JP2008018153A - Separation method of albumin from biomolecule bound to albumin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating albumin from biomolecules by removing the bonding of the biomolecules bounded to albumin. <P>SOLUTION: This separation method includes to extract plasma component to the outside of a living body, electrodialyze it under a specific pH, selectively remove albumin-binding biomolecule therefrom, and return albumin to the body again. This separation method has superior removal capacity of albumin-binding toxin, especially bilirubin and indoxylsulfuric acid, and is useful for the treatment of renal insufficiency and hepatic insufficiency. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for separating albumin and a biomolecule bound to albumin using electrodialysis.

  Albumin is a protein biosynthesized in the liver, and has functions such as maintaining osmotic pressure in blood and transporting various substances to each tissue.

  In normal cases, the albumin binding substance is taken into various tissues and metabolized / excreted, but in abnormal cases, the binding substance may remain bound to albumin and the uptake into the tissue may not proceed. In this case, the binding substance circulates in the blood and accumulates in the body, causing diseases such as liver dysfunction, and worsening the transfer of the drug to various tissues. Therefore, if the substance that binds to albumin is an unnecessary substance in the body (hereinafter referred to as “albumin-binding toxin”), it must be removed.

  Albumin-binding toxins include bile acids, bilirubin, aromatic compounds such as indoxyl sulfate / hippuric acid / phenol, furan carboxylic acid, etc., but these accumulate in the body in liver failure and kidney failure and are pathological. Many researches have been made on how to remove these substances.

  Conventionally, as a treatment for patients with chronic renal failure or liver failure, the patient's blood or plasma is contacted with an adsorbent such as activated carbon or anion exchange resin, and the toxin is removed using a physicochemical phenomenon. ing. However, adsorption of activated carbon or anion exchange resin is insufficient, and it cannot be said that it has sufficient removal ability particularly for albumin-binding toxins such as bilirubin and indoxyl sulfate, which are considered to cause renal failure and liver failure.

  Recently, the development of adsorbent materials has been underway. For example, as an adsorbent for bilirubin contained in a liquid such as blood or plasma, titanium oxide particles that physically adsorb bilirubin based on the crystal structure and the like are used. Has been proposed (see, for example, Patent Document 1).

  In addition, it is difficult to remove only the toxins bound to albumin. At present, the toxins that remain bound to albumin are removed together with an adsorbent, and the toxins are removed together with albumin by plasma exchange. However, since albumin is a useful substance in vivo as described above, an efficient method for removing only the toxin by removing the bond with albumin is desired.

  Treatment with MARS (Molecular Absorbent Recirculating System) takes out plasma outside the body, albumin dialysis (contact with clean albumin across a dialysis membrane) to remove toxins, and plasma containing purified albumin is put into the body again. It is a treatment method that returns, and is a few methods that remove only toxins (see, for example, Non-Patent Documents 1 to 5). However, since the toxin is basically removed in equilibrium with clean albumin, the efficiency is low, and the adsorbent is saturated within 6 hours from the start of treatment, resulting in a decrease in toxin removal ability and a decrease in platelets. Therefore, there is a problem that it cannot be used for patients with disseminated intravascular blood coagulation (DIC).

JP 2005-287701 A Blood Purification, 244-252, 2003 Artificial Organs 23, 319-330, 1999 Journal of American Society of Nephrology 12, s75-s82, 2001 Liver 22, 40-42, 2002 American Journal of Gastroenterology, 468-475, 2005

  In view of the above situation, the present invention provides a method for separating only biomolecules by removing the binding between albumin and biomolecules bound to albumin.

  The present inventors paid attention to the fact that albumin and albumin binding biomolecules have a charge, and a method for efficiently separating biomolecules by removing the binding between these biomolecules and albumin using electrodialysis. As a result, the present invention has been completed.

That is, the present invention relates to the following albumin-binding biomolecule separation method and the like.
1. A method of separating albumin and a biomolecule by electrodialyzing a solution containing albumin bound with a biomolecule.
2. 2. The method according to 1 above, wherein the biomolecule is a substance harmful to a living body.
3. 3. The method according to 2 above, wherein the harmful substance is a substance that is generated due to a disease or whose amount is increased due to a disease state.
4). 4. The method according to 3 above, wherein the disease is kidney disease or liver disease.
5. 5. The method according to 4 above, wherein the harmful substance is bilirubin and / or indoxyl sulfate.
6). A kidney disease or a disease characterized by removing a plasma component from the living body, removing harmful biomolecules from albumin using the method according to any one of 1 to 5, and returning the obtained albumin molecule to the living body. How to treat liver disease.
7). A method for treating liver disease or liver failure, wherein the harmful biomolecule is bilirubin.
8). A pump means for delivering a solution containing albumin between the ion exchange membranes, a potential difference applying means for applying a voltage between the ion exchange membranes, a means for recovering a liquid containing a biomolecule that has permeated the ion exchange membranes, and an ion exchange membrane. An albumin electrodialyzer comprising means for recovering a solution containing albumin flowing out.
9. 9. The apparatus according to 8, wherein the voltage is controlled so that a pH gradient is generated between the ion exchange membranes when a voltage containing a solution containing albumin is filled between the ion exchange membranes.
10. 10. The apparatus according to 8 or 9 above, comprising a plurality of ion exchange membrane flow paths comprising a plurality of pairs of ion exchange membranes, wherein a potential difference is applied between the ion exchange membranes constituting each ion exchange membrane pair.
11. 11. The apparatus according to any one of 8 to 10 above, comprising means for separating plasma components from blood and / or means for adding electrolyte and / or other blood components to a solution containing recovered albumin.

  According to the present invention, only biomolecules bound to albumin, such as albumin-binding toxin, can be selectively removed, and albumin can be returned to the body again, so that plasma can be purified efficiently. The present invention is particularly useful for diseases currently treated by various blood purification methods such as renal failure and liver failure because of its excellent ability to remove albumin-binding toxins such as bilirubin and indoxyl sulfate.

  In the method for separating albumin and biomolecule bound to albumin according to the present invention, the plasma component is taken out of the living body, electrodialyzed, and the biomolecule bound to albumin and albumin is removed and separated.

  Examples of biomolecules bound to albumin of the present invention include bile acids, bilirubin, aromatic compounds such as indoxyl sulfate, hippuric acid and phenol, furan carboxylic acid, AGE (late saccharification reaction product) and the like, bilirubin, Aromatic compounds such as indoxyl sulfate are substances that are harmful to living bodies (albumin-binding toxins), and are substances that cause serious diseases such as kidney diseases and liver diseases. In the present invention, a biomolecule refers to all molecules synthesized, metabolized and accumulated in a living body, and is not limited to a molecule unique to a living body, and its origin is not limited.

  Since blood cell components such as white blood cells and platelets in blood may be subject to irreversible changes when exposed to a strong electric field or subjected to physical stimulation, in the present invention, blood cell components and plasma components are separated in advance. Pretreatment is preferably performed. The method for separating the blood cell component and the plasma component is not particularly limited. A method such as a plasma separation method using a plasma separation membrane, a centrifugal separation method, or a generally used blood component blood collection device can be used.

  In electrodialysis, it is possible to remove the binding sufficiently efficiently without adjusting the pH, and in the vicinity of the pH 7.4 of the plasma. It is also possible to change the pH so that the bond is easily released. In particular, there are many biomolecules that are easily detached when the pH is lowered. However, lowering the pH to 4.0 near the isoelectric point of albumin is not suitable because albumin tends to aggregate. Further, even when the pH is extremely increased, denaturation is likely to occur and is not suitable. The method for adjusting the pH at this time is not particularly limited, but it can be carried out by adding hydrochloric acid, citric acid, sodium hydroxide, sodium hydrogen carbonate or the like.

  Electrodialysis will be described with reference to the schematic diagram (FIG. 1) of the electrodialysis tank portion of the albumin-binding biomolecule removal apparatus. Electrodialysis is performed by applying a voltage to plasma adjusted to around pH 7.4 and separating it through ion exchange membranes (anion exchange membrane (5) and cation exchange membrane (4)). In the vicinity of pH 7.4, albumin (9) and biomolecule (10) bound to albumin are negatively charged, and therefore are electrically attracted to the positive electrode when a voltage is applied. In addition, since the vicinity of the positive electrode (6) tends to be acidic (7), the binding between albumin and the biomolecule is easily released. Therefore, only an albumin-binding biomolecule can be separated by using an anion exchange membrane whose membrane pores allow only biomolecules to pass but not albumin.

  The applied voltage during electrodialysis can be operated at an arbitrary applied voltage. The suitable value varies depending on the size of the apparatus and the flow path width. For example, if the flow path width is about 0.5 to 2 mm, about 4 to 5 V is preferable. Moreover, when a voltage is applied by filling a solution containing albumin between the ion exchange membranes, electrodialysis is performed by controlling the voltage so that a pH gradient is generated between the ion exchange membranes.

  Anion exchange membranes and cation exchange membranes can be used with various membrane pore sizes (fractionated molecular weights). The larger the membrane pore size, the more efficiently and continuously separate biomolecules bound to albumin. Is possible and preferred. A membrane having a molecular weight cut-off of at least 500 and a molecular weight cut off as large as possible is preferable, but a membrane having a molecular weight cut-off of 50,000 or more that permeates albumin is not suitable.

  The apparatus used for the albumin-binding biomolecule (toxin) removal method of the present invention comprises a pump means for delivering a solution containing albumin between ion exchange membranes, a potential difference providing means for applying a voltage between the ion exchange membranes, and ion exchange. Means for recovering a liquid containing biomolecules permeated through the membrane and means for recovering a solution containing albumin flowing out between the ion exchange membranes.

In the pump means for delivering the albumin-containing solution between the ion exchange membranes, the method for obtaining the albumin-containing solution or the method for taking out the plasma component out of the living body is not particularly limited, but a commonly used blood component blood collecting device is used. It can be provided in the device of the present invention.
Further, in the apparatus of the present invention, a solution containing plasma components is circulated at a rate of about 1 to 200 ml / min. Therefore, if necessary, a plurality of circulation pumps are provided in the middle of the plasma solution flow path.

  In the potential difference applying means for applying a voltage between the ion exchange membranes, in order to remove albumin-binding biomolecules (toxins), it is necessary to include at least one set of anion exchange membranes and cation exchange membranes arranged alternately in parallel. However, in order to improve the removal capability, a plurality of pairs of ion exchange membrane channels may be provided in parallel between the potential difference applying means.

  Further, in the means for collecting the liquid containing the biomolecule that has permeated through the ion exchange membrane and the means for collecting the solution containing albumin flowing out between the ion exchange membranes, the electrolyte concentration is high in the liquid containing the biomolecule, Since the electrolyte concentration is reduced in the liquid containing albumin, it can be adjusted as necessary. The recovered plasma component after electrodialysis discharged from the electrodialysis tank, that is, the solution containing the recovered albumin, is added with an electrolyte and / or a pH adjuster, added with other blood components, and returned to the body. Is possible. When adjusting the electrolyte and pH, it is possible to perform dialysis using a dialysate whose electrolyte concentration and pH are adjusted, in which case complicated control becomes unnecessary.

  The method for separating albumin and biomolecules bound to albumin according to the present invention is applicable to treatment of diseases to which various blood purification methods such as renal failure, liver failure, respiratory failure, and sepsis are applied, particularly to continuous blood purification methods. It is effective as a method for treating diseases.

EXAMPLES Hereinafter, although this invention is demonstrated using an Example, the following examples are for demonstrating this invention, and do not limit this invention.
In this example, the electrodialysis tank used was an anion exchange membrane and an ion exchange membrane of a cation exchange membrane arranged alternately in parallel so as to have a channel width of 0.8 mm, and a voltage of 4 to 5 V was applied.
A molecular weight cut off of 1000 was used for the anion exchange membrane, and a molecular weight cut off of 300 (Astom Co., Ltd.) was used for the cation exchange membrane. Albumin is derived from bovine serum and is added to phosphate buffered saline (pH 7.4) containing 8 g sodium chloride, 3 g sodium monohydrogen phosphate, and 0.1 g sodium dihydrogen phosphate in 1 liter of solution. Dissolved to make an albumin aqueous solution.

Example 1:
Bilirubin (2.5 mg / dl) was dissolved as an albumin-binding toxin in an aqueous albumin solution (3 g / dl), and electrodialysis was performed at pH = 7.4.
The sample flow rate was 20 ml / min, and samples were collected over time. The bilirubin concentration was measured by absorbance at a wavelength of 600 nm using a coloring reagent to determine the binding concentration and the free concentration. The sample amount was 20 ml, and the removal amount was determined from the measured concentration. The results are shown in FIG.

Example 2:
The same procedure as in Example 1 was performed except that indoxyl sulfate (3.0 mg / dl) was used as the albumin-binding toxin instead of bilirubin. The indoxyl sulfate concentration was measured by absorbance at a wavelength of 230 nm. The results are shown in FIG.

[result]
Bilirubin initially had a large amount of free bilirubin removed, but with the passage of time, bound bilirubin was also removed in the same manner (FIG. 2; in the figure, ◆ represents bound bilirubin and □ represents free bilirubin). In indoxyl sulfate, a large amount of bound indoxyl sulfate was removed (FIG. 3; in the figure, ♦ represents bound indoxyl sulfate and □ represents free indoxyl sulfate). For any toxin, the toxin bound to albumin could be removed.

  In the operation at pH = 7.4, albumin and indoxyl sulfate are negatively charged, so they are electrically attracted to the positive electrode, and in electrodialysis, the positive electrode side is acidic, so that the binding between toxin and albumin is released. It becomes easy. The electrodialysis method removes the binding of albumin and toxin and removes the toxin efficiently by two mechanisms of electric force and pH change.

The schematic diagram of the electrodialysis tank part of an albumin binding toxin removal apparatus. The graph which shows a time-dependent change of the amount of bilirubin removed (mg) (♦ represents bound bilirubin, and □ represents free bilirubin). The graph which shows the time-dependent change of indoxyl sulfate removal amount (mg) (♦ represents bound indoxyl sulfate, and □ represents free indoxyl sulfate).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Electrodialysis tank part 2 of albumin binding toxin removal apparatus Electrodialysis tank 3 Negative electrode 4 Cation exchange membrane 5 Anion exchange membrane 6 Positive electrode 7 Acidic region 8 Plasma 9 Albumin 10 Toxin

Claims (11)

  A method of separating albumin and a biomolecule by electrodialyzing a solution containing albumin bound with a biomolecule.   The method according to claim 1, wherein the biomolecule is a substance harmful to a living body.   The method according to claim 2, wherein the harmful substance is a substance that is generated due to a disease or whose amount is increased due to a disease state.   The method according to claim 3, wherein the disease is kidney disease or liver disease.   The method according to claim 4, wherein the harmful substance is bilirubin and / or indoxyl sulfate.   A kidney disease characterized by removing plasma components from the living body, removing harmful biomolecules from albumin using the method according to any one of claims 1 to 5, and returning the obtained albumin molecule to the living body. Or a method for treating liver disease.   A method for treating liver disease or liver failure, wherein the harmful biomolecule is bilirubin.   A pump means for delivering a solution containing albumin between the ion exchange membranes, a potential difference applying means for applying a voltage between the ion exchange membranes, a means for recovering a liquid containing a biomolecule that has permeated the ion exchange membranes, and an ion exchange membrane. An albumin electrodialyzer comprising means for recovering a solution containing albumin flowing out.   The apparatus according to claim 8, wherein the voltage is controlled so that a pH gradient is generated between the ion exchange membranes when a voltage containing a solution containing albumin is filled between the ion exchange membranes.   The apparatus according to claim 8 or 9, comprising a plurality of ion exchange membrane flow paths composed of a plurality of pairs of ion exchange membranes, wherein a potential difference is applied between the ion exchange membranes constituting each ion exchange membrane pair. The apparatus according to any one of claims 8 to 10, comprising means for separating plasma components from blood and / or means for adding electrolytes and / or other blood components to a solution containing recovered albumin.

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