WO2000021591A1 - Method for production of stroma-free hemoglobin - Google Patents
Method for production of stroma-free hemoglobin Download PDFInfo
- Publication number
- WO2000021591A1 WO2000021591A1 PCT/US1999/024149 US9924149W WO0021591A1 WO 2000021591 A1 WO2000021591 A1 WO 2000021591A1 US 9924149 W US9924149 W US 9924149W WO 0021591 A1 WO0021591 A1 WO 0021591A1
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- WIPO (PCT)
- Prior art keywords
- solution
- hemoglobin
- red blood
- blood
- blood cells
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3692—Washing or rinsing blood or blood constituents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
- A61M1/3696—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging with means for adding or withdrawing liquid substances during the centrifugation, e.g. continuous centrifugation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
- A61M1/3698—Expressing processed fluid out from the turning rotor using another fluid compressing the treatment chamber; Variable volume rotors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
- A61M2202/0429—Red blood cells; Erythrocytes
- A61M2202/0433—Free haemoglobin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2205/00—General characteristics of the apparatus
- A61M2205/33—Controlling, regulating or measuring
- A61M2205/3306—Optical measuring means
- A61M2205/331—Optical measuring means used as turbidity change detectors, e.g. for priming-blood or plasma-hemoglubine-interface detection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2205/00—General characteristics of the apparatus
- A61M2205/33—Controlling, regulating or measuring
- A61M2205/3317—Electromagnetic, inductive or dielectric measuring means
Definitions
- the invention relates to a system and method, using an automated blood cell separator, to prepare a high-quality hemoglobin solution as the raw material for manufacture of hemoglobin-based therapeutic oxygen carriers ("blood substitutes").
- blood substitutes hemoglobin-based therapeutic oxygen carriers
- a blood substitute succeeds in the marketplace depends on several key factors. First, it must be effective. However, a clear-cut test for efficacy has not been established. One possible test would be whether the product can effectively reduce exposure of patients to allogenic blood. Second, the product must be safe. Safety issues that have arisen to date center around the known property of hemoglobin to be vasoconstrictive. At least part of this property is the very strong binding of nitric oxide (NO) to hemoglobin as a heme ligand and at sulfhydryl sites. Third, a red cell substitute must successfully compete with blood for clinical use. Blood substitute products currently under development have plasma retention times ranging from 12 to 58 hours (half-time). Thus, they will either be used only in temporary situations or in settings where repeated doses can be administered.
- NO nitric oxide
- Cow blood has the advantage that it can be obtained in large quantities.
- maintaining cows for this purpose requires strict health standards and veterinary care for the animals, frequent testing, and large amounts of land and food to support them.
- cow blood must be collected using special apparatus designed for that purpose.
- a recombinant source would be an ideal solution because of the reduced risk of contamination with human pathogens.
- recombinant hemoglobin requires extensive purification to separate the protein from other components of the fermentation, large volumes of water are utilized, and significant problems are encountered in handling the waste products. Such processing requirements result in a blood substitute product made with recombinant hemoglobin costing several times more than the cost of conventionally-banked blood.
- the method employs a commercially-available blood cell separator comprising a computer-controlled centrifuge having a rotor into which a blood processing bag containing donor blood is placed. Once the blood is collected, the process is performed entirely within the enclosed centrifuge bowl, preferably in situ at the donor collection site.
- the centrifuge rotor includes one or more processing chambers for receiving the processing bag(s).
- the blood is centrifuged to separate the plasma from the cellular components.
- Supernatant i.e., leukocytes, platelets, and plasma, is removed by using hydraulic fluid force through a flexible diaphragm in the blood processing bag and solenoid- controlled pinch valves, leaving the packed cells.
- the pinch valves function by pinching the tubing containing the solution, avoiding direct contact to keep the fluid path sterile.
- a rotating seal allows passage of fluids into and out of the blood processing bag while the centrifuge is rotating.
- the wash solution is removed by hydraulic force and transferred through tubing into a supernatant collection container.
- a photosensor is positioned to monitor the tubing leading to the supernatant collection container to detect the presence of red blood cells. If the red blood cells are detected, a stop or hold function is initiated by the system controller.
- red blood cells are then lysed by hypotonic shock and centrifugal force is used to separate the red cell membranes (stroma) from the lysate, which is collected into a sterile container, leaving only the stroma in the centrifuge bowl.
- the final product can be used as raw material for any of the hemoglobin-based oxygen carriers currently being developed as red cell substitutes. All of these steps are performed within the bowl of the cell separator or saver to maintain sterility.
- Figure 1 is a diagrammatic view of red blood cell processor for use in preparation of stroma-free hemoglobin
- Figure 2 is a flow diagram for production of stroma-free hemoglobin from packed red blood cells
- FIG. 3 is a block diagram of the process flow according to the present invention.
- Figure 4 shows a schematic diagram of one possible configuration of the invention to produce a "blood substitute”.
- the method for production of stroma-free hemoglobin uses a conventional commercial blood cell processing apparatus which is diagrammatically illustrated in Figure 1 .
- Such systems are used for processing of separation of whole blood from a patient or donor into components. The red blood cells are collected, and the remaining components can be returned to the donor or discarded.
- the apparatus comprises a centrifuge 2 for retaining a blood bag 6, a plurality of reservoirs 8 and 10 containing processing solutions, a reservoir for receiving supernatant 14, a plurality of valves and a sterile tube harness connecting the various reservoirs to the blood bag 6, and a controller 20 for controlling the centrifuge and the valves.
- a disposable rotating seal 34 is included within the tube harness to allow passage of fluids into and out of the blood processing bag while the centrifuge rotor is rotating.
- the seal prevents fluids from leaking out of the fluid path and air from leaking into the fluid.
- Blood is collected from the donor (arm 22 is shown) through tube 30 and valve 32 directly into the blood bag 6, preferably for immediate processing.
- blood bag 6 and the tube harness are parts of a pre-sterilized, disposable processing set.
- the valves function by pinching the tube so that there is no direct contact with the fluid in the tubes.
- Centrifuge rotor 24 is activated to separate the red blood cells from the plasma.
- the centrifugal force provides the pumping for expression of the plasma to supernatant reservoir 14 (or back to the patient or donor, if desired).
- a displacement chamber is included in the centrifuge rotor 24 comprising a hydraulically-operated diaphragm 4.
- the flow of a hydraulic fluid to and from the region under flexible diaphragm 4 is controlled by system controller 20 by controlling rotation of the centrifuge drive 36 and direction of a reversible hydraulic pump 38.
- Controller 20 causes valve 16 to open to permit the plasma to exit the blood bag through tube 18, then closes valve 16 after the plasma is removed. Complete removal of the plasma can be determined by use of optical detector 28, which detects the presence of red blood cells in the clear tubing.
- Saline solution from reservoir 8 is released through tube 26 into blood bag 6 by opening valve 12.
- an anti-bacterial agent, detergent or other appropriate cleansing solution can be used as the wash solution, or in addition to the saline solution, to remove any bacterial contaminants that might be in the blood.
- the centrifuge rotor rotates at a relatively low speed to provide agitation to wash the red blood cells. After washing, the rotational speed increases and controller 20 opens valve 16 allowing the centrifugal force and flexible diaphragm to express the wash solution into supernatant reservoir 14.
- red blood cells are lysed to separate the stroma from the hemoglobin by introducing distilled water to induce hypotonic shock.
- Distilled water which is stored in reservoir 10 is transferred through tube 1 1 by opening valve 13.
- red cells first come into contact with the distilled water, some of them lyse, liberating hemoglobin into solution.
- the cell-free component can be harvested at this time, and the distilled water infusion continues, liberating more and more hemoglobin as the ionic strength of the suspending medium decreases.
- the stroma-free hemoglobin With the centrifuge rotor rotating at a low speed to minimize damage to the hemoglobin, the stroma-free hemoglobin will be distributed throughout the bowl, while the red blood cells and membranes will become packed at the outer edge of the bowl. Since large amounts of distilled water may be required, several iterations will be required to liberate most of the hemoglobin. Removal of the hemoglobin is achieved via a sterilized port 40 in blood bag 6 through tube 42 into a sterile container 44. It may be desirable to include a filter 46 in the transfer pathway (tube) to remove the last traces of membrane particles. Given enough water and time, all of the hemoglobin should be removed, however, it may be necessary later to concentrate the hemoglobin solution, depending on the use for which it is intended.
- Determination of the completeness of hemoglobin extraction can be made by measurement of ion concentration in the hemoglobin solution.
- the conductivity meter By connecting the conductivity meter to system controller 20, when the ionic concentration drops below a predetermined level, the process can be terminated. Selection of the predetermined level is based upon a balancing of the time versus efficiency of hemoglobin extraction, i.e., a cost- benefit analysis.
- Other methods for inducing hypotonic shock for lysing the red blood cells are known and may be substituted for, or used in combination with, the distilled water rinse. Alternate lysing methods are described below in more detail.
- the sterile container into which the hemoglobin solution is transferred can contain pre-measured reagents for preparation of blood substitute, including, for example, buffer salts, Traut's reagents and activated polyethylene glycol (PEG).
- pre-measured reagents for preparation of blood substitute including, for example, buffer salts, Traut's reagents and activated polyethylene glycol (PEG).
- PEG polyethylene glycol
- Example 1 The method of processing takes place entirely within a disposable, pre- sterilized processing set, which preferably includes all tubing, reservoirs, centrifuge bowl and in-line filters. This closed system eliminates the risk of contamination.
- a disposable, pre- sterilized processing set which preferably includes all tubing, reservoirs, centrifuge bowl and in-line filters. This closed system eliminates the risk of contamination.
- An experimental apparatus for production of stroma-free hemoglobin from blood was constructed comprising a manifold for emptying bags of outdated blood; cross-flow filtration devices for washing, lysis and purification; a bioreactor for cross-linking hemoglobin; a preparative-scale HPLC apparatus; and a hood for final filling of product into sterile bags.
- the flow loops including PFW distribution loops, were closed. All connections were of the sanitary triclamp type and were made in a laminar flow environment. The entire process required approximately 1000 ft 2 of laboratory space, exclusive of offices. This capacity of this pilot plant was about 5 liters per week and the cost of production was about $1000/1 iter.
- the cross-flow filtration system consisted of three pumps, four tanks and four distinct filter packages. All construction materials were made from 316L stainless steel with internal finishes of 180 grit. Tubing was flexible and made of reinforced silicone. The hollow fiber filtration cartridges were nonhemolytic, nopyrogenic polysulfone membranes.
- the RBC's were washed with 7 volumes of chilled normal saline by diafiltration at a constant volume (80 liters) through three 0.65 ⁇ m hollow fiber membrane cartridges (total surface area 69 ft 2 ). To verify that plasma was removed, the filtrate was assayed for albumin as a marker for plasma proteins. During a typical wash, albumin concentration consistently decreased from more than 2 mg/ml to undetectable levels ( ⁇ 2 ⁇ g/ml). The RBC's were lysed slowly, and the stromata were removed by diafiltration with 5 volumes of chilled 10 mM NaHP0 4 , pH 7.6, at a constant volume (80 liters) through 0.1 ⁇ m hollow fiber membrane cartridges.
- the filtrate of the 0.1 ⁇ m cartridge was directed to a 100-liter stainless-steel tank. It was then diafiltered at a constant volume (80 liters) through a 500-kDa membrane cartridge to remove any stroma particles.
- the filtrate (SFH) from the 500-kDa cartridges was then concentrated to 10 g/dl by circulation through three 10-kDa hollow fiber membrane cartridges.
- the resulting SFH solution was diafiltered with 6 volumes of Ringer's acetate, pH 7.4, and then transferred from the stainless-steel concentration tank through a 0.2 ⁇ m, 10-inch filter to a 40 liter stainless-steel holding tank. Finally, the SFH product was transferred under sterile conditions into plastic bags and frozen at -80°C. Alternatively, the SFH was transferred to a 70-liter bioreactor for cross- linking.
- Stroma-free hemoglobin solutions produced using the above apparatus and procedure were formulated either in water or in phosphate buffer. Methemoglobin concentration was routinely less than 1 %, and the solutions could be stored indefinitely at - 80° C. The rabbit pyrogen test was negative and the solutions were not contaminated by bacteria.
- Blood collected in this way may be acceptable for remanufacture into a
- blood substitute such as that disclosed in Patent No. 5,814,601 .
- blood substitute such as that disclosed in Patent No. 5,814,601 .
- patients with hemachromatosis are currently prevented by the FDA from donating blood for transfusion. This has prompted these patients to seek private physicians to carry out phlebotomies, at considerable cost to the patient.
- the Iron Overload Disease Society estimates that there may be as many as 1 .5 million individuals in the U.S. alone with significant iron overload.
- the society has records of approximately 5,000 patients who are in regular phlebotomy programs, which remove about 6 units/year, or 30,000 units.
- the red cell substitute has established a dose- equivalence such that 1 g/dl of plasma hemoglobin is as effective in a hemorrhage model as 7 g/dl of red cell hemoglobin.
- this group of patients alone could supply the raw materials for as many as 150,000 units of artificial blood per year.
- a self-contained, portable unit designed to collect donor/patient blood at the bedside is used.
- Appropriate systems for this application include the Haemonetics MCS+ 8150 Blood Collection System (Haemonetics Corporation, Braintree, MA) and the COBE 2991 Cell Processor (COBE Laboratories, Inc., Lakewood, CO) .
- Blood is collected in a CP2D/AS-3 anticoagulant/additive system, at 1 :16 ratio of anticoagulant to anticoagulated blood.
- the machine is configured to collect either one or two units of red blood cells from a single donor in approximately 25 minutes. Each unit collected yields approximately 180 ml of packed red blood cells ("RBC”) and 400 ml of plasma.
- RBC count x post-process weight x 100 ⁇ % RBC recovery pre-process RBC count x pre-process weight
- the number of leukocytes remaining within the packed red blood cells should be less than 5 x 10 8 .
- the absolute white blood cell (“WBC”) count is:
- a tube harness Also included in this processing set are a tube harness, a rotating seal and a supernatant collection container.
- the rotating seal allows passage of fluids into and out of the blood processing bag while the centrifuge rotor is rotating. The seal prevents fluids from leaking out oi the fluid path and air from leaking into the fluid.
- the tube harness provides a sterile fluid path for introduction of wash solutions to the red cells.
- Three valves control the flow of blood or wash solutions into the blood processing bag. Each valve is a solenoid-controlled pinch valve, under the control of the system computer, and have no direct contact with the fluid ' in the tubing.
- a sterile outlet port at the outer edge of the processing bag enables transfusion of the processed cells directly from the bag into a sterile container.
- the bag is configured to be supported within a contoured processing chamber in the centrifuge rotor so that the second blood component travels along a short internal bag dimension to achieve separation.
- a displacement chamber having a hydraulically-operated diaphragm is also positioned within the blood processing chamber of the centrifuge rotor.
- the flow of hydraulic fluid to and from the region under the flexible diaphragm is controlled by rotation of the centrifuge drive and pinch valve solenoids.
- the hydraulic system consists of a positive displacement piston-type pump assembly, flow rate controls and switches, along with the fluid flow network of piping and reservoir (plastic bag.)
- the pump is driven by a variable-speed reversible motor. The volume capacity of the pump is adjusted for approximately 600 ml.
- Pump motor control is driven forward at a set rate to express the supernatant by exerting pressure on the blood processing bag, forcing the supernatant fluid out of the bag through one of the open valves to the supernatant collection container. This continues until red cells are sensed through a photosensor, the supernatant-out volume is reached, or a stop or hold function is initiated.
- the pump is driven in the reverse direction to draw fluid from the centrifuge hydraulic fluid chamber and from the reservoir. The centrifuge rotor can then be stopped to allow return of second blood component to the donor. Because all of the functions are under computer control, there is minimal intervention by the operator and, thus, little opportunity for operator error.
- the volume of the centrifuge bowl is approximately 250 ml, and the centrifuge speed is approximately 4,000 rpm.
- Blood entering the centrifugal field is diluted with sterile saline, and red cells are concentrated by continuous removal of non-red cell components and plasma.
- the goal is to reduce the amount of serum albumin to as low a level as possible, preferably to become undetectable.
- the non- red blood cell components can be discarded or used for other purposes.
- Evaluation of serum protein removal after the RBC wash step is conducted using procedures prescribed by the manufacturer of the blood cell processor. For example, in the COBE 2991 Cell Processor, after the red blood cells are washed, a 1 ml sample is extracted through the sterile port in the blood bag, is centrifuged in a laboratory centrifuge, and the supernatant is collected. A commercially- available protein chemistry dipstick wet pad can be used to test the supernatant, with the color change being indicative of protein level. Protein level should be trace (15-30 mg/dl) or less, representing a plasma reduction of greater than or equal to 96%.
- the saline is replaced by distilled water.
- distilled water Referring to Figure 3, it can be seen that when red cells first come into contact with the distilled water, some of them lyse, liberating hemoglobin into solution. Conditions for lysis are discussed below.
- the cell-free component can be harvested at this time, and the distilled water infusion continues, liberating more and more hemoglobin as the ionic strength of the suspending medium decreases. Given sufficient time, it is possible to remove all of the hemoglobin, leaving only red blood cell ghosts in the centrifuge bowl.
- the volume of the hemolysate depends on the amount of distilled water needed to achieve a desired level of lysis. However, the hemoglobin solution may need to be concentrated further, depending on the use for which it is intended. Because NaCI may accompany the hemoglobin solution, subsequent purification may be required. A rapid, sensitive measure of the ionic strength of the final solution can be obtained by conductivity measurement. Conditions for lysis of human red blood cells
- hemoglobin is a fragile protein. It is made up of 4 polypeptide subunits, 2 ⁇ and 2 ⁇ . An ⁇ and a ⁇ chain are tightly linked into an ⁇ subunit, and 2 ⁇ subunits form a less stable tetramer, the fully formed hemoglobin molecule.
- Each of the 4 subunits contains an iron-prosthetic group, heme. The iron atom is maintained in the reduced, Fe + + state in order to bind oxygen. Maintenance of this reduced state is by the presence of a number of enzyme systems within the red blood cell. When hemoglobin is liberated from the cell, this protection is no longer present, and a series of events occur which ultimately leads to degradation of the molecule.
- lyse human red cells there are three methods to lyse human red cells. First, they can be frozen and thawed repeatedly. The formation of ice within and around the cells disrupts the membranes and hemoglobin is liberated. However, lysis using this method is notoriously incomplete, and the hemoglobin denaturation can occur. In the second method, organic solvents, such as CCI 4 or toluene can be used. This method is more efficient, however, the solvents can also adversely affect the stability of the protein. A third method is osmotic lysis, such as described above. The susceptibility of red cells to osmotic lysis is well known. Estimated process efficiency
- hemoglobin production is protein denaturation. Hemoglobin is sensitive to the effects of dilution, pressure, shear, temperature and pH changes, these factors can lead to problems involving oxidation, precipitation, heme loss, and subunit dissociation. Thus, it is important that all process steps be performed at uniform and low temperature. For example, the COBE 2991 Cell Processor can be refrigerated to maintain a temperature of 4°C. Furthermore, agitation should not be too vigorous, pH changes should not be abrupt, and pressures should not reach high levels. Characterization and quality control
- Measurement of stromal contamination can be performed by using the total phosphate assay. Extraction of phospholipid from a hemoglobin solution can be difficult, so the total phosphate assay is an easier and less costly procedure.
- Intracellular enzymes constitute a second potential group of red blood cell contaminants, however, it is possible that some contamination of this type may be acceptable in blood substitutes.
- Analytical FPLC can be used to evaluate the homogeneity of stroma-free hemoglobin product using a 280 nm filter to resolve
- Endotoxin measurement can be performed using a kinetic turbidimetric assay based on the initiation of the Limulus amebocyte lysate (LAL) coagulation cascade.
- LAL Limulus amebocyte lysate
- Stroma-free hemoglobin solution pH and conductivity can be measured using conventional pH and conductivity meters, such as those in the Accumet line from Fisher Scientific Co., Pittsburgh, PA.
- Spectral analysis can provide estimates of hemoglobin concentration and the amount of methemoglobin in the solutions.
- Such analysis can be performed using a rapid scanning diode array spectrophotometer, such as the Milton Roy 3000, in the Soret and visible regions, with evaluation performed according to the multicomponent analysis technique 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), which is incorporated herein by reference.
- the most sensitive test to determine the functional state of a hemoglobin solution is to measure its oxygen binding curve.
- One such method entails the linear consumption of oxygen in a closed optical cuvette by a novel enzyme system. As the oxygen is depleted, repeated visible spectra are taken, while P0 2 is measured simultaneously with a Clark-type electrode.
- the hemoglobin is diluted in 0.1 M bis- Tris propane or phosphate buffer to a concentration of approximately 60 ⁇ m (in heme). The reaction takes approximately 15 minutes.
- the reaction uses protocatechuic acid (PCA, Sigma Chemical Co., St.
- the hemoglobin solutions produced using this invention are also characterized by isoelectric focusing (IEF) on agarose and polyacrylamide in the pH ranges 8.5 to 5.5.
- the subunit structure is evaluated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.
- Spectral analysis provides estimates of the hemoglobin concentration and the amount of methemoglobin and carboxyhemoglobin in the solutions.
- Spectra are collected on a rapid scanning diode array spectrophotometer in. the Soret and visible regions.
- Spectra are evaluated by multicomponent analysis.
- Other quality control measures include measurement of hemoglobin concentration, pH and conductivity. Additional quality control measures are instituted, as needed.
- Example 3 Preparation of modified hemoglobin using the described invention
- the present invention can further be used to prepare a modified hemoglobin solution ("the product") using the stroma-free hemoglobin (SFH) solution prepared in the described modified cell separator device as shown in Figure 4.
- the product stroma-free hemoglobin (SFH) solution prepared in the described modified cell separator device as shown in Figure 4.
- the red blood cells can be obtained from any source, including animal or human, stored or fresh, or even outdated human units obtained from a blood bank.
- the modification procedure can also use a hemoglobin solution prepared by any means, including the present invention, or by other methods, including recombinant hemoglobins. If donor blood is used, it is first mixed with an anticoagulant 50, washed in the centrifuge bowl 52 with normal saline 54 and lysed with distilled water 56. Plasma, platelets and white blood cells fractions are removed and stored in a separate container 58. The hemoglobin solution (SFH) 60 is placed either into a separate reservoir 62, or into the centrifuge bowl 52, where chemical modification is carried out. After filtration, the final product is collected in a sterile container 64 which can be stored for future use.
- FSH hemoglobin solution
- the SFH is collected into a reservoir which contains the premeasured reagents for the chemical modification.
- the reagents are buffer salts, iminothiolane and activated polyethylene glycol.
- the modified hemoglobin (PEG-Hb) is collected into a plastic bag or other storage receptacle and stored.
- any described modification can be carried out by the described apparatus of the present invention, and these procedures are well known in the field of blood substitute preparation.
- Such modifications might include, as examples, internal cross-linkers, polymerization reactions, or surface modification of hemoglobin with dextrans, starches, or other synthetic or natural polymers.
- the product can be further purified by passing it through filters 66 and 68 in Figure 4).
- filters 66 and 68 in Figure 4
- filters 66 and 68 in Figure 4
- filters 66 and 68 in Figure 4
- Such filters, and ultracentrifugation devices could be size exclusion filters, ion-exchange filters, mixed-bed ion exchangers, activated charcoal filters or other in-line filters used in protein purification or dialysis procedures.
- the product can be formulated with any solution of salts or other materials.
- Sterilization of the product can be performed in the same apparatus by any number of procedures, including solvent-detergent treatment, gamma irradiation, nanofiltration, methylene blue or similar derivative, or any other means for inactivating or removing organisms such as bacteria and viruses.
- the inventive method provides means for more rapid and less costly collection of red blood cells which are needed for producing blood substitutes that are capable of addressing the significant worldwide shortfall in available blood for transfusion.
- the inventive method can be used for processing of outdated blood, but is most advantageously used in situ at the donor/patient's bedside. Because the process is performed in a computer-controlled, fully self-contained apparatus, handling is minimized, and risk of contamination is eliminated. The procedure also eliminates the need to type and store units oi collected blood, and units collected for this purpose could be pooled to reduce the cost of testing for infectious agents.
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/807,565 US6773613B1 (en) | 1998-10-15 | 1999-10-15 | Method for production of stroma-free hemoglobin |
JP2000575563A JP2002527149A (ja) | 1998-10-15 | 1999-10-15 | 無ストロマヘモグロビンの産生のための方法 |
BR9915734-9A BR9915734A (pt) | 1998-10-15 | 1999-10-15 | Método para a produção de hemoglobina livre de estroma |
MXPA01003836A MXPA01003836A (es) | 1998-10-15 | 1999-10-15 | Metodo para `produccion de hemoglobina libre de estroma.. |
CA002346825A CA2346825C (en) | 1998-10-15 | 1999-10-15 | Method for production of stroma-free hemoglobin |
KR1020017004781A KR20010099700A (ko) | 1998-10-15 | 1999-10-15 | 무-간질 헤모글로빈의 제조 방법 |
EP99954950A EP1121165A1 (en) | 1998-10-15 | 1999-10-15 | Method for production of stroma-free hemoglobin |
AU11170/00A AU771465B2 (en) | 1998-10-15 | 1999-10-15 | Method for production of stroma-free hemoglobin |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10431998P | 1998-10-15 | 1998-10-15 | |
US60/104,319 | 1998-10-15 | ||
US12218099P | 1999-03-01 | 1999-03-01 | |
US60/122,180 | 1999-03-01 |
Publications (1)
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WO2000021591A1 true WO2000021591A1 (en) | 2000-04-20 |
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PCT/US1999/024149 WO2000021591A1 (en) | 1998-10-15 | 1999-10-15 | Method for production of stroma-free hemoglobin |
Country Status (9)
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EP (1) | EP1121165A1 (es) |
JP (1) | JP2002527149A (es) |
KR (1) | KR20010099700A (es) |
CN (1) | CN1332646A (es) |
AU (1) | AU771465B2 (es) |
BR (1) | BR9915734A (es) |
CA (1) | CA2346825C (es) |
MX (1) | MXPA01003836A (es) |
WO (1) | WO2000021591A1 (es) |
Cited By (5)
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---|---|---|---|---|
WO2005099858A1 (en) * | 2004-04-13 | 2005-10-27 | Sangart, Inc. | Methods and compositions for simultaneously isolating hemoglobin from red blood cells and inactivating viruses |
WO2009000763A1 (en) * | 2007-06-22 | 2008-12-31 | Kedrion S.P.A. | Process for purifying apyrogenic and virus-inactivated human hemoglobin |
US7901342B2 (en) | 2004-07-08 | 2011-03-08 | Hitachi Koki Co., Ltd. | Centrifugal separator with sterilizing apparatus |
CN109298171A (zh) * | 2018-11-15 | 2019-02-01 | 武汉中生毓晋生物医药有限责任公司 | 醛化红细胞中内毒素的去除方法与应用 |
US11472836B2 (en) | 2013-02-26 | 2022-10-18 | Emd Millipore Corporation | Selective removal of a protein from a mixture of proteins using activated carbon by adjusting solution conditions |
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EP2433713B1 (en) * | 2007-12-07 | 2017-07-26 | Miltenyi Biotec GmbH | Cell processing systems and methods |
US9493616B2 (en) * | 2010-02-25 | 2016-11-15 | Sangart, Inc. | Methods for preparing PEG-hemoglobin conjugates using reduced reactant ratios |
WO2014070209A1 (en) * | 2012-11-05 | 2014-05-08 | Haemonetics Corporation | System and method for continuous separation of whole blood |
CN103091149B (zh) * | 2013-01-10 | 2015-08-05 | 济南金域医学检验中心有限公司 | 血红蛋白电泳液的制备方法 |
EP3131589B1 (en) * | 2014-04-15 | 2023-05-17 | Boehringer Ingelheim International GmbH | Method and use for continuously inactivating a virus during manufacture of a biological product |
WO2019166998A1 (en) * | 2018-03-02 | 2019-09-06 | Thermo Electron Led Gmbh | Single-use centrifuge containers for separating biological suspensions and methods of use |
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-
1999
- 1999-10-15 EP EP99954950A patent/EP1121165A1/en not_active Withdrawn
- 1999-10-15 BR BR9915734-9A patent/BR9915734A/pt not_active IP Right Cessation
- 1999-10-15 JP JP2000575563A patent/JP2002527149A/ja not_active Withdrawn
- 1999-10-15 KR KR1020017004781A patent/KR20010099700A/ko not_active Application Discontinuation
- 1999-10-15 CA CA002346825A patent/CA2346825C/en not_active Expired - Fee Related
- 1999-10-15 CN CN99813429A patent/CN1332646A/zh active Pending
- 1999-10-15 AU AU11170/00A patent/AU771465B2/en not_active Ceased
- 1999-10-15 MX MXPA01003836A patent/MXPA01003836A/es unknown
- 1999-10-15 WO PCT/US1999/024149 patent/WO2000021591A1/en not_active Application Discontinuation
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US4303193A (en) | 1979-01-22 | 1981-12-01 | Haemonetics Corporation | Apparatus for separating blood into components thereof |
US4401652A (en) * | 1980-12-31 | 1983-08-30 | Allied Corporation | Process for the preparation of stroma-free hemoglobin solutions |
US5464814A (en) * | 1986-06-20 | 1995-11-07 | Northfield Laboratories, Inc. | Acellular red blood cell substitute |
US5296465A (en) | 1986-11-10 | 1994-03-22 | Biopure Corporation | Ultra pure hemoglobin solutions and blood-substitutes |
EP0367475A2 (en) * | 1988-10-29 | 1990-05-09 | Nippon Oil And Fats Company, Limited | Process for producing haemoglobin concentrate |
US5646252A (en) * | 1992-02-10 | 1997-07-08 | Staat Der Nederlanden, De Minister Van Defensie, Voor Deze: Het Hoofd Van De Afdeling Militair Geneeskundig Beleid | Hemoglobin composition and preparation thereof |
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US5814601A (en) | 1997-02-28 | 1998-09-29 | The Regents Of The University Of California | Methods and compositions for optimization of oxygen transport by cell-free systems |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005099858A1 (en) * | 2004-04-13 | 2005-10-27 | Sangart, Inc. | Methods and compositions for simultaneously isolating hemoglobin from red blood cells and inactivating viruses |
US7901342B2 (en) | 2004-07-08 | 2011-03-08 | Hitachi Koki Co., Ltd. | Centrifugal separator with sterilizing apparatus |
US7909751B2 (en) | 2004-07-08 | 2011-03-22 | Hitachi Koki Co., Ltd. | Method for sterilizing a centrifugal separator |
WO2009000763A1 (en) * | 2007-06-22 | 2008-12-31 | Kedrion S.P.A. | Process for purifying apyrogenic and virus-inactivated human hemoglobin |
US11472836B2 (en) | 2013-02-26 | 2022-10-18 | Emd Millipore Corporation | Selective removal of a protein from a mixture of proteins using activated carbon by adjusting solution conditions |
CN109298171A (zh) * | 2018-11-15 | 2019-02-01 | 武汉中生毓晋生物医药有限责任公司 | 醛化红细胞中内毒素的去除方法与应用 |
CN109298171B (zh) * | 2018-11-15 | 2022-06-28 | 武汉中生毓晋生物医药有限责任公司 | 醛化红细胞中内毒素的去除方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
BR9915734A (pt) | 2001-10-02 |
MXPA01003836A (es) | 2002-09-18 |
KR20010099700A (ko) | 2001-11-09 |
EP1121165A1 (en) | 2001-08-08 |
AU1117000A (en) | 2000-05-01 |
AU771465B2 (en) | 2004-03-25 |
JP2002527149A (ja) | 2002-08-27 |
CN1332646A (zh) | 2002-01-23 |
CA2346825A1 (en) | 2000-04-20 |
CA2346825C (en) | 2008-07-29 |
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