JP5308171B2 - Absorption column for body fluid purification treatment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adsorption column for body fluid purifying treatment, capable of significantly reducing the capacity of the column or significantly reducing the treatment time by eliminating the influence of a co-adsorbed substance whose molecular weight is smaller than that of a substance to be adsorbed. <P>SOLUTION: The adsorption column for body fluid purifying treatment includes adsorbers with functional groups anchored on the surface of porous carriers. The adsorption column includes an auxiliary adsorber 1 for adsorbing and removing the co-adsorbed substance from the body fluid to be treated in advance, and a main adsorber 2 for adsorbing and removing the substance to be adsorbed from the body fluid to be treated after the removal of the co-adsorbed substance by the auxiliary adsorber. The porous carrier of the respective adsorbers includes a scaffold of a three-dimensional mesh structure, three-dimensional mesh-like through holes formed in gaps of the scaffold, and pores dispersed on the surface of the scaffold and penetrating inward from the surface of the scaffold. The diameter of the center pore measured by the nitrogen adsorbing method in the porous carrier of the auxiliary adsorber is smaller than the maximum length of the substance to be adsorbed. The lower limit of the distribution range of the diameters of the pores measured by the nitrogen adsorbing method is not more than 2 nm, and the upper limit of the distribution range is larger than the maximum value of the maximum length of the co-adsorbed substance. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an adsorption column for body fluid purification treatment in which a functional group that specifically binds to a substance to be adsorbed is immobilized on the surface of a porous carrier, and in particular, etiology such as LDL (low density lipoprotein) in blood The present invention relates to an adsorption column for apheresis treatment for the purpose of adsorption removal of substances.

  Apheresis treatment involves humoral factors (proteins and immune-related substances such as cytokines that are present in the blood by binding to proteins) and cells (lymphocytes, granulocytes, This is a treatment method that aims to improve the pathological condition by removing viruses, etc.).

  It is well known that LDL in lipoproteins present in the blood contains a high amount of cholesterol, and hyper-LDLemia in which the blood concentration of LDL increases increases the risk of arteriosclerosis and the risk of myocardial infarction and cerebral infarction. ing. Hereinafter, unless otherwise specified, LDL is assumed to include cholesterol, and means LDL cholesterol which is a complex of low density lipoprotein and cholesterol. Although LDL apheresis treatment has been put to practical use as one of the treatment methods for hyper-LDLemia, it is mainly familial (hereditary) hyper-LDLemia and obstruction because of the high treatment costs and the burden on patients. Currently, it is performed only for severe patients who are not effective for drug treatment, such as atherosclerosis and focal glomerulosclerosis.

  The LDL apheresis treatment is an adsorption-type apheresis treatment in which blood or plasma of a patient is perfused through an adsorption column in which a functional group having affinity for LDL, which is an adsorption target substance, is immobilized on the surface of a carrier. This is a treatment method that adsorbs and removes LDL from the blood of a patient circulated extracorporeally. Examples of LDL adsorption columns include Kaneka Liposorba (registered trademark) (see Patent Literature 1 and Non-Patent Literature 1 below), DALI System of Fresenius (Germany) and others (see Patent Literature 2 below). These LDL adsorption columns have a bead-like porous cellulose gel having a large number of pores or a support surface made of polyacrylamide, dextran sulfate, polyacrylic acid, etc. as functional groups (ligands) having affinity for LDL. The chain polyvalent acid is surface-modified and immobilized, and the surface-modified beads are packed in a column container. The bead diameter is about 250 μm for the direct blood perfusion method and about 50 μm for the plasma perfusion method.

Japanese Examined Patent Publication No. 4-511430 Japanese Patent Publication No. 3-254756 Japanese Examined Patent Publication No.3-39736 JP 7-41374 A

Takashi Tani, “Development of blood adsorption device”, Medical Instrumentation, Vol. 58, no. 6, pp. 266-273, 1988

  However, the LDL adsorption column using porous cellulose gel beads and the like that are currently in practical use as a carrier has the following problems.

  First, the porous cellulose gel beads are thread-like with entangled cellulose fibers, and the pore size distribution in the beads is 0.1 to 1 μm, which is 4 to 40 times the diameter of spherical LDL molecules (about 26 to 27 μm). Since it is large and broad (see Non-Patent Document 1 (4.1 Support)), the surface area per pore volume decreases with the increase in pore diameter, and the adsorption efficiency decreases. In addition, since the beads are thread-like, it is difficult to control the distribution of the pore diameter. When the pore diameter is less than or equal to the diameter of the LDL molecule, LDL is not adsorbed on the pore surface and the adsorption efficiency decreases. To do.

  Secondly, the blood or plasma sent into the adsorption column flows by the blood flow through the gaps between the beads, but the movement of LDL, which is the adsorption target substance, to the pore surface inside the beads is diffused. Furthermore, since the carrier is in the form of beads and its diameter is larger than the pore diameter, LDL is adsorbed only on the pore surface near the bead surface, and all of the pore surface area extending from the bead surface to the deep part. Cannot be used effectively.

  Third, if the bead diameter is reduced in order to improve the second problem, the gap between the bead-shaped carriers is narrowed, the flow resistance of the blood flow is increased, the pressure loss is increased, and the blood flow is increased. The flow rate cannot be increased. Assuming that the bead-shaped carrier is packed tightly in the column container, the diameter of the flow path passing through the narrowed portion of the gap between each bead-shaped carrier is narrowed to about 15% of the bead diameter, and the narrowed portion is It is considered that the pressure loss increases because the channel surrounded by the bead-shaped carrier from four directions and the flow path entering the constricted portion from one direction is largely bent by the facing bead-shaped carrier.

  Fourth, a soft gel such as a porous cellulose gel has a higher pressure loss. When blood or plasma is fed at a high flow rate, consolidation occurs and blood or plasma does not flow above a certain flow rate.

  Fifth, since polymer carriers such as porous cellulose gel contain a small amount of additives, protein adsorption in the blood and activation of the blood coagulation mechanism are likely to occur.

  In summary, the LDL adsorption column using the porous cellulose gel beads that are currently in practical use as the carrier has problems caused by the carrier being porous cellulose gel (first, fourth, and fifth problems). ) And problems (first, second and third problems) caused by the support being in the form of beads.

  For the fourth problem caused by the carrier being a porous cellulose gel, that is, the problem that the pressure loss is high and blood or plasma does not flow above a certain flow rate, a solution in which the carrier is composed of an inorganic porous material, This is proposed in Patent Document 3 above. However, in Patent Document 3, the relationship between pressure and flow rate is compared for porous glass beads (particle size 80 to 120 mesh) and agarose gel beads (particle size 50 to 100 mesh) as carriers. The flow rate does not increase, but the flow rate increases with increasing pressure, whereas the agarose gel beads only reveal that the flow rate does not increase (see FIG. 1 of Patent Document 3). Here, the bead diameter of the porous glass bead carrier is as large as about 250 μm assuming a direct blood perfusion method, and the diameter of the flow path passing through the narrowed portion of the gap between each bead-like carrier is also large. ing. Therefore, no consideration or evaluation is made on the influence of the bead diameter on pressure loss or flow velocity. Furthermore, in Patent Document 3, although there is a description that “any shape such as granular, fibrous, membrane, or holofiber can be selected as the shape of the carrier”, the actual evaluation is a bead shape (spherical shape). ) It is carried out on the carrier, and no consideration or evaluation is made on the influence of the carrier shape on the pressure loss or flow velocity.

  Further, in Patent Document 3, performance evaluation as an LDL adsorbent using a bead-shaped carrier is performed, but in a state where blood or plasma is flowed in accordance with the use form of an LDL adsorption column that is currently in practical use. This is not a dynamic adsorption test, but is a static adsorption test in which plasma is added to a test tube containing a spherical adsorbent, stirred and left at 20 ° C. for 15 minutes, and then the LDL concentration of the supernatant is measured. The influence of the bead diameter of the carrier, that is, the influence of the pressure loss in a state where blood or plasma is passed through the adsorption column has not been sufficiently evaluated. Moreover, since evaluation by a dynamic adsorption test has not been performed at all, no consideration has been given to the influence of the bead diameter or the carrier shape on the adsorption performance in an actual use state as an LDL adsorption column.

  In addition, due to the first and second problems described above, in the current LDL adsorption column, the saturated adsorption amount of LDL is limited to about 6 mg / mL. Therefore, the non-regenerative type column capacity is 400 to 1000 mL. In the case of the regenerative type, 150 mL × 2 columns and a complicated regeneration system are required. Furthermore, since it takes a long time to reach saturated adsorption, the time required for the treatment is as long as 2 to 3 hours, which imposes a heavy burden on the patient.

  Therefore, as a result of diligent investigation focusing on the fact that there is room for improvement in the material and shape of the carrier of the current LDL adsorption column, a functional group that specifically binds to the substance to be adsorbed is fixed on the surface of the porous carrier. In the adsorption column for body fluid purification treatment, instead of the conventional configuration in which a bead-shaped adsorbent in which a functional group is fixed to a bead-shaped porous carrier is packed in the column, a porous carrier is used as a three-dimensional porous carrier. An inorganic skeleton made of silica gel or silica glass having a network structure, and a three-dimensional network-like through hole formed in the gap between the skeleton, and penetrates from the surface to the inside of the skeleton having a smaller diameter than the through hole. By using an integrated porous carrier with pores dispersed in the skeleton, and optimizing the average pore diameter of the through-holes and pores, the problems of conventional adsorption columns can be solved. Improve performance Found that are separately patents do patent application prior to the present application (Japanese Patent Application No. 2007-236564).

  Further, the inventors of the present application have a selective adsorption performance for the adsorption target substance due to the influence of the co-adsorption substance having a molecular weight smaller than that of the adsorption target substance contained in the body fluid to be treated in the novel adsorption column proposed in the above prior application. It was confirmed that the selective adsorption performance for the adsorption target substance can be greatly improved by finding the decreasing phenomenon and separating and adsorbing the adsorption target substance and the co-adsorption substance.

  Specifically, plasma has a variety of large and small molecules. In addition to LDL having a molecular weight of about 3500 kDa (kilodalton), peptides and small molecules having molecular weights in the range of about 5 kDa to 340 kDa are present in the LDL adsorption column. It is adsorbed specifically. Since these peptides and small molecule proteins are adsorbed together with LDL as a co-adsorbing substance of the LDL adsorption column and inhibit the adsorption of LDL, the amount of LDL adsorption per column volume tends to decrease. In addition, since the amount of coadsorbing substance in plasma varies depending on the patient, it is difficult to provide the column with a stable LDL removal capability.

  The present invention has been made in view of the newly found problems in the above-described novel adsorption column, and its purpose is to provide a functional group that specifically binds to at least a substance to be adsorbed including low-density lipoprotein. In an adsorption column for body fluid purification treatment that has an adsorbent that is fixed on the surface of a porous carrier, the effect of co-adsorbents with a molecular weight lower than that of the substance to be adsorbed can be eliminated, and the column capacity can be greatly reduced or treated. An object of the present invention is to provide an adsorption column for body fluid purification treatment that can significantly reduce the time.

  The adsorption column for body fluid purification treatment according to the present invention for achieving the above object is an adsorption column in which a functional group that specifically binds to a substance to be adsorbed containing at least low-density lipoprotein is immobilized on the surface of a porous carrier. An adsorption column for body fluid purification treatment comprising a body, wherein an adsorbent adsorbs and removes in advance a coadsorbent having a molecular weight smaller than that of the substance to be adsorbed from a body fluid to be treated, and the coadsorbent in the auxiliary adsorbent A main adsorbent that adsorbs and removes the substance to be adsorbed from the body fluid to be adsorbed and removed. The auxiliary adsorbent and the porous carrier of the main adsorbent are made of silica gel or silica glass having a three-dimensional network structure. A skeleton body, three-dimensional network-like through-holes formed in the gaps of the skeleton body, and pores penetrating from the surface of the skeleton body to the inside formed dispersed on the surface of the skeleton body. Shi The center pore diameter measured by the nitrogen adsorption method of the pores in the porous carrier of the auxiliary adsorbent is smaller than the maximum length of the substance to be adsorbed, and the pores measured by the nitrogen adsorption method of the pores The lower limit of the diameter distribution range is 2 nm or less, the upper limit of the pore diameter distribution range is larger than the maximum value of the maximum length of the coadsorbent, and the pore nitrogen in the porous support of the main adsorbent The first feature is that the central pore diameter measured by the adsorption method is larger than the maximum length of the substance to be adsorbed.

  Here, the surface of the porous carrier corresponds to the inner wall surfaces of the through holes and pores of the porous carrier, and the surface of the skeleton body agrees with the inner wall surfaces of the through holes. In the present invention, the central pore diameter is a value (dV / d) obtained by differentiating the pore volume (V) in the pore distribution measured by the nitrogen adsorption method from the common logarithm (logD) of the pore diameter (D). (LogD), hereinafter referred to as “differential pore volume”) is defined as the pore diameter showing the maximum peak of a curve (hereinafter referred to as “pore distribution curve”) plotted against pore diameter (D). The In the definition of the central pore diameter, not the pore diameter (D) but the common logarithm (logD) is used because the pore diameter (D) is in a relatively small region of about 2 nm to 15 nm. This is for accurately detecting a case where a change in volume (V) appears.

  Furthermore, in addition to the first feature, the adsorption column for body fluid purification treatment according to the present invention has a central pore diameter of 10 nm measured by the nitrogen adsorption method of the pores in the porous carrier of the auxiliary adsorbent. The second feature is that the thickness is 26 nm or more.

  Furthermore, the adsorption column for body fluid purification treatment according to the present invention, in addition to the first or second feature, has a central pore diameter measured by a nitrogen adsorption method of the pores in the porous carrier of the main adsorbent. The third feature is that is at least twice as large as the maximum length of the substance to be adsorbed.

  Furthermore, the adsorption column for body fluid purification treatment according to the present invention includes the auxiliary adsorbent on the upstream side in the path through which the body fluid in the single container flows, in addition to any of the first to third features. The fourth feature is that the main adsorbent is provided on the downstream side.

  Furthermore, in addition to any one of the first to third features, the adsorption column for body fluid purification treatment according to the present invention includes the auxiliary adsorbent in the single container upstream of the body fluid passage through which the body fluid flows. A fifth feature is that an auxiliary adsorption column is provided, and a main adsorption column is provided on the downstream side of the body fluid passage, and the main adsorbent is filled in another single container. .

  Furthermore, the adsorption column for body fluid purification treatment according to the present invention has a functional group that specifically binds to a substance to be adsorbed containing at least low-density lipoprotein fixed on the surface of a porous carrier, and the adsorbing column from the body fluid to be treated. An adsorbing column for body fluid purification treatment comprising an auxiliary adsorbent that previously adsorbs and removes a co-adsorbing substance having a molecular weight smaller than that of the target substance, wherein the porous carrier of the auxiliary adsorbent is a three-dimensional network structure silica gel or A skeleton body made of silica glass, a three-dimensional network-like through-hole formed in a gap between the skeleton bodies, and a pore penetrating from the surface of the skeleton body to the inside formed dispersed on the surface of the skeleton body A central pore diameter measured by a nitrogen adsorption method of the pores in the porous carrier of the auxiliary adsorbent is smaller than a maximum length of the substance to be adsorbed, and a nitrogen adsorption method of the pores Measured in The lower limit of the distribution range of the pore diameter of at 2nm or less, the upper limit of the distribution range of the pore diameter and the sixth aspect of the greater than the maximum value of the maximum length of the co-adsorbent material.

  Furthermore, in addition to the sixth feature, the adsorption column for body fluid purification treatment according to the present invention has a central pore diameter of 10 nm measured by a nitrogen adsorption method of the pores in the porous carrier of the auxiliary adsorbent. The seventh characteristic is that the thickness is 26 nm or more.

  Further, the adsorption column for body fluid purification treatment according to the present invention is a dextran sulfate having an affinity that the functional group specifically binds to low density lipoprotein in addition to any of the first to seventh features. Alternatively, the eighth feature is that it is a salt thereof, polyacrylic acid or a salt thereof, or other chain polyvalent acid or a salt thereof.

  Furthermore, the adsorption column for body fluid purification treatment according to the present invention is characterized in that, in addition to any of the first to eighth features, the porous carrier is synthesized by a spinodal decomposition sol-gel method. And

  According to the adsorption column for body fluid purification processing according to the first or second feature, the body fluid to be treated flows when the body fluid flows through the auxiliary adsorbent whose central pore diameter is smaller than the maximum length of the substance to be adsorbed. Most of the adsorption target substances cannot enter the pores of the auxiliary adsorbent and are not removed by adsorption. However, many coadsorbents with different molecular weights than the adsorption target substance have different pore diameter distributions of the auxiliary adsorbents. Since the range covers the maximum length (molecular size) of each co-adsorbing substance, it enters the pores of the auxiliary adsorbent and is adsorbed and removed. Therefore, in the auxiliary adsorbent, the adsorption target substance with a large molecular weight is prevented from unnecessarily entering the pores and blocking the entrance of the pores, so that the co-adsorbed substance with a small molecular weight enters the deep pores. Thus, the co-adsorbing substance having a molecular weight smaller than that of the substance to be adsorbed is selectively adsorbed and removed from the liquid to be treated flowing through the auxiliary adsorbent. Further, comparing the case where the central pore diameter of the auxiliary adsorbent is smaller than the maximum length of the substance to be adsorbed, the surface area of the porous carrier to which the functional group is fixed is larger in the case where it is smaller than in the case where it is smaller. Therefore, the adsorption capacity of the co-adsorbing substance is increased. Further, since the co-adsorbing material has been selectively adsorbed and removed by the auxiliary adsorbing body from the liquid to be treated flowing through the main adsorbing body, the co-adsorbing material enters the pores of the main adsorbing body and is shared. Since it is not adsorbed, it is reduced that the adsorption of the adsorption target substance on the main adsorbent is hindered by the co-adsorption substance, and the adsorption removal efficiency of the adsorption target substance on the main adsorbent is improved.

  The relationship between the central pore diameters of the auxiliary adsorbent and the main adsorbent and the maximum length of the adsorbed substances (adsorption target substance and coadsorbed substance) will be described in more detail.

  Since the pore diameter of the pores formed by being dispersed in the skeleton of the auxiliary adsorbent is distributed from 2 nm or less, which is the measurement lower limit of the nitrogen adsorption method, to the maximum value of the maximum length of the coadsorbent, The distribution range of the pore diameter covers all the maximum lengths of large and small coadsorbents with different molecular weights (or the diameter or maximum diameter if the coadsorbent is spherical, elliptical spherical, or disk-shaped). . Therefore, the auxiliary adsorbent was fixed on two types of surfaces, the surface exposed toward the through-hole of the skeleton (through-hole surface) and the surface exposed toward the pore of the skeleton (pore surface). The functional group can be used for adsorption of the co-adsorbing substance. As a result, the auxiliary adsorbent can effectively adsorb large and small coadsorbents having different molecular weights by effectively utilizing the pore surface. Furthermore, since the central pore diameter of the auxiliary adsorbent is set smaller than the maximum length of the adsorption target substance, the adsorption target substance cannot move into the pore or becomes extremely difficult, and the adsorption of the adsorption target substance is exclusively through-holes. It depends on the functional group fixed on the surface, and the functional group fixed on the surface of the pore having a larger surface area than the surface of the through-hole is exclusively used for the adsorption of the co-adsorbing substance. Increases adsorption capacity for substances. Here, if the diameter of the central pore of the auxiliary adsorbent is set to be greater than or equal to the maximum value of the maximum length of the coadsorbent within a range smaller than the maximum length of the substance to be adsorbed, the coadsorbent moves to the deep part in the pore. It is possible to improve the adsorption performance by using the functional group fixed on the pore surface more efficiently, but if the central pore diameter exceeds the maximum length of the adsorption target substance, Since the surface area is reduced and the number of functional groups used for the adsorption of the co-adsorbing substance is reduced, the adsorption performance for the co-adsorbing substance is lowered.

  When the adsorption target substance is LDL, the molecular weight is 3500 kDa and the molecular diameter is 26 to 27 nm. Therefore, it is preferable that the central pore diameter of the auxiliary adsorbent is 26 nm or less. In addition, as a co-adsorption substance when the adsorption target substance is LDL, as will be described later, peptides and small molecule proteins having a molecular weight of about 5 kDa to 340 kDa and a maximum molecular length of about 3 nm to 13 nm are adsorbed (see FIG. 7, see FIG. 8), and the center pore diameter of the auxiliary adsorbent is preferably 10 nm or more. When the pore diameter is measured by the nitrogen adsorption method, the pore diameter distribution extends to about + 50% of the central pore diameter, and when the central pore diameter is 10 nm, the pore diameter distribution. The upper limit of the range is about 15 nm, and the pore surface can be effectively used for adsorption of the co-adsorbing substance having a molecular weight of 340 kDa. When the central pore diameter is 26 nm, the upper limit of the pore diameter distribution range is about 40 nm, and it is expected that LDL is adsorbed to some extent in such large pores. Most of the pore volume is concentrated in the pore diameter near the central pore diameter (see FIG. 5B), and in the region where the pore diameter is larger than the central pore diameter, the pore volume is small and is used for adsorption. Therefore, it is considered that the influence of LDL adsorption in large pores can be ignored.

  Next, the central pore diameter of the main adsorbent is set to be larger than the maximum length of the adsorption target substance, so that the functional groups fixed on the two kinds of surfaces, the through-hole surface and the pore surface, are absorbed by the adsorption target substance. It becomes possible to use for adsorption. That is, if the central pore diameter is smaller than the maximum length of the substance to be adsorbed (if the substance to be adsorbed is a sphere, an ellipsoidal sphere or a disk, the diameter or maximum diameter corresponds to the case where the substance to be adsorbed is LDL). The substance to be adsorbed cannot move into the pores or becomes extremely difficult, and the adsorption of the substance to be adsorbed is solely due to the functional group fixed on the surface of the through hole. On the other hand, if the central pore diameter is larger than the maximum length of the substance to be adsorbed, at least one substance to be adsorbed moves into the pore and can be adsorbed to the functional group fixed on the surface of the pore. The surface area of the effective skeletal body that is subjected to is increased.

  Furthermore, according to the adsorption column for purifying body fluid according to the first or second feature, since the inorganic silica gel or silica glass is used as the porous carrier, the porous carrier in the conventional adsorption column described above is high. The problem caused by the molecule or soft gel and the problem caused by the porous carrier being in the form of beads are solved at the same time.

  Specifically, by using an integrated porous carrier having a three-dimensional network structure skeleton body and a three-dimensional network-like through-hole formed outside the skeleton body fluid (for example, body fluid purification target) Since the through-holes that serve as the flow channels for the plasma) communicate with each other in a three-dimensional network, the diameter of the through-holes is the narrowing of the gaps between the bead-like carriers when the bead-like carriers are closely packed in the column container. Even if the diameter of the passage is the same as the passage diameter, the passage resistance can be kept low, and the pressure loss can be kept low compared to the bead-shaped carrier. Moreover, in both the bead-like support and the three-dimensional network structure, the movement of the substance to be adsorbed in the pores is caused by diffusion, and the functional groups fixed to the pores near the surface of each support are exclusively effective. Assuming that it functions, the three-dimensional network structure with a larger surface area per unit volume has a pore surface area where the functional group functions more effectively than a bead shape (spherical) with a smaller surface area. You can make it bigger. Therefore, with the same flow path diameter, the three-dimensional network structure has lower pressure loss and larger effective pore area than the bead-shaped carrier. That is, the three-dimensional network structure can further reduce the channel diameter and further increase the effective pore area than the bead-like carrier.

  Here, when the pore diameter is increased to the same level as the through-hole diameter, it is no longer regarded as a pore but equivalent to a through-hole, and does not meet the condition that the pores are dispersed on the surface of the skeleton. Accordingly, the porous support having a three-dimensional network structure used in the auxiliary adsorbent and the main adsorbent of the present invention is characterized by a three-dimensional network-like through-hole and a hierarchy of two types of pore sizes smaller than that. This is a typical porous structure. In other words, by utilizing such a characteristic of the porous structure, it is possible to set conditions for greatly improving the performance of an adsorption column using a conventional bead-shaped porous body.

  In addition, when the flow path diameter and the through-hole diameter are the same when the bead-shaped carrier is closely packed in the column container, the three-dimensional network structure skeleton body can be made smaller in diameter than the bead-shaped carrier. The stretch length extending from the support surface to the inside is shorter in the three-dimensional network structure than in the bead-like support, and the movement distance due to the diffusion of the substance to be adsorbed into the pore is shortened. The adsorption efficiency to the functional group is improved. In other words, compared to conventional adsorption columns using bead-like carriers, the time to saturation adsorption is shortened, and a high-performance adsorption column can be used to the maximum in a short time, reducing the time required for treatment. The burden on the patient can be greatly reduced.

  Further, according to the adsorption column for body fluid purification processing of the third feature, the central pore diameter of the main adsorbent is set to be twice or more larger than the maximum length of the substance to be adsorbed, so that the pore surface The fixed functional group can be more efficiently used for adsorption of the adsorption target substance. In other words, if the central pore diameter is more than twice as large as the maximum length of the substance to be adsorbed, other substances to be adsorbed move into the same pore by the substance to be adsorbed by the functional group fixed on the pore surface. Therefore, the larger the pore diameter, the greater the depth of the pore (distance from the surface of the skeleton body) that is effectively used for adsorption. The substance to be adsorbed increases by being moved to. However, if the central pore diameter is large, the number of pores per unit surface area of the skeleton decreases, so even if the central pore diameter is increased beyond a certain range, the adsorption performance does not increase. Although the upper limit of the certain range depends on the through-hole diameter and the size of the substance to be adsorbed, it is not uniquely determined, but the measurement of the examples described in the specification of the above-mentioned prior application (Japanese Patent Application No. 2007-236564) From the result, it is considered to be about 6 to 10 times the maximum length of the substance to be adsorbed. However, the central pore diameter can be increased beyond the certain range as long as the adsorption performance is not significantly reduced.

  Furthermore, according to the adsorption column for body fluid purification treatment of the fourth feature, the auxiliary adsorbent and the main adsorbent are provided in the single container, so that the adsorption column having only the main adsorbent in the apheresis treatment is provided. It can be handled. Since the auxiliary adsorbent has a smaller central pore diameter than the main adsorbent, a large pore surface area can be secured with a smaller capacity than the main adsorbent, so that the volume of the auxiliary adsorbent can be significantly smaller than that of the main adsorbent. Therefore, it is convenient to accommodate the auxiliary adsorbent and the main adsorbent in the single container.

  Furthermore, according to the adsorption column for body fluid purification processing of the fifth feature, the auxiliary adsorption column provided with the auxiliary adsorbent and the main adsorption column provided with the main adsorbent are provided separately. Utilization methods such as exchanging the main adsorption column individually become possible. As an example, when only the adsorption performance of one of the auxiliary adsorption column and the main adsorption column is lower than that of the other, only the one adsorption column may be replaced.

  According to the adsorption column for body fluid purification treatment of the sixth or seventh feature, when the body fluid to be treated flows through the auxiliary adsorbent having a center pore diameter smaller than the maximum length of the substance to be adsorbed, Most of the adsorption target substances in the body fluid cannot enter the pores of the auxiliary adsorbent and are not removed by adsorption. However, a large number of coadsorbents having different molecular weights than the adsorption target substance have a pore diameter of the auxiliary adsorbent. Since the distribution range covers the maximum length (molecular size) of each co-adsorbing substance, it enters the pores of the auxiliary adsorbent and is removed by adsorption. Therefore, in the auxiliary adsorbent, the adsorption target substance with a large molecular weight is prevented from unnecessarily entering the pores and blocking the entrance of the pores, so that the co-adsorbed substance with a small molecular weight enters the deep pores. Thus, the co-adsorbing substance having a molecular weight smaller than that of the substance to be adsorbed is selectively adsorbed and removed from the liquid to be treated flowing through the auxiliary adsorbent. Further, comparing the case where the central pore diameter of the auxiliary adsorbent is smaller than the maximum length of the substance to be adsorbed, the surface area of the porous carrier to which the functional group is fixed is larger in the case where it is smaller than in the case where it is smaller. Therefore, the adsorption capacity of the co-adsorbing substance is increased. Therefore, since the co-adsorbed substance is adsorbed and removed from the liquid to be treated after flowing through the auxiliary adsorbent, the adsorption target substance is removed by adsorbing and removing the substance to be adsorbed on the liquid to be treated. It is reduced that the adsorption is hindered by the co-adsorbing substance, and the adsorption removal efficiency of the adsorption target substance is improved. The relationship between the center pore diameter of the auxiliary adsorbent and the maximum length of the adsorbed substances (adsorption target substance and coadsorbed substance) and the point of using inorganic silica gel or silica glass as the porous carrier are as described above. Because there is, the explanation which overlaps is omitted.

  Furthermore, according to the adsorption column for body fluid purification processing of the eighth feature, when the adsorption target substance is LDL (molecular diameter is about 26 to 27 nm), the auxiliary adsorbent is more than the LDL that is the co-adsorption substance. Low molecular weight peptides and small molecule proteins are effectively adsorbed and removed.

  Furthermore, according to the adsorption column for body fluid purification treatment of the ninth feature, by using the spinodal decomposition sol-gel method, controllability of the through-hole diameter and the pore diameter is improved, so that the through-hole diameter and the pore diameter are within an appropriate range. An adjusted high-performance adsorption column can be provided.

The block diagram which shows typically the schematic structure in 1st Embodiment of the adsorption column for bodily fluid purification processes which concerns on this invention. Sectional drawing which shows the principal part which shows typically the structure of the adsorption body of the adsorption column for bodily fluid purification processes which concerns on this invention Sectional drawing which shows the principal part which shows typically the structure of the porous support | carrier which comprises the adsorption body of the adsorption column for body fluid purification processes which concerns on this invention SEM photograph of the porous carrier constituting the adsorbent of the adsorption column for body fluid purification treatment according to the present invention Table of summary of pore measurement results by nitrogen adsorption method and pore distribution curve showing pore diameter distribution range of three kinds of samples having different pore diameters of the adsorbent of the adsorption column for body fluid purification treatment according to the present invention Graph The figure which shows the result of having comparatively evaluated the LDL adsorption | suction performance of five types of test columns filled with the adsorption body of the adsorption column of the bodily fluid purification process which concerns on this invention The figure which shows the analysis result by the electrophoresis of the co-adsorption substance adsorbed by the auxiliary | assistant adsorption body of the adsorption column for bodily fluid purification processing which concerns on this invention The figure which shows the mass spectrometry result of the co-adsorption substance adsorb | sucked by the auxiliary | assistant adsorption body of the adsorption column for body fluid purification processes which concerns on this invention Table and figure showing evaluation results of adsorption selectivity in auxiliary adsorbent and main adsorbent of adsorption column for body fluid purification treatment according to the present invention The block diagram which shows typically the schematic structure in 2nd Embodiment of the adsorption column for bodily fluid purification | cleaning processes which concerns on this invention. The block diagram which shows typically the schematic structure in another embodiment with respect to 1st Embodiment of the adsorption column for bodily fluid purification | cleaning processes which concerns on this invention. The block diagram which shows typically the schematic structure in another embodiment with respect to 2nd Embodiment of the adsorption column for bodily fluid purification | cleaning processes which concerns on this invention.

  An embodiment of an adsorption column for body fluid purification treatment according to the present invention (hereinafter, referred to as “the device of the present invention” as appropriate) will be described with reference to the drawings.

<First Embodiment>
As shown in FIG. 1, the device 10 of the present invention is configured such that a cylindrical auxiliary adsorbent 1 and a main adsorbent 2 are accommodated in a cylindrical container 3 with a partition plate 4 interposed therebetween. The auxiliary adsorbing body 1 and the main adsorbing body 2 are formed in the shape of a hollow cylinder having axial center portions 5 and 6 respectively. Openings 7 and 8 are formed on the respective end surfaces of the cylindrical container 3, and one opening 7 serves as an inlet for a body fluid to be treated (in this embodiment, blood plasma is assumed), and the other opening 8. Becomes the outlet for the body fluid after treatment. In addition, the arrow in FIG. 1 has shown the flow of the bodily fluid typically. In the device 10 of the present invention, the body fluid fed into the cylindrical container 3 from the opening 7 is blocked by the partition plate 4 from the inside to the outside of the cylindrical auxiliary adsorbing body 1 in the cylindrical container 3. Further, the body fluid that has passed through the auxiliary adsorbent 1 passes through the outer peripheral flow path 9 formed in the space between the outer walls of the auxiliary adsorbent 1 and the main adsorber 2 and the inner wall of the cylindrical container 3, and is cylindrical. The body fluid that flows from the outside to the inside of the main adsorbent 2 and passes through the main adsorbent 2 is sent out from the opening 8.

  As schematically shown in FIG. 2, the auxiliary adsorbent 1 and the main adsorbent 2 have functional groups 12 that specifically bind to the adsorption target substance on the surface of an inorganic porous carrier 11 formed into a cylindrical shape. The surface is modified and fixed. In this embodiment, LDL (molecular weight of about 3500 kDa) in blood is assumed as an adsorption target substance, and dextran sulfate or a salt thereof having affinity for LDL, polyacrylic acid or a salt thereof, as functional group 12; Alternatively, other chain polyvalent acids or salts thereof are used.

  An inorganic porous carrier 11 constituting the auxiliary adsorbent 1 and the main adsorbent 2 is formed in a gap between the three-dimensional network-like skeleton 13 and the skeleton 13 as schematically shown in FIG. The three-dimensional network-like through-holes 14 having an average pore diameter of 1 μm or more and less than 20 μm are formed, and the auxiliary adsorbent 1 and the main adsorbent 2 are different on the surface of the skeleton 13. The pores 15 having a pore diameter distribution are formed dispersed on the surface of the skeleton body 13. The pore 15 is a hole penetrating from the surface of the skeletal body 13 toward the inside. In the auxiliary adsorbent 1, the central pore diameter measured by the nitrogen adsorption method is the maximum length (26 mm to 27 nm), a lower limit of the distribution range of the pore diameter measured by the nitrogen adsorption method is 2 nm or less, and an upper limit is a coadsorbent having a lower molecular weight than LDL adsorbed to the main adsorbent 2 together with LDL (molecular weight is about 5 kDa). The pore diameter is adjusted to be larger than the maximum value (about 13 nm) of the maximum length (about 3 nm to 13 nm) of molecules of peptides and small molecule proteins in the range of ~ 340 kDa. The pore diameter is adjusted so that the center pore diameter measured in (1) is larger than the maximum length (27 nm) of LDL as the adsorption target substance. More specifically, the central pore diameter of each adsorbent measured by the nitrogen adsorption method is adjusted within the range of 10 nm to 26 nm for the auxiliary adsorbent 1, and 27 nm to 200 nm for the main adsorbent 2, more preferably , Adjusted to 60 nm to 120 nm. In the case of the auxiliary adsorbent 1, if the center pore diameter is in the range of 10 nm to 26 nm, the upper limit of the pore diameter distribution range is always equal to or greater than the maximum value (about 13 nm) of the maximum length of the coadsorbent substance. The pore diameter distribution range covers each maximum length of co-adsorbents of different molecular weights.

  Further, the porous carrier 11 of the auxiliary adsorbent 1 and the main adsorbent 2 used in the apparatus 10 of the present invention has a double pore structure composed of two kinds of pores (through holes 14 and 15) having different average pore diameters. It has become. Therefore, the upper limit of the pore diameter of the pores 15 is clearly smaller than the diameter distribution range of the through-holes 14 from a structural viewpoint. In FIG. 4, an example of the SEM (scanning electron microscope) photograph of the porous support | carrier 11 used with this invention apparatus 10 is shown.

  Next, examples of the auxiliary adsorbent 1 and the main adsorbent 2 in which dextran sulfate is immobilized as the functional group 12 on the surface of the porous carrier 11 and a method for producing the same will be described. First, a method for synthesizing the porous carrier 11 will be described. The method for synthesizing the porous carrier 11 used in the present embodiment uses the spinodal decomposition sol-gel method based on the principle disclosed in the above-mentioned “Method for producing an inorganic porous material” in Patent Document 4. .

<Synthesis of porous carrier: auxiliary adsorbent>
First, polyethylene oxide was dissolved in a range of 0.9 g to 1.1 g in 9 ml of 1.0 M (= mol / dm ³ ) nitric acid aqueous solution, 7 ml of tetraethoxysilane was added, and the mixture was stirred until it became homogeneous. The gel is left overnight in a constant temperature bath (step 1). Then, the obtained gel is immersed in 0.1M aqueous ammonia and reacted at 80 ° C. for 20 hours (step 2). Thereafter, the gel is dried and calcined at 600 ° C. for 5 hours to obtain a porous carrier made of silica gel or silica glass (step 3). When a porous carrier is produced under these conditions, the average pore diameter of the through holes of the porous carrier (sample # 1) is about 1.5 μm as measured by mercury porosimetry, and the nitrogen adsorption of the pores The pore diameter distribution range by the method is 2 nm to 30 nm, and the central pore diameter is about 21 nm.

<Synthesis of porous carrier: Sample for comparison of auxiliary adsorbent>
This is the same as the synthesis of the auxiliary adsorbent except that 1.0 M ammonia water is used for the treatment in the ammonia water in the step 2 of the synthesis of the porous carrier in the auxiliary adsorbent. When a porous carrier is produced under these conditions, the average pore diameter of the through holes of the porous carrier (sample # 2) is about 1.5 μm as measured by mercury porosimetry, and the nitrogen adsorption of the pores The pore diameter distribution range by the method is 10 nm to 40 nm, and the central pore diameter is about 33 nm. This comparative sample deviates from the condition that the center pore diameter of the auxiliary adsorbent is smaller than the maximum length (26 mm to 27 nm) of the LDL that is the adsorption target substance.

<Synthesis of porous carrier: main adsorbent>
This is the same as the synthesis of the auxiliary adsorbent except that the treatment temperature in the ammonia water in Step 2 of the synthesis of the porous carrier in the auxiliary adsorbent is 200 ° C. In addition, when a porous carrier is produced under these conditions, the average pore diameter of the through holes of the porous carrier (sample # 3) is about 1 μm to 5 μm as measured by mercury porosimetry, and the nitrogen adsorption of the pores The pore diameter distribution range by the method is 60 nm to 120 nm, and the central pore diameter is about 110 nm.

The results of pore measurement by the nitrogen adsorption method of samples # 1 to # 3 produced through the above steps 1 to 3 are shown in the list of FIG. 5A and the pore distribution curve graph of FIG. Show. Pore surface area by BET method, respectively in sample ^{# 1~ # 3, 560m 2 /} g, was 195m ² /g,45.9m 2 / g. Further, the pore volume by the BJH method, respectively in samples # 1 to # ^3, a ^{1.18cm 3 /g,1.06cm 3 /g,0.279cm 3 / g} , the distribution of the pore diameter by the BJH method The ranges are 2 to 30 nm, 10 to 40 nm, and 60 to 120 nm for Samples # 1 to # 3, respectively, and the central pore diameter is determined from the pore distribution curve of FIG. The value obtained by differentiating the pore volume with the common logarithm of the pore diameter) was given by the pore diameter showing the maximum peak, and was 21 nm, 33 nm, and 110 nm in Samples # 1 to # 3, respectively. FIG. 5B shows that most of the pore volume is concentrated in the pore diameter near the central pore diameter. The reason why the nitrogen adsorption method is used for the pore measurement is that the porous carrier with a small pore diameter whose pore diameter distribution reaches the measurement lower limit (about 2 nm) of the nitrogen adsorption method as in sample # 1. This is to avoid the use of mercury intrusion, which can destroy the skeleton. Therefore, in this example, the nitrogen adsorption method is used to compare the pore measurement results of samples # 1 to # 3 under the same conditions. In addition, in the pore measurement of a porous carrier having a large pore diameter like sample # 3, the mercury intrusion method may be used individually.

  In addition, in the synthesis of samples # 1 to # 3, the pore diameter distribution range by the nitrogen adsorption method can be changed by changing the processing conditions in step 2, and the central pore diameter is adjusted to the optimum range. can do. From the measurement results of Samples # 1 to # 3, it can be seen that the pore diameter increases by increasing the treatment temperature in ammonia water or increasing the concentration of ammonia water.

  Furthermore, in the synthesis of Samples # 1 to # 3, when the amount of polyethylene oxide added in Step 1 is increased or decreased in the range of 0.9 g to 1.1 g, the average pore diameter of the through holes decreases as the amount of polyethylene oxide added decreases. The average pore diameter of the through holes can be adjusted by adjusting the amount of polyethylene oxide added to the aqueous nitric acid solution.

  In the above synthesis method, a solution of organic and inorganic compounds is mixed, gelation is advanced by hydrolysis reaction and dehydration condensation reaction of alkoxide, and the gel is dried and heated to produce an oxide solid. The gel method is used. Furthermore, by mixing the organic polymer with the starting solution of the sol-gel method, the phase separation structure formed by spinodal decomposition of the silica polymer produced with the progress of gelation and the solvent containing the organic polymer is obtained. This utilizes the characteristic that a porous gel having pores of the order of μm is formed by gelation. That is, according to the above method, a porous carrier can be produced by using a sol-gel method and causing spinodal decomposition (spinodal decomposition sol-gel method).

  In the case of sample # 3 (main adsorbent), the total porosity (measured by the mercury intrusion method) of the through holes and the pores of the porous carrier produced through steps 1 to 3 is about 50%. It is in the range of ˜70%, and is about 1.5 times larger than the porosity (about 40%) of the Kaneka Liposorber (model number LA-15), which is a conventional LDL adsorption column.

  Next, the surface of the porous carrier (samples # 1 to # 3) produced through the above steps 1 to 3 is surface-modified with dextran sulfate sodium having affinity for LDL as a functional group and immobilized. How to do will be described.

  First, the porous carrier prepared through the above steps 1 to 3 was refluxed in a 10% toluene solution of γ-aminopropyltriethoxylane for 3 hours, washed with ethanol, and γ-aminopropylated porous. A carrier is obtained (step 4). The amount of γ-aminopropyl group immobilized by the organic trace element analysis under the above conditions is 0.3 mmol / mL.

Next, 10 mL of 1/300 M phosphate buffer aqueous solution (pH 7. 5) in which 100 mg of dextran sulfate sodium (500 mg in the prior application) is dissolved in 5 mL of the γ-aminopropylated porous carrier obtained through the above step 4. 0), γ-aminopropylated porous carrier is immersed in the aqueous solution, and shaken at 60 ° C. for 3 days (step 5). After the reaction, the porous carrier is immersed in 1% NaBH ₄ aqueous solution for 15 minutes, and then washed sequentially with pure water and physiological saline (in the order of description) to immobilize sodium dextran sulfate on the surface of the porous carrier. Thus obtained adsorbent (auxiliary adsorbent, auxiliary adsorbent comparative sample, main adsorbent) is obtained (step 6). The amount of sodium dextran sulfate immobilized by the fluorescent X-ray diffraction method under the above conditions is 10 mg / mL.

  Next, in order to compare and evaluate the LDL adsorption performance of the three types of adsorbents (auxiliary adsorbent, comparative sample, main adsorbent) produced through the above steps 1 to 6, the following five types of tests # 1 Since ~ # 5 was performed, the test method and test results will be described.

Test # 1:
Prepare a test column (column # 1) in which 1 mL of the auxiliary adsorbent is packed in a small glass column container, wash 10 mL of physiological saline through column # 1, and then pass 5 mL of fresh human plasma. The whole amount of fresh human plasma passed through the outlet of column # 1 was collected.

Test # 2:
Prepare a test column (column # 2) filled with 1 mL of a sample for comparison in a small glass column container, wash 10 mL of physiological saline through column # 2, and then pass 5 mL of fresh human plasma. Then, the whole amount of fresh human plasma passed through the outlet of column # 2 was collected.

Test # 3:
Prepare a test column (column # 3) in which 1 mL of the main adsorbent is packed in a small glass column container, wash 10 mL of physiological saline through column # 3, and then pass 5 mL of fresh human plasma. The whole amount of fresh human plasma passed through the outlet of column # 3 was collected.

Test # 4:
Prepare a test column (column # 4) in which 1 mL each of the auxiliary adsorbent and the main adsorbent is packed in a small glass column container, and wash by passing 10 mL of physiological saline through column # 4. After 5 mL of human plasma was passed through the auxiliary adsorbent of column # 4, it was subsequently passed through the main adsorbent, and the total amount of fresh human plasma passed through the outlet of column # 4 was recovered.

Test # 5:
Prepare a test column (column # 5) in which 1 mL each of the sample for comparison and the main adsorbent are packed in a small glass column container, and wash by passing 10 mL of physiological saline through column # 5 and then fresh. After 5 mL of human plasma was passed through the auxiliary adsorbent of column # 5, it was subsequently passed through the main adsorbent, and the total amount of fresh human plasma passed through the outlet of column # 5 was recovered.

  For the above tests # 1 to # 5, LDL concentrations Ci and Co (mg / dL) in plasma before and after passing were measured, and LDL per 1 mL of each adsorbent in each test column (columns # 1 to # 5) The adsorption capacity A (mg / mLgel) was calculated by the calculation formula shown in the following formula 1. The LDL concentrations Ci, Co and LDL adsorption capacity A in the plasma before and after passing through are summarized in the list of FIG.

[Equation 1]
A = 10 × (Ci-Co) / 100

  From FIG. 6, in tests # 1 and # 2, LDL adsorption is not confirmed in column # 1 filled with only the auxiliary adsorbent and column # 2 filled with only the comparative sample. In Test # 1, the center pore diameter of the auxiliary adsorbent (sample # 1) is 21 nm, which is smaller than the maximum length of LDL, and thus it is considered that the pore surface hardly contributes to LDL adsorption. For test # 2, the center pore diameter of the comparative sample (sample # 2) is 33 nm, which is larger than the maximum length of LDL, and satisfies the condition of the center pore diameter of the main adsorbent, but the center pore diameter is Since it is very close to the maximum length of LDL, once it is adsorbed on the pore surface, it is not adsorbed in the deep part of the pore, so the chelating effect (pinching effect) is weak. It is considered that LDL adsorbed on the surface was subsequently separated and contained and recovered in the plasma after passing through. In column # 3 packed with only the main adsorbent of test # 3, the LDL adsorption amount is 7.9 mg / mLgel, and the LDL adsorption amount is significantly increased as compared with test # 2. This is because the central pore diameter of the main adsorbent of column # 3 (sample # 3) is about 110 nm, which is about four times the maximum length of LDL, so that LDL penetrates deep into the pores and the pore surface becomes LDL. It is thought that it contributes greatly to the adsorption. Also in Test # 3, LDL adsorbed on the surface of the through-hole of the main adsorbent and the pore surface near the inlet of the pore is subsequently separated and contained in the plasma after passing through, as in Column # 2. Although it is thought that it was recovered, it is considered that the LDL that penetrated and adsorbed to the deep pores did not leave due to the chelate effect. Also from this point, the central pore diameter of the main adsorbent is preferably about 2 to 6 times the maximum length of LDL.

  From FIG. 6, in the comparison of tests # 3 to # 5, column # 3 filled with only the main adsorbent, column # 4 filled with the auxiliary adsorbent and main adsorbent, and the sample for comparison and main adsorbent filled In column # 5, column # 4 had a larger amount of LDL adsorption than column # 3 and column # 5, and the coadsorbent was adsorbed and removed in advance in the auxiliary adsorbent provided before the main adsorbent. This shows that the amount of LDL adsorption in the main adsorbent is increased. Also in column # 5, the amount of LDL adsorption slightly increased compared to column # 3, but this is not a significant difference. That is, in the case of column # 5 packed with a comparative sample whose central pore diameter is larger than the maximum length of LDL, LDL temporarily adsorbed on the pore surface near the pore inlet of the comparative sample is a coadsorbing substance. It is considered that the amount of adsorption of the co-adsorbing substance of the comparative sample is reduced, and the amount of LDL adsorption on the main adsorbent is reduced accordingly. Further, the comparative sample has a smaller pore surface area due to the larger pore diameter as compared with the auxiliary adsorbent, and the amount of coadsorbed substance adsorbed decreases.

  In addition, in order to verify the effect of providing an auxiliary adsorbent before the main adsorbent as in column # 4 and preliminarily adsorbing and removing the coadsorbent, the LDL adsorbed amounts were compared in tests # 1 to # 5 above. It is considered that it is preferable to verify the difference in the adsorption amount of the co-adsorbing substance in the tests # 1 and # 2, and the co-adsorbing substance has various molecular weights from about 5 kDa to about 340 kDa. It is difficult to accurately grasp the amount of co-adsorbed substances individually and as a whole. Therefore, in this embodiment, the effect of the auxiliary adsorbent is verified by comparing the LDL adsorption amounts in tests # 1 to # 5.

  Furthermore, by washing column # 4 used in test # 4 with 1.0 M saline, the protein once adsorbed to the auxiliary adsorbent and main adsorbent in column # 4 flows out, It was confirmed that LDL was adsorbed again when plasma was passed through column # 4 after washing with 1.0 M saline. Thereby, it turns out that this invention apparatus 10 is activated by salt solution washing | cleaning.

  Next, the analysis result of the coadsorbed substance in the auxiliary adsorbent produced through the above steps 1 to 6 will be described.

  First, two types of analysis results of the coadsorbed material adsorbed on the auxiliary adsorbent will be described. In the first analysis, another column # 1A similar to the column # 1 of the test # 1 described above is prepared, 10 mL of physiological saline is passed through the column # 1A and washed, and 5 mL of fresh human plasma is passed through. Then, after washing column # 1A with physiological saline, it was further washed with 1.0M saline, and the 1.0M saline solution was recovered from the column outlet and electrophoresed on a polyacrylamide gel for CBB staining. . FIG. 7 shows the result of the electrophoresis. In FIG. 7, four electropherograms are shown, and the right end is a molecular weight marker. The second and third from the right end show electrophoresis of fresh human plasma and albumin solution, respectively, for reference. In the case of an albumin solution, the vicinity of 66 kDa is dark and albumin can be confirmed. At the left end, a band corresponding to a co-adsorbed component was detected over a range of about 35 kDa to over 300 kDa by electrophoresis of a 1.0 M saline solution recovered from the auxiliary adsorbent.

  As the second analysis, since it is difficult to analyze a molecular weight of 35 kDa or less in the electrophoretic analysis, the 1.0 M saline washing solution of the first analysis is desalted and MALDI-TOF / sinapinate is used as a matrix. Mass spectrometry by MS was performed. FIG. 8 shows the mass spectrometry result. As shown in FIG. 8, as a result of mass spectrometry, a large number of peaks considered to correspond to the co-adsorbed component were confirmed at 20 kDa or less.

  Next, an analysis of the following five types of substances in the auxiliary adsorbent and the main adsorbent produced through the above steps 1 to 6 and the measurement results of the adsorption amount of each substance will be described.

  Prepare another column # 1B similar to column # 1 of test # 1 above, collect 5 mL of serum through column # 1B, collect the total amount of serum that passed through the outlet of column # 1B, and use lipoprotein. (A) Analysis of apolipoprotein B (third analysis) was performed. Also, another column # 1C similar to column # 1 was prepared, and 5 mL of fresh human plasma anticoagulated with citric acid was passed through another column # 1C and passed through the outlet of column # 1C. The total amount of plasma was collected and analyzed for fibrinogen, coagulation factor factor VII, and coagulation factor factor VIII (fourth analysis). Furthermore, another column # 2A and column # 2B similar to the column # 2 of the test # 2 described above were prepared, and the above-described two analyzes (third and fourth analyzes) were performed, respectively. Furthermore, another column # 3A and column # 3B similar to column # 3 of test # 3 described above were prepared, and the above-described two analyzes (third and fourth analyzes) were performed, respectively. The third and fourth analysis results for column # 1B and column # 1C, the third and fourth analysis results for column # 2A and column # 2B, and the third and fourth analysis results for column # 3A and column # 3B The analysis results are shown in the analysis result table of FIG. 9 (a), which shows the concentration of each substance in serum or plasma before and after passage and the concentration ratio before and after passage. In addition, the concentration ratio of each substance before and after the liquid flow is adjusted so that the column (column # 1B and column # 1C) packed with the auxiliary adsorbent, the column (column # 2A and column # 2B) packed with the comparative sample, and the main adsorbent. FIG. 9B shows a graph comparing the columns packed with (column # 3A, column # 3B).

  From FIG. 9, the main adsorbent has components other than fibrinogen (molecular weight 340 kDa), coagulation factor factor VII (molecular weight 50 kDa), coagulation factor factor VIII (molecular weight 330 kDa), apolipoprotein B (molecular weight 515 kDa), lipoprotein ( a) While macromolecules with a molecular weight of 3200 kDa are preferentially adsorbed, the adsorbent and the comparative sample adsorb fibrinogen, coagulation factor VII, and coagulation factor factor VIII, but apolipoprotein B And the amount of lipoprotein (a) adsorbed is small.

  It is considered that the selectivity of the adsorbing material is influenced by the influence of size exclusion by pores and the strength of electrostatic interaction by surface modification. Apolipoprotein B and lipoprotein are adsorbed to the main adsorbent, but not adsorbed to the auxiliary adsorbent and the comparative sample. Apolipoprotein B and lipoprotein are proteins contained in LDL, which is a macromolecule. Therefore, it is considered that the effect of size exclusion due to the pores appears because the auxiliary adsorbent and the comparative sample cannot enter the pores. The main adsorbent adsorbs coagulation factor factor VII and coagulation factor VIII and hardly adsorbs fibrinogen, whereas the auxiliary adsorbent adsorbs coagulation factor VII, coagulation factor factor VIII, and fibrinogen. The This is because fibrinogen is adsorbed in the pores in the comparative sample having a pore diameter distribution range of 10 nm to 40 nm, rather than in the pores in the pore diameter distribution range of 60 nm to 120 nm of the main adsorbent. Furthermore, when the adsorbent is adsorbed in pores having a pore diameter distribution range of 2 nm to 30 nm, the fibrinogen molecule receives electrostatic interaction with dextran sulfate on the adsorbent surface. In general, it is considered that it appears as a difference in the amount of adsorption because it becomes stronger and the number of adsorption sites increases as the pore diameter distribution range becomes smaller. In addition, the adsorption of coagulation factor factor VIII is influenced by the strength of electrostatic interaction by dextran sulfate on the adsorbent surface related to the pore size. It is thought that the influence of the strength of the electrostatic interaction by dextran sulfate on the adsorbent surface related to the pore size is small.

  From the above analysis results of the coadsorbent, it can be seen that the coadsorbent in the auxiliary adsorbent, the comparative sample, and the main adsorbent is a protein having a molecular weight in the range of about 5 kDa to 340 kDa.

Second Embodiment
Next, a second embodiment of the device of the present invention will be described. In the first embodiment, the device 10 of the present invention has a single column structure in which the auxiliary adsorbent 1 and the main adsorbent 2 are accommodated in one cylindrical container 3, but for example, the book according to the second embodiment. As shown in FIG. 10, the inventive device 20 accommodates the auxiliary adsorbent 1 and the main adsorbent 2 in separate cylindrical containers 21 and 22, respectively, and the outlet of the first cylindrical container 21 filled with the auxiliary adsorbent 1. The inlet of the second cylindrical container 22 filled with the main adsorbent 2 may be connected by a tube 23. As a result, the device 20 of the present invention has a two-stage configuration of the auxiliary adsorption column 24 filled with the auxiliary adsorbent 1 and the main adsorption column 25 filled with the main adsorbent 2. In addition, since the production method of the auxiliary | assistant adsorption body 1 and the main adsorption body 2 is the same as 1st Embodiment, the overlapping description is omitted.

  Also in the second embodiment, since the structure in which the plasma previously passed through the auxiliary adsorbent body 1 is passed through the main adsorbent body 2 is the same as in the first embodiment, it is the same as in the first embodiment. It is clear that the amount of LDL adsorption on the main adsorbent 2 is increased by reducing the influence of the coadsorbing substance.

  Next, another embodiment of the device of the present invention will be described.

  <1> In each of the embodiments described above, the axial center portions 5 and 6 are formed as hollow cylinders as the auxiliary adsorbing body 1 and the main adsorbing body 2, respectively. Also, it may be a rectangular tube shape or a prismatic shape instead of a cylindrical shape or a columnar shape. In the case where the auxiliary adsorbent 1 and the main adsorbent 2 are formed in a cylindrical shape, the adsorption column of each of the above embodiments is, for example, FIG. 11 (another embodiment of the first embodiment), FIG. Unlike the above embodiments, the flow direction of each body fluid (plasma) is the axial direction of each column.

  <2> Furthermore, in the second embodiment, the auxiliary adsorption column 24 filled with the auxiliary adsorbent 1 and the main adsorption column 25 filled with the main adsorbent 2 have a two-stage configuration. The auxiliary adsorption column 24 filled with is not limited to the form used by being arranged in the previous stage of the main adsorption column 25, and can be used alone. When the auxiliary adsorption column 24 is used alone, as is apparent from the third and fourth analysis results shown in FIG. 9 of the first embodiment, as an adsorption column for removing blood proteins such as fibrinogen and coagulation factors. Can be used.

  <3> In each of the above embodiments, the case where dextran sulfate is fixed as the functional group 12 on the surface of the porous carrier 11 constituting the auxiliary adsorbent 1 and the main adsorbent 2 has been described. Specific functional groups include dextran sulfate and its salt, polyacrylic acid or its salt, polycarboxylic acid or its salt, SDS (sodium dodecyl sulfate), heparin and other negatively charged compounds. Various functional groups can be used as long as they are functionally bonded.

  The adsorption column for body fluid purification treatment according to the present invention can be used as an adsorption column for apheresis treatment for the purpose of adsorption removal of pathogenic substances such as LDL in blood.

1: Auxiliary adsorbent 2: Main adsorbent 3: Cylindrical container 4: Partition plate 5, 6: Axial portion 7: Opening (feeding port)
8: Opening (discharge port)
9: Peripheral flow channel 10, 20: Adsorption column for body fluid purification treatment according to the present invention 11: Porous carrier 12: Functional group 13: Skeletal body 14: Through hole 15: Pore 21, 22: Cylindrical container 23: Tube 24 : Auxiliary adsorption column 25: Main adsorption column

Claims (9)

An adsorption column for body fluid purification treatment comprising an adsorbent formed by fixing a functional group that specifically binds to a substance to be adsorbed containing at least low-density lipoprotein on the surface of a porous carrier,
An adsorbent that adsorbs and removes a coadsorbed substance having a molecular weight smaller than that of the substance to be adsorbed in advance from the body fluid to be treated, and adsorbs the substance to be adsorbed from the body fluid that has been adsorbed and removed by the auxiliary adsorbent. With a main adsorber to remove,
The porous carrier of the auxiliary adsorbent and the main adsorbent is a skeleton made of silica gel or silica glass having a three-dimensional network structure, a three-dimensional network-like through-hole formed in a gap between the skeleton, Having fine pores penetrating from the surface of the skeleton body to the inside formed dispersed on the surface of the skeleton body,
The central pore diameter measured by the nitrogen adsorption method of the pore in the porous carrier of the auxiliary adsorbent is smaller than the maximum length of the substance to be adsorbed, and the pore diameter measured by the nitrogen adsorption method of the pore The upper limit of the distribution range of the pore diameter is larger than the maximum value of the maximum length of the coadsorbing substance,
An adsorption column for body fluid purification treatment, wherein a central pore diameter measured by a nitrogen adsorption method of the pores in the porous carrier of the main adsorbent is larger than a maximum length of the adsorption target substance.   2. The adsorption column for body fluid purification treatment according to claim 1, wherein a central pore diameter measured by a nitrogen adsorption method of the pores in the porous carrier of the auxiliary adsorbent is 10 nm or more and 26 nm or less.   The center pore diameter measured by the nitrogen adsorption method of the pores in the porous carrier of the main adsorbent is at least twice as large as the maximum length of the substance to be adsorbed. Adsorption column for body fluid purification treatment.   4. The apparatus according to claim 1, wherein the auxiliary adsorbent is provided on an upstream side in a path through which the body fluid to be processed in a single container flows, and the main adsorbent is provided on a downstream side. The adsorption column for body fluid purification treatment according to Item.   Provided with an auxiliary adsorption column formed by filling the auxiliary adsorbent in a single container upstream of the body fluid flow path through which the body fluid flows, and in another single container downstream of the body fluid flow path The adsorption column for body fluid purification treatment according to any one of claims 1 to 3, further comprising a main adsorption column filled with the main adsorbent. Auxiliary for adsorbing and removing in advance the coadsorbed substance having a molecular weight smaller than that of the substance to be adsorbed from the body fluid to be treated by immobilizing a functional group that specifically binds to the substance to be adsorbed including at least low-density lipoprotein on the surface of the porous carrier. An adsorption column for body fluid purification treatment comprising an adsorbent,
The porous carrier of the auxiliary adsorbent is a skeleton made of silica gel or silica glass having a three-dimensional network structure, a three-dimensional network-like through-hole formed in a gap between the skeleton, and a surface of the skeleton. Having pores penetrating from the surface to the inside of the skeleton formed by dispersion,
The central pore diameter measured by the nitrogen adsorption method of the pore in the porous carrier of the auxiliary adsorbent is smaller than the maximum length of the substance to be adsorbed, and the pore diameter measured by the nitrogen adsorption method of the pore An adsorption column for bodily fluid purification, wherein the lower limit of the distribution range is 2 nm or less and the upper limit of the distribution range of the pore diameter is larger than the maximum value of the maximum length of the coadsorbent.   The adsorption column for body fluid purification treatment according to claim 6, wherein the pore diameter of the auxiliary adsorbent in the porous carrier measured by a nitrogen adsorption method is 10 nm or more and 26 nm or less.   The functional group is dextran sulfate or a salt thereof, polyacrylic acid or a salt thereof, or other chain polyvalent acid or a salt thereof having an affinity for specifically binding to low density lipoprotein. The adsorption column for body fluid purification treatment according to any one of claims 1 to 7.   The adsorption column for body fluid purification treatment according to any one of claims 1 to 8, wherein the porous carrier is synthesized by a spinodal decomposition sol-gel method.