JP6141256B2 - Fluoroapatite, adsorption device and separation method - Google Patents

Description

  The present invention relates to a fluorapatite, an adsorption device, and a separation method for separating a 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.

  Since this fluorapatite is a more stable substance compared to hydroxyapatite, it has a property of high acid resistance. For this reason, fluorapatite has attracted attention in recent years because it has the advantage of being highly resistant to acidic solutions and capable of separating adsorbed substances such as proteins in acidic solutions (for example, Patent Document 1). reference.).

  However, fluorapatite has almost the same crystal structure as hydroxyapatite. For this reason, the adsorption characteristic (adsorption ability) of fluorapatite with respect to an adsorbent is almost equal to that of hydroxyapatite. However, due to the fact that at least a part of the hydroxyl groups are 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 or a positively charged substance such as a basic protein is selected as an adsorbing substance and these charged substances are separated using fluorapatite, the following problems arise. Depending on the substitution rate between the hydroxyl group and the fluorine atom in the fluorapatite, the adsorption characteristics of the negatively charged substance and the positively charged substance change. Thereby, the separation ability and adsorption amount of the negatively charged substance and the positively charged substance are changed.

JP 2004-330113 A

  An object of the present invention is a fluorine apatite that is excellent in acid resistance and that can separate a charged substance with high accuracy from a sample solution containing a charged substance and a substance other than the charged substance, and such a fluorapatite. It is an object of the present invention to provide a method for separating a charged substance using such a fluorine apatite.

Such an object is achieved by the present inventions (1) to ( 5 ) below.

(1) Fluoroapatite used for separating the negatively charged substance from a sample solution containing a negatively charged substance and a substance other than the negatively charged substance,
The fluorapatite is represented by the following general formula:
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X
In the general formula, x satisfies a relationship of 0.05 ≦ x ≦ 0.40 ,
The fluorapatite has a granular shape, a specific surface area of 30 m ² / g to 50 m ² / g, and an average particle size of 0.5 μm to 150 μm .

As a result, the negatively charged substance is obtained by adsorbing or desorbing the negatively charged substance to the fluorapatite from a sample liquid that has excellent acid resistance and contains a negatively charged substance and a substance other than the negatively charged substance. Can be separated with high accuracy.

( 2 ) The fluorapatite according to ( 1 ), wherein the negatively charged substance is an acidic protein.

  The fluorapatite of the present invention is preferably used when separating acidic protein from a sample solution.

(3) The fluorapatite according to the above (1) or (2), wherein x satisfies the relationship of 0.05 ≦ x ≦ 0.25 in the general formula .

  If the specific surface area is within this range, it can be said that the specific surface area is wide, and the separation ability and adsorption amount of the negatively charged substance can be improved.

( 4 ) An adsorption device comprising the fluorapatite according to any one of (1) to (3) as an adsorbent.

  As a result, the adsorption device includes an adsorbent that has high acid resistance and is excellent in the ability to separate and adsorb charged substances.

(5) A separation method for separating the negatively charged substance from a sample liquid containing a negatively charged substance and a substance other than the negatively charged substance,
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X represented by the general formula, wherein x is composed of fluorine apatite satisfying the relationship of 0.05 ≦ x ≦ 0.40 , In the adsorbing device formed by filling at least part of the filling space with a filler having a specific surface area of 30 m ² / g to 50 m ² / g and an average particle size of 0.5 μm to 150 μm , Supplying a sample solution;
Supplying a buffer solution into the adsorption device to obtain an effluent flowing out of the adsorption device;
Separating the negatively charged substance from the sample liquid in the effluent of each fractionated fraction by fractionating the effluent by a predetermined amount.

Thereby, a low pH buffer solution can be used as an eluent. Furthermore, the negatively charged substance can be adsorbed to the adsorbent with excellent adsorbability from a sample liquid containing a negatively charged substance and a substance other than the negatively charged substance . Therefore, the negatively charged substance can be separated from the sample liquid with high accuracy.

  According to the present invention, it is possible to obtain fluorapatite that has excellent acid resistance and can separate a charged substance with high accuracy from a sample liquid containing a charged substance and a substance other than the charged substance.

  In particular, when the charged substance is a negatively charged substance, it is possible to provide fluorapatite that has excellent acid resistance and can separate the negatively charged substance from the sample liquid with high accuracy.

  Further, when the charged substance is a positively charged substance, it is possible to provide fluorapatite that has excellent acid resistance and can separate the positively charged substance from the sample liquid with high accuracy.

FIG. 1 is a longitudinal sectional view showing an example of an adsorption device used in the present invention. FIG. 2 is a diagram showing a relationship between the Ca elution amount from the sintered particles 1 to 5 and the fluorine atom substitution rate in the sintered particles 1 to 5. FIG. 3 is a diagram showing the relationship between the amount of acidic protein adsorbed on the sintered particles 1 to 5, the specific surface area of the sintered particles 1 to 5, and the substitution rate of fluorine atoms in the sintered particles 1 to 6. FIG. 4 is a diagram showing the relationship between the basic protein adsorption amount to the sintered particles 1 to 5, the specific surface area of the sintered particles 1 to 5, and the substitution rate of fluorine atoms in the sintered particles 1 to 5.

  Hereinafter, the fluorapatite, the adsorption device and 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 fluorapatite of the present invention, an example of an adsorption device (adsorption device of the present invention) used in the present invention will be described.

  In the following, the charged substance separated using the adsorption device is exemplified by acidic protein as a negatively charged substance and basic protein as a positively charged substance. First, the case where this acidic protein is isolate | separated from the sample solution containing acidic protein using an adsorption | suction apparatus is demonstrated typically. Next, a case where the basic protein is separated from the sample solution containing the basic protein using an adsorption device 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 or basic protein is separated (purified), the inflow side refers to, for example, a liquid such as a sample solution (liquid containing a sample) or a phosphate buffer solution as an eluate. , Refers to the side supplied into the adsorption device, while the outflow side refers to the side opposite to the inflow side, that is, the side from which the liquid flows out from the adsorption device.

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

  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 solution (sample) and the substance other than the acidic protein (which may contain a basic protein) are different in the adsorptivity to the adsorbent 3 and the phosphate buffer solution. Separation based on affinity differences. In addition, the basic protein contained in the sample solution and the substance other than the basic protein (which may contain acidic protein) are different in the adsorptivity to the adsorbent 3 and the affinity for the phosphate buffer solution. Separation based on differences.

  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 composed of the fluorapatite of the present invention. That is, the adsorbent 3 is represented by a chemical formula of Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X , where x is composed of fluorapatite with 0.05 ≦ x ≦ 0.76. Yes.

  This fluorapatite is one in which at least a part of the hydroxyl groups of hydroxyapatite is substituted with fluorine atoms. Specifically, the substitution rate of the hydroxyl group by the fluorine atom is 5% or more and 76% or less.

  Here, in the present invention, the fluorapatite used when separating acidic protein from the sample solution and the fluorapatite used when separating basic protein from the sample are: The range is different from each other. Hereinafter, the fluorapatite used when separating acidic protein from the sample solution and the fluorapatite used when separating basic protein from the sample will be described.

[1] Fluoroapatite for separating acidic protein Fluoroapatite has superior acid resistance compared to hydroxyapatite because at least part of the hydroxyl groups of hydroxyapatite are substituted with fluorine atoms. It will be a thing. 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 contrary, it has been found by the present inventors that if the substitution rate becomes too high, the adsorption ability of the acidic protein to the adsorbent 3 composed of fluorapatite 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.

  Accordingly, the acidic protein generally binds (adsorbs) to the fluorapatite by forming an ionic bond with the Ca site of the fluorapatite. However, if the substitution rate of the hydroxyl group with a fluorine atom becomes too high, the fluorine atom having negative chargeability has some influence on the Ca site having positive chargeability, and as a result, the adsorbent 3 composed of fluorine apatite. It is presumed that the adsorption ability of acidic protein to the water decreases.

  The present inventor pays attention to this point, and as a result of intensive studies, the above problem can be solved by setting the substitution rate of the hydroxyl group of hydroxyapatite with a fluorine atom to 5% or more and 55% or less. As a result, the present invention has been completed.

  That is, by making the substitution rate of the hydroxyl group of hydroxyapatite with a fluorine atom 5% or more and 55% or less, the fluorapatite is excellent in acid resistance and has an acidic protein and a substance other than the acidic protein. Acidic proteins can be separated with high accuracy from a sample solution containing.

  Further, the substitution rate of the hydroxyl group with a fluorine atom may be 5% or more and 55% or less, but is preferably 20% or more and 40% or less, and more preferably about 25%. That is, in the chemical formula, x may be 0.05 ≦ x ≦ 0.55, but is preferably 0.20 ≦ x ≦ 0.40, and more preferably about 0.25. Thereby, the improvement of the acid resistance of the adsorbent (fluorapatite) 3 and the separation of the acidic protein with the adsorbent 3 with high accuracy can be achieved more reliably.

  The production method for producing the above fluorapatite will be described in detail later.

  Further, 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, Honeycomb shape) and the like. Especially, it is preferable that the form of the adsorbent 3 is 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.

Furthermore, the specific surface area of the granular adsorbent 3 is preferably wider. When the fluorapatite is produced using a production method as described later, the specific surface area is preferably 30 m ² / g or more and 50 m ² / g or less, and is 42 m ² / g or more and 50 m ² / g or less. More preferably. In the production method described later, the substitution rate of the hydroxyl group with a fluorine atom is set to 5% to 55%, preferably 20% to 40%. By doing so, since the specific surface area can be within the above range, the separation ability of acidic proteins can be improved.

  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.

[2] Fluoroapatite for separating basic proteins Fluoroapatite is more resistant to acid than hydroxyapatite because hydroxyapatite has at least a part of hydroxyl groups substituted with fluorine atoms. It will be excellent. As a result, when the basic protein adsorbed on the adsorbent 3 is eluted with the eluent, the range of selection of the eluent to be used is expanded. That is, since the pH value of the eluate can be set in a wide range, the eluate can be more reliably set under conditions for purifying the basic protein with high accuracy. Furthermore, even if the pH value of the eluate temporarily changes to the acidic side during the purification of the basic protein, alteration (dissolution, etc.) of the adsorbent 3 can be suitably prevented. Thus, by replacing the hydroxyl group with a fluorine atom, the acid resistance of the fluorapatite is improved.

  Furthermore, it has been found by the inventor's examination that the adsorption ability of the basic protein to the fluorapatite improves as the substitution rate of the hydroxyl group of the fluorapatite with the fluorine atom increases.

  By the way, a basic protein generally contains a relatively large amount of basic amino acids as its constituent amino acids, and is a protein that is positively charged due to this (positively 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, the basic protein generally binds (adsorbs) to the fluorapatite by forming an ionic bond with the phosphate site of the fluorapatite. It is considered that the fluorine atom having negative chargeability has some influence on the Ca site having positive chargeability as the substitution rate of the hydroxyl group with fluorine atoms increases. Therefore, the repulsive force (repulsive force) with respect to the basic protein in Ca site falls, and it is guessed that it results from this and the adsorbability of the basic protein with respect to fluorapatite improves.

  On the other hand, it has been found that if the substitution rate of the hydroxyl groups of fluorapatite with fluorine atoms becomes too high, the specific surface area of the adsorbent 3 composed of fluorapatite becomes narrow (small). When the specific surface area of the adsorbent 3 becomes narrow in this way, naturally, the contact opportunity between the adsorbent 3 and the basic protein decreases, so that the adsorbing ability of the basic protein by the adsorbent 3 composed of fluorapatite decreases. .

  As described above, substitution of the hydroxyl group of fluorapatite with a fluorine atom is favorable from the viewpoint of improving the acid resistance and basic protein adsorption ability of fluorapatite. However, from the viewpoint of widening (enlarging) the specific surface area of the adsorbent 3 composed of fluorapatite, that is, from the viewpoint of increasing the chance of contact of the basic protein with the adsorbent 3, it is likely to be defective. .

  The present inventor has paid attention to these points, and has intensively studied the range of the substitution rate with fluorine atoms, which is excellent in both acid resistance and basic protein adsorption ability. As a result, the present inventors have found that the above problem can be solved by setting the substitution rate of the hydroxyl group of hydroxyapatite with a fluorine atom to 6% or more and 76% or less, and to complete the present invention. It came.

  That is, the substitution rate of the hydroxyl group of hydroxyapatite with a fluorine atom is 6% or more and 76% or less. By doing so, fluorapatite has excellent acid resistance and can separate basic protein with high accuracy from a sample solution containing basic protein and a substance other than basic protein. Become.

  The substitution rate of the hydroxyl group with a fluorine atom may be 6% or more and 76% or less, but is preferably 25% or more and 60% or less, and more preferably about 25%. That is, in the chemical formula, x may be 0.06 ≦ x ≦ 0.76, but is preferably 0.25 ≦ x ≦ 0.60, and more preferably about 0.25. Thereby, the improvement of the acid resistance of the adsorbent (fluorapatite) 3 and the high-precision separation of the basic protein by the adsorbent 3 can be achieved more reliably.

  The production method for producing the above fluorapatite will be described in detail later.

  Further, 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, Honeycomb shape) and the like. Especially, it is preferable that the form of the adsorbent 3 is granular (granular). Thereby, the surface area can be increased and the separation ability of basic proteins 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.

Furthermore, it is preferable that the specific surface area of the adsorbent 3 is wider. Specifically, the specific surface area, ^{25 m} 2 / g or more, below ^{50 m} 2 / g, preferably ^{30 m} 2 / g or more, below ^{50 m} 2 / g, more preferably ^35m 2 / g or more, ^{50 m} 2 / g or less. In particular, in the production method for producing fluorapatite, the substitution rate of the hydroxyl group of the fluorapatite with a fluorine atom is preferably set to 6% to 76%, more preferably 25% to 60%. By doing so, since the specific surface area can be within the above range, the adsorption ability of the basic protein can be made excellent.

  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 be improved further and it can be set as the adsorption agent 3 suitable for the use at the time of isolate | separating a lot of basic 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) basic 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 A>
Next, a method for separating acidic protein using the adsorption device 1 (the separation method of the present invention) will be described.

[1A] Preparation Step First, a sample solution containing an acidic protein (such as an antibody) to be purified and a contaminant (substance) other than the acidic protein 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. And animal cells such as CHO cells derived from Chinese hamster ovary cells, secretions from microorganisms such as Escherichia coli, cytoplasmic components thereof, and the like.

  When the acidic protein is a natural protein, examples of the sample liquid include body fluids such as blood (plasma) derived from various animals, lymph, saliva, and nasal discharge.

  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.

  As the sample solution, secretions from the microorganisms or body fluids derived from animals may be used as they are, but those diluted in a neutral buffer such as water or physiological saline, and filters You may make it use what was filtered with the filter membranes. In addition, the sample solution may contain a plurality of types of acidic proteins.

[2A] Supplying 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 adsorbed on the adsorbent 3. Make contact.

  As a result, an acidic protein having a high adsorptive capacity for the adsorbent 3 and a contaminant (protein) having a relatively high adsorbing capacity for the adsorbent 3 among the contaminants other than the acidic protein (contaminating proteins, etc.) Held in. 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.

  At this time, in the present invention, the adsorbent 3 is composed of fluorapatite, and the substitution rate of the hydroxyl group by the fluorine atom is 5% or more and 55% or less. Can be retained.

[3A] Fractionation step Next, for example, a phosphate buffer solution is supplied from the inflow tube 24 into the column 2 as an eluent for eluting the acidic protein. Then, the effluent flowing out from the column 2 through the effluent pipe 25 is fractionated (collected) 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 dissolved in the respective fractions 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.

  As the buffer solution, a phosphate buffer solution is preferably used. Examples of the phosphate buffer include sodium phosphate, potassium phosphate, lithium phosphate, and ammonium phosphate. The buffer solution may be MES or sulfuric acid 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. Further, the adsorbent 3 is composed of fluorapatite, and the substitution rate of the hydroxyl group by the fluorine atom is 5% or more and 55% or less, and the adsorbent 3 has excellent acid resistance. Therefore, even if a phosphate buffer solution in such a pH range is used, alteration (dissolution, etc.) of the adsorbent 3 can be suitably prevented, and a change in separation ability in the adsorption device 1 can be reliably prevented. You can also.

  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.

  Therefore, the recovery rate of the target acidic protein can be improved by using a phosphate buffer in such a pH range and temperature range.

  In addition, the salt concentration of the phosphate buffer is preferably 500 mM or less, and more preferably 400 mM or less. By performing separation of acidic protein using a phosphate buffer solution having such a salt concentration, adverse effects on the acidic protein due to metal ions in the phosphate buffer solution can be prevented.

  Specifically, a phosphate buffer solution having a salt concentration of about 1 to 400 mM can be used. Moreover, it is preferable to change the salt concentration of the phosphate buffer solution continuously or stepwise during the separation operation of the acidic protein. Thereby, the efficiency of separation operation of acidic protein can be achieved.

  Furthermore, the flow rate of the phosphate buffer is preferably about 0.1 to 10 mL / min, and 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.

  In the above description, acidic protein is an example of a negatively charged substance, but examples of other negatively charged substances include acidic amino acids, DNA, RNA, and negatively charged liposomes. According to the present invention, these negatively charged substances can be separated easily and with high purity, like acidic proteins.

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

[1B] Preparation Step First, a sample solution containing a basic protein (such as an antibody) to be purified and a contaminant (substance) other than the basic protein is prepared.

  When the basic protein is a recombinant 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 basic protein, or an insect such as a silkworm. Animal, animal cells such as CHO cells derived from Chinese hamster ovary cells, secretions from microorganisms such as Escherichia coli, and their cytoplasmic components.

  When the basic protein is a natural protein, examples of the sample solution include blood (plasma) derived from various animals, body fluids such as lymph, saliva, and nasal discharge.

  Furthermore, examples of the basic protein include lysozyme, thaumatin, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, histone, protamine, polylysine and monelin.

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

  As the sample solution, secretions from the microorganisms or body fluids derived from animals may be used as they are, but those diluted in a neutral buffer such as water or physiological saline, and filters You may make it use what was filtered with the filter membranes. In addition, the sample solution may contain a plurality of types of basic proteins.

[2B] Supplying 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 adsorbed on the adsorbent 3. Make contact.

  As a result, a basic protein having a high adsorbing ability with respect to the adsorbent 3 and a contaminant (protein) having a relatively high adsorbing ability with respect to the adsorbent 3 among contaminants other than the basic protein (such as contaminating proteins) Retained in column 2; Then, contaminants having a low adsorption capacity with respect to the adsorbent 3 flow out from the column 2 through the filter member 5 and the outflow pipe 25.

  At this time, in the present invention, the adsorbent 3 is composed of fluorapatite, and the substitution rate of the hydroxyl group by the fluorine atom is 6% or more and 76% or less, so that the basic protein is more surely adsorbed. Can be held.

[3B] Fractionation step Next, for example, a phosphate buffer solution is supplied from the inflow tube 24 into the column 2 as an eluent for eluting the basic protein. Then, the effluent flowing out from the column 2 through the effluent pipe 25 is fractionated (collected) by a predetermined amount. As a result, the basic protein and contaminants adsorbed on the adsorbent 3 are recovered (separated) in a state of being dissolved in the respective fractions according to the difference in adsorption force with respect to the adsorbent 3 respectively. The

  That is, since the basic protein and the contaminant are adsorbed specifically to the adsorbent 3 with their own adsorption (supporting) force, the basic protein and the contaminant are determined according to the difference in the adsorption force. Are separated and purified.

  As the buffer, a phosphate buffer is preferably used. Examples of the phosphate buffer include sodium phosphate, potassium phosphate, lithium phosphate, and ammonium phosphate. The buffer solution may be MES or sulfuric acid 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, the basic protein adsorbed on the adsorbent 3 can be surely eluted without dissolving the adsorbent 3 composed of fluorapatite, and the purification rate can be improved. Further, the adsorbent 3 is made of fluorapatite, and the substitution rate of the hydroxyl group by the fluorine atom is 6% or more and 76% or less, and the adsorbent 3 has excellent acid resistance. Therefore, even if a phosphate buffer solution in such a pH range is used, alteration (dissolution, etc.) of the adsorbent 3 can be suitably prevented, and a change in separation ability in the adsorption device 1 can be reliably prevented. You can also. Furthermore, even if the pH in the column 2 temporarily changes to the acidic side during the transition from the supply step [2B] to the main step [3B], alteration (dissolution) of the adsorbent 3 is suitably prevented. be able to.

  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 basic protein to be separated can be prevented.

  Therefore, by using a phosphate buffer in such a pH range and temperature range, the recovery rate of the target basic protein can be improved.

  In addition, the salt concentration of the phosphate buffer is preferably 500 mM or less, and more preferably 400 mM or less. By performing separation of the basic protein using the phosphate buffer solution having such a salt concentration, it is possible to prevent an adverse effect on the basic protein due to metal ions in the phosphate buffer solution.

  Specifically, a phosphate buffer solution having a salt concentration of about 1 to 400 mM can be used. The salt concentration of the phosphate buffer solution is preferably changed continuously or stepwise during the separation operation of the basic protein. Thereby, efficiency of separation operation of basic protein can be aimed at.

  Furthermore, the flow rate of the phosphate buffer is preferably about 0.1 to 10 mL / min, and more preferably about 1 to 5 mL / min. By separating the basic protein at such a flow rate, it is possible to reliably separate the target basic protein without taking a long time for the separation operation, that is, to obtain a highly pure basic protein. Can do.

  By the operation as described above, basic protein is recovered in a predetermined fraction.

  In the above description, the basic protein is an example of a positively charged substance, but examples of the positively charged substance include basic amino acids, positively charged cholesterol, and positively charged liposomes. According to the present invention, these positively charged substances can be separated easily and with high purity, as with basic proteins.

<Method for producing fluorapatite>
Next, the fluorapatite as described above may be produced by any production method, and can be produced, for example, by the following method I or II.

  I: Hydroxyapatite and hydrogen fluoride are reacted in a mixed liquid obtained by mixing a slurry containing hydroxyapatite and a hydrogen fluoride-containing liquid containing hydrogen fluoride. Thereby, at least a part of the hydroxyl group of hydroxyapatite is substituted with a fluorine atom to obtain fluorine apatite.

  II: A first liquid containing a calcium-based compound containing calcium, a second liquid containing hydrogen fluoride, and a third liquid containing phosphoric acid are respectively prepared. Thereafter, the first liquid, the second liquid, and the third liquid are mixed to obtain a reaction liquid. In this reaction solution, fluorapatite is obtained by reacting calcium-based compound, hydrogen fluoride and phosphoric acid.

  According to these methods, for example, hydrogen fluoride is used as a fluorine source compared to a method of synthesizing fluorine apatite by adding ammonium hydrogen fluoride as a fluorine source in a slurry containing hydroxyapatite. Fluorine apatite with little or no impurities remaining can be obtained.

  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 adsorbent can be used for separation of acidic proteins and basic proteins, in which an eluate having a relatively low pH must be used for separation. Separation can be carried out without dissolution of 3. For this reason, the separation of the acidic protein and the basic protein can be reliably performed.

  Among these, the method I is a relatively simple operation of appropriately setting the addition amount of the hydrogen fluoride-containing liquid, and the substitution rate of the hydroxyl group in the fluorine apatite with a fluorine atom is in the range of 5% or more and 76% or less. Can be set within.

  Hereinafter, the method I will be described in detail.

<Method I>
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-based compound 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, etc. can be used for the phosphoric acid compound as a phosphoric acid source. Among these, particularly, as a calcium-based compound as a calcium source, those containing calcium hydroxide or calcium oxide as a main component are preferable. Moreover, what has phosphoric acid as a main component is preferable as a phosphoric acid compound as a phosphoric acid source.

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. By stirring and mixing this, hydroxyapatite is synthesized and primary hydroxyapatite particles are produced. A slurry is thus 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, the prepared slurry and the prepared hydrogen fluoride-containing liquid are mixed. By doing so, in the slurry (reaction liquid) containing the hydrogen fluoride-containing liquid, the hydroxyapatite primary particles and hydrogen fluoride are reacted to obtain fluorapatite primary particles.

  That is, by bringing hydrogen fluoride into contact with the hydroxyapatite primary particles, at least a part of the hydroxyl groups of hydroxyapatite is substituted with fluorine atoms as shown in the following formula. As a result, hydroxyapatite is converted to fluorapatite to obtain primary fluorapatite particles.

Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ →
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X
[However, in the formula, x is 0.05 ≦ x ≦ 0.76. ]

  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 in the range of 5% or more and 76% or less of the substitution rate of the hydroxyl group by the fluorine atom in the fluorapatite primary particle is set between the prepared slurry and the prepared hydrogen fluoride-containing liquid in this step A3. It can be easily performed by appropriately setting the mixing ratio (that is, the amount of hydrogen fluoride-containing liquid added).

Moreover, since hydrogen fluoride (HF) is used as the fluorine compound as the fluorine source, ammonium hydrogen fluoride (NH ₄ F), lithium fluoride (LiF), sodium fluoride (NaF), potassium fluoride (KF) As compared with the case of using magnesium fluoride (MgF ₂ ), calcium fluoride (CaF ₂ ) or the like, no or very little side reaction product is generated.

  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, by mixing a hydrogen fluoride-containing liquid with the slurry, the pH of the slurry is preferably adjusted within the range of about 2.5 to 5, more preferably about 2.7 to 4. In this state, hydroxyapatite (primary particles) is 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 dissolve. Therefore, it may be difficult to obtain primary particles by converting hydroxyapatite to fluorapatite. 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 into the slurry 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, the total amount of the slurry becomes extremely large, and the yield of the fluorapatite primary particles relative to the total amount of the slurry may be reduced. Therefore, it is also disadvantageous industrially.

  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. Further, the substitution rate of fluorine atoms between the obtained primary fluorapatite particles can be made more uniform. For example, when the adsorbent (dried particles or sintered particles) 3 is produced using such fluorapatite primary particles, the variation in the characteristics becomes small, and a more reliable one 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 the primary fluorapatite particles obtained by the method I as described above is dried or granulated to obtain dry particles (secondary particles), and the dried particles are fired to obtain sintered particles. It can be.

  As the adsorbent 3, the sintered particles are preferably used from the viewpoint of mechanical strength. However, dry particles can be used as the adsorbent 3 when the load on the adsorbent is relatively small.

  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, and more preferably about 400 to 700 ° C. By setting the firing temperature within such a range, the adsorbent 3 having excellent mechanical strength can be obtained while leaving gaps (holes) formed in the primary particles or between the primary particles (aggregates). Can do.

  When the sintered particles are used as the adsorbent 3, the specific surface area is preferably wider from the viewpoint of improving the separation ability of acidic protein by the adsorbent 3. The following points are clarified by the study of the present inventor.

That is, when the fluorapatite primary particles are produced using the method I described here, the substitution rate of the hydroxyl group of the fluorapatite with a fluorine atom is 5% or more and 55% or less, preferably 20% or more, 40 Set to% or less. By doing so, it has been found that the specific surface area of the adsorbent 3 can be 30 m ² / g or more and 50 m ² / g or less, preferably 42 m ² / g or more and 50 m ² / g or less.

  The fluorapatite, the adsorption device, and the separation method of the present invention have been described above, but 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 [1] First, calcium hydroxide was suspended in pure water, and an aqueous phosphoric acid solution was dropped therein, and the mixture was sufficiently stirred. As a result, 200 L of slurry containing 10 wt% primary particles of hydroxyapatite was obtained.

  The obtained composite (primary particles) was confirmed to be hydroxyapatite by a powder X-ray diffraction method.

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

  [2] Next, in a state where the slurry was stirred with a stirring power of 1 kW, a hydrogen fluoride-containing liquid was dropped into the slurry at a speed of 5 L / hour.

  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 in which the hydrogen fluoride-containing liquid was dropped was stirred at a temperature of 30 ° C. for 48 hours with a stirring power of 1 kW. 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.

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

  [4] Next, a part of the dried particles was classified so as to have a center particle size of about 40 μm. Thereafter, the classified dry particles were fired in an electric furnace at 400 ° C. for 4 hours. As a result, sintered particles (fluorine apatite) 1 having a fluorine atom substitution rate of 100% were obtained.

  Further, by appropriately changing the dropping amount of the hydrogen fluoride-containing liquid in the step [2], sintered particles (fluorapatite) 2 to 4 having fluorine atom substitution rates of 75, 50, and 25% were obtained, respectively. . Further, by omitting the dropping of the hydrogen fluoride-containing liquid in the step [2], sintered particles (hydroxyapatite) 5 having a fluorine atom substitution rate of 0% were obtained.

  In addition, the substitution rate of the fluorine atom in each sintered particle 1-5 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 2-1. Acid resistance evaluation by Ca elution First, each of the sintered particles 1 to 5 is packed in a packed space of a stainless steel column (inner diameter: 4.0 mm × length: 100 mm) so as to be almost full. Sample No. 1-5 columns packed with ˜5 were produced.

  The volume of the column filling space was 1.256 mL, and the amount of the sintered particles 1 to 5 packed in each column was measured.

  Each column was washed by passing 20 mL of 400 mM sodium phosphate buffer (pH 5, temperature 25 ° C.) through each sample No. column. Thereafter, another 10 mL of 400 mM sodium phosphate buffer was passed through to recover the effluent (eluate).

  Next, the calcium concentration in the effluent was measured for each effluent collected from the column of each sample No. using an ICP emission spectrometer (“ICPS-7500”, manufactured by Shimadzu Corporation). Then, by dividing the effluent of the column of each sample No. by the amount of each sintered particle packed in each column measured in advance, the amount of Ca elution from each sintered particle [ppm / g].

  FIG. 2 shows the relationship between the Ca elution amount from each of the sintered particles 1 to 5 determined as described above and the fluorine atom substitution rate in each of the sintered particles 1 to 5.

  Here, it can be said that the sintered particles (fluorapatite) have excellent acid resistance when the Ca elution amount is less than about 80 ppm / g. For this reason, it has been clarified that by setting the substitution rate of fluorine atoms in the sintered particles to 5% or more, the sintered particles (fluorapatite) can have excellent acid resistance.

2-2. Evaluation of Acidic Protein Adsorption Capacity First, each of the sintered particles 1 to 5 is packed so as to be almost full in a packing space of a stainless steel column (inner diameter 4.6 mm × length 35 mm). Sample No. 1-5 columns packed with ˜5 were produced.

  In addition, the specific surface area of the sintered particles 1 to 5 packed in each column was previously measured using a fully automatic BET specific surface area measuring device (manufactured by Mountech, “Macsorb model-1201”).

  Next, Albumin, bovine serum,> 96% Essentially Fatty Acid Free (SIGMA, “A6003”) was added to 10 mM sodium phosphate buffer (pH 6) so that BSA (bovine serum albumin) was 0.5 mg / mL. 8) to obtain a mixed solution. Thereafter, the mixed solution was filtered through a 0.22 μm filter, and further deaerated for 30 minutes to obtain a sample solution.

  The absorbance value (280 nm, reference: pure water) and molar extinction coefficient value of the sample solution were measured using an absorptiometer (manufactured by Shimadzu Corporation, “UV Spectrophotometer UV-1800”). By doing so, the actual amount of protein adsorption in the sample solution was determined.

  Next, a 10 mM sodium phosphate buffer solution (pH 6.8, temperature 25 ° C.) is passed through the column of each sample No. packed with each sintered particle at a liquid passing rate of 0.42 mL / min. To equilibrate the column.

  Next, each column of sample No. was attached to a chromatographic apparatus. Thereafter, the sample solution was fed from the pump of the chromatographic apparatus while measuring the absorbance value (280 nm) of the sample solution with the chromatographic apparatus.

  And in the chromatogram obtained from the chromatograph equipped with the column of each sample No., until the time when the absorbance value (280 nm) of the elution peak becomes 10% of the absorbance value (280 nm) of the sample solution (stock solution) The liquid was sent. Thereafter, the amount of acidic protein adsorbed was determined from the fed sample solution.

  FIG. 3 shows the relationship between the amount of acidic protein adsorbed on each of the sintered particles 1 to 5 and the substitution rate of fluorine atoms in each of the sintered particles 1 to 5 obtained as described above. Furthermore, the relationship between the specific surface area of each sintered particle 1-5 and the fluorine atom substitution rate in each sintered particle 1-5 is also shown in FIG.

  Here, according to the study by the present inventors, it can be said that when the acidic protein adsorption amount exceeds about 2 mg / mL, the sintered particles (fluorapatite) have excellent acidic protein adsorption ability. For this reason, it has been clarified that by setting the substitution rate of fluorine atoms in the sintered particles to 55% or less, the sintered particles (fluorine apatite) can have excellent acidic protein adsorption ability.

Furthermore, by setting the substitution rate of fluorine atoms in the sintered particles to 55% or less, the specific surface area of the sintered particles can be easily set to 30 m ² / g or more. Since the specific surface area of the sintered particles is wide, it was speculated that the acidic protein adsorbing ability of the sintered particles was improved from this viewpoint.

2-3. Evaluation of basic protein adsorption capacity First, the sintered particles 1 to 5 are packed in a packed space of a stainless steel column (inner diameter: 4.6 mm × length: 35 mm) so as to be almost full, and the sintered particles are obtained. Sample No. 1-5 columns packed with 1-5 were manufactured.

  In addition, the specific surface area of the sintered particles 1 to 5 packed in each column was previously measured using a fully automatic BET specific surface area measuring device (manufactured by Mountech, “Macsorb model-1201”).

  Next, Lysozyme from Chicken Egg White (manufactured by SIGMA, “L6876”) is dissolved in 10 mM sodium phosphate buffer (pH 6.8) so as to be 0.5 mg / mL of lysozyme (Lyso). Got. Thereafter, the mixed solution was filtered with a 0.22 μm filter, and further deaerated for 30 minutes to obtain a sample solution.

  The absorbance value (280 nm, reference: pure water) and molar extinction coefficient value of the sample solution were measured using an absorptiometer (manufactured by Shimadzu Corporation, “UV Spectrophotometer UV-1800”). By doing so, the actual amount of protein adsorption in the sample solution was determined.

  Next, a 10 mM sodium phosphate buffer solution (pH 6.8, temperature 25 ° C.) was passed through the column of each sample No. packed with each sintered particle at a liquid passing rate of 0.42 mL / min. The column was equilibrated.

  Next, each column of sample No. was attached to a chromatographic apparatus. Thereafter, the sample solution was fed from the pump of the chromatographic apparatus while measuring the absorbance value (280 nm) of the sample solution with the chromatographic apparatus.

  And in the chromatogram obtained from the chromatograph equipped with the column of each sample No., until the time when the absorbance value (280 nm) of the elution peak becomes 10% of the absorbance value (280 nm) of the sample solution (stock solution) The liquid was sent. Then, the basic protein adsorption amount was calculated | required from the sent sample liquid.

  FIG. 4 shows the relationship between the basic protein adsorption amount to each of the sintered particles 1 to 5 and the fluorine atom substitution rate in each of the sintered particles 1 to 5 determined as described above. Furthermore, the relationship between the specific surface area of each of the sintered particles 1 to 5 and the substitution rate of fluorine atoms in each of the sintered particles 1 to 5 is also shown in FIG.

  Here, according to the study by the present inventors, it can be said that when the basic protein adsorption amount exceeds about 30 mg / mL, the sintered particles (fluorapatite) have excellent basic protein adsorption ability. Therefore, it becomes clear that the sintered particles (fluorine apatite) can have excellent basic protein adsorption ability by setting the substitution rate of fluorine atoms in the sintered particles to 6% or more and 76% or less. It was.

  This is because as the substitution rate by fluorine atoms in the sintered particles (fluorapatite) increases, the fluorine atom having negative chargeability has some influence on the Ca site having positive chargeability, and as a result, fluorine apatite is obtained. The adsorption ability of the basic protein to the adsorbent composed of is improved. On the other hand, the specific surface area of the sintered particles (fluorine apatite) tends to become narrower with a peak when the fluorine atom substitution rate is 25% as the substitution rate by fluorine atoms in the fluorine apatite increases. Due to this, the adsorption ability of the basic protein to the adsorbent composed of fluorapatite is lowered. When these two facts overlap, when the fluorine atom substitution rate in the sintered particles is 6% or more and 76% or less, the sintered particles (fluorapatite) exhibit excellent basic protein adsorption ability. It was guessed.

2-4. Summary From the above, the sintered particles (fluorine apatite) can be made to have excellent acid resistance by setting the substitution rate of fluorine atoms in the sintered particles to 5% or more. Furthermore, by setting the substitution rate to 6% or more and 76% or less, the sintered particles (fluorapatite) can have excellent basic protein adsorption ability. Therefore, by setting the substitution rate of fluorine atoms in the sintered particles to 6% or more and 76% or less, as a result, the sintered particles (fluorine apatite) are excellent in both acid resistance and basic protein adsorption ability. It will exhibit its characteristics.

The fluorapatite of the present invention is a fluorapatite used for separating the charged substance from a sample solution containing a chargeable substance and a substance other than the charged substance, and the fluorapatite is generally It is represented by the formula Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X , wherein x satisfies the relationship of 0.05 ≦ x ≦ 0.76. Thereby, it is excellent in acid resistance, and it can isolate | separate a charged substance with high precision from the sample liquid containing a charged substance and substances other than this charged substance. Therefore, the present invention has industrial applicability.

Claims (5)

Fluorapatite used for separating the negatively charged substance from a sample solution containing a negatively charged substance and a substance other than the negatively charged substance,
The fluorapatite is represented by the following general formula:
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X
In the general formula, x satisfies a relationship of 0.05 ≦ x ≦ 0.40 ,
The fluorapatite has a granular shape, a specific surface area of 30 m ² / g to 50 m ² / g, and an average particle size of 0.5 μm to 150 μm .   The fluorapatite according to claim 1, wherein the negatively charged substance is an acidic protein. 3. The fluorapatite according to claim 1 , wherein x satisfies the relationship of 0.05 ≦ x ≦ 0.25 in the general formula .   An adsorption device comprising the fluorapatite according to any one of claims 1 to 3 as an adsorbent. A separation method for separating the negatively charged substance from a sample liquid containing a negatively charged substance and a substance other than the negatively charged substance,
Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X represented by the general formula, wherein x is composed of fluorine apatite satisfying the relationship of 0.05 ≦ x ≦ 0.40 , In the adsorbing device formed by filling at least part of the filling space with a filler having a specific surface area of 30 m ² / g to 50 m ² / g and an average particle size of 0.5 μm to 150 μm , Supplying a sample solution;
Supplying a buffer solution into the adsorption device to obtain an effluent flowing out of the adsorption device;
Separating the negatively charged substance from the sample liquid in the effluent of each fractionated fraction by fractionating the effluent by a predetermined amount.

Images