JP2002045686A - Adsorbent for basic protein and production process of the same adsorbent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a basic protein adsorbent which has such an introduced surface structure that basic protein can efficiently be adsorbed on the (c)face of the adsorbent compound, and also has a high specific surface area. SOLUTION: This adsorbent is a boron-containing calcium phosphate compound represented by the formula [Ca9.5+0.5X (PO4)6-X(BO3)X) (BO2)1-XOX}] (wherein X is a numerical value of 0-1.0) and has a >=10 m2/g specific surface area. The production process of the adsorbent compound comprises forming a raw material solution containing calcium oxide, phosphorus oxide, boric acid and nitric acid into fine liquid droplets, thereafter, introducing the fine droplets into an atmosphere of 300-1,200 deg.C while atomizing the fine droplet to synthesize the objective boron-containing calcium phosphate compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a basic protein adsorbent capable of specifically adsorbing and separating biological substances (cells, proteins, nucleic acids, etc.) in the medical or biochemical field.

[0002]

2. Description of the Related Art When separating and purifying useful substances derived from living organisms in the production of pharmaceuticals and the like, it is desired to efficiently recover target substances. For that purpose, a material which specifically adsorbs only the target substance or which does not specifically adsorb it is necessary. BACKGROUND ART Hydroxyapatite has been known as a material for efficiently adsorbing and separating a biological substance without deactivating it. Hydroxyapatite chromatography is a kind of affinity chromatography, and its adsorption mechanism is considered to be ion exchange. The surface of hydroxyapatite is composed of a positively charged surface a and a negatively charged surface c. Therefore, it is estimated that the acidic substance is adsorbed on the a-plane and the basic substance is adsorbed on the c-plane. For this reason, in hydroxyapatite chromatography, an acidic substance and a basic substance are adsorbed without distinction, and the acidic substance and the basic substance must be separated in the subsequent elution process.

Therefore, in order to specifically adsorb an acidic substance or a basic substance to hydroxyapatite, it is necessary to create a surface structure suitable for the purpose. As one of such methods, it is conceivable to adopt a plate-like structure in which a c-plane is grown in order to specifically adsorb a basic substance to hydroxyapatite. In addition, a method has been proposed in which hydroxyapatite is calcined at a high temperature to adsorb only basic proteins (see, for example, JP-A-8-71412).

[0004]

However, as described above, if only a basic substance is adsorbed by using hydroxyapatite, a special structure of a plate-like structure having a c-plane grown as described above is required. Must be synthesized. The most common method of synthesizing this plate-like hydroxyapatite is a method of hydrothermally treating low-crystalline hydroxyapatite in the coexistence of the same amount of methanol. It takes time and is not suitable for mass production. Further, high-temperature calcined hydroxyapatite, which is said to be capable of specifically adsorbing basic proteins, has the disadvantage that the specific surface area becomes extremely small as a result of calcining, and the amount of basic protein that can be specifically adsorbed is small.

In view of the above-mentioned drawbacks, the inventors of the present invention have continued research on the adsorption of basic proteins to apatite.
The present inventors have found that a basic protein can be specifically adsorbed by using a boron-containing calcium phosphate compound having a surface structure capable of adsorbing a basic protein efficiently on a surface, and the present invention has been accomplished. Accordingly, an object of the present invention is to provide a basic protein adsorbent having a specific surface area by introducing a surface structure capable of efficiently adsorbing a basic protein to the c-plane.

[0006]

The above object of the present invention is achieved by the following inventions.

[1] A basic protein adsorbent, which is a boron-containing calcium phosphate compound. [2] The boron-containing calcium phosphate compound is represented by the general formula [I].
4. The basic protein adsorbent according to 1. General formula [I] [Ca _{9.5 + 0.5X} {(PO ₄ ) _6-X (BO ₃ ) _X } (B
_{O 2) 1-X O X} } ] (where, X is 0 to 1.0.) [Specific surface area of claim 3] The boron-containing calcium phosphate compound is 10 m ²
/ G or more.
4. The basic protein adsorbent according to 1. [Claim 4] A calcium compound, a phosphoric acid compound and boric acid are dissolved in a nitric acid solution, and the obtained raw material solution is formed into fine droplets.
A method for producing a basic protein adsorbent, characterized in that a boron-containing calcium phosphate compound is synthesized by spraying in a 0 to 1200 ° C atmosphere. [5] The method for producing a basic protein adsorbent according to [4], wherein the calcium compound is a nitrate and the phosphate compound is diammonium hydrogen phosphate.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below, but the present invention is not limited to these embodiments.

According to the first aspect of the present invention, the basic protein adsorbent is a boron-containing calcium phosphate compound, whereby a surface structure capable of adsorbing the basic protein efficiently on the c-plane is obtained. Moreover, a basic protein adsorbent having a high specific surface area can be obtained. In the invention according to claim 2, the boron-containing calcium phosphate compound is represented by the general formula [I].

[Ca _{9.5 + 0.5X} ＰＯ (PO ₄ ) _6-X (B
O ₃ ) _X ｝ ｛(BO ₂ ) _1-X O _X ｝]

The boron-containing calcium phosphate compound used in the present invention is a compound in which the PO4 and OH groups of hydroxyapatite are substituted with BO3 and BO2 groups, as is apparent from the above-mentioned general formula [I]. Since boric acid is introduced into the c-plane, which is assumed to be adsorbed, the c-plane is more strongly negatively charged than in the case of hydroxyapatite. It is considered that this increases the basic substance adsorption power. In the general formula [I], the range of x is 0 to 1.0, and a large amount of the basic substance is adsorbed in this range. Preferably, more basic substance is absorbed in the range of 0 to 0.8.
In particular, when x is in the range of 0 to 0.3, it is preferable because more basic substances are adsorbed. Further, in the invention according to claim 3, the specific surface area of the boron-containing calcium phosphate compound is set to 1 because the protein or the like is adsorbed more.
It is preferably 0 m ² / g or more.

In order to synthesize the boric acid-containing calcium phosphate compound, a solid phase reaction method in which the raw material is heated and pulverized in a dry method, and an ultrasonic spray heat in which an electric furnace is spray-heated while applying ultrasonic waves in a wet method There is a decomposition method. In the present invention, the ultrasonic spray pyrolysis method, in which spherical particles particularly convenient for protein adsorption separation are formed, is preferable as a method for synthesizing a basic protein adsorbent. The invention described in claim 4 is
In this ultrasonic spray pyrolysis method, a calcium compound, a phosphoric acid compound and boric acid are dissolved in a nitric acid solution, and the obtained raw material solution is formed into fine droplets.
A method for producing a basic protein adsorbent, comprising synthesizing a boron-containing calcium phosphate compound by spraying in an atmosphere of 00 ° C. The atmosphere temperature is preferably 850 to 1100 ° C., so that the basic protein adsorbent can be efficiently produced. The conditions for producing fine droplets are not particularly limited, but a method using ultrasonic waves is preferable.

Specifically, the synthesizing apparatus shown in FIG. 1 is used. That is, the operation is performed by operating the ultrasonic oscillator 3 installed at the lower part of the synthesizer at 2.4 MHz. The raw material solution 6 is put in the container 2 covered with the polyethylene film 12,
With the film interposed, the raw material solution 6 continuously generates droplets by ultrasonic waves generated by the ultrasonic oscillator 3. The droplet generated by this method is extremely fine, and its size depends on the solute concentration (density) and surface tension of the raw material solution, but is generally about 1 μm. The droplets thus generated are drawn into the reaction system by an aspirator and introduced into a reaction tube 7 in an electric furnace 8, where they are dried and thermally decomposed and collected as powder. This series of operations is performed continuously. The composition of the raw material solution suitable for synthesizing the protein adsorbent used in the present invention has a Ca ²⁺ ion concentration of ^0.1 %.
A 1~0.9mol · dm ^-3, PO ₄ ^3- ion concentration is 0.05~0.58mol · dm ^-3, also H
_{The 3} BO ₃ concentration is 0.01 to 0.1 mol · dm ⁻³ . These solutions were used with HNO ₃ concentrations ranging from 0.1 to 0.
Dissolve using a solution up to 8 mol · dm ^-3 . Further, in the invention of the method for producing a basic protein adsorbent according to claim 5, it is preferable that the calcium compound is a nitrate and the phosphate compound is diammonium hydrogen phosphate.

[0014]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[Example 1] [Method for producing basic protein adsorbent] The raw material solution was 0.50 mol of calcium nitrate.
Dm ^-3 , 0.30 mol of diammonium hydrogen phosphate
dm ^-3, boric acid 0.052 mol · dm ^-3 to prepare by dissolving in nitric acid solution. On the other hand, in FIG. 1, water 4 is put in a water tank 1 in which an ultrasonic oscillator 3 is set at a lower part, and a reaction tank 2 is immersed in the water. After the sample solution 6 was put into the reaction tank 2, the air was sucked by an aspirator 11 at an air flow rate of 1.0 dm ³ / min. 1 m in length;
(Internal diameter 3 cm). In this example, x = 0.2
Was synthesized.

Similarly, a sample solution is prepared by mixing calcium nitrate, diammonium hydrogen phosphate and boric acid so that x becomes a boron-containing calcium phosphate compound of 0.4 and 0.8. The powder was synthesized with an ultrasonic spray pyrolysis device. Hydroxyapatite was also synthesized. 0.2 g of each powder is added to 10 cm ³ of a phosphate buffer solution containing lysozyme (isoelectric point 11.0 to 11.4) at pH 6, 7, or 8 and shaken at 25 ° C. at 100 times / minute for 72 hours. Thereafter, the absorbance at 280 nm of the supernatant was measured. Table 1 shows the results of calculating the saturated adsorption amount of the protein from the Langmuir plot.

[0017]

[Table 1]

As apparent from Table 1, the calcium phosphate compound containing boric acid adsorbed lysozyme (LSZ) at least three times that of hydroxyapatite.

Further, when the specific surface area of the protein adsorbent obtained by the above production method was measured, the specific surface area was 10 to 40 m ² · g ⁻¹ regardless of the value of x. On the other hand, when the compound having the same composition was prepared by a usual solid-phase reaction method in which the raw material was heated and pulverized under a dry system, the specific surface area was 1 m ² · g ⁻¹ or less. As described above, the specific surface area is an important factor for protein adsorption. Therefore, the method for producing boron-containing calcium phosphate of the present invention is the most suitable method when used as a basic protein adsorbent.

Comparative Example 1 An adsorption test was performed in the same manner as in Example 1 except that bovine serum albumin (BSA; isoelectric point of 4.7 to 5.2) was used as an adsorbing substance. It is shown in FIG.

[0021]

[Table 2]

As is clear from Table 2, no difference was observed in the amount of adsorption between the boric acid-containing calcium phosphate compound and the hydroxyapatite.

COMPARATIVE EXAMPLE 2 0.2 g each of a boric acid-containing calcium phosphate compound having x of 0.2 and 0.2 g of hydroxyapatite
In addition to glucose 10 cm ³ of 00ppm, 25 ℃, 1
After shaking at 00 times / minute for 24 hours, the amount of glucose in the supernatant was quantified by the Tauber-Kleiner method. FIG. 2 shows the amount of glucose adsorbed. In FIG. 2, no significant difference was observed in the amount of adsorption between the boric acid-containing calcium phosphate compound and the hydroxyapatite.

[0024]

According to the present invention, the boron-containing calcium phosphate compound makes it possible to increase the adsorptivity of the c-plane for adsorbing basic proteins, and furthermore, it can adsorb a large amount of basic proteins because of its high specific surface area. An adsorbent is provided.

[Brief description of the drawings]

FIG. 1 is a sectional view showing an ultrasonic spray pyrolysis apparatus used in the present invention.

FIG. 2 is a graph showing the amount of glucose adsorbed.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Water tank 7 Reaction tube 2 Reaction tank 8 Thermocouple 3 Ultrasonic transmitter 9 Powder collection part 4 Water 10 Cooling tube 5 Sample reservoir 11 Aspirator 6 Sample solution 12 Film

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Hiroyasu Takeuchi 1-5-1, Otemachi, Chiyoda-ku, Tokyo F-term in the Technology & Development Division, Mitsubishi Materials Corporation 4G066 AA17A AA17B AA50A AA50B AA52A AA53A AA56A AA56B BA26 BA36 CA54 DA11 FA03 FA26 FA34 FA39

Claims (5)

[Claims] 1. A basic protein adsorbent, which is a boron-containing calcium phosphate compound. 2. The basic protein adsorbent according to claim 1, wherein the boron-containing calcium phosphate compound is represented by the general formula [I]. General formula [I] [Ca _{9.5 + 0.5X} {(PO ₄ ) _6-X (BO ₃ ) _X } (B
O ₂ ) _1-X O _X } (where X is 0 to 1.0) 3. The basic protein adsorbent according to claim 1, wherein the boron-containing calcium phosphate compound has a specific surface area of 10 m ² / g or more. 4. A calcium compound, a phosphoric acid compound and boric acid are dissolved in a nitric acid solution, and the obtained raw material solution is formed into fine droplets, and then sprayed in an atmosphere at a temperature of 300 to 1200 ° C. to form a boron-containing calcium phosphate. A method for producing a basic protein adsorbent, comprising synthesizing a compound. 5. The method for producing a basic protein adsorbent according to claim 4, wherein the calcium compound is a nitrate and the phosphate compound is diammonium hydrogen phosphate.