JPWO2019224660A1 - Adsorbent production method - Google Patents

Abstract

A method for producing an adsorbent for obtaining an adsorbent containing at least a part of a calcium phosphate-based compound and having a separable ability. The production method includes a step of preparing an adsorbent, a first solvent and a first addition. The first step of immersing the adsorbent in the first solution containing the substance, and the second step of bringing the adsorbent into contact with the second solution containing the second solvent and the second additive. The first additive has the ability to affect the adsorbing power of the calcium element constituting the adsorbent on the adsorbent when added to the first solvent. The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution, and the second step is the first step. A production method that takes place after.

Description

The present invention relates to a method for producing an adsorbent (filler) containing a calcium phosphate-based compound. More specifically, the present invention relates to a method for producing an adsorbent containing a calcium phosphate-based compound, which suppresses or prevents fluctuations in the resolution of the obtained adsorbent.

Since calcium phosphate-based compounds such as hydroxyapatite have excellent biocompatibility, they have been conventionally used for adsorption and separation of charged substances such as proteins contained in a sample solution for a liquid chromatography column (adsorption device). It is widely used as an agent (see, for example, Patent Document 1).

In this liquid chromatography column, the charged substance contained in the sample liquid is adsorbed on an adsorbent composed of a calcium phosphate-based compound and then separated by being separated from the adsorbent, and this adsorption / separation is repeated. By carrying out this, the charged substance contained in the sample solution can be separated a plurality of times.

By the way, the separation of the charged substance using the adsorbent containing the calcium phosphate compound may be repeatedly carried out without a period of time, or a certain period (storage period) such as 7 days or more from the execution of the separation. ) May be implemented after opening. For a certain period of time until the charged substances are separated, the adsorbent is contained in a storage solution such as a high-concentration sodium hydroxide solution (for example, about 1.0 M) or a phosphate buffer solution (for example, about 0.4 M). May be stored in.

When the present inventor separates the charged substance using an adsorbent containing a calcium phosphate-based compound after storage in such a storage solution for a certain period of time, it looks like a basic protein that binds to a phosphate site. The separation position (elution time) of the positively charged substance separated from the adsorbent does not change, but the separation position (elution time) of the negatively charged substance such as acidic protein that binds to calcium sites is separated from the adsorbent. I found that time) changes.

By fractionating the effluent flowing out of the liquid chromatography column by a predetermined amount, when a negatively charged substance is to be separated into the effluent of each fractionated fraction, the negatively charged substance is generated. The separated fractions change before and after the storage period. Therefore, in order to recover the desired negatively charged substance, it is necessary to reset the fraction to be recovered before and after the storage period, which causes a problem that it takes time and labor. The present inventor has found such a problem.

Japanese Patent Application Laid-Open No. 2013-534252

An object of the present invention is to suppress or prevent fluctuations in the resolution of the obtained adsorbent in the production of an adsorbent containing a calcium phosphate-based compound.

The present invention has any of the following aspects in one preferred embodiment.
(A1) A method for producing an adsorbent, which comprises at least a part of a calcium phosphate compound and has a separable ability.
The process of preparing the adsorbent and
A first step of immersing the adsorbent in a first solution containing a first solvent and a first additive, and
A second step of bringing the adsorbent into contact with a second solution containing the second solvent and the second additive, and
Including
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
The production method, wherein the second step is performed after the first step.
(A2)
The production method according to A1, wherein the second solution contains a pH buffer.
(A3)
The production method according to A1 or A2, wherein the first additive comprises one or more selected from the group consisting of bases, salts and alcohols.
(A4)
The production method according to any one of A1 to A3, wherein the charged substance has a negatively charged portion.
(A5)
The production method according to any one of A1 to A4, wherein the concentration of the first additive is about 0.01 M or more and about 2.0 M or less.
(A6)
When the concentration of the second additive is A [M] and the concentration of the first additive is B [M], the A / B is about 0.001 or more and about 0.1 or less. The production method according to any one of A1 to A5.
(A7)
The second step includes a standing step of allowing the adsorbent to stand in the second solution.
The production method according to any one of A1 to A6, wherein the standing step is about 0.1 hour or more and about 24 hours or less.
(A8)
The production method according to any one of A1 to A7, wherein the calcium phosphate-based compound contains hydroxyapatite as a main component.
(A9)
The first additive comprises sodium hydroxide
The production method according to any one of A1 to A8, wherein the second additive contains a phosphate buffer.
(A10)
The production method according to any one of A1 to A8, wherein the first additive and the second additive contain a phosphate buffer.
(A11)
The production method according to any one of A1 to A8, wherein the first additive comprises a combination of a hydroxyl group-containing compound and a pH buffer.
(A12)
The production method according to any one of A1 to A8, wherein the first additive comprises a combination of a hydroxyl group-containing compound and a buffer having a sulfo group.
(A13)
A solution set containing at least a part of a calcium phosphate-based compound and used in a method for producing an adsorbent for obtaining an adsorbent having a separable ability.
The solution set includes a first solution containing a first solvent and a first additive, and a second solution containing a second solvent and a second additive.
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
The solution set, wherein the production method is the production method according to any one of A1 to A13.
(A14)
The use of a solution set in the method of producing an adsorbent for obtaining an adsorbent containing at least a part of a calcium phosphate-based compound and having a separable ability.
The solution set includes a first solution containing a first solvent and a first additive, and a second solution containing a second solvent and a second additive.
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
Use, wherein the production method is the production method according to any one of A1 to A13.
(A15)
An adsorbent produced by the production method according to any one of A1 to A13.
(A16)
A column for liquid chromatography filled with an adsorbent produced by the production method according to any one of A1 to A13.

Since the technique of the production method of the present invention can suppress or prevent a change in the adsorptive capacity of the adsorbent and stabilize it, it can also be used as a stabilization method. The stabilizing method of the present invention preferably has the following aspects. The following description of the stabilization method is also applicable to the production method of the present invention.
(1) Stable that stabilizes the separability of an adsorbent whose surface is composed of a calcium phosphate-based compound at least, which is stored in a preservative solution containing water and at least one additive of a base, a salt and an alcohol. It is a method of conversion
By storing the adsorbent in the preservative solution, the elution time of the negatively charged substance adsorbed on the adsorbent is shorter than the elution time of the negatively charged substance before storage.
A stabilization method comprising a treatment step of bringing the adsorbent into contact with a treatment liquid containing the additive, which has a content lower than the content of the additive in the storage liquid.

As a result, the separation ability of the adsorbent is stabilized, the elution time for separating the negatively charged substance from the adsorbent becomes longer, and as a result, the elution time can be restored to almost the same as before storage with the preservation solution. it can. Therefore, it is possible to accurately suppress or prevent a change in the separation position (elution time) of the negatively charged substance as well as the positively charged substance before and after storage in the storage solution.

Therefore, by fractionating the effluent flowing out of the adsorbent filled with the adsorbent by a predetermined amount, negatively charged substances are separated into the effluent of each fractionated fraction. It is possible to accurately suppress or prevent the fraction from which the substance is separated from being displaced before and after storage with the storage solution. Therefore, since it is not necessary to reset the fraction to be recovered from the negatively charged substance, the negatively charged substance can be separated (purified) from the sample liquid without requiring time and labor.

(2) The stabilization method according to (1) above, wherein in the preservation solution, the additive is the base and the base is sodium hydroxide.

As a result, it is possible to further elute other substances such as contaminating proteins remaining in the adsorbent, and further, it is possible to accurately suppress or prevent the growth of bacteria and the like during storage in the adsorbent.

(3) The stabilization method according to (1) or (2) above, wherein the concentration of the additive in the preservation solution is about 0.01 M or more and about 2.0 M or less.

This makes it possible to further elute other substances such as contaminating proteins remaining in the adsorbent.

(4) When the concentration of the additive in the treatment solution is A [M] and the concentration of the additive in the preservation solution is B [M], the A / B is about 0.001 or more and about 0. The stabilization method according to any one of (1) to (3) above, which is 1 or less.

As a result, the elution time of the negatively charged substance by the adsorbent can be reliably restored to almost the same time as before the storage with the storage solution.

(5) In the treatment step, the adsorbent is allowed to stand in the treatment liquid, and the treatment agent is allowed to stand for about 0.1 hour or more and about 24 hours or less, as described in (1) to (4). ). The stabilization method described in any one of.

As a result, the elution time of the negatively charged substance by the adsorbent can be reliably restored to almost the same time as before the storage with the storage solution.

(6) The stabilization method according to any one of (1) to (5) above, wherein in the treatment liquid, the additive is the salt and the treatment liquid is a phosphate buffer solution.

As a result, the elution time of the negatively charged substance by the adsorbent can be reliably restored to almost the same time as before the storage with the storage solution.

(7) The stabilizing method according to (6) above, wherein the phosphate buffer solution has a pH of about 6.0 or more and about 7.5 or less.

As a result, the elution time of the negatively charged substance by the adsorbent can be reliably restored to almost the same time as before the storage with the storage solution.

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

As a result, the elution time of the acidic protein by the adsorbent can be reliably restored to almost the same time as before the storage with the storage solution.

(9) The stabilization method according to any one of (1) to (8) above, wherein the calcium phosphate compound is hydroxyapatite.

Since hydroxyapatite is close to a component constituting a living body, it is possible to preferably prevent such protein from being denatured (denatured), particularly when adsorbing or separating a protein as a charged substance.

The production method of the present invention can suppress or prevent fluctuations in the resolution of the obtained adsorbent in the production of an adsorbent composed of a calcium phosphate-based compound.

It is a vertical cross-sectional view which shows an example of the adsorption apparatus provided with the adsorbent to which the stabilization method of this invention can be applied. It is a graph which shows the relationship between the elution time and the absorbance before and after storage by a preservation solution in Example 1. FIG. It is a graph which shows the relationship between the elution time and the absorbance before and after storage by a preservation solution in Example 2. FIG. It is a graph which shows the relationship between the elution time and the absorbance before and after storage by a preservation solution in Example 3. FIG. 6 is a graph showing the relationship between the elution time and the absorbance before and after storage with a storage solution in Comparative Example 1. It is a graph which shows the relationship between the elution time and the absorbance before and after storage by a preservation solution in Comparative Example 2. It is a graph which shows the difference between the elution time after using each of the phosphate buffer solutions of different concentrations as a preservation solution, and the elution time before using the preservation solution in Example 4. It is a graph which shows the difference between the elution time after using each of the phosphate buffers of different pH as a preservation solution, and the elution time before using the preservation solution. In Comparative Example 3, it is a graph which shows the difference between the elution time after using 20% ethanol as a preservation solution, and the elution time before using the preservation solution. 6 is a graph showing the difference between the elution time after using a 10 mM sodium phosphate buffer (pH 6.5) containing 20% ethanol as a preservation solution and the dissolution time before using the preservation solution in Example 6. In Example 7, the graph which shows the difference between the elution time after using the phosphate buffer solution containing 20% ethanol and each having a different concentration as a preservation solution, and the elution time before using the preservation solution. is there. In Example 8, the graph which shows the difference between the elution time after using the phosphate buffer solution containing 20% ethanol and each different pH as a preservation solution, and the elution time before using the preservation solution. is there. In Example 9, a graph showing the difference between the elution time after using a phosphate buffer solution containing 20% ethanol and having different pH values as a preservation solution and the elution time before using the preservation solution. Is. It is a flowchart which shows each process of the production method of this invention.

Hereinafter, the production method and the stabilization method of the present invention will be described in detail based on the preferred embodiments shown in the accompanying drawings. First, prior to explaining the production method and stabilization method of the present invention, an example of an adsorbent including an adsorbent to which the production method and stabilization method of the present invention can be applied will be described.

In the following, a case where the charged substance separated by using the adsorption device is a protein, that is, a case where the negatively charged substance is an acidic protein and the positively charged substance is a basic protein will be described as an example.

<Adsorption device>
FIG. 1 is a vertical cross-sectional view showing an example of an adsorbent including an adsorbent to which the production method and the stabilization method of the present invention can be applied. In the following description, the upper side in FIG. 1 is referred to as an “inflow side” and the lower side is referred to as an “outflow side”.

Here, the inflow side means that, when separating (purifying) the target protein, for example, a liquid such as a sample liquid (a liquid containing a sample) or a phosphoric acid-based buffer solution which is an eluate is introduced into the adsorbent. The supply side is referred to, while the outflow side is the side opposite to the inflow side, that is, the side on which the liquid flows out from the adsorption device.

The adsorbent 1 shown in FIG. 1, which separates (purifies) the protein from the sample solution, has a column 2, a granular adsorbent (filler) 3, and two filter members 4 and 5.

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

The column body 21 is composed of, for example, a cylindrical member. The constituent materials of each part (each member) constituting the column 2 including the column body 21 may be, for example, various glass materials, various resin materials, various metal materials, various ceramic materials, or the like, or these. It may be any combination of.

In the column main body 21, the filter members 4 and 5 are arranged so as to close the inflow side opening and the outflow side opening, respectively, and caps 22 and 23 are screwed to the inflow side end portion and the outflow side end portion, respectively. It will be installed depending on the situation.

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

The volume of the adsorbent filling space 20 is appropriately set according to the volume of the sample solution, and is not particularly limited, but is preferably about 0.1 mL or more and about 100 mL or less, and about 1 mL or more and about 50 mL or less with respect to 1 mL of the sample solution. More preferred.

Further, with the caps 22 and 23 attached to the column body 21, the liquidtightness between them is ensured.

The inflow pipe 24 and the outflow pipe 25 are liquid-tightly fixed (fixed) to the centers of the caps 22 and 23, respectively. The liquid is supplied to the adsorbent 3 via the inflow pipe 24 and the filter member 4. Further, the liquid supplied to the adsorbent 3 passes between the adsorbents 3 (gap) and flows out of the column 2 through the filter member 5 and the outflow pipe 25. At this time, a protein to be separated (hereinafter, may be simply referred to as “protein”) contained in the sample solution (sample) and a substance other than the protein to be separated (a contaminating protein other than the protein to be separated (hereinafter, simply referred to as “protein”). It may also contain (sometimes referred to as "contamination protein")) and is separated based on the difference in adsorptivity to the adsorbent 3 and the difference in affinity for the phosphate buffer.

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

The adsorbent 3 contains at least a part of the calcium phosphate-based compound. More preferably, at least the surface of the adsorbent 3 is composed of a calcium phosphate-based compound. The protein to be separated is specifically adsorbed on the adsorbent 3 by the adsorption (supporting) force peculiar to this protein, and depending on the difference in the adsorption force, a substance other than the protein to be separated (contamination protein). (Including) and separated / purified.

Calcium phosphate compounds include, for example, hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), TCP (Ca ₃ (PO ₄ ) ₂ ), Ca ₂ P ₂ O ₇ , Ca (PO ₃ ) ₂ , DCPD ( _{CaHPO 4 · 2H 2 O),} Ca 4 O (PO 4) 2, or may be a such as that some of these have been replaced with another atom or atomic group, or one of these It may be a species or a combination of two or more species.

Here, proteins are generally classified into acidic proteins that contain a relatively large amount of acidic amino acids and are negatively charged as constituent amino acids, and basic proteins that contain a relatively large amount of basic amino acids and are positively charged.

The calcium phosphate-based compound has a positively charged Ca site and a negatively charged phosphoric acid site in its crystal structure.

Therefore, the acidic protein is adsorbed on the calcium phosphate-based compound by forming an ionic bond between the Ca site of the calcium phosphate-based compound and the acidic amino acid residue of the acidic protein. The basic protein is adsorbed on the calcium phosphate-based compound by forming an ionic bond between the phosphate site of the calcium phosphate-based compound and the basic amino acid residue. The strength of binding (adsorption) of a protein to a calcium phosphate-based compound differs depending on the amount of charge of each protein (acidic protein and basic protein).

Therefore, if an adsorbent 3 whose at least surface is composed of a calcium phosphate-based compound is used, proteins (acidic proteins and basic proteins) and other substances (for example, contaminating proteins) can be adsorbed by these adsorbents 3. It is possible to separate by utilizing the difference in adsorption force with respect to.

Among the above-mentioned compounds, the calcium phosphate-based compound preferably contains hydroxyapatite as a main component. In particular, since hydroxyapatite is close to the components constituting the living body, it is possible to preferably prevent the protein from being denatured (denatured) when adsorbing and separating the protein. It also has an advantage that the protein can be specifically removed from the adsorbent 3 by using the phosphoric acid-based buffer solution as the eluate by changing the salt concentration.

Here, the phrase "the calcium phosphate-based compound contains hydroxyapatite as a main component" means that the mass of hydroxyapatite is about 50% or more when the total mass of the calcium phosphate-based compound is 100% by mass. This mass is more preferably about 60% or more, still more preferably about 70% or more, even more preferably about 80% or more, and most preferably about 90% or more.

Further, the hydroxyapatite may have at least a part of the hydroxyl group contained therein replaced with a fluorine atom. Hydroxiapatite substituted with a fluorine atom (hereinafter referred to as "fluorine apatite") has a calcium atom (calcium ion) released from the fluorine apatite due to the presence of a fluorine atom (fluorine ion) in its crystal structure. It can be prevented more reliably.

In the following, hydroxyapatite and hydroxyapatite substituted with a fluorine atom may be collectively referred to as "apatite".

As shown in FIG. 1, the adsorbent 3 preferably has a granular (granular) form (shape). The form of the adsorbent 3 may be, for example, a pellet shape (small lump shape), a block shape (for example, a porous body in which adjacent pores communicate with each other, a honeycomb shape) or the like. By making the adsorbent 3 granular, the surface area thereof can be increased, and the separation characteristics of the protein can be improved.

The average particle size of the granular adsorbent 3 is not particularly limited, but is preferably about 0.5 μm to about 150 μm, and more preferably about 10 μm to about 80 μm. By using the adsorbent 3 having such an average particle size, it is possible to secure a sufficient surface area of the adsorbent 3 while surely preventing clogging of the filter member 5.

The adsorbent 3 may be entirely composed of a calcium phosphate-based compound, or the surface of a carrier (base) may be coated with a calcium phosphate-based compound. Among these, it is preferable that the whole is composed of a calcium phosphate-based compound. Thereby, the strength of the adsorbent 3 can be further improved. In addition, a column 2 suitable for use when separating a large amount of protein can be obtained.

The adsorbent 3, which is entirely composed of a calcium phosphate-based compound, obtains calcium phosphate-based compound particles (primary particles) by using, for example, a wet synthesis method or a dry synthesis method, and prepares a slurry containing the calcium phosphate-based compound particles. It can be obtained by drying or granulating to obtain dried particles, and further by firing the dried particles.

On the other hand, the adsorbent 3 in which the surface of the carrier is coated with a calcium phosphate compound can be obtained, for example, by a method of colliding (hybridizing) the dry particles with a carrier composed of a resin or the like.

When the adsorbent 3 is filled in the adsorbent filling space 20 in a substantially full amount as in the present embodiment, the adsorbent 3 has substantially the same composition in each part of the adsorbent filling space 20. Is preferable. As a result, the adsorption device 1 has a particularly excellent protein separation (purification) ability.

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

<Separation method>
Next, a method for separating proteins using such an adsorption device 1 will be described.

[1A] Preparation step First, a sample solution containing a protein to be separated (purified) and a substance (mixture) other than the protein to be separated is prepared.

When the protein is a recombinant protein (monoclonal antibody, etc.), the sample solution is a mammal such as a sheep, a rabbit, or a chicken into which a nucleic acid containing a gene encoding the protein has been introduced, an animal such as an insect such as a silkworm, or a Chinese hamster. Animal cells such as CHO cells derived from ovarian cells, secretions from microorganisms such as Escherichia coli, their cytoplasmic components, etc., or any combination thereof may be used.

When the protein is a natural protein, the sample solution may be, for example, blood (plasma) derived from various animals, lymph, saliva, body fluid such as nasal discharge, or the like.

Among the proteins, the acidic protein may be, for example, BSA, HSA, fibrinogen, pepsinogen, α-globulin, β-globulin, γ-globulin, or the like. The basic protein may be, for example, lysozyme, thaumatin, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, histone, protamine, polylysine, monellin or the like.

In addition, these proteins (acidic protein and basic protein) may be variants in which some amino acids in the amino acid sequence are replaced with other amino acids.

As the sample solution, secretions from the above-mentioned microorganisms and body fluids derived from animals may be used as they are. Alternatively, the sample solution may be one obtained by diluting these in a neutral buffer solution such as water or physiological saline, or one obtained by filtering with a filtration membrane such as a filter. In addition, the sample solution may contain a plurality of types of proteins.

[2A] Supply Step Next, the obtained sample liquid is supplied to the adsorbent 3 via the inflow pipe 24 and the filter member 4, passed through the column 2 (adsorption device 1), and becomes the adsorbent 3. Make contact.

As a result, proteins having a high adsorptive capacity to the adsorbent 3 and contaminants (contamination proteins, etc.) other than the proteins to be separated (purified) have a relatively high adsorbing capacity to the adsorbent 3 (protein). Is held in column 2. Then, the contaminant having a low adsorption ability with respect to the adsorbent 3 flows out from the column 2 through the filter member 5 and the outflow pipe 25.

[3A] Fractionation Step Next, for example, a phosphoric acid-based buffer solution is supplied from the inflow pipe 24 into the column 2 as an eluate for eluting the protein. Then, the effluent flowing out from the column 2 through the outflow pipe 25 is fractionated (collected) by a predetermined amount. As a result, the proteins and contaminants adsorbed on the adsorbent 3 flow out at different timings according to the difference in the adsorbing power of the adsorbent 3 and each of them is dissolved in each fraction. Collected (separated / purified).

That is, proteins and contaminants are specifically adsorbed on the adsorbent 3 with the adsorbing (supporting) force peculiar to each. That is, they have different adsorption powers for the adsorbent 3. Proteins and contaminants are separated and purified according to this difference in adsorption power. A purified protein can be obtained by selectively recovering the fraction in which the protein to be separated (purified) is dissolved.

The buffer used in the eluate is preferably Good's buffers such as HEPES and MES, sulfates, or phosphates. Among these, buffers and phosphates having a sulfo group are more preferable. The buffer having a sulfo group is preferably MOPS, MES, or HEPES. The phosphate may be, for example, sodium phosphate, potassium phosphate, lithium phosphate, ammonium phosphate or the like. Of these, sodium phosphate is the most preferred.

The pH of the eluate containing the Good's buffer may be in the range where the pH buffering function of each Good's buffer can be exhibited, but it is more preferably "Preferred pH" described in the table below.

The pH of the phosphoric acid-based buffer (solution in which the phosphate is dissolved) is preferably set to about 5.5 to 8.5, more preferably about 6.5 to 7.5. As a result, the protein can be reliably eluted, and the purification rate thereof can be improved.

The temperature of the buffer solution (eluate) is preferably about 10 to 50 ° C, more preferably about 20 to 35 ° C. This makes it possible to prevent alteration (degeneration) of the protein to be separated.

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

The salt concentration of the buffer solution is preferably 500 mM or less, more preferably 400 mM or less. By separating the protein using a buffer solution having such a salt concentration, it is possible to prevent adverse effects on the protein due to metal ions in the re-impact solution.

A buffer solution having a salt concentration of about 1 to 400 mM is more preferably used. In addition, the salt concentration of the buffer solution is preferably changed continuously or stepwise during the protein separation operation. This makes it possible to streamline the protein separation operation.

The flow rate of the buffer solution is preferably about 0.1 to 10 mL / min, more preferably about 1 to 5 mL / min. By separating the proteins at such a flow velocity, it is possible to reliably separate the target protein without requiring a long time for the separation operation, that is, to obtain a high-purity protein.

By the above operation, the protein is recovered in a predetermined fraction. This gives the purified protein to be separated in a given fraction.

The eluate may further contain alcohol in addition to the buffer. If the eluate contains alcohol in addition to the buffer, the alcohol may be, for example, methanol, ethanol, or isopropanol. Of these, ethanol is particularly preferable.

The concentration of alcohol in the eluate may be, for example, 1% or more when the total mass of the eluate is 100%. It is more preferably 2% to 50%, even more preferably 5% to 40%, even more preferably 10% to 30%, and most preferably 15% to 25%. When the concentration of alcohol in the eluate is within the above range, the effect of further elution of other substances and / or suppression of the generation of bacteria and the like is more remarkable, and the adsorptive ability of the adsorbent is adversely affected. Can be minimized.

Next, the stabilization method of the present invention is applied to the columns subjected to the above steps 1A to 3A, as described below. Therefore, it can be understood that the above steps 1A to 3A are steps for preparing a column for applying the stabilization method of the present invention. Further, in the production method of the present invention, it can be understood that the above steps 1A to 3A are steps of preparing a column as a material of the production method of the present invention. The "material column" of the production method of the present invention is a column containing (possibly) a substance that changes the adsorptive capacity of the adsorbent by being used already, or is unused. Is also a column having an adsorbent whose adsorptive capacity has changed due to some reason such as the presence of impurities. This step corresponds to S101 in FIG.

<Stabilization Method> [4A] Preservation Step Here, in the protein separation method using the adsorption device 1, generally, after the above-mentioned protein separation (purification), a high concentration of the protein is maintained without a delay. By eluting and washing other substances (contamination proteins, etc.) adsorbed on the adsorbent 3 using a buffer solution (phosphate-based buffer solution, etc.), the adsorbent 1 is provided again for protein separation. , Protein separation is repeated.

Alternatively, the adsorbent 3 may be used for protein separation in the adsorbent 1 after a certain period (storage period) such as about 1 to 7 days. In this case, in a certain period until the separation of the charged substance, a high-concentration sodium hydroxide solution (for example, about 1.0 M) or the like is usually used for the purpose of further elution of other substances and / or suppression of the generation of bacteria and the like. The adsorbent 3 is stored in a storage solution such as a phosphate buffer solution (for example, about 0.4 M) and an aqueous ethanol solution (for example, about 20% (0.3 M)). That is, the adsorbent 3 is a preservative solution containing water and at least one additive (also referred to as "first additive" in the present specification) of a base, a salt and an alcohol for a certain period of time. It is stored in (also referred to as "first solution") in the specification. The storage step (4A step) is a step of immersing the adsorbent in the preservative solution, and corresponds to S102 in FIG.

However, when the protein is separated using the adsorption device 1 after such a certain period of time, the basic protein that binds to the phosphate site among the proteins is the separation position (elution time) of the basic protein. However, the separation position (elution time) of the acidic protein that binds to the calcium site may change. More specifically, the elution time for separating the acidic protein from the adsorbent 3 is shorter than the elution time before storage in the storage solution.

The concentration of the additive (first additive in the first solution) in the preservation solution is preferably about 0.01 M or more and about 2.0 M or less, more preferably about 0.1 M or more and about 1.0 M or less. Is. For example, when the additive is sodium hydroxide as a base, the concentration of sodium hydroxide may be about 0.1 M or more and about 1.0 M or less. When the preservation solution is a phosphate buffer solution containing a base as an additive, the concentration of the base may be about 0.1 M or more and about 0.5 M or less. Further, when the additive is alcohol, the concentration of alcohol may be about 0.2 M or more and about 0.6 M or less. By setting the concentration of sodium hydroxide in such a concentration range, both further elution of other substances (contamination proteins) and suppression of the growth of bacteria and the like can be further enhanced. Further, by setting the concentration of the phosphate buffer solution in such a concentration range, other substances can be further eluted. Furthermore, by setting the concentration of alcohol in such a concentration range, the outbreak of bacteria and the like can be further suppressed.

The additive contained in the preservation solution may be a base, a salt, or an alcohol. Of these, it is preferably a base. Since it is a base, it is particularly preferably sodium hydroxide. This makes it possible to further elute other substances (contamination proteins). Furthermore, the growth of bacteria and the like in the adsorbent 3 can be more strongly suppressed.

The stabilization method of the present invention suppresses or prevents a change in the adsorption ability (separation ability) of an acidic protein adsorbed on the calcium site of the adsorbent 3 in the adsorbent 3 after a certain period of time as described above. To do. This suppresses or prevents the elution time for separating the acidic protein from the adsorbent 3 from being shortened. The stabilizing method of the present invention has the treatment step shown in the following [1B].

[1B] Treatment Step In the treatment step, the adsorbent 3 is brought into contact with a second solution (also referred to as “treatment liquid” in the present specification) containing the second additive (preferably in the second solution). It is a process (to stand still). The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution. Step 1B (processing step) corresponds to S103 in FIG.

More specifically, the treatment liquid is supplied from the inflow pipe 24 into the column 2 with respect to the adsorbent 3 stored in the storage liquid in the column 2. Then, when the storage liquid flows out from the outflow pipe 25 and the entire inside of the column 2 is replaced with the treatment liquid, the supply of the treatment liquid from the inflow pipe 24 is stopped. By stopping the supply of the treatment liquid into the column 2 in this way, the adsorbent 3 is allowed to stand in the treatment liquid.

As described above, before the treatment by this step [1B], the elution time for separating the acidic protein from the adsorbent 3 by storage in the preservation solution is shortened, but in this step [1B], the adsorbent 3 is used. By allowing it to stand in the treatment liquid, its mechanism of action is not clear, but the separating ability of the adsorbent 3 is stabilized, and the elution time for separating the acidic protein from the adsorbent 3 becomes longer. As a result, the elution time can be restored to almost the same elution time as before storage with the preservation solution. That is, it is possible to suppress or prevent the separation position (elution time) of the acidic protein from changing before and after storage in the storage solution.

Therefore, it is possible to suppress or prevent a deviation in the elution timing of the acidic protein before and after the storage period. Therefore, since it is not necessary to reset the fraction to be recovered from the acidic protein, the acidic protein can be separated (purified) from the sample solution without requiring time and labor.

In the present specification, the stationary state of the adsorbent 3 in the treatment liquid means the supply of the treatment liquid from the inflow pipe 24 and the treatment liquid from the outflow pipe 25 in a state where the adsorbent 3 is immersed in the treatment liquid. It means the state where the discharge of is stopped. For the stationary state, for example, vibration by ultrasonic waves or vibration using a shaker may be applied.

The treatment liquid preferably has a lower molar concentration of the additive than the molar concentration of the additive in the preservation liquid. The type of the additive (first additive) contained in the preservation solution and the type of the additive (second additive) contained in the treatment solution may be the same or different. The first additive and the second additive may be one kind or any combination thereof as long as they contain at least one kind of base, salt and alcohol. The first additive and the second additive are preferably phosphate buffers containing a salt as an additive. As a result, the elution time can be restored to almost the same as before the storage with the storage solution. Further, since the replacement of the adsorbent 3 with the buffer solution in the next step [2B] described later can be omitted, the step can be simplified. As the phosphate buffer solution, the same one as in the above step [3A] can be used.

When a phosphate buffer solution is used as the treatment solution, the pH of the phosphate buffer solution is preferably about 6.0 or more and about 7.5 or less, and more preferably about 6.5. As a result, it can be restored with almost the same elution time as before storage with the preservation solution.

When the concentration of the additive in the treatment liquid is A [M] and the concentration of the additive in the preservation liquid is B [M], the A / B is preferably about 0.001 or more and about 0.1 or less. , More preferably about 0.01 or more and about 0.05 or less.

The time for allowing the adsorbent 3 to stand in the treatment liquid is preferably about 0.1 hours or more and about 24 hours or less, and more preferably about 0.5 hours or more and about 2 hours or less.

By setting the concentration of the additive in the treatment liquid and the time for allowing the adsorbent 3 to stand in the treatment liquid within the above-mentioned ranges, the elution time before storage with the storage liquid can be restored closer. ..

In the above description, the case where the adsorbent 3 is allowed to stand in the treatment liquid in the present step [1B] has been described, but in the present step [1B], the adsorbent 3 may be in contact with the treatment liquid. The adsorbent 3 can also be treated by continuing to supply the treatment liquid from the inflow pipe 24 to the inside of the column 2 and maintaining the state in which the treatment liquid is passed through the adsorbent 3. However, by selecting a method in which the adsorbent 3 is allowed to stand in the treatment liquid, wasteful treatment liquid can be reduced. When the method of passing the treatment liquid through the adsorbent 3 is selected, the flow rate of the treatment liquid is preferably about 0.1 mL / min or more and about 10 mL / min or less, and more preferably about 1 mL / min or more. It is 5 mL / min or less. Further, the time for passing the treatment liquid is preferably about 0.1 hour or more and about 24 hours or less, and more preferably about 0.5 hours or more and about 2 hours or less.

[2B] Replacement Step Next, by supplying a buffer solution from the inflow pipe 24 into the column 2, the inside of the column 2 is replaced with this buffer solution.

As a result, the adsorbent 3 is immersed in the buffer solution, and the adsorbent 1 can be prepared again so that it can be used for the separation method described above. From the viewpoint of enabling the unusable adsorbent 3 to be subjected to the separation method again, these treatments are a method for producing the adsorbent 3. After these treatments, the step of subjecting the adsorbent 3 to the separation method again corresponds to S104 of FIG.

This buffer solution may be, for example, a phosphate buffer solution. As the phosphate buffer solution, for example, the same one as in the above step [3A] can be used.

When a phosphate buffer solution is used as the treatment liquid in the treatment step [1B], this step [2B] can be omitted.

By the above operation, the change in the separating ability of the acidic protein bound to the calcium site of the adsorbent 3 is suppressed or prevented. As a result, the adsorbent 3 can be subjected to the above-mentioned separation (purification).

In the above description, the protein is used as an example of the charged substance, but the negatively charged substance may be an acidic amino acid, DNA, RNA, a negatively charged liposome, or the like in addition to the acidic protein. The positively charged substance may be a basic amino acid, positively charged cholesterol, positively charged liposome, or the like, in addition to the basic protein.

Although the stabilization method and the production method of the present invention have been described above, the present invention is not limited thereto.

In the stabilization method and the production method of the present invention, one or more steps may be added for any purpose.

Next, specific examples of the present invention will be described.
1. 1. Manufacture of Adsorber Hydroxyapatite (CHT 40 μm Type I, manufactured by BIO-RAD) as an adsorbent (filler) is used to fill the filling space of a stainless steel column (inner diameter 4.0 mm x length 100 mm). By filling, an adsorption device was manufactured.

2. Separation of proteins (acidic protein and basic protein) by an adsorbent (Example 1)
2-1. Separation of protein by adsorber before storage with preservative solution <1A> First, the content of BSA (bovine serum albumin) as an acidic protein is 10 mg / mL, and the content of α-chymotrypsinogen A as a basic protein is 5 mg. BSA and α-chymotrypsinogen A were each dissolved in 1.0 mM sodium phosphate buffer (pH 6.8) so as to be / mL to obtain a mixed solution. Then, the mixed solution was filtered through a 0.22 μm filter to obtain a sample solution.

<2A> Next, the adsorption device was attached to the chromatographic device. Then, a 10 mM sodium phosphate buffer solution (pH 6.5, temperature 25 ° C.) was passed through the adsorbent at a liquid passing rate of 1.0 mL / min to equilibrate the adsorber.

<3A> Next, 50 μL of the sample solution was supplied into the adsorber at a flow rate of 1 mL / min, and then the phosphate buffer solution (pH 6.5) was gradient to 10 mM to 400 mM (75%) in 15 minutes. The absorbance (280 nm) of the effluent (eluent) flowing out of the adsorption device was measured.

2-2. Separation of protein by adsorber after storage with preservative solution <1B> First, pass the preservative solution of 1.0 M sodium hydroxide solution (temperature 25 ° C.) through the adsorber at a liquid flow rate of 1.0 mL / min. Then, the adsorbent filled in the adsorbent was immersed in the sodium hydroxide aqueous solution (preservative solution). Then, in this state, the filler was stored for 7 days.

<2B> Next, a treatment solution of 10 mM sodium phosphate buffer (temperature 25 ° C.) is passed through the adsorbent at a liquid passing speed of 1.0 mL / min to phosphorus the adsorbent filled in the adsorbent. It was immersed in a sodium phosphate buffer solution (treatment solution). Then, in this state, the filler was stored (standing) for 1 hour.

<3B> Next, the adsorption device was attached to the chromatographic device. Then, a 10 mM sodium phosphate buffer solution (pH 6.5, temperature 25 ° C.) was passed through the adsorbent at a liquid passing rate of 1.0 mL / min to equilibrate the adsorber.

<4B> Next, 50 μL of the same sample solution as prepared in the above step <1A> was supplied into the adsorption device at a flow rate of 1 mL / min, and then a phosphate buffer solution (pH 6.5) was applied in 15 minutes. The gradient was applied so as to be 10 mM to 400 mM (75%), and the absorbance (280 nm) of the effluent (eluate) flowing out of the adsorption device was measured.

(Example 2)
In the examination of protein separation (2-2.) After storage with a preservative solution, 0.4 M phosphorus was replaced with 1.0 M sodium hydroxide solution (temperature 25 ° C.) in the step <1B>. Protein separation by an adsorbent before and after storage with the preservative solution (above 2-1 and the above) in the same manner as in Example 1 except that the adsorbent was immersed in the sodium phosphate buffer solution (temperature 25 ° C.). 2-2.) Was examined.

(Example 3)
In the examination of protein separation (2-2.) After storage with a storage solution, the pH was adjusted by substituting with 0.4 M sodium phosphate buffer (temperature 25 ° C.) after the step <1B>. After setting the pH to 6.5, further, in the step <2B>, after filling the adsorber with a 10 mM sodium phosphate buffer solution (temperature 25 ° C.; treatment solution), the flow rate of this treatment solution is 1.0 mL / min. Protein separation by an adsorbent before and after storage with the preservation solution (above 2-1) in the same manner as in Example 1 except that the treatment solution was brought into contact with the packing material by continuing the supply in 1 hour. And the above 2-2.) Were examined.

(Comparative Example 1)
Before and after storage with the preservative solution, as in Example 1 except that the step <2B> was omitted in the examination of protein separation (2-2.) After storage with the preservative solution. The separation of proteins by an adsorber (2-1. And 2-2.) Was examined.

(Comparative Example 2)
In the examination of protein separation (2-2.) After storage with a preservative solution, 0.4 M phosphorus was replaced with 1.0 M sodium hydroxide solution (temperature 25 ° C.) in the step <1B>. The protein by the adsorbent before and after storage with the preservative solution is the same as in Example 1 except that the adsorbent is immersed in the sodium phosphate buffer (temperature 25 ° C.) and the step <2B> is omitted. (2-1. And 2-2.) Were examined.

(Example 4)
After the step <1B>, in the step <2B>, the mixture was immersed in a phosphate buffer solution (treatment solution) having different concentrations of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM. Then, in this state, the test was carried out in the same manner as in Example 1 except that the filler was stored (standing) for 1 hour. The difference between the elution time after using these preservation solutions and the elution time before using the preservation solution was measured. The results are shown in FIG.

(Example 5)
After the step <1B>, in the step <2B>, the mixture is immersed in a 10 mM phosphate buffer solution (treatment solution) having pH values different from pH 6.0, pH 6.5, pH 7.0, and pH 7.5. It was. Then, in this state, the test was carried out in the same manner as in Example 1 except that the filler was stored (standing) for 1 hour. The difference between the elution time after using these preservation solutions and the elution time before using the preservation solution was measured. The results are shown in FIG.

(Comparative Example 3)
In the step <1B>, the test was carried out in the same manner as in Example 1 except that 20% ethanol was used as a preservative solution instead of the 1.0 M sodium hydroxide solution. The difference between the elution time after using 20% ethanol as the preservation solution and the elution time before using the preservation solution was measured. The results are shown in FIG. The solid line indicated by "Run-1" is the elution time before storage. The dashed line "Run-2" is the elution time after storage.

(Example 6)
In the step <1B>, the same as in Example 1 except that a 10 mM NaP solution (pH 6.5) containing 20% ethanol was used as a preservation solution instead of the 1.0 M sodium hydroxide solution and stored for 7 days. The test was conducted. The difference between the elution time after using this preservation solution and the elution time before using the preservation solution was measured. The results are shown in FIG. The solid line indicated by "Run-1" is the elution time before storage. The dashed line "Run-2" is the elution time after storage.

(Example 7)
Same as in Example 1 except that, in the step <1B>, phosphate buffer solutions having different concentrations of 5 mM, 10 mM, 15 mM, and 20 mM were used as storage solutions instead of the 1.0 M sodium hydroxide solution. Was tested. The difference between the elution time after using these preservation solutions and the elution time before using the preservation solution was measured. The results are shown in FIG.

(Example 8)
In the step <1B>, a 10 mM phosphate buffer solution containing 20% ethanol instead of the 1.0 M sodium hydroxide solution, which has a pH of 6.0, pH 6.5, pH 7.0, and pH 7.5. The test was carried out in the same manner as in Example 1 except that those having different pH values were used as the preservation solutions. The difference between the elution time after using these preservation solutions and the elution time before using the preservation solution was measured. The results are shown in FIG.

(Example 9)
In the step <1B>, 10 mM HEPES (pH 7.5 and pH 8.0) containing 20% ethanol and MES buffer solution (pH 6.5 and pH 7) containing 20% ethanol instead of the 1.0 M sodium hydroxide solution. The test was carried out in the same manner as in Example 1 except that 0.0) was used as a preservation solution. The difference between the elution time after using these preservation solutions and the elution time before using the preservation solution was measured. The results are shown in FIG.

3. 3. Evaluation In Examples 1, 2 and 3 and Comparative Examples 1 and 2, graphs showing the relationship between the elution time of the eluate and the absorbance of the eluate measured before and after storage with the preservation solution were created. , These graphs are shown in FIGS. 2, 3, 4 and 5, 6.

As is clear from FIGS. 2 and 3, in Examples 1 and 2, by preserving the adsorbent with the preservative solution and then preserving the adsorbent with the treatment solution (standing), before and after storage with the preservative solution. In, the elution time of α-chymotrypsinogen A as a basic protein and the elution time of BSA as an acidic protein were almost the same. Therefore, it was found that the separability of the adsorbents for basic protein and acidic protein did not change.

Further, as is clear from FIG. 4, also in Example 3 in which the contact with the adsorbent by the treatment liquid was replaced with the passage of the treatment liquid to the adsorbent, the adsorbent was stored (standing) in the treatment liquid. The same results as in Examples 1 and 2 were obtained.

On the other hand, in Comparative Examples 1 and 2, the storage (standing) of the adsorbent by the treatment liquid was omitted after the storage of the adsorbent by the preservative liquid. As is clear from FIGS. 5 and 6, the elution times of α-chymotrypsinogen A as a basic protein are almost the same before and after storage with the preservation solution, but the elution time of BSA, which is an acidic protein, is after storage. Was shortened in. That is, in Comparative Examples 1 and 2, the separability of the adsorbent of the acidic protein changed after the storage of the adsorbent.

In Example 4 shown in FIG. 7, the change in the adsorptive capacity of the adsorbent was the smallest when the phosphate buffer had a concentration of 10 mM. In Example 5 shown in FIG. 8, the change in the adsorptive capacity of the adsorbent was the smallest when the pH of the 10 mM phosphate buffer was 6.0 or 6.5. In Comparative Example 3 shown in FIG. 9, the adsorbent after storage in 20% ethanol for 7 days had a shorter elution time of BSA, which is an acidic protein, after storage.

In Example 6 shown in FIG. 10, the adsorbent after being stored in a 10 mM NaP solution (pH 6.5) containing 20% ethanol for 7 days had an elution time of BSA, which is an acidic protein, after storage. Has hardly changed. In Example 7 shown in FIG. 11, the adsorbent after being stored in phosphate buffers having different concentrations of 0 to 15 mM had the largest change in BSA elution time at a concentration of 10 mM. It was small.

In Example 8 shown in FIG. 12, the adsorbent after being stored in 10 mM phosphate buffer, each containing 20% ethanol and having different pH values, was of BSA when the pH was 6.5. The change in elution time was the smallest. In Example 9 shown in FIG. 13, the adsorbent after storage in 10 mM HEPES buffer, each containing 20% ethanol and having different pH values, had a pH of 7.5 or 8.0. In addition, the change in the elution time of BSA was the smallest.

According to the production method safety and the standardization method of the present invention, for example, a high-concentration sodium hydroxide solution (for example, about 1.0 M) or a phosphate buffer solution (phosphate buffer solution, (for example, about 0.4 M)). ), Etc., for an adsorbent composed of a calcium phosphate-based compound after being stored in a preservative solution such as), the ability to separate negatively charged substances bound to calcium sites of the calcium phosphate-based compound (adsorbent) should be stabilized. It becomes.

Therefore, the separation position (elution time) for separating from the adsorbent can be kept substantially constant before and after storage with the preservative solution. Therefore, by fractionating the effluent flowing out from the liquid chromatography column filled with the adsorbent by a predetermined amount, when separating the negatively charged substance into the effluent of each fractionated fraction. It is possible to accurately suppress or prevent the fraction from which the negatively charged substance is separated from being displaced before and after storage by the preservative solution. Therefore, since it is not necessary to reset the fraction to be collected, the negatively charged substance can be separated from the sample liquid without requiring time and labor.

In the present specification, the numerical values with "about" indicate that the numerical values may be different within the range where no contradiction occurs. The numerical value with "about" may be, for example, a range including an error of plus or minus 10%. This range is more preferably a range of plus or minus 5%, further preferably a range of plus or minus 1%, and most preferably a range of no error (the numerical value itself).

1 Adsorbent 2 Column 3 Adsorbent 4 Filter member 5 Filter member 20 Adsorbent filling space 21 Column body 22 Cap 23 Cap 24 Inflow pipe 25 Outflow pipe

Claims (16)

A method for producing an adsorbent, which comprises at least a part of a calcium phosphate compound and has a separable ability.
The process of preparing the adsorbent and
A first step of immersing the adsorbent in a first solution containing a first solvent and a first additive, and
A second step of bringing the adsorbent into contact with a second solution containing the second solvent and the second additive, and
Including
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
The production method, wherein the second step is performed after the first step. The production method according to claim 1, wherein the second solution contains a pH buffer. The production method according to claim 1, wherein the first additive comprises one or more selected from the group consisting of bases, salts and alcohols. The production method according to claim 1, wherein the charged substance has a negatively charged portion. The production method according to claim 1, wherein the concentration of the first additive is about 0.01 M or more and about 2.0 M or less. When the concentration of the second additive is A [M] and the concentration of the first additive is B [M], the A / B is about 0.001 or more and about 0.1 or less. The production method according to claim 1. The second step includes a standing step of allowing the adsorbent to stand in the second solution.
The production method according to claim 1, wherein the standing step is about 0.1 hour or more and about 24 hours or less. The settlement method according to claim 1, wherein the calcium phosphate-based compound contains hydroxyapatite as a main component. The first additive comprises sodium hydroxide
The production method according to claim 1, wherein the second additive comprises a phosphate buffer. The production method according to claim 1, wherein the first additive and the second additive contain a phosphate buffer. The production method according to claim 1, wherein the first additive comprises a combination of a hydroxyl group-containing compound and a pH buffer. The production method according to claim 1, wherein the first additive comprises a combination of a hydroxyl group-containing compound and a buffer having a sulfo group. A solution set containing at least a part of a calcium phosphate-based compound and used in a method for producing an adsorbent for obtaining an adsorbent having a separable ability.
The solution set includes a first solution containing a first solvent and a first additive, and a second solution containing a second solvent and a second additive.
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
A solution set in which the production method is the production method according to claim 1. The use of a solution set in the method of producing an adsorbent for obtaining an adsorbent containing at least a part of a calcium phosphate-based compound and having a separable ability.
The solution set includes a first solution containing a first solvent and a first additive, and a second solution containing a second solvent and a second additive.
The first additive has an ability to affect the adsorption force of the calcium element constituting the adsorbent to the adsorbent when added to the first solvent.
The molar concentration of the second additive in the second solution is lower than the molar concentration of the first additive in the first solution.
The production method according to claim 1, wherein the production method is used. An adsorbent produced by the production method according to claim 1. A column for liquid chromatography filled with an adsorbent produced by the production method according to claim 1.

Images