WO2021260772A1

WO2021260772A1 - Aluminum phosphate compound and method for producing same, carrier for purifying protein and method for purifying protein which uses same

Info

Publication number: WO2021260772A1
Application number: PCT/JP2020/024422
Authority: WO
Inventors: 一気鎌井; 明人板東
Original assignee: 富田製薬株式会社
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2021-12-30
Also published as: JPWO2021260772A1; JP6882816B1

Abstract

Provided are an aluminum phosphate compound exhibiting an excellent ability to purify, a method for producing the same, a carrier for purifying protein, and a method for purifying protein which uses the same.　This aluminum phosphate compound exhibits a pore mode diameter in the range of 100-300nm, said pore mode diameter corresponding to the peaks appearing when the pore diameter (D) is in the range of 2,000nm or less in a Log differential pore volume distribution (dV/dlogD) obtained by the mercury press-in method.

Description

Aluminum phosphate compound and its production method, protein purification carrier and protein purification method using it

The present invention relates to an aluminum phosphate compound and a method for producing the same, a carrier for protein purification, and a protein purification method using the same.

As a protein purification technique, purification methods such as salting out method, centrifugation method, and column chromatography method have been widely used. Among proteins, antibody proteins for pharmaceuticals and antigen proteins for vaccines require particularly advanced purification technology due to the reduction of side effects when inoculated to humans and the stricter formulation standards in recent years.

Patent Document 1 focuses on aluminum phosphate from among inorganic compounds as a carrier for purifying proteins useful for purifying proteins such as vaccines, and uses an aqueous solution further containing specific ions that are not constituent elements of aluminum phosphate. A method for producing an aluminum phosphate compound is disclosed.

Japanese Patent No. 66774428

As a result of diligent research by the present inventors, the aluminum phosphate compound obtained by using the production method disclosed in Patent Document 1 has a strong adsorption power to the protein, but it is difficult to purify the protein with a high recovery rate. It was found that further improvement of purification ability is required for use as a carrier for protein purification.

Therefore, an object of the present invention is to provide an aluminum phosphate compound having excellent purification ability and a method for producing the same, a carrier for protein purification, and a protein purification method using the same.

An embodiment of the present invention (hereinafter referred to as “the present embodiment”) is, for example, as follows.
[1] In the Log differential pore volume distribution (dV / dlogD) obtained by the mercury intrusion method, the pore mode diameter corresponding to the peak in which the pore diameter (D) appears in the range of 2000 nm or less is in the range of 100 nm to 300 nm. An aluminum phosphate compound.
[2] The aluminum phosphate compound according to [1], wherein the pore mode diameter is in the range of 100 nm to 200 nm.
[3] The aluminum phosphate compound according to [1] or [2], wherein the average particle size (D _{50) is in the range of 50 μm to 80 μm.}

[4] The aluminum phosphate compound according to [1] or [2], wherein the average particle size (D _{50) is in the range of 55 μm to 70 μm.}
[5] The aluminum phosphate compound according to any one of [1] to [4], which is amorphous.
[6] The aluminum phosphate compound according to any one of [1] to [5], which contains sulfur and potassium.
[7] The aluminum phosphate compound according to any one of [1] to [6], wherein magnesium is not contained, or if it is contained, the content thereof is less than 0.01% by mass.

[8] A carrier for purifying a protein containing the aluminum phosphate compound according to any one of [1] to [7].
[9] A protein purification carrier used in a protein purification method comprising a step of filtering a solution containing the protein purification carrier according to [8] on which a protein is adsorbed using a column, a cross-flow filter or a dead-end filter.
[10] The protein-containing solution is brought into contact with the protein purification carrier according to [8] or [9], and the protein is adsorbed on the protein purification carrier.
Separation of the protein-adsorbed carrier for protein purification by filtration using a column, a cross-flow filter or a dead-end filter, and elution of the protein from the separated protein purification carrier with an eluent. Protein purification method including.

[11] A method for producing an aluminum phosphate compound, which comprises mixing an aqueous solution A containing a phosphate ion and an aqueous solution B containing an aluminum ion, a sulfate ion and a potassium ion to obtain a mixed solution containing an aluminum phosphate compound. Therefore, the concentration of the phosphate ion in the aqueous solution A is more than 0.6 mol / L, the concentration of the aluminum ion in the aqueous solution B is more than 0.3 mol / L, and the concentration of the potassium ion is more than 0.3 mol / L. A manufacturing method in which the amount is more than 0.2 mol / L.
[12] The production method according to [11], wherein the aluminum phosphate compound is the aluminum phosphate compound according to any one of [1] to [7].
[13] Further comprising separating the aluminum phosphate compound from the mixture, washing the separated aluminum phosphate compound, and drying the washed aluminum phosphate compound using a spray dryer. , [11] or [12].

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to provide an aluminum phosphate compound having excellent purification ability and a method for producing the same, a carrier for protein purification, and a protein purification method using the same.

FIG. 1 is a graph showing the Log differential pore volume distribution of the aluminum phosphate compounds of Examples 1 and 2 and Comparative Examples 1 and 2 by the mercury intrusion method. FIG. 2 is a graph showing a powder X-ray diffraction spectrum of the aluminum phosphate compound of Example 1. FIG. 3 is a scanning electron microscope (SEM) image of the aluminum phosphate compound of Example 1. FIG. 4 is a scanning electron microscope (SEM) image of the aluminum phosphate compound of Comparative Example 1.

Hereinafter, embodiments of the present invention will be described in detail.
<First Embodiment: Aluminum Phosphate Compound>
-Pore mode diameter The aluminum phosphate compound according to this embodiment is amorphous having a specific pore distribution in which the pore mode diameter of the Log differential pore volume distribution (dV / dlogD) is in the range of 100 nm to 300 nm. It is a quality aluminum phosphate compound.

In the present embodiment, the pore mode diameter of the Log differential pore volume distribution is measured by the mercury intrusion method. Specifically, based on the measurement result by the mercury intrusion method, a distribution curve is obtained by plotting the pore diameter (D) of the sample on the horizontal axis and the Log differential pore volume (dV / dlogD) on the vertical axis. The Log differential pore volume (dV / dlogD) on the vertical axis is the logarithmic value (dlogD) of the difference in pore volume between measurement points (differential pore volume (dV)) and the difference in pore diameter (D). The value divided. In this Log differential pore volume distribution, the pore diameter corresponding to the peak top (the maximum value of dV / dlogD, which represents the maximum frequency) of the peak appearing in the pore diameter range of 2000 nm or less is the pore mode diameter. It is called.

Comparative Example 1 and Comparative Example 2 described later are retests of the methods for producing an aluminum phosphate compound described in Examples 1 and 2 of Patent Document 1 under substantially the same conditions, but the obtained aluminum phosphate was obtained. The pore mode diameters of the compounds are as small as 42.9 nm and 48.3 nm, respectively. The aluminum phosphate compound obtained by using the production method disclosed in Patent Document 1 through diligent research by the present inventors has a small pore mode diameter, so that it has a strong adsorptive power to the protein, and the protein from the carrier can be obtained. It was found that it is difficult to purify the protein with a high recovery rate because the target protein cannot be eluted with the general elution agent used for elution.

The aluminum phosphate compound according to the present embodiment has a specific pore distribution in which the pore diameter is controlled, and as described above, the pore mode diameter of the Log differential pore volume distribution (dV / dlogD) is The first feature is that it is in the range of 100 nm to 300 nm. When the pore mode diameter of the aluminum phosphate compound is in the range of 100 nm to 300 nm, the adsorptive power to the object to be purified such as protein is optimized, and both the adsorptive power to the object to be purified and the elution property to the eluent are excellent. Since the aluminum phosphate compound is obtained, the target product to be purified can be purified with a high recovery rate. The pore mode diameter of the aluminum phosphate compound is preferably 200 nm to 300 nm.

-Average particle size (D ₅₀ )
The aluminum phosphate compound according to the present embodiment has an average particle size (D ₅₀ ) of preferably 50 μm to 80 μm, and more preferably 55 μm to 70 μm. The average particle diameter (D ₅₀ ) is the particle diameter (volume median diameter) with respect to the integrated frequency of 50% by volume in the integrated distribution curve showing the relationship between the particle diameter and the integrated frequency from the small particle side. Obtained by the laser diffraction scattering method.

In the protein purification method described later using the aluminum phosphate compound according to the present embodiment as a carrier for protein purification, the aluminum phosphate compound to which the protein is adsorbed by contact with the protein in the solution is separated from other components. To be filtered. When the average particle size (D ₅₀ ) of the aluminum phosphate compound is in the above range, the filterability of the aluminum phosphate compound on which the protein is adsorbed is improved, and the productivity is also improved.

-Cumulative pore volume, average pore diameter, bulk density, apparent density, pore ratio, and skeletal density The aluminum phosphate compound according to this embodiment has better adsorptivity to objects to be purified such as proteins. From the viewpoint, the cumulative pore volume is preferably 1.5 to 3.0 mL / g, more preferably 2.0 to 2.5 mL / g, and the average pore diameter is preferably 150 to 500 nm, more preferably 250. It is about 400 nm, the bulk density is preferably 0.2 to 1.0 g / mL, more preferably 0.3 to 0.5 g / mL, and the apparent density (true density) is preferably 0.3 to 1. It is 0 g / mL, more preferably 0.5 to 0.7 g / mL, the porosity is preferably 30 to 70%, more preferably 40 to 50%, and the skeletal density is preferably 1.0 to 3. It is 0 g / mL, more preferably 1.5 to 2.5 g / mL. Cumulative pore volume, average pore diameter, bulk density, apparent density, porosity, and skeletal density can be measured by the mercury intrusion method as well as the pore mode diameter.

-Composition of Aluminum Phosphate Compound The aluminum phosphate compound according to the present embodiment is produced by a production method using an aqueous solution A containing a phosphate ion described later and an aqueous solution B containing a sulfate ion, an aluminum ion and a potassium ion. Therefore, it contains not only aluminum phosphate but also sulfur and potassium. Since the aluminum phosphate compound is composed of sulfur and potassium, its adsorptivity to a substance to be purified such as protein is improved, and it becomes a useful substance as a carrier for purification.

The content of sulfur (sulfur atom) in the aluminum phosphate compound is not particularly limited, but is preferably 0.3 to 5.0% by mass, more preferably from the viewpoint of improving protein adsorptivity. It is 1.0 to 2.0% by mass.

The content of potassium (potassium atom) in the aluminum phosphate compound is preferably 0.3 to 5.0% by mass, more preferably 0.5 to 5.0% by mass, from the viewpoint of improving protein adsorption. It is 2.0% by mass.

The aluminum phosphate compound may further contain other components. Examples of other components include water and calcium.

However, in the production method disclosed in Patent Document 1, an aqueous solution containing magnesium ions as an essential component in addition to the above ions is used as the aqueous solution B, and the obtained aluminum phosphate compound contains 0.01% by mass or more of magnesium. Although it is contained in terms of content (see claims 1, 3, 6, etc. of Patent Document 1), it is preferable that the aluminum phosphate compound according to the present embodiment does not substantially contain magnesium. The mechanism is not clear, but the presence of magnesium adversely affects the purification ability of the aluminum phosphate compound according to the present embodiment.

Here, "substantially free of magnesium" means that it does not exclude the case where magnesium is inevitably mixed as an impurity in the process of producing the aluminum phosphate compound. Specifically, the aluminum phosphate compound according to the present embodiment does not contain magnesium, or if it contains magnesium, it is preferably less than 0.01% by mass, and more preferably 0.003% by mass or less. preferable.

The content of aluminum in the aluminum phosphate compound is not particularly limited as long as it contains the above-mentioned other components, but is preferably 8 to 35% by mass from the viewpoint of improving protein adsorption. , More preferably 10 to 16% by mass.

The molar ratio of aluminum to phosphorus in the aluminum phosphate compound (Al (mol) / P (mol)) is preferably 0.3 to 1.5, more preferably 0.5 to 1.0. ..

<Second Embodiment: Method for producing aluminum phosphate compound>
The aluminum phosphate compound of the present embodiment having a pore mode diameter in the range of 100 nm to 300 nm and having a specific pore distribution is an aqueous solution A containing phosphate ion and an aqueous solution containing sulfate ion, aluminum ion and potassium ion. In the method for producing an aluminum phosphate compound including mixing with B, it can be obtained by setting the phosphate ion concentration in the aqueous solution A and the aluminum ion concentration and the potassium ion concentration in the aqueous solution B to high concentrations. can.

・ Aqueous solution A
Aqueous solution A contains phosphate ions. The aqueous solution A can be prepared as an aqueous solution in which the phosphoric acid-containing compound is dissolved. Examples of the phosphoric acid-containing compound include phosphoric acid, phosphoric acid-free salt, phosphoric acid monohydrogen salt, and phosphoric acid dihydrogen salt.

Phosphoric acid is orthophosphoric acid. Phosphate-free salts are phosphates that are not hydrogen salts, such as ammonium phosphate ((NH ₄ ) ₃ PO ₄ ), trisodium phosphate (Na ₃ PO ₄ ), and tripotassium phosphate (K ₃ PO _4). ), Hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) and the like. Dicalcium phosphate is a phosphate of monohydrogen salt, diammonium hydrogen phosphate ((NH ₄ ) ₂ HPO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), dipotassium hydrogen phosphate ((NH 4) 2 HPO 4). Examples thereof include K ₂ HPO ₄ ) and calcium monohydrogen phosphate (CaHPO ₄ ). Dihydrogen phosphate is a phosphate dihydrogen, ammonium dihydrogen phosphate _{_{_{(NH 4 H 2 PO 4)}}} , sodium dihydrogen phosphate _(NaH 2 _PO 4), potassium dihydrogen phosphate (KH ₂ PO ₄ ) and the like can be mentioned.

The phosphoric acid-containing compound may be anhydrous or a hydrate. Further, the hydration number of the hydrate is not particularly limited. These phosphoric acid-containing compounds may be used alone or in combination of two or more.

Among the above-mentioned phosphoric acid-containing compounds, monohydrogen phosphate is preferably mentioned, and disodium hydrogen phosphate (Na ₂ HPO) is more preferable, from the viewpoint of improving the protein adsorptivity of the aluminum phosphate compound. ₄ ) can be mentioned.

The phosphate ion concentration in the aqueous solution A is 0.6 mol / mol / mol as the total amount of the phosphate ions constituting the above-mentioned phosphate-containing compound used in the preparation of the aqueous solution A from the viewpoint of controlling the pore mode diameter within the above range. It is more than L, preferably 0.7 mol / L or more, and more preferably 0.8 mol / L or more. The upper limit of the phosphate ion concentration in the aqueous solution A may be, for example, 1.5 mol / L or less as the total amount of phosphate ions.

The liquid property of the aqueous solution A is not particularly limited, but can be prepared, for example, as a basic solution. The aqueous solution A as a basic solution may be prepared by dissolving the above-mentioned phosphoric acid-containing compound in water, or by dissolving the above-mentioned phosphoric acid compound in water, sodium hydroxide, potassium hydroxide, and water. It may be prepared by adjusting the pH using one or more kinds of metal hydroxides such as calcium oxide, or the above-mentioned phosphoric acid compound is dissolved in a pre-prepared basic aqueous solution of the above-mentioned metal hydroxide. It may be prepared by letting it.

The specific pH of the aqueous solution A (23 ° C., the same applies hereinafter) is preferably such that the pH of the mixture obtained by mixing with the aqueous solution B described later is 2.0 to 4.0. It can be determined in consideration of pH. Specifically, the pH of the aqueous solution A may be, for example, 7.5 to 10.5 and may be 8.5 to 9.5.

The method for dissolving the phosphoric acid-containing compound is not particularly limited, and examples thereof include stirring, shaking, and ultrasonic dissolution. Further, the temperature and pressure conditions for dissolving the phosphoric acid-containing compound are not particularly limited, and examples thereof include conditions such as normal temperature and normal pressure and warming and normal pressure.

The aqueous solution A is preferably a prepared solution filtered through a filter. The pore diameter of the filter used may be, for example, 0.2 to 3 μm, or 0.45 to 1 μm.

・ Aqueous solution B
Aqueous solution B contains aluminum ions, sulfate ions, and potassium ions. As described above, by using an aqueous solution containing potassium ion which is not a constituent element of aluminum phosphate, the obtained aluminum phosphate compound can be obtained as a useful substance as a carrier for protein purification which has an excellent adsorptive action on proteins. can.

Aqueous solution B can be prepared as an aqueous solution in which one or more salts containing aluminum ion, sulfate ion and potassium ion are dissolved. The salt may be a single salt or a double salt.

As the salt for preparing the aqueous solution B, a plurality of types of salts containing aluminum ions, sulfate ions and potassium ions alone may be combined, or salts containing a plurality of types of ions may be used alone or in combination of a plurality of types. May be good. When two or more salts for preparing the aqueous solution B are used, it is preferable that each salt has a sulfate ion as an anion from the viewpoint of obtaining better protein adsorption of the aluminum phosphate compound. ..

Examples of the salt containing aluminum ions include aluminum hydroxide (Al (OH) ₃ ), aluminum oxide (Al ₂ O ₃ ), aluminum sulfate (Al ₂ (SO ₄ ) ₃ ), aluminum chloride (AlCl ₃ ), and nitrate. Examples thereof include aluminum (Al (NO ₃ ) ₃ ), potassium aluminum sulfate (AlK (SO ₄ ) ₂ ), and the like, preferably aluminum sulfate (Al ₂ (SO ₄ ) ₃ ), potassium aluminum sulfate (AlK (SO ₄ )). ₂ ) is mentioned, and more preferably, potassium aluminum sulfate (AlK (SO ₄ ) ₂ ) is mentioned.

Examples of the salt containing sulfate ion include potassium sulfate and the like in addition to the _{above-mentioned aluminum sulfate (Al 2} (SO ₄ ) ₃ ) and potassium aluminum sulfate (AlK (SO ₄ ) _{2), and more preferably, sulfate.} Potassium aluminum (AlK (SO ₄ ) ₂ ) can be mentioned.

Examples of the salt containing potassium ion include potassium hydroxide (KOH), potassium chloride (KCl), potassium nitrate (KNO ₃ ) and the like in addition to the above-mentioned potassium aluminum sulfate (AlK (SO ₄ ) _{2) and potassium sulfate.} Preferred are potassium aluminum sulfate (AlK (SO ₄ ) ₂ ), potassium sulfate, and more preferably potassium aluminum sulfate (AlK (SO ₄ ) ₂ ).

The above-mentioned salt may be anhydrous or a hydrate. Further, the hydration number of the hydrate is not particularly limited.

The aluminum ion concentration in the aqueous solution B is more than 0.3 mol / L as the total amount of aluminum ions constituting the above-mentioned salt used in the preparation of the aqueous solution B from the viewpoint of controlling the pore mode diameter within the above range. It is preferably 0.4 mol / L or more, and more preferably 0.5 mol / L or more. The upper limit of the aluminum ion concentration in the aqueous solution B may be, for example, 2.4 mol / L or less as the total amount of aluminum ions.

The amount of aluminum ions in the aqueous solution B is set as the lower limit of the total amount of aluminum ions constituting the above-mentioned salt used in the preparation of the aqueous solution B from the viewpoint of improving the protein adsorptivity of the aluminum phosphate compound. , 0.1 mol or more, more preferably 0.2 mol or more, per 1 mol of the phosphate ion constituting the phosphoric acid-containing compound used in the preparation of the aqueous solution A. As an upper limit, it is preferably 0.5 mol or less, more preferably 0.3 mol or less, per 1 mol of the phosphate ion constituting the phosphoric acid-containing compound used in the preparation of the aqueous solution A.

The potassium ion concentration in the aqueous solution B is more than 0.2 mol / L as the lower limit of the total amount of potassium ions constituting the above-mentioned salt used in the preparation of the aqueous solution B from the viewpoint of controlling the pore mode diameter within the above range. Yes, preferably 0.3 mol / L or more, and more preferably 0.5 mol / L or more. The upper limit of the potassium ion concentration in the aqueous solution B may be, for example, 2.4 mol / L or less as the total amount of potassium ions.

Regarding the concentration of sulfate ion in the aqueous solution B, from the viewpoint of controlling the pore mode diameter within the above range, and / or from the viewpoint of reacting with the aqueous solution A to efficiently obtain an aluminum phosphate compound, and / or phosphoric acid. From the viewpoint of obtaining better protein adsorption of the aluminum compound, the lower limit of the total amount of sulfate ions constituting the above-mentioned salt used in the preparation of the aqueous solution B is, for example, 1.0 mol / L or more, preferably 1. It is 0.3 mol / L or more, and the upper limit is, for example, 4.8 mol / L or less, preferably 3.0 mol / L or less, and more preferably 2.0 mol / L or less.

The liquid property of the aqueous solution B is not particularly limited, but can be prepared, for example, as an acidic solution. The aqueous solution B as an acidic solution may be prepared by dissolving the above-mentioned salt in water, or the above-mentioned salt may be dissolved in water and one of mineral acids such as hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid. Alternatively, it may be prepared by adjusting the pH using a plurality of types, or it may be prepared by dissolving the above-mentioned salt in an acidic aqueous solution of the above-mentioned mineral acid prepared in advance. The specific pH of the aqueous solution B is preferably determined in consideration of the pH of the aqueous solution A so that the pH of the mixture obtained by mixing with the above-mentioned aqueous solution A is 2.0 to 4.0. Can be done. Specifically, the pH of the aqueous solution B may be, for example, 1.5 to 4.0, and may be 2.0 to 3.5.

The method for dissolving the above-mentioned salt is not particularly limited, and examples thereof include stirring, shaking, and ultrasonic dissolution. Further, the temperature and pressure conditions for dissolving the above-mentioned salt are not particularly limited, and examples thereof include conditions such as normal temperature and normal pressure and warming and normal pressure.

The aqueous solution B is preferably a prepared solution filtered through a filter. The pore diameter of the filter used may be, for example, 0.2 to 3 μm, or 0.4 to 1 μm.

-Mixing Aqueous solution A and aqueous solution B produce an aluminum phosphate compound by mixing. From the viewpoint of improving the protein adsorptivity of the aluminum phosphate compound, it can be preferably mixed by adding the aqueous solution B to the aqueous solution A. The method of addition is not particularly limited, and examples thereof include batch addition, drop addition, partial addition, and any combination thereof. However, from the viewpoint of improving the protein adsorptivity of the aluminum phosphate compound, it is preferable. Examples thereof include dripping addition, divided addition, and combinations thereof, and more preferably dripping addition.

The pH of the obtained mixture of the aqueous solution A and the aqueous solution B is not particularly limited, but is preferably 2.0 to 4.0 from the viewpoint of improving the protein adsorptivity of the aluminum phosphate compound. By mixing the aqueous solution A and the aqueous solution B, the aluminum phosphate compound can be obtained as a precipitate or a gel.

-Post-treatment The produced aluminum phosphate compound is subjected to post-treatment such as separation from the mixture, washing and drying. The method of separation from the mixture, washing, and drying is not particularly limited. Examples of the method of separation from the mixture include filtration and centrifugation. The separated aluminum phosphate compound can be obtained in the form of a precipitate or a gel. Examples of the washing method include washing with water, suspension in an aqueous solution, and centrifugation. Such a cleaning operation can be performed once or a plurality of times. The washed aluminum phosphate compound can be obtained in the form of a precipitate or a gel. Examples of the drying method include a method using a spray dryer and a shelf-type dryer. Among them, according to the spray dryer, the aluminum phosphate compound _{can be granulated into a substantially spherical shape having an average particle diameter (D 50} ) of 50 μm to 80 μm, which is preferable because the filtering ability described later is improved.

The aluminum phosphate compound according to this embodiment can be suitably used in a wide range of fields as a material such as a solid catalyst, a catalyst carrier, an adsorbent, and a separating material.

<Third Embodiment: Carrier for protein purification>
The aluminum phosphate compound according to this embodiment is suitably used as a carrier for protein purification. As described above, the aluminum phosphate compound according to the present embodiment has an optimized adsorption power to the protein and is excellent in both the adsorption power to the protein and the elution property to the eluent. Therefore, the target protein should be purified with a high recovery rate. Can be done.

The aluminum phosphate compound according to the present embodiment is, for example, a protein synthesized by a cell-free system and / or a protein expressed using bacteria, yeast, algae, insect cells, mammalian cells, cultured animal cells and the like as hosts. It can be used as a carrier for purification.

The type of protein is not particularly limited, and examples thereof include antibody proteins for pharmaceuticals and antigen proteins for vaccines.
Examples of the antibody protein for pharmaceuticals include antitumor antibodies (eg, trussumab, beltzumab, rituximab, ofatsumumab, setuximab, vanitummab, alemtuzumab, gemtuzumab, ibritsumomab, brentuximab), immunomodulatory antibodies (eg, infliximab, adalimumab, adalimumab). Sertrizumab, tosirizumab, omalizumab, ecrizumab, basiliximab, natalyzumab, mogamurizumab, nibolumab), anti-interleukin antibodies (eg, ustequinumab, canaquinumab, sekkinumab), anti-cardiovascular regulatory antibodies (eg, bebasimab) , Denosumab), anti-viral antibody (eg, basiliximab) and the like.

Examples of the antigen protein for vaccine include those for the following vaccines. Specifically, influenza vaccine, cholera vaccine, mad dog disease vaccine, diphtheria vaccine, meningitis vaccine, tick-mediated encephalitis vaccine, carbon bacillus vaccine, intestinal typhoid vaccine, pneumonia vaccine, tetanus vaccine, human papillomavirus vaccine, pertussis vaccine, Examples thereof include a polio vaccine, a hepatitis A virus vaccine, a hepatitis B virus vaccine, and a corona virus vaccine.

Other proteins used in pharmaceuticals include, for example, albumin, antithrombin, insulins, interferons, interleukins, erythropoetins, granulocyte colony stimulating factors, granulocyte macrophage colony stimulating factors, glucagon, blood coagulation factor VII, etc. Examples thereof include blood coagulation factor VIII, blood coagulation factor IX, growth factors, thrombin, thrombomodulin, uric acid degrading enzyme, fibrinogen, fibrin, parathyroid hormone, follicular stimulating hormone, lysosome enzyme, leptin, DNA degrading enzyme and the like.

Examples of proteins used for other research purposes include CBD tags, CBP tags, FLAG tags, GFP tags, GST tags, HA tags, His tags, MBP tags, myc tags, RFP tags, and Thioredoxin tags.

<Fourth Embodiment: Protein purification method>
As described above, the aluminum phosphate compound according to the present embodiment can be suitably used as a carrier for protein purification. As a method for purifying a protein using the carrier for purifying a protein according to this embodiment, a known method can be used.

The protein purification method using the protein purification carrier according to the present embodiment is, in one embodiment, contacting a solution containing a protein with the protein purification carrier to adsorb the protein to the protein purification carrier. Separation of the protein purification carrier adsorbed by the protein by filtration using a column, a cross-flow filter or a dead-end filter, and elution of the protein from the separated protein purification carrier with an eluent. including.

Since the carrier for protein purification according to the present embodiment has an optimized adsorption power for proteins and is excellent not only in adsorption power but also in elution property for eluents, a protein that is widely used as an eluent is used in the protein purification method. be able to. Further, since a commercially available column, cross-flow filter or dead-end filter can be used in the filtration step included in the protein purification method, the protein purification method using the protein purification carrier according to the present embodiment is simple. Has excellent protein purification ability.

<Manufacturing of aluminum phosphate compound>
Example 1
Purified water is added to 56.4 kg of disodium hydrogen phosphate dodecahydrate, stirred, heated to 60 ° C. to dissolve, and a total of 135 L of aqueous solution (phosphate ion concentration: 1.2 mol / L) is prepared. did. This aqueous solution was filtered through a filter having a pore size of 1 μm to obtain a disodium hydrogen phosphate aqueous solution (aqueous solution A).

Next, purified water was added to 44.8 kg of potassium aluminum sulfate dodecahydrate, stirred, heated to 60 ° C. to dissolve, and a total of 123 L of aqueous solution (aluminum ion concentration: 0.77 mol / L, sulfate ion concentration). : 1.5 mol / L, potassium ion concentration: 0.77 mol / L) was prepared. This liquid was filtered through a filter having a pore size of 1 μm to obtain an aqueous potassium alum sulfate solution (aqueous solution B).

While maintaining the disodium hydrogen phosphate aqueous solution (aqueous solution A) at 60 ° C. and stirring at 150 rpm, the potassium aluminum sulfate aqueous solution (aqueous solution B) was added dropwise over 1 hour. The resulting mixture was filtered through a filter press. Further, the filtered cake was washed with purified water until the filtrate conductivity became 5 mS / cm.

The obtained cake is suspended so as to have a solid content of 10% by mass, and dried and granulated with a spray dryer (OOLD-16 manufactured by Okawara Kakoki) under the conditions of an inlet temperature of 250 ° C., an outlet temperature of 100 ° C., and an atomizer rotation speed of 11000 rpm. Then, the aluminum phosphate compound a was obtained.

Example 2
The phosphate ion concentration in the disodium hydrogen phosphate aqueous solution (aqueous solution A) was changed (phosphate ion concentration: 0.82 mol / L) with respect to the production method shown in Example 1, and the potassium aluminum sulfate aqueous solution (aqueous solution B) was changed. ), Other than changing the aluminum ion concentration, sulfate ion concentration, and potassium ion concentration (aluminum ion concentration: 0.52 mol / L, sulfate ion concentration: 1.0 mol / L, potassium ion concentration: 0.52 mol / L) Obtained an aluminum phosphate compound b in the same manner as in Example 1.

Comparative Example 1
Water was added to 35.9 g of disodium hydrogen phosphate dodecahydrate and dissolved by stirring to prepare a 1000 mL aqueous solution (phosphate ion concentration: 0.25 mol / L) in total. This aqueous solution was filtered through a filter having a pore size of 0.22 μm to obtain a disodium hydrogen phosphate aqueous solution (aqueous solution A).

Next, water was added to 28.4 g of potassium aluminum sulfate dodecahydrate and 9.86 g of magnesium sulfate heptahydrate and stirred to dissolve them, and a total of 1000 mL of an aqueous solution (aluminum ion concentration: 0.06 mol / L, sulfuric acid) was added. Ion concentration: 0.2 mol / L, potassium ion concentration: 0.06 mol / L, magnesium ion concentration: 0.08 mol / L) were prepared. This aqueous solution was filtered through a filter having a pore size of 0.22 μm to obtain an aqueous solution (aqueous solution B) having the above composition.

The aqueous solution of disodium hydrogen phosphate (aqueous solution A) having the above composition was added dropwise over 1 hour while stirring at 700 rpm. The resulting mixture was centrifuged at 2070 xg for 15 minutes and a gelled precipitate was collected. The recovered gel-like substance was put into 0.9 mass / v% physiological saline, suspended using an ultrasonic generator, and washed. Then, centrifugation was performed at 2070 × g for 15 minutes, and the gel-like substance was recovered. The gel was suspended in physiological saline and centrifuged again under the same conditions. The obtained gel was spread on a tray and dried at 60 ° C. overnight. As a result, a dry gel which is a white crystal was obtained. The dried gel was manually crushed and pulverized to obtain aluminum phosphate compound c.

Comparative Example 2
Water was added to 35.9 g of disodium hydrogen phosphate dodecahydrate and dissolved by stirring to prepare a 1000 mL aqueous solution (phosphate ion concentration: 0.25 mol / L) in total. This aqueous solution was filtered through a filter having a pore size of 0.22 μm to obtain a disodium hydrogen phosphate aqueous solution (aqueous solution A).
Next, water was added to 28.4 g of potassium aluminum sulfate dodecahydrate and 9.86 g of magnesium sulfate heptahydrate and stirred to dissolve them, and a total of 1000 mL of an aqueous solution (aluminum ion concentration: 0.06 mol / L, sulfuric acid) was added. Ion concentration: 0.2 mol / L, potassium ion concentration: 0.06 mol / L, magnesium ion concentration: 0.08 mol / L) were prepared. This aqueous solution was filtered through a filter having a pore size of 0.22 μm to obtain an aqueous solution (aqueous solution B) having the above composition.

While stirring the disodium hydrogen phosphate aqueous solution (aqueous solution A) at 200 rpm, the aqueous solution (aqueous solution B) having the above composition was added in about 2 seconds. The resulting mixture was centrifuged at 2070 xg for 15 minutes and a gelled precipitate was collected. The recovered gel-like substance is put into 0.9 mass / v% physiological saline, suspended and washed using an ultrasonic generator, and then centrifuged at 2070 × g for 15 minutes to carry out the gel-like substance. Was recovered. The gel was suspended in physiological saline and centrifuged again under the same conditions. The obtained gel was spread on a tray and dried at 60 ° C. overnight. As a result, a dry gel which is a white crystal was obtained. The dried gel was manually crushed and pulverized to obtain aluminum phosphate compound d.

In addition, Comparative Examples 1 and 2 described above are retests of the methods for producing an aluminum phosphate compound described in Examples 1 and 2 of Patent Document 1 (Japanese Patent No. 66774428) under substantially the same conditions. be.

The physical properties of the aluminum phosphate compounds a to d obtained in Examples 1 and 2 and Comparative Examples 1 and 2 were measured by the methods shown below, and the performance was evaluated. The appearance of Example 1 and Comparative Example 1 was also observed.

<Pore characteristics by mercury injection method>
The aluminum phosphate compounds a to d obtained in Examples 1 and 2 and Comparative Examples 1 and 2 were used as measurement samples of pore characteristics by the mercury intrusion method. The pore distribution of each sample was measured in the range of pore diameter of about 3.6 nm to 200,000 nm by the mercury intrusion method, and the pore mode diameter, cumulative pore volume, average pore diameter, bulk density, apparent density, and porosity were measured. , And skeletal density were obtained. A Polemaster 60GT (manufactured by Quantachrome) was used as an apparatus.

[Pore mode diameter]
Accurately weigh 0.1 to 0.3 g of each sample, enclose it in a measurement cell, set the contact angle of mercury to 140 °, and set the surface tension of mercury to 480 dyn / cm, and the measurement results of each sample obtained by the mercury intrusion method. FIG. 1 shows a Log differential pore volume distribution plotted with the horizontal axis as the pore diameter (D) (nm) and the vertical axis as the Log differential pore volume distribution (dV / dragD) (cc / g). In the Log differential pore volume distribution shown in FIG. 1, the mode diameter corresponding to the peak top (maximum value of dV / dlogD) of the first peak appearing in the range of the pore diameter (D) of 3.6 nm to 2000 nm or less is set. , Determined as the pore mode diameter of the aluminum phosphate compound. The results are shown in Table 2 below.

In the fine Log differential pore volume distribution (dV / dlogD) shown in FIG. 1, the mode diameter corresponding to the peak top of the second peak in which the pore diameter D appears in the range of more than 2000 nm to 200,000 nm is the gap between particles. The void mode diameter of.

Table 2 below shows the measurement results of the pore mode diameter, cumulative pore volume, average pore diameter, bulk density, apparent density, porosity and skeletal density of each aluminum phosphate compound.

<Average particle size (D ₅₀ )>
Each aluminum phosphate compound was used as a measurement sample of the average particle size (D _{50) by the laser diffraction method.} Each sample was put into a laser diffraction type particle size distribution meter (MT3300EXII: manufactured by Microtrac) so as to have a DV value of 0.1 to 0.6, circulated at a flow rate of 32.5 mL / sec for 10 seconds, and then measured for 20 seconds. sec as measured number two conditions, was measured in purified water, the average of two measured D ₅₀ value was calculated as the average particle size. The measurement results of the average particle size (D ₅₀ ) are shown in Table 2 below.

<Powder X-ray diffraction (XRD)>
The aluminum phosphate compound a of Example 1 was pulverized in an agate mortar, filled in a sample cell (made of glass), and the powder X-ray diffraction spectrum was measured under the following measurement conditions. A graph showing the obtained powder X-ray diffraction spectrum is shown in FIG. From FIG. 2, the aluminum phosphate compound a is determined to be amorphous.

[Measurement condition]
X-ray tube: CuKα
Optical system: Centralized method Tube voltage / tube current: 40kV-30mA
Scan range: 5-90deg
Scan step: 0.02 deg
Scan speed: 40 deg / min Detector: 1-dimensional semiconductor detector Measuring device: X-ray diffraction analyzer SmartLab (manufactured by Rigaku)

<Electron microscope observation>
The appearance of the aluminum phosphate compound a of Example 1 and the aluminum phosphate c of Comparative Example 1 was observed by a field emission scanning electron microscope (FE-SEM) method. Each sample was collected, and a 1000x field of view was observed and photographed by FE-SEM. As an apparatus, FE-SEM SU8220 (manufactured by Hitachi High-Tech) was used. The SEM photograph of the aluminum phosphate compound a of Example 1 is shown in FIG. 3, and the SEM photograph of the aluminum phosphate compound c of Comparative Example 1 is shown in FIG.

As shown in FIG. 3, the particle shape of the aluminum phosphate compound a of Example 1 is substantially spherical, whereas as shown in FIG. 4, the particles of the aluminum phosphate compound c of Comparative Example 1 which is a pulverized product. It was confirmed that the shape was irregular (non-spherical).

<Elemental analysis>
Elemental analysis was performed on the aluminum phosphate compound a of Example 1, the aluminum phosphate c of Comparative Example 1 and the aluminum phosphate d of Comparative Example 2 by the acid decomposition / ICP-OES method. SPECTRO ARCOS (manufactured by SPECTRO Analytical Instruments) was used for the device. The measurement results are shown in Table 1.

Test Example 1 [Evaluation of purification ability of GFP protein]
After expressing GFP (Green Fluorescent protein) in Escherichia coli (E. coli HB101 K-12 strain: manufactured by BIO-RAD), suspend it in BactYast Lysis Buffer (manufactured by ATTO) to dissolve the cells, and centrifuge. Qing was recovered. As a result, a crude protein solution was obtained. 50 μL of this crude protein solution was mixed with 6.5 mg of each aluminum phosphate compound prepared in Examples 1 and 2 and Comparative Examples 1 and 2 as a carrier, and the protein was adsorbed.

The protein solution in which each carrier was suspended was separated into a carrier and a supernatant by centrifugation at 2000 × g for 5 minutes, and the supernatant was removed. Each carrier was washed 3 times with 50 μL of a washing solution (BactYast Lysis Buffer). Then, each carrier was washed twice with 50 μL of an eluate (BactYeast Lysis Buffer to which 80 mM EDTA-2NA was added), and the solution was collected and used as an eluate fraction. The protein concentration of the crude protein solution and the recovered eluted fraction was quantified by the BCA method. Further, the crude protein solution and the recovered eluted fraction were mixed with the same amount of 2 × sample buffer and boiled at 95 ° C. for 3 minutes. This solution was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As the gel, a gel having a uniform concentration of acrylamide (15% gel) was used, and 4 μL of a crude protein solution and 8 μL of an elution fraction were applied.

The obtained gel was stained with a CBB staining solution and then decolorized, the GFP concentration in each lane was calculated by densitometry, and the GFP recovery rate was calculated by the following formula. The results are shown in Table 2.
GFP recovery rate (%) = [GFP concentration of eluted fraction x 2 / GFP concentration of crude protein solution] x 100

Test Example 2 [Evaluation of filterability]
5 g of each aluminum phosphate compound prepared in Examples 1 and 2 was suspended in 500 mL of water. To set the membrane filter (Mixed Cellulose Ester φ47 mm pore diameter 0.2 μm manufactured by Advantec) in the filter holder and suction filter the suspension using a suction filtration bell and an aspirator (PSA152AB manufactured by Advantec) to filter the entire amount. I recorded the time it took. The results are shown in Table 2.

From Table 2, for example, Examples 1 and 2 having a pore mode diameter in the range of 100 nm to 300 nm have a higher GPF recovery rate (%) than Comparative Examples 1 and 2 having a pore mode diameter of less than 100 nm. It turns out to be expensive.

The present invention is not limited to the above embodiment, and can be variously modified at the implementation stage without departing from the gist thereof. In addition, each embodiment may be carried out in combination as appropriate, in which case the combined effect can be obtained. Further, the above-described embodiment includes various inventions, and various inventions can be extracted by a combination selected from a plurality of disclosed constituent requirements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, if the problem can be solved and the effect is obtained, the configuration in which the constituent elements are deleted can be extracted as an invention.

Claims

Phosphorus having a pore mode diameter in the range of 100 nm to 300 nm corresponding to a peak in which the pore diameter (D) appears in the range of 2000 nm or less in the Log differential pore volume distribution (dV / dlogD) obtained by the mercury intrusion method. Aluminum compound.
The aluminum phosphate compound according to claim 1, wherein the pore mode diameter is in the range of 100 nm to 200 nm.
The aluminum phosphate compound according to claim 1 or 2, wherein the average particle size (D 50) is in the range of 50 μm to 80 μm.
The aluminum phosphate compound according to claim 1 or 2, wherein the average particle size (D 50) is in the range of 55 μm to 70 μm.
The aluminum phosphate compound according to any one of claims 1 to 4, which is amorphous.
A carrier for protein purification containing the aluminum phosphate compound according to any one of claims 1 to 5.
A protein purification carrier used in a protein purification method comprising a step of filtering a solution containing the protein purification carrier according to claim 6 on which a protein is adsorbed using a column, a cross-flow filter or a dead-end filter.
Contacting the protein-containing solution with the protein purification carrier according to claim 6 or 7, and adsorbing the protein on the protein purification carrier.
Separation of the protein-adsorbed carrier for protein purification by filtration using a column, a cross-flow filter or a dead-end filter, and elution of the protein from the separated protein purification carrier with an eluent. Protein purification method including.
A method for producing an aluminum phosphate compound, which comprises mixing an aqueous solution A containing a phosphate ion and an aqueous solution B containing an aluminum ion, a sulfate ion and a potassium ion to obtain a mixed solution containing an aluminum phosphate compound. The concentration of the phosphate ion in the aqueous solution A is more than 0.6 mol / L, the concentration of the aluminum ion in the aqueous solution B is more than 0.3 mol / L, and the concentration of the potassium ion is 0.2 mol. A manufacturing method that exceeds / L.
The production method according to claim 9, wherein the aluminum phosphate compound is the aluminum phosphate compound according to any one of claims 1 to 5.