CN100348267C - Purification of red blood cells by separation and infiltration - Google Patents

Abstract

The present invention relates to a process for preparing red blood cell components and liquid components by separating the whole human blood, such as centrifugating and purifying red blood cells. The whole human blood can be processed by blood coagulation prevention before being defibrinated or separated, and the whole human blood is preferably selected from the calf blood. Then, the red blood cells are percolated and purified, the purified red blood cells are dissolved to form dissolved matter of the purified red blood cells, and the purified red blood cells and the dissolved matter of the purified red blood cells are suitable for preparing hemoglobinemia substitutes.

Description

By separating and diafiltration purification Red blood corpuscle

Background of invention

Development has concentrated on the oxygen that is used for pharmaceutical treatment based on the emphasis of the Red blood corpuscle carrier of oxygen and has sent, as the production of blood transfusion and blood products.The carrier of oxygen based on Red blood corpuscle can be used for preventing or treating and (for example lose blood, owing to acute hemorrhage or in surgical procedures), because anemia (for example, pernicious anemia or sickle cell anemia), or owing to (for example suffer a shock, volume deficiency shock, anaphylactic shock, septic shock or allergic shock) hypoxia that causes.

The existing carrier of oxygen based on Red blood corpuscle comprises high fluorine chemistry thing, synthetic hemoglobin analogues, and the methemoglobinemia of liposomes enclose, the methemoglobinemia of chemical modification and based on the carrier of oxygen of methemoglobinemia, wherein the Red blood corpuscle molecule is crosslinked.Preparation based on the carrier of oxygen of Red blood corpuscle comprises several purification steps.In order from whole blood, to remove plasma proteins, utilize the method for micro-filtration to wash the cell of whole blood.The cell washing operation utilizes diafiltration from the whole blood of calf, by the micro-filtration film of 0.2um, ooze salt/citric acid solution with grade and remove plasma proteins.Diafiltration is successive filter operation, wherein adds salt/citric acid solution and keep a volume in the recycling tank in filtering retentate.By filter paper recirculation in blood circulation solution, the filtrate that will contain plasma proteins abandons.

Utilize the filtration washing blood solution to cause process time very variable, it influences the product circulation unfriendly.In addition, the prolongation of the time of cell washing process can cause biological load unacceptable level growth and cause cytolysis, thereby the output in the reduction process.

Summary of the invention

The present invention relates generally to purification and be used to make the method for the blood cell of blood substituent.This method comprises separating whole blood, thereby forms red blood cell fraction and liquid component.Thereby the red blood cell fraction diafiltration is formed the Red blood corpuscle of purification.Then, can further process the Red blood corpuscle of purification, separate red hemocyte molecule.

In one embodiment, the present invention relates to be formed for the solute of Red blood corpuscle of the purification of Red blood corpuscle blood substituent.This method comprises, separating whole blood, thus form red blood cell fraction and liquid component.The diafiltration red blood cell fraction forms the Red blood corpuscle of purification.The Red blood corpuscle of dissolving purification forms the solute of the Red blood corpuscle of purification.

In another embodiment, this method branch comprises by centrifugalize and goes Fibrotic whole bovine blood, thereby forms red blood cell fraction and liquid component.Thereby the diafiltration red blood cell fraction forms the Red blood corpuscle of purification.Mechanically dissolve the Red blood corpuscle of purification.

The present invention has many advantages.For example, separation of whole blood is become blood cell component and liquid component, make and removed many potential membrane contaminants in the red blood cell fraction that obtains, allow more effectively to process red blood cell fraction.Specifically, separating whole blood forms red blood cell fraction and liquid component, and diafiltration red blood cell fraction then can shorten by diafiltration or only by the centrifugal time span that obtains the Red blood corpuscle needs of purification.Usually, thus the diafiltration red blood cell fraction is formed the time span normalization of needs of the Red blood corpuscle of purification; The time that the sample of the relative purification of the Red blood corpuscle that suspend near diafiltration diafiltration period of red blood cell fraction approximately needs.The shortening that obtains the time that the blood cell of purification needs is that the whole blood with whole blood and defiberization reaches.Time that reduce or normalized cell washing method has been reduced the potentiality of growth of the unacceptable level of biological load and cytolysis thing.Successively, the reduction of the growth of unacceptable biological load and cytolytic level has improved the output of method.

Brief description of drawings

Accompanying drawing is to be applicable to the diagram of carrying out method of the present invention.

The detailed description of invention

Front of the present invention with other purpose, feature and advantage are with understanding in the explanation of the more specifically preferred embodiment below of the present invention, described in accompanying drawing. Accompanying drawing is not necessarily pro rata, and emphasis is to be to illustrate principle of the present invention.

The present invention relates generally to the method for coming the purifying red blood cell by separating whole blood generation red blood cell fraction and liquid component. Believe that potential membrane contaminant can separate from the red blood cell fraction that obtains. The liquid that comprises basic pollutant part and the formation of classification separating whole blood partly separates from red blood cell fraction, and then red blood cell fraction is carried out diafiltration.

Referring to accompanying drawing, what wherein show is instrument 10, is an embodiment that is applicable to implement the instrument of method of the present invention. In vessel 12, collect whole blood. The whole blood that is applicable to use among the present invention can be fresh collection, or collect from other existing sources, such as the human blood of from the blood storehouse, paying. In addition, although preferably, whole blood did not have freezing before this method of use, and whole blood can maintain in the state of freezing and/or liquid. The example of suitable full blood comprises, people calf, sheep, pig and other vertebrates, with the methemoglobinemia that transgenosis produces, the transgenosis Hb described in BIO/TECHNOLOGY. 12:55-59 (1994), the document is by being incorporated in full this paper in this citation. Can collect whole blood from the animal donor of butchering that live or fresh. The disclosed U.S. Patent number of the people such as Rausch has been narrated a method of collecting bovine blood in 5,084,558, and the document is by being incorporated in this paper in this citation.

In one embodiment, in vessel 12, whole blood is gone fibrillatable by suitable method. Go to finish described in the U. S. application that fibrillatable can submit such as people such as Gawryl on February calendar year 2001 28 number 09/795,821, the document is by being incorporated in full this paper in this citation. Go fibrillatable blood to start coagulation cascade, manually removed the cellulosic molecule that comprises in the formation of blood clotting. Go fibrillatable to induce by chemistry or mechanical method. The chemistry clotting reagent is defined as inducing the material of blood coagulation in this article. For example, collagen-induced blood coagulation, so that when external wound is arranged, the fiber sludged blood will stop blood flow. Contact the formation that will cause fibrin clots with blood and collagen are artificial, can remove to produce and remove fibrillatable blood. The example of other clotting reagents is tissue extractor things, tissue factor, tissue thromoboplastin, anionic phospholipid, calcium, negative band electric charge material (for example, glass, kaolin, the plastics that some are synthetic, fiber). Preferred clotting reagent is collagen. By partly being commonly referred to " serum " from the liquid that goes Fibrotic separation of whole blood blood cell to obtain.

Whole blood can contact one with clotting reagent is enough to cause substantially that all fibres element in the blood is transformed into the period of fibrin clots.The point that obviously stops multimerization by cellulosic molecule can be determined reasonable time.To go fibrosis be to contact with chemical clotting reagent and inductive as being defined as Fibrotic chemistry herein, is to carry out under suitable temperature, and preferably temperature range is about 4 ℃ to about 40 ℃.

In another embodiment, mechanical agitation also can be used to start coagulation cascade as stirring.Whole blood can stir up to the fiber multimerization and obviously stop.Remove the cellulose of accumulation and finish fibrosis.Inductive as to go the machinery of Fibrotic this paper definition to go fibrosis be to carry out in suitable temperature by intensifying blood solution, preferably temperature range is at about 4 ℃ to about 40 ℃.

Or the choosing embodiment in, whole blood can processedly be prevented hemostasis-coagulation.For example, whole blood can be used anticoagulation reagent such as sodium citrate, heparin, and ethylenediaminetetraacetic acid (EDTA) and Disodium oxalate. are handled, and the concentration of utilization is enough to suppress solidifying of whole blood.In one embodiment, the sodium citrate (34g/l) of 5L being added the final concentration that the whole blood of the fresh collection of 15L produced in the whole blood solution is the citric acid of 8.5g/l.In another embodiment, in the whole blood of fresh separated, add EDTA, produce last concentration 0.18%.The liquid component that obtains by the whole blood separation blood cell with the anticoagulation agent treated is commonly referred to " blood plasma ".

Same possible to being gone celluloseization by the blood of Citrated, as by blood, then, remove fibrosis solution with the saturated Citrated of bivalent cation, method is similar in appearance to the processing method of the blood of non-Citrated.Preferred bivalent cation is a calcium.

When blood is treated when inducing blood coagulation, remove fibrin clots from whole blood by suitable method.The example that has shown suitable method among the figure.The whole blood of inducing cellulose and fibrin clots is from vessel 12, is to carry out with filter 16 by 14.The screening washers of 60 nets are examples of suitable filter.Perhaps, substitute and be suitable for filter, cheese cloth or polypropylene filter also can be used to remove big fragment, comprise fibrin clots.Collect fibrin clots in filter 16, the residue of whole blood is sent to vessel 18.

When with the anticoagulant processing blood, the blood of processing can be in centrifuge 27, by 14 be, filter 16, vessel 18 are 20, pump 22, filter 24 and filter 26 carry out.But the blood of processing also can pass through 14 lines, filter 16, and vessel 18,20 lines, pump 22, filter 24 and filter 26 carry out.The whole blood that anticoagulation is handled can be used for removing any big fragment that exists in the whole blood of processing by filter 16 and filter 14 and 26.

As shown in FIG., whole blood (through or not through inducing blood coagulation to handle) be by pump 22 from vessel 18 through lines 20, undertaken by first filter 24 and second filter 26 centrifugal 27.In one embodiment, first filter 24 and second filter 26 are polypropylene filters.In particularly preferred embodiments, first filter 24 has the permeability of about 800um, and second filter has the permeability of about 50um.When whole blood has been handled when starting coagulation cascade, remove all fiber factors substantially by first filter 24 and second filter 26 and finished and go the fibrosis step.

Behind the filter 26, whole blood goes fibrosis or does not have, and separates, and forms red blood cell fraction and liquid component." separation " also is also referred to as " fractionated " herein, comprises the separation formation liquid component separately from the Red blood corpuscle of serum or blood plasma, preferably serum or blood plasma.As used herein, isolation technics be basically come by density isolating, as by centrifugal, as from distinguishing as the separation of diafiltration according to size.

As used herein, " liquid component " comprises the liquid that obtains from separation of whole blood, with the liquid that goes obtain separating of Fibrotic whole blood.In one embodiment, liquid component is " serum ", for example from the branch liquid component that Fibrotic whole blood obtains of leaving away.In another embodiment, liquid component is " blood plasma ", as the liquid component that obtains from the treated anticoagulative whole blood of separation.In one embodiment, with anticoagulant such as sodium citrate or heparin processing whole blood.

In one embodiment, behind filter 26, go fibrosis or do not go Fibrotic whole blood in centrifuge 27, to separate.Usually, in centrifugal process, whole blood and G-power is about 1,000 and about 12, contact in the scope between the 000XG, and purpose is a separating whole blood, thereby forms blood cell component (blood cell component) and liquid component.Usually, centrifugal is to carry out in time period in the scope between about 30 seconds to about 4 minutes.Preferably, centrifugal is that 000-10 has carried out among the 000XG about 3 minutes about 8.The temperature of the whole blood in separating step is the scope between about 4 to about 15 ℃ normally.

In one embodiment, red blood cell fraction comprises Red blood corpuscle and the leukocyte in the whole blood, and liquid component comprises the platelet of whole blood.Equally, preferably, red blood cell fraction comprises most of Red blood corpuscles (RBCs) (for example, at least about 90%, or at least about 95%, or at least about 99%) of whole blood.

Liquid component is to remove from red blood cell fraction substantially.Usually, liquid component is removed from red blood cell fraction simultaneously centrifugal.In one embodiment, liquid component utilizes for example tubular bowl centrifuge simultaneously in centrifugal process, remove continuously in the successive mode of feeding.In another embodiment, tubular bowl centrifuge can mode in batches be used.In another embodiment, liquid component is removed from red blood cell fraction after separating red blood cell fraction and liquid component by abandon liquid component from red blood cell fraction.

The centrifuge 27 that red blood cell fraction relates among the figure arrives vessel 28.Red blood cell fraction is suspended in the suitable solution of vessel 28 (for example, diafiltration buffer).Suitable isosmotic solution is known in the art, comprises that solution as having the citrate/saline of a pH and infiltration molar concentration, do not catch the cell membrane of Red blood corpuscle in this solution, has substituted the liquid part of whole blood.Preferred isosmotic solution has neutral pH and waits osmol concentration, between 285-315mOsm.An example of suitable solution is to wait to ooze citric acid/salt buffer (sodium citrate hydrate-6.0g/L, sodium chloride-8.0g/L).Or the embodiment of choosing in, red blood cell fraction can resuspending in suitable arbitrarily isosmotic solution, for example in 5% the glucosan.Red blood cell fraction can about 20 to about 200g/L concentration resuspending.

In one embodiment, red blood cell fraction is suspended in the volume of isosmotic solution, so that has recovered to obtain the initial volume of the whole blood of red blood cell fraction.Blood cell component with resuspending is called " blood solution " later on.

Blood solution maintains in suitable temperature and the vessel 28.Preferably, between about 4 ℃ and about 5 ℃ of the temperature range kept of blood solution.The temperature of blood solution is by at circulation suitable culture medium such as ethylene glycol in vessel 28, keeps by bottle 30 and vessel 28.By bottle 30 recirculation culture medium is by line 32, bin 34, and pump 36,38 and cryoprobe or frozen units 40 are kept.

Then, filtering blood solution, thereby purification Red blood corpuscle.Preferably, blood solution is to come filtering by diafiltration.In one embodiment, diafiltration be by instruct blood solution from vessel 28 to line 42 and pump 44 carry out to diafiltration module 46.Diafiltration module 46 comprises inlet 48, is detained outlet 50 and permeant outlet 52.Film 54 has separated the retentate part 56 of diafiltration module 46 from the infiltration component 58 from diafiltration module 46.Preferably, film 54 has a permeation limit, and scope is between about 5um at about 0.01um.In one embodiment, blood solution is passed diafiltration, has a permeation limit, and scope is between about 2.0um at 0.2um.Or the suitable percolation filter of choosing comprises microporous membrane, and hole size will be from littler basically blood solution component separating RBC, as 0.1um to the filter of 0.5um (for example, 0.2um empty fibrous filters, Microgon Krosflo II micro-filtration post, Laguna Hills, CA).In an especially preferred embodiment, to have a permeation limit scope be to about 2um about 0.1 to film 54.

The part of the liquid component of the blood solution in the diafiltration module 46 is passed through film 54 from being detained part 56 to permeate portion 58, thereby purification is detained the Red blood corpuscle of part 56.The composition of blood solution such as blood plasma, or obviously littler than RBC composition forms permeate by the diafiltration wall in diameter.

Get back to the Red blood corpuscle that vessel 28 relate to the purification of blood solution by retentate outlet 50 and line 60.The Red blood corpuscle that can collect purification to line 64 by valve 62 is used for further processing.The membrane permeation liquid component contains any residual liquid part of whole blood (for example, blood plasma or serum) and diafiltration buffer.The liquid component that sees through film 54 may relate to from the permeate portion 58 of diafiltration module 46 to be collected by 66 lines with from vessel 68.The recirculation of the blood solution by vessel 28 and diafiltration module 46 can online 42 or line 60 in the sampling of sampling place (not shown).

Preferably, when diafiltration blood solution washing Red blood corpuscle, a liquid relates to from vessel 70 to line 72 as isosmotic solution, and the blood solution in the vessel 28 has been diluted the concentration of blood solution.In one embodiment, blood solution is diluted to a concentration, and scope is in the suspension concentration of about 25% to about 75% initial Red blood corpuscle, and this percent is to volume.Then, the concentration in infiltration process can reduce back volume original concentration or higher.Usually, liquid is added in the Red blood corpuscle that suspends, the method for removing the liquid of at least a portion then is called " cell washing ".Preferably, isosmotic solution comprises solion, or aqueous solution.Suitable isosmotic solution as mentioned above.One or the choosing embodiment in, blood is by the dilution and the concentration step washing of a series of orders (or opposite sequence), wherein blood solution is to add at least one isosmotic solution dilution, and is spissated by flow filter, thereby forms the blood solution of dialysis.

The Red blood corpuscle of blood solution is washing as mentioned above usually, and residual extracellular plasma proteins from be retained in red blood cell fraction such as serum albumin or antibody (for example, immunoglobulin (IgG)) come the separate red hemocyte.The result is that the amount of the amount (comprising the permeable plasma proteins of film) of the micro-filtration membrane permeation kind in the blood solution has reduced.

When the level of blood plasma that has polluted Red blood corpuscle or serum proteins has reduced substantially, it has been generally acknowledged that cell washing is (usually to be present in the red blood cell fraction before washing at least about 90% blood plasma or serum proteins) completely.Other washings can be further from RBCs plasma proteins outside the isolated cell.For example, the fibrosis of going with isosmotic solution of 6 volumes may be enough to remove IgG at least about 99% from blood solution.

Method of the present invention has reduced the existence of the potential membrane contaminant of the manufacture process that can slow down.For example, if in the infiltrative film surface accumulation with 0.1 to 5um, little cellulosic molecule may be a problem, and so possible polluted membrane filter paper is blind hole.The narrow scope that pollutant may be problems is 0.2 to 0.4um.But shown in embodiment 4, cellulose does not illustrate whole potential membrane contaminants separately.Shown in embodiment 4, in 2 in 3 experiments, go Fibrotic whole blood to have time greater than 100 minutes cell washing process.But, go fibrosis blood red blood cell fraction that produce and that isotonic buffer solution, be diluted to initial volume to have 45 minutes or still less cell washing time from equal volume.

In addition, go fibrosis can cause some hemolysis.Cracked Red blood corpuscle, leukocyte or platelet can attach on the filter paper.Be sure of, from liquid component the whole blood of fractionated with separate red blood cell fraction and removed the part of tangible such potential pollutant, thereby the time normalization that diafiltration is needed, or " washing " Red blood corpuscle purification they be used to make the blood substituent.

For the Red blood corpuscle from purification prepares methemoglobinemia blood substituent, the Red blood corpuscle of the purification of the blood solution of having washed can further be processed and separate the hyperglobulinemia molecule.The blood solution of a washing that obtains can be used for from blood cell and hematoblastic washed blood solution as the method by the red blood cell of centrifugalize.Be appreciated that from the common known method in this area of other blood cell component separate red hemocytees be utilizable.For example, one embodiment of the invention are by settlement separate Red blood corpuscle, and wherein isolating method is not caught the obvious amount of RBC, as are less than the cell membrane of about 5% RBC, separate red hemocyte from other blood constituents then.

Behind the separate red hemocyte, dissolving RBC has caused generation hyperglobulinemia (Hb) solution the composition of the red blood cell fraction of having washed from other.Dissolved method comprises mechanical lysis, chemolysis, and hypotonic or isoosmotic dissolving or other known dissolving methods, transportation and release oxygen discharge hyperglobulinemia under the situation of the ability of not damaging Hb.

After dissolving, then bigger cell debris is removed in the ultrafiltration of dissolved Red blood corpuscle phase, as molecular weight at about 100,000 daltonian protein.Then, hyperglobulinemia separates from the non-Hb composition of filtrate.

The method of ultrafiltration and by merging in full in this citation in the United States Patent (USP) 5,691,452 with this paper narration is being arranged in addition from the method for non-Hb component separating Hb by pH gradient and chromatography.

Preferably, figure oxidation Hb eluate forms deoxygenated Hb solution before polymerization, further is processed into the carrier of oxygen based on hyperglobulinemia.In a preferred embodiment, the basic deoxygenated Hb of deoxygenated, and do not have obviously to reduce the Hb transportation in the Hb eluate and discharge the ability of oxygen, as what will in the formation of oxidation hyperglobulinemia (met Hb), take place.Perhaps, can be by comprising N-ethyl-L-cysteine (NAC) with being selected from, cysteine, the Reducing agent of the group of two sodium sulfate or ascorbic acid can deoxygenated hemoglobin solution.The suitable method of deoxygenated has been narrated the suitable method of deoxygenated in the U.S. Patent number of submitting July 7 nineteen ninety-five 5,895,810, this full patent texts is by merging and this paper in this citation.

Deoxygenated hemoglobin solution can further be processed into the hyperglobulinemia based on the carrier of oxygen.As defined herein, " based on the carrier of oxygen of hyperglobulinemia " is to be applicable to the people, mammal, with other vertebrate compositionss based on hyperglobulinemia, can transport and shift oxygen in the organ and tissue of life, at least can keep enough endovascular carcinous pressures, wherein hyperglobulinemia separates from Red blood corpuscle." vertebrates " comprises the people, or other any in blood circulation, utilize blood transfer oxygen to the tissue in vertebrates.

" stable polymerization hyperglobulinemia " as defined herein, is based on the composition of the compositions of the carrier of oxygen, do not increase or reduce the distribution of molecular weight substantially, and/or about 2 years of the high content of hemoglobin in the storage process in period of suitable storage temperature or more.Store 1 year or longer suitable storage temperature between about 0 ℃ and about 40 ℃.Preferred storage temperature range is between 0 ℃ to about 25 ℃.

Suitable low-oxygen environment, or the environment of basic anaerobic was defined as in bimestrial storage at least period, preferably at least one year, or more preferably the storage period in months 2 years and the amount of gathering based on the carrier-bound oxygen of sample of hyperglobulinemia at least, this will cause based on metahemoglobin concentration in the carrier of oxygen of metahemoglobin at least about 15% weight.The amount of gathering of oxygen comprises based on the primary oxygen content of the carrier of oxygen of hyperglobulinemia and the packing except entering the oxygen that obtains based on the oxygen seepage in the packing of the carrier of oxygen of hyperglobulinemia.

By this method, reclaim up to hemoglobin polymerization from RBC, blood solution, RBC and hyperglobulinemia all are maintained at is enough to reduce growth of microorganism, or reduce under the condition of biological load, as maintain and be lower than about 20 ℃ and under the condition of the temperature more than 0 ℃.Preferably, temperature maintenance is at about 15 ℃ or still less.More preferably, temperature maintenance is at 10 ± 2 ℃.

In this method, the component portion based on the method for the carrier of oxygen of hyperglobulinemia of preparation stable polymerization is to carry out sufficient sanitized to produce aseptic product.In particular at USP XXII, the 71st part during the aseptic united states drug that the 1483-1488 page or leaf provides requires, defines as this area " aseptic ".In addition, the part of the composition that contacts with the stream of this method, normally Fibrotic, or come separately with streamlined material nonreactive or that pollute this method.Such material can comprise rustless steel and other steel alloys, as Hasteloy.

In one embodiment, intramolecular crosslinked Hb has caused polymerization.The blended amount of sulfhydryl compound and deoxidation Hb be high enough to can improve the crosslinked of intramolecular Hb in the polymerization process and enough be low to moderate can not obvious reduction because the Hb molecule of high ionic strength intermolecular crosslinked.Usually, (what be about 0.25mole between the deoxidation Hb of about 5mole SH) is oxidation-stabilized needed for the sulfur hydrogen functional group of about 1mole.

Perhaps, before polymerization oxidation stabilized deoxy Hb, in polymer reactor, add the water of appropriate amount.In one embodiment, the suitable water yield is to cause producing when adding oxidation-stabilized deoxidation Hb in polymer reactor the solution of the concentration of concentration about 10 to about 100g/lHb.Preferably, water is oxygen depletion.

The temperature of oxidation-stabilized deoxidation Hb solution is the temperature that rises to when contacting with cross-linking agent the polymerization the best that is oxidation-stabilized deoxidation Hb in polymer reactor.Usually, the temperature of oxidation-stabilized deoxidation Hb is about 25 to about 45 ℃, preferably in the polymerization process about 41 to about 43 ℃.The example of the acceptable thermal transfer method of heated polymerizable reactor is the hot system in bottle, is meant that conducting crosses the hot ethylene glycol of bottle and heat.

Then, oxidation-stabilized deoxidation Hb is contacted with suitable cross-linking agent, the temperature of contact is enough to the solution of polymerization oxidation stabilized deoxy Hb at the about 2 hours polymeric hyperglobulinemias of interim formation (poly-(Hb)) when about 6 hours.The cross-linking agent of appropriate amount is the polymer that allows intramolecular crosslinked stable Hb and allow the intermolecular Hb of being cross-linked to form, thereby strengthens the amount of endovascular delay.Usually, the amount of suitable cross-linking agent is that the molar ratio of cross-linking agent and Hb surpasses about 2: 1 amount.Preferably, the molar ratio of cross-linking agent and Hb is between about 20: 1 to 40: 1.

The example of suitable cross-linking agent comprises the proteinic poly functional reagent with crosslinked Hb; as glutaraldehyde; the succinyl dialdehyde; the activated form of polyoxyethylene and glucosan; Alpha-hydroxy acetaldehyde; as glycerol acetaldehyde; the amino caproyl of N maleic amide-6--(2 '-nitro; 4 ' sulphuric acid)-phenylester; m-maleic amide benzoic acid-N-hydroxyl succinyl ester; succinimido 4-(N-maleic amide methyl)-cyclohexyl-1-carboxylic acid; thiosuccimide 4-(N-maleic amide methyl) cyclohexane extraction-1-carboxylic acid; m-maleic amide benzoic acid-N-hydroxyl-succinimide ester; m-maleic amide benzoic acid-N-hydroxy thiosuccinimide ester; N-butanimide (4-iodo ethyl) amino benzoic Acid; thiosuccimide (4-iodo ethyl) amino benzoic Acid; butanimide 4-(p-Malaysia imines phenyl) butanoic acid; thiosuccimide 4-(p-Malaysia imines phenyl) butanoic acid; 1-ethyl-3-(3-dimethylaminopropyl) carboxyl diimine hydrochloric acid, N, N '-phenyl two Malaysia imines; with belong to the diimine class; ethyl two nitrine classes, or aryl two halogens and other chemical compound.

If the essential part of Hb molecule (at least about 50%) is chemically combined in poly-(Hb), so poly-(Hb) is defined as and has tangible intramolecular crosslinking.

In preferred embodiments, glutaraldehyde is as cross-linking agent.Usually, utilize about 10 glutaraldehydes among the oxidation-stabilized deoxidation Hb of every kg to about 70 grams.More preferably, add glutaraldehyde adds about 29-31g in the oxidation-stabilized deoxidation Hb of every kg glutaraldehyde 5 hours period.

When the cross-linking agent that utilizes was not aldehyde, poly-(Hb) that forms usually was stable poly-(Hb).When the cross-linking agent that utilizes was aldehyde, poly-(Hb) that forms usually is normally unsettled to form more stable key up to the more unsettled key that has reduced in poly-(Hb) with suitable reduction reagent mix.The example of suitable Reducing agent comprises sodium borate, cyanogen sodium borate, sodium dithionite, Trimethylamine, t-butylamine, boric acid morpholine, boric acid pyridine.Be increased to about 75 to about 85g/l by ultrafiltration or concentrated poly-(Hb) solution up to the concentration of poly-(Hb) solution.For example, suitable ultrafiltration is 30,000 dalton filter (for example, Millipore  Helicon ^TMCat#CDUF050LT; Amicon  Cat#540430, Bedford, MA).

Then, the pH of poly-(Hb) solution adjusts to the alkaline pH scope, the protection Reducing agent, the formation of protection hydrogen, can be in reduction process subsequently degeneration Hb.Usually, will gather (Hb) purification and remove the hyperglobulinemia of non-polymericization.This can finish by diafiltration and hydroxyapatite chromatography (referring to for example, the United States Patent (USP) 5,691,453 that submit July 7 nineteen ninety-five, this full patent texts is by merging and this paper in this citation).After pH adjusts, at least one Reducing agent, preferably dobell's solution adds polymerization procedure.Then, the pH of poly-(Hb) that can stable storing and electrolyte be to the physiological level of the carrier of oxygen on the hyperglobulinemia basis that forms stable polymerization, and method is by with the diafiltration solution diafiltration with suitable pH and physiological electrolyte level stable poly-(Hb).

Gone through the proper method of the carrier of oxygen on crosslinked hyperglobulinemia and protection hyperglobulinemia basis in the laid-open U.S. Patents 5,691,452 on November 25th, 1997, this patent is by merging in full and this paper in this citation.

The vertebrates that can accept the carrier of oxygen on the hyperglobulinemia basis that method of the present invention forms comprises mammal, as the people, and non-human primate, Canis familiaris L., cat, rat, horse, or sheep.In addition, the vertebrates that can accept the carrier of oxygen on described hyperglobulinemia basis comprises fetus (neonate vertebrates), postnatal vertebrates, or connatae vertebrates.

The carrier of oxygen based on hyperglobulinemia of the present invention can arrive vertebrate blood circulation by the carrier of oxygen on direct and/or indirect injection hyperglobulinemia basis, is administered in the blood circulation by one or more injecting methods.The example of the method for direct injection comprises intravascular injection, as intravenous and intra-arterial injection, and intracardiac injection.The example of the method for indirect injection comprises peritoneal injection, subcutaneous injection, so that will be transported to blood circulation or the side by joint or conduit is injected into bone marrow by lymphsystem based on the carrier of oxygen of hyperglobulinemia.Preferably, the carrier of oxygen on hyperglobulinemia basis is an intravenous administration.

Pending vertebrates can be before the carrier of oxygen on input hyperglobulinemia basis, in the process, and/or is later on normal volemic, over-drastic volemic, or low volemic.The carrier of oxygen based on hyperglobulinemia can be directly by entering blood circulation as the method for top-loaded with by the method that exchanges.

Carrier of oxygen administration remedially based on hyperglobulinemia, so that comprise anemia in many different reasons, shock and or by the part of blood circulation in RBC stream reduce hypoxic organizing in addition in the vertebrates that causes, based on the carrier of oxygen of hyperglobulinemia prophylactically administration prevent the oxygen depletion in vertebrates, organized, this may be that RBC flows to tissue or by reduction possible in the vertebrate blood circulation or expectation.Therapeutic ground or prophylactically treat the obstacle of part tremulous pulse or intercept other discussion of the administration of the hypoxic hyperglobulinemia that causes from microcirculatory part, wherein the dosage of Li Yonging is the United States Patent (USP) of submitting March 23 nineteen ninety-five 5,854, provide in 209, this patent is by merging in full and this paper in this citation.

Usually, suitable dosage or be the amount of concentration between about 0.1 to about 10gHb/dl that causes total hyperglobulinemia in the vertebrates blood when containing at blood plasma based on the combination of the dosage of the carrier of oxygen of hyperglobulinemia, remedy a large amount of blood loss if desired, then need more amount.

The present invention will further and particularly be narrated in the following embodiments.

Embodiment

The laboratory scale experiment of embodiment 1-

Reference diagram, in the container that anticoagulant (EDTA) arranged, reclaim whole bovine blood, and 2,600rpm (1,200XG), at 4 ℃, whole blood is being separated in the centrifuge 27 (Beckman J2-21 utilizes the JA-10 centrifuge) of heavy phase (red blood cell fraction, or cell component) and light phase (liquid component) centrifugal 30 minutes.The volume of the beginning of blood is 200ml.Separately red blood cell fraction and liquid component, each phase of processing in laboratory scale cell washing system.Direct observation of anion.Red blood cell fraction is instructed recirculation vessel 28, and with etc. ooze citric acid/salt buffer (sodium citrate hydrate, 6.0g/L, sodium chloride 8g/L) and be diluted to its initial volume.The recirculation vessel are by at the recirculation bottle, and the suitable culture medium of recirculation in 30 remains in the suitable temperature of recirculation vessel.By filter unit, 46 (Microgon Minikros sampler) separation of blood solution becomes penetrant and retentate to carry out the separating of Red blood corpuscle (for example, blood solution) of dilution.In a cylinder progressively, reclaim penetrant.Directly retentate is revert in the recirculation vessel by line 60.By the pressure input pressing tube (0-30PSI) of line 42 and outlet pressure syringe (0-15PSI) detected pressures of line 60.Ooze citric acid/salt buffer with waiting, in the system of bench scale the washed cell composition up to, obtain the membrane permeate (2 retentate volumes) of 400ml, data are summarised in table 1.

Table 1

Sample	The time of the penetrant of recovery 400ML (minute: second)
		Red blood cell fraction	34:58
Liquid component	52:20

As seeing in the table 1, liquid component contains the micro-filtration membrane contaminant, because the processing of liquid component is slower than the processing of red blood cell fraction.

The laboratory scale experiment of embodiment 2-II

In the stainless steel container of the aseptic process that contains the sodium citrate anticoagulant, reclaim bovine blood, and second centrifuge type, (NewBrunswick Scientific company, Edison NJ) carry out centrifugal production red blood cell fraction of batch and liquid component to CEPA pipe intestinal centrifuge.In two configurations of feeding, carry out centrifugal.In a configuration, pump into food with a peripteral pump, and in second configuration, utilize siphon to add blood.Then, the red blood cell fraction that obtains from each configuration is diluted to the initial volume of bovine blood, produces the blood solution of oozing citric acid/salt buffer such as to contain.In contrast, there is not the whole blood of centrifugal Citrated to be included as the sample that separates.Sample is washed up to the penetrant that obtains three retentate volumes (about 600ml) in the washing instrument of bench scale.Data are summarised in table 2.

Table 2

Experimental design	The centrifugal blood carrying method	Reclaim the time of the penetrant of 600ML
			Centrifuge #1	All post pumps	36:56
Centrifuge #2	Siphon	38:08
			The citric acid contrast	Do not have centrifugal	1:20:53

As seeing in the table 2, cause from red blood cell fraction, removing the centrifugal speed that increases cell washing of liquid component.

The experiment of embodiment 3-pilot-scale

From three animals, merge blood, and with anticoagulant as processing as described in the embodiment 2.(Northvale, NJ) blood of the merging of centrifugal treating has produced red blood cell fraction and liquid component in SA-1 or the SB-7 centrifuge, or directly washing in contrast at Westfalia.Utilize the washing system washing red blood cell fraction of pilot-scale.

(Watson-Marlow pump, Wilmington MA), carry out the separation of Red blood corpuscle by filter to utilize pump.By behind the filter, carry out the separation of Red blood corpuscle by the recirculation vessel, and with etc. ooze citric acid/salt buffer and dilute.The volume of recirculation vessel is 9.6 liters.The recirculation vessel are remained under the suitable temperature by the recirculation bottle recycled ethylene glycol around the recirculation vessel.(Waukesha, Delavan WI) carry out red blood cell fraction/buffer solution mixture separation red blood cell fraction/buffer solution mixture by filter (Microgon) and become penetrant and retentate to utilize pump.In the recovery of permeate container of floor scale, permeate.Retentate is directly got back in the recirculation vessel.Utilize food pressure syringe and retentate pressure syringe detected pressures.Three diafiltration volumes cells altogether by keeping.Data are summarised in the table 3.

Table 3

Sample	Process time (minute)
		The whole blood contrast of citric acid (1)	285
The whole blood contrast of citric acid (2)	＞400
		Red blood cell fraction WestfaliaSA-1 (1)	88
Red blood cell fraction WestfaliaSA-1 (2)	72
		Red blood cell fraction WestfaliaSB-7 (1)	134
Red blood cell fraction WestfaliaSB-7 (2)	141

Can see as table 3, come the red blood cell fraction of the centrifugal blood in comfortable SA-1 or the SB-7 centrifuge to greatly reduce the process time of comparing with the whole blood of citric acid.When not wishing binding isotherm, when comparing with SB-7, when centrifugal, reduce greatly by the cell washing time in the SA-1 centrifuge for whole blood, because SB-7 has caused the obvious degradation of cell, its substrate can cause that the microstrainer film pollutes.

Embodiment 4-goes fibrosis to there being and not having the effect of the time of centrifugal process

To merge from about 4 liters blood of two milch cows, go fibrosis by mechanical agitation.Blood contains the hyperglobulinemia of the 400g that has an appointment.The fibrosis blood that goes that obtains contains the hyperglobulinemia of 12g/L.To remove the fibrosis centrifugal blood, the red blood cell fraction of the blood plasma residue of the 5-10% that obtains having an appointment, or not centrifugal.Go the fibrosis red blood cell fraction and remove the whole blood of fibrosis as washing as described in the embodiment 3, in addition, reclaimed 5 diafiltration volumes.The volume of the beginning of two samples is 7 liters.

Table 4

Sample	The time of process (minute)
		The centrifugal fibrosis blood (1) that goes	37
Go the whole blood of fibrosis-contrast (1)	28
		The centrifugal fibrosis blood (2) that goes	45
Go the whole blood of fibrosis-contrast (2)	150
		The centrifugal fibrosis blood (3) that goes	25
Go the whole blood of fibrosis-contrast (3)	135

In first experiment, as shown in table 4, it is very fast going the fibrosis red blood cell fraction and going the cell washing speed of the whole blood of fibrosis, the centrifugal time that can not improve process, but, in experiment 2 and 3, the speed of cell washing relatively is for going Fibrotic red blood cell fraction improved, proving that membrane contaminant is not by going fibrosis to remove with removing Fibrotic whole blood.So, the centrifugal cell washing efficient that has improved whole blood and removed Fibrotic blood.

Equivalent

Person of skill in the art will appreciate that, maybe can utilize the approach that is no more than normal experiment to determine many equivalents of specific embodiment of the present invention as described herein.These and every other such equivalent all plan to be included in the following claim.

Claims (24)

1. the method for a purification Red blood corpuscle comprises step:

A) divide the Fibrotic whole blood of leaving away, thereby form red blood cell fraction and liquid component; With

B) thus the diafiltration red blood cell fraction forms the Red blood corpuscle of purification.

2. the described method of claim 1, wherein whole blood is isolating by sedimentation Red blood corpuscle in whole blood.

3. the described method of claim 2, wherein the sedimentation of Red blood corpuscle obtains by centrifugal whole blood.

4. the method for claim 3, what wherein whole blood centrifugal caused that red blood cell fraction comprises substantially all is Red blood corpuscle.

5. the described method of claim 1, wherein whole blood be by with whole blood with at 10 * G to 12, the G power contact in the scope between 000 * G and fractionated.

6. the described method of claim 1, wherein liquid component is by abandoning after step (a), removing from red blood cell fraction.

7. the described method of claim 1, wherein liquid component utilizes that separately liquid component and red blood cell fraction are removed simultaneously from red blood cell fraction.

8. the described method of claim 1, wherein whole blood mechanically goes fibrosis.

9. the described method of claim 1, wherein whole blood is handled with anticoagulant.

10. the described method of claim 9, wherein anticoagulant is selected from and comprises sodium citrate, heparin, the group of ethylenediaminetetraacetic acid (EDTA) and Disodium oxalate..

11. the described method of claim 10, wherein anticoagulant is a sodium citrate.

12. the described method of claim 10, wherein anticoagulant is a heparin.

13. the described method of claim 1 comprises the step of the Red blood corpuscle that dissolves purification in addition.

14. the described method of claim 13, wherein the Red blood corpuscle of purification is a mechanical lysis.

15. the described method of claim 13, wherein dissolving such as the Red blood corpuscle of purification with oozing.

16. the described method of claim 1, wherein red blood cell fraction comprises the most leukocyte and the platelet of whole blood.

17. the described method of claim 1, wherein red blood cell fraction is with having infiltrative diafiltration, and the scope of film is that 0.1um is between the 5um.

18. the described method of claim 1, wherein whole blood is a whole bovine blood.

19. the method for the hemolysis thing of a purification that is formed for hyperglobulinemia blood substituent comprises step:

A) separately remove Fibrotic whole blood, thereby form red blood cell fraction and liquid component;

B) diafiltration red blood cell fraction, thereby the Red blood corpuscle of formation purification; With

C) Red blood corpuscle of dissolving purification, thereby the solute of the Red blood corpuscle of formation purification.

20. the described method of claim 19, wherein to be that machinery goes Fibrotic for whole blood.

21. the described method of claim 19, wherein whole blood comes fractionated by centrifugal.

22. the described method of claim 19, wherein the Red blood corpuscle of purification is a mechanical lysis.

23. the described method of claim 19, wherein whole blood is a whole bovine blood.

24. the method for the hemolysis thing of a purification that is formed for hyperglobulinemia blood substituent comprises step:

A) separately go Fibrotic whole bovine blood by centrifugal, thereby form red blood cell fraction and liquid component;

B) diafiltration red blood cell fraction, thereby the Red blood corpuscle of formation purification; With

C) Red blood corpuscle of mechanical lysis purification, thereby the solute of the Red blood corpuscle of formation purification.

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