JP2017064668A - Separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation method of a negative electrification substance capable of isolating the negative electrification substance, by adsorbing the negative electrification substance by much adsorption quantity to a filler from a simple liquid of including the negative electrification substance and a contaminant composed of a substance except for the negative electrification substance, by using fluorine apatite excellent in acid resistance as the filler.SOLUTION: A separation method of the present invention is a method for separating a negative electrification substance from a sample liquid of including the objective negative electrification substance and a contaminant, and comprises a supply step of supplying the sample liquid of including the negative electrification substance, the contaminant, a monovalent or bivalent cation and a buffer solution of not including a phosphoric acid in an adsorption device of filling at least part of filling space with a filler constituted of fluorine apatite expressed by Ca(PO)(OH)F[in an expression, x is 0<x≤1] and a fractionation step of isolating the negative electrification substance by fractionating an outflow liquid of flowing out of the device inside by supplying the buffer solution in the device.SELECTED DRAWING: None

Description

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

  Fluorine apatite is one in which at least a part of the hydroxyl groups of hydroxyapatite is substituted with fluorine atoms.

  This fluorapatite has a property of high acid resistance because fluorapatite is a more stable substance than hydroxyapatite. For this reason, since fluorapatite has the advantage that it has high durability against acidic solutions and can separate adsorbed substances such as proteins in acidic solutions, an adsorbent (filler) provided in an adsorbing device in recent years. (For example, refer to Patent Document 1).

  However, fluorapatite has almost the same crystal structure as that of hydroxyapatite. Therefore, although the adsorption characteristic (adsorption capacity) for adsorbed substances is almost equal to that of hydroxyapatite, it is still at least one of the hydroxyl groups. Due to the fact that the part is substituted with fluorine atoms, the adsorption characteristics are slightly different from those of hydroxyapatite.

  In particular, when a negatively charged substance such as an acidic protein is selected as an adsorbent and the negatively charged substance is separated (isolated) using fluorapatite, the adsorption amount of the negatively charged substance on the adsorbent cannot be obtained sufficiently. There was a problem.

JP 2004-330113 A

  The object of the present invention is to use fluorapatite with excellent acid resistance as a filler, and to adsorb a large amount of such a filler from a sample solution containing a negatively charged substance and contaminants other than the negatively charged substance. An object of the present invention is to provide a method for separating a negatively charged substance that can adsorb a negatively charged substance in an amount and isolate the negatively charged substance.

Such an object is achieved by the present inventions (1) to (7) below.
(1) A separation method for separating the negatively charged substance from a sample solution 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 0 <x ≦ 1. ] In the adsorption device formed by filling at least a part of the filling space with a filler composed of fluorapatite represented by the chemical formula: A supplying step of supplying a sample solution containing a cation and a buffer solution not containing phosphate;
And a fractionation step of isolating the negatively charged substance by supplying a buffer solution into the device and fractionating the effluent flowing out of the device.

  As a result, a negatively charged substance is adsorbed in a large amount of adsorption to a filler composed of fluorapatite from a sample solution containing a negatively charged substance and contaminants other than the negatively charged substance. The material can be isolated.

  (2) The separation method according to (1), wherein the phosphate-free buffer solution contains at least one of hepes, females, tris, mops, pipes, tapus, and tess.

  Thereby, it can prevent reliably that the phosphoric acid derived from a buffer comes to adsorb | suck with respect to Ca site with which a fluoroapatite is equipped.

(3) The cation is described in (1) or (2) above, wherein the cation is at least one of Na ⁺ , K ⁺ , NH ₄ ⁺ , Li ⁺ , Mg ²⁺ , Zn ²⁺ , Fe ²⁺ and Sr ^2+. Separation method.

  Thereby, an ionic bond can be reliably formed between the phosphate site with which fluorapatite is provided, and a cation. Therefore, even if the fluorine atom has an influence on the Ca site provided in the fluorapatite, the repulsive force between the phosphate site and the acidic protein is reduced. An ionic bond can be formed. Therefore, it is possible to reliably suppress or prevent a decrease in the adsorption ability of acidic proteins to fluorapatite.

  (4) The separation method according to any one of (1) to (3), wherein in the chemical formula, x is 0.05 ≦ x ≦ 1.00.

  In the fluorapatite substituted with fluorine atoms at such a substitution rate, the absolute amount of fluorine atoms affecting the Ca site of the filler increases, and as a result, the adsorptive capacity of acidic proteins to the filler decreases. Since it is remarkably recognized, the separation method of the present invention is more suitably applied.

  (5) The separation according to any one of (1) to (4), wherein the filler is sintered particles obtained by firing secondary particles of fluorapatite at a firing temperature of 450 ° C. or less. Method.

  When sintered particles sintered at such a sintering temperature are used as a filler, the absolute amount of fluorine atoms affecting the Ca sites of the filler increases, and as a result, the filler for acidic proteins. Since the decrease in the adsorption capacity with respect to is remarkably recognized, the separation method of the present invention is more suitably applied.

  (6) The separation method according to any one of (1) to (5), wherein the pH of the sample solution is 5.0 or more and 9.0 or less.

  Thereby, when the sample solution is brought into contact with the filler without causing denaturation of the acidic protein, an ionic bond can be more reliably formed between the Ca site of the fluorapatite and the acidic protein.

  (7) The separation method according to any one of (1) to (6), wherein the negatively charged substance is an acidic protein.

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

  According to the separation method of the present invention, fluorine apatite having excellent acid resistance is used as a filler, and a sample solution containing a negatively charged substance and a contaminant other than the negatively charged substance is used as the filler. Negatively charged substances can be isolated by adsorbing negatively charged substances with a large amount of adsorption. Therefore, a negatively chargeable substance can be efficiently isolated from the contaminant (sample solution).

It is a longitudinal cross-sectional view which shows an example of the adsorption | suction apparatus used by this invention. It is a graph which shows the change of the adsorption amount of the acidic protein to the sintered particles 3 and 4 when each sample liquid 1-3 is used. It is a graph which shows the change of the adsorption amount of the 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 the acidic protein to the sintered particles 2 and 3 when using each sample liquid 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 explaining the separation method of the present invention, an example of an adsorption apparatus used in the present invention will be described.

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

<Adsorption device>
FIG. 1 is a longitudinal 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 referred to as “inflow side” and the lower side is referred to as “outflow side”.

  Here, when the target acidic protein is separated (purified), the inflow side is, for example, a liquid such as a sample solution (liquid containing the sample) or a buffer solution such as a phosphate buffer that is an eluate. Is the side that supplies the liquid into the adsorption device, while the outflow side is the side opposite to the inflow side, that is, the side from which the liquid flows out of the adsorption device.

  The adsorption apparatus 1 shown in FIG. 1 that separates (purifies) acidic protein has a column 2, a granular adsorbent (filler) 3, and two filter members 4 and 5.

  The column 2 includes a column main body 21 and caps (lid bodies) 22 and 23 attached to the inflow side end portion and the outflow side end portion of the column main body 21, respectively.

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

  In the column body 21, caps 22, 23 are screwed into the inflow side end and the outflow side end, respectively, in a state where the filter members 4, 5 are arranged so as to block the inflow side opening and the outflow side opening, respectively. It is attached by the match.

  In the column 2 having such a configuration, an adsorbent filling space 20 is defined by the column main body 21 and the filter members 4 and 5. The adsorbent 3 is filled in at least a part of the adsorbent filling space 20 (almost full in this embodiment).

  The volume of the adsorbent filling 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.

  In addition, in a state where the caps 22 and 23 are attached to the column main body 21, the liquid-tightness between them is ensured.

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

  Each of the filter members 4 and 5 has a function of preventing the adsorbent 3 from flowing out of the adsorbent filling space 20. These filter members 4 and 5 are made of, for example, a nonwoven fabric made of a synthetic resin such as polyurethane, polyvinyl alcohol, polypropylene, polyether polyamide, polyethylene terephthalate, polybutylene terephthalate, or a foam (a sponge-like porous body having communication holes). ), Woven fabric, mesh or the like.

The adsorbent 3 is Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X [wherein x is 0 <x ≦ 1. ], And in this embodiment, it is composed of sintered particles of fluorapatite.

  This fluorapatite is one in which at least a part of the hydroxyl group of hydroxyapatite is substituted with fluorine atoms. Specifically, the substitution rate by fluorine atoms is more than 0% and 100% or less. It is.

  Here, the fluorapatite has excellent acid resistance compared to hydroxyapatite because at least a part of the hydroxyl groups of the hydroxyapatite is substituted with fluorine atoms. As a result, when the acidic protein adsorbed on the adsorbent 3 is eluted with the eluent, the selection range 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.

  Thus, by replacing the hydroxyl group with a fluorine atom, the acid resistance of the fluorapatite is improved. On the other hand, when the hydroxyl group is substituted with a fluorine atom, the adsorbent 3 composed of fluorapatite is adsorbed. The inventors have found that the adsorption ability (adsorption rate) of acidic proteins decreases.

  By the way, an acidic protein generally contains a relatively large amount of acidic amino acids as constituent amino acids, and is a protein 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, and forms positively charged Ca sites and negatively charged phosphate sites. Yes.

  Therefore, acidic protein generally binds (adsorbs) to fluorapatite by forming an ionic bond with the Ca site of fluorapatite, but the hydroxyl group is replaced with a fluorine atom in fluorapatite. As a result, the negatively charged fluorine atom adjacent to the Ca site has some influence on the positively charged Ca site. As a result, the acidic protein is adsorbed to the adsorbent 3 composed of fluorine apatite. It is assumed that the adsorptive capacity decreases. In addition, it is thought that a certain influence is that a fluorine atom couple | bonds with Ca site, and, therefore, inhibits adsorption | suction of acidic protein and Ca site.

  Further, such a decrease in the adsorption ability of acidic protein is remarkable when the sintering temperature at the time of producing the sintered particles of fluorapatite constituting the adsorbent 3 is as low as 450 ° C. or less, particularly 400 ° C. or less. It has been found by further study by the present inventor that it is not remarkably observed when the temperature is higher than 400 ° C. (more than 450 ° C., particularly more than 650 ° C.). This shows that the higher the sintering temperature, the more the fluorine atoms constituting the fluorapatite tend to be taken into the sintered particles, and as a result, the Ca sites involved in the adsorption of acidic amino acids, that is, the surfaces of the sintered particles are exposed. This is considered to be due to a decrease in the absolute amount of fluorine atoms that affect the Ca site. That is, when sintered particles sintered at a sintering temperature of 400 ° C. or lower are used as the adsorbent 3, a tendency that the adsorbing ability of the acidic protein to the adsorbent 3 is significantly reduced is recognized. In addition, when sintering temperature is too low, since sintering becomes inadequate and there exists a possibility that durability of a sintered particle may be inferior, it is preferable that sintering temperature is 300 degreeC or more.

  Further, the substitution rate of the hydroxyl group with a 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 more preferably 25% or more and 75%. More preferably, it is% or less. That is, in the chemical formula, x is 0 <x ≦ 1, but is preferably 0.05 ≦ x ≦ 1.00, more preferably 0.20 ≦ x ≦ 1.00, and More preferably, 25 ≦ x ≦ 0.75. In the fluorapatite substituted with fluorine atoms at such a substitution rate, the absolute amount of fluorine atoms affecting the Ca sites included in the sintered particles increases, and as a result, the adsorption ability of the acidic protein to the adsorbent 3 is increased. The decrease is noticeable.

When the above-described fluorapatite is used as the adsorbent 3 in the acidic protein separation method, by applying the separation method of the present invention, the acidic protein is adsorbed on the adsorbent 3 in a large amount of adsorption. Although it can be adsorbed, a detailed description thereof will be given later.
Further, a production method for producing fluorapatite will be described in detail later.

  The form (shape) of the adsorbent 3 is not particularly limited, and is, for example, granular (granular), pellet (small block), block (for example, a porous body in which adjacent pores communicate with each other, a honeycomb shape, or the like. Among them, it is preferable to be granular (granular). Thereby, the surface area can be increased and the separation ability of acidic protein can be improved.

  The average particle size 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 ensure a sufficient surface area of the adsorbent 3 while reliably preventing the filter member 5 from being clogged.

Further, the specific surface area of the granular adsorbent 3 is preferably wider, but when the fluorapatite is produced using a production method as described later, the specific surface area is 30 m ² / g or more and 50 m ² / g or less. It is preferable that it is 42 m ² / g or more and 50 m ² / g or less. In the production method described later, since the specific surface area can be within the above range by setting the substitution rate of the hydroxyl group with a fluorine atom to preferably 20% or more and 100% or less, the separation performance of acidic proteins is improved. Can be achieved.

  The adsorbent 3 may be entirely composed of fluorapatite, or may be one in which the surface of the carrier (substrate) is coated with fluorapatite, but is entirely composed of fluorapatite. It is preferred that Thereby, the intensity | strength of the adsorption agent 3 can further be improved and it can be set as the adsorption agent 3 suitable for the use at the time of isolate | separating a lot of acidic proteins.

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

  Alternatively, 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 other adsorbents may be filled in other parts.

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

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

  When the acidic protein is a genetically engineered protein (monoclonal antibody, etc.), the sample solution is a mammal such as a sheep, rabbit, or chicken introduced with a nucleic acid containing a gene encoding the acidic protein, or an animal such as an insect such as a silkworm. Buffers that do not contain monovalent or divalent cations and phosphate in animal cells such as CHO cells derived from Chinese hamster ovary cells, secretions from microorganisms such as E. coli, and their cytoplasmic components What added the liquid is mentioned.

  When the acidic protein is a natural protein, examples of the sample solution include blood (plasma) derived from various animals, body fluids such as lymph, saliva, nasal discharge, and monovalent or divalent cations, What added the buffer solution which does not contain phosphoric acid is mentioned.

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

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

  In addition, secretions from the microorganisms and body fluids derived from animals may be used as they are, but those prepared by filtering them with a filter membrane such as a filter are prepared, and then a monovalent or divalent cation. It is preferable to prepare a sample solution by adding a phosphate-free buffer solution.

  Furthermore, examples of 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 the target acidic protein.

  The buffer solution not containing 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-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 solution and Tes (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) buffer solution, etc. Hepes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, female (2-morpholinoethanesulfonic acid) buffer and Tris-hydro xymethylammonium) buffer and the like, and one or more of these may be used in combination. Thereby, it can prevent reliably that the phosphoric acid derived from a buffer comes to adsorb | suck with respect to Ca site with which a fluoroapatite is equipped.

  The concentration of the buffer solution not containing phosphate in the sample solution is preferably set to about 1.0 mM to 100 mM, more preferably about 5.0 mM to 50 mM. By setting the concentration of the buffer solution in the sample solution within this range, when the sample solution is brought into contact with the adsorbent 3 in the next step [2], the pH change in the sample solution is accurately suppressed or prevented. Therefore, acidic protein can be isolated (purified) with high accuracy.

Furthermore, 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. These may be used, and one or more of these may be used in combination. Thereby, an ionic bond can be reliably formed between the phosphate site with which fluorapatite is provided, and a cation.

In addition, these cations are added as salts when preparing a sample solution, and thus exist as cations in the sample solution. Such a salt is not particularly limited, and examples thereof include NaCl, KCl, NH ₄ Cl, MgCl _{2 and the} like.

  The concentration of the monovalent or divalent cation in the sample solution is preferably set to about 0.01M to 1.0M, more preferably about 0.1M to 0.5M. 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 phosphate sites included in the fluorapatite and the cation Ion bonds can be more reliably formed between them.

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

[2] Supplying process 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 (adsorbing device 1) to be adsorbed on the adsorbent 3. Make contact.

  As a result, acidic proteins having a high adsorptive capacity for the adsorbent 3 and those having a relatively high adsorbing capacity for the adsorbent 3 among the contaminants (contaminating proteins, etc.) other than the acidic protein are retained in the column 2. Is done. Then, contaminants having a low adsorbability with respect to the adsorbent 3 flow out from the column 2 through the filter member 5 and the outflow pipe 25.

  In this case, in the present invention, in addition to the acidic protein to be isolated and the contaminant (contaminant), the monovalent or divalent cation and the phosphate-free buffer are used as the sample liquid (adsorbed liquid). What contains the liquid is supplied to the adsorbent 3 composed of fluorapatite.

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

  To solve this problem, first, if the sample solution contains a buffer solution not containing phosphoric acid, the following advantages can be obtained. That is, when phosphoric acid derived from a buffer solution is contained in the sample solution, the phosphoric acid and acidic protein compete with the Ca site included in the sintered particles. In the fluorapatite, the fluorine atom has an influence on the Ca site, and furthermore, the phosphate derived from the buffer competes with the acidic protein, so that the adsorption ability of the acidic protein to the adsorbent 3 is lowered. In contrast, by selecting a buffer solution that does not contain phosphate as the buffer solution contained in the sample solution, competition between phosphate derived from the buffer solution and acidic protein is prevented. Moreover, the fall of the adsorption ability with respect to the adsorption agent 3 of acidic protein can be suppressed.

  However, even if competition between the phosphate derived from the buffer solution and the acidic protein is prevented, the fluorine atom still affects the Ca site included in the sintered particles, and phosphate is included as the buffer solution. This is not solved by simply using a non-existing one.

  In contrast, in the present invention, the sample solution further contains monovalent or divalent cations. As a result, the cation forms an ionic bond with the negatively charged phosphate site of the fluorapatite, so the repulsive force generated between the negatively charged acidic protein is reduced. Can be reduced. Therefore, even if the fluorine atom has an influence on the Ca site included in the sintered particles, the repulsive force between the phosphate site and the acidic protein is reduced. An ionic bond can be formed. Therefore, it is possible to reliably suppress or prevent a decrease in the adsorption ability of the acidic protein to the adsorbent 3.

  From the above, the sample solution contains a monovalent or divalent cation and a buffer solution not containing phosphoric acid, so that the adsorbent 3 is adsorbed with acidic protein in a large amount of adsorption. be able to.

  Prior to this step, an equilibration solution containing a monovalent or divalent cation and a buffer solution not containing phosphoric acid is prepared, and this equilibration solution is supplied to the adsorbent 3 in advance. Also good. As a result, the fluorapatite (adsorbent 3) is stabilized (equilibrium), and the adsorption of the acidic protein by the adsorbent 3 in this step is performed more smoothly.

[3] Fractionation step Next, for example, a phosphate buffer solution is supplied from the inflow pipe 24 into the column 2 as an eluent for eluting the acidic protein, and the column 2 is passed through the outflow pipe 25. Fractionate (collect) the effluent flowing out by a predetermined amount. As a result, the acidic protein and contaminants adsorbed on the adsorbent 3 are recovered (separated) in a state of being eluted in each fraction according to the difference in adsorption force with respect to the adsorbent 3 respectively. .

  That is, since the acidic protein and the contaminant are specifically adsorbed to the adsorbent 3 by the inherent adsorption (supporting) force, the acidic protein and the contaminant are separated according to the difference in the adsorption force. Separation and purification (isolation).

  As the buffer solution, a phosphate buffer solution is preferably used. Examples of the phosphate buffer solution include sodium phosphate, potassium phosphate, lithium phosphate, and ammonium phosphate. The buffer solution may be a phosphate-free buffer solution such as MES or HEPES described above, in addition to the phosphate buffer solution.

  When using a phosphate buffer, the pH is not particularly limited, but is preferably set to about 5.5 to 8.5, more preferably about 6.5 to 7.5. Thereby, an acidic protein can be eluted reliably and the improvement of the purification rate can be aimed at.

  The temperature of the phosphate buffer is not particularly limited, but is preferably about 10 to 50 ° C, more preferably about 20 to 35 ° C. Thereby, alteration (denaturation) of the acidic protein to be separated can be prevented.

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 acidic protein at such a flow rate, the target acidic protein can be reliably separated without requiring a long time for the separation operation, that is, a high-purity acidic protein can be obtained.
By the above operation, acidic protein is recovered in a predetermined fraction.

  When 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 with a large amount of adsorption with respect to the adsorbent (filler) 3, the acidic protein can be efficiently isolated in this step.

  In the above description, acidic protein is an example of a negatively charged substance, but examples of the negatively charged substance include acidic amino acids, DNA, RNA, negatively charged liposomes, and the like. The negatively charged substance can be separated easily and with high purity in the same manner as the acidic protein.

<Method for producing fluorapatite>
Next, the fluorine apatite 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, fluoride Produced by a method in which at least a part of the hydroxyl group is substituted with a fluorine atom by reacting hydroxyapatite and hydrogen fluoride in a mixed liquid containing a hydrogen fluoride-containing liquid containing hydrogen. Can do.

  According to this method, for example, hydrogen fluoride is used as a fluorine source in a slurry containing hydroxyapatite, compared with a case where synthesis is performed by adding ammonium hydrogen fluoride as a fluorine source. There is no or very little fluorine apatite.

  For this reason, the crystallinity is high, resulting in a fluorapatite with excellent acid resistance. Therefore, if the adsorbent 3 composed of such fluorapatite is used, the separation of the acidic protein for which the eluate having a relatively low pH must be used for the separation without the dissolution of the adsorbent 3. Can do. For this reason, separation of such acidic proteins can be performed reliably.

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 hydroxyapatite primary particles and aggregates thereof are dispersed will be described as this slurry.

  Hydroxyapatite primary particles can be obtained using various synthesis methods, but are preferably synthesized by a wet synthesis method using at least one of a calcium source and a phosphate source as a solution.

  The hydroxyapatite thus obtained becomes fine primary particles. Since such hydroxyapatite primary particles have a small shape, the reactivity with hydrogen fluoride is extremely high.

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

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

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

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

  Any solvent 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 these can be used as a mixture. Among them, water is particularly preferable.

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

  A3: Next, by mixing the prepared slurry and the prepared hydrogen fluoride-containing liquid, in the slurry (reaction liquid) containing the hydrogen fluoride-containing liquid, the hydroxyapatite primary particles and hydrogen fluoride are mixed. By reacting, primary fluorapatite particles are obtained.

  That is, by bringing hydrogen fluoride into contact with the hydroxyapatite primary particles, as shown in the following formula, at least a part of the hydroxyl groups of hydroxyapatite are substituted with fluorine atoms, and converted into fluorine apatite, and then fluorine apatite is obtained. Obtain primary particles.

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

  Thus, a fluorapatite primary particle can be easily manufactured by making a hydroxyapatite primary particle and hydrogen fluoride react in the slurry containing a hydroxyapatite primary particle.

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

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 There are no or very few side reaction products compared to the case of using (MgF ₂ ), calcium fluoride (CaF ₂ ), or the like.

  Specifically, the impurity concentration in the fluorapatite is preferably as low as possible, preferably 300 ppm or less, and more preferably 100 ppm or less. As a result, the primary particles of fluorapatite have a higher acid resistance due to the reduced impurity concentration, thereby preventing the release of fluorine atoms from the fluorapatite.

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

  In particular, the pH of the slurry is preferably adjusted to a range of about 2.5 to 5, more preferably about 2.7 to 4 by mixing a hydrogen fluoride-containing liquid. In this state, the hydroxyapatite ( Primary particles) are reacted with hydrogen fluoride. Thereby, it is possible to set the concentration within the range more reliably. In this 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, when 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 obtain primary particles by converting into hydroxyapatite. Furthermore, there may be a problem that the constituent components of the apparatus used when the hydrogen fluoride-containing liquid is mixed with the primary particles are eluted and the purity of the resulting primary particles is lowered. Furthermore, it is technically difficult to adjust the slurry to a low pH, which is less than pH 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 a hydrogen fluoride-containing liquid, a large amount of water must be added to the slurry. For this reason, since the whole quantity of a slurry becomes very large and there exists a possibility that the yield of the fluorapatite primary particle with respect to the whole quantity of a slurry may fall, it is industrially disadvantageous.

  On the other hand, by adjusting the pH of the slurry to 2.5 to 5, the fluorapatite (primary particles) generated by the reaction once shows a tendency to dissolve and then recrystallizes. For this reason, highly crystalline fluorapatite primary particles can be obtained.

  The slurry and the hydrogen fluoride-containing liquid may be mixed at a time (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 performed while stirring the slurry. By stirring, the primary particles of hydroxyapatite and hydrogen fluoride come into uniform contact, and the reaction can proceed efficiently. Moreover, the substitution rate of the fluorine atoms between the obtained fluorapatite primary particles can be made more uniform. For example, the adsorbent (dry particles or sintered particles) 3 can be formed using the fluorapatite primary particles. When manufactured, the variation in characteristics is reduced, and a more reliable product can be obtained.

  In this case, the stirring force for stirring the slurry is preferably about 0.1 to 3 W, more preferably about 0.5 to 1.8 W, with respect to 1 L of the slurry.

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

  Although the temperature at the time of making a hydroxyapatite primary particle and hydrogen fluoride react is not specifically limited, It is preferable that it is about 5-50 degreeC, and it is more preferable that it is about 20-40 degreeC.

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

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

  Moreover, it does not specifically limit as a method of drying and granulating the slurry containing primary particles, For example, the method of spray-drying a slurry with a spray dryer etc. is mentioned.

  Furthermore, the firing temperature when firing the dried particles is preferably about 200 to 800 ° C. By setting within this range, it is possible to obtain the adsorbent 3 having excellent mechanical strength while leaving gaps (holes) formed in the primary particles or between the primary particles (aggregates).

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

  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, and an aqueous phosphoric acid solution was dropped therein and stirred sufficiently. As a result, 200 L of slurry containing 10 wt% primary particles of hydroxyapatite was obtained.

  It was confirmed by powder X-ray diffractometry that the resultant composite was hydroxyapatite.

  On the other hand, 4.2 wt% hydrogen fluoride (Morita Chemical Industries, Ltd., “HF-20”) was prepared as a hydrogen fluoride-containing liquid.

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

  Note that when the dropping of the hydrogen fluoride-containing liquid was completed, the pH of the slurry was 3.00. In addition, the amount of hydrogen fluoride mixed was about 1.05 times the amount of fluorine with respect to the amount of hydroxyl groups of hydroxyapatite.

  Subsequently, the slurry was stirred with a stirring power of 1 kW at a temperature of 30 ° C. for 48 hours. Thereby, the hydroxyapatite primary particle and hydrogen fluoride were made to react, and the slurry containing a fluorapatite primary particle was obtained.

  The reaction product in the slurry was confirmed to be fluorapatite by powder X-ray diffraction. Further, 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 product other than fluorapatite could be confirmed in the fluorapatite dry particles.

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

  [4A] Next, after classifying a part of the dried particles with a center particle size of about 40 μm, the particles were baked 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 dripping 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 dripping 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.

  In addition, the substitution rate of the fluorine atom in each sintered particle 1-4 was calculated | required by measuring the fluorine density | concentration in a fluorine apatite using an ion electrode, and converting the obtained measured value into a 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 is filled in a filling space of a stainless steel column (inner diameter 6.0 mm × length 35 mm) so that the sintered particles 1 to 4 are almost full. Columns of packed sample Nos. 1 to 4 were manufactured.

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

  [2B] Next, Albumin, bovine serum,> 96% Essentially Fatty Acid Free (SIGMA, “A6003”) was added to a 10 mM sodium phosphate buffer so that BSA (bovine serum albumin) was 0.1 mg / mL. After being dissolved in the liquid (pH 6.0), it was filtered through a 0.22 μm filter, and further deaerated for 30 minutes to obtain 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) used for preparing the sample solution 1 was used. ° C)) was equilibrated by passing at a flow rate of 0.71 mL / min.

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

  For each sample No. column, from the sample solution sent until the absorbance value (280 nm) of the elution peak in the chromatographic apparatus becomes 10% of the absorbance value (280 nm) of the sample solution (stock solution). The amount of acidic protein adsorbed was determined.

  In addition, in the sample liquid 1 by measuring the absorbance value (280 nm, reference: pure water) and molar extinction coefficient value of the sample liquid using an absorptiometer (manufactured by Shimadzu Corporation, “UV Spectrophotometer UV-1800”). The actual protein adsorption amount was determined.

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

  In the case of using each sample solution obtained as described above, the amount of acidic protein adsorbed to each sintered particle 1 to 4 is compared in FIG. 2 with the sintered particle 3 and the sintered particle 4. 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.

As shown in FIGS. 2 to 4 (particularly FIG. 2), by using the sample solution 3 as the sample solution, that is, by using the MES buffer solution not containing phosphoric acid and the sample solution containing Na ⁺ as the cation. Even if the sintered particles 3 made of fluorapatite are selected as the sintered particles, the sintered particles have almost the same ability to adsorb acidic proteins as when the sintered particles 4 made of hydroxyapatite are selected. It became clear to show.

  Moreover, the effect obtained by using such a sample solution 3 is that, as shown in FIGS. 3 and 4, when the sintering temperature of the sintered particles composed of fluorapatite is 400 ° C., that is, the sintered particles When the absolute amount of fluorine atoms affecting the Ca site included in is increased, it has been found that this is remarkably exhibited.

DESCRIPTION OF SYMBOLS 1 Adsorber 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 (7)

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 desired negatively charged substance,
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X [wherein x is 0 <x ≦ 1. ] In the adsorption device formed by filling at least a part of the filling space with a filler composed of fluorapatite represented by the chemical formula of the above formula, the negatively charged substance, the contaminant, and the monovalent or divalent A supplying step of supplying a sample solution containing a cation and a buffer solution not containing phosphate;
And a fractionation step of isolating the negatively charged substance by supplying a buffer solution into the device and fractionating the effluent flowing out of the device.   2. The separation method according to claim 1, wherein the phosphate-free buffer solution contains at least one of hepes, female, tris, mops, pipes, tapus, and tess. The separation method according to claim 1, wherein the cation is at least one of Na ⁺ , K ⁺ , NH ₄ ⁺ , Li ⁺ , Mg ²⁺ , Zn ²⁺ , Fe ^2+, and Sr ²⁺ .   4. The separation method according to claim 1, wherein x in the chemical formula satisfies 0.05 ≦ x ≦ 1.00. 5.   5. The separation method according to claim 1, wherein the filler is sintered particles obtained by firing secondary particles of fluorapatite at a firing temperature of 450 ° C. or less.   The separation method according to any one of claims 1 to 5, wherein the pH of the sample solution is 5.0 or more and 9.0 or less.   The separation method according to claim 1, wherein the negatively charged substance is an acidic protein.

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