JP2006081759A - Biological substance adsorbing agent and method of manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological substance adsorbing agent capable of selectively adsorbing and removing an etiologic substance such as β<SB>2</SB>-microglobulin without adsorbing useful protein in blood or blood plasma. <P>SOLUTION: The biological substance adsorbing agent comprises calcium phosphate hydroxide including 0.01-20 weight% metal M other than calcium with a mole ratio of the number of moles of phosphorus to the total number of moles of calcium and the metal M [(Ca+M)/P] of 1.3-1.55. The biological substance adsorbing agent can selectively adsorb β<SB>2</SB>-microglobulin in blood while inhibiting the adsorption of albumin. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a biological material adsorbent that adsorbs biological materials, particularly proteins such as β ₂ -microglobulin, and a method for producing the same.

  At present, there are already less than 200,000 patients with chronic renal failure in Japan, and it is certain that the number of patients will increase further as the population ages rapidly. Despite such a large number of patients with chronic renal failure, there is no fundamental treatment, and there is currently only symptomatic hemodialysis for the purpose of prolonging life. Hemodialysis therapy was produced and accumulated in the patient's blood using a hollow fiber membrane type artificial kidney made of a cellulose-based natural polymer or a synthetic polymer such as polysulfone or polymethyl methacrylate. It is common to remove harmful substances such as uremic substances. The dialysis membrane wall is formed so that fine pores having a diameter of about 10 to 100 nm penetrate, and harmful substances are transferred from the blood side to the dialysate by the osmotic pressure difference. However, there is an upper limit to the material permeability of the through-holes in the membrane wall, and it is usually designed so that the permeation blocking rate of albumin (molecular weight: 66000) is about 99.5% as the upper limit of the membrane performance evaluation method. Yes. Moreover, it is technically very difficult to make the diameters of the holes uniform, and it is inevitable that the holes have a certain degree of distribution. Therefore, when the albumin rejection is 100%, the substance permeability in the low-molecular region also decreases.

On the other hand, improvement in dialysis treatment techniques has increased the survival rate of patients and extended the dialysis treatment years. As a result, many complications have been observed. Among them, most patients have various complications such as pruritus associated with cutaneous amyloidosis, bone pain, joint pain, and root canal syndrome. It is said that β ₂ -microglobulin (β ₂ -MG, molecular weight: 12000) in the medium is abnormally produced and deposited in each tissue in the body. At present, attempts have been made to reduce the thickness of the dialysis membrane to improve the removal rate of this substance. However, reducing the thickness of the dialysis membrane reduces the strength of the membrane, and backfilling of pyrogenic substances from the dialysate side. There is also a negative effect such as torsion, and it has reached the limit of sufficiently removing β ₂ -MG in the patient's blood with a dialysis membrane.

As described above, patients with dialysis complications may continue to increase unless β ₂ -MG, which cannot be sufficiently removed by current dialysis membrane therapy, is removed by some method. One countermeasure is β ₂ -MG removal therapy using an adsorbent. The selective removal of etiological agents by adsorption is an established treatment, and it is excellent by using appropriate adsorbents for patients with hyperlipidemia, myasthenia gravis, hyperbilirubinemia, etc. Clinical results have been obtained. However, these are all plasma perfusion methods in which blood is once separated into formed components (red blood cells, white blood cells, platelets) and plasma, and pathogenic substances in plasma are selectively adsorbed and removed.

The use of calcium hydroxide phosphate (hydroxyapatite) as an adsorbent for removing β ₂ -MG in blood is described in Patent Documents 1 to 5. In these patent documents, it is described that β ₂ -MG can be removed by using various forms of hydroxyapatite. However, it has been difficult to selectively remove β ₂ -MG as a pathogenic substance while suppressing adsorption of useful components (eg, albumin) in blood.

On the other hand, as described in Patent Document 6 or Non-Patent Document 1, by using calcium hydroxide with a carbonate radical introduced therein, β ₂ -MG is selectively suppressed while suppressing the amount of albumin adsorbed. It became possible to adsorb to. However, selectivity was not always sufficient.

Japanese Unexamined Patent Publication No. 63-15960 JP-A 63-159591 Japanese Patent Laid-Open No. 2-180273 Japanese Patent Laid-Open No. 6-262065 JP-A-7-88361 JP 2003-126248 A “Journal of Biomedical Materials Research”, Wiley Periodicals, Inc., April 6, 2004, 69A, p. 544-551

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide an adsorbent capable of selectively adsorbing a biological substance and a method for producing the same.

  The said subject is solved by providing the biological material adsorption agent which consists of calcium hydroxide phosphate which contains 0.01-20 weight% of metals (M) other than calcium. At this time, the metal (M) is lithium, sodium, potassium, rubidium, cesium, magnesium, strontium, barium, zinc, aluminum, silicon, germanium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium. , Niobium, molybdenum, silver and tantalum are preferable, and the metal (M) is particularly preferably zinc. Further, at this time, it is also preferable that the ratio [(Ca + M) / P] of the number of moles of phosphorus to the total number of moles of calcium and metal (M) is 1.3 to 1.55.

  The above-mentioned problem can also be solved by providing a biological material adsorbent comprising calcium phosphate having a ratio of the number of moles of phosphorus to the number of moles of calcium [Ca / P] of 1.3 to 1.55. .

In the biological material adsorbent of the present invention, it is preferable that the specific surface area is 80 m ² / g or more. Further, it is preferable that the β ₂ -microglobulin adsorption amount per unit weight is 200 μg / g or more, and the albumin adsorption amount per unit weight is 30 mg / g or less.

Preferred embodiments of the biological material adsorbent of the present invention include an adsorbent for purifying blood or plasma, and a β ₂ -microglobulin adsorbent. A dialysis apparatus incorporating an adsorbing means using the biological material adsorbent is also a preferred embodiment. A biological material separation column packed with the biological material adsorbent is also a preferred embodiment.

  A preferred method for producing the biological material adsorbent of the present invention comprises mixing an aqueous solution (A) containing calcium ions and ions of a metal (M) other than calcium with an aqueous solution (B) containing phosphate ions. This is a method for producing a solid content composed of calcium hydroxide phosphate. At this time, the aqueous solution (A) preferably contains calcium nitrate and metal (M) nitrate. It is also preferable that the pH of the aqueous solution (A) is 4.5-7. Moreover, it is also suitable that pH of aqueous solution (B) is 8-12.

The biological material adsorbent of the present invention is excellent in biocompatibility, and can selectively adsorb biological materials such as proteins. In particular, it can selectively adsorb and remove pathogenic substances such as β ₂ -microglobulin without adsorbing useful proteins in blood or plasma, and also has excellent blood compatibility. It is suitable for removing the pathogenic substances accumulated in the blood of the receiving patient.

  The biological material adsorbent of the present invention is made of calcium hydroxide phosphate containing 0.01 to 20% by weight of a metal (M) other than calcium. The calcium hydroxide phosphate preferably has a hydroxyapatite structure, and it can be confirmed by an X-ray diffraction peak that it has the structure. Hydroxyapatite is excellent in biocompatibility, particularly blood compatibility.

By containing a metal (M) other than calcium, the selectivity when adsorbing biological substances such as proteins is greatly improved. In particular, it is possible to suppress adsorption of albumin, which is a useful substance in blood, while efficiently adsorbing β ₂ -MG, which is a pathogenic substance accumulated in the blood of patients undergoing hemodialysis therapy. . The reason why the selectivity is greatly improved by the presence of a metal (M) other than calcium is not necessarily clear, but the presence of a metal different from calcium changes the charge distribution on the surface of the calcium hydroxide phosphate particles to exhibit the selectivity. It is estimated that it contributes to.

  The metal (M) other than calcium is not particularly limited, but lithium, sodium, potassium, rubidium, cesium, magnesium are considered in consideration of safety to the living body, chemical reactivity, and availability. Strontium, barium, zinc, aluminum, silicon, germanium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, molybdenum, silver and tantalum are preferred examples. Among these, zinc is a particularly preferable metal because it is relatively safe to living bodies and easily becomes a divalent cation and is easily replaced with calcium. A plurality of types of metals may be used in combination as the metal (M).

The preferred content of metal (M) other than calcium is 0.01 to 20% by weight. As shown in the examples described later, from the viewpoint of increasing the adsorption amount of β ₂ -MG, it is more preferably 0.1% by weight or more, and further preferably 0.5% by weight or more. Particularly preferably, it is 2% by weight or more. On the other hand, from the viewpoint of reducing the amount of albumin adsorbed, the metal (M) content is preferably higher, more preferably 3% by weight or more, and further preferably 4% by weight or more. On the other hand, when the content of the metal (M) exceeds 20% by weight, it becomes difficult to incorporate the metal (M) into the crystal structure of hydroxyapatite. The metal (M) content is more preferably 15% by weight or less, and even more preferably 10% by weight or less. However, since the biological substance is adsorbed on the surface of calcium hydroxide phosphate, when the metal (M) other than calcium is unevenly distributed on the surface, even if the content of the metal (M) is small, In some cases, the effect can be efficiently achieved.

At this time, the ratio [(Ca + M) / P] of the number of moles of phosphorus to the total number of moles of calcium and metal (M) is preferably 1.3 to 1.55. The theoretical chemical formula of calcium hydroxide phosphate constituting hydroxyapatite is represented by Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , and the molar ratio of calcium to phosphorus [Ca / P] is 1.67. In contrast, a so-called calcium deficient chemical composition is preferred. The higher the metal (M) content, the smaller the ratio [(Ca + M) / P] value is observed, and the smaller the ratio [(Ca + M) / P], the better the adsorption selectivity of proteins and the like. To do. The ratio [(Ca + M) / P] is more preferably 1.5 or less. On the other hand, if the ratio [(Ca + M) / P] is too small, it is difficult to maintain the crystal structure of hydroxyapatite, and the ratio [(Ca + M) / P] is more preferably 1.4 or more. is there.

  As described above, the selectivity for adsorption of proteins and the like was greatly improved by increasing the content of metals (M) other than calcium and simultaneously reducing the ratio [(Ca + M) / P]. This effect is considered to be achieved by the calcium hydroxide phosphate containing a metal (M) and also by having a calcium-deficient chemical composition.

  Therefore, another aspect of the biological material adsorbent of the present invention is composed of calcium hydroxide phosphate having a ratio [Ca / P] of the number of moles of phosphorus to the number of moles of calcium of 1.3 to 1.55. That is, the selectivity of adsorption of proteins and the like is improved by the chemical composition of the calcium deficient type. As the ratio [Ca / P] decreases, the adsorption selectivity for proteins and the like improves. The ratio [Ca / P] is more preferably 1.5 or less. On the other hand, when the ratio [Ca / P] is too small, it is difficult to maintain the crystal structure of hydroxyapatite, and the ratio [Ca / P] is more preferably 1.4 or more.

  As described above, when calcium hydroxide phosphate contains a metal (M) other than calcium or has a calcium-deficient chemical composition, the selectivity for the adsorption of biological substances is improved. All of these aspects are considered to produce the effects of the present invention independently and synergistically.

  As described above, the crystallite diameter of the calcium hydroxide phosphate of the present invention is preferably 35 nm or less. In the present invention, the higher the content of metal (M), the smaller the value of the ratio [(Ca + M) / P] is observed. At this time, the tendency for the crystallite diameter to decrease is also recognized. Yes. The small crystallite size is also considered to contribute to the improvement of the biological material adsorption selectivity, and it seems preferable to form secondary particles in which the small crystallites (primary particles) are randomly aggregated. . At this time, the crystallite diameter is more preferably 30 nm or less, and further preferably 25 nm or less. Moreover, in order to show the effect as hydroxyapatite, the crystallite diameter is usually 10 nm or more.

Moreover, in order to improve adsorption efficiency, it is preferable that a specific surface area is 80 m < ² > / g or more. As described above, particles having a large specific surface area can be easily obtained by using calcium phosphate particles having a small crystallite diameter. The specific surface area is more preferably 120 m ² / g or more, and even more preferably 150 m ² / g or more. The specific surface area is usually 500 m ² / g or less.

It is preferable that the β ₂ -MG adsorption amount per unit weight of the biological material adsorbent of the present invention is 200 μg / g or more. The biological material adsorbent of the present invention can adsorb more β ₂ -MG than conventional hydroxyapatite. The β ₂ -MG adsorption amount is more preferably 250 μg / g or more, and even more preferably 300 μg / g or more. In the biological material adsorbent of the present invention, the amount of β ₂ -MG adsorbed tended to increase as the content of metal (M) other than calcium increased, and as the degree of calcium deficiency became more prominent. .

Moreover, it is preferable that the albumin adsorption amount per unit weight of the biological material adsorbent of the present invention is 30 mg / g or less. The biological material adsorbent of the present invention can suppress the adsorption of albumin as compared with conventional hydroxyapatite. The albumin adsorption amount is more preferably 20 mg / g or less, and even more preferably 10 mg / g or less. In the biomaterial adsorbent of the present invention, the amount of albumin adsorbed once increases as the content of metal (M) other than calcium increases, but when the content of metal (M) exceeds a certain amount, albumin adsorbed The quantity was found to be very low. In this case, β ₂ -MG can be efficiently adsorbed while suppressing the adsorption of albumin, and a high degree of selectivity can be achieved.

The β ₂ -MG adsorption amount (μg / g) and the albumin adsorption amount (mg / g) are physiological saline containing both β ₂ -MG having a concentration of 2.0 mg / dL and albumin having a concentration of 3.5 g / dL. These were obtained by multiplying the weight of β ₂ -MG and albumin adsorbed by 0.1 g of calcium hydroxide phosphate sample in 2 mL of water by 10 times, respectively.

  The method for producing the biological material adsorbent of the present invention is not particularly limited. As a preferable method, an aqueous solution (A) containing calcium ions and ions of a metal (M) other than calcium and an aqueous solution (B) containing phosphate ions are mixed to obtain a solid content composed of calcium hydroxide phosphate. The method of generating is mentioned. According to this method, it is easy to form calcium hydroxide phosphate having a hydroxyapatite lattice structure while taking in both calcium ions and ions of metal (M) other than calcium.

  The calcium salt contained in the aqueous solution (A) is not particularly limited as long as it dissolves in water, and calcium nitrate, calcium acetate, calcium chloride, and the like can be used, but the solubility of the salt and the obtained aqueous solution (A ) Calcium nitrate is preferably used. Further, the salt of the metal (M) contained in the aqueous solution (A) is not particularly limited, and nitrates, acetates, sulfates, chlorides and the like can be used, but the solubility of the salt and the obtained aqueous solution (A) Nitrate is preferably used in consideration of the pH and the like. That is, it is particularly preferable that the aqueous solution (A) contains calcium nitrate and metal (M) nitrate.

  The content of the calcium salt in the aqueous solution (A) is preferably 0.01 to 1 mol / L, and more preferably 0.1 to 0.8 mol / L. The content of the metal (M) salt in the aqueous solution (A) is preferably 0.002 to 0.16 mol / L, more preferably 0.02 mol / L or more, and 0.03 mol / L or more. More preferably. Moreover, it is suitable that pH of aqueous solution (A) is 4.5-7. By making the pH relatively low, it is easy to form particles having a large specific surface area. The pH of the aqueous solution (A) is more preferably 5 or more. The pH of the aqueous solution (A) is more preferably 6 or less. The pH here is a value measured at 60 ° C.

The phosphate contained in the aqueous solution (B) is not particularly limited as long as it dissolves in water. The anion species may be contained in any form of phosphate anion (PO ₄ ³⁻ ), hydrogen phosphate anion (HPO ₄ ²⁻ ), or dihydrogen phosphate anion (H ₂ PO ₄ ⁻ ). The cation species is not particularly limited, and ammonium salts, sodium salts, potassium salts, and the like can be used. However, ammonium salts that can prevent the mixing of metal elements into the resulting calcium hydroxide phosphate are preferably used. At this time, the aqueous solution in which the ammonium salt is dissolved may be prepared by mixing phosphoric acid (orthophosphoric acid: H ₃ PO ₄ ) and aqueous ammonia.

  The phosphate content in the aqueous solution (B) is preferably 0.01 to 0.5 mol / L. Moreover, it is suitable that pH of aqueous solution (B) is 8-12. By setting the pH relatively high, it is easy to form particles having a large specific surface area. The pH of the aqueous solution (B) is more preferably 9 or more. The pH of the aqueous solution (A) is more preferably 11 or less. In order to adjust the pH of the aqueous solution (B), it is preferable to add ammonia. The pH here is a value measured at room temperature.

  As a method of mixing the aqueous solution (A) and the aqueous solution (B), either the method of adding the aqueous solution (B) into the aqueous solution (A) or the method of adding the aqueous solution (A) into the aqueous solution (B). However, a method of adding the aqueous solution (B) to the aqueous solution (A) is desirable in order to suppress the pH fluctuation during the mixing operation. In mixing, it is preferable to mix gradually while stirring sufficiently. It is preferable that the temperature at the time of mixing is 15-100 degreeC. In particular, when it is desired to increase the content of metal (M) other than calcium, it is preferable to increase the temperature, more preferably 30 ° C. or higher, and even more preferably 50 ° C. or higher. When the inventors mixed at room temperature, it was difficult to contain 1% by weight or more when zinc was introduced as the metal (M).

  The pH of the mixture after mixing the aqueous solution (A) and the aqueous solution (B) usually takes a value in the range of 4 to 12, but a lower pH value has a larger specific surface area and a biological material. A biological material adsorbent with good adsorption selectivity can be obtained. Therefore, the pH of the mixed solution is more preferably 7 or less. The pH here is a value measured at 60 ° C.

  As described above, in addition to the method of mixing the aqueous solution (A) and the aqueous solution (B), a solid solution made of calcium hydroxide phosphate by mixing an aqueous solution containing calcium ions and an aqueous solution containing phosphate ions. A method of adding an aqueous solution (A) containing metal (M) ions during the operation of generating a minute can also be employed. In this case, it is possible to produce particles in which the metal (M) element is unevenly distributed only in the surface layer on which the biological component is directly adsorbed.

  Thus, the produced hydroxyapatite crystals can be ripened by continuing the stirring even after the mixing operation is completed. As the temperature at this time, a temperature comparable to the temperature at the time of mixing can be adopted, and the stirring time is usually about 1 to 168 hours. The obtained solid content is separated from the aqueous solution by an operation such as filtration or centrifugation, and a washing operation or a drying operation is appropriately performed. Furthermore, a hydroxyapatite crystal can be grown by heat treatment.

  The object to be adsorbed by the biological material adsorbent of the present invention may be a biological material, and may adsorb not only substances dissolved in water but also viruses and bacteria. Among biological materials, proteins are preferably adsorbed and particularly suitable for adsorbing proteins having a relatively low molecular weight, for example, water-soluble proteins having a molecular weight of 20000 or less.

A particularly suitable application of the biological material adsorbent of the present invention is an adsorbent for purifying blood or plasma. The biological material in the blood may be directly adsorbed, or the plasma may be once separated and the biological material in the plasma may be adsorbed. Since the calcium phosphate phosphate constituting the biological material adsorbent of the present invention is excellent in biocompatibility and blood compatibility, it is suitable for such applications. Among these, a β ₂ -MG adsorbent is particularly preferable. According to the biological material adsorbent of the present invention, it is possible to minimize loss of useful proteins such as albumin while efficiently removing β ₂ -MG accumulated in the blood of a patient undergoing dialysis treatment. it can. As a result, the occurrence of complications associated with dialysis treatment such as cutaneous pruritus, bone pain, joint pain, and root canal syndrome can be suppressed.

When used as an adsorbent for purifying blood or plasma, a dialysis apparatus incorporating an adsorbing means using the biological material adsorbent of the present invention is preferable. By incorporating it into a dialysis machine, it is possible to efficiently remove pathogenic substances such as β ₂ -MG while reducing the burden on the patient. The method of incorporation is not particularly limited, and it may be filled in a container such as a column or may be supported on various carriers.

  A biological material separation column packed with the biological material adsorbent of the present invention is also a preferred embodiment of the present invention. Although the separation column can be used by being incorporated in a dialysis apparatus as described above, it is also suitably used as a separation column incorporated in an analytical instrument such as affinity chromatography. In this case, taking advantage of the high selective adsorption property for a specific low molecular weight protein, it is particularly suitably used as a protein separation column.

  The usage form of the biological material adsorbent of the present invention is not limited to a powder state, and a material obtained by processing the adsorbent of the present invention into a pellet or the like using a binder or the like may be used. It is also possible to cover plastic, metal, or ceramic substrates formed in various forms such as beads, rods, fibers, nonwovens, wires, and coils. In addition, since adsorption characteristics such as viruses and bacteria are also expected, it can be used as an adsorbent in non-woven fabric for masks, applied to ventilation fans and filters for air purifiers, and diagnoses, inspections and collections of viruses and bacteria. It is also promising as a material for use.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

・ Synthesis of calcium hydroxide phosphate:
The calcium phosphate ceramic according to the present invention was synthesized by the following procedures (1) to (5).
(1) Nitrogen gas was circulated at 3.0 L / min for 30 minutes, and 200 g of distilled water from which dissolved oxygen and dissolved carbon dioxide gas were removed was added to 23 g of calcium nitrate tetrahydrate (Ca (NO ₃ ) ₂ / 4H. ₂ O) was added and dissolved by stirring to prepare a 0.5 mol / L calcium nitrate aqueous solution. In this aqueous solution, zinc nitrate hexahydrate (Zn (NO ₃ ) ₂ / 6H ₂ O) was dissolved so that the concentration of zinc nitrate was 0 to 0.048 mol / L, and nine types of zinc nitrate concentrations were dissolved. 60 ° C. aqueous solutions having The pH of the obtained aqueous solution is 6 when the concentration of zinc nitrate is 0 mol / L, and is 5 when the concentration of zinc nitrate is 0.048 mol / L. The pH value is shown.
(2) 7.9 g of diammonium hydrogen phosphate ((NH ₄ ) ₂ HPO ₄ ) was added to 200 mL of distilled water from which nitrogen gas was passed at 3.0 L / min for 30 minutes to remove dissolved oxygen and dissolved carbon dioxide gas. It melt | dissolved and 0.3 mol / L diammonium hydrogenphosphate aqueous solution was prepared. To this aqueous solution, 28 wt% aqueous ammonia was added to obtain a room temperature aqueous solution whose pH was adjusted to 10.
(3) Using a liquid feed pump, add the phosphate-containing aqueous solution obtained in (2) at a flow rate of 3.0 mL / min to the aqueous calcium nitrate solution having a different zinc ion concentration obtained in (1). The mixture was stirred and mixed while maintaining the temperature at 60 ° C. The pH of the mixed solution when zinc nitrate was contained at 0.048 mol / L was 5.4.
(4) The obtained mixed solution was aged by stirring at 60 ° C. for 24 hours. The solid content thus produced was separated with a centrifuge, the supernatant liquid was discarded, distilled water was added, stirred and washed, and then centrifuged again. This washing operation was repeated three times.
(5) The obtained solid content was dried at 105 ° C. for 24 hours. After drying, the powder obtained by grinding in an alumina mortar was used as a sample for analysis and adsorption experiments.

  Table 1 summarizes the chemical composition, crystallite diameter and specific surface area of the nine types of calcium hydroxide phosphate samples obtained as described above.

The contents of zinc, calcium and phosphorus were determined by high frequency inductively coupled plasma (ICP) emission spectroscopy. FIG. 1 shows the result of X-ray diffraction measurement of calcium hydroxide phosphates having different zinc contents. As can be seen from FIG. 1, all the X-ray diffraction patterns were attributed to hydroxyapatite, and the diffraction peak width increased with increasing zinc content. FIG. 2 shows the relationship between the crystallite diameter calculated from the half width of the peak attributed to diffraction on the (002) plane and the zinc content. As the zinc content increased, the crystallite size decreased from 42 nm to 16 nm. The specific surface area was calculated by BET method by measuring nitrogen adsorption isotherm at liquid nitrogen temperature using “GEMINI2370 Micromeritics” manufactured by Shimadzu Corporation. As the zinc content increased, the specific surface area increased from 57 m ² / g to 202 m ² / g. Furthermore, the size and form of the calcium hydroxide phosphate particles were also observed with a transmission electron microscope. The result is shown in FIG. It can be seen that the crystal size decreases as the zinc content increases.

The albumin and beta ₂ -MG absorptive rating concentration 4.0 mg / dL of beta ₂ -MG and concentration 7.0 g / dL of bovine serum albumin (BSA) containing saline 1 mL (liquid 1), 0 1 mL of physiological saline (Liquid 2) containing 1 g of calcium hydroxide phosphate sample was mixed, β ₂ -MG having a concentration of 2.0 mg / dL, bovine serum albumin having a concentration of 3.5 g / dL, and hydroxylated A 2 mL mixed solution containing 0.1 g of calcium phosphate sample was prepared. The mixture thus obtained was shaken in a hot water bath maintained at 37 ° C. for 6 hours, passed through a cellulose acetate filter having a pore diameter of 0.45 μm to separate calcium hydroxide phosphate, and an aqueous solution for evaluation (6 hours). ) Further, for blank test, 1 mL of the above (Liquid 1) and 1 mL of physiological saline not containing a calcium hydroxide phosphate sample were mixed, β ₂ -MG having a concentration of 2.0 mg / dL, and a concentration of 3.5 g / A 2 mL blank aqueous solution (0 min) containing dL bovine serum albumin was made. The blank aqueous solution (0 minutes) thus obtained was shaken in a hot water bath maintained at 37 ° C. for 6 hours to obtain a blank aqueous solution (6 hours). By quantifying β ₂ -MG and BSA in the aqueous solution for evaluation (6 hours) and the blank aqueous solution (0 minutes) thus obtained, their adsorption amounts were determined.

The quantification of the β ₂ -MG, Sanyo Chemical Industries, Ltd. "Gurazaimu β ₂ -Microgloburin - EIA TEST" was used. The aqueous solution of the sample was developed by an enzyme immunoassay (EIA method), the absorbance at a wavelength of 492 nm was examined, and the β ₂ -MG concentration was calculated from a calibration curve. beta ₂ -MG adsorption amount, after confirming that there is no density difference of beta ₂ -MG between the blank aqueous solution (0 minute) and the blank solution (6 hours), the evaluation solution (6 hours) beta ₂ It calculated | required from the difference of -MG density | concentration and (beta) _2- MG density | concentration of blank aqueous solution (0 minute). For determination of BSA, “Albumin B-Test Wako” manufactured by Wako Pure Chemical Industries, Ltd. was used. The aqueous solution of the sample was colored by the Bromcresol green assay (BCG method), the absorbance at a wavelength of 630 nm was examined, and the BSA concentration was calculated from the calibration curve. After confirming that there is no difference in BSA concentration between the blank aqueous solution (0 minutes) and the blank aqueous solution (6 hours), the BSA adsorption amount was compared with the BSA concentration of the evaluation aqueous solution (6 hours) and the blank aqueous solution (0 Min) from the difference in BSA concentration. The experiment was repeated three times for the same sample (n = 3), and the average value and standard deviation were determined. For the measurement of absorbance, an ultraviolet-visible spectrophotometer “UV-2550” manufactured by Shimadzu Corporation was used.

Further, the ratio of β ₂ -MG and albumin that were adsorbed and removed in each protein solution was determined as the respective adsorption rate (%). Then, the ratio of the adsorption rate of β ₂ -MG to the adsorption rate of albumin was obtained as selectivity. Table 2 summarizes the β ₂ -MG adsorption amount, β ₂ -MG adsorption rate, albumin adsorption amount, albumin adsorption rate and selectivity. Moreover, the graph which plotted (beta) _2- MG adsorption rate and albumin adsorption rate with respect to zinc content is shown in FIG.

The physical property required for the selective blood adsorbent is that the amount of adsorption of useful components in the blood is as small as possible, and the target pathogenic substance is efficiently removed. In the above test, albumin used in the evaluation of the dialysis membrane was selected from many useful substances in blood, and its adsorption rate was evaluated together with the pathogenic substance β ₂ -MG. As is clear from FIG. 4, compared with conventional hydroxyapatite (zinc content 0 wt%), calcium hydroxide containing zinc has an albumin adsorption rate of up to 2 wt% zinc, Slightly increased, but significantly decreased above 2% by weight. As a result, it was confirmed that clinical improvement was achieved in the desired direction. The cause of this slight increase in albumin adsorption rate is not clear, but according to observation with a transmission electron microscope, only when the zinc content is around 2% by weight, apatite particles with two different forms, acicular and granular, are present. From the confirmation, it is considered that there is an influence of the difference in crystal form. Furthermore, the adsorption rate of β ₂ -MG, which is the main object of the present invention, was significantly improved to 90% or more as the zinc content increased. As described above, it was revealed that ideal selective adsorptivity is expressed.

Although the reason for this is not clear, it is thought that the electrostatic interaction between albumin and the carboxyl group at the end of the molecule is weakened by the zinc ion distribution on the surface of hydroxyapatite compared to conventional hydroxyapatite. It is done. On the contrary, it was advantageous for the adsorption of β ₂ -MG, and the adsorbing rate was improved. Moreover, by adding zinc ions during the synthesis of hydroxyapatite, the crystallite size of the apatite to be produced was reduced and the specific surface area was increased. Thereby, the adsorption capacity of β ₂ -MG is improved by increasing the effective adsorption area per unit weight. Further, when considering the crystal morphological homogeneity observed by transmission electron microscope observation, it is considered that the condition that the zinc content is 2% by weight or more is desirable.

In the above-mentioned Patent Document 6, β ₂ -MG adsorption amount is 183 μg / g, β ₂ -MG adsorption rate is 40% or less, albumin adsorption amount is 7.5 mg / g, albumin adsorption rate is about 10 to 20%, and The selectivity between the two was about 1.5 times. On the other hand, in the biomaterial adsorbent of the present invention, in the sample that gave the most excellent result, the β ₂ -MG adsorption amount was 380 μg / g or more, the β ₂ -MG adsorption rate was 90% or more, and the albumin adsorption amount was About 2 mg / g, the albumin adsorption rate was about several percent, and the selectivity of both was several tens to several hundred times. By comparing these, it is clear that the adsorption characteristics are greatly improved, and it can be seen that the biological material adsorbent of the present invention is extremely useful.

It is an X-ray-diffraction measurement chart about calcium hydroxide with different zinc contents. It is the graph which showed the relationship between a crystallite diameter and zinc content. 2 is a photograph of calcium hydroxide phosphate particles observed with a transmission electron microscope. It is the graph which plotted (beta) _2- MG adsorption rate and albumin adsorption rate with respect to zinc content.

Claims (16)

A biological material adsorbent comprising calcium hydroxide phosphate containing 0.01 to 20% by weight of a metal (M) other than calcium. Metal (M) is lithium, sodium, potassium, rubidium, cesium, magnesium, strontium, barium, zinc, aluminum, silicon, germanium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, The biological material adsorbent according to claim 1, which is at least one selected from the group consisting of molybdenum, silver and tantalum. The biological material adsorbent according to claim 2, wherein the metal (M) is zinc. The biological material adsorbent according to any one of claims 1 to 3, wherein a ratio [(Ca + M) / P] of the number of moles of phosphorus to the total number of moles of calcium and metal (M) is 1.3 to 1.55. A biological material adsorbent comprising calcium hydroxide phosphate having a ratio of the number of moles of phosphorus to the number of moles of calcium [Ca / P] of 1.3 to 1.55. Biological material adsorbent according to any one of claims 1 to 5 specific surface area of ^{80 m} 2 / g or more. The biological material adsorbent according to any one of claims 1 to 6, wherein the adsorption amount of β ₂ -microglobulin per unit weight is 200 µg / g or more. The biological material adsorbent according to any one of claims 1 to 7, wherein the albumin adsorption amount per unit weight is 30 mg / g or less. The biological material adsorbent according to any one of claims 1 to 8, which is an adsorbent for purifying blood or plasma. The biological material adsorbent according to claim 1, which is a β ₂ -microglobulin adsorbent. A dialysis apparatus incorporating an adsorption means using the biological material adsorbent according to any one of claims 1 to 10. A biological material separation column packed with the biological material adsorbent according to claim 1. 2. An aqueous solution (A) containing calcium ions and ions of a metal (M) other than calcium and an aqueous solution (B) containing phosphate ions are mixed to produce a solid content comprising calcium hydroxide phosphate. The manufacturing method of the biological material adsorption agent in any one of 10-10. The method for producing a biological material adsorbent according to claim 13, wherein the aqueous solution (A) contains a nitrate of calcium and a nitrate of metal (M). The method for producing a biological material adsorbent according to claim 13 or 14, wherein the pH of the aqueous solution (A) is 4.5 to 7. The method for producing a biological material adsorbent according to any one of claims 13 to 15, wherein the pH of the aqueous solution (B) is 8 to 12.