JPH0365989B2 - - Google Patents

Description

〔Technical field〕

The present invention relates to a separating agent and an apparatus using the same for separating and removing high molecular weight proteins of plasma components from blood excluding blood cell components. [Prior art and problems] It has become clear that various intractable diseases develop due to the accumulation of certain components, particularly high molecular weight proteins, such as immunoglobulins, immune complexes, complement, and fibrinogen in the blood. It's here. In order to treat this type of intractable disease, a therapeutic method (plasma exchange therapy) in which a patient's plasma is exchanged with a replacement solution such as fresh frozen plasma or an albumin preparation has recently been widely attempted. However, with the spread of this therapy, the consumption of replacement fluid has increased,
Problems such as tight supply and increasing costs have arisen. In addition, these replacement solutions have started to cause side effects such as hepatitis and allergic diseases. Therefore, it is possible to selectively remove high molecular weight proteins, which are pathogenic substances, from a patient's plasma without using such an exchange solution, and return purified plasma containing albumin and other useful autologous plasma components to the patient. Research on plasma purification methods is beginning to take place. Two known plasma purification methods include a method in which high molecular weight proteins are removed using a membrane with a smaller pore size than the plasma separation membrane used to separate plasma from blood, and a method in which high molecular weight proteins are adsorbed and removed using an adsorbent. It is being However, the former method is inferior in selectivity in the sense of removing specific proteins, and the latter method has the disadvantage that a large amount of plasma cannot be processed at one time. In view of these points, the applicant has filed a patent application filed in 1983-
No. 207463 (Unexamined Japanese Patent Publication No. 1983-99265) and Patent Application No. 1983
In No. 207464 (Japanese Unexamined Patent Publication No. 60-99266), an application was filed which purported to use an alkali metal chloride, such as sodium chloride, as a fractionation separation agent by salting out plasma proteins. This fractionation separation agent has a relatively weak salting-out effect and low solubility in plasma, so it does not precipitate useful low-molecular-weight proteins such as albumin even when added to plasma at a rate exceeding saturation. It has the effect of specifically precipitating only high molecular weight proteins such as fibrinogen and immunoglobulin. Purpose of the Invention The purpose of the present invention is to provide a separation agent that can efficiently precipitate and remove high-molecular proteins among the proteins contained in the whole plasma component obtained by removing blood cell components from blood, and in particular, to provide a separation agent that can efficiently precipitate and remove high-molecular proteins contained in the entire plasma component from which blood cell components have been removed. It has a low precipitation rate for low molecular weight proteins such as globulin,
The present invention provides a separation agent that has a high precipitation rate for high molecular weight proteins such as fibrinogen and can specifically precipitate and remove high molecular weight proteins. Another object of the present invention is to provide an apparatus for separating and removing high molecular weight proteins from plasma using such a separating agent. The term "plasma protein" as used herein refers to a protein present in a liquid component obtained by removing the formed components from blood, ie, blood cells (red blood cells, white blood cells, and platelets). These plasma proteins include albumin, globulin (α ₁ , α ₂ ,
β, γ) and fibrinogen,
Further detailed analysis revealed that transferrin,
It can be subdivided into more than 80 types, including haptoglobin, hemopexin, glycoproteins, lipoproteins, immunoglobulins, complement, and immune complex enzymes. Plasma proteins are roughly divided into high molecular weight proteins and low molecular weight proteins.High molecular weight proteins include immunoglobulins, immune complex complements, fibrinogen, etc., and low molecular weight proteins include albumin and the like. The separation agent and device of the present invention are used to remove high molecular weight proteins from plasma components. What achieves the above object is a separating agent for precipitating and removing high-molecular proteins in the plasma component containing albumin from blood cell components and albumin-containing plasma components that are fractionated from blood. The separation agent comprises an alkali metal salt and at least an amino acid selected from the group consisting of neutral amino acids and N-acetyl tryptophan, which promotes the precipitation action of the alkali metal salt on high molecular weight proteins in plasma. This is a separation agent for separating and removing high-molecular proteins from plasma components, which contains a mixture of one type of protein as an active ingredient. Preferably, the alkali metal salt is an alkali metal chloride.
Furthermore, it is preferable that the alkali metal salt is sodium chloride or potassium chloride. The amount of the amino acid is preferably 5 to 50% by weight of the total weight of the mixture of the alkali metal salt and amino acid. Furthermore, it is preferable that the neutral amino acid is glycine. Furthermore, what achieves the above object is a separation agent for precipitating and removing high molecular weight proteins in blood cell components and albumin-containing plasma components that are fractionated from blood. A metal salt and at least one amino acid selected from the group consisting of neutral amino acids and N-acetyltryptophan that promotes the precipitation action of the alkali metal salt on high molecular weight proteins in plasma. a container containing a separating agent having a mixture of the plasma as an active ingredient; a means for introducing a plasma component into the container; and a means for introducing a plasma component into the container; An apparatus for separating and removing high molecular weight proteins from plasma components, comprising means for separating and removing and means for removing at least a part of the separating agent component from the plasma components after removing the precipitate. . DETAILED DESCRIPTION OF THE INVENTION As a result of intensive research into alkali metal chlorides such as sodium chloride in order to improve their plasma protein separation effect, the present inventor discovered that alkali metal chlorides are more effective than neutral amino acids and N-acetyltryptophan. It has been found that by incorporating at least one amino acid selected from the group consisting of the following, the above-mentioned advantages of alkali metal chlorides are not impaired at all, and the plasma protein separation effect thereof is significantly improved. It has also been found that these amino acids improve the plasma protein separation effect not only against alkali metal chlorides but also against other alkali metal salts. The alkali metal salts constituting the active ingredients of the separating agent of this invention include sodium chloride, potassium chloride,
Lithium chloride, rubidium chloride, cesium chloride,
These include chlorides such as francium chloride, and even sulfates such as sodium sulfate. Among these alkali metal salts, chlorides exhibit the most remarkable effect of adding amino acids, which will be described in detail below. As mentioned above, alkali metal chlorides do not precipitate useful protein components such as albumin even when added to plasma at a quantitative ratio exceeding the saturation amount, and do not cause immunity to harmful high molecular weight proteins. It has the effect of selectively precipitating only globulin, and is very convenient in terms of concentration control in plasma. On the other hand, sodium sulfate itself has a strong salting-out effect, but if the proportion of sodium sulfate in the plasma is too high, it will also precipitate useful protein components such as albumin, so the added concentration must be strictly controlled. . However, by adding an amino acid to this sodium sulfate according to the present invention, its salting-out effect can be further improved. By the way, as will be explained in detail later, after harmful proteins are precipitated from plasma and the precipitate is removed, the separating agent used is removed from the purified plasma components and then returned to the patient. Therefore, it is desired that the alkali metal salt used be easy to remove from purified plasma components and be safe for living organisms. From this point of view, the alkali metal salt used is most preferably a chloride, particularly sodium chloride and potassium chloride, which exist in living organisms. The amino acid added to the alkali metal salt is selected from neutral amino acids (e.g., glycine, alanine, valine, leucine, isoleucine) and N-acetyltryptophan,
A mixture of two or more of these may also be used. Among these amino acids, glycine is the most effective. The above-mentioned amino acid exhibits an effect of improving the separation effect when incorporated in a proportion of 5 to 50% by weight based on the total weight of the mixture of the above-mentioned alkali metal salt and the above-mentioned amino acid. The blending ratio of amino acids is preferably 10 to 45% of the total weight of the mixture of alkali metal salt and amino acid. The ratio of the separation agent added to plasma is determined by the above-mentioned alkali metal salt's protein separation effect (selective precipitation effect).
The amount is sufficient to express this. Specifically, for alkali metal salts, the concentration is at least the saturation concentration in plasma. It is generally believed that salting out occurs by neutralizing the charge of colloids in an aqueous column. Alkali metal salts added to plasma proteins
It dissociates into ions in plasma and selectively neutralizes and precipitates high molecular weight proteins, especially globulins, which are harmful components. Although the principle of the effect of the addition of amino acids on improving the salting-out effect is currently unknown, it is thought that the amino acids have some effect on plasma protein components, thereby further improving the salting-out effect. Next, referring to FIG. 1, the basic configuration of an apparatus for selectively precipitating and removing high molecular weight proteins in plasma using the separation agent of the present invention will be explained together with a system for returning purified plasma components to the patient. explain. First, patient blood is supplied to the plasma filtration separator 11 via the line 20 by the blood pump 18 . Blood is separated into blood cell components and plasma components by the plasma filtration separator 11, and the separated plasma components are transported by the plasma pump 19 through the line 21 to the container 12 containing the separating agent of the present invention. be done. The plasma components introduced into container 12 are there mixed with the separation agent of the present invention, and macromolecular proteins are precipitated. After standing for 1 to 200 hours, the entire contents of the container are sent via line 22 to a sedimentation separator, such as a filter. The precipitate is separated and removed by this filter, and the purified plasma components are sent to the line 23.
It is accommodated in the secondary container 14 via. Connected to this secondary vessel 14 via lines 24 and 25 are means for removing the separating agent, for example a hollow fiber dialyzer. The purified plasma component contained in the secondary container 14 is introduced into the hollow fiber dialyzer 15 via a line 24 and flows into the secondary container 14 via a line 25 again. This circulation of purified plasma components is repeated as desired. In the dialyzer 15, purified plasma components are passed through the dialyzer 15 through line 26 and line 2.
The dialysate flowing through the membrane 7 is dialyzed with, for example, a commercially available dialysate used in general artificial dialysis to remove the separation agent and purify the membrane. In this case, if sodium chloride and/or potassium chloride, which are also components in living organisms, are used as alkali metal components, it is not necessary to completely remove them by dialysis, but only to remove them to the level of their concentration in living blood. That's enough. The secondary container containing the purified plasma component thus purified is replaced with a replacement fluid container 16, which will be described in detail hereinafter, and returned to the patient via the same route as the replacement fluid. By the way, the blood cell components separated by the plasma separator 11 are returned to the patient via the line 30, but before that, a replacement fluid such as physiological saline, 5% It is mixed in a mixer 17 with a replacement liquid sent from a container 16 containing an albumin solution, etc., and then returned to the patient via a line 30. This is because purified plasma has not yet been prepared at the initial stage of the plasma purification operation. As mentioned above, once the purified plasma is prepared, a container 14 containing the purified plasma is placed in place of the replacement fluid container 16, and is returned to the patient through the same route as the replacement fluid. FIG. 2 shows more specifically the separation apparatus of the present invention, the basic configuration of which has been described above. That is, this separation device is equipped with a flexible bag 41 as a container for storing plasma.
The separation agent of the present invention described above is stored in advance. This bag 41 includes a tube L1 as a plasma inflow path and a tube L2 as a plasma outflow path. The tip of the tube L1 inside the bag 41 is closed, and there is a notch (not shown) in the tube wall at a location slightly away from the tip.
is provided, and communication between the tube L1 and the inside of the container 41 is established by cutting it out at this notch portion during use. Further, the tube L2 is closed at the end on the outside of the container 41, and a notch similar to the above is formed in the tube wall portion at a location slightly away from the tip. This tube L2 and the filter 4 described later
The tube L4 that communicates with the tube L4 is connected to the tube L3 which has a relatively large diameter. This tube L3 is arranged so that the notch portion of the tube L2 is located inside. During use, by cutting out the tube L2 at the notch portion, communication between the tube L2 and the tube L4 can be established. Tube L3 is connected to settling filter 42 via tubes L4 and L5, as mentioned above. The tube L4 and the tube L5 are sterilely connected by a connector C1 which will be described in detail later. The sedimentation filter 42 filters the precipitate generated when the plasma components in the bag 41 come into contact with the protein separation agent of the present invention. The filtrate is
It is stored in the secondary bag 43 from the filter 42 via the tube L6. A tube L14 is inserted deep inside the secondary bag 43, and the other end of this tube L14 is
It is connected to a hollow fiber dialyzer 44.
Further, a tube L8 is connected to the other end of the dialyzer 44, and the other end of the tube L8 is connected to the secondary bag 43. In this way, tubes L14 and L8 constitute a closed circuit. The filtrate in the secondary bag 43 is circulated through this closed circuit to complete the desired degree of dialysis. The dialysate is introduced into the dialyzer 44 through the inlet 44a and flows out through the outlet 44b. Further, the external tip of the bag 41 of the tube L1 is connected to a tube L9 via a connector C2, which will be described in detail later, and this tube L9 is connected to a tube L10 via a connector C3, which will be explained in detail later. Plasma outflow tubes L11 and L12 of the plasma separation filter 45 merge into tube L10. Blood introduced from the blood inlet 45b of the plasma filter is separated into plasma components and blood cell components by this filter 45, and the blood cell components are
It is discharged from the discharge port 45a of the filter 45. Note that a tube L13 for discharging internal air or introducing air is connected to the tube L5, and at the tip of this tube L13,
A sterilization filter F1 that allows air to pass through but not liquid is connected to prevent bacteria from entering the outside air. In addition, inside tube L5,
An elastic tube E1 is provided which is pumped to drain the contents of the bag 41. A similar sterilization filter F2 is also connected to the sedimentation filter 42 via a tube L4. Furthermore, a priming tube P1 having a filter F3 disposed in the middle thereof is connected to the tube L7, and an elastic tube E2 similar to the above is disposed downstream thereof. On the other hand, the tube L8 is provided with a pressure monitor M1 for monitoring the pressure inside the tube, and the tube L8 is further provided with a pressure monitor M1 for monitoring the pressure inside the tube.
A priming tube P2, in which a filter F6 is disposed in the middle, is connected to the priming tube P2. The pressure monitor M1 is equipped with a sterilization filter F4 similar to the above.
and F5 are connected. Similarly, the tube L9 is connected to a priming tube P3 having a filter F7 disposed in the middle thereof, and an elastic tube E3 and a pressure monitor M2 similar to those described above are disposed on the upstream side thereof. There is. Now, the connector C1 that connects the tubes L4 and L5, the connector C2 that connects the tubes L1 and L9, and the connector C3 that connects the tubes L9 and L10 connect the respective tubes in a sterile manner. This type of connector is disclosed in Japanese Patent Laid-Open No. 57-211353. This connector is made of heat-resistant and corrosion-resistant material (ceramic,
A short tubular first connector part made of (stainless steel, titanium, etc.), and a female engagement part with a female insertion end formed into a female mold that is inserted into the male engagement part, and a short length made of heat-resistant and corrosion-resistant material. and a tubular second connector part. Both of these connector parts are
It is locked by a locking structure that can be locked without screwing, and there is a cylindrical support made of a heat insulating material (silicone rubber, Teflon resin, cork, etc.) on the side where both connectors partially connect with the tube. is fitted. Since this type of connector can connect the first and second connector parts while directly heating them with flame, a sterile connection can be achieved in the sense that sterilization can be reliably performed. Among the connectors disclosed in the above-mentioned publication, those shown in FIGS. 5 and 11 are preferably used as the connectors C1, C2, and C3 of the separating device of the present invention. An example of these connectors will be explained with reference to FIG. The illustrated connector includes a first cylindrical body 51 made of a heat-resistant and corrosion-resistant material, which corresponds to the first connector part, and a second cylindrical body 53, which is made of a heat-resistant and corrosion-resistant material, which corresponds to the second connector part. It consists of The inner diameter of the first cylindrical body is uniform, and the surface thereof is formed by a flat portion 51a parallel to the inner surface and a tapered portion 51b whose diameter decreases toward the tip. A small annular projection 52 is formed at the boundary between the flat portion 51a and the tapered portion 51b. Second
The cylindrical body 53 has a uniform outer diameter, but has a tip portion 53a that has an inner diameter larger than the outer diameter of the flat portion 51a of the cylindrical body 51 and expands in diameter toward the outside, and a tapered portion 51b of the cylindrical body 51. It is constituted by a reverse tapered portion 53b having an inner surface corresponding to the surface, and a flat portion 51e having an inner diameter equal to the inner diameter of the cylindrical body 51. Furthermore, an annular groove 54 corresponding to the projection 52 of the cylinder 51 is formed within the inner surface of the cylinder 53 . When the cylinder 51 is pushed into the cylinder 53 from the direction of the arrow shown in the figure, the diameter of the tip 53a of the cylinder 53 expands slightly to accommodate the cylinder 51, and the protrusion 52 of the cylinder 51 is inserted into the annular shape of the cylinder 53. It is fitted into the groove 54 and locked. Examples of this invention will be described below. Example 1 Human plasma was mixed with a mixture of sodium chloride and glycine, left to stand for 16 hours, and the resulting precipitate was filtered. In this example, the amount of sodium chloride used was 30 g/dl plasma, and the amount of glycine added (g/dl) was varied. Table 1 shows the amounts of total protein (TP), albumin (Alb), and globulin (Glo) in plasma before and after this treatment. Among globulins, the amounts of immunoglobulins IgG, IgA, and IgM are also shown.

[Table] From the results in Table 1, it can be seen that by incorporating sodium chloride with glycine, the selective precipitation and separation effect of high molecular weight proteins in plasma is significantly improved. Example 2 Human plasma was treated in the same manner as in Example 1 with a mixture of 30 g/dl sodium chloride plasma and 8 g/dl alanine plasma or 8 g/dl N-acetyltryptophan plasma. Total protein amount, albumin amount, globulin amount, and ratio of albumin to globulin (A/G) before and after this treatment
are shown in Table 2. The results obtained when glycine was added at the same concentration as alanine and N-acetyltryptophan are also shown.

[Table] From the results in Table 2, it can be seen that among the amino acids used in this invention, glycine is particularly effective. Specific Effects of the Invention As described above, according to the separation agent of the present invention, high molecular weight proteins, such as immunoglobulin and fibrinogen, among proteins contained in plasma can be efficiently and in large quantities with high selectivity. Can be removed. When a chloride is used as the alkali metal salt, it is preferable because even if it is used at a high concentration, a large amount of useful proteins such as albumin will not be precipitated, so there is no need to strictly control the concentration. Furthermore, when an alkali metal chloride, sodium chloride, or potassium chloride is used, safety is particularly high because these compounds exist in living organisms. Furthermore, although the separation apparatus of the present invention has a simple configuration, it is possible to selectively precipitate and separate high molecular weight proteins since it uses the above-mentioned separation agent. Also,
The dialysis machine for removing the separation agent simultaneously removes low-molecular-weight toxic substances in the plasma with molecular weights of several thousand to tens of thousands, thereby further enhancing the plasma purification effect.

[Brief explanation of drawings]

FIG. 1 is a block diagram for explaining the basic configuration of the separation device of the present invention, FIG. 2 is a plan view showing the separation device of the invention in more detail, and FIG.
FIG. 2 is a sectional view of a connector for aseptically connecting tubes in the separation device of the present invention. 11,45...Plasma separator, 12,45...Container, 13,42...Precipitation separation filter, 15,
44...Dylyzer.

Claims (1)

[Scope of Claims] 1. A separating agent for precipitating and removing high-molecular proteins in blood cell components and albumin-containing plasma components that are fractionated from blood, the separating agent comprising: , an alkali metal salt, and at least one amino acid selected from the group consisting of neutral amino acids and N-acetyltryptophan, which promotes the precipitation action of the alkali metal salt on high molecular weight proteins in plasma. A separating agent for separating and removing high molecular weight proteins from plasma components, characterized in that the active ingredient is a mixture of certain substances. 2. The separating agent according to claim 1, wherein the alkali metal salt is an alkali metal chloride. 3. The separating agent according to claim 2, wherein the alkali metal salt is sodium chloride or potassium chloride. 4. The separating agent according to claim 1, wherein the amount of the amino acid is 5 to 50% by weight of the total weight of the mixture of the alkali metal salt and amino acid. 5. The separating agent according to any one of claims 1 to 4, wherein the neutral amino acid is glycine. 6. A separating agent for precipitating and removing high molecular proteins in blood cell components and albumin-containing plasma components that are fractionated from blood, and which separates alkali metal salts and high molecular proteins in plasma. Separation containing as an active ingredient a mixture with at least one amino acid selected from the group consisting of neutral amino acids and N-acetyltryptophan, which promotes the precipitation action of the alkali metal salt on proteins. a container for accommodating an agent, a means for introducing a plasma component into the container, a means for separating and removing a precipitate produced by contact between the separating agent and the plasma in the container; 1. An apparatus for separating and removing high molecular weight proteins from plasma components, comprising means for removing at least a part of the separating agent component from the plasma components after precipitate has been removed.