Columns and Adsorption Apparatus

Abstract

The present invention provides a column for use in an adsorption device, which is capable of purifying and isolating an adsorbate contained in a sample liquid at an excellent purification rate, and an adsorption device equipped with the column. [Solution] A column 2 of the present invention comprises a column body 21, filter members 4, 5 (first partition, second partition) that define an adsorbent filling space 20, a lid 26 (first lid) having a flow path 41 (first flow path) that defines a space 262 (first space) between the filter member 4, and a lid 27 (second lid) having a flow path 51 (second flow path) that defines a space 272 (second space) between the filter member 5, and the lids 26, 27 have first and second lid surfaces facing the spaces 262, 272, respectively, and the angle θ1 [°] between the first partition surface of the filter member 4 and the first lid surface and the angle θ2 between the second partition surface of the filter member 5 and the second lid surface satisfy at least one of 7.5°≦θ1≦20° and 7.5°≦θ2≦20°.

Description

[Technical field]

[0001] The present invention relates to a column used in an adsorption device capable of purifying and isolating an adsorbate contained in a sample liquid at an excellent purification rate, and to an adsorption device equipped with this column. [Background technology]

[0002] In recent years, hydroxyapatite has been widely used as a stationary phase material in chromatography, i.e., as an adsorbent, for the reasons of its high biocompatibility and excellent safety, for example, when purifying and isolating biopharmaceuticals such as antibodies and vaccines as an adsorbent substance.

[0003] As an adsorption device having, as an adsorbent, a powder of hydroxyapatite (HAP) used as a stationary phase material in chromatography, for example, one having the following configuration is known.

[0004] That is, an adsorption device has been proposed in which unsintered powder consisting of particles including secondary particles of hydroxyapatite, or sintered powder obtained by firing (sintering) this unsintered powder, is filled into an adsorbent filling space provided in the column body of a column, etc., and the unsintered powder or sintered powder is used as a stationary phase material (adsorbent) (see, for example, Patent Document 1).

[0005] When a sample liquid is supplied to an adsorption device having such a configuration, the adsorbed substance contained in the sample liquid as an isolated substance is specifically adsorbed by its own inherent adsorption force (supporting force), and depending on the difference in adsorption force, it flows out of the column in a state where it is eluted in a fraction different from the impurities other than the isolated substance contained in the sample liquid. Therefore, by recovering the fraction containing the adsorbed substance (isolated substance), the adsorbed substance can be purified and isolated.

[0006] Incidentally, it is known that the adsorbed substance separated and purified using the above-mentioned adsorption device is eluted according to a Gaussian distribution in the fraction flowing out of the column. Therefore, in order to purify and isolate the adsorbed substance (isolated product) with high accuracy, it is necessary to adjust the adsorbed substance to a Gaussian distribution showing a steep peak in the fraction and elute it. However, particularly in this peak, tailing occurs in which the rear part of the peak is receded, and due to the large tailing, a problem occurs in which the purification rate of the adsorbed substance cannot be sufficiently improved. The present inventor discovered the existence of such a problem. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2011-68539 A Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a column for use in an adsorption device capable of purifying and isolating an adsorbate contained in a sample liquid at an excellent purification rate, and an adsorption device equipped with this column. [Means for solving the problem]

[0009] Such an object can be achieved by the present invention described in the following (1) to (11). (1) a cylindrical column body; a first partition wall and a second partition wall disposed in the column body and spaced apart from each other along a tube axis direction of the column body and defining an adsorbent filling space therebetween; a first lid defining a first space between itself and the first partition wall and having a first flow path communicating with the first space; a second lid defining a second space between itself and the second partition wall and having a second flow path communicating with the second space, the first lid has a first lid surface facing the first space, the second lid has a second lid surface facing the second space, A column characterized in that at least one of the following formula (1) and formula (2) is satisfied, when an angle between a first partition surface of the first partition facing the first space and the first lid surface is θ1 [°], and an angle between a second partition surface of the second partition facing the second space and the second lid surface is θ2 [°]: 7.5°≦θ1≦20° (1) 7.5°≦θ2≦20° (2)

[0010] This makes it possible to reduce the tailing observed in the peaks indicating the content of the eluted adsorbed substance (isolated product) in the fractions flowing out from the adsorption device. Therefore, the column can be used in an adsorption device that can purify and isolate the adsorbed substance contained in the sample liquid with an excellent purification rate.

[0011] (2) The column according to (1) above, wherein when θ1 satisfies formula (1), the first lid surface is an inclined surface that is inclined with respect to the transverse cross section of the column body. The column of the present invention is suitably applied to a column having such a configuration.

[0012] (3) The column according to (2) above, wherein the first lid surface is a surface corresponding to a peripheral surface of a truncated cone having the first flow path as a central axis. The column of the present invention is suitably applied to a column having such a configuration.

[0013] (4) The column according to (1) above, wherein when θ2 satisfies formula (2), the second lid surface is an inclined surface inclined with respect to the transverse cross section of the column body. The column of the present invention is suitably applied to a column having such a configuration.

[0014] (5) The column according to (4) above, wherein the second lid surface is a surface corresponding to the peripheral surface of a truncated cone having the second flow path as a central axis. The column of the present invention is suitably applied to a column having such a configuration.

[0015] (6) The column according to any one of (1) to (5) above, wherein the first partition and the second partition have liquid permeability. The column of the present invention is suitably applied to a column having such a configuration.

[0016] (7) The column according to (6) above, wherein the first partition and the second partition are filter members. The column of the present invention is suitably applied to a column having such a configuration.

[0017] (8) a cylindrical column body; a first partition wall and a second partition wall disposed in the column body and spaced apart from each other along a tube axis direction of the column body and defining an adsorbent filling space therebetween; a first lid defining a first space between itself and the first partition wall and having a first flow path communicating with the first space; a second lid defining a second space between itself and the second partition wall and having a second flow path communicating with the second space, the first lid has a first lid surface facing the first space, the second lid has a second lid surface facing the second space, The first lid surface is inclined from the center of the first lid surface toward the outside so as to approach the center of the longitudinal direction of the column body, and has an area of ​​100.40% to 130.00% of the unit cross-sectional area of ​​the column body in the horizontal direction perpendicular to the longitudinal direction of the column body that occupies an area vertically below the first lid surface, or A column characterized in that the second lid surface is inclined from the center of the second lid surface toward the outside so as to approach the center of the longitudinal direction of the column body, and has an area of ​​100.40% or more and 130.00% or less of the unit cross-sectional area of ​​the column body in a horizontal direction perpendicular to the longitudinal direction of the column body, occupying an area vertically below the second lid surface.

[0018] This makes it possible to reduce the tailing observed in the peaks indicating the content of the eluted adsorbed substance (isolated product) in the fractions flowing out from the adsorption device. Therefore, the column can be used in an adsorption device that can purify and isolate the adsorbed substance contained in the sample liquid with an excellent purification rate.

[0019] (9) A column according to any one of (1) to (8) above; and hydroxyapatite powder filled in the adsorbent filling space of the column.

[0020] The adsorption device of the present invention is suitably applied to an adsorption device having hydroxyapatite powder as an adsorbent, and this makes it possible to purify and isolate the adsorbed substance contained in the sample liquid with an excellent purification rate.

[0021] (10) In a state in which the first lid is positioned vertically above the second lid, The adsorption device according to (9) above, in which a sample liquid containing an adsorbent is supplied to the adsorbent filling space via the first flow path of the first lid, and the adsorbent is adsorbed by the powder.

[0022] The adsorption device of the present invention is preferably applied to an adsorption device in which adsorption of an adsorbent is performed under the above-mentioned conditions, and thereby the adsorbent contained in the sample liquid can be purified and isolated with an excellent purification rate.

[0023] (11) Purification of the adsorbent is carried out by supplying a buffer solution to the adsorbent filling space; The adsorption device according to (10) above, wherein the supply rate of the buffer solution to the adsorbent filling space is 40 cm / h or more and 200 cm / h or less.

[0024] The adsorption device of the present invention is preferably applied to an adsorption device in which a buffer solution is supplied to the adsorbent filling space at the above-mentioned supply rate, thereby enabling the adsorbent contained in the sample liquid to be purified and isolated with an excellent purification rate. Effect of the Invention

[0025] According to the present invention, it is possible to provide a column for use in an adsorption device capable of purifying and isolating an adsorbate contained in a sample liquid at an excellent purification rate, and an adsorption device including this column. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a vertical sectional view showing an example of an adsorption apparatus of the present invention. [Diagram 2] 2 is a partially enlarged longitudinal sectional view of a portion A shown in FIG. 1. [Diagram 3] 2 is a partially enlarged longitudinal sectional view of a portion B shown in FIG. 1. FIG. [Figure 4] 3A to 3C are vertical cross-sectional views for explaining a manufacturing method of the suction device of the first embodiment. [Diagram 5] 3A to 3C are vertical cross-sectional views for explaining a manufacturing method of the suction device of the first embodiment. [Figure 6] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption apparatuses of Examples 1 to 5 and Comparative Example 1. [Figure 7] FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption apparatuses of Examples 1 to 5 and Comparative Example 1. [Figure 8] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 1 and Comparative Example 1. [Figure 9]FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 1 and Comparative Example 1. [Figure 10] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 2 and Comparative Example 1. [Figure 11] FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 2 and Comparative Example 1. [Figure 12] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 3 and Comparative Example 1. [Figure 13] FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 3 and Comparative Example 1. [Figure 14] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 4 and Comparative Example 1. [Figure 15] FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 4 and Comparative Example 1. [Figure 16] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 5 and Comparative Example 1. [Figure 17] FIG. 1 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption devices of Example 5 and Comparative Example 1. [Figure 18] 1 is a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption apparatuses of Comparative Examples 2 and 1. [Figure 19]FIG. 2 is an enlarged view of a graph showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent in the adsorption apparatuses of Comparative Examples 2 and 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a column and an adsorption apparatus according to the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.

[0028] Fig. 1 is a vertical cross-sectional view showing an example of an adsorption apparatus according to an embodiment of the present invention. In the following description, the upper side in Fig. 1 is referred to as the "inlet side" and the lower side is referred to as the "outlet side".

[0029] Here, the "inlet side" refers to the side where, for example, a sample liquid (liquid containing the adsorbent) and an eluent such as a buffer solution or water are supplied into the adsorption device when purifying and isolating the adsorbent as an isolated product such as a biopharmaceutical or the like to be separated, i.e., a protein, while the "outlet side" refers to the side opposite the inlet side, i.e., the side where the liquid flows out of the adsorption device as an effluent.

[0030] An adsorption apparatus 1 shown in FIG. 1, which purifies and isolates substances such as proteins from a sample liquid, has a column 2 and a granular adsorbent (packing material) 3.

[0031] Column 2 (a column according to one embodiment of the present invention) has a column body 21, a lid portion 22 (first port) and a lid portion 23 (second port) attached to the inlet end and the outlet end of this column body 21, respectively, and a filter member 4 and a filter member 5 arranged between the column body 21 and the lid portions 22 and 23, respectively.

[0032] The column body 21 is composed of, for example, a cylindrical member. Examples of materials constituting the column 2 including the column body 21 and each part (each member) except for the filter members 4 and 5 include various glass materials, various resin materials, various metal materials, and various ceramic materials.

[0033] In the column body 21, filter members 4, 5 are arranged at a distance from each other along the axial direction of the column body 21 in correspondence with the inlet side opening and the outlet side opening, so that the inlet side opening and the outlet side opening are blocked by the filter members 4, 5, respectively. In this state, lid portions 22, 23 are attached to the inlet side end and the outlet side end of the column body 21.

[0034] In the column 2 having such a configuration, an adsorbent filling space 20 is defined inside the column body 21 between the filter member 4 and the filter member 5 by the column body 21, and the filter member 4 (first partition) and the filter member 5 (second partition).

[0035] Furthermore, the column body 21 and the lids 22 and 23 ensure liquid-tightness in the adsorbent filling space 20. At least a portion of the adsorbent filling space 20 is filled with the adsorbent 3 (in this embodiment, almost to the full extent).

[0036] In a preferred embodiment of the present invention, the inner diameter (column inner diameter) of the adsorbent filling space 20 is appropriately set depending on the volume of the sample liquid and is not particularly limited, but is preferably about 10.0 mm to 100.0 mm, more preferably about 20.0 mm to 70.0 mm. The length (column length) of the adsorbent filling space 20 is, for example, preferably about 10.0 mm to 300.0 mm, more preferably about 20.0 mm to 200.0 mm.

[0037] By setting the dimensions of the adsorbent filling space 20 as described above and the dimensions of the adsorbent 3 described below as well, it is possible to selectively isolate (purify) a target substance from a sample liquid, i.e., to reliably separate an adsorbed substance (isolated substance) such as a protein from impurities other than the isolated substance contained in the sample liquid. The present invention is preferably applied to an adsorption device 1 including a column 2 in which the dimensions of the adsorbent filling space 20 are set as described above. That is, by satisfying at least one of the following formulas (1) and (2) for the adsorption device 1 having such a configuration, it is possible to reliably improve the purification rate of the adsorbed substance separated and purified in the fractions recovered by the adsorption device 1.

[0038] The isolated substances isolated (purified) using the adsorbent 3 are not limited to proteins such as acidic proteins, such as albumin and antibodies, and basic proteins, but include, for example, negatively charged substances such as acidic amino acids, DNA, RNA, and negatively charged liposomes, as well as positively charged substances such as basic amino acids, positively charged cholesterol, and positively charged liposomes. In other words, various substances such as antibodies and biopharmaceuticals such as vaccines can be purified and isolated using the adsorbent 3 as adsorbents.

[0039] Moreover, by attaching each of the lids 22, 23 to the column body 21, liquid-tightness between them is ensured.

[0040] The lid portion 22 (first port) and the lid portion 23 (second port) each have a cap 28 and a cap 29, an inlet pipe 24 and an outlet pipe 25, and a lid 26 and a lid 27, respectively.

[0041] The caps 28, 29 are attached by screwing to the inlet end (one end) and the outlet end (the other end) of the column body 21, respectively, with the inlet end of the column body 21 vertically upward and the outlet end vertically downward, and the caps 28, 29 and the column body 21 ensure liquid-tightness between them.

[0042] Further, the inlet pipe 24 and the outlet pipe 25 are constituted by a pipe body through which the liquid flows, and are fixed (secured) in a liquid-tight manner to the approximate center of the caps 28 and 29, respectively.

[0043] Furthermore, as shown in Figures 2 and 3, the lids 26, 27 are respectively arranged between the filter members 4, 5 and the caps 28, 29, and have a flow path 41 (first flow path) and a flow path 51 (second flow path) that communicate with the inlet pipe 24 and the outlet pipe 25, respectively, and a lid surface 261 (first lid surface) and a lid surface 271 (second lid surface) that face the filter members 4 and 5, respectively.

[0044] In such lids 26, 27, a space 262 (first space) is defined by the lid surface 261 and the filter member 4, and a space 272 (second space) is defined by the lid surface 271 and the filter member 5.

[0045] The lid surfaces 261, 271 are surfaces corresponding to the peripheral surfaces of a truncated cone with the flow paths 41, 51 as the central axis, respectively, and as a result, the lid surfaces 261, 271 form inclined surfaces that are inclined with respect to the cross section of the column body 21, i.e., the flat surfaces 45, 55 facing the lid surfaces 261, 271 of the filter members 4, 5.

[0046] The lid surfaces 261, 271 of the lids 26, 27 thus configured and the flat surfaces 45, 55 of the filter members 4, 5 define truncated cone-shaped spaces 262, 272, respectively.

[0047] An inflow passage defined by the inflow pipe 24 and the flow passage 41 communicates with this space 262, and the liquid is supplied through this inflow passage and then supplied from the space 262 to the adsorbent 3 via the filter member 4. That is, the liquid is supplied to the adsorbent 3 from the space 262 side via the filter member 4 in a state in which the flow passage through which the liquid flows expands in a funnel shape from the inflow pipe 24 toward the space 262.

[0048] The liquid supplied to the adsorbent 3 passes between the adsorbents 3 (gaps) and is supplied to the space 272 via the filter member 5, and then flows out of the column 2 from the space 272 via an outflow path defined by the flow path 51 communicating with the space 272 and the outflow pipe 25. That is, in a state where the flow path through which the liquid flows from the space 272 toward the outflow pipe 25 is reduced in diameter like a funnel, the liquid is supplied from the adsorbent 3 to the space 272 side via the filter member 5. At this time, when a sample liquid is supplied to the adsorbent 3 as the liquid, the adsorbed substance (isolated product) contained in the sample liquid (sample) and impurities other than the adsorbed substance (isolated product) are separated based on the difference in adsorptivity to the adsorbent 3 and the difference in affinity to the eluate.

[0049] That is, with the adsorption device 1 in a position such that the lid portion 22 is positioned vertically above the lid portion 23, the liquid is supplied to the adsorbent filling space 20 via the inlet pipe 24, the adsorbent 3 filled in the adsorbent filling space 20 adsorbs the adsorbent, and the adsorption of the adsorbent is utilized to purify and isolate the adsorbed substance (isolated product).

[0050] The filter member 4 (first partition wall) and the filter member 5 (second partition wall) have liquid permeability (liquid permeability) that allows the liquid to pass uniformly in the thickness direction between the space 262 and the adsorbent filling space 20 and between the space 272 and the adsorbent filling space 20. That is, they have a function of supplying the liquid from the space 262 to the adsorbent filling space 20 (adsorbent 3) and further allowing the liquid to flow out from the adsorbent filling space 20 (adsorbent 3) to the space 272. In addition to this function, they also have a function of preventing the adsorbent 3 from flowing out from the adsorbent filling space 20, that is, a function of retaining the adsorbent 3 in the adsorbent filling space 20.

[0051] These filter members 4 and 5 are respectively constituted by, for example, a polypropylene mesh, a sintered filter of polyethylene particles, a stainless steel mesh filter, and a sintered filter of stainless steel particles.

[0052] In the adsorption device 1, the adsorbent 3 includes a powder consisting of an aggregate of fine particles having a non-uniform particle size, and has an ability to adsorb isolated substances (adsorbed substances) contained in a sample liquid (sample), and is preferably composed of unsintered powder consisting of particles including secondary particles of hydroxyapatite, or sintered powder thereof. In this specification, the unsintered powder and sintered powder of hydroxyapatite are collectively referred to as "hydroxyapatite powder". Therefore, in the following, a case where hydroxyapatite powder is filled in the adsorbent filling space 20 of the column body 21 as the adsorbent 3 will be described as an example.

[0053] This hydroxyapatite powder (unsintered powder) includes secondary particles, as well as primary and multi-secondary particles, and is mainly composed of the secondary particles.

[0054] In addition, hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 The secondary particles of hydroxyapatite are obtained by drying a slurry containing the primary particles and their aggregates and granulating the resulting mixture, and are mainly composed of hydroxyapatite. Hydroxyapatite has a chemically stable apatite structure, and powder containing the secondary particles of hydroxyapatite is particularly suitable for use as the adsorbent 3 in the adsorption device 1. The hydroxyapatite intended has a Ca / P ratio of about 1.64 or more and 1.70 or less.

[0055] Furthermore, the secondary particles are not particularly limited, but preferably have a bulk density of 0.65 g / mL or more and a specific surface area of ​​70 m 2 / g or more. Such secondary particles can be more suitably used for the adsorbent 3 provided in the adsorption device 1.

[0056] When a sample liquid is supplied to the adsorbent 3 having such a configuration, the adsorbable substance (isolated product) contained in the sample liquid is specifically adsorbed by the adsorbing (supporting) power inherent to the adsorbent, and depending on the difference in the adsorbing power, it is purified and isolated from impurities other than the adsorbable substance (isolated product) contained in the sample liquid.

[0057] As described above, the secondary particles of hydroxyapatite preferably have a bulk density of 0.65 g / mL or more, more preferably 0.70 g / mL or more and 0.95 g / mL or less. Secondary particles having a bulk density within this range are considered to be heavy and have fewer voids within the particles, and can be said to have a high packing density, and therefore exhibit high strength. Therefore, when unsintered powder containing these secondary particles or sintered powder thereof is used as the adsorbent 3, it is possible to extend the life of the adsorbent.

[0058] As described above, in this embodiment, the specific surface area is 70 m 2 It is preferable that it is set to 75m / g or more. 2 / g or more 100m 2 / g or less is more preferable. When the unsintered powder or the sintered powder containing secondary particles having a high specific surface area in this range is used as the adsorbent 3, the opportunity for the adsorbed substance to come into contact with the adsorbent 3 increases, and the interaction between the adsorbed substance and the adsorbent 3 is improved, so that the adsorbent 3 exhibits excellent adsorption ability for the adsorbed substance.

[0059] 1, the secondary particles preferably have a granular form (shape), more preferably a sphericity of about 0.95 to 1.00, and even more preferably a sphericity of 0.97 to 1.00. When an unsintered powder or a sintered powder thereof containing secondary particles with high sphericity is used as the adsorbent 3, the filling rate of the adsorbent 3 in the adsorbent filling space 20 can be improved.

[0060] The angle of repose of such secondary particles, when classified into a particle size of 40±4 μm, is preferably 27° or less, and more preferably about 22° to 25°. Secondary particles with such a low angle of repose have high fluidity, and can improve operability (filling efficiency) when filling the adsorbent filling space 20 with unsintered powder containing secondary particles or sintered powder thereof as the adsorbent 3.

[0061] In addition, the sintered powder obtained by sintering the unsintered powder containing secondary particles has an average pore size of 0.07 μm or less, and more preferably 0.04 μm to 0.06 μm, when sintered at 700° C. In addition, the average pore size of 0.05 μm or less, and more preferably 0.02 μm to 0.04 μm, when sintered at 400° C., is preferably 0.05 μm or less, and more preferably 0.02 μm to 0.04 μm. By keeping the average pore size within this range, the specific surface area of ​​the sintered powder can be reliably increased.

[0062] In this embodiment, the adsorbent 3 filled in the adsorbent filling space 20 of the adsorption device 1 according to the embodiment of the present invention is a powder of hydroxyapatite, but this adsorbent 3 includes a powder consisting of an aggregate of fine particles having non-uniform particle diameters. In a preferred embodiment of the present invention, the aggregate of fine particles preferably has a predetermined most frequent particle diameter (mode diameter) and has a mountain-shaped particle diameter distribution centered on the value of the most frequent particle diameter. In another preferred embodiment of the present invention, the aggregate of fine particles preferably has particle diameters distributed above and below the value of the most frequent particle diameter such that the frequency of occurrence decreases the further away from the value of the most frequent particle diameter.

[0063] The powder constituting the adsorbent 3 filled in the adsorbent filling space 20 of the adsorption device 1 according to one embodiment of the present invention may be, in addition to the hydroxyapatite powder shown in this embodiment, a powder of an inorganic compound such as silica, fluoroapatite, calcium pyrophosphate, magnesium pyrophosphate, alumina, or zirconia, or a powder of an organic compound such as polymer beads, or a polymer such as sepharose or agarose, or a mixture of these. The effect of the present invention is more effective when the adsorbent 3 contains a powder consisting of an aggregate of fine particles having non-uniform particle diameters.

[0064] The hydroxyapatite powder may be a commercially available product, such as CHT (USA registered trademark) Type I, CHT (USA registered trademark) Type II, or CHT (USA registered trademark) XT, MPC, or CFT, manufactured by Bio-Rad.

[0065] When producing hydroxyapatite powder, the hydroxyapatite powder can be produced according to the method described in, for example, JP-B-7-88205 and Japanese Patent No. 5724050. Specifically, in order to produce hydroxyapatite powder, a production method can be carried out that includes a step [S1A] of reacting a first liquid containing a calcium source such as calcium hydroxide with a second liquid containing a phosphorus source such as phosphoric acid while stirring to obtain a slurry containing primary particles of hydroxyapatite and their aggregates, a step [S2A] of physically crushing the aggregates contained in the slurry and dispersing the crushed aggregates in the slurry, and a step [S3A] of drying the slurry and granulating the crushed aggregates to obtain a powder mainly composed of particles containing secondary particles of hydroxyapatite.

[0066] The powder constituting the adsorbent 3 filled in the adsorbent filling space 20 of the adsorption device according to one embodiment of the present invention may be an unsintered powder or a sintered powder. Moreover, the powder constituting the adsorbent 3 filled in the adsorbent filling space 20 of the adsorption device 1 according to one embodiment of the present invention may be classified or unclassified. When the powder is unclassified, the powder has a wider particle size distribution, and therefore the effect of the present invention may be more significantly exhibited.

[0067] The powder constituting the adsorbent 3 filled in the adsorbent filling space 20 of the adsorption device 1 according to one embodiment of the present invention, when classified, may have an average particle size and particle size distribution, for example, a particle size of 20±4 μm, a particle size of 40±4 μm, a particle size of 60±4 μm, a particle size of 80±4 μm, etc.

[0068] Next, a separation method for purifying and isolating the adsorbed substance using the adsorption apparatus 1 will be described. The method for separating an adsorbed substance in this embodiment includes a step [S1B] of contacting a sample liquid with an adsorbent 3, and a step [S2B] of fractionating the effluent from the column 2 into predetermined amounts.

[0069] These steps will be described in order below. [S1B: Step of contacting the feed solution with the adsorbent 3] In this step, the sample liquid (liquid) containing the adsorbent as the isolated product to be purified is supplied to the adsorbent 3 via the inlet pipe 24, the flow path 41 of the lid 26, the space 262 and the filter member 4, i.e., by passing through the adsorbent filling space 20 of the column 2 (adsorption device 1), it is brought into contact with the adsorbent 3 filled in the adsorbent filling space 20.

[0070] As a result, adsorbable substances (isolated substances) having a high adsorption capacity for the adsorbent 3 and impurities other than the adsorbable substances (isolated substances) that have a relatively high adsorption capacity for the adsorbent 3 are adsorbed to the adsorbent 3 and are thereby retained in the adsorbent filling space 20 of the column 2. Impurities having a low adsorption capacity for the adsorbent 3 flow out from the adsorbent filling space 20 of the column 2 via the filter member 5, the space 272, the flow path 51 of the lid 27, and the outflow pipe 25.

[0071] [S2B: A step of fractionating the effluent from column 2 into predetermined amounts] In this step, a buffer solution is supplied from the inlet pipe 24 into the adsorbent filling space 20 of the column 2 as an eluent (liquid) for eluting the adsorbed substance, and the effluent flowing out from the adsorbent filling space 20 of the column 2 via the outlet pipe 25 is fractionated (collected) in predetermined amounts.

[0072] As a result, the adsorbed substance and impurities adsorbed to the adsorbent 3 are recovered (separated) in a state in which they are eluted in each fraction as the effluent, depending on the difference in the adsorption power of each substance to the adsorbent 3. Therefore, by recovering the fraction containing the adsorbed substance (isolated product) from each of the recovered fractions, the adsorbed substance (isolated product) can be purified and isolated.

[0073] The buffer is not particularly limited, but for example, a phosphate buffer is preferably used, and examples of the phosphate buffer include sodium phosphate, potassium phosphate, and lithium phosphate.

[0074] The pH of the buffer solution is not particularly limited, but is preferably about 6 or more and 8 or less, and more preferably about 6.5 or more and 7.5 or less. This makes it possible to prevent the alteration and deterioration of the adsorbent to be separated. In addition, since the alteration (dissolution, etc.) of the adsorbent 3 can be suitably prevented, it is also possible to appropriately suppress or prevent a change in the separation ability in the adsorption device 1.

[0075] The temperature of the buffer solution is not particularly limited, but is preferably about 30° C. to 50° C., and more preferably about 35° C. to 45° C. This makes it possible to appropriately suppress or prevent alteration or deterioration of the adsorbent to be separated.

[0076] Therefore, by using a buffer solution within such pH and temperature ranges, it is possible to improve the recovery rate of the target adsorbed substance.

[0077] In addition, the supply rate of the buffer solution as the eluent to the adsorbent filling space 20 of the column 2 is not particularly limited, but is set, for example, preferably to about 40 cm / h to 200 cm / h, more preferably to about 80 cm / h to 150 cm / h. By setting the supply rate within the above range, the target adsorbent can be efficiently recovered. In addition, when the supply rate is set within the above range, the present invention is preferably applied. That is, the effect obtained by satisfying at least one of the following formula (1) and formula (2) described later can be more significantly exhibited.

[0078] In the above-described operation, the fractions containing the adsorbed substance (isolated product) are collected, and the adsorbed substance is purified and isolated. As described above, it is known that the adsorbed substance is eluted according to a Gaussian distribution in each fraction of the effluent discharged from the outlet pipe 25. Therefore, in order to purify and isolate the adsorbed substance with high accuracy, it is necessary to adjust the elution of the adsorbed substance to a Gaussian distribution showing a steep peak in the collected fraction. However, particularly in this peak, tailing occurs, in which the rear part of the peak is pulled back, and the large tailing causes a problem in that the purification rate of the adsorbed substance cannot be sufficiently improved.

[0079] As a result of extensive research by the inventors into this problem, it has been found that the occurrence of the tailing in the peak is related to the fact that when the effluent is supplied from space 262 to filter member 4 in step [S2B], and when the effluent is supplied from filter member 5 to space 272, the passing speed of the eluate and effluent passing through the filter members 4, 5 in the thickness direction is not uniform between the center side and the edge side of the filter members 4, 5, and a difference occurs.

[0080] As a result of further investigation by the inventors thereafter, it was found that the occurrence of the difference in the passing speed is closely related to the magnitude of the angles θ1, θ2 formed between the lid surfaces 261, 271 of the lids 26, 27 and the flat surfaces 45, 55 (first partition surface, second partition surface) of the filter members 4, 5 in the spaces 262, 272. Then, by setting the magnitude of the angles θ1, θ2 so as to satisfy at least one of the following formula (1) and formula (2), the occurrence of the difference in the passing speed can be appropriately suppressed, and the passing speed can be set to be almost uniform from the center side of the filter members 4, 5 to the edge side thereof. Therefore, it was found that the magnitude of the tailing at the peak can be reliably reduced, that is, the adsorbed substance can be purified and isolated with an excellent purification rate using the adsorption device 1, and the present invention was completed. 7.5°≦θ1≦20° …(1), 7.5°≦θ2≦20° …(2)

[0081] Here, when the angle θ1 between the lid surface 261 of the lid 26 and the flat surface 45 of the filter member 4 satisfies 7.5°≦θ1≦20°, the angle θ1 may satisfy 7.5°≦θ1≦20°, but it is preferable for the angle θ1 to satisfy 7.5°≦θ1≦13.5°, and it is more preferable for the angle θ1 to satisfy 8.0°≦θ1≦12.0°.

[0082] Furthermore, when the angle θ2 between the lid surface 271 of the lid 27 and the flat surface 55 of the filter member 5 satisfies 7.5°≦θ2≦20°, the angle θ2 may satisfy 7.5°≦θ2≦20°, but it is preferable for the angle θ2 to satisfy 7.5°≦θ2≦13.5°, and it is more preferable for the angle θ2 to satisfy 8.0°≦θ2≦12.0°.

[0083] When the angles θ1 and θ2 satisfy the above relationship, the adsorbent can be purified and isolated with a higher purification rate. When the angles θ1 and θ2 are equal to or greater than the lower limit, a predetermined volume or more is ensured in the spaces 262 and 272, so that the space through which the liquid moves can be sufficiently ensured, and the passing speed can be made close between the center side and the edge side of the filter members 4 and 5. When the angles θ1 and θ2 are equal to or less than the upper limit, the volumes of the spaces 262 and 272 can be kept within an appropriate range, and a difference in the passing speed between the center side and the edge side of the filter members 4 and 5 due to convection occurring when the liquid moves in the spaces 262 and 272 can be prevented.

[0084] The angles θ1 and θ2 may satisfy at least one of the above formula (1) and formula (2), but if they satisfy either one, it is preferable that they satisfy the above formula (1), and it is more preferable that they satisfy both the above formula (1) and formula (2). This allows the adsorbent to be purified and isolated with a higher purification rate. The angles θ1 and θ2 may be the same or different.

[0085] The ratio of the area L1 of the lid surface 261 to the area B1 of the flat surface 45 occupying the area vertically below the lid surface 261 is preferably 100.40% or more, and this ratio is preferably 130.00% or less. Furthermore, at the lower limit, this ratio is more preferably 100.85% or more. Further, this ratio is more preferably 101.00% or more, even more preferably 101.25% or more, and even more preferably 101.50% or more. Further, at the upper limit, this ratio is more preferably 125.00% or less. Further, this ratio is more preferably 120.00% or less, even more preferably 115.00% or less, and even more preferably 110.00% or less.

[0086] The above-mentioned area ratio is, in other words, the area of ​​the lid surface 261 relative to the unit cross-sectional area of ​​the column 2 in the horizontal direction perpendicular to the length direction of the column 2. For example, in FIG. 2, when θ1=0°, the unit cross-sectional area of ​​the column 2 in the horizontal direction is the same as the area of ​​the lid surface 261, so the above-mentioned ratio is 100%. When θ1=45°, the above-mentioned ratio is root 2 / 1×100=141.42%. In addition to having the above-mentioned area ratio, the lid surface 261 preferably has a shape that slopes downward from the center to the periphery. As a result, even when the lid surface 261 is not flat, it is expected to achieve the same effect as when the lid surface 261 is flat and has a preferred angle of θ1. The same applies to the relationship between the lid surface 271 and the flat surface 55.

[0087] Although the column and the adsorption device according to one embodiment of the present invention have been described above, the present invention is not limited thereto.

[0088] For example, in the column and adsorption device according to one embodiment of the present invention, each component can be replaced with any component that can exert a similar function, or any component can be added.

[0089] In addition, in the above embodiment, the column body of the column is described as being cylindrical, but the column body may be cylindrical in shape and may be polygonal, such as hexagonal or heptagonal. EXAMPLES

[0090] Next, specific examples of the present invention will be described. However, the present invention is not limited to the descriptions in these examples.

[0091] 1. Fabrication of the Adsorption Apparatus Example 1 First, a column 2 was prepared having a column body 21 with an inner diameter of φ50 mm and a column length of 200 mm, cover portions 22, 23 equipped with lids 26, 27 having lid surfaces 261, 271 with θ1, θ2 = 10°, and filter members 4, 5 with a diameter of φ45 mm and a thickness of 5 mm. Furthermore, an extension column 200 was prepared having a column passing portion 221 with an inner diameter of φ50 mm and a column length of 230 mm, and a column connector 250 was obtained by screwing the connecting portion 229 of the extension column 200 to the inlet end of the column body 21 of the column 2 (see Figure 4).

[0092] Next, 248 g of sintered powder of hydroxyapatite (manufactured by BIO-RAD, CHT 40 μm Type I) was weighed out as adsorbent 3, and this adsorbent 3 was dispersed in 737 mL of 0.2 mM sodium phosphate buffer by stirring at 25° C. to prepare slurry 30.

[0093] Next, as shown in FIG. 4, the column coupler 250 was arranged so that the column 2 was vertically downward and the extension column 200 was vertically upward. After that, the slurry 30 was supplied from an opening at the upper end of the column passing section 221 of the extension column 200 to the adsorbent passing space 220 of the column passing section 221, thereby filling the adsorbent passing space 220 with the slurry 30. Then, the cap 228 was attached to the upper end of the column passing section 221 by screwing. Then, the column coupler 250 was inverted and stirred several times in the vertical direction to suspend the packing material that had settled due to gravity, and the column coupler 250 was maintained in an attitude in which the column 2 was vertically upward and the extension column 200 was vertically downward. At the same time, the supply of water as the packing liquid 66 stored in the container 65 to the adsorbent passing space 220 of the column passing section 221 through the inflow pipe 224 of the cap 228 was started using the pump 60 (see FIG. 5). At this time, the flow rate of water through the adsorbent passing space 220 of the column passing portion 221 using the pump 60 was set to 160 mL / min.

[0094] Next, the pump 60 was used to supply water to the adsorbent passing space 220 of the column passing section 221 until the adsorbent 3 rose sequentially through the adsorbent passing space 220 of the column passing section 221 and the adsorbent filling space 20 of the column body 21, and the adsorbent filling space 20 of the column body 21 was filled with almost the entire amount of the adsorbent 3. Thereafter, the column was returned to its original state and filled with the entire amount.

[0095] Next, the operation of the pump 60 was stopped to terminate the supply of water to the adsorbent passing space 220 of the column passing section 221, and then the extension column 200 was detached from the column 2 by detaching the connecting part 229, which was attached by screwing to the inlet end of the column body 21 of the column 2, from the column body 21. Next, with the filter member 4 interposed on the inlet end side of the column body 21, the lid part 22 was attached to the inlet end of the column body 21, thereby obtaining the adsorption device 1 of Example 1.

[0096] (Examples 2 to 5, Comparative Examples 1 and 2) The adsorption devices 1 of Examples 2 to 5 and Comparative Example 1 were obtained in the same manner as Example 1, except that cover portions 22, 23 were prepared, each equipped with lids 26, 27 having lid surfaces 261, 271 with lid angles θ1, θ2 = 7.5° (Example 2), θ1, θ2 = 13.5° (Example 3), θ1, θ2 = 20° (Example 4), θ1, θ2 = 5°, 10° (Example 5), θ1, θ2 = 5° (Comparative Example 1), and θ1, θ2 = 40° (Comparative Example 2).

[0097] 2. Purification and isolation of adsorbed substances using an adsorption device First, sodium chloride was dissolved in ion-exchanged water to a concentration of 1M to prepare a sample solution.

[0098] Next, for the adsorption apparatus 1 of Examples 2 to 5 and Comparative Example 1, the column was equilibrated with a 0.1 M NaCl solution at a rate of 100 cm / h. Then, 40 mL of the sample solution was supplied (applied) into the adsorption apparatus 1. Then, while flowing 500 mL of the 0.1 M NaCl solution into the adsorption apparatus 1 at the same rate, the electrical conductivity of the effluent coming out of the adsorption apparatus 1 was measured.

[0099] The electrical conductivity (mS / cm) of the effluent flowing out from column 2 was measured over time using an electrical conductivity meter attached to a liquid chromatography device (BioRad, "BioLogic DuoFlow 40").

[0100] For the adsorption apparatus 1 of Comparative Example 2, the electrical conductivity of the effluent was measured in the same manner as for the adsorption apparatus 1 of Examples 2 to 5 and Comparative Example 1, except that the rate at which the 0.1 M NaCl solution was introduced was set to 50 cm / h.

[0101] Graphs showing the relationship between the elution time (min) of the effluent and the electrical conductivity (mS / cm) of the effluent obtained by measuring the electrical conductivity are shown in Figs. 6 to 19 for the adsorption apparatus 1 of each Example and each Comparative Example. Figs. 6 and 7 show the graphs of the relationship between Examples 1 to 5 and Comparative Example 1, Figs. 8 to 17 show the graphs of the relationship between one of Examples 1 to 5 and Comparative Example 1, and Figs. 18 and 19 show the graphs of the relationship between Comparative Example 2 and Comparative Example 1.

[0102] As is clear from Figures 6 to 17, in each Example, compared to each Comparative Example, the occurrence of tailing was appropriately suppressed in the peak indicating the content of NaCl as a sample in the effluent (fraction) flowing out from column 2.

[0103] That is, the adsorption apparatus of each Example showed results that the adsorbed substance could be purified and isolated at a higher purification rate than the adsorption apparatus of each Comparative Example. [Explanation of symbols]

[0104] 1 Adsorption device 2 Columns 3. Adsorbents 4 Filter material 5 Filter material 20 Adsorbent filling space 21 Column body 22 Lid 23 Lid 24 Inflow pipe 25 Outflow pipe 26 Lid 27 Lid 28 Cap 29 Cap 30 Slurry 41 Flow Path 51 Flow Path 45 Flat surface 55 Flat surface 60 Pump 65 Container 66 Filling liquid 200 Extension Column 220 Adsorbent passage space 221 Column passage 224 Inflow pipe 228 Cap 229 Connecting part 250 Column Connection 261 Lid surface 271 Lid surface 262 Space 272 Space θ1 angle θ2 angle

Claims

1. A cylindrical column body; a first partition wall and a second partition wall disposed in the column body and spaced apart from each other along a tube axial direction of the column body and defining an adsorbent filling space therebetween; a first lid defining a first space between itself and the first partition wall and having a first flow path communicating with the first space; a second lid defining a second space between itself and the second partition wall and having a second flow path communicating with the second space, the first lid has a first lid surface facing the first space, the second lid has a second lid surface facing the second space, a column, characterized in that at least one of the following formula (1) and formula (2) is satisfied, when an angle between a first partition surface of the first partition facing the first space and the first lid surface is θ1 [°], and an angle between a second partition surface of the second partition facing the second space and the second lid surface is θ2 [°]: 7.5°≦θ1≦20° (1) 7.5°≦θ2≦20° (2)

2. The column according to claim 1 , wherein when θ1 satisfies the formula (1), the first lid surface is an inclined surface that is inclined with respect to a transverse cross section of the column body.

3. The column according to claim 2 , wherein the first lid surface is a surface corresponding to a peripheral surface of a truncated cone having the first flow path as a central axis.

4. The column according to claim 1 , wherein when θ2 satisfies the formula (2), the second lid surface is an inclined surface that is inclined with respect to the transverse cross section of the column body.

5. The column according to claim 4 , wherein the second lid surface is a surface corresponding to a peripheral surface of a truncated cone having the second flow path as a central axis.

6. 6. The column according to claim 1, wherein the first partition and the second partition have liquid permeability.

7. The column according to claim 6 , wherein the first partition and the second partition are filter members.

8. A cylindrical column body; a first partition wall and a second partition wall disposed in the column body and spaced apart from each other along a tube axial direction of the column body and defining an adsorbent filling space therebetween; a first lid defining a first space between itself and the first partition wall and having a first flow path communicating with the first space; a second lid defining a second space between itself and the second partition wall and having a second flow path communicating with the second space, the first lid has a first lid surface facing the first space, the second lid has a second lid surface facing the second space, The first lid surface is inclined from the center of the first lid surface toward the outside so as to approach the center of the longitudinal direction of the column body, and has an area of ​​100.40% to 130.00% of the unit cross-sectional area of ​​the column body in a horizontal direction perpendicular to the longitudinal direction of the column body that occupies a region vertically below the first lid surface, or A column characterized in that the second lid surface is inclined from the center of the second lid surface toward the outside so as to approach the center of the longitudinal direction of the column body, and has an area of ​​100.40% or more and 130.00% or less of the unit cross-sectional area of ​​the column body in a horizontal direction perpendicular to the longitudinal direction of the column body, occupying an area vertically below the second lid surface.

9. A column according to any one of claims 1 to 8; and hydroxyapatite powder filled in the adsorbent filling space of the column.

10. In a state in which the first lid is positioned vertically above the second lid, 10. The adsorption device according to claim 9, wherein a sample liquid containing an adsorbent is supplied to the adsorbent filling space via the first flow path of the first lid, and the adsorbent is adsorbed by the powder.

11. The adsorbent is purified by supplying a buffer solution to the adsorbent filling space; 11. The adsorption device according to claim 10, wherein the supply rate of the buffer solution to the adsorbent filling space is 40 cm / h or more and 200 cm / h or less.

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