JP6736738B2 - Separation method - Google Patents

Description

The present invention relates to a separation method for separating a negatively charged substance from a sample solution.

Fluorapatite is one in which at least part of the hydroxyl groups of hydroxyapatite are replaced with fluorine atoms.

This fluoroapatite has a property of high acid resistance because fluoroapatite is a more stable substance than hydroxyapatite. For this reason, fluoroapatite has the advantage that it has high durability against an acidic solution and can separate adsorbed substances such as proteins in an acidic solution. Has been focused on (see, for example, Patent Document 1).

However, fluorapatite has almost the same crystal structure as hydroxyapatite, and therefore, although its adsorption property (adsorption capacity) for adsorbing substances is almost equal to that of hydroxyapatite, it still has at least one of the hydroxyl groups. Due to the substitution of a part by a fluorine atom, its adsorption characteristics are slightly different from those of hydroxyapatite.

In particular, when a negatively charged substance such as acidic protein is selected as an adsorbent and fluoroapatite is used to separate (isolate) the negatively charged substance, the amount of adsorption of the negatively charged substance to the adsorbent cannot be sufficiently obtained. There was a problem.

JP, 2004-330113, A

The object of the present invention is to use a fluoroapatite excellent in acid resistance as a filler, and from a sample liquid containing a negatively charged substance and a contaminant other than the negatively charged substance, to a large amount of adsorption to the filler. An object of the present invention is to provide a method for separating a negatively charged substance, which is capable of adsorbing the negatively charged substance in an amount and isolating the negatively charged substance.

Such an object can be achieved by the embodiment of the present invention described in (1) or (2) below.
(1) A separation method for separating the negatively charged substance from a sample solution containing a desired negatively charged substance and a contaminant other than the desired negatively charged substance, which is Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X [In the formula, x is 0<x≦1. ] A sample containing the negatively charged substance, the contaminant, and a buffer solution in an adsorption device in which at least a part of the filling space is filled with a filler composed of fluoroapatite represented by the chemical formula A supply step of supplying a liquid, and a fractionation step of isolating the negatively charged substance by supplying a buffer solution into the device and fractionating an outflow liquid flowing out of the device. The separation method, wherein the buffer solution contained in the sample solution does not contain phosphoric acid.
(2) A method for producing a negatively charged substance, comprising a step of isolating the negatively charged substance by the separation method described in (1) above.

The separation method of the present invention is preferably used when separating acidic proteins from a sample solution.

According to the separation method of the present invention, by using fluoroapatite excellent in acid resistance as a filler, from a sample liquid containing a negatively charged substance and a contaminant other than the negatively charged substance, the filler is The negatively charged substance can be isolated by adsorbing the negatively charged substance with a large adsorption amount. Therefore, the negatively chargeable substance can be efficiently isolated from the contaminant (sample solution).

It is a longitudinal cross-sectional view showing an example of an adsorption device used in the present invention. It is a graph which shows the change of the adsorption amount of acidic protein to the sintered particles 3 and 4 when using each sample liquid 1-3. It is a graph which shows the change of the adsorption amount of acidic protein to the sintered particles 1 and 3 when using each sample liquid 1-3. It is a graph which shows the change of the adsorption amount of acidic protein to sintered particles 2 and 3 when using each sample liquids 1-3.

Hereinafter, the separation method of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.
First, before describing the separation method of the present invention, an example of the adsorption device used in the present invention will be described.

In the following, as a negatively charged substance to be separated by using an adsorption device, an acidic protein will be mentioned as a representative, and this acidic protein will be separated from a sample solution containing the acidic protein and contaminants using an adsorption device. The case of separation (separation) will be described as a representative.

<Adsorption device>
FIG. 1 is a vertical sectional view showing an example of an adsorption device used in the present invention. In the following description, the upper side in FIG. 1 is called the “inflow side” and the lower side is called the “outflow side”.

The term “inflow side” as used herein refers to a liquid such as a sample solution (liquid containing the sample) or a buffer solution such as a phosphate buffer solution that is an eluent when the target acidic protein is separated (purified). On the other hand, the outflow side means the side opposite to the inflow side, that is, the side on which the liquid flows out of the adsorption device.

The adsorption device 1 shown in FIG. 1 for separating (purifying) acidic proteins has a column 2, a granular adsorbent (filler) 3, and two filter members 4 and 5.

The column 2 is composed of a column main body 21 and caps (lids) 22 and 23 attached to the inflow side end and the outflow side end of the column main body 21, respectively.

The column body 21 is made of, for example, a cylindrical member. Examples of the constituent material of each part (each member) that configures the column 2 including the column body 21 include various glass materials, various resin materials, various metal materials, various ceramic materials, and the like.

Caps 22 and 23 are respectively screwed onto the inflow-side end and the outflow-side end of the column body 21 with the filter members 4 and 5 arranged so as to close the inflow-side opening and the outflow-side opening, respectively. It will be installed depending on the combination.

In the column 2 having such a configuration, the adsorbent filling space 20 is defined by the column body 21 and the filter members 4 and 5. Then, the adsorbent 3 is filled into at least a part of the adsorbent filling space 20 (substantially full amount in the present embodiment).

The volume of the adsorbent-filled space 20 is appropriately set according to the volume of the sample liquid and is not particularly limited, but is preferably about 0.1 to 100 mL, more preferably about 1 to 50 mL with respect to 1 mL of the sample liquid.

Further, in the state where the caps 22 and 23 are attached to the column body 21, the liquid tightness between them is ensured.

An inflow pipe 24 and an outflow pipe 25 are liquid-tightly fixed (fixed) to substantially the centers of the caps 22 and 23, respectively. The liquid is supplied to the adsorbent 3 via the inflow pipe 24 and the filter member 4. Further, the liquid supplied to the adsorbent 3 passes between the adsorbents 3 (gap) and flows out of the column 2 via the filter member 5 and the outflow pipe 25. At this time, the acidic protein contained in the sample solution (sample) and the contaminant (contaminant) composed of a substance other than the acidic protein differ in the adsorptivity to the adsorbent 3 and the affinity to the phosphate buffer solution. Are separated based on.

Each of the filter members 4 and 5 has a function of preventing the adsorbent 3 from flowing out from the adsorbent filling space 20. The filter members 4 and 5 are, for example, non-woven fabrics and foams (sponge-like porous bodies having communicating holes) made of synthetic resin such as polyurethane, polyvinyl alcohol, polypropylene, polyether polyamide, polyethylene terephthalate, and polybutylene terephthalate. ), woven fabric, mesh, etc.

The adsorbent 3 is Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X [where x is 0<x≦1. ] It is comprised with the fluoroapatite represented by the chemical formula, and in this embodiment, it is comprised with the sintered particle of a fluoroapatite.

This fluoroapatite is one in which at least a part of the hydroxyl groups of hydroxyapatite are substituted with fluorine atoms, and specifically, the substitution rate by the fluorine atoms is more than 0% and 100% or less. Is.

Here, fluorapatite becomes excellent in acid resistance as compared with hydroxyapatite due to at least part of the hydroxyl groups of hydroxyapatite being substituted with fluorine atoms. As a result, when eluting the acidic protein adsorbed on the adsorbent 3 with the eluent, the range of selection of the eluent to be used is widened. That is, since the pH value of the eluate can be set in a wide range, the eluate can be set more reliably under the condition that the acidic protein is purified with high accuracy.

As described above, by substituting the hydroxyl group with the fluorine atom, the acid resistance of the fluoroapatite is improved. On the contrary, when the hydroxyl group is replaced with the fluorine atom, the adsorption to the adsorbent 3 composed of the fluoroapatite is prevented. The inventors of the present invention have found that the adsorption capacity (adsorption rate) of acidic proteins decreases.

By the way, an acidic protein is a protein that generally contains a relatively large amount of acidic amino acids as its constituent amino acids and that is negatively charged due to this (negatively charged protein). On the other hand, fluorapatite contains a large amount of calcium atoms (calcium ions) and phosphate groups in its crystal structure, forming positively charged Ca sites and negatively charged phosphate sites. There is.

Therefore, an acidic protein generally binds (adsorbs) to fluoroapatite by forming an ionic bond with the Ca site of fluoroapatite, but in the fluoroapatite, the hydroxyl group is replaced with a fluorine atom. By doing so, the negatively charged fluorine atom in the vicinity of the Ca site exerts some influence on the positively charged Ca site, and as a result, the amount of acidic protein to the adsorbent 3 composed of fluoroapatite is increased. It is presumed that the adsorptivity is reduced. It should be noted that some influence is that a fluorine atom binds to the Ca site, and therefore it is considered that the adsorption of the acidic protein and the Ca site is inhibited.

Further, such a decrease in the adsorption capacity of acidic proteins is remarkable when the sintering temperature at the time of producing the sintered particles of fluoroapatite constituting the adsorbent 3 is 450° C. or lower, particularly 400° C. or lower. It has been found by further study by the present inventor that it is not observed remarkably at a high temperature such as above 400° C. (more than 450° C., especially above 650° C.). This shows that the higher the sintering temperature, the more the fluorine atoms constituting the fluoroapatite tend to be taken into the inside of the sintered particles, and as a result, the Ca sites involved in the adsorption of acidic amino acids, that is, the surface of the sintered particles, is exposed. It is considered that this is due to a decrease in the absolute amount of fluorine atoms that affects the Ca site. That is, when the sintered particles sintered at a sintering temperature of 400° C. or lower are used as the adsorbent 3, it is noticeable that the adsorbability of the acidic protein to the adsorbent 3 tends to decrease. If the sintering temperature is too low, the sintering may be insufficient and the durability of the sintered particles may be deteriorated. Therefore, the sintering temperature is preferably 300° C. or higher.

Further, the substitution ratio of the hydroxyl group with the fluorine atom is more than 0% and 100% or less, preferably 5% or more and 100% or less, more preferably 20% or more and 100% or less, and 25% or more and 75% or more. % Or less is more preferable. That is, in the above chemical formula, x is 0<x≦1, but preferably 0.05≦x≦1.00, more preferably 0.20≦x≦1.00, and 0. It is more preferable that 25≦x≦0.75.
In the fluoroapatite substituted with the fluorine atom at such a substitution rate, the absolute amount of the fluorine atom that has an influence on the Ca site included in the sintered particles is increased, and as a result, the adsorption capacity of the acidic protein to the adsorbent 3 is increased. The decrease is noticeable.

In addition, when the above-mentioned fluoroapatite is used as the adsorbent 3 in the method for separating the acidic protein, by applying the separation method of the present invention, the acidic protein can be adsorbed on the adsorbent 3 in a large amount. It can be adsorbed, but its detailed description will be given later.
The manufacturing method for manufacturing fluoroapatite will also be described in detail later.

The form (shape) of the adsorbent 3 is not particularly limited, and is, for example, granular (granular), pellet (small lump), block (for example, a porous body in which adjacent pores communicate with each other, a honeycomb shape). ) Or the like, but it is preferably granular (granular). As a result, the surface area can be increased and the separability of acidic proteins can be improved.

The average particle diameter of the granular adsorbent 3 is not particularly limited, but is preferably about 0.5 to 150 μm, more preferably about 10 to 80 μm. By using the adsorbent 3 having such an average particle diameter, it is possible to reliably prevent the filter member 5 from being clogged and to ensure a sufficient surface area of the adsorbent 3.

Further, it is preferable that the specific surface area of the granular adsorbent 3 is wider, but when the fluoroapatite is produced by the production method described below, the specific surface area is 30 m ² /g or more and 50 m ² /g or less. It is preferable that the amount is 42 m ² /g or more and 50 m ² /g or less. In the production method described later, by setting the substitution rate of the hydroxyl group by the fluorine atom to preferably 20% or more and 100% or less, the specific surface area can be set within the above range, and thus the separation ability of acidic proteins is improved. Can be planned.

The adsorbent 3 may be wholly composed of fluoroapatite, or may be one in which the surface of the carrier (base) is coated with fluoroapatite, but the whole is composed of fluoroapatite. It is preferable that the Thereby, the strength of the adsorbent 3 can be further improved, and the adsorbent 3 suitable for use in separating a large amount of acidic proteins can be obtained.

Further, when the adsorbent 3 is filled in the adsorbent filling space 20 almost completely as in the present embodiment, the adsorbent 3 has substantially the same composition in each part of the adsorbent filling space 20. Is preferred. This makes the adsorption device 1 particularly excellent in the ability to separate (purify) acidic proteins.

The adsorbent 3 may be filled in a part of the adsorbent filling space 20 (for example, a part on the inflow pipe 24 side), and the other part may be filled with another adsorbent.

<Separation method>
Next, a method for separating acidic protein (separation method of the present invention) using such an adsorption device 1 will be described.

[1] Preparation Step First, an acidic protein to be purified (target) (albumin, antibody, etc.), a contaminant (contaminant) composed of a substance other than the acidic protein, and a monovalent or divalent cation, A sample solution containing a phosphate-free buffer solution is prepared.

When the acidic protein is a recombinant protein (monoclonal antibody, etc.), the sample solution is a mammal such as sheep, rabbit, chicken or the like into which a nucleic acid containing a gene encoding the acidic protein has been introduced, or an animal such as an insect such as silkworm. , A buffer not containing monovalent or divalent cations and phosphate in secretions from animal cells such as CHO cells derived from Chinese hamster ovary cells, microorganisms such as Escherichia coli, and their cytoplasmic components The liquid may be added.

When the acidic protein is a natural protein, the sample liquid may be, for example, blood (plasma) derived from various animals, body fluids such as lymph, saliva, and nasal discharge, and a monovalent or divalent cation. A buffer solution containing no phosphoric acid may be added.

Furthermore, examples of acidic proteins include BSA, HSA, fibrinogen, pepsinogen, α-globulin, β-globulin and γ-globulin.

Note that these acidic proteins may be modified forms in which some amino acids in the amino acid sequence are replaced with other amino acids.

Moreover, the secretions from the microorganisms and the body fluids of animal origin may be used as they are, but those obtained by filtering these with a filtration membrane such as a filter may be prepared.

Further, the contaminants include basic proteins, nucleic acids such as amino acids, peptides, DNA and RNA, and acidic proteins having a molecular weight or isoelectric point different from that of the target acidic protein.

The buffer solution containing no phosphoric acid is not particularly limited as long as it does not contain phosphoric acid. For example, hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, female (2- Morpholinoethanesulfonic acid) buffer, Tris (Tris-hydroxymethylammonium) buffer, mops (3-(N-morpholino)propanesulfonic acid) buffer, Pipes (piperazine-1,4-diethanesulfonic acid) buffer, Taps (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, Tess (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer and the like can be mentioned. Of these, preferred Examples include Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, female (2-morpholinoethanesulfonic acid) buffer solution, Tris (Tris-hydroxymethylammonium) buffer solution, and the like. They may be used alone or in combination of two or more. As a result, it is possible to reliably prevent the phosphoric acid derived from the buffer solution from adsorbing to the Ca site of the fluoroapatite.

The concentration of the buffer solution containing no phosphate in the sample solution is preferably set to about 1.0 mM or more and 100 mM or less, more preferably 5.0 mM or more and 50 mM or less. By setting the concentration of the buffer solution in the sample solution within such a range, when the sample solution is brought into contact with the adsorbent 3 in the next step [2], the change in the pH of the sample solution is appropriately suppressed or prevented. Therefore, the acidic protein can be isolated (purified) with high accuracy.

Further, the cation is not particularly limited as long as it is monovalent or divalent, but preferably Na ⁺ , K ⁺ , NH ₄ ⁺ , Li ⁺ , Mg ²⁺ , Zn ²⁺ , Fe ^2+, Sr ^{2+ and the} like. Among these, one kind or a combination of two or more kinds is used. As a result, an ionic bond can be reliably formed between the phosphate site of the fluoroapatite and the cation.

Further, these cations are added as salts when preparing the sample solution, and thus, they are present as cations in the sample solution. Examples of such salts include, but are not limited to, NaCl, KCl, NH ₄ Cl, MgCl _{2, and the} like.

The concentration of monovalent or divalent cations in the sample solution is preferably set to about 0.01 M or more and 1.0 M or less, more preferably about 0.1 M or more and 0.5 M or less. By setting the concentration of the buffer solution in the sample solution to such a range, in the next step [2], when the sample solution is brought into contact with the adsorbent 3, the phosphate site of the fluoroapatite and the cation are provided. An ionic bond can be formed more reliably between them.

The pH of the sample solution is preferably 5.0 or more and 9.0 or less, more preferably 5.0 or more and 7.0 or less, and further preferably about 6.0. As a result, when the sample solution is brought into contact with the adsorbent 3 in the next step [2] without causing denaturation of the acidic protein, the ionic bond between the Ca site of the fluoroapatite and the acidic protein is further improved. It can be reliably formed.

[2] Supply Step Next, the obtained sample solution is supplied to the adsorbent 3 through the inflow pipe 24 and the filter member 4, and is passed through the column 2 (adsorption device 1) to be adsorbent 3. Contact.

As a result, the acidic protein having a high adsorbing ability to the adsorbent 3 and the contaminants (contaminant proteins and the like) other than the acidic protein having a relatively high adsorbing ability to the adsorbent 3 are retained in the column 2. To be done. Then, the contaminant having a low adsorptivity with respect to the adsorbent 3 flows out from the inside of the column 2 through the filter member 5 and the outflow pipe 25.

At this time, in the present invention, as a sample liquid (adsorption liquid), in addition to an acidic protein to be isolated and a contaminant (contaminant), a monovalent or divalent cation and a phosphate-free buffer are used. Those containing the liquid are supplied to the adsorbent 3 composed of fluoroapatite.

Here, when the adsorbent 3, that is, the sintered particles are composed of fluoroapatite, the fluorine atoms have an influence on the Ca sites included in the sintered particles as described above. There is a problem in that a decrease in the adsorptivity of the protein to the adsorbent 3 is observed.

In order to solve this problem, first, when the sample solution contains a buffer solution containing no phosphoric acid, the following advantages can be obtained. That is, when the sample solution contains phosphoric acid derived from the buffer solution, the phosphoric acid competes with the acidic protein for the Ca site of the sintered particles. In the fluoroapatite, the fluorine atom affects the Ca site, and the phosphoric acid derived from the buffer competes with the acidic protein on the Ca site, so that the adsorptivity of the acidic protein to the adsorbent 3 decreases. On the other hand, by selecting a buffer solution containing no phosphoric acid as the buffer solution contained in the sample solution, the competition between the phosphoric acid derived from the buffer solution and the acidic protein is prevented. It is possible to suppress a decrease in the adsorption ability of the acidic protein to the adsorbent 3.

[3] Fractionation step Next, for example, a phosphate-based buffer solution is supplied from the inflow pipe 24 into the column 2 as an eluent for eluting the acidic protein, and the eluate is supplied from the column 2 via the outflow pipe 25. The effluent flowing out is fractionated (collected) by a predetermined amount. As a result, the acidic protein and the contaminant adsorbed on the adsorbent 3 are collected (separated) in a state of being eluted in each fraction according to the difference in the adsorbing power of the adsorbent 3 to the adsorbent 3. ..

That is, since the adsorbent 3 specifically adsorbs the acidic protein and the contaminant by their own adsorption (supporting) forces, the acidic protein and the contaminant are separated according to the difference in the adsorption force. Separated and purified (isolated).

A phosphate buffer is preferably used as the buffer, and examples of the phosphate buffer include sodium phosphate, potassium phosphate, lithium phosphate, ammonium phosphate and the like. In addition to the phosphate buffer, the buffer may be the above-mentioned buffer containing no phosphate such as MES or HEPES.

When a phosphate buffer is used, its pH is not particularly limited, but is preferably set to about 5.5 to 8.5, more preferably 6.5 to 7.5. As a result, the acidic protein can be surely eluted, and the purification rate thereof can be improved.

The temperature of the phosphate buffer is also not particularly limited, but is preferably about 10 to 50°C, more preferably about 20 to 35°C. This makes it possible to prevent alteration (denaturation) of the separated acidic protein.

Further, the flow rate of the phosphate buffer is preferably about 0.1 to 10 mL/min, more preferably about 1 to 5 mL/min. By separating the acidic protein at such a flow rate, it is possible to reliably separate the target acidic protein without requiring a long time for the separation operation, that is, to obtain a highly pure acidic protein.
By the above operation, the acidic protein is collected in a predetermined fraction.

At the time of recovering such an acidic protein, in the step [2], a sample solution containing a monovalent or divalent cation and a phosphate-free buffer solution is used. Since the acidic protein is adsorbed on the adsorbent (filler) 3 in a large amount, the acidic protein can be efficiently isolated in this step.

In the above description, the acidic protein is taken as an example of the negatively charged substance, but the negatively charged substance may be, for example, acidic amino acids, DNA, RNA, and negatively charged liposomes. Like the acidic protein, the negatively charged substance can be easily separated with high purity.

<Method for producing fluoroapatite>
Next, the fluoroapatite as described above may be produced by any production method, and a known wet synthesis method or dry synthesis method can be used. For example, a slurry containing hydroxyapatite and fluorinated apatite are used. In a mixed solution obtained by mixing a hydrogen fluoride-containing solution containing hydrogen, by reacting hydroxyapatite with hydrogen fluoride, at least a part of the hydroxyl groups, by a method obtained by replacing with a fluorine atom You can

According to this method, for example, since hydrogen fluoride is used as the fluorine source compared with the case where the compound is prepared by adding ammonium hydrogen fluoride as the fluorine source in the slurry containing hydroxyapatite, the residual impurities It is possible to obtain fluoroapatite with no or very little.

Therefore, the fluoroapatite has high crystallinity and, due to this, is excellent in acid resistance. Therefore, if the adsorbent 3 composed of such fluoroapatite is used, the separation of acidic proteins, which must use an eluate having a relatively low pH for separation, can also be performed without dissolution of the adsorbent 3. You can Therefore, it becomes possible to reliably perform the separation of such acidic proteins.

Hereinafter, this method will be described in detail.
A1: First, a slurry containing hydroxyapatite is prepared.

Hereinafter, a method for preparing a slurry in which the hydroxyapatite primary particles and the aggregates thereof are dispersed will be described as this slurry.

The hydroxyapatite primary particles can be obtained using various synthetic methods, but it is preferable to synthesize them by a wet synthetic method using at least one of a calcium source and a phosphoric acid source as a solution.

The hydroxyapatite obtained as a result becomes fine primary particles. Since such hydroxyapatite primary particles have a small shape, they have extremely high reactivity with hydrogen fluoride.

As the calcium source, for example, calcium hydroxide, calcium oxide, calcium nitrate or the like can be used. On the other hand, as the phosphoric acid source, phosphoric acid, ammonium phosphate or the like can be used. Among these, those containing calcium hydroxide or calcium oxide as the main component as the calcium source and those containing phosphoric acid as the main component as the phosphoric acid source are particularly preferable.

Specifically, for example, in a container, a phosphoric acid (H ₃ PO ₄ ) solution is dropped into a suspension of calcium hydroxide (Ca(OH) ₂ ) or calcium oxide (CaO) and mixed with stirring. As a result, hydroxyapatite is synthesized, hydroxyapatite primary particles are generated, and a slurry is obtained.

The content of the hydroxyapatite primary particles in the slurry is 1 to 20 wt.
%, preferably about 5 to 12 wt %.

A2: On the other hand, a hydrogen fluoride-containing liquid containing hydrogen fluoride is prepared separately from the slurry containing hydroxyapatite.

Any solvent that dissolves hydrogen fluoride can be used as long as it does not inhibit the reaction between hydroxyapatite and hydrogen fluoride.

Examples of such a solvent include water, alcohols such as methanol and ethanol, and the like, and a mixture of these may be used. Among them, water is particularly preferable.

The content of hydrogen fluoride in the hydrogen fluoride-containing liquid is preferably about 1 to 60 wt%, more preferably about 2.5 to 10 wt%.

A3: Next, by mixing the prepared slurry and the prepared hydrogen fluoride-containing liquid, the hydroxyapatite primary particles and hydrogen fluoride are mixed in a slurry (reaction liquid) containing the hydrogen fluoride-containing liquid. The reaction is performed to obtain fluoroapatite primary particles.

That is, by contacting hydrogen fluoride with the hydroxyapatite primary particles, as shown in the following formula, at least a part of the hydroxyl groups of the hydroxyapatite is replaced by fluorine atoms, and converted into fluoroapatite, and fluoroapatite. Obtain primary particles.

Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ →
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X
[In the formula, x is 0<x≦1. ]

Thus, by reacting the hydroxyapatite primary particles with hydrogen fluoride in the slurry containing the hydroxyapatite primary particles, the fluoroapatite primary particles can be easily produced.

In addition, the setting of the substitution rate of the hydroxyl group of the hydroxyl group in the fluorapatite primary particles by the mixing ratio of the prepared slurry and the prepared hydrogen fluoride-containing liquid in this step A3 (that is, addition of the hydrogen fluoride-containing liquid It can be easily performed by appropriately setting (amount).

Further, since hydrogen fluoride (HF) is used as the fluorine source, ammonium hydrogen fluoride (NH ₄ F), lithium fluoride (LiF), sodium fluoride (NaF), potassium fluoride (KF), magnesium fluoride. Compared with the case of using (MgF ₂ ) or calcium fluoride (CaF ₂ ), the production of side reaction products is little or very little.

Specifically, the concentration of impurities in the fluoroapatite is preferably as low as possible, preferably 300 ppm or less, more preferably 100 ppm or less. As a result, the fluorine apatite primary particles have higher acid resistance because the release of fluorine atoms from the fluorine apatite is suppressed due to the lower impurity concentration.

In addition, by adjusting the reaction conditions (eg, pH, temperature, time, etc.) between hydroxyapatite (primary particles) and hydrogen fluoride, the impurity concentration in the fluoroapatite primary particles falls within the above range, and thus the supernatant. It is possible to reliably set the concentration of fluorine released in the liquid within the above range.

In particular, the pH of the slurry is adjusted to preferably in the range of about 2.5 to 5, more preferably about 2.7 to 4 by mixing the liquid containing hydrogen fluoride, and in this state, the hydroxyapatite ( Primary particles) are reacted with hydrogen fluoride. This makes it possible to set the concentration within the range more reliably. In the present specification, the pH of the slurry is the pH at the time when the entire amount of the hydrogen fluoride-containing liquid is mixed with the slurry.

Here, if the pH of the slurry is adjusted to less than 2.5, the hydroxyapatite itself tends to be dissolved, and it may be difficult to convert it into fluoroapatite to obtain primary particles. Further, there is a possibility that the constituent components of the apparatus used when mixing the hydrogen fluoride-containing liquid with the primary particles are eluted and the purity of the obtained primary particles is lowered. Furthermore, it is technically very difficult to adjust the slurry to a low pH of less than 2.5 using a hydrogen fluoride-containing liquid.

On the other hand, in order to adjust the pH of the slurry to more than 5 using the hydrogen fluoride-containing liquid, there is no choice but to add a large amount of water to the slurry. Therefore, the total amount of the slurry becomes extremely large, and the yield of the fluorapatite primary particles with respect to the total amount of the slurry may be reduced, which is industrially disadvantageous.

On the other hand, by adjusting the pH of the slurry to 2.5 to 5, the fluoroapatite (primary particles) produced by the reaction once shows a tendency to dissolve and then recrystallizes. Therefore, it is possible to obtain fluoroapatite primary particles having high crystallinity.

Further, the slurry and the hydrogen fluoride-containing liquid may be mixed temporarily (simultaneously), but it is preferable to mix them by dropping the hydrogen fluoride-containing liquid into the slurry.

The rate of dropping the hydrogen fluoride-containing liquid is preferably about 1 to 100 L/hour, more preferably about 3 to 100 L/hour.

The reaction between the hydroxyapatite primary particles and hydrogen fluoride is preferably carried out while stirring the slurry. By stirring, the hydroxyapatite primary particles and hydrogen fluoride are brought into uniform contact with each other, and the reaction can proceed efficiently. Further, the substitution ratio of fluorine atoms among the obtained fluorapatite primary particles can be made more uniform. For example, by using such fluorapatite primary particles, the adsorbent (dry particles or sintered particles) 3 can be obtained. When manufactured, the variation in the characteristics is reduced, and a more reliable product can be obtained.

In this case, the stirring force for stirring the slurry is preferably an output of about 0.1 to 3 W, and more preferably an output of about 0.5 to 1.8 W, relative to 1 L of the slurry.

Further, the amount of hydrogen fluoride mixed is preferably such that the amount of fluorine is about 0.05 to 0.55 times the amount of hydroxyl groups contained in hydroxyapatite, and about 0.15 to 0.35 times. It is more preferable that

The temperature at which the hydroxyapatite primary particles are reacted with hydrogen fluoride is not particularly limited, but is preferably about 5 to 50°C, more preferably about 20 to 40°C.

In this case, the time for adding (adding) hydrogen fluoride to the hydroxyapatite primary particles is preferably about 30 minutes to 16 hours, more preferably about 1 to 8 hours.

The slurry containing the fluorapatite primary particles obtained by the above method is dried or granulated to obtain dried particles (secondary particles), and the dried particles are fired to obtain sintered particles. The sintered particles can be used as the adsorbent 3.

The method of drying or granulating the slurry containing primary particles is not particularly limited, and examples thereof include a method of spray drying the slurry with a spray dryer or the like.

Further, the firing temperature for firing the dry particles is preferably about 200 to 800°C. By setting it within such a range, it is possible to obtain the adsorbent 3 which is excellent in mechanical strength while leaving gaps (holes) formed inside the primary particles or between the primary particles (aggregates).

In addition, the separation method of the present invention, when the sintered particles obtained by firing in the above temperature range and fired at a firing temperature of 400° C. or less are used as the adsorbent 3, as described above, It is more preferably applied because the absolute amount of fluorine atoms that affect Ca sites involved in the adsorption of acidic amino acids increases.

Although the separation method of the present invention has been described above, the present invention is not limited to this.

For example, in the separation method of the present invention, one or more steps can be added for any purpose.

Next, specific examples of the present invention will be described.
1. Production of Fluorapatite [Sintered particles (Fluorapatite) 1]
[1A] First, calcium hydroxide was suspended in pure water, an aqueous phosphoric acid solution was added dropwise thereto, and the mixture was sufficiently stirred. As a result, 200 L of a slurry containing 10 wt% of hydroxyapatite primary particles was obtained.

It was confirmed by powder X-ray diffractometry that the obtained compound was hydroxyapatite.

On the other hand, 4.2 wt% hydrogen fluoride (manufactured by Morita Chemical Industry Co., Ltd., "HF-20") was prepared as a hydrogen fluoride-containing liquid.

[2A] Next, while the slurry was stirred with a stirring force of 1 kW, a hydrogen fluoride-containing liquid was added dropwise at a rate of 5 L/hour.

The pH of the slurry was 3.00 when the dropping of the hydrogen fluoride-containing liquid was completed. Further, the amount of hydrogen fluoride mixed was about 1.05 times the amount of fluorine with respect to the amount of hydroxyl groups contained in hydroxyapatite.

Subsequently, the slurry was stirred at a temperature of 30° C. for 48 hours with a stirring power of 1 kW. As a result, the hydroxyapatite primary particles were reacted with hydrogen fluoride to obtain a slurry containing the fluoroapatite primary particles.

The powder X-ray diffraction method confirmed that the reaction product in the slurry was fluoroapatite. As a result of powder X-ray diffraction, the substitution rate of fluorine atoms in the fluorapatite primary particles was 100%.

As a result of powder X-ray diffraction, no products other than fluoroapatite could be confirmed in the fluoroapatite dry particles.

[3A] Next, the slurry containing the fluorapatite primary particles was spray-dried at 150° C. using a spray dryer (“OC-20” manufactured by Okawara Kakohki Co., Ltd.) to produce spherical dry particles. ..

[4A] Next, after classifying a part of the dried particles with a central particle size of about 40 μm, the particles were fired in an electric furnace at 650° C. for 4 hours, the substitution rate of fluorine atoms was 100%, and the sintering temperature was Sintered particles (fluorapatite) 1 at 650° C. were obtained.

[Sintered particles (fluorapatite) 2]
By changing the dropping amount of the hydrogen fluoride-containing liquid in the step [2A] and setting the sintering condition of the dry particles in the step [4A] to 700° C.×4 hours, the substitution rate of fluorine atoms is 25. %, and sintered particles (fluorapatite) 2 having a sintering temperature of 700° C. were obtained.

[Sintered particles (fluorapatite) 3]
By changing the dropping amount of the hydrogen fluoride-containing liquid in the step [2A] and setting the sintering condition of the dry particles in the step [4A] to 400° C.×4 hours, the substitution rate of fluorine atoms is 25. %, and sintered particles (fluorapatite) 3 having a sintering temperature of 400° C. were obtained.

[Sintered particles (hydroxyapatite) 4]
By omitting the dropping of the hydrogen fluoride-containing liquid in the step [2A], sintered particles (hydroxyapatite) 4 having a fluorine atom substitution rate of 0% were obtained.

The substitution rate of fluorine atoms in each of the sintered particles 1 to 4 was determined by measuring the fluorine concentration in the fluoroapatite using an ion electrode and converting the obtained measurement value into the substitution rate.

2. Evaluation of acidic protein adsorption capacity [Sample solution (10 mM NaP) 1]
[1B] First, each of the sintered particles 1 to 4 was packed into a packed space of a stainless steel column (inner diameter 6.0 mm×length 35 mm) so that the packed space was almost full, and the sintered particles 1 to 4 were packed. Packed sample No. 1-4 columns were manufactured.

The specific surface area of the sintered particles 1 to 4 packed in each column was measured in advance using a fully automatic BET specific surface area measuring device (“Macsorb model-1201” manufactured by Mountech Co., Ltd.).

[2B] Next, Albumin, bovine serum, >96% Essentially Fatty Acid Free (SIGMA, "A6003") was added to 10 mM sodium phosphate buffer so that BSA (bovine serum albumin) was 0.1 mg/mL. After dissolving in a liquid (pH 6.0), it was filtered through a 0.22 μm filter and degassed for 30 minutes to obtain a sample liquid 1.

[3B] Next, for each sample No. column packed with each sintered particle, the buffer solution (10 mM sodium phosphate buffer solution (pH 6.0, temperature 25 Was carried out at a liquid flow rate of 0.71 mL/min for equilibration.

[4B] Next, each sample No. column was attached to a chromatographic apparatus, and while measuring the absorbance value (280 nm) in the chromatographic apparatus, the sample solution 1 was sent from the pump of the chromatographic apparatus.

Then, for each sample No. column, from the sample solution sent until the absorbance value (280 nm) of the elution peak in the chromatograph becomes 10% of the absorbance value (280 nm) of the sample solution (stock solution) The amount of adsorption of acidic protein was determined.

In addition, by measuring the absorbance value (280 nm, reference: pure water) and the molar extinction coefficient value of the sample solution using an absorptiometer (“UV Spectrophotometer UV-1800” manufactured by Shimadzu Corp.), The actual amount of adsorbed protein was determined.

[Sample solution (10 mM MES) 2]
In the step [2B], 10 mM MES buffer (pH 6.0) was used in place of the sodium phosphate buffer to prepare the sample solution 2, and in the step [3B], the sodium phosphate buffer was used. Each sample No. was used in the same manner as in the case of using the sample liquid 1 except that 10 mM MES buffer (pH 6.0) was used in place of the solution to perform equilibration in the column of each sample No. The amount of acidic protein adsorbed on the column was determined.

[Sample solution (10 mM MES+0.1 M NaCl) 3]
In the step [2B], a sample solution 3 was prepared using a (10 mM MES+0.1M NaCl) buffer solution (pH 6.0) instead of the sodium phosphate buffer solution, and in the step [3B] , Sample solution 1 was used, except that (10 mM MES+0.1 M NaCl) buffer solution (pH 6.0) was used instead of the sodium phosphate buffer solution to perform equilibration in the column of each sample No. Similarly to the case, the adsorption amount of the acidic protein on the column of each sample No. was obtained.

The amount of acidic protein adsorbed on each of the sintered particles 1 to 4 in the case of using each sample solution obtained as described above is compared between the sintered particles 3 and the sintered particles 4 in FIG. Further, FIG. 3 shows a comparison between the sintered particles 1 and the sintered particles 3, and FIG. 4 shows a comparison between the sintered particles 2 and the sintered particles 3.

Further, the effect obtained by using such a sample liquid 3 is, as shown in FIGS. 3 and 4, when the sintering temperature of the sintered particles composed of fluoroapatite is 400° C., that is, the sintered particles are It was found that when the absolute amount of fluorine atoms, which has an effect on the Ca site provided in, increases, it is remarkably exhibited.

DESCRIPTION OF SYMBOLS 1 Adsorption device 2 Column 20 Adsorbent filling space 21 Column main body 22, 23 Cap 24 Inflow pipe 25 Outflow pipe 3 Adsorbent 4, 5 Filter member

Claims (2)

A separation method for separating the negatively charged substance from a sample liquid containing a target negatively charged substance and a contaminant other than the target negatively charged substance,
_{Ca 10 (PO 4) 6 (} OH) 2-2x F 2X [ wherein, x is a 0 <x ≦ 1. ] A sample containing the negatively charged substance, the contaminant, and a buffer solution in an adsorption device in which at least a part of the filling space is filled with a filler composed of fluoroapatite represented by the chemical formula A supply step of supplying a liquid,
By supplying a buffer solution into the device and fractionating the effluent flowing out of the device,
And a fractionation step of isolating the negatively charged substance,
The separation method, wherein the buffer solution contained in the sample solution does not contain phosphoric acid. A method for producing a negatively charged substance, comprising the step of isolating the negatively charged substance by the separation method according to claim 1.

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