HU218159B - Displacement chromatography process for production of purified hemoglobin product - Google Patents

Abstract

The invention relates to the chromatographic purification of hemoglobin. The method for purifying hemoglobin from the raw solution containing the admixture of proteinaceous origin provides for the use of ion-exchange chromatography. The raw solution containing 0.1-20% hemoglobin. At the first stage, anion-exchanging column is used when the admixtures are more acidic than hemoglobin, or cation-exchanging column is used when the admixtures are more alkaline than hemoglobin. The continuous flow of the solution results first in the complete saturation of the column with hemoglobin and the admixtures with high affinity to the column, then the column is overloaded following the substitution of hemoglobin. At the second stage, the ion-exchanger alternative to that used at the first stage is employed. Again, upon the complete saturation and overloading of the column, hemoglobin is substituted.

Description

The invention relates to displacement chromatography for the purification of hemoglobin, more particularly to the purification of a commercially available hemoglobin, whereby a solution having a hemoglobin content of at least 99% can be prepared.

The development of hemoglobin-based blood substitutes is an ongoing and constant need and recent findings suggest that hemoglobin obtained from animal blood cells, after appropriate modifications such as intramolecular cross-linking and some degree of polymerization, offers very good hopes of being used as blood substitutes. However, with the advancements, the requirements for hemoglobin purity are constantly increasing. At one time, it was believed that hemoglobin was required to be stromal-free, and this could be achieved by gentle washing and lysis of red blood cells and filtration of the resulting lysate. Subsequently, it has been found that the presence of traces of certain impurities, such as phospholipids, also leads to certain specific reactions, such as vasoconstriction (vasoconstriction) in experimental animals. Even after the product had undergone certain diafiltration steps, the product still contained an unexpected amount of potentially harmful impurities such as erythrocytes, modified and various forms of hemoglobin, phospholipids and surface antigens.

One of the most urgent tasks in the field of blood substitutes, and more particularly in the field of hemoglobin-based oxygen carriers, is to develop a cost-effective method for the purification of commercially available hemoglobin in an efficient and economical manner. This should be competitive with other potential blood volume expanders and oxygen carriers used as blood substitutes.

While the times required for dissolution and analysis are very important in analytical separation procedures, the critical parameters for preparative chromatography in industrial and semi-industrial cases are as follows:

amount of material separated per unit time for a specific purity (power), and

economics of the process, such as column size, recirculation and reuse of media, buffers, equipment, components and reagents, etc.

There are no known purification procedures for hemoglobins that meet the criteria of a cost-effective production process.

A hemoglobin-based blood substitute should be a single controlled form of hemoglobin or, if multiple forms are present, a well-controlled composition of known hemoglobin information. Accordingly, an effective hemoglobin purification procedure should be capable of separating one hemoglobin from another and the desired hemoglobin from contaminating components of red blood cells such as erythrocyte enzymes, proteins, phospholipids, and antigens.

Chromatographic techniques have already been used to purify hemoglobin solutions. USP 4,925,474 discloses an affinity chromatography method for purifying hemoglobin in a column in which a ligand with a preferred chemical binding affinity for the hemoglobin DPG site is bound to the stationary phase of the column.

Ion exchange chromatography was also used to purify hemoglobin. The principles of ion exchange chromatography are well known. A solution of a mixture of different materials is added to a suitably prepared ion exchange column. Each of the substances in the mixture has different affinities for the groups of chemical reagents in the column. By changing the column parameters, such as the pH of the solution, it is possible to selectively bind or elute each substance from the column so that the individual substances in the mixture can be separated. The use of this method to purify proteins such as hemoglobin is not economically attractive unless it is used only for small amounts or for analytical work. If hemoglobin is to be manufactured on an industrial scale, such as for use as an oxygen carrier for resuscitation (resuscitation) fluid (blood substitute), the conventional method is impractical. The amount of hemoglobin that is to be absorbed and subsequently eluted from the chromatography column is so large that the column size that satisfies this requirement is already too large and too expensive.

Christensen et al., Christensen et al., J. Biochem. Phys. 17, (1988), 143-154] discloses a chromatographic method for purifying human hemoglobin. The method described is a standard ion exchange chromatography method which is not suitable for industrial scale production levels.

Winslow and Chapman (Winslow and Chapman, Piloi-Scale Preparation of Hemoglobin Solutions, Methods in Enzymology, 231, 3, 1994) disclose the production of stromal hemoglobin (SFH), high purity hemoglobin A0, and crosslinked hemoglobin as starting material outdated blood is used. SHF is produced by filtration. Hemoglobin Ao is obtained from SHF by continuous chromatography using a strong anion exchange medium and an ionic gradient.

Both Christensen et al. And Winslow et al. Performed the described work at the Letterman Army Institute for Forward Research (LAIR), now known as the Walter Reed Army Institute of Research.

USP 5,084,588 discloses a standard anion and cation exchange chromatography procedure for the separation and purification of hemoglobin. Three approaches to anion-exchange chromatography are described in this description:

(a) hemoglobin (Hb) is bound at an elevated pH and eluted with a decreasing pH gradient or a lower pH gradient;

b) the Hb is bound at a higher pH and eluted with a salt gradient at low ionic strength;

HU 218 159 Β

c) The hemoglobin is applied under pH conditions where it is not bound to the anion exchanger but passes through the column without any residue, while the impurities (more strongly acidic impurities) are bound to the column.

Solutions (a) and (b) above have been described in detail, but are not favorable due to the low load capacity required for large scale production to properly release hemoglobin products. This loading capacity is usually only 20-30 mg / ml, which means the need for a large and expensive column on an industrial scale to purify hemoglobin. For example, providing a single hemoglobin-based oxygen delivery vehicle (HBOC) at 50 g would require a column of 1.5 to 2.5 liters.

Although the solution in (c) above seems to be a more practical solution in practice, it has been found that the chromatographic properties of unmodified hemoglobin Ao from normal human adults and certain major contaminants (e.g. HbAlc, a chemically modified human hemoglobin) do not differ in practice. to a satisfactory degree.

There are similar limitations to purification of mammalian hemoglobin by cation exchange chromatography.

U.S. Pat. No. 5,083,588 discloses bovine hemoglobin solutions in which more than 99.9% of the proteins present are bovine hemoglobin but do not disclose the identity of the protein constituents.

It is an object of the present invention to provide a new chromatographic method for purifying hemoglobin.

It is a further object of the present invention to provide a chromatographic process suitable for large scale production and economical preparation of aqueous solutions of hemoglobin of greater than 99% purity starting from contaminated aqueous solutions containing at least 60% hemoglobin.

More particularly, it is an object of the present invention to provide a commercially available hemoglobin product containing at least 99% hemoglobin or crosslinked hemoglobin in aqueous solution, which is pure, suitable for use as a blood substitute, and has in vivo properties not previously disclosed.

Accordingly, the present invention provides a process for purifying hemoglobin comprising subjecting an aqueous solution of a pre-selected hemoglobin (Hb) having an Hb content of 60-99% to two-step ion exchange chromatography. One of the two steps is anion exchange chromatography and the other is cation exchange chromatography. The two types of ion exchange chromatography can be carried out in any order. The crude solution containing the pre-selected hemoglobin (e.g., HbAo) is subjected to a first chromatographic step, such as anion exchange chromatography, under conditions such that initially all of the constituents in the solution are adsorbed onto the solid phase of the chromatography column. This requires a relatively high pH in the case of anion exchange. The conditions are further selected to provide a very high effective loading on the column using a low ionic strength medium, which results in all available sites in the solid chromatography medium being occupied by the substances in the feed solution. The eluate obtained in this step is free of protein products. The displacement of HbAo and impurities with lower affinity to the column under selected conditions is then ensured by adding further aqueous media as described above. The conditions thus achieved, hereinafter referred to as the overload condition, result in impurities having a higher affinity for the column displace Hb, and impurities with a lower affinity for the column are eluted from the column. If this first chromatographic separation step is an anion exchange chromatography step, impurities that are more acidic than the selected Hb material will adsorb to the solid phase of the chromatographic column and thus be separated from the selected Hb material, which in turn appears in the eluate. with more basic pollutants. If this is the first step cation exchange chromatography, the more basic impurities are separated by having higher affinity for the column and the more acidic impurities and selected hemoglobin material appear in the eluate.

Then, in a second chromatographic step, a second ion exchange chromatography is performed on the partially purified hemoglobin material, which is in the eluate from the first column under different pH conditions. If the eluate from the first column contains hemoglobin and basic impurities, the second chromatographic step is a cation exchange chromatography step or vice versa. The solution containing these materials is again fed to the second column until a saturation load is reached and then overloaded with column conditions. At this overload, the pre-selected hemoglobin material is eluted by displacement from the column, which is produced by a higher affinity for impurities under the conditions used, so that the hemoglobin material is from the second column in very high purity. Thus, the process of the invention is generally referred to as "two-step, self-displacement chromatography".

As a result of the process according to the invention, the Hb material eluted from the column is of extremely pure form and generally of greater than 99% purity with commercially favorable yield. Impurities with a more acidic property than the selected Hb material will remain bound to the resin in one column, while impurities with a more basic Hb substance will bind to the resin in the other column. The columns can be easily regenerated by known cleaning and regeneration procedures.

It is a very important feature of the process according to the invention for the efficient operation, especially in the case of large-scale production, that the pollutants bind selectively and in large amounts to the

EN 218 159 Β and it is possible to isolate the Hb material in the eluate.

When the process of the present invention is used to purify human hemoglobin Ao, new human hemoglobin products are obtained whose composition and properties have not been known with respect to human hemoglobin Ao, and are particularly advantageous when used as blood substitutes. These properties include the substantially complete absence of viral particles, particularly viral particles encapsulated in lipid; and a substantially complete lack of vasoactivity when administered in vivo; and a complete lack of endothelial cell toxicity in vitro incubation, as evidenced by reduced lactate dehydrogenase release compared to similar doses of other hemoglobin formulations. In addition, the general properties required for hemoglobins used as blood substitutes, such as the absence of endotoxins and other antipyretics, are also superior to similar hemoglobin formulations of the present invention.

Figure 1 is a diagrammatic representation of an initial anion exchange portion of the process of the invention.

Figure 2 is a diagrammatic representation of a second cationic portion of the process of the invention.

Figure 3 shows the analytical anion exchange chromatogram of Examples 2 and 3.

Figure 4 is an analytical anion exchange chromatogram of Example 4.

Figure 5 is a graphical representation of the results of Example 8.

Figure 6 shows isoelectric focusing lines for analysis of the product of Example 13.

Figure 7 shows the results of staining and blotting experiments of the product of Example 13.

Figure 8 is a graphical representation of the results of the product of Example 14.

9 and 9A. FIG. 3B is a graphical representation of the results of the product of Example 15.

Figure 10 is a graphical representation of the results of the product of Example 16.

By-pass chromatography or displacement elution are common concepts in the separation of proteins. This is one of three methods for obtaining a sample from a chromatographic column. When a particular protein is separated from a solution of a mixture of proteins, the higher affinity protein displaces the lower affinity protein on the matrix, so that the proteins appear on the column in descending order from inlet to outlet. When a protein displaces another protein, a sharp demarcation line between the different substances is rarely formed. Thus, the process is not recommended for the preparation of high purity single protein solutions such as hemoglobin. If a separate, non-proteinaceous displacement agent is selected, the result may be that the chromatographic matrix is bound to very high affinity substances that are very difficult to regenerate for repeated use.

The process of the present invention is particularly economically applicable to purification of adult human hemoglobin Ao (HbAo) and, for the sake of simplicity, hereinafter, the process will be described using it. However, the process of the present invention is by no means limited to purifying HbAo, but may also be used to purify additional hemoglobin substances such as cross-linked hemoglobin derivatives, selected bovine hemoglobin variants and other hemoglobin variants such as fetal hemoglobin, sickle cell hemoglobin and the like.

The process of the present invention is particularly advantageous economically for the commercial purification of HbAo hemoglobin for the production of blood substitutes. Highly effective column loading capacity can be achieved. As a result, relatively small columns can be used to purify relatively large volumes of contaminated hemoglobin solutions. The process may be performed to purify intermittently or continuously given hemoglobin solutions obtained from red blood cells by known methods and generally containing about 80% hemoglobin with a wide variety of impurities, whether proteinaceous or non-proteinaceous. Further, the contaminated hemoglobin solution itself is used as a displacement medium. In this way, the use of a separate displacement medium can be avoided and the associated inconvenience, complications and costs can be avoided.

The process for the preparation of purified HbAo solutions according to the invention generally starts from normal adult human red blood cells. These are pooled, optionally filtered to remove leukocytes. The cells are washed to remove serum proteins and lysed to extract hemoglobin and other cellular components. The lysate was filtered to remove red blood cell membranes, and the resulting hemoglobin solution was diafiltered and concentrated. The contaminated hemoglobin extract thus obtained is then treated with carbon monoxide to form COHb complexes and then heated to disinfect, thereby inactivating the viral impurities in solution. The resulting solution is then centrifuged to collect any remaining residues and cellular debris. The material thus obtained is then ready for use in the self-displacement process of the invention.

In a preferred embodiment of the process of the invention, the first chromatographic step is an anion exchange step and the second is a cation exchange step. Preferably, the first anion exchange step is carried out at a high pH such as pH 7-10, preferably pH 8.5-9 with low ionic strength, i.e. low conductivity. The conductivity is preferably less than 3 mS (milliseconds), more preferably less than 1 mS. Such conditions are provided by a substantially saline-free buffer. The desired HbAo material

HU218 159 Β is first bonded to the column material, together with the other materials. As the column is fed with contaminated HbAo solution, substances that exhibit higher affinity for the matrix than HbAo, generally more acidic impurities, gradually displace HbAo and more basic impurities from the column. Ultimately, the column is overloaded by displacing all HbAo and the more basic impurities, leaving only the acidic impurities on the column. In most cases, this load is well above the values recommended by the column manufacturers. Chromatographic separation of the more acidic impurities from HbAo was thus ensured at this step.

The contaminated hemoglobin solution is fed at a low linear rate not greater than 10 cm / min and preferably around 1 cm / min. This allows the column to be kinetically balanced. The concentration of the feed material is not critical but is suitably between 0.1 and 20%, preferably between 2 and 7%.

The achievement of overload conditions is demonstrated by analyzing the column effluent, if no acidic contamination is detected in the eluate composition or if it is present only in traces, the maximum practical loading of the column is achieved.

Figure 1 is a diagrammatic representation of the anion exchange portion of the process of the invention. The ion exchange column containing -N ⁺ R ₃ groups (typically anion exchange resin, but by way of example only) and shown in Figure 1A is fed with a solution of a mixture of acidic impurities (denoted by an asterisk), HbAo. (denoted by ellipse) and more basic impurities (denoted by squares). At the capacity load (Figure IB), substantially all of the active NR ₃ groups are bonded to one of the materials in the mixture by electrostatic charge.

To provide the overload conditions, another identical solution was fed to the capacitance column (Fig. IC). At this point, all the basic impurities (squares) and a certain amount of HbAo (substance elliptical) are displaced by the more acidic components (stars), which have a higher affinity for the column under the given conditions. When an additional solution is added to the overloaded column (Figure ID), the acidic substances will displace substantially all of the HbAo from the column, since they have a higher affinity for the matrix under the given conditions. Thus, the effluent material is substantially free of detectable acidic impurities. However, the completely pure HbAo solution thus obtained contains some more basic impurities.

In the second step of the preferred process of the present invention, a cation exchange column is used and the material flowing from the first column is fed to it under similar overload conditions. The desired HbAo and other undesirable basic impurities are bound by overload. The partially purified hemoglobin solution from the first column was fed to the second column. Relatively low feed rates are used repeatedly at a rate of 10 cm / min or less, preferably around 1 cm / min, to achieve a kinetic balance between solid and liquid phases. The pH of the eluent from the first column is preferably adjusted to a value of 5-9, more preferably 6.5-8.5, particularly preferably 7-8, before applying the eluent to the cation exchange column. A low ionic strength solution is used, i.e. a conductivity value of less than 3 mS, preferably less than 1 mS. A value of about 2.5 mS is used only at very low feed rates and very high nutrient dilution. The overload conditions are then repeated. Higher affinity proteinaceous impurities, i.e. more HbAo-based substances, displace HbAo from the column and the HbAo thus eluted is of exceptionally pure form.

Figure 2 is a diagrammatic representation, similarly to Figure 1, of a process on a cation exchange column which contains SO ₃ groups on the cation exchange column. The eluent from the first column, after having been adjusted to the appropriate pH, is fed to the column, whereby, at step A, the capacity load achieves electrostatic binding of both the HbAo (ellipse) and the basic impurities (squares) to the column. Although the matrix contains sulfone groups as shown in the figure, other groups such as carboxyl groups can be used in a similar manner. Subsequently, the eluent solution was added to the column to provide overload conditions (Fig. 2B), whereby the more basic impurity displaces HbAo from the column due to its greater affinity for the column. When additional solution is applied to the overloaded column (Figure 2C), HbAo of almost 100% purity is displaced and recovered from the column. The higher the column load, the greater the percentage of purified HbAo that can be recovered from the column. The eluent is assayed for the presence of basic substances (squares). At this point no further HbAo displacement was observed and maximum load was determined.

The more basic impurities according to the invention are contaminants which can be eluted from the column at a higher level of ρΗ than is necessary for the elution of HbAo. Conversely, the term acidic impurities refers to impurities that can be eluted from the column at a lower pH than is necessary to elute HbAo. In a preferred embodiment of the process of the invention, the anion-exchange chromatography is performed first, the more basic impurities are eluted before HbAo and the more acidic impurities are after HbAo. Depending on the ion exchange medium used in the chromatography, different elution profiles can be observed with slight changes in the order of elution.

For both the first and second columns, the rate of addition of the contaminated hemoglobin solution should be chosen according to the concentration of the contaminated hemoglobin solution. Linear flow rates of about 1 cm / min or less are optimal for hemoglobin solutions having a concentration of 2 to 7%.

A flow rate of about 159 Β cm / min can be used for dilute solutions, such as when the Hb content is less than 1%. In ion-exchange chromatography, dissolution at low loading capacities is generally insensitive to linear flow rate, but kinetic equilibrium during chromatography in the process of the invention is highly dependent on the flow rate. However, the concentration of the feed is not critical at any of the steps.

In the displacement chromatography method of the invention, first using anion exchange chromatography to remove the more acidic components and then a cationic chromatography to remove the more basic components, HbAo is swallowed with very high yield and exceptionally high purity, which is substantially free of For example, serum proteins and membrane proteins, and the process is commercially applicable. The purity as determined by analytical anion exchange chromatography is greater than 99%, generally greater than 99.5%. The actual capacity of the steps is at least one order of magnitude greater than the capacity values observed for normal chromatography. In addition to the removal of proteinaceous impurities, the process of the present invention also allows substantially complete removal of substantially modified and other undesirable forms of hemoglobins to yield a substantially pure (> 99.5%) HbAo product. Further, as will be apparent from the following detailed description, complete removal of the active or inactivated enveloped viral particles is also provided.

Comparing the dimensional advantages of the process of the invention with standard ion-exchange adsorption / elution chromatography methods, the following

Table 1 shows. The data given in the column "Self-displacement" are the following data obtained in the process according to the invention, the data in the column "Adsorption / elution" are the data mentioned above by Winslow et al.

Table 1

Operational parameters used for purification of hemoglobin Ao using adsorption / elution chromatography or self-displacement chromatography

self Containment Adsorption / clúció Volume column 5 and 31 120 L column Capacity 200-300 mg / ml 15 mg / ml g Hb, entered 1234 1818 % yield 81 55 g Hb, won 1000 1000 Puffer vasculature 132 L 900 L

From the above data, it is apparent that the self-displacement chromatographic method of the invention can be run faster with advantageously smaller columns. Lower pressures and ambient temperatures can be used. The effective loading capacity, which is 10-20 times higher than the capacities of known chromatographic methods, allows the use of a relatively small column volume. This. leading to a reduction in the amount of water required, thus reducing the amount of wastewater and the amount of buffer material, each of which significantly increases the economics of the process.

The purified hemoglobin solution coming from the cation exchange column can be diafiltered into a buffer of choice and the desired HbAo concentration adjusted.

The choice of ion exchange gels to be used as the stationary phase is by no means critical to the process of the invention and is within the skill of the art. Any known commercially available gels may be used, provided that they can be converted to a material that operates in the ion exchange mode under the affinity conditions selected for the materials to be separated. Common carriers for such ion exchange agents are macroporous, macrophages and non-porous, divinylbenzene crosslinked polystyrene or polymethacrylate, polyalkyleneamine, cellulose, dextran, agarose, silica, ceramic, glass, alumina, etc. Commercially available carriers include, for example, POROS media, Fractogel, HyperD, Dowex, Amberlite, Doulite, Bio-Rex, Chelex, Sephadex, Sepharose, Toyopearl, Macro-prep, Bio-protocol, Accell, Mono types, etc. . Functional groups attached to these supports to provide ion exchange include, for example, sulfonate, sulfopropyl, carboxymethyl, carboxylate, phospho, quatemeric amine, quatemeric aminoethyl, polyethyleneimine, trimethylamino- ethyl, diethylaminoethyl, dimethylaminoethyl, aspertamide and the like.

The process of the present invention provides a solution of HbAo of at least 99.5% purity, which is unexpectedly free from active or inactivated viral particles, in particular lipid-enveloped viruses such as HIV, herpes simplex virus (HSV) or bovine diarrhea virus. in many cases, a model of the human hepatitis C virus. These are the most common viruses that make up the most serious and dangerous contaminants in the blood. This is a very important additional advantage of the process according to the invention, which was unpredictable and unpredictable before the process was tested and the results analyzed. Although a preferred embodiment of the method of the invention comprises a pasteurization step for inactivating viruses prior to purification, as mentioned above, removal of viral particles as part of the method of the invention is particularly preferred. The process of the invention removes all active viral particles that have survived the pasteurization from the blood substitute and also removes any remaining inactivated viral particles. All this without any further specific viral particle removal step and additional special reagents or other

EN 218 159 Β can be provided without dosing. Substantial complete removal of lipid-coated viruses is assured, and a significant reduction in the amount of other viruses is assured.

The hemoglobin products of the present invention have a higher purity than any of the previously described hemoglobin products as far as is known. Perhaps due to the exceptionally high purity or other factor that has not yet been fully explained, the products of the present invention have unexpectedly very beneficial properties not previously described for hemoglobin products, at least on industrial scale.

In particular, the HbAo products of the invention are free of any red blood cell membrane protein that can be detected by isoelectric focusing and immunoblot analysis. Trace amounts of human serum albumin (HSA), less than 4 pg, generally 2-4 pg HSA / 100 mg Hb, are detected by immunoabsorption. The only other detectable proteins that are sometimes present in the products of the present invention are fragments of the globulin and a globulin chains of hemoglobin, the amount of which does not exceed 1 pg / 100 mg of hemoglobin. Such an extremely high purity product of the invention exhibits unique and unexpected properties as described below. One of these is reduced or completely eliminated vasoconstrictor activity. Another such property is the lack of toxicity to cultured endothelial cells.

The high purity hemoglobins described above, both crosslinked and noncrosslinked, result in an increased blood pressure response when infused (Hess et al., Systemic & Pulmonary Hypertension After Resuscitation with Cell-Free Hemoglobin, 1993; Chang et al., Effect of Single Replacement). ..., BIOMAT, Art Cells and Immob., Biotech, 20 (2-4), 503-510, (1992)] Since the products were apparently of very high purity, according to current theories, the hemoglobin In particular, it was thought that hemoglobin is able to bind to nitric oxide, which is responsible for this effect. Nitric oxide, also called endothelial relaxation factor, is an effective vasodilator and is formed from endothelial cells Because hemoglobin molecules bind with high affinity to nitric oxide, It is currently accepted that intravenous infusion of hemoglobin solutions, particularly non-crosslinked hemoglobin solutions, binds this nitric oxide and induces a hypertensive response with vasoconstriction.

Our finding that the products of the invention are devoid of vasopressor activity in models of isovolemic change and hemorrhagic shock is completely unexpected and contradictory to the present theories and assumptions regarding the intrinsic properties of hemoglobin. The present invention suggests that it is not due to the intrinsic property of hemoglobin that it is responsible for the vasoconstrictor effect as previously described, but that this effect is due, at least in part, to the presence of extremely small amounts of hemoglobin impurities not previously known. completely removed, even though the references to earlier hemoglobins also mention extreme purity.

The advantage of the products according to the invention in that they do not have vasopressor activity is retained for the crosslinked and polymerized hemoglobin products obtained from the products of the invention, the preparation of which is described, for example, in U.S. Patent No. 5,532,352.

Furthermore, the products of the present invention exhibit a particularly high degree of toxicity to endothelial cells when administered in vitro compared to the previously described hemoglobins. Studies with previously described oxyhemoglobin solutions, including chromatographically purified hemoglobins, have shown cellular damage as indicated by significant increases in lactate dehydrogenase in endothelial cell culture media compared to the lymphocyte endothelial deac

Unexpectedly and to a significant extent, it has been found that the hemoglobin of the present invention does not mediate lactate dehydrogenase release. However, in contrast to the hemoglobin of the invention, significant levels of lactate dehydrogenase release were observed during incubation of stromal hemoglobin solutions with endothelial cells, which hemoglobin was purified by other chromatographic techniques.

It is generally accepted and in fact obvious that any blood substitute and any component or raw material used in a blood substitute should be of sufficient purity, i.e., free from harmful impurities. It has previously been found that such harmful impurities include cellular stroma, endotoxins and other pyrogens, non-hemoglobin proteins, phospholipids. Prior art discloses blood substitutes that are "substantially free" of one or more of these contaminants. However, the term "substantially free" is a relative and subjective term, the interpretation and limitations of which will depend on how the particular parameter is determined and which are described in the literature according to the state of the art. Improved purification and analytical techniques have been reported on blood substitutes and hemoglobin products of greater and greater purity. For example, there is a reference in the literature to a hemoglobin solution having a hemoglobin content of more than 99.9% in one form or another (see USP 5,084,588). It is contemplated by the present invention that even this purity is not sufficient to be used as a suitable blood substitute, since the nature of residual contamination can be a cause of vasoconstrictive effects and cellular toxicity.

HU 218 159 Β

Stromal hemoglobin is available from the Walter Reed Army Research Institute (WRAIR) and is generally accepted as the industry standard for purity; from this it can be shown that it contains a large number (> 10) of various impurity proteins, many of which are less than 0.01% of the total protein content. Although this is a very small amount, the fact that hemoglobin as a blood substitute is administered to patients in relatively large amounts and that it is harmful or non-harmful is determined by the total amount of impurities added and not by the concentration in any given mixture. One unit of blood substitute solution contains 25-50 g of hemoglobin, so 0.01% impurity means 2.5-5 mg of impurity. Of the very different proteins that are present, one or two can be very, very damaging in such amounts. The present invention provides a hemoglobin product having a non-hemoglobin protein content of less than 0.5%, preferably less than 0.1%, and presumably, but not necessarily, due to its lack of vasoconstrictor activity and no significant toxicity to endothelial cells. Thus, the present invention provides a process by which such hemoglobin solutions are commercially available.

Another very important advantage of the products according to the invention is the reduced fibroblast activity. The products of the present invention appear to contain significantly reduced amounts of substances that may promote blood coagulation compared to other stromal-free, previously known hemoglobin products.

The invention is further illustrated by the following non-limiting examples.

Example 1

Generation of crude Hb lysate Units (about 30 L) of packed red blood cells (RBCs) from the same blood type were pooled and diluted with 2410.9% saline. The resulting material is then filtered through a 20 µm and 8 µm polypropylene filter to remove residual leukocytes.

After filtering off the leukocytes RBC were washed with six volumes of 0.9% saline constant volume diafiltration, which used a hollow filter membranes of 3.5 m ^{1 2} 0.3 micron Sepracor polyethersulphone. The wash buffer was then replaced with 50 mM Tris lysis buffer and RBC was gradually lysed into 6 volumes of such lysis buffer.

The crude lysate is then collected and concentrated from about 2.5% total hemoglobin (Total Hemoglobin, THb) to about 9% THb using a 30K Molecular Weight Cut Off (MWCO) Millipore PTTK membrane. After concentration, the vessel is filled with CO gas to produce the COHb form of hemoglobin. The lysate was then pasteurized at 62 ± 2 ° C for 10 hours in a jacketed container, cooled and centrifuged. Further filtration was performed on Millipore 0.8 µm filters and the pasteurized lysate was diafiltered on another Millipore 30K PTTK membrane. The material was diafiltered with 5 mM Tris buffer at pH 8.9 until its conductivity was <0.3 mS at pH 8.9 ± 0.2. The material is then diluted to a concentration of 4.5-5% THb, which is suitable for the next chromatographic purification step.

Example 2

Self displacement chromatography on anion exchange resin

A 1 x 10 cm column was killed with PerSeptive Poros HQ-50 anion exchange resin, then washed with a column volume of 1 N NaCl solution and equilibrated with mmol of Tris buffer (pH 8.8). After equilibration, 50 mL of 3% (3 g / 100 mL) crude hemoglobin lysate (~ 85% HbAo) in 5 mM Tris buffer prepared as in Example 1 at pH 8.8 was added to the column at 0.78 mL / min ( 1 cm / min). Ultimately, this results in column overload, 200 mg solution / ml resin, while the manufacturer's loading capacity is 50 mg / ml. The eluent was collected. The column was then washed with 2 column volumes of 5 mM Tris buffer (pH 8.8) and the eluent was combined with the eluent flowing over the load. The column was then washed with 1 N NaCl to elute the retained proteins and discard this eluent. The column was regenerated with 3 column volumes of 1M NaCl / HCl and 4 column volumes of 1M NaOH. Hemoglobin eluate descending from the anion exchange column is analyzed with an analytical anion exchange resin using a pH gradient as discussed below. Examples are described in detail in Example 1. The assay determined that about 95-96% HbAo was obtained per column (see Figure 3) with a 90% yield per column added.

Example 3

Self displacement chromatography on anion exchange resin

A 1 x 10 cm column was filled with Merck Fractogel-TMAE resin and washed with 4 column volumes of 1 N NaCl solution and equilibrated with 5 mM Tris buffer (pH 8.8). After equilibration, 50 mL of 3% (3 g / 100 mL) crude hemoglobin lysate (~ 85% HbAo) in 5 mM Tris buffer, prepared as in Example 1, at pH 8.8 at 0.78 mL / min was added. 1 cm / min). Likewise, this eventually results in column overload. The eluent was collected. The column was then washed with 2 column volumes of 5 mM Tris buffer (pH 8.8) and this eluate was combined with the eluate collected during loading. The column was then washed with 1 N NaCl to elute the retained proteins and discarded this eluate. The column was regenerated with 3 column volumes of 0.5 N HCl and 4 column volumes of 1 M NaOH. Hemoglobin descending from the anion exchange column is analyzed by analytical anion exchange.

HU 218 159 Β column and pH gradient all. as described in Example 3, this value was determined to be 95-96% as shown in Figure 3, and the yield was 73% HbAo per column applied.

Example 4

Self displacement chromatography on cation exchange resin

A 1 x 10 cm column was filled with PerSeptive Poros HS-50 resin and washed with 4 volumes of 1N NaCl solution and equilibrated with 5 mM Tris pH 5.5. After equilibration, 120 ml of a 2% (2 g / 100 ml) anion exchange column purified hemoglobin solution (about 95% HbAo) in 5 mM Tris buffer, pH 7.5, prepared as described in Examples 2 and 3 was added. At a rate of 78 ml / min (1 cm / min). This addition essentially results in overload of the column, which is about five times the amount recommended by the manufacturer. The eluate was collected. The column was then washed with 2 column volumes of 5 mM Tris buffer (pH 7.5) and the eluate was combined with the eluate under load. The column was then washed with 1 N NaCl to elute residual proteins and discarded this eluate. The column was regenerated with 3 column volumes of 0.5 N HCl and 4 column volumes of 1 M NaOH. Hemoglobin eluate from the anion exchange resin was analyzed on an analytical anion exchange column using a pH gradient, determined to be> 99% HbAo as shown in Figure 4, and yield 90% based on HbAo applied to the column.

Example 5

The experiment investigates the effect of conductivity in self displacement chromatography on anion exchange resin (first step).

Effective displacement chromatography is critically dependent on the ionic strength of the buffer and / or sample as determined by the conductivity. The following experiment summarized in Table 2 was performed by adding the lysate obtained in Example 1 to the Poros HQ-50 anion exchange resin from PerSeptive Biosystems Inc. The column eluate was analyzed by analytical anion exchange chromatography. All fractions obtained in Experiment 1 were free of detectable acidic impurities, while the first fraction in Experiment 2 was contaminated with acidic material.

Table 2

Experiment Load capacity (Mg / ml) THb PH mS Column (length χ diameter) Buffer Rationing speed (Cm / min) acidic contamination removal First 200 3.0 8.8 0.2 / 0.4 10x1 tris 1 100% Second 200 2.8 8.8 2.85 10x1 tris 1 0%

Example 6

Yield versus Protein Load Overload of the column results in a higher yield of purified HbAo. The experimental results summarized in Table 3 below show that the very high overload of the 10 mm column filled with the cation-exchanging Porous HS-50 resin ensures that all bonding sites are occupied by the contaminants and allows a high yield of purified HbAo. In the first experiment, the overload was 13.5 times the manufacturer's recommended value (308 mg protein / ml). Overload in the second experiment was five times the manufacturer's recommended value. Higher overload leads to a significant increase in the amount of HbAo recovered.

Table 3

Workload (mg protein / ml) THb PH mS Column (length χ diameter) Buffer Feed rate (cm / min) basic contamination-free 308 3.1 7.5 0.5 9,5x1 tris 0.6 100% 88% recovery 216 2.9 7.4 0.6 9,5x1 tris 0.6 100% 77% recovery

Example 7

Comparison of Small and Large Columns The displacement chromatography method of the invention can be applied to both small and large columns. For the Poros HQ-50 anion exchange resin and the Poros HS-50 cation exchange resin, this comparison was made with 15 and 10 liter columns using 10 liter columns. The solution prepared in Example 1 was added to the Poros HQ-50 column. The eluate from the HQ-50 column was added to the HS-50 column. The following Table 4 summarizes the conditions and the results obtained with the Poros HQ-50 columns. Table 5 shows the results obtained with Poros HS-50 filled columns. The results show that at each step of the process, a volume of at least 10-15 liters can be switched to column size to achieve industrial size.

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Table 4

Load altogether Load (Mg / ml) THb pH Conductivity (mS) Column (length χ diameter) Buffer Rationing speed (Cm / min) Amount of acidic impurities removed (%) 1.6 g 210 5.6 8.9 0.5 10x1 tris 1 100% 3.2 kg 200 4.7 8.8 0.3 10x ^ 5 tris 1 100%

Table 5

Load altogether Load (Mg / ml) THb PH Conductivity (mS) Column (length χ diameter) Buffer Rationing speed (Cm / min) % Of more basic impurities removed (%) 2.4 kg 308 3.1 7.5 0.5 9.5 χ 1 tris 0.6 100% 1.7 kg 254 3.7 7.6 0.4 9,5x30 tris 0.6 100%

Example 8

Analysis of crude hemoglobin lysate and purified hemoglobin by isoelectric focusing (IEF)

Isoelectric focusing analysis (IEF) was performed using an agarose-based gel in which a stable pH gradient was introduced with commercially available ampholytes. 90% of the ampholytes used were between pH 5-8 and 10% were between pH 3.5-10. The final amount of agarose in the gel was 1.25% and the final amount of ampholytes was about 2%. The gels were heavily overloaded (1 mg protein per lane) to determine purity. Electrophoresis was performed at low current values to ensure that the proteins migrated in a uniform straight band. After steady state was reached, the voltage and current were briefly increased to further sharpen the bands. After electrophoresis, the proteins were fixed in agarose gel using solutions of trichloroacetic acid and sulfosalicylic acid. After the fixation step, the gel was stained with Coomassie-Blue to visualize protein bands. For scanning, the gels were air-dried and then the electrofocused bands containing crude hemoglobin lysate and purified hemoglobin lysate were detected using a laser densitometer. Figure 5 shows a comparison of IEF values for the hemoglobin lysate prepared in Example 1 and the HbAo purified according to the invention. Purification of the hemoglobin solutions was carried out on the Poros HQ-50 column as in Example 2 and then on

Example 4 was performed on a Poros HS-50 column.

Example 9

Immunoassay of crude hemoglobin lysate and purified hemoglobin

Immunostaining was used to determine the various impurities present in the crude hemoglobin lysate prepared in Example 1 and in the hemoglobin purified by the process of the invention. After electrofocusing the proteins on an agarose gel, the proteins were transferred to a nitrocellulose paper using capillary blotting (suction). The remaining free binding sites on the paper were blocked by incubating the spots with hydrolyzed fish gelatin solution. After incubation, the spots were washed with tris-buffered saline (TBS) and incubated in the presence of primary antibody. [Pri20 mer antibody is an antibody against which the sample is to be tested, for example, anti-human serum albumin (antiHSA) is used to determine HSA in hemoglobin solutions.] conjugated (e.g., horseradish peroxidase), which antibody is the primary antibody. After incubation with the second antibody, the stain was repeatedly washed with TBS buffer and a suitable substrate was added to the marker enzyme. A colored precipitate appears where the secondary antibody is bound to the primary antibody bound to its antigen, thus indicating the amount and position of the primary antigen on the antigen on the IEF gel.

The results obtained are summarized in the following Table 7, where "+ + + + +" indicates a strong signal, "+" indicates a barely detectable signal, and indicates no signal.

Table 6

Antibody used Crude HB lysate hemodialysis globin Human serum albumin +++++++++++ + ^a Red blood cell (RBC) +++++++ + ^b RBC membrane + + + + + - Human plasma +++++++++++ 3 ^C Carbonic anhydrase I +++ - spectrin + + + + - glycophorin ++ -

HSA present in the purified HbAo was ^using Λζ immunoblottokon HSA standards amount to about <5 pg / mg Hb (<50 ppm) was estimated ^b Trace amounts of impurities was 5.2 pl ~ antiRBC detectable cross-reaction with an antibody. Other researchers have also found similar protein in their purified Hb solutions (see Christensen et al., Supra), the anti-HSA antibody present in ^c

In HbAo, he detected the presence of HSA

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Example 10

Human Serum Albumin Immunodetection (HSA-ID)

Chromatography was used to determine the amount of HSA present in the hemoglobin solutions. The method utilized an antibody (anti-HSA) covalently bound to the column matrix. Once the sample is injected onto the column, all but the antibody (HSA) antigen passes through the column at the load / wash step. Using the elution buffer, the bound antigen (HSA) is eluted from the column and the antigen present in the sample is quantified based on the area of the peaks.

The immunodetection charge (ImmunoDetection *, ID) used in the experiment was from PerSeptive Biosystems. A dose rate of 2 ml / min was used for the experiment. The loading column (1.6 mm diameter χ 26 mm in length) was equilibrated with loading buffer (10 mmol phosphate buffer + 300 mmol NaCl, pH 7.2) and 50 μΐ of sample was injected. The loading buffer was also used to wash the column for 0.25 minutes, and then the elution buffer (3.5 minutes) was applied to elute the bound HSA on the column. The eluent was assayed with a Beckmandio Array Detector Kit at 220 nm and 414 nm. The column was then re-equilibrated with the loading buffer for the next injection. For the application of this method, the amount of HSA present in purified hemoglobin was found to be no greater than 0.0005% by weight of hemoglobin.

Example 11

Analytical anion exchange chromatography

Chromatographic analysis of the purity of the hemoglobin products obtained in Examples 2 and 4 was carried out as follows. The assay is based on the method reported by Huisman and Dozy (Huisman et al., Studies on Heterogeneity of Hemoglobins, IX - The Use of Tris-HCl Buffers in Hemoglobins, J. Chromatog., 19, 160-169). (1965)], whereby the sample is added to the anion exchange column at a pH such as pH 8.5 that all protein is bound to the column, and a gradient of pH 8.5-6.5 is applied to the protein. This chromatographic assay distinguishes very similar proteins (e.g., various hemoglobin variants) using an analytical anion exchange column 4.6 mm x 100 mm (PerSeptive Biosystems) The following buffers are used: A) 25 mM Tris + mmol bis-tris, pH 8.5 and B) 25 mmol tris-25 mmol bis-tris, pH 6.5. The assay was performed at a dose rate of 5 ml / min. After equilibrating the column with buffer B, 10 μΐ of sample (concentration 10 mg THb / ml) was injected onto the column and the gradient (100% buffer A) to 100% buffer B) was started at 25 column volumes. The eluent was monitored by UV detection at 280 nm to detect protein peaks. Figure 3 shows the chromatographic profile of the anion-exchange purified hemoglobin (product of Example 2) and Figure 4 shows the profile of purified HbAo (product of example 4) based on the analysis performed with this assay.

This method is a standard test procedure for checking the purity of hemoglobin solutions. The HbAo purified by the process of the present invention is free of any impurities that can be observed in stromal hemoglobins. The small shoulder at the HbAo peak occurs only occasionally and is probably due to the oxidized form of HbAo (MetHbAo). The purity of the product of the invention is routinely greater than 99.5% as determined by this analytical method.

Example 12

Virus Removal

The method of the invention has been investigated to determine how it removes viruses from raw hemoglobin.

For samples of crude lysates prepared as in Example 1 and partially purified hemoglobin from the anion exchange column, i.

The following viruses were added to the products of Examples 2 and 3 in active form in assay amounts:

Poliovirus Type 1,

Human Immunodeficiency (HIV),

Bovine Diarrhea Virus (a model for the human hepatitis virus),

Herpes Simplex Virus Type 1 (HSV-1).

An aliquot of the eluates from the appropriate columns were added to the assay cell system. In the case of Poliovirus, Herpes Virus and Bovine Diarrhea Virus, the active virus present interacts with the test cells and forms plaques. Known standard tests were performed. In the case of HIV, an aliquot was added to tissue culture. The amount of virus present was determined by measuring the change in cell line growth. This change is TCID ₅₀ (tissue culture infectious dose 50%). The results obtained are summarized in Table 7 below.

Table 7

Decrease in viral log by chromatography

Virus Poliovirus Type 1 Human immunodeficiency Bovine Diarrhea Virus herpes genome RNA RNA RNA RNA wrap no Yes Yes Yes test System Vero cells MT-4 cells Ebtr cells Vero cells

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Table 7 (continued)

Virus Poliovirus Type 1 Human Immunodcficicrcy Bovine Diarrhea Virus herpes Anionterhelés 1.4 χ 10 ⁷ pfu / ml 1.4 χ 10 ⁵ TCID _Sll / ml lJx10 ⁵ pfu / ml 5.3x10 µfu / ml Anioneluátum 8.5 χ 10 ⁶ pfu / ml <3 TCID ₅₀ ML <20 pfu / ml <3 pfu / ml Log reduction <1 > 2 > 4 > 6 Kationterhelés 3.1 χ 10 ⁷ pfu / ml 3.2χ 10 «TCID _S0 / ML 2.8 x 105 pfu / ml 2.0 χ 10 p pfu / ml Kationeluátum 7.5 χ 10 p pfu / ml 5.6 χ 10 ² TCID ₅ / ml 2.1 χ 10 ⁴ pfu / ml 4.1 x ^{10 3} pfu / ml Log reduction <1 > 3 > 1 > 3

Experimental results show a slight decrease in Poliovirus in either the anion exchange self-displacement or cation exchange self-exclusion steps of the present invention, but there is a noticeable reduction in viral load following HIV, HSV and Bovine Diarrhea viruses and lower anion exchange rates. decrease after cation exchange chromatography. This was completely unexpected, but represents a significant advantage in the use of the present invention.

Example 13 25

Purity test by isoelectric focusing and

Western blot analysis

The isoelectric focusing experiment (IEF) was performed by the method of Saravis and Zamcheck (Savavis and Zamcheck). 30

IEF gels were cast using agarose (1% final concentration) and a pH gradient was generated using ampholytes (Pharmacia Biotech Inc., Piscataway, NJ) such that 90% of the ampholytes were at pH 5-8 and 10%. % was in the pH range 3.5 to 10. Generally, 10 μ 35 of Hb solution at 100 mg / ml was added to the 35 IEF gels. After focusing the proteins, the gels were fixed with 10% trichloroacetic acid (TCA), dried, and stained with CoomassieBlue. Generally, the total protein load is 1 mg per 40 bands.

Isoelectric focusing analysis (IEF) was performed to compare the purity of SFH with the various purified HbAo materials of the present invention. 50 µg total Hb or 1 mg 45 total Hb were applied per lane. The results obtained are shown in Figure 6. Lane 20 shows stromal hemoglobin at 50 pg total Hb load, and lanes 22,24.26 and 28 correspond to purified Hb materials according to the invention at the same load value. Lane 30 shows 50 SFH at 1 mg total load and lanes 32, 34, 36 and 38 represent different HbAo samples at the same load. A sample of 50 pg shows that purified HbAo and SFH are of the same purity. However, at 1 mg total Hb / band load, various 55 red blood cell proteins (plasma proteins or membrane proteins) were observed in the SFH band, but were not present in the purified HbAo band. A very weak p1 band was observed around 5.2 in the purified HbAo band, probably due to the globin chain phage. The bands above the HbAo band in the 50 pg sample samples are likely to correspond to the higher positively charged MetHb materials generated during the pre-electron focus.

Following isoelectric focusing, Westemblot analysis was performed, which included antibody staining (immunostaining), which indicated the purity of the product of the invention. Thus, the pyoteins from the IEF gel were transferred to nitrocellulose paper by capillary blotting. The remaining free sites on the nitrocellulose paper were blocked by incubation of the spots with hydrolyzed fish gelatin (Hipure Liquid Gelatin, Norland Products Inc., New Brunswick, N.J.). The blots were then incubated with rabbit anti-human plasma antibody (Dako Corp., Santa Barbara, CA, lot 00) and diluted 1: 500 in tris buffer buffered saline. After overnight incubation, the spots were washed with tris-buffered saline (TBS) containing 1% fish gelatin and incubated with goat rabbit IgG-horseradish peroxidase (HRP) (1: 1000 dilution in TBS). After incubation for 1 hour, the stain was repeatedly washed with TBS containing 1% fish gelatin. The arithigenic antibody-HRP complex was then visualized with 4-chloro-1-naphthol (30 mg / 20 mL methanol) and hydrogen peroxide (50 pl in 100 mL TBS). Dark purple precipitate bands indicate the presence of antigens, particularly plasma proteins.

The following samples were focused on the IEF gel in parallel: stromal hemoglobin, Hb of the invention, Hb of the IEF, purified Hb from Waltzer Reed Army Institute (WRAIR) (purification as described in Winslow et al., Supra). and purified Hb from Sigma Corporation (purified as described in their catalog of diagnostic reagents, Hemoglobin Ao, Ref. H 0267). Half of the gels were stained with Coomassie-Blue and the other half was aspirated onto nitrocellulose paper. The spots were then stained for visualization of human plasma-human plasma antibody complexes.

The results obtained are shown in Figure 7. In the figure, lane 40 is the IEF Coomassie-Blue stained sample of stromal hemoglobin, and lane 42,44,46 are the corresponding lanes for the HbAo samples of the present invention and the WRAIR Hb and Sigma Hb samples. Lane 48 is the streak of Westst blot-free human plasma antibody,

The bands 50, 52, and 54 are the corresponding bands for the HbAo, WRAIR Hb, and Sigma Hb samples of the present invention.

Significant amounts of plasma protein were detected in SFH. In contrast, human serum albumin (HSA) is only detected in trace amounts in the band corresponding to the product of the invention. No other plasma protein was detected in the product of the invention. No HSA was detected in the WRAIR HbAo sample, but several other plasma proteins were detected below the HbAo bands. The Sigma HbAo standard sample contains a number of plasma contaminants, including significant amounts of HSA.

Example 14

Acute hypovolaemic study in alert rats

The assay was performed to detect the vasopressor activity of the product of the invention.

Sprague-Dawley rats were randomized into six groups and allowed to acclimate for 4 days, followed by chronic cannulation according to Tabata and Chang (Tabata and Chang, Artificial Organs 6, 213-214). (1982)]. Properly cannulated animals were dosed intravenously with 150 IU / kg heparin. An arterial cannula was attached to a Stratham pressure transducer to measure blood pressure and heart rate. These parameters were continuously recorded using a Grass polygraph writing device. After obtaining a constant baseline signal, fatal haemorrhagic shock was induced by Wiggers and 67% of the total blood volume was removed via the other arterial catheter at a rate of 0.5 ml / min in 2 steps. Immediately after the shock was induced, each animal was dosed with a test substance at a rate of 0.5 mL / min, i.e., homologous blood, the HbAo of the invention or the cross-linked HbAo of the invention, which was reacted with the U.S. Patent No. 5,532,352 (3x volume). Mean arterial pressure (MAP) and heart rate were recorded for 30 minutes after completion of the infusion. Each rat was housed in a separate cage and monitored for body weight, hematocrit, and survival for 14 days.

The results obtained are shown in Figure 8, where MAP values are plotted versus time.

From the data obtained, it can be seen that the product of the present invention is a highly purified hemoglobin-Ao, which is effective in restoring mean arterial pressure in rat experimental animals after hemorrhagic shock. There was no difference in restored arterial pressure or heart rate in animals receiving whole blood or HbAo product.

Example 15

Haemodynamic effects of isovolemic exchange transfusions in rats

In this experiment, the effect of the HbAo of the invention was evaluated to evaluate the hemodynamic effect of isovolemic exchange transfusions in alert rats.

The external iliac vein and artery of rats were cannulated through the femoral section according to Keipert and Chang (Effect of Partial ..., 1987, Vox. Sang 53: 7-14). During the exchange transfusion, blood volume was removed through the iliac artery and replaced with the test solution through the iliac vein at a rate of 0.5 ml / min using an infusion pump (1). 30 minutes before replacement, 2) during replacement, and 3) 2-3 hours after replacement. Mean arterial pressure and heart rate were calculated from blood pressure traces.

One group of rats (4) was transfused with human serum albumin, HSA. Another group (6) stromal hemoglobin (SFH) was added as test substance. Another group (8) of the inventive HbAo was added as a test solution.

The results obtained are shown graphically in Figures 9 and 9A. Figures 4 to 5 are shown.

For each test solution, the mean arterial pressure increased by 10-20 mmHg during the exchange transfusion. For the albumin control solution, the mean arterial pressure decreased by 15 mmHg below the baseline during the post-replacement period and remained at this level throughout the post-exchange test period for 2.5 hours (Figure 9). This observation is consistent with those previously observed by Keipert and Chang (1987, Effects of Partial ..., Vox. Sang. 53, 7-14).

For stromal hemoglobin, mean arterial pressure decreased to below baseline after the exchange period, but increased to at least about 15 mmHg above baseline 30-40 minutes after exchange (Figure 9A).

In contrast, in the case of the HbAo of the invention, the mean arterial pressure returned to baseline after replacement, increased by no more than 5 mmHg after replacement, thus maintaining the MAP within the normal range.

Experimental results show that the HbAo product of the present invention does not possess the potent vasoactivity associated with exchange transfusions in stromal hemoglobins.

Example 16

Examination of in vitro tissue cultures for oxidative damage to endothelial cells previously exposed to hemoglobin preparations

In this experiment, the HbA product of the invention was tested for its ability to induce oxidative damage in culture of endothelial cells. The release of lactate dehydrogenase activity in the medium indicates cellular damage and cytotoxicity.

Porcine thoracic aortic endothelial cells were cultured to confluence and re-cultured at a ratio of 1: 3

HU 218 159 Β passes were made. Within 48 hours, cells obtained by passage 7 were used for the experiment. Cellular damage was monitored by measuring the amount of lactate dehydrogenase released from the cells in the medium. Before the hemoglobin solutions were added, the medium was removed and the cells were washed twice with phosphate buffered saline. Cells were incubated with hemoglobin (250 pmol heme) in complete EGM medium for 6 or 24 hours at 37 ° C (n = 4-6). Cells incubated in complete EGM medium served as a negative control. At the end of the incubation period, 50 µl of media was removed from each well and lactate dehydrogenase activity was determined by a lactate dehydrogenase assay (Sigma). The lactate dehydrogenase activity measured in the wells was compared to the dehydrogenase activity released from the cells after administration of 2% Triton X-100 (total LDH release) and expressed as a percentage of the total release.

After incubation for 1 h, significant lactate dehydrogenase release was observed with stromal hemoglobin SFH (74.4 ± 4.2%, p <0.005). Similar but slightly smaller increases were observed for purified hemoglobin from the Walter Reed Army Institute of Research. No increase was observed in the purified HbAo sample of the invention (39.8 ± 2.3%) compared to the control (37.5 ± 2.2%). Lactate dehydrogenase release was further increased after 24 hours of incubation with stromal hemoglobin (99.6 ± 3.5%), while no change was observed with the HbAo of the invention (41.6 ± 4.6%) compared to control (34 , 1 ± 2.6%).

Cultured endothelial cells incubated with HbAo as described herein do not mediate the release of lactate dehydrogenase activity, indicative of cellular injury. Consequently, cytotoxicity against cultured endothelial cells is not an intrinsic property of hemoglobin solutions, and purification of HbAo removes cytotoxic components present in stromal hemoglobin.

Example 7

Removal of procoagulant activity

The experiment was conducted to determine the amount of a component of the present invention that promotes blood coagulation. The study was performed according to Biessels et al., Annual Report of the Kari Landsteiner Foundation, 1991.

Guinea pigs were anesthetized by administration of Hyponorm ™ and a silicone cannula was introduced into the carotid artery. After cannulation, 30% of the total blood volume was removed (2% of body weight) and replaced with an equal volume of test solution. 1% human albumin solution is infused at a rate of 2.5 ml / h to maintain the cannula. The initiation of albumin infusion was considered the starting time of the experiment. Factor Xa was administered by infusion at 8-9 pg / kg, this dose alone did not induce coagulation, and the amount of fibrinopeptide A (FPA), known to be formed from fibrinogen by blood coagulation, was measured before and after dosing. Blood samples were collected in a mixture of heparin (1000 U / ml), Trasylol (1000 U / ml) and sodium citrate (3.8%), separated and the plasma stored on ice.

Plasma samples were then assayed for FPA by the Leessma method on serine (Leessma et al., Blood, 67, 650-654, 1986), and the resulting FPA was measured according to the RIA method previously described by Gerrits et al., Thromb. Rés. 5, 197. 1974). Plasma proteins were precipitated with an equal volume of 40% PEG 6000 / phosphate buffered saline. The supernatant solutions were heated in a hot water bath, centrifuged and stored until assayed. The results are summarized in Table 8 below.

Table 8

test samples description AUC _IO * SFH Rapid, transient increase in FPA from 1.4 ng / ml to 13.8 ± 3 ng / ml in the first 10 minutes, followed by a fall to baseline after 40 minutes 87.6 ± 12 HSA The increase in FPA from the 1.4 ng / ml baseline is maximal 5.5 ± 1.5 ng / ml 45.7 ± 8.2 HbAo The increase in FPA from 1.4 ng / ml base value was maximal 5 ± 1.2 ng / ml 31.8 ± 2.9

* Area under the curve during the first 10 minutes after Factor Xa administration

It is apparent from the above data that the product of the present invention is essentially devoid of procoagulant activity as compared to stromal hemoglobin. Such procoagulant activity is known to be largely due to lipid phospholipid impurities in the test solutions.

Example 18

Determination of human serum protein contaminants

ELISA

In this experiment, they tested the amount of serum protein contaminants in the purified hemoglobin products of the present invention compared to the currently available hemoglobin products by sandwich ELISA.

The sandwich ELISA method described by Butler J. E. (Butler, J. E., ELISA and other Solid Phase Immunoassays, D.M. Chimney and

S. J. Challacombe, John Wiley and Sons Ltd., 1988, 1-180.).

Dynatech-type 96-well 1 mm 4 microtiter plates were coated with rabbit anti-human serum antibody (Dako Corpo14) diluted 1:40 in Tris-buffered saline and absorbed into solid phase goat serum.

HU 218 159 Β ration, Santa Barbara, CA, Lot # 072). The plates were then incubated for 2 hours at 37 ° C, then washed with tris-buffered saline (TBS) and the remaining free sites blocked by incubation with hydrolyzed fish gelatin (Hipure Liquid Gelatin, Norland Products Inc., New Brunswick, N.J.). After incubation for 1 hour, the plates were washed with TBS and the test substances added twice in serial dilution. The plates were then incubated for 1 hour and washed again with TBS and incubated with solid phase serum goat anti-human serum antibody, IgG fraction (Accurate Chemical and Scientific Corporation, Westbury Ν Y., Lot # H036) containing 1% fish gelatin. and diluted 1: 1000 with TBS. After incubation for 1 hour, the plates were washed with TBS and incubated with affinity purified rabbit antibody / IgG fraction (Horseradish peroxidase HRP conjugated) (BioRad Lot # 75312) (1: 250 dilution in TBS). After incubation for 2 hours, the plates were washed with TBS. The antigen-antibody-HRP complex was visualized using o-phenyl diamine dichloride (OPD-26 mg / 15 mL citric acid, pH 5). The appearance of a pale yellow color indicated the appearance of the antigen, specifically the serum protein. Its absorbance was determined using a Molecular Devices Thermomax Microplate at 490 nm.

The assay used two replicate samples: normal human serum (Dimension Laboratories Inc., Mississauga, Ontario, Lot # 30317), purified HbAo product from Sigma Corporation (H-0267), purified HbAo from Walter Reed Army Institute of Research (WRAIR). (Lot # 92092) and the purified HbAo product of the invention. The results obtained are summarized in Table 9 below. The results show that the product according to the invention contains no more than 0.0004% of serum protein, and this amount is at least an order of magnitude higher in the other samples tested.

Table 9

Sample Serum protein content (w / w) for Hb relative Serum protein in solution (mg / ml) HbAo according to the invention (8% total Hb in solution) 0.0004% 0.000328 WRAIR HbAo (total Hb 6.7%) 0.0039% 0.00263 Sigma HbAo (total Hb 1.1%) 0.0029% 0.000328

The only detectable serum protein in the product of the invention was human serum albumin. In the other products, the detected serum proteins appeared to be a heterogeneous mixture of HSA and other serum proteins.

Claims (11)

PATENT CLAIMS A process for separating a pre-selected hemoglobin from a crude solution thereof which also contains proteinaceous impurities, characterized in that: - in a first chromatographic step, the crude solution is added to the chromatography column and either chromatographed on anion under conditions whereby the acidic components of the crude solution, which are more acidic than the pre-selected hemoglobin, are bound, or components with more basic properties bind with preferential affinity, - continuing to feed the crude solution in the first chromatographic step until the column is substantially saturated with hemoglobin and the higher affinity component, - continue to inject the crude solution into the first chromatographic column until the column is overloaded and subsequently hemoglobin displacement is performed, - in a second chromatographic step, eluting the hemoglobin eluate from the chromatographic column used in the first step to a chromatographic column and chromatographing it on anion exchange or cation exchange, the operation being different from that used in the first step, - continuing to add said eluent to the second chromatographic column until the column is substantially completely saturated with hemoglobin and a higher affinity component under the second step conditions, - continuing to add said eluent to the second chromatography column until the column is overloaded and thereafter until the hemoglobin is expelled from the column. Process according to claim 1, characterized in that the first step uses anion-exchange chromatography and the second step uses cation-exchange chromatography. 3. The method of claim 2 wherein the pre-selected hemoglobin material is normal, adult, human hemoglobin HbAo. The process according to claim 3, wherein the first step is carried out at an pH of 7-10 and with a conductivity of less than 3 mS in the medium. The process of claim 4, wherein the first step is anion exchange chromatography at a pH of 8.5 to 9.0 and a conductivity of less than 1 mS in the medium. 6. The method of claim 4, wherein the feed to the column is carried out at a rate of not more than 10 cm / min. 7. The method of claim 6 wherein the concentration is 0.1-20%. The process of claim 4, wherein the second step is a cation exchange chromatography HU 218 159 Β was carried out at a pH of 6.5 to 8.5 and with a conductivity of less than 3 mS. 9. The process of claim 8, wherein the second step is cation exchange chromatography at a pH of 7-7.5 and a conductivity of less than 1 mS. The method of claim 8, wherein the medium is added to the cation exchange column at a rate of not more than 10 cm / min in the second step. 11. The process of claim 8, wherein in the second step, a cation exchange column is added at a concentration of 2% to 6%.