment WBC count × post-treatment weight × 100 =% remaining WBC

WBC coefficient before treatment-× weight before treatment

to be.

Commercially available basic blood cell processing systems are provided in Latham, Jr. Patent 4,303,193 and Tovas et al. Patent 5,921,950. The technical content of these patents is hereby incorporated by reference. Commercially available blood cell processing systems use a self-balancing centrifuge to separate the blood from the donor into two components, the plasma-rich and cellular components. This device is intended to be used when the blood donor / patient is in close proximity. The blood flow path is a complete disposable treatment set comprising a venous incision needle and a blood-compatible tube connecting the venous incision needle to a flexible blood treatment bag of about 630 ml capacity. The treatment set also includes a tube fixture, a rotary seal and a supernatant collection container. The rotary seal allows fluid to enter and exit the blood treatment bag while the centrifuge rotor is rotating. This seal prevents water from leaking out of the fluid path and air from entering the fluid. The tube facility provides a sterile fluid pathway for introducing the wash solution into the red blood cells. Three valves regulate the flow of blood or wash solution into the blood treatment bag. Each valve is a solenoid-regulated pinch valve under the control of the system computer and is not in direct contact with the fluid in the tube. A sterile discharge passage at the discharge end of the treatment bag allows the treated cells to move directly from the bag to the sterile container.

The bag is configured such that the second blood component travels along a short inner bag scale and is held in an enclosed treatment chamber positioned in a centrifuge rotor for separation. A hydraulically-operated diaphragm replacement chamber is also located in the blood treatment chamber of the centrifuge rotor. Hydraulic fluid flow into and out of the region under the flexible diaphragm is controlled by the rotation of the centrifuge driver and the pinch valve solenoid. The hydraulic system consists of a fluid flow network of pipes and reservoirs (plastic bags) with volumetric piston-type pump assembly, flow rate regulators and switches. The pump is driven by a multi-speed reversible motor. The volume capacity of this pump is adjusted to about 600 ml. Pressure is applied to the blood treatment bag to drive the pump motor adjustment forward at a set rate that squeezes the supernatant and allows the supernatant fluid from the bag to move to the supernatant collection container through one open valve. This is continued until red blood cells are detected through the light sensor, reach the supernatant-extinction volume, or until the stop or hold function is activated. The pump works in the reverse direction to withdraw fluid from the hydraulic fluid chamber and reservoir of the centrifuge. The centrifuge rotor can then be stopped to return the second blood component to the donor. Since all functions are under computer control, there is only minimal adjustment by the operator, so there can be little operator error.

The volume of the centrifuge bowl is about 250 ml and the speed of the centrifuge is about 4,000 rpm. Blood entering the centrifuge compartment is diluted with sterile saline, and erythrocytes are concentrated through continuous removal of non-erythrocyte components and plasma. The purpose of this is to reduce the amount of serum albumin to the lowest possible level, preferably to an undetectable level. Non-erythrocyte components may be discarded or used for other purposes.

The extent of removal of serum protein after the RBC wash step is assessed by methods established by the manufacturer of the blood cell processor. For example, in the case of COBE 2991 Cell Processor, after washing the red blood cells, 1 ml of the sample is extracted through a sterile outlet present in the blood bag, and the supernatant is collected after centrifugation in an experimental centrifuge. Commercially available protein chemistry dipstick wet pads can be used to test the supernatant with color changes indicating protein levels. Protein levels should be traces (15-30 mg / ml) or less, with plasma levels of equivalent or higher than 96%.

After the initial washing step, distilled water replaces saline. As shown in FIG. 3, it can be seen that when red blood cells first contact distilled water, some of them are dissolved and released from the hemoglobin into solution. Dissolution conditions are described later. At this time, the cell-free component can be recovered, and more hemoglobin is released by continuing injection of distilled water, resulting in a lower ionic strength of the suspension medium. Given enough time, all hemoglobin can be removed while leaving only traces of red blood cells in the centrifuge bowl.

The volume of hemolyte depends on the amount of distilled water required to achieve the desired level of dissolution. However, depending on the intended use, the hemoglobin solution can be further concentrated. The hemoglobin solution is accompanied by NaCl and subsequent purification may be required. Rapid and delicate measurements of the ionic strength of the final solution can be carried out by conductivity measurement.

Conditions for Lysing Human Red Blood Cells

Despite the long lifespan of red blood cells, hemoglobin is a fragile protein. This includes 4 polypeptide subunits, 2α and 2β. α and aβ chains bind tightly to αβ subunits, and the 2αβ subunits provide less stable tetramer, fully formed hemoglobin molecules. Each of the 4 subunits includes iron-substituents, heme. Iron atoms remain in the reduced Fe ⁺⁺ state to bond with oxygen. This reduced state is maintained by the presence of numerous enzyme systems in the red blood cells. When hemoglobin is released from the cell, this protection no longer exists and a series of reactions occur that ultimately result in the disintegration of the molecule. These reactions are oxidation of iron, relaxation of heme-globin linkage, release of heme, separation of tetramers into dimers, denaturation of globins and eventually precipitation. Therefore, what is ultimately important in the production of red blood cell substitutes is the very careful handling of proteins to inhibit the disintegration step of one or more of these. Otherwise raw material will be lost and a precipitate will be formed which must be filtered. In addition, even partially denatured or degraded hemoglobin molecules do not reverse oxygenate bonds and thus become useless as oxygen carriers.

In general, there are three ways to dissolve human red blood cells. First, it can be repeatedly frozen and thawed. Freezing in and around the cells disrupts the membrane and releases hemoglobin. However, it is well known that dissolution using this method is incomplete and hemoglobin denaturation may occur. In the second method, an organic solvent such as CCl ₄ , or toluene can be used. This method is more effective, but solvents can also adversely affect the stability of the protein.

The third method is osmotic dissolution, as described above. The tolerability of red blood cells for osmotic lysis is well known.

Estimated Processing Efficiency

According to the protocol of COBE 2991 Cell Processor, the average RBC recovery 82%, plasma 99% and leukocytes 93.4% were removed. Using the method of the present invention, washing by centrifugation and lysing red blood cells, about 40 g of interstitial hemoglobin ("SFH") were obtained from 50 g of starting mass and the yield is estimated to be about 80%. Thus, the final yield is estimated to be about 66% mass from the starting material for the SFH treated.

The problem with hemoglobin production is protein denaturation. Hemoglobin is sensitive to dilution, pressure, shear stress, temperature and pH changes. Therefore, it is important that all processing steps be performed at a uniform and low temperature. For example, the COBE 2991 Cell Processor can be refrigerated to maintain 4 ° C temperatures. In addition, the agitation should not be too vigorous, the pH change should not be rapid and the pressure should not rise to high levels.

Properties and quality control

Contamination measurements of epilepsy can be performed using total phosphate analysis. Extracting phospholipids from hemoglobin solutions is difficult and therefore uses total phosphate analysis in an easier and less expensive way. Although fairly delicate, this analysis cannot identify the source of phosphate.

Although intracellular enzymes make up the second potential group of erythrocyte contaminants, some contaminants of this type can be tolerated in blood substitutes. Analytical FPLC using a 280 nm filter to dissolve SFH from interstitial hemoglobin protein can be used to assess the homogeneity of the interstitial hemoglobin product.

Endotoxin measurements can be performed by dynamic turbidity analysis based on the initialization of the Limulus amebocyte lysate (LAL) coagulation cascade. Such assays are well known in the art. (See, eg, Cohen et al., Biomedical Applicatiions of the Horseshoe Crab (Limulidae) , 1979, Alan R. Liss, Inc., New York.)

The pH and conductivity of the interstitial hemoglobin solution can be measured with conventional pH groups and conductivity meters, such as, for example, Accumet lines from Fisher Scientific Co., Pittsburgh, PA. Spectral analysis makes it possible to estimate the hemoglobin concentration and the amount of metemoglobin in solution. Such analysis can be performed in the Soret and visible region using a rapid scanning diode aligned spectrophotometer, such as Milton Roy 3000, for example. The evaluation can be performed according to multicomponent analysis techniques as described by Vandegriff and Shrager ("Evaluation of oxygen equilibrium binding to hemoglobin by rapid scanning spectrophotometry and singular value decomposition", Meth. Enzymol . 232: 460-485, 1994). , This relates here by reference.

Most sensitivity tests to determine the functional state of the hemoglobin solution are to measure its oxygen binding curve. One such method involves linear oxygen consumption in an optical cuvette closed by a novel enzymatic system. Because oxygen is depleted, repeated visible spectroscopy appears while PO ₄ is measured simultaneously with the Clark-type electrode. Hemoglobin is diluted in 0.1 M bis-tris propane or phosphate buffer to a concentration of about 60 μm (in heme). The reaction occurs for about 15 minutes. The reaction uses protocatecuic acid (PCA, Sigma Chemical Co., St. Louis) as the substrate, consumes one mole of O ₂ for each mole of PCA, and the enzyme protocatecuic acid 3,4-dioxygenase (PCD, Sigma Chemical Co., St. Lous) to the product. After the experiment, the PO ₂ values matched the spectroscopy. To determine the parameters of the oxygen binding curve (P50, Hill's variable, n), biscopy is provided for multi-component analysis and curve-like methods. During the analysis of these spectra, the relative proportions of metemoglobin are calculated at each stage of deoxygenation. In addition, this method and analysis reveal the presence of any additional hemoglobin components such as denaturation and disintegration products.

Hemoglobin solutions provided by the present invention are also characterized by isoelectric focusing (IEF) on agarose and polyacrylamides having a pH range of 8.5 to 5.5. Subunit structure is assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spectroscopic analysis makes it possible to estimate the hemoglobin concentration and the amount of metemoglobin and carboxyhemoglobin in solution. Spectroscopy is collected in the Soret and visible region on a fast scanning diode aligned spectrophotometer. Spectroscopy is evaluated by multicomponent analysis. Other quality control measures include measuring hemoglobin concentration, pH and conductivity. If necessary, additional quality control measures are presented.

Example 3 Preparation of Modified Hemoglobin Using the Present Invention

In addition, the present invention can be used to prepare modified hemoglobin solutions (“products”) using non-interstitial hemoglobin (SFH) solutions prepared in the modified cell separator device shown in FIG. 4.

Erythrocytes may be obtained from any source, including animals or human units, human units that have been stored or fresh, or even older than those obtained from blood banks. Modification methods can also utilize hemoglobin solutions prepared with the present invention or prepared by other methods, including recombinant hemoglobin. If using donated blood, first mix with anticoagulant 50, wash in centrifuge bowl 52 with standard saline 54 and dissolve with distilled water 56. Plasma, platelet and leukocyte sections are removed and stored in a separate container 58. Hemoglobin solution (SFH) 60 is placed in a separate reservoir 62, or centrifuge bowl 52, where chemical modification is performed. After filtration, the final product is collected in a sterile container 64 that can be stored for future use.

When further treating the SFH produced in the above apparatus with the product, this SFH is collected into a reservoir containing a reagent intended for chemical modification. In a preferred method the reagents are buffer salts, iminothiolanes and activated polyethylene glycols. At the end of the reaction, the modified hemoglobin (PEG-Hb) is collected and stored in a plastic bag or other storage container.

Although reaction with PEG is preferred for hemoglobin modification, all modifications can be carried out by the device of the present invention and these methods are well known in the art of blood substitute preparation. Such modifications include, for example, surface modification of hemoglobin using internal cross-linking agents, polymerization reactions or dextran, starch or other synthetic or natural polymers.

This product can be further purified via filters 66 and 68 (in FIG. 4). Such filters, and ultrafiltration devices, can be, for example, size exclusion filters, ion-exchange filters, mixed-bed ion exchangers, activated carbon filters, or other in-line filters used in protein purification or dialysis methods. This product can be combined with any salt solution or other materials.

Sterilization of the product can be accomplished in the same apparatus by a number of methods such as solvent-washing treatment, gamma irradiation, nanofiltration, methylene blue or similar derivatives, or any other method of inactivating or removing organisms such as bacteria and viruses.

The method of the present invention provides a faster and less expensive collection of red blood cells required for the production of blood substitutes that can address a significant shortage of blood available for transfusion worldwide. The method of the invention can be used for the treatment of out-of-date blood, but most advantageously it can be used in the clinical location of a donor / patient. Since this treatment is performed under a computer-controlled, completely self contained device, direct manipulation is minimized and the risk of contamination is eliminated. The method also does not require a blood type or storage unit of the collected blood and the units collected for this purpose can be mixed to reduce the cost of testing for infectious agents.

Although not specifically included in the detailed description, additional embodiments and applications exist, and are obviously included within the scope and spirit of the present invention. This specification is not intended to be limiting and the scope of the invention will be limited only by the appended claims.

Claims (18)

(a) separating red blood cells in solution, (b) removing the supernatant produced during the separation step, washing the red blood cells with the wash solution, (c) lysing red blood cells to produce hemolysates, (d) separating the erythrocytes epilepsy from the hemolytes, wherein the hemoglobin solution is prepared from a solution comprising red blood cells in an automated blood separator comprising a centrifuge. The method of claim 1 wherein the wash solution comprises a standard saline solution. The method of claim 1 wherein the wash solution comprises a bacterial fungicide. The method of claim 1 wherein the wash solution comprises the inactive of the virus. The process of claim 1 wherein steps (a) to (d) are performed in a single container for processing located in a centrifuge. 2. The method of claim 1, further comprising the step of measuring the ion concentration of the hemolyte while repeating steps (c) and (d) until it decreases to a predetermined level of ion concentration. The solution is collected in a sterile treatment set comprising treatment bags and tube equipment, wherein the treatment bags are placed in the centrifuge of the cell treatment device, Separate the red blood cells from the plasma by rotating the treatment bag in a centrifuge, Compresses the plasma of the treated bag, Introduce washing solution into the treatment bag to wash red blood cells, After washing, compress the supernatant, Hemoglobin is released into the solution by introducing distilled water into the treatment bag to dissolve red blood cells, Rotate the treatment bag in a centrifuge to separate the erythrocyte membrane from the hemoglobin solution, Removing the hemoglobin solution through a sterile outlet of the treatment bag, wherein the method is performed in a cell processing apparatus for producing hemoglobin from a solution comprising red blood cells and plasma. 8. The method of claim 7, wherein the step of separating the erythrocyte membrane from hemoglobin removes the first-generated hemoglobin when distilled water first contacts the erythrocytes and continuously removes additional hemoglobin produced as the ionic strength of the solution decreases. How to include. The method according to claim 8, wherein the ionic strength of the hemoglobin solution produced by the step of removing hemoglobin from the solution, Add additional distilled water, Repeating the step of further removing hemoglobin and adding additional distilled water until the ionic strength reaches a predetermined limit. Erythrocytes are washed with a solution which has been alive in the treatment container, Dissolve red blood cells in the treatment container, Separating the red blood cell membrane and the undissolved red blood cells from the hemoglobin in the treatment container, Extracting hemoglobin in solution from the treatment container is performed in the apparatus, wherein the hemoglobin is separated from the red blood cells packed in the treatment container of the cell treatment apparatus. The method of claim 10, wherein the separating step comprises centrifugation of the treatment container in the apparatus to pack the red blood cell membrane and the undissolved red blood cells. The method of claim 10 wherein the washing step comprises the addition of a cleaning agent, antimicrobial agent or antiviral agent to the live solution. The method of claim 10, further comprising measuring the ionic strength of the hemoglobin solution, wherein the dissolving, separating, and extracting steps are performed repeatedly until the ionic strength decreases to a predetermined level. The packed red blood cells are mixed with a standard saline solution, Washing with a solution using red blood cells, Dissolve red blood cells, Dialysis of red blood cells to remove epilepsy, Dialysed filtered red blood cells, Collect the hemoglobin solution produced in the container, wherein the container contains a predetermined agent that is subjected to chemical modification of the hemoglobin solution, React the hemoglobin solution with the agent, A method of making a modified hemoglobin solution comprising storing a solution reacted in a storage container. The method of claim 14, wherein the predetermined agent comprises a buffer salt. The method of claim 15 wherein the buffer salt is iminothiolane and activated polyethylene glycol. 15. The method of claim 14, further comprising filtering the reacted solution. The method of claim 14, further comprising sterilizing the reacted solution to remove or inactivate the organism.