JP5643938B2 - Endotoxin removal method and endotoxin adsorbent - Google Patents

Description

  The present invention relates to an endotoxin adsorbent comprising an apatite / collagen porous material, and a method for removing endotoxin using the same.

  Endotoxin is an outer membrane constituent of gram-negative bacteria and exhibits various physiological activities on living organisms. In particular, endotoxin is considered to be a relatively strong pyrogen in pyrogens, and is known to cause fever, diarrhea, bleeding, leukocyte fluctuation, and the like. However, antitoxins against endotoxins are not easily formed in vivo, and even if formed, endotoxins cannot always be effectively inactivated.

  The structural component of endotoxin is lipopolysaccharide, and is contaminated by, for example, bacteria that inhabit during the production process of pharmaceuticals being produced and killed. Conventionally known methods for removing endotoxins include adsorption methods using activated carbon and ion exchangers, filtration methods using membranes and membrane filters, high-temperature / high-pressure treatments, and decomposition methods using acids / alkalis.

  In recent years, various adsorbents and methods have been disclosed for endotoxin removal on an industrial scale such as a pharmaceutical production process. For example, Japanese Patent Publication No. 6-18443 (Patent Document 1) discloses an adsorbent composed of a polyamino acid containing a modifying group having an aliphatic group and / or aryl at the end of the side chain and / or main chain. JP-A 1-127039 (Patent Document 2) discloses an adsorbent obtained by binding an imidazole derivative to a carrier of polyamino acid spherical particles. Japanese Patent Laid-Open No. 2002-263486 (Patent Document 3) discloses a method for selectively adsorbing endotoxin by controlling the pore size of an adsorbent on which a basic substance is immobilized.

  However, endotoxin removal using these adsorbents can be accomplished by a chromatographic method in which the target solution is passed through a column packed with granular adsorbents to adsorb and remove endotoxins, or adsorbent particles are added to the target solution and stirred. Thereafter, the adsorbent particles are separated and removed by a method such as filtration and centrifugation. Such a method is convenient when processing a large amount of solution at a time, which is performed industrially, but requires expensive equipment and complicated operations. A simple method is desired, not suitable for the treatment of water and the like used for the appliance.

Japanese Patent Publication No.6-16843 Japanese Unexamined Patent Publication No. 1-127039 JP 2002-263486 A

  Accordingly, an object of the present invention is to provide an endotoxin adsorbent capable of easily and efficiently removing endotoxin from a small amount of endotoxin-containing solution, and an endotoxin removal method.

  As a result of diligent research in view of the above object, the present inventors have found that a porous body comprising an apatite / collagen composite fiber is excellent in endotoxin adsorption ability and easy to handle when used as an adsorbent. I came up with the invention.

  That is, the method for adsorbing and removing endotoxin of the present invention is characterized in that the apatite / collagen porous material is brought into contact with an endotoxin-containing solution.

  It is preferable to immerse the apatite / collagen porous body in an endotoxin-containing solution, adsorb endotoxin on the porous body, and then remove the porous body.

  The apatite / collagen porous body is preferably used as a block body having a size of 5 mm × 5 mm × 5 mm to 50 mm × 50 mm × 50 mm.

  The porous body is preferably irradiated with gamma rays.

  The endotoxin adsorbent of the present invention comprises an apatite / collagen porous body and is a sponge-like block body in a wet state.

  The block body preferably has a size of 5 mm × 5 mm × 5 mm to 50 mm × 50 mm × 50 mm.

  The method of adsorbing and removing endotoxin according to the present invention uses a sponge-like block composed of apatite / collagen porous material as the adsorbent, so that the endotoxin-containing solution can be easily absorbed and discharged, and endotoxin can be easily removed from a small amount of solution. Can be removed. Moreover, compared with the chromatography method using a column, an expensive apparatus and complicated operation are unnecessary.

[1] Apatite / Collagen Porous Body In the present invention, an apatite / collagen porous body is used as an endotoxin adsorbent. The porous body is preferably made of an apatite / collagen composite fiber and preferably has a porosity of 90 to 95% and an average pore diameter of about 10 to 500 μm.

  Apatite / collagen porous body is uncalcined hydroxyapatite, has an exchange function for magnesium ions, phosphate ions, etc., and the phosphate group in the lipid A part of endotoxin adsorbs to apatite, so it has excellent endotoxin adsorption capacity. Have. Since the apatite / collagen porous body is sponge-like when wet, the contact area with the endotoxin-containing solution is large, and endotoxin can be efficiently removed even by a batch method.

  In particular, when treating a trace amount of endotoxin-containing solution, if a non-sponge-like porous body is used as the adsorbent, the adsorbent may absorb most of the endotoxin-containing solution, and a sufficient amount of solution may not be recovered. When the sponge-like adsorbent is used in the wet state of the present invention, the endotoxin-containing solution once absorbed can be easily discharged by crushing the block body, and a sufficient amount of solution can be secured even after endotoxin removal.

  The apatite / collagen porous body is preferably used as a block body. The block body may be of a size that can be easily recovered from the endotoxin-containing solution, and preferably has a size of 5 mm × 5 mm × 5 mm to 50 mm × 50 mm × 50 mm, 10 mm Those having a size of × 10 mm × 10 mm to 10 mm × 20 mm × 30 mm are more preferable. The block body does not necessarily have to be a cube or a rectangular parallelepiped, and may have a shape such as a cylinder, a triangular prism, or a hexagonal column, a flat plate shape, a spherical shape, an elliptical shape, or the like.

[2] Method for producing apatite / collagen porous material
(1) Apatite / collagen composite fiber
(a) Raw material The apatite / collagen composite fiber is made from collagen, phosphoric acid or a salt thereof, and a calcium salt. It does not specifically limit as collagen, What was extracted from the animal etc. can be used. The species, tissue site, age, etc. of the derived animal are not particularly limited. Generally, collagen obtained from the skin, bone, cartilage, tendon, organ, etc. of mammals (eg, cows, pigs, horses, rabbits and mice) and birds (eg, chickens) can be used. Collagen-like proteins obtained from the skin, bones, cartilage, fins, scales, organs, etc. of fish (eg, cod, flounder, flounder, salmon, trout, tuna, mackerel, Thailand, sardine and shark) may also be used. The method for extracting collagen is not particularly limited, and can be based on a general extraction method. In addition, collagen obtained by gene recombination techniques may be used instead of those extracted from animal tissues.

  Examples of phosphoric acid or a salt thereof (hereinafter simply referred to as “phosphoric acid (salt)”) include phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate. Examples of calcium salts include calcium carbonate, calcium acetate, and calcium hydroxide. The phosphate and calcium salt are preferably added in the form of a uniform aqueous solution or suspension, respectively.

  The mass ratio of apatite / collagen in the product can be controlled by the mass ratio of the apatite raw material [phosphoric acid (salt) and calcium salt] to be used and collagen. For this reason, the mass ratio of the apatite raw material to be used and collagen is appropriately determined depending on the composition ratio of the target apatite / collagen composite fiber. The apatite / collagen ratio in the apatite / collagen composite fiber is preferably 9/1 to 6/4 (mass ratio), particularly preferably about 8/2 (mass ratio).

(b) Preparation of solution The concentration of the phosphoric acid (salt) aqueous solution and the calcium salt aqueous solution is not particularly limited as long as the phosphoric acid (salt) and the calcium salt are in a desired mixing ratio, but for the convenience of the dropping operation described below, The concentration of the phosphoric acid (salt) aqueous solution is preferably about 50 to 250 mM, and the concentration of the calcium salt aqueous solution is preferably about 200 to 600 mM. Collagen is generally added in advance to the aforementioned phosphoric acid (salt) aqueous solution in a phosphoric acid aqueous solution state. The collagen aqueous phosphoric acid solution preferably has a collagen concentration of 0.5 to 1% by mass and a phosphoric acid concentration of 10 to 30 mM. More preferably, the concentration of collagen is 0.8 to 0.9% by mass and the concentration of phosphoric acid is 15 to 25 mM, and particularly preferably, the concentration of collagen is about 0.85% by mass and the concentration of phosphoric acid is about 20 mM.

(c) Production of apatite / collagen composite fiber Approximately the same amount of calcium salt aqueous solution to be added (preferably 0.5 to 2 times, more preferably 0.8 to 1.2 times the calcium salt aqueous solution to be added) Put in a reaction vessel in advance and heat to about 40 ° C. There, the phosphoric acid (salt) aqueous solution and calcium salt aqueous solution which contain collagen are dripped simultaneously. The length of the apatite / collagen composite fiber to be synthesized can be controlled by the dripping conditions. The dropping speed is preferably about 10 to 50 ml / min, and the reaction solution is preferably stirred at about 50 to 300 rpm. During the dropping, it is preferable to maintain the calcium ion concentration in the reaction solution at 3.75 mM or less and the phosphate ion concentration at 2.25 mM or less. Thereby, the pH of the reaction solution is maintained at 8.9 to 9.1. When the concentration of calcium ions and / or phosphate ions exceeds the above range, self-assembly of the complex is prevented. In this specification, “self-organization” means that hydroxyapatite (calcium phosphate having an apatite structure) is oriented along the collagen fiber, that is, the C axis of hydroxyapatite is along the collagen fiber. It means that it is oriented. With the above dropping conditions, the apatite / collagen composite fiber is self-organized with a length of 1 mm or less suitable as a raw material for the porous body.

  After completion of the dropwise addition, the slurry-like apatite / collagen composite fiber dispersion is freeze-dried. Freeze-drying is carried out by drawing a vacuum in a state of being frozen at −10 ° C. or lower and drying rapidly.

(2) Preparation of dispersion containing apatite / collagen composite fiber Water, phosphoric acid aqueous solution or the like is added to the apatite / collagen composite fiber and stirred to prepare a paste-like dispersion. The content of the liquid contained in the dispersion is preferably 80 to 99% by volume, and more preferably 90 to 97% by volume. That is, the content of the composite fiber is preferably 1 to 20% by volume, and more preferably 3 to 10% by volume.

The porosity P (%) of the porous material to be produced depends on the volume ratio of the apatite / collagen composite fiber and the liquid in the dispersion, and the following formula (1):
P = Y / (X + Y) × 100 (1)
[Wherein X represents the volume of the apatite / collagen composite fiber in the dispersion, and Y represents the volume of the liquid in the dispersion. ]. Therefore, the porosity P of the porous body can be controlled by the amount of liquid added. By stirring the dispersion after adding the liquid, the apatite / collagen composite fiber is cut and the fiber length distribution width is increased, so that the strength of the porous body to be produced is improved.

  Collagen as a binder is added to the dispersion of the composite and further stirred. The amount of collagen added is preferably 1 to 10% by mass and more preferably 3 to 6% by mass with respect to 100% by mass of the apatite / collagen composite fiber. As in the case of the complex, the collagen is preferably added in the form of an aqueous phosphoric acid solution. The concentration of the phosphoric acid aqueous solution of collagen is not particularly limited, but practically the collagen concentration is 0.8 to 0.9 mass% (for example, 0.85 mass%), and the phosphoric acid concentration is 15 to 25 mM (for example, 20 mM). .

(3) Gelation of the dispersion The sodium hydroxide solution is added to adjust the pH to about 7 to the dispersion which has been made acidic by the addition of an aqueous phosphoric acid (salt) solution of collagen. The pH of the dispersion is preferably 6.8 to 7.6, more preferably 7.0 to 7.4. By setting the pH of the dispersion to 6.8 to 7.6, the fiberization of collagen added as a binder can be promoted.

  A concentrated solution of about 2.5 to 10 times the phosphate buffer solution (PBS) is added to the dispersion and stirred to adjust the ionic strength to 0.2 to 0.8. A more preferable ionic strength is the same ionic strength as PBS (about 0.2 to 0.8). By increasing the ionic strength of the dispersion, it is possible to promote the fiberization of collagen added as a binder.

  After putting the dispersion in a mold, the dispersion is gelled by maintaining the temperature at 35 to 43 ° C. The holding temperature is more preferably 35 to 40 ° C. In order to sufficiently gel the dispersion, the holding time is preferably 0.5 to 3.5 hours, and more preferably 1 to 3 hours. By maintaining the temperature of the dispersion at 35 to 43 ° C., collagen added as a binder is fibrillated, and the dispersion becomes a gel. By gelling the dispersion, it is possible to prevent the apatite / collagen composite fibers from being settled in the dispersion, and it is possible to produce a uniform porous body.

(4) Freezing and drying of the gel body The gel body containing the apatite / collagen composite fiber is frozen. The average pore size of the target apatite / collagen porous body depends on the time required for freezing the gel body. The freezing temperature is preferably −100 to 0 ° C., more preferably −100 to −10 ° C., and particularly preferably −80 to −20 ° C. Below −100 ° C., the average pore size of the resulting apatite / collagen porous body is too small. If it exceeds 0 ° C., it does not freeze or it takes a long time to freeze, and the average pore diameter of the porous body is too large.

  The frozen gel body is dried by freeze-drying to make a porous body. That is, as in the case of the apatite / collagen composite fiber, it is evacuated in a state of being frozen at −10 ° C. or lower and rapidly dried. Freeze-drying may be performed until the dispersion is sufficiently dried, and the time is not particularly limited, but is generally about 24 to 72 hours.

(5) Collagen cross-linking Collagen cross-linking may be either physical cross-linking (method using gamma rays, ultraviolet rays, thermal dehydration, electron beam, etc.) or chemical cross-linking (method using cross-linking agent or condensing agent). In the case of chemical crosslinking, the porous body is immersed in a solution of a crosslinking agent. Examples of crosslinking agents include aldehyde-based crosslinking agents (such as glutaraldehyde and formaldehyde), isocyanate-based crosslinking agents (such as hexamethylene diisocyanate), and carbodide-based crosslinking agents (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. Etc.), polyepoxy crosslinking agents (ethylene glycol diethyl ether, etc.), transglutaminase and the like. Of these crosslinking agents, glutaraldehyde is particularly preferable from the viewpoint of easy control of the degree of crosslinking and the biocompatibility of the resulting porous body.

  When crosslinking is performed using glutaraldehyde, the concentration of the glutaraldehyde solution is preferably 0.005 to 0.015% by mass, and more preferably 0.005 to 0.01% by mass. When an alcohol such as ethanol is used as a solvent for the glutaraldehyde solution, the porous body can be dehydrated simultaneously with the crosslinking of collagen. By performing dehydration at the same time as crosslinking, a crosslinking reaction can be caused in a contracted state of the apatite / collagen composite fiber, and the elasticity of the resulting porous body can be improved.

  After the crosslinking treatment, the porous body is immersed in an aqueous solution of about 2% by mass of glycine to remove unreacted glutaraldehyde, and then washed with water. Further, the porous body is dehydrated by dipping in ethanol and then dried at room temperature.

  Thermal dehydration crosslinking is performed by holding the lyophilized porous body in a vacuum oven at 100 to 160 ° C. and 0 to 100 hPa for 10 to 12 hours.

(6) Gamma-ray treatment Since apatite collagen porous bodies using animal-derived collagen may contain endotoxin in itself, 16-35 kGy [unit is Gy (gray)] in the porous body after crosslinking Preferably, a dose of gamma rays is irradiated. Endotoxin activity can be reduced by gamma irradiation. The higher the gamma ray dose, the lower the endotoxin activity, but the elasticity of the apatite / collagen porous material is lost and the block shape cannot be maintained. Therefore, the irradiation dose is preferably 35 kGy or less. If it is less than 16 kGy, inactivation of endotoxin is insufficient. The irradiation dose of gamma rays is more preferably 20 to 30 kGy, and further preferably 22 to 25 kGy. Cobalt 60 is generally used as the radiation source. As an irradiation method, enter the irradiation chamber with a belt conveyor, go out after a certain period of time, and then enter the irradiation chamber again until it reaches the fixed absorbed dose. There is static irradiation.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
(A) Synthesis of apatite / collagen composite
To 200 ml of a 120 mM phosphoric acid aqueous solution, 2020 g of a collagen phosphoric acid aqueous solution (collagen concentration: 0.56 mass%, phosphoric acid concentration: 20 mM) was added and stirred to prepare a diluted collagen phosphoric acid aqueous solution. On the other hand, 1123 ml of 400 mM calcium hydroxide suspension was prepared. 200 ml of pure water was placed in the reaction vessel and heated to 38 ° C. A dilute collagen phosphate aqueous solution and a calcium hydroxide suspension are simultaneously added dropwise to the reaction vessel at a rate of about 30 ml / min, and the resulting reaction solution is stirred at 200 rpm to obtain an apatite / collagen composite fiber. A slurry containing was prepared. The pH of the reaction solution during the dropping was kept at 8.9 to 9.1. The slurry containing the apatite / collagen complex was lyophilized. The fiber length of the produced apatite / collagen composite was approximately 1 mm or less. The blending ratio of apatite / collagen in the apatite / collagen composite was 8/2 on a mass basis.

(B) Preparation of apatite / collagen crosslinked porous body 1 g of freeze-dried apatite / collagen composite was added with 6.3 ml of PBS (phosphate buffered saline) and stirred to obtain a paste-like dispersion. After adding and stirring 1.7 g of phosphoric acid aqueous solution of collagen to this paste-like dispersion, 1 N NaOH aqueous solution was added until the pH became approximately 7, thereby obtaining a dispersion. The concentration of the apatite / collagen complex in the dispersion was 5% by volume.

  The obtained dispersion was put into a mold and kept at 37 ° C. for 2 hours for gelation to obtain a jelly-like molded body. This molded body was frozen at −20 ° C., then dried using a freeze dryer, and then thermally dehydrated at 140 ° C. to obtain an apatite / collagen porous body.

(c) Gamma ray treatment The obtained apatite / collagen porous body was cut into blocks of 10 mm × 10 mm × 10 mm and irradiated with gamma rays at a dose of 25 kGy. This block body was 0.16 g (hydroxyapatite: 0.128 g, collagen: 0.032 g).

[Endotoxin adsorption removal test]
The endotoxin adsorption removal performance of the apatite / collagen porous material was measured. Endotoxin standards were diluted to prepare an endotoxin-containing solution at a concentration of 1.0 EU / ml. 2 ml of this endotoxin-containing solution was placed in a pyrogen-free nylon bag, and the block of apatite / collagen porous material irradiated with gamma rays was placed therein. After the endotoxin-containing solution is absorbed into the porous body, the operation of pushing the porous body from the outside of the bag and discharging the absorbed solution is repeated 5 times (total time: 5 minutes), and the finally discharged solution is tested. Liquid. The endotoxin concentration of the test solution was measured using an endotoxin measuring apparatus (Toxinometer ET2000: Wako Pure Chemical Industries). The results are shown in Table 1.

Comparative Example 1
Hydroxyapatite synthesized by mixing calcium hydroxide and phosphoric acid was calcined at 700 ° C. for 4 hours to obtain hydroxyapatite powder. Hydroxyapatite powder 0.128 g treated with gamma rays in the same manner as in Example 1 was placed in an endotoxin solution adjusted to a concentration of 1.0 EU / ml in the same manner as in Example 1 and stirred for 5 minutes. Thereafter, the hydroxyapatite powder was removed with a filter, and the endotoxin concentration of the collected test solution was measured in the same manner as in Example 1. Since the process of filtering the hydroxyapatite powder was added, the operation was complicated as compared with Example 1. The results are shown in Table 1.

Reference example 1
The endotoxin adsorption removal test was carried out in the same manner as in Example 1 using apatite / collagen porous material (10 mm × 10 mm × 10 mm) irradiated with gamma rays at a dose of 50 kGy. However, the apatite / collagen porous material collapsed during the discharging operation, and measurement was impossible.

Claims (3)

A method for adsorbing and removing endotoxin by bringing the apatite / collagen porous body into contact with an endotoxin-containing solution , comprising preparing an apatite / collagen porous body, which is a sponge-like block body having elasticity in a wet state. The endotoxin absorbed in the porous body by immersing the collagen porous body in the endotoxin-containing solution, absorbing the endotoxin-containing solution in the porous body, adsorbing the endotoxin to the porous body, and crushing the porous body A method for removing endotoxin, comprising: removing the porous body after performing an operation of discharging the contained solution from the porous body. 2. The endotoxin removal method according to claim 1, wherein the block of the apatite / collagen porous body has a size of 5 mm × 5 mm × 5 mm to 50 mm × 50 mm × 50 mm. Removal method. 3. The endotoxin removal method according to claim 1 or 2, wherein the porous body is irradiated with gamma rays at an irradiation dose of 35 kGy or less .