JP2018145437A - Cleaning composition, protein purification method and protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cleaning composition used in a process for separation purifying a target protein and excellent in impurity removal performance.SOLUTION: There is provided a cleaning composition used in a process for separation purifying a target protein from a mixture containing the target protein and impurities, and containing an amide-based solvent.SELECTED DRAWING: None

Description

  The present invention relates to a cleaning composition, a protein purification method, and a protein.

  Antibodies are an important component of the immune system of many animals, particularly the human. Recent advances in recombination technology have made it possible to produce antibodies against cancer cells, bacteria, and viruses. For example, antibodies are produced using cell lines that are designed to be expressed at high levels. The engineered cell lines are also cultured in cultures containing various proteins including saccharides, amino acids, complex mixtures of growth factors, serum proteins and the like.

  In order to separate proteins such as antibodies from cell by-products and culture components and use them for research purposes or as therapeutic / diagnostic agents, it is necessary to increase the purity to a sufficient level. Purification of antibody molecules is particularly important when the antibody is to be used as a drug for human administration. As conventional antibody purification methods, methods described in Patent Documents 1 to 4 are known.

US Pat. No. 6870034 Japanese Patent No. 4031049 Japanese Patent No. 4460302 Japanese Patent No. 5150488

  The subject of this invention is providing the composition for washing | cleaning excellent in the impurity removal performance used for the process of isolate | separating and purifying a target protein.

The above problems have been solved by the following means.
<1> A cleaning composition used in a process for separating and purifying a target protein from a mixed solution containing the target protein and impurities, and one type selected from the following components (A), (B) and (C) Or the composition for washing | cleaning characterized by including 2 or more types.
(A) A cationic surfactant selected from a quaternary ammonium salt type cationic surfactant having an organic group having 5 or more carbon atoms and an amine salt type cationic surfactant having an organic group having 5 or more carbon atoms. (B) Hydroxy acid ester (C) Amide solvent

<2> a binding step in which a ligand immobilized on a solid phase is brought into contact with a mixed solution containing the target protein and impurities, and the ligand and the protein in the mixed solution are bound;
The washing composition of <1> above, washing the solid phase on which the protein-bound ligand is immobilized to remove impurities, and using the elution buffer from the solid phase washed in the washing step A recovery process for recovering the target protein;
A protein purification method comprising:

  <3> A protein of interest purified by the purification method of <2> above.

The cleaning composition of the present invention exhibits excellent impurity removal performance when used in a process for separating and purifying a target protein.
Therefore, according to the protein purification method of the present invention, impurities can be efficiently removed. In addition, the target protein purified by the purification method has few impurities.

  Hereinafter, the present invention will be described in detail. In addition, the notation such as “a to b” representing a numerical range and the like includes “a” and “b” in the numerical range unless otherwise specified, and has the same meaning as “a or more and b or less”.

[Cleaning composition]
The cleaning composition of the present invention is a cleaning composition used in a process for separating and purifying a target protein from a mixed solution containing the target protein and impurities, and includes the following components (A), (B) and (C) 1) or 2 or more types selected from the above.
(A) A cationic surfactant selected from a quaternary ammonium salt type cationic surfactant having an organic group having 5 or more carbon atoms and an amine salt type cationic surfactant having an organic group having 5 or more carbon atoms. (B) Hydroxy acid ester (C) Amide solvent

(Component (A): Cationic surfactant)
The cationic surfactant of component (A) is a quaternary ammonium salt type cationic surfactant having an organic group having 5 or more carbon atoms, and an amine salt type cationic surfactant having an organic group having 5 or more carbon atoms. It is selected from agents. The amine salt type cationic surfactant may be any of primary to tertiary amine salt types.

  As the cationic surfactant, from the viewpoint of bactericidal properties, storage stability, impurity removal performance, and efficient recovery of the target protein, a quaternary ammonium salt type cationic having an organic group having 5 or more carbon atoms. A surfactant is preferred, and a quaternary ammonium salt type cationic surfactant represented by the following formula (1) (hereinafter also referred to as a cationic surfactant (1)) is more preferred.

[In Formula (1),
R ¹ represents an organic group having 5 or more carbon atoms,
R ² , R ³ and R ⁴ each independently represents an organic group,
n represents an integer of 1 or more,
Z ⁿ⁻ represents an n-valent anion. ]

In formula (1), R ¹ represents an organic group having 5 or more carbon atoms. Here, when the organic group represented by R ¹ is an organic group having 2 or more carbon atoms, the carbon number means an average carbon number. The average carbon number can be calculated by calculating the total number of carbon atoms of R ¹ of all molecules corresponding to the cationic surfactant (1) and converting the number of carbon atoms per molecule (R ¹ of all molecules). Total number of carbons / number of molecules).

Examples of the organic group represented by R ¹ include a hydrocarbon group having 5 to 30 carbon atoms, a hydroxyalkyl group having 5 to 30 carbon atoms, and a total carbon in which some of hydrogen atoms of the hydroxyalkyl group are substituted with hydrocarbon groups. A group having a number of 5 to 30, an average addition mole number of 10 or less and a polyoxyalkylene group having 5 or more carbon atoms, and a part of hydrogen atoms of the polyoxyalkylene group having an average addition mole number of 10 or less are hydrocarbon groups. A substituted group having 5 to 30 total carbon atoms is preferred.

The carbon number of the hydrocarbon group having 5 to 30 carbon atoms in R ¹ is preferably from the viewpoint of impurity removal performance and from the viewpoint of facilitating reuse of the solid phase washed with the cleaning composition of the present invention. It is 6-24, More preferably, it is 8-22, More preferably, it is 9-20, More preferably, it is 10-18, Most preferably, it is 10-16.

Examples of the hydrocarbon group include an alkyl group, an alkenyl group, and an aralkyl group.
The alkyl group may be linear or branched. For example, octyl group, nonyl group, decyl group, undecyl group, 1-methyldecyl group, dodecyl group, 1-methylundecyl group, 1-ethyldecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, octadecyl group, etc. Can be mentioned.
The alkenyl group may be linear or branched. For example, a decenyl group, an undecenyl group, a dodecenyl group, a tetradecenyl group, a hexadecenyl group, etc. are mentioned.
Examples of the aralkyl group include a benzyl group and a phenethyl group.

The hydroxyalkyl group in R ¹ has 5 to 30 carbon atoms. From the viewpoint of impurity removal performance, the viewpoint of facilitating the reuse of the solid phase washed with the cleaning composition of the present invention, the recovered target protein From the viewpoint of suppressing the contamination of the cleaning composition to the low level, it is preferably 6 to 24, more preferably 8 to 22, more preferably 9 to 20, still more preferably 10 to 18, particularly preferably 10 to 16. is there. Examples of the alkyl group contained in the hydroxyalkyl group include the same alkyl groups as those described above.

The polyoxyalkylene group having an average added mole number of R ¹ of 10 or less and a carbon number of 5 or more is — (CH ₂ CH ₂ O) _p H (p represents an integer of 3 to 10; From the viewpoint of performance, the viewpoint of facilitating the reuse of the solid phase washed with the cleaning composition of the present invention, and the viewpoint of suppressing the contamination of the cleaning composition into the recovered target protein, preferably 5 A polyoxyethylene group represented by the formula (1) to (8).

A group in which part of the hydrogen atoms of the hydroxyalkyl group in R ¹ is substituted with a hydrocarbon group, and a group in which part of the hydrogen atoms of a polyoxyalkylene group having an average addition mole number of 10 or less are substituted with a hydrocarbon group Although the total number of carbon atoms is 5 to 30, the viewpoint of removing impurities, the viewpoint of facilitating the reuse of the solid phase washed with the cleaning composition of the present invention, the cleaning composition for the recovered target protein From the viewpoint of suppressing the contamination of the product to a low level, it is preferably 8 to 26, more preferably 9 to 25, still more preferably 12 to 24, still more preferably 14 to 22, and particularly preferably 16 to 20. Further, examples of the polyoxyalkylene _{group, - (CH 2 CH 2 O} ) q H (q is an integer from 1 to 10, preferably 1 to 7 integer, more preferably an integer of 1 to 4, further A polyoxyethylene group represented by 1 or 2 is preferred.

Moreover, as a substituted position of the hydrocarbon group substituted by the hydroxyalkyl group and the polyoxyalkylene group, the hydrogen atom in the hydroxyl group contained in a hydroxyalkyl group or a polyoxyethylene group is preferable. Moreover, as said hydrocarbon group, a C1-C10 alkyl group, a C6-C20 aryl group, and an alkylaryl group are preferable, and an alkylaryl group is more preferable. The alkylaryl group is preferably an alkylaryl group having 7 to 20 carbon atoms, more preferably an alkylphenyl group having 10 to 18 carbon atoms, and still more preferably an alkylphenyl group having 12 to 16 carbon atoms. For example, (1,1,3,3-tetramethylbutyl) phenyl group and the like can be mentioned.
Examples of the group in which a part of the hydrogen atoms of the polyoxyalkylene group having an average addition mole number of 10 or less are substituted with a hydrocarbon group include those represented by the following formula. In addition, q in a formula is synonymous with the above.

In formula (1), R ² , R ³ and R ⁴ each independently represents an organic group.
R ² , R ³ and R ⁴ include a hydrocarbon group having 1 to 30 carbon atoms, a hydroxyalkyl group having 1 to 30 carbon atoms, and a total carbon in which some of the hydrogen atoms of the hydroxyalkyl group are substituted with hydrocarbon groups. 2 or more groups, polyoxyalkylene group having an average addition mole number of 10 or less, polyoxyalkylene group having an average addition mole number of 10 or less, and a total of 2 or more carbon atoms in which some hydrogen atoms are substituted with hydrocarbon groups Are preferred.

Examples of the hydrocarbon group having 1 to 30 carbon atoms in R ^2, an alkyl group, an alkenyl group, an aralkyl group.

The number of carbon atoms of the alkyl group in R ² is preferably 1 to 12, more preferably 1 to 8, and still more preferably 1 to 4, from the viewpoint of suppressing the contamination of the washing composition into the recovered target protein to a low level. 1 to 3 are more preferable, and 1 or 2 is particularly preferable. The alkyl group may be linear or branched. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group and the like can be mentioned.

The number of carbon atoms of the alkenyl group in R ² is preferably 2 to 12, more preferably 2 to 8, and even more preferably 2 to 4 from the viewpoint of suppressing the contamination of the washing composition into the recovered target protein to a low level. 2 or 3 is more preferable. The alkenyl group may be linear or branched. For example, an ethenyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group and the like can be mentioned.

The number of carbon atoms of the aralkyl group of R ^2, preferably from 7 to 20, more preferably from 7 to 10, 7 or 8 are more preferred. Examples of the aralkyl group include a benzyl group and a phenethyl group.

The number of carbon atoms of the hydroxyalkyl group in R ² is 1 to 30, but 1 to 12 is preferable, and 1 to 8 is more preferable, from the viewpoint of suppressing mixing of the cleaning composition into the recovered target protein. 1-4 are more preferable, 1-3 are more preferable, and 1 or 2 is especially preferable. Examples of the alkyl group contained in the hydroxyalkyl group include the same alkyl groups as those represented by R ² above.
Examples of the hydroxyalkyl group contained in a group in which part of the hydrogen atoms of the hydroxyalkyl group are substituted with a hydrocarbon group include the same hydroxyalkyl groups as those described above.

As a polyoxyalkylene group having an average number of added moles of R ² of 10 or less, — (CH ₂ CH ₂ O) _r H (r represents an integer of 1 to 10, preferably an integer of 1 to 7, A polyoxyethylene group represented by an integer of preferably 1 to 4 and more preferably 1 or 2 is preferable.
In addition, examples of the polyoxyalkylene group contained in a group in which part of the hydrogen atoms of the polyoxyalkylene group having an average addition mole number of 10 or less are substituted with a hydrocarbon group include the same polyoxyalkylene groups as those described above. .

In R ² , the substitution position of the hydrocarbon group substituted with a hydroxyalkyl group or a polyoxyalkylene group is preferably a hydrogen atom in a hydroxyl group contained in the hydroxyalkyl group or polyoxyethylene group. Moreover, as said hydrocarbon group, a C1-C10 alkyl group, a C6-C20 aryl group, and an alkylaryl group are preferable, and an alkylaryl group is more preferable. The alkylaryl group is preferably an alkylaryl group having 7 to 20 carbon atoms, more preferably an alkylphenyl group having 10 to 18 carbon atoms, and still more preferably an alkylphenyl group having 12 to 16 carbon atoms. An example is (1,1,3,3-tetramethylbutyl) phenyl group.

Among the above R ² , it is easy to reuse the solid phase washed with the washing composition of the present invention from the viewpoint of suppressing the contamination of the washing composition into the recovered target protein to a low level, the viewpoint of impurity removal performance, and the washing composition of the present invention. From the viewpoint of making it, a hydrocarbon group having 1 to 30 carbon atoms, a hydroxyalkyl group having 1 to 30 carbon atoms, a polyoxyalkylene group having an average addition mole number of 10 or less is preferable, and a hydrocarbon group having 1 to 30 carbon atoms Are more preferable, and an alkyl group and an aralkyl group are particularly preferable.

As the R ³ and R ^4, preferably a hydrocarbon group having 1 to 30 carbon atoms. As carbon number of the hydrocarbon group in R < ³ > and R < ⁴ >, 1-12 are preferable from a viewpoint of suppressing mixing of the composition for washing | cleaning to the collect | recovered target protein to a low level, 1-8 are more preferable, 1- 4 is more preferable, 1-3 are more preferable, and 1 or 2 is particularly preferable.
Moreover, as a hydrocarbon group in R < ³ > and R < ⁴ >, an alkyl group is preferable. The alkyl group may be linear or branched. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group and the like can be mentioned.

Further, the relationship between the carbon numbers of R ^{1 to} R ⁴ depends on the impurity removal performance, the point of facilitating the reuse of the solid phase washed with the cleaning composition of the present invention, and the washing of the recovered target protein. From the viewpoint of suppressing the mixing of the composition for use to a low level, the carbon number of R ² ≧ R ^{3 and} the carbon number of R ² ≧ R ⁴ , and the carbon number of R ¹ and R ² Is preferably 10 or more. From the same viewpoint as described above, the total number of carbon atoms of R ¹ and R ² is preferably 10 to 26, more preferably 12 to 24, and particularly preferably 12 to 22.

  Moreover, in Formula (1), n means the valence of the atom or atomic group used as a counter anion, and shows an integer greater than or equal to 1. n is preferably 1.

In the formula (1), as Z (atom or atomic group), specifically, in addition to a halogen atom, CH ₃ SO ₄ , CH ₃ CH ₂ SO ₄ , OH, an atomic group derived from an amino acid, a fatty acid An atomic group derived from the above, an atomic group derived from a phosphate ester having a linear or branched alkyl or alkenyl group having 1 to 30 carbon atoms, an atomic group derived from a phosphonic acid ester, an atom derived from a sulfonic acid ester An anion containing a formalin condensate of a sulfonated product of a polycyclic aromatic compound having a group, an atomic group derived from a sulfate ester, a styrenesulfonic acid having a degree of polymerization of 3 or more, or a hydrocarbon group as a substituent Group derived from a functional oligomer or polymer.
Among these, as Z, a halogen atom is preferable, a chlorine atom and a bromine atom are more preferable, and a chlorine atom is particularly preferable.

  Among the cationic surfactants (1), those represented by the following formula (2) or (3) are preferable from the viewpoint of impurity removal performance. Among these, from the viewpoint of facilitating the reuse of the solid phase washed with the cleaning composition of the present invention, from the viewpoint of suppressing the contamination of the washing composition into the recovered target protein to a low level, and from the viewpoint of safety What is represented by Formula (2) is more preferable.

[In Formula (2),
R ⁵ is a hydrocarbon group having 5 to 30 carbon atoms or

A group represented by
R ⁶ and R ⁷ each independently represent a hydrocarbon group having 1 to 4 carbon atoms,
n and ^Zn are as defined above. ]

[In Formula (3),
R ⁸ represents an alkyl group having 9 to 30 carbon atoms,
R ^{9 to} R ¹¹ each independently represents a hydrocarbon group having 1 to 4 carbon atoms,
n and ^Zn are as defined above. ]

In formula (2), R ⁵ is preferably a hydrocarbon group having 5 to 30 carbon atoms from the viewpoint of impurity removal performance. Here, when the hydrocarbon group represented by R ⁵ is a hydrocarbon group having two or more carbon atoms, the carbon number means an average carbon number. The average number of carbon atoms can be calculated by calculating the total number of carbon atoms of R ⁵ of all molecules corresponding to the cationic surfactant (2) and converting the number of carbon atoms per molecule (of R ⁵ of all molecules). Total number of carbons / number of molecules).
The number of carbon atoms of the hydrocarbon group represented by R ⁵ is determined from the viewpoint of impurity removal performance, the viewpoint of facilitating the reuse of the solid phase washed with the washing composition of the present invention, and washing to the recovered target protein. From the viewpoint of keeping the composition for use at a low level, it is preferably 6-24, more preferably 8-22, still more preferably 9-20, still more preferably 10-18, and particularly preferably 10-16.
Moreover, as said hydrocarbon group, an alkyl group is preferable. The alkyl group may be linear or branched. For example, octyl group, nonyl group, decyl group, undecyl group, 1-methyldecyl group, dodecyl group, 1-methylundecyl group, 1-ethyldecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, octadecyl group, etc. Can be mentioned.

In formula (2), R ⁶ and R ⁷ each independently represent a hydrocarbon group having 1 to 4 carbon atoms. The number of carbon atoms of the hydrocarbon group is preferably 1 to 3, and particularly preferably 1 or 2, from the viewpoint of suppressing the contamination of the washing composition into the collected target protein to a low level.
Further, the hydrocarbon group represented by R ⁶ and R ⁷ is preferably an alkyl group. The alkyl group may be linear or branched. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group and the like can be mentioned.

In formula (3), R ⁸ represents an alkyl group having 9 to 30 carbon atoms. The number of carbon atoms of the alkyl group is determined in terms of impurity removal performance, easy reuse of the solid phase washed with the washing composition of the present invention, and contamination of the washing composition into the recovered target protein. From the viewpoint of suppressing to a low level, it is preferably 6-24, more preferably 8-22, still more preferably 9-20, still more preferably 10-18, still more preferably 10-16, and even more preferably 10-14. Yes, particularly preferably 11-13.
Further, the alkyl group represented by R ⁸ may be linear or branched. For example, octyl group, nonyl group, decyl group, undecyl group, 1-methyldecyl group, dodecyl group, 1-methylundecyl group, 1-ethyldecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, octadecyl group, etc. Can be mentioned.

In formula (3), R < ⁹ > -R < ¹¹ > shows a C1-C4 hydrocarbon group each independently. As the said hydrocarbon group, the same thing as R < ⁶ > in Formula (2) is mentioned.

Specific examples of the cationic surfactant include octyltrimethylammonium chloride, decyltrimethylammonium chloride, didecyldimethylammonium chloride, dodecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride. Hexadecyltrimethylammonium bromide, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, benzyltrimethylammonium chloride, benzyltriethylammonium chloride, benzalkonium chloride, benzalkonium bromide, benzethonium chloride and the like.
Among these, benzalkonium chloride and benzethonium chloride are preferable from the viewpoint that they are pharmaceutical additives that can be administered intravenously or intramuscularly and are safe for humans. Among these, benzalkonium chloride is particularly preferable from the viewpoint of achieving both safety and impurity removal performance.
In addition, in the formula (1), benzalkonium chloride is R ¹ = an alkyl group having an average carbon number of 10 to 15, R ² = benzyl group, R ³ = R ⁴ = methyl group, Z ⁿ⁻ = It is a compound of Cl ⁻ .

  Moreover, you may use the said cationic surfactant individually by 1 type or in combination of 2 or more types.

When the cationic surfactant is used, the concentration thereof is preferably 0.01% (w / v) or more, more preferably 0.05, in the cleaning composition of the present invention, from the viewpoint of impurity removal performance. % (W / v) or more, more preferably 0.1% (w / v) or more, more preferably 0.5% (w / v) or more, particularly preferably 1% (w / v) or more, Further, from the viewpoint of suppressing an increase in pressure during separation and purification, in the cleaning composition of the present invention, it is preferably 20% (w / v) or less, more preferably 10% (w / v) or less, and still more preferably 8 % (W / v) or less, more preferably 6.5% (w / v) or less, more preferably 5% (w / v) or less, more preferably 3.5% (w / v) or less, particularly preferably Is 2% (w / v) or less.
By setting the concentration of the cationic surfactant in the above range, an increase in pressure during separation and purification can be significantly suppressed, and excellent impurity removal performance can be obtained.

(Component (B): Hydroxy acid ester)
The hydroxy acid ester is preferably a hydroxy acid ester having 1 to 3 hydroxy groups and 1 to 3 ester bonds in the molecule, and preferably has 1 or 2 hydroxy groups and 1 or 2 ester bonds in the molecule. The hydroxy acid ester possessed by is preferred.
Moreover, as a hydroxy acid ester, the hydroxy acid ester derived from the hydroxy acid which has 1-3 hydroxy groups and 1-3 carboxy groups in a molecule | numerator is preferable, or 1 or 2 hydroxy groups and 1 or 2 A hydroxy acid ester derived from a hydroxy acid having one carboxy group in the molecule is more preferred.

Examples of the hydroxy acid ester include glycolic acid ester, lactic acid ester, tartronic acid ester, glyceric acid ester, hydroxybutyric acid ester, malic acid ester, tartaric acid ester, and citric acid ester.
Among the hydroxy acid esters, α-hydroxy acid esters are preferable, glycolic acid and lactic acid esters are more preferable, and lactic acid esters are more preferable.

  The hydroxy acid ester is preferably a hydroxy acid alkyl ester. As said alkyl group, a C1-C10 alkyl group is preferable, a C1-C5 alkyl group is more preferable, and a C1-C3 alkyl group is further more preferable. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. It is done.

  Among the hydroxy acid esters as described above, ethyl lactate is particularly preferable because it is a pharmaceutical additive that can be administered intravenously or intramuscularly and is safe for humans.

  In addition, you may use the said hydroxy acid ester individually by 1 type or in combination of 2 or more types.

  When the hydroxy acid ester is used, its concentration is preferably 1% (v / v) or more, more preferably 5% (v / v) in the cleaning composition of the present invention, from the viewpoint of impurity removal performance. Or more, more preferably 10% (v / v) or more, particularly preferably 15% (v / v) or more, preferably 40% (v / v) or less, more preferably 30% (v / v). ) Or less, more preferably 20% (v / v) or less. Specifically, 1 to 40% (v / v) is preferable, 5 to 30% (v / v) is more preferable, 10 to 20% (v / v) is further preferable, and 15 to 20% (v / v). Is particularly preferred.

(Component (C): Amide solvent)
As the amide solvent, a solvent represented by the following formula (4) is preferable.

[In Formula (4), R ²¹ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ²² and R ²³ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or In the case where an aryl group is shown and both R ²¹ and R ²² are alkyl groups having 1 to 4 carbon atoms, they may be bonded to each other to form a ring structure. ]

R ²¹ is preferably an alkyl group having 1 to 4 carbon atoms. Moreover, as R <^22> and R < ²³ >, a C1-C4 alkyl group and an aryl group are preferable.
In addition, the alkyl group having 1 to 4 carbon atoms represented by R ²¹ , R ²² and R ²³ may be linear or branched. For example, a methyl group, ethyl group, n-propyl group, isopropyl group, n- A butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group are mentioned.
The aryl group represented by R ²² and R ²³ is preferably an aryl group having 6 to 12 carbon atoms. Specific examples include a phenyl group and a naphthyl group.
Examples of the ring formed by combining the alkyl groups having 1 to 4 carbon atoms represented by R ²¹ and R ²² with each other include a 2-pyrrolidone ring, a 2-piperidone ring, a 2-caprolactam (ε-caprolactam) ring, and the like. .

Examples of the amide solvent include N, N-dimethylacetamide, N, N-diethylacetamide, N, N-dipropylacetamide, N-methyl-N-ethylacetamide, N-butyl-N-phenylacetamide, N , N-dimethylpropanamide, N, N-diethylpropanamide, N-methyl-N-ethylpropanamide, N-methyl-2-pyrrolidone, N-methyl-2-caprolactam, N-ethyl-2-caprolactam, etc. Can be mentioned.
Among these, N, N-dimethylacetamide is particularly preferable because it is a pharmaceutical additive that can be administered intravenously or intramuscularly and is safe for humans.

  In addition, the said amide solvent may be used individually by 1 type or in combination of 2 or more types.

  When the amide solvent is used, the concentration thereof is preferably 1% (v / v) or more, more preferably 5% (v / v) in the cleaning composition of the present invention from the viewpoint of impurity removal performance. Or more, more preferably 10% (v / v) or more, particularly preferably 15% (v / v) or more, preferably 40% (v / v) or less, more preferably 30% (v / v). ) Or less, more preferably 20% (v / v) or less. Specifically, 1 to 40% (v / v) is preferable, 5 to 30% (v / v) is more preferable, 10 to 20% (v / v) is further preferable, and 15 to 20% (v / v). Is particularly preferred.

In the cleaning composition of the present invention, the component (A), the component (B), and the component (C) may be used alone or in combination of two or more of these components. However, when two or more kinds are combined, the impurity removal performance is further improved.
Combinations of these components include combinations of component (A) and component (B), combinations of component (A) and component (C), combinations of component (B) and component (C), component (A) and component ( The combination of B) and a component (C) is mentioned. Among cleaning compositions containing such a combination, from the viewpoint of impurity removal performance, the cleaning composition contains at least component (A) and further contains at least one selected from component (B) and component (C). Compositions are preferred.

  Moreover, when using both a component (A) and a component (B) among said each component, the content rate [(A) / (B)] of a component (A) and a component (B) is 0.01-10. (W / v) is preferred, 0.05 to 1 (w / v) is more preferred, and 0.05 to 0.5 (w / v) is even more preferred.

  Moreover, when using both a component (A) and a component (C) among said each component, the content rate [(A) / (C)] of a component (A) and a component (C) is 0.01-10. (W / v) is preferred, 0.05 to 1 (w / v) is more preferred, and 0.05 to 0.5 (w / v) is even more preferred.

(Other ingredients)
In addition to the above components, the cleaning composition of the present invention may contain water; inorganic salts such as sodium phosphate, sodium chloride and sodium sulfate; amino acids; sugars and the like. In addition, these other components may be used individually by 1 type, or may be used in combination of 2 or more type.

  The concentration of the inorganic salt is preferably 1 to 1500 mM, more preferably 5 to 1000 mM, still more preferably 10 to 750 mM, and particularly preferably 200 to 750 mM in the cleaning composition from the viewpoint of impurity removal performance. .

The pH at 23 ° C. of the cleaning composition of the present invention is preferably 5 or more, more preferably 6 or more, still more preferably 7 or more, and preferably 9 or less, from the viewpoint of the stability of the target protein. Preferably it is 8 or less.
Moreover, as a viscosity at 23 degrees C of the cleaning composition of this invention, 0.8-2.0 mPa * s is preferable and 0.8-1.6 mPa * s is more preferable.
Moreover, it is preferable that the cleaning composition of the present invention is a cleaning liquid composition.

The cleaning composition of the present invention is used in a process for separating and purifying a target protein from a mixed solution containing the target protein and impurities. For example, it is used by filling a column container with a solid phase on which a ligand to which a target protein is bound is fixed, and passing a washing composition through it.
(Separation and purification process)
Separation and purification processes include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and so on. Is preferred.
In particular, the cleaning composition of the present invention is suitable for antibody purification by affinity chromatography using protein A as a ligand, and is particularly useful as an intermediate cleaning composition in the antibody purification. When the cleaning composition of the present invention is used for such cleaning, the impurities can be removed very effectively and the antibody is hardly dissociated from protein A, so that the antibody can be recovered very efficiently.
Intermediate washing refers to washing the solid phase in a state in which the target protein is retained in the ligand in order to wash away impurities in the separation and purification process of the target protein such as an antibody.

(Target protein)
The target protein is preferably an antibody, and more preferably an IgG type antibody containing an Fc region (CH2 / CH3 region (CH2 region and CH3 region)). Moreover, you may use Fc fusion protein and a recombinant protein. The antibodies include antibodies produced in animal cells, chimeric antibodies, and humanized antibodies.

[Protein purification method]
The protein purification method of the present invention comprises:
A binding step of bringing a ligand immobilized on a solid phase into contact with a mixed solution containing a target protein and impurities, and binding the ligand and the protein in the mixed solution;
In the cleaning composition of the present invention, the solid phase on which the protein-bound ligand is immobilized is washed to remove impurities, and the target protein is washed from the solid phase washed in the washing step using an elution buffer. Recovery process to recover,
It is characterized by including.
Hereinafter, each step will be described.

(Joining process)
The binding step in the present invention is a step in which a ligand immobilized on a solid phase is brought into contact with a mixed solution containing the target protein and impurities to bind the ligand and the protein in the mixed solution.

The material of the solid phase is not particularly limited because the cleaning composition of the present invention can be used for purification using a solid phase of various materials. In addition to synthetic polymers, natural polymers, silica, glass, etc. Is mentioned.
Among these, synthetic polymers and natural polymers are preferable. Examples of synthetic polymers include polystyrene resins and methacrylate resins, and examples of natural polymers include those composed of polysaccharides such as agarose, dextran, and cellulose. Among these, agarose and methacrylate resin are preferable, and methacrylate resin is more preferable because it is easy to fill the column and scale up.

  Examples of the shape of the solid phase include particles, hollow fibers, nonwoven fabrics, filters, and monoliths, but particles are preferable.

The ligand may be selected in consideration of the affinity with the target protein. For example, proteins such as protein A, protein G, avidin, His tag, GST tag and the like can be mentioned.
Among these, protein A is particularly preferable because it can be used in the production of an antibody drug. Since the cleaning composition of the present invention hardly dissociates the antibody from protein A, the antibody is recovered very efficiently when the solid phase having protein A as a ligand is washed using the cleaning composition of the present invention. be able to.

  Examples of the mixed solution containing the target protein and impurities include a culture solution of CHO cells (Chinese hamster ovary protein) containing impurities, a culture solution of hybridoma containing impurities, and a cell disruption solution of recombinant E. coli. In addition to impurities, the CHO cell culture medium contains an IgG type antibody containing an Fc region produced by CHO cells.

  The method for binding the ligand and the target protein in the mixed solution is not particularly limited. For example, a particulate carrier on which the ligand is immobilized is filled in a column container, the mixed solution is added to the column, and the target protein and the ligand are then added. The method of making it couple | bond by making it contact.

(Washing process)
The washing step in the present invention is a step of removing impurities by washing the solid phase on which the protein-bound ligand is immobilized with the washing composition of the present invention.
The washing step may be performed in the same manner as in the conventional intermediate washing except that the washing composition of the present invention is used. For example, the washing composition of the present invention is applied to the column while the antibody is retained in the ligand. The solid phase can be washed to remove impurities by passing through the liquid.

  Examples of impurities include HCP (Host Cell Protein), DNA, lipids, medium-derived components, and the like. HCP includes proteins derived from cell fragments of antibody-producing cells such as CHO cells.

The amount of the cleaning composition of the present invention is usually 1 to 10 column volumes when a column is used, but 3 to 5 column volumes are preferred.
The washing temperature is usually 0 to 50 ° C., preferably 5 to 35 ° C., and more preferably normal temperature.

  Before and after this washing step, the solid phase may be further washed with a washing solution in which an inorganic salt such as sodium chloride or sodium sulfate is added to a neutral buffer such as sodium phosphate or Tris-HCl buffer. Good.

(Recovery process)
The recovery step in the present invention is a step of recovering the target protein from the solid phase washed in the washing step using an elution buffer. For example, a method of eluting the target protein with an elution buffer and recovering the target protein using a fraction collector of a chromatography system can be mentioned.

  Examples of the elution buffer include an acidic buffer solution when the ligand is protein A or the like, an imidazole-containing buffer solution when the ligand is a His tag or the like, and a glutathione-containing buffer solution when the ligand is a GST tag or the like.

  As above, the target protein is preferably an antibody, and more preferably an IgG type antibody containing an Fc region (CH2 / CH3 region (CH2 region and CH3 region)). Moreover, you may use Fc fusion protein and a recombinant protein.

  In addition, when component (A)-(C) is contained in the solution containing the target protein isolate | separated and purified, you may remove component (A)-(C) after a collection | recovery process. As the removal method, for example, the components (A) to (C) are separated from the target protein by the difference in adsorption behavior using a carrier for ion exchange chromatography, a carrier for hydrophobic interaction chromatography or a carrier for multimodal chromatography. The method of separating and removing is mentioned.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples. Examples 1 to 19 and 23 are reference examples.

(Reference Example 1)
Affinity chromatography carrier JX02 mainly composed of a methacrylate resin in which the protein ligand (protein A) described in the examples of WO 2012/088660 is immobilized in a column container (Tricorn 10/50 column manufactured by GE Healthcare) A column was prepared by packing PB2 at a packing height of about 5 cm. The obtained column was connected to AK Healthcare's AKTA Prime Plus, and 20 mM sodium phosphate buffer (pH 7.5) was passed through 5 column volumes (5 times the column volume) at a flow rate of 1 mL / min for equilibration. I let you.
Next, CHO cell culture supernatant containing the biosimilar of monoclonal antibody Trastuzumab was passed through the column at a flow rate of 1 mL / min with a loading amount of about 24 g antibody / mL carrier.
Next, 20 mM sodium phosphate buffer (pH 7.5) was passed through the column as the washing solution 1 at a column volume of 5 and a flow rate of 1 mL / min.
Next, 20 mM sodium phosphate / 1M sodium chloride buffer (pH 7.5) was passed through the column as washing solution 2 at 5 column volumes at a flow rate of 1 mL / min.
Subsequently, 20 mM sodium phosphate buffer (pH 7.5) was passed through the column as the washing solution 3 at 5 column volumes and a flow rate of 1 mL / min.
Thereafter, 100 mM glycine hydrochloride buffer (pH 3.0) was passed through the column at 5 column volumes and a flow rate of 1 mL / min to elute the monoclonal antibody captured in the column, and the Abs. An elution fraction of 280> 100 mAu was collected.
Then, using a CHO HCP ELISA kit, 3G manufactured by Cygnus Technologies, the concentration of Host Cell Protein (HCP) contained in the collected fraction was measured. The results are shown in Table 2.
In addition, using a spectrophotometer (SmartSpecPlus manufactured by Bio-Rad Laboratories), the antibody concentration contained in the collected fraction is measured, and the amount of antibody recovered is calculated by multiplying the amount of recovered solution, and further divided by the amount of added antibody. The antibody recovery rate was measured. The results are shown in Table 2.

(Reference Example 2)
The same treatment as in Reference Example 1 was performed except that the methacrylate resin carrier (JX02-PB2) was changed to an agarose carrier (MabSelect SuRe (trademark)), and the HCP concentration and the antibody recovery rate were evaluated. The results are shown in Table 2.

(Examples 1-10 and Comparative Examples 1-2)
Except that the cleaning solution 1 in Reference Example 1 was changed to each cleaning solution shown in Table 1, the same treatment as in Reference Example 1 was performed to evaluate the HCP concentration and antibody recovery rate. The results are shown in Table 2.

(Example 11)
Except having changed the washing | cleaning liquid 1 in the reference example 2 into the washing | cleaning liquid A-2 of Table 1, the process similar to the reference example 2 was performed and HCP density | concentration and the antibody recovery rate were evaluated. The results are shown in Table 2.

(Reference Example 3)
The CHO cell culture supernatant (about 24 g antibody / mL carrier loading) containing the monoclonal antibody Trastuzumab biosimilar was added to the CHO cell culture supernatant (about 6.5 g antibody / Except for changing to (load amount of mL carrier), the same treatment as in Reference Example 1 was performed to evaluate the HCP concentration and the antibody recovery rate. The results are shown in Table 3.

(Examples 12 to 13 and Comparative Example 3)
Reference Example 3 was changed except that the cleaning liquid 1 in Reference Example 3 was changed to the cleaning liquid A-2 (Example 12), the cleaning liquid A-4 (Example 13), and the cleaning liquid B-1 (Comparative Example 3) shown in Table 1. The same treatment as in Example 3 was performed to evaluate the HCP concentration and antibody recovery rate. The results are shown in Table 3.

As shown in Tables 2 and 3 above, HCP could be efficiently cleaned and removed by using the cleaning liquids A-1 to A-10.

(Reference Example 4)
Affinity chromatography carrier JX02 mainly composed of a methacrylate resin in which the protein ligand (protein A) described in the example of WO 2012/088660 is immobilized on a column container (Tricorn 5/200 column manufactured by GE Healthcare) -A column was prepared by packing PB2 at a packing height of about 20 cm. The obtained column was connected to AK Healthcare's AKTA Prime Plus, and 20 mM sodium phosphate buffer (pH 7.5) was passed through 5 column volumes (5 times the column volume) at a flow rate of 1 mL / min for equilibration. I let you. Thereafter, the column was removed from AKTA Prime Plus manufactured by GE Healthcare.
Next, 20 mM sodium phosphate / 500 mM sodium chloride buffer (pH 7.5) (hereinafter also referred to as measurement solution 11) is passed through AKTA Prime Plus at 5 column volumes at a flow rate of 1 mL / min, and the pressure when the column is not connected. The value was measured.
Next, the column was connected, and again the measurement solution 11 was passed through AKTA Prime Plus at 5 column volumes and a flow rate of 1 mL / min, and the pressure value at the time of column connection was measured. The pressure value when the column was not connected was subtracted from the pressure value when the column was connected to obtain a pressure loss value when the measurement liquid 11 was passed. The results are shown in Table 5.
The pressure value when the column was not connected and the pressure value when the column was connected were measured with a pressure gauge attached to AKTA Prime Plus.

(Examples 14 to 16 and Comparative Examples 4 to 5)
Except that the measurement liquid 11 in Reference Example 4 was changed to each measurement liquid shown in Table 4, the same treatment as in Reference Example 4 was performed, and the pressure loss value was evaluated. The results are shown in Table 5.

(Examples 17 to 22)
Except that the cleaning liquid 1 in Reference Example 1 was changed to the cleaning liquids A-21 to A23 and A31 to A33 shown in Table 6, respectively, the same treatment as in Reference Example 1 was performed to evaluate the HCP concentration and antibody recovery rate. did. The results are shown in Table 7.

(Examples 23 and 24)
Except having changed the washing | cleaning liquid 1 in the reference example 1 into the washing | cleaning liquid A-41 and A-42 of Table 8, respectively, the process similar to the reference example 1 was performed and HCP density | concentration and the antibody recovery rate were evaluated. The results are shown in Table 9.

Claims (8)

A cleaning composition used in a process for separating and purifying a target protein from a mixed solution containing the target protein and impurities,
Including an amide solvent,
Cleaning composition. The amide solvent is a solvent represented by the following formula (4).
The cleaning composition according to claim 1.

[In Formula (4),
R ²¹ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ²² and R ²³ each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or an aryl group,
When both R ²¹ and R ²² are alkyl groups having 1 to 4 carbon atoms, they may be bonded to each other to form a ring structure. ] R ²¹ is an alkyl group having 1 to 4 carbon atoms, and R ²² and R ²³ are each independently an alkyl group having 1 to 4 carbon atoms or an aryl group having 6 to 12 carbon atoms (provided that R ^{21 When} both ²¹ and R ²² are alkyl groups having 1 to 4 carbon atoms, they may be bonded to each other to form a ring structure),
The cleaning composition according to claim 2. The process of separating and purifying the target protein is affinity chromatography.
The cleaning composition according to any one of claims 1 to 3. The target protein is an antibody;
The cleaning composition according to any one of claims 1 to 4. Separation and purification of the target protein is separation and purification by affinity chromatography using a synthetic polymer or a natural polymer as a solid phase,
The cleaning composition according to any one of claims 1 to 5. Separation and purification of the target protein is separation and purification by affinity chromatography using agarose or methacrylate resin as a solid phase.
The cleaning composition according to any one of claims 1 to 6. A binding step of bringing a ligand immobilized on a solid phase into contact with a mixed solution containing a target protein and impurities, and binding the ligand and the protein in the mixed solution;
The washing composition according to any one of claims 1 to 7, wherein a washing step of washing a solid phase on which the protein-bound ligand is fixed to remove impurities, and using an elution buffer, A recovery step for recovering the target protein from the solid phase washed in the washing step;
A protein purification method comprising: