Agent for separating proteins from blood plasma

Abstract: An agent is provided for the precipitative separation of proteins from blood plasma comprising, as an active ingredient, a mixture of an alkaline metal salt and an amino acid in an amount sufficient to promote a fractional precipitating effect of said alkaline metal salt with respect to plasma protein, said amino acid being at least one selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acytltryptophan and tyrosine.

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention: This invention relates to an agent for the separation and removal of proteins from blood plasma by the salting-out effect, to separate and remove specific proteins such as immune complexes, immunoglobulins, fibrinogen and other soluble macromolecular proteins from blood plasma. 2. Description of the Prior Art: It is becoming clear that immunoglobulins, immune complexes, complement, fibrinogen and other soluble macromolecular substances contained in blood play a role in causing autoimmune diseases, rheumatoid arthritis and other antigenic diseases. These diseases are treated by a plasma exchange process which includes removing plasma containing the harmful substances from the patient's blood and replacing the plasma with a substitute fluid. This process was first used in 1963 for the treatment of macroglobulinemia and since then has been tried in the treatment of many illnesses. With the development in recent years of membrane-type plasma separators, plasma exchange has become simpler and, hence, more widely practiced. However, the wider application of this treatment has been accompanied by increased consumption of the substitute fluids (e.g., FFP, agents made of albumin and the like) the supply of which has, as a result, become limited and higher in cost. An additional problem is that the infusion of a large quantity of human plasma can bring about unwelcome side effects such as hepatitis, alergic reactions and serum sickness. A method now undergoing research for dealing with the foregoing problems is so-called plasma purification or cleansing which, rather than relying upon a substitute fluid, selectively removes the macromolecular proteins causing the particular illness from the patient's blood plasma and reinfuses the patient with his or her own cleansed blood plasma containing albumin and other useful plasma ingredients. Plasma purification processes known so far include a process or removing macromolecular protein from blood plasma by using a membrane having a pore diameter smaller than that of a plasma separating membrane, and a process for the adsorptive removal of macromolecular protein from blood plasma by relying upon an adsorbent. While the former has tentatively shown some efficacy in clinical use and has won some standing as a curative means, the latter is still in the basic research stage and only partial clinical use has been reported. Both processes have drawbacks in that the former exhibits insufficient selectivity in terms of removing specific proteins and the latter is incapable of treating a large quantity of plasma at one time. Thus, neither process is truly adequate. Accordingly, there is a need to develop a more effective and efficient agent for purifying blood plasma. In view of these circumstances, the applicant has filed patent applications for inventions the gist of which is to use a chloride of an alkaline metal, e.g., sodium chloride, as an agent, or precipitating medium, for separating proteins from plasma by the salting-out effect. The applications filed are Japanese Patent Application Nos. 58-207463 and 58-207464. The proposed separating agent, owing to its weak salting-out action and low solubility in blood plasma, is effective in causing the specific precipitation solely of macromolecular proteins such as fibrinogen and immuno-globulins without resulting in the precipitation of such useful proteins of low molecular weight as albumin, even if the rate at which the agent is added to plasma is greater than that required for saturation. Another method for treatment of blood plasma in which alkaline metal salts (Na₂SO₄) are used for the precipitative separation of proteins from blood plasma is known from FR-A-2 552 329. SUMMARY OF THE INVENTION An object of the present invention is to provide an agent for the precipitative separation of proteins from blood plasma, which agent retains the advantages of the abovementioned alkaline metal chloride while exhibiting an improved separating action against plasma proteins. According to embodiments of the present invention, the precipitating medium solution is a solution of various inorganic salts, a solution of various organic solvents, or a solution of an inorganic or organic acid. It should be noted that the plasma proteins mentioned herein refer to proteins present in the liquid fraction that results when tangible constituents, namely blood cells (red blood cells, white blood cells and platelets) are removed from blood. Though these plasma proteins may be broadly classified into albumin, globulins (α₁, α₂, β, γ) and fibrinogen, a further subdivision into some 80 types now known can be made by a more detailed analysis, these proteins including transferrin, haptoglobin, hemopexin, glocoprotein, riboprotein, immunoprotein, compliment and immune complex enzyme. Furthermore, plasma proteins can also be broadly classified into proteins of high and low molecular weight. The former, namely macromolecular protein, refers to immunoglobulins, immune complexes, compliment, fibrinogen and the like, while the latter refers to albumin, etc. The plasma protein separating agent of the present invention is used for the extraction mainly of macromolecular proteins. According to the present invention, the object is attained by providing an agent for the precipitative separation of proteins from blood plasma comprising, as an active ingredient, a mixture of an alkaline metal salt or an ammonium salt and an amino acid in an amount sufficient to promote the fractional precipitating effect of the alkaline metal salt with respect to plasma protein, the amino acid being at least one selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan and tyrosine. Other features and advantages of the present invention will be apparent from the following description taken in conjunction with the accompanying drawing. BRIEF DESCRIPTION OF THE DRAWING Fig. 1 is a block diagram illustrating an exemplary embodiment of a blood purifying apparatus in which the agent of the present invention can be used. DESCRIPTION OF THE PREFERRED EMBODIMENT Blood plasma contains proteins of a high molecular weight, such as immune complexes, immunoglobulins and fibrinogen, as well as proteins of a comparatively low molecular weight, such as albumin. There is a need in self-purifying plasma treatment to selectively remove only the proteins of high molecular weight and leave the plasma with the useful proteins of low molecular weight, such as albumin. The inventors have conducted extensive research with regard to media capable of precipitating proteins in plasma and as a result have found that alkaline metal chlorides, particularly sodium chloride, owing to their comparatively weak salting-out action and low solubility, cause specific precipitation solely of the macromolecular proteins such as fibrinogen and immunoglobulins with hardly any precipitation of albumin, even if the medium, say sodium chloride, is added to the plasma in an amount greater than that required for saturation. More specifically, the inventors have discovered that if the sodium chloride added for separating and removing the precipitate formed by mixing the precipitating medium with plasma is maintained in the plasma at an amount above that needed for saturation, then strict control is unnecessary, and that there is no change in the effectiveness of precipitation or in the composition of the precipitate, as well as no change in the properties of the protein, even at the high level of sodium chloride addition. Based on the fruits of this research, the inventors have also perfected an apparatus which is capable of purifying blood by separating solely the macromolecular proteins from a large quantity of blood plasma by on-line implementation of the steps of plasma separation, precipitate formation, removal of the precipitate and recovery of the processed plasma. Preferred embodiments of the present invention will now be described with reference to the accompanying drawing. Fig. 1 is a block diagram illustrating a blood purifying system in which the agent of the invention for the precipitative separation and removal of a protein constituent from blood plasma can be used. A blood pump 1 delivers blood collected from a patient into a main conduit 101 made of, e.g., vinyl chloride. The main conduit 101 is connected to a plasma separator 2 which, by centrifugal- or membrane-type separation, separates the blood entering from the conduit into plasma and blood cell fractions. The separated plasma from the separator 2 is fed into a secondary conduit 102, also made of vinyl chloride, by a double roller-type plasma pump 3 which, at the same time, returns processed blood plasma in amount equivalent to the plasma drawn from the patient. A vessel 4 containing a solution of a precipitating medium is connected to a precipitating medium solution pump 5 operatively associated with the plasma pump 3 for delivering the solution of precipitating medium from the vessel 4 at a fixed feed ratio. A mixer 6 receives plasma delivered by the plasma pump 3 and the solution of precipitating medium delivered by the pump 5 for mixing the two together. The outlet of the mixer 6 is connected to a plasma filtering unit 7 for removing a protein fraction which has precipitated inside the mixer 6. The outlet of the plasma filtering unit 7 is in turn connected to a plasma constituent adjusting unit 8 for removing the precipitating medium from the filtered plasma and for adjusting water content and electrolyte. The processed plasma following its adjustment in the adjusting unit 8 is fed by the pump 3 into a plasma mixing unit 9, where the plasma is mixed and rejoined with the concentrated blood comprising the blood cells originally separated from the incoming blood by the plasma separator 2. A control unit 10 acts through the blood pump 1, plasma pump 3 and precipitating medium solution pump 5 to control the amounts of blood, plasma and precipitating medium solution delivered. With the above-described arrangement, the patient's blood introduced from a blood inlet a is cleansed automatically and continuously before being returned to the patient from a blood outlet b. More specifically, blood drawn from the patient is introduced by the blood pump 1 into the plasma separator 1, where the blood is separated into blood cell and plasma fractions. The plasma resulting from the separation is led into the secondary conduit 102 by the plasma pump 3 and flows into the mixing unit 6. Meanwhile, the solution of precipitating medium in the vessel 4 is delivered to the mixing unit 6, in which mixing with the plasma takes place, by the plasma medium solution pump 5 rotated at a fixed ratio in operative association with the plasma pump 3. A precipitate (mainly fibrinogen and globulin fractions) that forms in the mixing unit 6 is filtered out by the plasma filtering unit 7 for precipitate removal. The resulting filtered plasma, which still contains the precipitating medium, flows from the filtering unit 7 into the plasma constituent adjusting unit 8. Here the processed plasma is submitted to dewatering, removal of the precipitating medium and adjustment of electrolyte before being delivered to the plasma mixing unit 9 by the double roller-type plasma pump 3 in an amount equivalent to that of the plasma collected from the patient. The processed plasma mixes with the concentrated blood delivered by the plasma separator 2 and the mixture of processed plasma and concentrated blood is reinfused into the patient from the blood outlet b . Examples of the solution of the precipitating medium for protein separation are solutions of a variety of salts, namely alkaline metal salts such as sodium chloride, potassium chloride, sodium sulfate, potassium phosphate and sodium citrate, ammonium salts such as ammonium sulfate, and the like. In particular, when an alkaline metal salt is used, the amount of salt added need not be strictly controlled, for it will suffice if control is exercised such that the amount of salt added is maintained above that necessary for causing the macromolecular proteins to precipitate out of the plasma. In addition, though it is possible to use an organic solvent such as ethanol, an inorganic acid such as hydrochloric acid or sulfuric acid or an organic acid such as ascorbic acid, there is the possibility of a change in properties of the proteins when these substances are employed. It is therefore necessary to provide a set-up in which close attention is paid to temperature measurement and control of pH at mixing. The control unit 10 sets and regulates the rotating ratios of the pumps 3 and 5 mainly in dependence upon both the concentration of the precipitating medium solution and the protein that is desired to be removed from the plasma and is adapted to control the average flow rates of these motors so as to mix in an amount of the precipitating medium solution prescribed in accordance with the amount of plasma. For example, if ammonium sulfate is used as the precipitating medium solution, fibrinogen will substantially precipitate at an ammonium sulfate concentration in plasma of about 10 g/dl, and both fibrinogen and γ-globulin will substantially precipitate at an ammonium concentration in plasma of about 20 g/dl. Therefore, in case of a saturated solution (about 54 g/dl), each of the proteins can be made to precipitate and separate from the plasma if the settings are such as to add the ammonium sulfate at rates of about 23 ml and 59 ml, respectively, with respect to 100 ml of plasma. It takes about 10 to 20 ms for this chemical reaction. Flow rate control of this kind can readily be executed under the control of a microprocessor equipped with a control program. A dialyzer used in ordinary dialysis is adopted as the plasma constituent adjusting unit 8. Any dialyzer configuration will suffice providing that it has a dewatering capability that enables removal of the added amount of precipitating medium solution at less than the usable pressures, as well as a dialyzing capability that enables the concentration of the precipitating medium to be brought below a stipulated value. Preferably, an alkaline metal chloride such as sodium chloride or potassium chloride is used as the protein precipitating medium. A precipitating medium of this type will neither alter the effectiveness of precipitation nor change the properties of the protein in the plasma even if the medium is added to the plasma in large quantities. This provides a high level of stability even when large quantities of plasma are submitted to the purification treatment. In addition, since the precipitating medium selectively precipitates solely the proteins of large molecular weight such as immunoglobulins and fibrinogen but causes almost no precipitation of the useful proteins of low molecular weight such as albumin, the useful constituents (albumin, etc.) can be reinfused into the patient following the self-purification of the patient's blood; hence, a substitute fluid used in plasma exchange treatment is unnecessary. This solves the problems encountered in the prior art, namely the side effects such as hepatitis and allergic reactions, the high cost and the scarcity of substitute fluid supplies. Further, the process for separating and removing protein consituents from blood plasma can be practiced on-line, thereby shortening overall processing time and reducing the danger of bacterial infection. The inventors have conducted extensive research with regard to alkaline metal chorides such as sodium chloride in an effort to improve the precipitative separating action thereof with respect to plasma proteins. As the result of this research, the inventors have found that the precipitative separating action of these chlorides can be greatly improved, without detracting from the earlier mentioned advantages thereof, by blending with an alkaline metal chloride at least one aminio acid selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan and tyrosine. The inventors have also found that these amino acids enhance the plasma protein-separating action not only of alkaline metal chlorides but also of other salts of alkaline metals. The alkaline metal salt constituting the effective ingredient of the plasma protein-precipitative separating agent of the present invention includes chlorides such as sodium chloride, potassium chloride, lithium chloride, rubidium chloride, cesium chloride and francium chloride, and further includes sulfates such as sodium sulfate. Among these alkaline metal salts, the effect of adding the amino acid is best exhibited for the chlorides, as will be set forth below. It has been mentioned earlier that alkaline metal chlorides have the effect of selectively precipitating only harmful macromolecular proteins, especially immunoglobulins, without precipitating useful plasma constituents such as albumin even when the chlorides are added in an amount in excess of that needed for saturation. This is of great convenience in terms of controlling the concentration of the separating agent with respect to plasma. Sodium sulfate, on the other hand, exhibits a strong salting-out effect and can result in the precipitation of the useful plasma constituents such as albumin if mixed with plasma at too high a percentage. This therefore necessitates strict control of the added sodium sulfate concentration. However, the salting-out effect even of sodium sulfate can be enhanced by blending it with an amino acid in accordance with the present invention. It has been set forth earlier in the specification that harmful proteins are precipitated out of blood plasma, the precipitate is removed, the plasma protein-separating agent used is subsequently removed from the purified plasma fraction, and the resulting plasma is returned to the patient. Therefore, it is preferred that the alkaline metal salt used be readily removable from the purified plasma fraction and safe as far as the patient is concerned. Seen in this light, the most desirable alkaline metal salts that can be used are chlorides, especially sodium chloride and potassium chloride, which exist naturally in the human body. According to the invention, the amino acids blended with the abovementioned alkaline metal salts are neutral amino acids (e.g., glycine, alanine, valine, leucine, isoleucine), aspartic acid, tyrosine, N-acetyltryptophan and cystine. A mixture of any two or more of these may also be used. The neutral amino acids are the most effective among these amino acids, with glycine being the most useful. Enhancement of the plasma protein-separating effect of the alkaline metal salt is brought about by the addition thereto of the abovementioned amino acid at a proportion of 5 to 50 wt-% with respect to the total weight of the alkaline metal chloride - amino acid mixture. It is preferred that the amino acid be blended with the alkaline metal salt at a proportion of 10 to 45% with respect to the total weight of the alkaline metal chloride - amino acid mixture. As to the ratio of added separating agent to plasma, the amount of separating agent added should be enough for the alkaline metal salt to manifest its plasma separating effect, namely its selective precipitating effect. More specifically, the amount of separating agent added is more than that needed to raise the alkaline metal salt concentration in plasma to saturation. It is generally considered that salting out occurs due to neutralization of the electric charges of the colloid in the aqueous solution. The alkaline metal salt added to the plasma protein dissociates into ions in the plasma and selectively neutralizes and precipitates the harmful macromolecular proteins, especially the globulins. Though the mechanism through which the addition of amino acid enhances the salting-out effect is not yet known in terms of theory, it seems that the amino acid acts in some way upon the plasma protein constituent to thereby raise the effectiveness at which salting-out takes place. EXAMPLE 1 Human plasma was mixed with sodium chloride and glycine and was left standing for 16 h, followed by filtering the precipitate that formed. The amount of sodium chloride used was 30 g/dl plasma, and the amount of glycine added (g/dl) was varied with respect to the amount of sodium chloride. The total amount of protein (TP) and the amounts of albumin (Alb) and globulins (Glo) contained in the plasma before and after treatment are shown in Table 1. The amounts of immunoglobulins (Ig)G, IgA and IgM contained in the globulins are also shown in the table. TP (g/dl) Alb (g/dl) Glo (g/dl) IgG (mg/dl) IgA (mg/dl) IgM (mg/dl) BEFORE PROCESSING 6.4 3.8 2.6 1270 216 134 AMOUNT OF GLYCINE ADDED 0 5.7 3.5 2.2 1180 196 124 5 4.8 3.2 1.6 680 136 96 10 4.2 3.0 1.2 380 100 90 15 3.8 2.9 0.9 240 72 66 20 3.3 2.5 0.8 90 38 60 The tabulated results show that mixing glycine with sodium chloride greatly enhances the effectiveness of selective precipitative separation of macromolecular protein from plasma. EXAMPLE 2 Human plasma was treated by using the methodology of Example 1 with a mixture of 30 g/dl (plasma) of sodium chloride and 8 g/dl (plasma) of cystine, 8 g/dl (plasma) of tyrosine or 8 g/dl (plasma) of N-acetyltryptophan. The total amount of protein, the amounts of albumin and globulin, and the ratio (A/G) of albumin to globulin are shown in Table 2. The results of adding glycine in the same concentration as cystine, tyrosine or N-acetyltryptophan are also shown. T P (g/dl) Alb (g/dl) Glo (g/dl) A/G BEFORE PROCESSING 5.8 2.5 3.3 0.76 AMINO ACID ADDED NONE 4.0 2.1 1.9 1.11 CYSTINE 3.9 2.1 1.8 1.17 TYROSINE 3.8 2.0 1.7 1.17 GLYCINE 3.0 2.0 1.0 2.00 N-ACETYLTRYPTOPHAN 3.1 1.8 1.3 1.38 It will be appreciated from Table 2 that glycine is particularly effective among the amino acids used in the present invention. As set forth above, the agent for the precipitative separation of proteins from blood plasma according to the present invention enables macromolecular proteins, e.g., immunoglobulins and fibrinogen, to be efficiently removed in large quantity with a high degree of selectivity. When a chloride is used as the alkaline metal salt, even in high concentration, large quantities of such useful proteins as albumin do not precipitate. This is particularly advantageous because it eliminates the need for strict control of concentration. In addition, among the alkaline metal chlorides, use of sodium chloride or potassium chloride is particularly safe since these compounds are present in body fluid. Further, though an apparatus for separating proteins from blood plasma can be simple in construction, the selective precipitative separation of macromolecular protein is possible owing to use of the above-described separating agent. Moreover, by using of a dialyzer for removing the plasma-protein separating agent from the plasma, the effectiveness of plasma purification is enhanced even further since toxic substances having a molecular weight of from several thousand to tens of thousands are removed simultaneously. It is obvious that if the agent for the precipitative separation of proteins from plasma according to the present invention is used as the solution of the precipitating medium employed in the separating apparatus of Fig. 1, the precipitative separating effect can be greatly enhanced.

Claims: An agent for the precipitative separation of proteins from blood plasma comprising, as an active ingredient, a mixture of an alkaline metal salt or an ammonium salt and an amino acid in an amount sufficient to promote a fractional precipitating effect of said alkaline metal salt with respect to plasma protein, said amino acid being at least one selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan and tyrosine. The agent according to claim 1, wherein the alkaline metal salt is an alkaline metal chloride. The agent according to claim 2, wherein the alkaline metal chloride is sodium chloride. The agent according to claim 2, wherein the alkaline metal chloride is potassium chloride. The agent according to claim 1, wherein the amount of the amino acid is 5 to 50 wt-% with respect to the total weight of said mixture.

Inventors: OHE HIROAKI, WATANABE MASAHARU, OHE, HIROAKI, WATANABE, MASAHARU
Applicants: TERUMO CORP, TERUMO KABUSHIKI KAISHA TRADING AS TERUMO CORPORATION
Application year: 1985
Main classification: B01D15
IPC codes: A61M1, C07K1