CA2049275A1 - Purification of sf hemoglobin - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Hemoglobin substantially free of leukocyte residues and substantially stromal free is prepared by a process using a leukocyte filter and a delipid filter, followed by ultrafiltration through a membrane filter, thereby avoiding the use of cumbersome centrifugation processes and the use of harmful solvents as extractants.

Description

2~9~7.' This invention relates to hemoglobin preparation, and more particularly to methods of preparing substantially pure, stromal free hemoglobin suitable for use in prepara-tion of blood substitutes.

Hemoglobin in its natural form is a non-cross-linked protein constituent of red blood cells (erythrocytes), possessing in solution the fundamental property of reversible oxygen binding. Thus it functions in the body as an oxygen transporter, delivering oxygen from the lungs to other parts of the body where it is required.

There is a continuing need, and frequent short-age, of whole blood for administration to patients during surgical procedures. Because of the difficulties in obtaining, storing, typing and administering whole blood, research and development directed towards providing an acceptable blood substitute has been pursued vigorously over the past 20 years or so. To date, however, only limited success has been achieved.

Because an acceptable blood substitute should have oxygen binding and release characteristics approximat-ing that of natural blood, hemoglobin presents itself as a natural candidate on which to base a blood substitute.
Indeed, much effort has been devoted to the development of blood substitutes based on hemoglobin, as reflected in the patent and scientific literature. Hemoglobin is, however, a relatively complicated macromolecule, consisting of a tetramer of two pairs of sub-units, ~ sub-units and B sub-units. Each sub-unit has a globin polypeptide chain capable of adopting various conformations, and a heme group. The molecular weight of the tetramer is about 64,000, and it is necessary to ensure that, in a blood substitute, the tetramer does not dissociate into its sub-units, since these are of too low a molecular weight to avoid excretion from the kidneys.

The principal source of hemoglobin is, of course, natural blood, where it is found in the red blood cells in association with other compounds such as phospholipids, cholesterol and other minor proteins. Before using it as a basis of a clinically acceptable blood substitute, hemoglobin should be obtained in as pure form as possible, or at least in a form in which it is substantially free from biologically harmful impurities present in a condition which the impurities can harm the patient. Extraction of hemoglobin from whole blood, and purification thereof, in an economically satisfactory manner for use on a production scale, presents difficult problems.

U.S. patent 4.001.200 Bonsen et al, and the related patents 4,001,401; 4,053,590 and 4,061,736, all of which have substantially identical disclosures, stress the importance of obtaining stromal free hemoglobin as a basis for a blood substitute. Stroma is essentially the cellular debris from the erythrocytes, obtained after lysing the cells, mainly comprising proteins, lipids and phospholipids derived from the cell components and the cell walls. In the Bonsen et al process, the red blood cells obtained from centrifugation of whole blood are lysed in cold aqueous medium. Then the red blood cell suspension is shaken, and cold toluene is added to it. The mixture is shaken, then left to stand for 24 - 72 hours to produce a three phase mixture. The lower, clear red layer is isolated and centrifuged at 40,000 - 50,000 g for at least 60 minutes at 4 - 6C. Then the upper clear supernatant is separated and filtered through a diatomaceous earth filter. This filtra-tion is reported to remove any traces of stroma. Residual low molecular weight salts and metabolites are removed by dialysis against an appropriate buffer, e.g. using hollow cellulosic fihres as the semi-permeable membrane.

2 ~ i~ (3 ), There are serious drawbacks to this process when it is considered from a production point of view, as opposed to a laboratory procedure. During the process, there is created a three-phase system, consisting of two liquid phases and one solid phase, which presents a diffi-cult processing problem. A discontinuous, small-batch approach has to be adopted.

Also, the process of Bonsen et al involves the use of toluene, as a solvent for removing lipids and phospholipids. Whilst toluene is a convenient and efficient solvent for laboratory use, since it readily and efficiently extracts the lipids and phospholipids and forms clean phase separations from water, its use is undesirable on a production scale. It poses a fire hazard. Its fumes create an unacceptable working environment unless strict and expensive precautions are taken in its handling.
Noreover, any residual contamination of the hemoglobin product with toluene can present physiological problems to the patient.

It is an ob~ect of the present invention to provide a novel process for purification of hemoglobin.

It is a further object to provide such a process which is capable of operation on a production scale, to yield hemoglobin and hemoglobin solutions of exceptionally high purity.

It is a further object of the in~vention to provide a novel, substantially pure hemoglobin product.

In the process according to this invention, centrifugation is avoided, and the use of toluene and similar organic solvents is avoided. The process of the present invention involves the sequential steps of passage 3.`' . ' of a suspension of red blood cells through a coarse filter, to remove debris and fibrinogen clots, then passage of the suspension through a leukocyte filter, to remove white blood cells (leukocytes) and their residues. Then the red blood cells in the suspension are washed and lysed, passed through a delipid filter to remove lipids and stroma, and then concentrated by ultrafiltration through a membrane filter.

The use of a leukocyte filter at the stage described above enables centrifugation to be eliminated.
Accordingly, the process of the present invention is readily adaptable to scale up to production size, to run on a continuous basis to treat a large batch of red blood cell suspension to obtain substantially pure hemoglobin there-from. The use of the delipid filter allows one to avoid the use of toluene or any other similarly objectionable solvent, thereby eliminating a solvent removal step from the process. Substantially reduced quantities of biologi-cally contaminated waste liquids are formed, as compared with processes involving toluene extraction and centrifugation, thereby reducing disposal problems.

In addition the above practical advantages of the process of the invention, however, it has been found that a superior product, in terms of bio-acceptability of the purified hemoglobin produced is obtained by this invention.
As mentioned above, the product is totally free from any aromatic organic solvent residues. It is also substan-tially totally free, or indeed even totally free, of leukocyte residues. It has not previously been recognized, in prior art descriptions of blood substitutes based on hemoglobin, that it is very important to remove leukocyte residues. If they are present to any significant extent, they will cause human leukocyte antigen reactions in the patient to whom the blood substitute is administered. One 2 ~ L~ 9 ,~, 7 :`

such reaction is the generation of Complement Factor 3A
(C3A) from Complement Factor 3, a protein constituent of the natural immune system. When C3A is freed from C3, it acts as an anaphylactic toxin. By the practice of the present invention, involving the use of a leukocyte filter at the appropriate stage, a product is produced which is so low in leukocyte residues, that the risk of causing human leukocyte antigen reactions is reduced to a minimal level.

The single accompanying Figure of drawings is a diagrammatic process flow sheet of the presently most preferred embodiment of the present invention.

The starting material for the process of the present invention is a suspension of red blood cells obtained from whole blood, by conventional processes.
Thus, the whole blood is treated with an anti-coagulant~
centrifuged, and the plasma withdrawn. This yields a red blood cell concentrate containing all the cellular elements of blood along with some residual plasma. The concentrate is mixed to form a suspension with an aqueous electrolyte, of a concentration suitable to be substantially isotonic and at a pH of about 7 - 7.2, preferably, to reduce the risk of premature lysis of the cells. The suspension is formed by gentle agitation, so that cell rupture is reduced to a minimum. It is preferred in the process of the present invention to maintain the integrity of the red blood cells in which the hemoglobin is contained, whilst white cell residues and other debris are removed. The entire process is suitably conducted at a temperature of from about 2 - 10C, to avoid premature cell rupturing, and under aseptic conditions. Preferably, the red blood cell suspension is mixed with the aqueous electrolyte at about a 1:1 volume ratio.

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Next, the suspension of red blood cells and other materials is passed through a coarse filter, at which point some cellular debris and fibrinogen clots are removed.
Suitably, the coarse filter is of polymeric material, e.g.
polyester, polysulfone etc., or of stainless steel, and suitably has a passage cutoff size of about 50 microns.
The material which is collected on the filter is biological waste material, for careful disposal in an environmentally acceptable mannerO Passing through the coarse filter is a cellular suspension of white blood cells, red blood cells and platelets, along with some residual plasma. This material is next passed to the leukocyte filter. Without the preliminary use of the coarse filter, the leukocyte filter may clog and be unsatisfactory for use on a continu-ous or semi-continuous basis.

Leukocyte filters for use in the process of the present invention are available on the market, and have previously been used to filter whole blood at a patient's bedside as the blood is being administered to the patient by a nurse. They normally comprise charged polyester filter material through which the suspension is passed. On the basis of electrokinetic absorption and depth filtra-tion, the leukocyte filter material selectively retains white blood cells as opposed to red blood cells, according to the different electrostatic charge characteristics of the two types of cell. At least a three log reduction in the quantity of white blood cells is achieved by a single passage through the leukocyte filter. Inevitably, a small quantity of red blood cells are filtered out as well, but not to any practically significant extent. It is possible to recover any such retained red blood cells from the filter material by elution, but the amounts of red blood cells retained by the filters are usually sufficiently small that such a process step is not necessary. The filter material, after use, is discarded. Passage of the suspension through a leukocyte filter can be repeated if traces of leukocyte residues are detectable therein, for an additional three log (1000 fold) reduction therein.

One example of a commercially available leukocyte filter useful in the process of the present invention is that manufactured by Asahi Medical Co. Ltd. and sold under the trade-mark "SEPACELL" by the Fenwal Division, Baxter Health Care Corporation. Another is that marketed by Pall Biomedical Products Corporation under the trade-marks "PALL
RC 50" and "PALL RC 100" leukocyte removal filters. Both are sold as recommended for use for blood transfusion at bedside.

From the outlet of the leukocyte filter, there is obtained a suspension of red blood cells and residual plasma proteins in aqueous electrolyte, and this suspension is now preferably subjected to one or more washing steps, to remove all extra-cellular contaminants including plasma.
This is preferably done by washing the suspension in a tangential flow microfiltration device of 0.1 - 0.45 micron size with isotonic sodium chloride solution. It is pre-ferred to conduct at least three separate, sequential such washes, using in each case a 3:1 v/v ratio of sodium chloride solution to red blood cell suspension. The sodium chloride stabilizes the proteins and maintains them in solution. A substantially pure red blood cell suspension has now been obtained.

Next, the red blood cells are lysed, to free the hemoglobin and other contents thereon. As is well known in the art, there are a wide variety of methods for lysing red blood cells. In the present process, it is preferred to conduct a gradual decrease of the ionic strength and hence osmotic pressure of the solution, by slow addition of a solution of 5-20 mmolar phosphate at pH 7. Too rapid a '0~'~'7;"

lysis of the cell causes it to break into very small components, which can create complications in subsequent steps of separation of cellular components.

During the lysing procedure, the mixture is subjected to a tangential flow micro filter, suitably of 0.1 - 0.45 micron size, which are readily available, standard units. This separates the cell wall residues, "ghosts". Passing through the microfilter are the red cell intracellular proteins, along with some residual debris and some lipids. The steps of washing, lysing and microfilter-ing are suitably all combined in a single unit.

The filtrate from the micro filter is preferably fed directly to the delipid filter. Preferred forms of delipid filters are those containing fumed silica embedded in cellulose. Lipids, phospholipids and stroma particles are removed by such delipid filters by affinity mechanisms.
Also, the delipid filter will remove some if not all of the contaminating pyrogens present in the suspension at this stage.

Delipid filters commonly require a stabilization period during start up of a process, so that it is pre-ferred to arrange for a recycle line in the process of the present invention, whereby initial quantities of filtrate from the lipid filter are recycled through the filter until stabilization occurs.

The filtrate from the delipid filter, once it has stabilized, is dilute stromal free hemoglobin suspension.
It is nex~ subjected to ultrafiltration, whereby soluble molecules from the suspension below a certain size and molecular weight are removed in the filtrate, and soluble and insoluble molecules above the cut off molecular weight are retained. The ultrafiltration medium is preferably an ~ a ~

asymmetric membrane with pores of a size sufficient to provide a cut-off of about 30,000 molecular weight, i.e. a membrane which will retain 95% of all molecules of molecu-lar weight of 30,000 or higher. This preferred choice of 30,000 molecular weight cut-off is made on the basis of the likely presence in the material of carbonic anhydrase, one of the intracellular proteins present in red blood cells along with hemoglobin, in small but significant amounts.
By this choice of molecular weight cut-off, a substantial amount of carbonic anhydrase can be separated from the hemoglobin, it being understood that even initially carbon anhydrase is present in only very minor amounts, compared with the amount of hemoglobin. The ultrafiltration is performed until the desired final hemoglobin concentration of 8 - 15% w/w is achieved.

The preferred embodiment of the process of the present invention next subjects the concentrated suspension of stroma free hemoglobin which has not passed through the ultrafilter to a micro pre-filtration to remove any aggre-gates which may have been formed during the ultrafiltration process. This suitably uses a 0.6 micron filter membrane.
Then it is subjected to a sterilizing micro filter, with a 0.2 micron size, to obtain the final, purified, hemoglobin product.

In the alternative, a step of ion exchange chromatography may be used, further to reduce traces of other proteins which may be present, for example carbonic anhydrase. When chromatography is used, it is preferred to arrange for hemoglobin to pass through the column, whilst other proteins are retained, using conditions based upon the isoelectric point of hemoglobin. Such a chromatography step can be used in addition to the micro pre-filter and the sterilizing micro-filtration.

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The resulting product is one which contains no detectable leukocyte residues, and no organic solvent contaminants. Whilst the possibility of the presence of microscopic amounts of other protein contaminants cannot be ruled out, the product produced by the process of the present invention can justifiably be termed substantially pure hemoglobin. The process is operable on a continuous basis for a given batch of red blood cell suspension, avoiding centrifugation of a three phase system.

With reference to the accompanying Figure, red blood cell concentrate RBC obtained in the normal way from whole blood is contained as a batch in vessel 10, and from there it is fed to a ~essel 12, where it is mixed with an equal volume of isotonic aqueous sodium chloride solution from line 14 and gently agitated at 4 C, to form a re-suspended dilute RBC suspension. This and all other steps in the process are conducted under aseptic conditions, using incoming solutions which have all passed through a sterilizing filter prior to use.

From the vessel 12, the dilute suspension passes to a coarse filter 14 which is a 50 micron cut-off polyester membrane, and removes the fibrinogen clots from the suspension. The clots so removed are waste material, for careful disposal. The temperature of the suspension is maintained at about 4C through this step.

Issuing from the coarse filter is a cellular suspension with residual plasma, and this is passed into the leukocyte filter 16, which is a SEPACELL filter, and serves to absorb the white blood cells preferentially over the red blood cells, resulting in a 1000 fold reduction in 2 ~ t~

the amount of white cell residues, in the product issuing from the leukocyte filter 16.

Next, the red blood cell suspension is washed three separate times with isotonic aqueous sodium chloride solution, again maintaining the temperature at 4C, as diagrammatically illustrated by inlet lines 18, 20 and 22 from saline storage facility 24. This is conducted in a single tangential flow filtration device, the red blood cell suspension being passed tangentially across the filter membrane, under pressure, with the saline on the same side of the membrane.

Next, 20 mmolar potassium phosphate solution is added to the suspension via line 26, and the mixture passes into a microfilter unit 28 in which lysis of the cells takes place, the ghosts being removed via line 30 and the resulting proteinaceous material and accompanying cell contents passing through the 0.1 - 0.45 micron microfilter membrane 32.

The lysed material next passes into the delipid filter 34, which contains fumed silica embedded in cellu-lose as the affinity removal medium, to which stroma particles, lipids and phospholipids adhere. During initial start-up, recycle line 36 is utilized to pass filtrate back through the delipid filter a second or third time, until the operation of the delipid filter has stabilized.

The output from the delipid filter is fed into a 30 K ultrafiltration unit 3>3, which removes from the solution, by passage through the filter membrane, molecular constituents of molecular weight about 30,000 or lower, to waste line 40, whilst higher molecular weight materials in the solution pass on to a 0.6 micro pre-filter 42 where aggregates formed during the ultrafiltration process are ,`' 0 '.~ ~ -' 7 ~`

removed, and then to a 0.2 micro sterilizing filter 44 for final treatment. The product issuing from sterilizing microfilter 44 via outlet line 46 is substantially pure stromal free hemoglobin product, in aqueous solution.

Claims (11)

1. A process of preparing hemoglobin which is sub-stantially stromal free and substantially totally free of leukocyte residues, which comprises:

passing a suspension of whole red blood cells, substantially free of solid debris and fibrinogen clots, through a leukocyte filter;

lysing the cells in the filtered suspension to release hemoglobin and other cellular contents therefrom into the suspension;

passing the suspension so obtained through a delipid filter, to remove lipids and stroma therefrom;

and concentrating the hemoglobin by ultrafiltration through a membrane filter.

2. The process of claim 1, including the additional step of washing the filtrate from the leukocyte filter with a substantially isotonic solution, prior to lysing the cells, to remove from the suspension extracellular contaminants.

3. The process of claim 2, wherein the washing is conducted by tangential flow filtration, using isotonic saline.

4. The process of claim 2, including the additional step of subjecting the suspension containing the lysed cells, prior to passage through the delipid filter, to microfiltration to remove therefrom cell wall residues.

5. The process of claim 4, wherein the microfiltra-tion is through a microfilter of size 0.1 - 0.45 microns.

6. The process of claim 4, wherein the delipid filter comprises fumed silica embedded in cellulose.

7. The process of claim 4, wherein the concentration of the filtrate from the delipid filter by ultrafiltration utilizes an ultrafiltration medium which is an asymmetric membrane with pores of a size sufficient to provide a cut-off of about 30,000 daltons molecular weight.

8. The process of claim 7, wherein the concentrated suspension of hemoglobin from the ultrafiltration is subjected to micro pre-filtration to remove aggregates formed during ultrafiltration.

9. The process of claim 8, wherein said micro pre-filtration utilizes a 0.6 micron filter membrane.

10. The process of claim 8, wherein the micro pre-filtration is followed by step of sterilizing microfiltra-tion, utilizing a 0.2 micron size filter.

11. The process of claim 7, wherein the concentrated suspension from the ultrafiltration is subjected to ion exchange chromatography.