WO2022202611A1

WO2022202611A1 - Method for producing useful substance

Info

Publication number: WO2022202611A1
Application number: PCT/JP2022/012299
Authority: WO
Inventors: 鉄馬岸; 和信水口; 正克西八條; 史憲鴻池
Original assignee: 株式会社カネカ
Priority date: 2021-03-26
Filing date: 2022-03-17
Publication date: 2022-09-29

Abstract

A purpose of the present invention is to provide a method for efficiently producing a useful substance while suppressing an increase in turbidity of a liquid sample. The method for producing a useful substance according to the present invention is characterized by including: a step for obtaining a liquid sample including the useful substance using cells; a step for treating the liquid sample by a water-insoluble inorganic compound including one or more elements selected from magnesium, calcium, and aluminum to obtain an adsorption-treated liquid sample; a step for removing the water-insoluble inorganic compound from the adsorption-treated liquid sample; and a step for lowering the pH of the adsorption-treated liquid sample from which the water-insoluble inorganic compound has been removed to 6 or less, then raising the pH 0.1 or more.

Description

Method for producing useful substances

The present invention relates to a method for efficiently producing useful substances while suppressing an increase in turbidity of a liquid sample.

In recent years, many useful substances such as proteins and small molecules that act on target substances with pinpoint accuracy have been developed. These useful substances are mainly produced using microorganisms, animal cells, and the like as hosts. In addition to the medium, the culture medium of the cells contains impurities such as host-derived proteins (HCP) and nucleic acids (DNA). Refined by

However, if the culture solution subjected to chromatography contains a large amount of impurities, the load on the column increases, leading to a decrease in the adsorption capacity, separation ability, and processing speed of the column. As a result, there are problems such as a shortened column life and an increased risk of trouble due to an increase in back pressure. Therefore, reducing impurities from the culture solution is important for reducing the load in the subsequent processes.

Techniques for treating a useful substance solution containing impurities to reduce impurities include polyamines (Non-Patent Document 1); chitosan (Non-Patent Document 2); and free divalent cations at low pH (Patent Document 1). Poly (diallyldimethyl-ammonium chloride) (pDADMAC) (Non-Patent Document 3); Impurities are removed by a method of adding water-soluble additives such as endonuclease (Non-Patent Document 4) or heat treatment (Patent Document 2). Methods for precipitating are known.

Since the purification of useful substances is usually carried out in an aqueous system, the removal of water-soluble additives in the latter process is an issue. In addition, there is a problem of a decrease in useful substance recovery rate, such as precipitation of additives and impurities involving useful substances. In addition, endonuclease is excellent because it can be easily removed by setting the conditions of chromatography when removing impurities such as other contaminant proteins, but it is not effective for removing contaminant proteins, etc., and is not sufficient as an impurity removal method. be. Furthermore, heat treatment has a high risk of functional deterioration, denaturation, aggregation, and decomposition of the intended useful substance.

Therefore, the present inventors have developed a technique for easily adsorbing and removing host-derived proteins and nucleic acids from the culture solution using a water-insoluble inorganic compound (Patent Document 3).

Japanese Patent Publication No. 2010-508352 JP-A-63-275600 WO2020/045290 pamphlet

As described above, various techniques have been developed for removing impurities from liquid samples containing useful substances. Apart from this, the present inventors have found that in the process of purifying useful substances from cell culture media, insoluble components precipitate from liquid samples, which sometimes places a burden on columns and filters.
Specifically, when purifying useful substances from cell culture media, the pH may be adjusted, for example, for virus inactivation, and the pH of liquid samples may also be adjusted prior to ion-exchange chromatography. Sometimes. The present inventors have found that when the pH is adjusted, if the pH of the liquid sample is changed probably near the isoelectric point of the impurity or via the isoelectric point of the impurity, the impurity precipitates and the turbidity of the liquid sample increases. I have found that it increases.
SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for efficiently producing a useful substance while suppressing an increase in turbidity of a liquid sample.

The present inventors have made intensive studies to solve the above problems. As a result, by adding a specific water-insoluble inorganic compound to a liquid sample containing impurities in addition to the intended useful substance, the intended useful substance is maintained in the aqueous solution, and the impurities presumably precipitated due to changes in pH can be removed from the water-insoluble inorganic compound. The present invention has been completed by discovering that it can be adsorbed by a compound to suppress an increase in turbidity of a liquid sample due to a change in pH.
The present invention is shown below.

[1] A method for producing a useful substance, comprising:
using cells to obtain a liquid sample containing the useful substance;
a step of treating the liquid sample with a water-insoluble inorganic compound containing one or more elements selected from magnesium, calcium, and aluminum to obtain an adsorption-treated liquid sample;
removing the water-insoluble inorganic compound from the adsorption-treated liquid sample;
A method comprising the step of lowering the pH of said adsorption-treated liquid sample from which said water-insoluble inorganic compounds have been removed to 6 or less and then raising it by 0.1 or more.
[2] The method according to [1], further comprising a step of filtering the adsorption-treated liquid sample after the step of lowering and then raising the pH.
[3] After the step of removing the water-insoluble inorganic compound from the adsorption-treated liquid sample and before the step of lowering and then raising the pH, the step of subjecting the adsorption-treated liquid sample to chromatography. The method according to [1] or [2] above.
[4] The method according to any one of [1] to [3], further comprising a step of subjecting the adsorption-treated liquid sample to chromatography after the step of lowering and then raising the pH.
[5] The OD600nm value of the liquid sample that has undergone the step of lowering and then raising the pH is the same as that of the liquid sample that has undergone the step of lowering and then raising the pH without the step of treating with the water-insoluble inorganic compound. The method according to any one of [1] to [4], wherein the OD600nm value is 0.01 or more smaller.
[6] Any of the above [1] to [5], wherein the water-insoluble inorganic compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium phosphate, calcium sulfate, and aluminum oxide. described method.

According to the present invention, by using a specific water-insoluble inorganic compound, it is possible to remove impurities that precipitate due to changes in pH, and to suppress an increase in turbidity of a liquid sample due to subsequent pH adjustment. Therefore, it is possible to reduce the load on filters and columns for chromatography.

In the present invention, a liquid sample containing impurities in addition to the intended useful substance is treated with a specific water-insoluble inorganic compound to adsorb the impurities, thereby suppressing precipitation of the impurities due to pH adjustment in the subsequent process, and improving the quality of the liquid sample. Reduces turbidity build-up. The method of the present invention will be described below by dividing it into steps, but the present invention is not limited to the following specific examples.

1. Step of Preparing Liquid Sample Containing Useful Substance In this step, cells are used to prepare a liquid sample containing the desired useful substance. The production target of the method of the present invention is not particularly limited as long as it is a useful substance produced by cells. For example, the intended useful substance may be released extracellularly, may be accumulated intracellularly, or may be accumulated in the periplasmic space. . The periplasm of Gram-positive bacteria is sometimes called the "inner wall zone (IWZ)". Useful substances of interest include, for example, antibodies, antibody-binding proteins, enzymes, growth factors, vaccines, hormones, cytokines, blood proteins, and the like.

Antibodies may be monoclonal antibodies or minibodies. Antibodies include, for example, IgM, IgD, IgG, IgA, and IgE; Fab; F(ab′) ₂ as Fab dimer; F(ab′) ₃ as Fab trimer; dsFv bound by disulfide bond; single-chain antibody (scFv) in which VH chain and VL chain are bound by a peptide linker; scFv diabody, scFv trimer tribody, scFv Multimers of single chain antibodies can be mentioned, such as Tetrabody, which is a tetramer. In addition, multimers in which antibody fragments are not covalently associated may also be purified by the method of the present invention, depending on the conditions. Examples of such multimers include Fv in which a heavy chain variable region and a κ chain variable region are associated.

Peptide linkers for binding antibody fragments are not particularly limited, but examples include peptide linkers containing 5 or more and 25 or less amino acid residues. Such peptide linkers include, for example, GS linkers having repeating sequences of glycine and serine.

The antibody-binding protein is not particularly limited as long as it is a protein having a specific binding ability to an antibody. For example, protein A, protein G, protein L, Fcγ receptor, these antibody binding domains, and antibodies and those mutants that maintain or improve their ability to bind to .

Enzymes include, for example, lipase, protease, steroidogenic enzyme, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.

Growth factors include, for example, epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial cell proliferation factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast proliferation factor (FGF), hepatocyte growth factor (HGF).

Hormones include, for example, insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin. Cytokines include, for example, interleukins, interferons (IFNα, IFNβ, IFNγ), tumor necrosis factor (TNF).

Blood proteins include, for example, thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

Cells that produce the above useful substances may be naturally derived cells, but are preferably recombinant host cells. Here, the host is a cell used to produce a useful substance, which is transformed with an expression vector or gene fragment containing DNA encoding the useful substance, and expresses the introduced DNA to produce the useful substance. There are no particular limitations so long as they are animal cells, plant cells, insect cells, or microorganisms that can be infected with viruses. A genetic recombinant in the present invention is transformed with an expression vector or gene fragment containing a nucleotide sequence encoding an amino acid sequence of a useful substance and a promoter operably linked to the nucleotide sequence and capable of functioning in a host. It is a host cell that has been transformed into

Useful substances can be manufactured using conventional methods. For example, among antibodies, a monoclonal antibody extracts B cells from the spleen or lymph node of an animal immunized with an antigen, and selects cells that produce antibodies that react with the antigen from hybridomas obtained by fusing the B cells with myeloma. It can be produced by culturing. Among antibodies, low-molecular-weight antibodies can be produced using bacterial cells or yeast cells. Specifically, for example, after designing the amino acid sequence of each chain constituting the minibody, the base sequence encoding the amino acid sequence is designed by reverse translation. At this time, a nucleotide sequence encoding the amino acid sequence of a minibody in which each chain is covalently bound by a peptide linker may be designed. A DNA encoding the base sequence is chemically synthesized and inserted into a vector such as a plasmid. The vector is introduced into a cell to obtain a transformant, and the transformant is cultured to produce the desired low-molecular-weight antibody. Alternatively, after producing IgG or the like, a low-molecular-weight antibody may be produced by an enzymatic reaction using papain, pepsin, or the like.

The liquid sample containing a useful substance is not particularly limited as long as it contains a useful substance and is liquid. Examples include supernatants from which insoluble components such as body fluids have been removed. In addition, when producing cell-derived useful substances by enzymatic reaction, or when producing useful substances using cells themselves or cell-derived enzymes, the reaction solution itself after the reaction or insoluble components should be removed from the reaction solution. The resulting solution may be used as a liquid sample.

When the useful substance is divided into two or more parts and the cells are made to produce each part, the useful substances may be produced by chemically combining the parts in the above-mentioned culture supernatant. Also in this case, the reaction solution itself after the reaction, or a solution obtained by removing insoluble components from the reaction solution may be used as the liquid sample.

The pH of the useful substance-containing liquid sample obtained using cells is generally in the range of 4 or more and 12 or less, although it depends on the medium components. The pH is preferably 5 or more, more preferably 6 or more or more than 6, still more preferably 6.5 or more, and preferably 10 or less, more preferably 8 or less, and even more preferably 7.5 or less. The pH may be adjusted to a suitable range at this stage.

2. Treatment step with a water-insoluble inorganic compound In this step, a liquid sample containing impurities in addition to useful substances is purified by treating it with a water-insoluble inorganic compound containing one or more elements selected from magnesium, calcium, and aluminum. Impurities other than the target useful substance are selectively adsorbed onto a water-insoluble inorganic compound to purify the useful substance. The term "purification" means that the ratio of impurities to useful substances in a liquid sample is reduced before it is brought into contact with a water-insoluble inorganic compound. "Treatment" means bringing a liquid sample of a useful substance containing impurities into contact with a water-insoluble inorganic compound. In the present disclosure, a liquid sample treated with a water-insoluble inorganic compound is referred to as an "adsorption-treated liquid sample."

In the present disclosure, water-insoluble refers to the amount of purified water required to dissolve within 30 minutes when 1 g of powder of an inorganic compound is put in purified water and vigorously shaken for 30 seconds every 5 minutes at 20 ± 5 ° C. 100 mL or more.

The water-insoluble inorganic compound contains one or more elements selected from magnesium, calcium, and aluminum, and these water-insoluble carbonates, water-insoluble sulfates, water-insoluble phosphates, water-insoluble oxides, etc. preferably one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, calcium sulfate, magnesium phosphate, and aluminum oxide, more preferably a water-insoluble magnesium salt. For example, basic magnesium carbonate, which is a double salt of magnesium hydroxide and magnesium carbonate, is also preferably used. However, phosphates other than magnesium phosphate, such as calcium phosphate, are not preferable because they have relatively high water solubility and may adsorb the intended useful substance.

In particular, magnesium carbonate is effective for gastric/duodenal ulcer, gastritis (including acute/chronic gastritis, drug-induced gastritis), upper gastrointestinal dysfunction (including anorexia nervosa, so-called gastroptosis, hyperacidity), and constipation. As a medical drug, several grams are administered daily, and its safety has been confirmed. It is also excellent in that it can be easily separated and removed from the target useful substance by chromatographic treatment.

The size of the water-insoluble inorganic compound may be adjusted as appropriate. For example, the average particle size can be 0.1 μm or more and 1000 μm or less. If the average particle size is 1000 μm or less, the specific surface area of the water-insoluble inorganic compound is sufficiently large, and impurities can be adsorbed more efficiently. no energy required. From the viewpoint of handleability when filling a column, etc., the average particle size is preferably 1 μm or more. In the present disclosure, the average particle size is measured by a laser diffraction particle size distribution analyzer, and the average particle size may be measured on a volume basis, a weight basis, or a number basis, but the volume basis is preferred.

The amount of the water-insoluble inorganic compound used may be adjusted according to the concentration of the useful substance-containing liquid sample. good. The ratio is preferably 15 g/100 mL or less. Moreover, the water-insoluble inorganic compound can be used in an amount of 0.1% by mass or more and 20% by mass or less relative to the useful substance-containing liquid sample, and the ratio is preferably 1% by mass or more and 15% by mass or less.

The method of contacting the liquid sample containing the useful substance with the water-insoluble inorganic compound should be selected as appropriate. For example, a water-insoluble inorganic compound may be added to a useful substance-containing liquid sample and shaken or stirred. Alternatively, a water-insoluble inorganic compound may be packed in a column, and impurities other than the useful substance may be adsorbed on the water-insoluble inorganic compound by circulating the useful substance-containing liquid sample. In this case, the adsorption of impurities and the separation of the liquid from the water-insoluble inorganic compound can be performed simultaneously. The amount of the water-insoluble inorganic compound packed in the column and the flow rate of the useful substance-containing liquid sample are preferably adjusted within a range in which impurities are sufficiently adsorbed by the water-insoluble inorganic compound.

The temperature at which the useful substance-containing liquid sample and the water-insoluble inorganic compound are brought into contact may be normal temperature, specifically 0°C or higher and 40°C or lower. The temperature is preferably 1° C. or higher, more preferably 10° C. or higher or 15° C. or higher, and preferably 30° C. or lower and more preferably 25° C. or lower. In addition, normal temperature means 20±5 degreeC. Also, the contact time can be 1 second or more and 10 hours or less.

Impurities adsorbed to water-insoluble inorganic compounds in this step are not particularly limited as long as they are compounds other than useful substances, but examples include aggregated antibodies, host cell contaminants, and cell culture contaminants. Host cell contaminants include host cell-derived nucleic acids, plasmids, and host cell-derived proteins. Cell culture contaminants include media components, plasmid DNA for transfection.

In addition, many of the water-insoluble inorganic compounds used in the present invention are neutral to weakly basic, and the pH of the adsorption-treated liquid sample treated with the water-insoluble inorganic compound may be about 7 or more and 10 or less.

3. Separation Step In this step, after step 2 of treating a liquid sample containing useful substances with a water-insoluble inorganic compound, the water-insoluble inorganic compound that has adsorbed impurities is removed from the adsorption-treated liquid sample. Separation means are not particularly limited, but include, for example, filtration and centrifugation. In the present disclosure, the liquid sample after being treated with the water-insoluble inorganic compound is referred to as an adsorption-treated liquid sample. In addition, as described above, by filling a column with a water-insoluble inorganic compound and circulating a liquid sample containing a useful substance, impurities other than the useful substance are adsorbed on the water-insoluble inorganic compound, whereby the water-insoluble inorganic compound treatment step 2 is performed. and main separation step 3 can be carried out at the same time.

Due to the treatment process described above, the useful substances are mainly dispersed in the liquid, and all or part of the other impurities are mainly adsorbed by the water-insoluble inorganic compounds. In addition, part of the useful substances may be adsorbed by the water-insoluble inorganic compound, and part of the other impurities may be dissolved in the liquid, but at least the total amount of impurities in the liquid can be reduced, The useful substances in the liquid part are concentrated. In this step, the useful substance can be roughly purified by removing the water-insoluble inorganic compound that adsorbs the impurities.

This step can be carried out without any particular restrictions so long as it is after the step of treating with a water-insoluble inorganic compound. However, since at least part of the water-insoluble inorganic compound may be dissolved by, for example, pH adjustment, the separation step 3 is preferably carried out after the treatment step 2 with the water-insoluble inorganic compound.

4. pH adjustment step In this step, in order to purify useful substances, the pH of the adsorption-treated liquid sample obtained by removing the water-insoluble inorganic compound after treatment with the water-insoluble inorganic compound is lowered to 6 or less, and then raised by 0.1 or more. Let

For example, it is known that viruses can be inactivated by reducing the pH of the virus-containing liquid. Therefore, in the present invention, it is preferable to inactivate the virus by lowering the pH of the adsorption-treated liquid sample, since there is a possibility that the virus may be present at the stage of the culture solution, for example. The pH can be, for example, 4 or less, preferably 3.8 or less, and more preferably 3.5 or less. On the other hand, if the pH is too low, the useful substance may be damaged, so the pH is preferably 2 or higher, more preferably 2.5 or higher, and even more preferably 3 or higher.

The acid for lowering the pH is not particularly limited, and examples thereof include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, and organic acids such as formic acid, acetic acid and citric acid. , and hydrochloric acid are preferred.

The conditions for virus inactivation can be adjusted as appropriate. For example, after lowering the pH, the adsorption-treated liquid sample may be maintained at a temperature of about 10° C. or higher and 35° C. or lower for 1 minute or longer and 10 hours or shorter. The temperature for virus inactivation treatment may be normal temperature.

In this step, after the pH of the adsorption-treated liquid sample is lowered to 6 or less, the pH of the liquid sample is increased by 0.1 or more for subsequent processes. The range of the pH is not particularly limited as long as it is 0.1 or more higher than the once lowered pH. The pH is preferably 4.5 or higher, more preferably 5 or higher, and even more preferably 6 or higher or 6.1 or higher. For such pH adjustment, for example, trishydroxymethylaminomethane or 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid can be used.

For example, when further purifying the intended useful substance by ion exchange chromatography, it is preferable to adjust the pH of the liquid sample according to the ion exchange column to be used before ion exchange chromatography. For example, the main impurities contained in the culture solution are, in addition to the medium components, proteins and DNA derived from host cells. charge varies with pH. Specifically, the charge of proteins and DNA is positive at pH below the isoelectric point and negative at pH above the isoelectric point. Therefore, for example, if the pH of a liquid sample is lowered and then raised to 4 or more and less than 6, the charge of many proteins and DNA becomes positive. Impurities can be separated. Conversely, if the pH of the liquid sample is lowered and then adjusted to 6 or more and 9 or less, the charge of many proteins and DNA becomes negative. can be separated.

In addition, it is preferable to adjust the pH and ionic strength of the equilibration buffer and washing solution so that the target useful substance is bound to the carrier of the ion exchange column without fail and impurities are not bound as much as possible. After the desired useful substance is adsorbed on the ion exchange column, the desired useful substance is eluted from the column with an eluent having a high salt concentration or a neutral eluent. For example, sodium chloride can be used as the salt contained in the eluate, and the pH of the eluate can be adjusted to, for example, 6 or more and 8 or less.

In the present invention, since the impurities in the liquid sample are adsorbed and removed by the water-insoluble inorganic compound in the treatment step using the water-insoluble inorganic compound, the concentration of impurities in the liquid sample subjected to this step is reduced. there is As a result, even if the pH of the liquid sample is changed in this step, precipitation of impurities is suppressed and an increase in turbidity of the liquid sample is suppressed. For example, after the treatment step with the water-insoluble inorganic compound described above, the OD600nm value of the liquid sample was measured after changing the pH in this step, and the present step was performed in the same manner except that the treatment was not performed with the water-insoluble inorganic compound described above. When the OD600nm value is measured later, it is preferable that the OD600nm value of the liquid sample that has undergone both the treatment step with the water-insoluble inorganic compound and this step is lower by 0.01 or more. Such a difference is more preferably 0.02 or more, and even more preferably 0.03 or more. The upper limit of the difference is not particularly limited, but the OD600nm value of the liquid sample that has undergone both the treatment step with the water-insoluble inorganic compound and this step is preferably the same as the blank value or within ±5% of the blank value. The difference is preferably 10 or less.

5. Filtration Step In this step, the adsorption-treated liquid sample is filtered after step 4 in which the pH is lowered and then raised. Although the implementation of this step is optional, even if the amount of impurities is reduced by the treatment using the water-insoluble inorganic compound in the present invention, the impurities may be precipitated by adjusting the pH. Such precipitates can be removed by this step.

Centrifugal separation may be used in addition to filtration to separate precipitates, but in the present invention, the amount of impurities is reduced by treatment using a water-insoluble inorganic compound, and as a result, impurities are precipitated by pH adjustment. It is preferable to separate such precipitates by filtration because they are fine or the amount thereof is small.

6. Chromatography step In this step, at least, the adsorption-treated liquid sample whose impurities have been reduced through the step 2 of treating with a water-insoluble inorganic compound and the step 3 of separating the water-insoluble inorganic compound is subjected to chromatography to obtain the intended useful substance. Purify further. Chromatography is not particularly limited, but examples thereof include affinity chromatography, hydrophobic chromatography, hydroxyapatite chromatography, and mixed mode chromatography.

The ligand used for affinity chromatography may be appropriately selected according to the desired useful substance. For example, when the useful substance of interest is an antibody, ligands include protein A, protein G, and protein L. Other chromatographies may also be carried out by conventional methods. Also, two or more chromatographies may be combined.

The order and combination of chromatographies are not particularly limited after the liquid sample is prepared, but since impurities can be removed with a simple operation by the treatment step with the above-mentioned water-insoluble inorganic compound, it is also possible from the viewpoint of reducing the chromatographic load. , chromatography is preferably carried out after step 2 of treatment with a water-insoluble inorganic compound and step 3 of separation of the water-insoluble inorganic compound. Also, one or more of the chromatographies may be performed before or after the pH adjustment step described above, and the pH adjustment step described above may be a prior pH adjustment performed for these operations. Therefore, the above pH adjustment step may be performed twice or more.

7. Other Purification Steps In the present invention, other general purification procedures such as activated charcoal treatment, flocculant treatment, and endonuclease treatment may be performed.

The activated carbon used for the activated carbon treatment may be selected as appropriate. For example, the activated carbon may have a specific surface area of 800 m ² /g or more and 2500 m ² /g or less, and the activated carbon may have an average pore diameter of 0.1 nm or more and 20 nm. You can: The average pore diameter is preferably 0.5 nm or more, more preferably 2.0 nm or more, still more preferably 3.0 nm or more, and preferably 5.0 nm or less. The average pore diameter of activated carbon can be calculated from a nitrogen adsorption isotherm curve using the BJH method.

Although the shape of the activated carbon is not particularly limited, for example, granular activated carbon such as pulverized carbon, granulated carbon, spherical carbon, and pelletized carbon; fibrous activated carbon such as fiber and cloth; special molding such as sheet, molded body and honeycomb. activated carbon; and powdered activated carbon.

The purification operation using activated carbon is not particularly limited, but includes, for example, a batch method, a membrane treatment method, a column chromatography method, and the like, and an appropriate shape of activated carbon is selected according to each method. If necessary, in the form of particles in which activated carbon is encapsulated in a porous polymer or gel, in the form of a membrane in which activated carbon is adsorbed, fixed or formed using a support agent such as polypropylene or cellulose or fibers, or in the form of a cartridge. You can also use

The amount of activated carbon used may be adjusted according to the concentration of the useful substance-containing liquid sample, etc. For example, 0.5 g or more and 5 g or less of activated carbon may be used per 100 mL of the liquid sample.

Examples of flocculants used for flocculant treatment include caprylic acid, polyamines, divalent cations, polyetherimine, chitosan, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, pDADMAC, and the like. Divalent cations include, for example, Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , Co ²⁺ , Mn ²⁺ , Ni ²⁺ , Be ²⁺ , Sr ²⁺ , Ba ²⁺ , Ra ²⁺ , Zn ²⁺ , Cd ²⁺ , Ag ²⁺ , Pd ²⁺ , Rh ²⁺ , and these divalent cations can be used in free form or as hydrochloride, sulfate, citrate and the like.

The amount of flocculant used may be adjusted depending on the concentration of the liquid sample, etc. For example, when the flocculant is polyamine or polyetherimine, 0.01 w/v% or more and 10 w/v% or less of flocculant may be used. More preferably, flocculant of 0.1 w/v% or more and 1 w/v% or less may be used. When the flocculant is caprylic acid, chitosan, polyethylene glycol, polyvinyl alcohol or polyvinylpyrrolidone, the floculant should be used in an amount of 0.01 w/v% or more and 10 w/v% or less, more preferably 1 w/v% or more and 5 w. /v% or less flocculant may be used. When the flocculant is pDACMAC, 0.01 w/v% or more and 0.1 w/v% or less of floculant may be used, and more preferably 0.1 w/v% or more and 0.5 w/v% or less of flocculant. should be used. When the flocculant is a divalent cation, it may be added in an amount such that the divalent cation concentration is 1 mM or more and 100 mM or less, more preferably 2 mM or more and 50 mM or less.

As for the method of contacting the liquid sample with the flocculant, the floculant may be added to the liquid sample and shaken or stirred, or the column may be filled with the floculant, as in the case of water-insoluble inorganic compounds. When the activated carbon treatment and the flocculant treatment are performed at the same time, a mixture of the flocculant and the activated carbon may be used.

An endonuclease is one of the enzymes that degrade DNA, and has the function of degrading even the center of the base sequence. The amount of endonuclease to be used may be adjusted depending on the concentration of the liquid sample, but is preferably 10 U/mL or more, more preferably 100 U/mL or more, for the liquid sample. On the other hand, although the upper limit of the ratio is not particularly limited, it is preferably 10,000 U/mL or less.

8. Post-treatment step, etc. After the useful substance is purified, the amount of the solvent may be reduced to concentrate the useful substance, or the solvent may be exchanged.

The amount of impurities can be measured using commercially available assay kits by absorbance analysis, electrophoresis, HPLC, etc. at any stage. For example, a CHO HCP ELISA kit (manufactured by Cygnus) can be used to quantify host-derived proteins derived from CHO cells. If a desired impurity quantification kit is not available, a desired detection system can be created by immunizing animals such as chickens with impurities. In addition, impurities such as DNA can be quantified by techniques such as qPCR.

This application claims the benefit of priority based on Japanese Patent Application No. 2021-53477 filed on March 26, 2021. The entire contents of the specification of Japanese Patent Application No. 2021-53477 filed on March 26, 2021 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be carried out with appropriate modifications within the scope of the gist of the present invention. Of course, it is also possible to do so, and all of them are included in the technical scope of the present invention. In addition, commercial products were used for the reagents used in the present examples unless otherwise specified.

Example 1: Suppression of increase in turbidity by pH manipulation of culture solution (1) Preparation of IgG-expressing CHO culture solution Culture was performed according to the protocol. The host cells, medium, and transfection reagent used were those attached to the kit, and culture was terminated 7 days after transfection. The cell density at the end of culture was 5.5×10 ⁷ cells/mL, and the viability was 90%. The obtained cell culture solution was centrifuged at 3000×g for 5 minutes, and the obtained supernatant was filtered through a filter with a pore size of 0.22 μm. This culture supernatant was used as an IgG-expressing CHO cell culture medium.

(2) Adsorption Treatment To the IgG-expressing CHO culture solution obtained in (1) above, an adsorbent shown in Table 1 was added at a concentration of 1 w/v%, and the mixture was stirred at room temperature with a mix rotor for 1 hour. Then, it was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected as an adsorption-treated liquid sample. The recovered supernatant was filtered through a membrane with a pore size of 0.22 μm, and the turbidity of the filtrate was measured as the turbidity before pH change. Silica gel was manufactured by Merck, basic magnesium carbonate granules were manufactured by Kishida Chemical Co., light basic magnesium carbonate was manufactured by Fujifilm Wako Pure Chemical Industries, and magnesium oxide and magnesium hydroxide were manufactured by Tomita Pharmaceutical. For comparison, an IgG-expressing CHO cell culture was treated in the same manner, except that no adsorbent was added.

(3) Virus inactivation treatment While thoroughly stirring the adsorption-treated liquid sample obtained in (2) above, 1M hydrochloric acid was slowly added dropwise to adjust the pH to 4.0 and allowed to stand at room temperature for several minutes. After that, 1M trishydroxymethylaminomethane was slowly added dropwise to adjust the pH to 7.0. Next, the undiluted solution of the resulting mixed solution was dispensed into a general quartz cell for absorbance measurement, and the turbidity (OD 600 nm) was measured at room temperature as the turbidity after changing the pH. Table 1 shows the results.

As the results shown in Table 1, turbidity after pH manipulation was significantly reduced when silica gel, a common adsorbent, was treated with a water-insoluble magnesium compound without adsorbent treatment. The reason for this is thought to be that the adsorbent removed substances that were insolubilized by the pH adjustment operation and caused an increase in turbidity.

(4) Measurement of IgG recovery rate After the adsorption treatment in (2) above and the culture solution after the virus inactivation treatment in (3) above, Protein A affinity column ("TSKgel Protein A-5PW" Tosoh The amount of IgG contained was determined by analysis using a general chromatography system including (manufactured by Co., Ltd.). Next, the ratio of the amount of purified IgG to the amount of initial IgG contained in the IgG-expressing CHO culture was calculated. Table 2 shows the results.

(5) Measurement of Amount of Impurities Next, changes in the amounts of protein and DNA in the culture solution before and after treatment with the adsorbent were checked. The amount of host cell-derived protein (HCP) contained in the culture solution after the adsorption treatment in (2) above and after the virus inactivation treatment in (3) above was measured using a host cell-derived protein detection kit (" Measurement was performed using CHO Host Cell Protein ELISA Kit, 3rd Generation (manufactured by Cygnus) according to the attached protocol. Table 2 shows the results.
In addition, the DNA content contained in the culture medium after the adsorption treatment in (2) above and after the virus inactivation treatment in (3) above was measured using a host cell-derived DNA detection kit ("CHO DNA Amplification Kit in Tubes ” (manufactured by Cygnus), and measured according to the attached protocol. Table 2 shows the results.

As the results shown in Table 2, in the case of no adsorbent treatment or in the case of treatment with silica gel, which is a general adsorbent,
・HCP and DNA content are high even after adsorption treatment,
・After the virus inactivation treatment by pH adjustment, the concentration of HCP and DNA in the culture medium decreased significantly, and they were insolubilized and precipitated.
On the other hand, when adsorption treatment is performed with a water-insoluble magnesium compound,
- Both HCP and DNA can be removed to a relatively large extent while the target IgG recovery rate is maintained at 95% or higher, and
・There was little change in the concentration of HCP and DNA in the solution before and after pH adjustment, and HCP and DNA were hardly precipitated.
From this result, it is considered that at least part of the mechanism by which the pretreatment with a water-insoluble magnesium compound reduces the increase in turbidity after the latter pH adjustment is due to the removal of HCP and DNA.

Example 2: Turbidity reduction effect at various pH As in Example 1, the turbidity was measured after acidification and after adjusting the pH to 7 with 1M trishydroxymethylaminomethane. For comparison, turbidity was measured in the same manner except that light basic magnesium carbonate was not used. Table 3 shows the results.

As the results shown in Table 3 show, pretreatment with basic magnesium carbonate can suppress precipitation of insoluble components and reduce turbidity at any post-acidification pH. .

Example 3: Turbidity reduction in virus inactivation treatment (1) Adsorption treatment To the IgG-expressing CHO culture solution, the adsorbent shown in Table 5 was added at a concentration of 1 w/v%, and the mixture was stirred at room temperature with a mix rotor for 1 hour. did. Then, it was centrifuged at 5,000 rpm for 1 minute, and the supernatant was collected as a treated liquid. For comparison, a supernatant was obtained in the same manner except that the adsorbent was not used.

(2) pH adjustment While thoroughly stirring the treatment liquid obtained in (1) above, 1M sodium citrate aqueous solution (pH 5.5) is slowly added dropwise to adjust the pH to 7.0, and then left to stand for several minutes. did.

(3) Protein A purification Using a column packed with a protein A carrier ("KANEKA KanCapA ^™ 3G" manufactured by Kaneka) (1 mL), IgG was purified from the treated liquid obtained in (2) above according to the program in Table 4. did. "CV", which is a unit of flow rate in Table 4, indicates "column volume" and represents a relative amount to the volume of the column.

(4) Virus Inactivation Treatment The eluate obtained in (3) above was slowly added dropwise with 1M hydrochloric acid while stirring well to adjust the pH to 3.4, and allowed to stand at room temperature for 1 hour. Then, while stirring the eluate well, 1M trishydroxymethylaminomethane was slowly added dropwise to adjust the pH to 7.0.

(5) Turbidity measurement The turbidity (OD600 nm) of the liquid was measured before and after the virus inactivation treatment in (4) above. Table 5 shows the results.

As shown in Table 5, although there is no significant difference in the turbidity before the virus inactivation treatment, the turbidity after the virus inactivation treatment is derived from the culture solution treated with a water-insoluble magnesium compound or a water-insoluble aluminum compound. was clearly lower than that of These results indicate that the adsorption treatment with a water-insoluble magnesium compound or aluminum oxide can reduce not only the liquid immediately after the treatment, but also the turbidity after the subsequent pH adjustment.

Example 4: Improvement of Filterability after Virus Inactivation Treatment The processes from adsorption treatment to virus inactivation treatment were performed in the same manner as in Examples 3 (1) to (4). The resulting virus inactivation solution (6 mL) was filled in a syringe, and a syringe pump (manufactured by YMC) was passed through a silicone tube at a flow rate of 0.2 mL/min. manufactured by PVDF, pore size: 0.22 μm, membrane area: 0.1 cm ² ) until the back pressure reaches 0.1 MPa, and the filtrate compared the quantities. Table 6 shows the results.

As the results shown in Table 6, when the virus-inactivated solution derived from the culture medium was filtered, the amount of filtrate was clearly greater in the case of treatment with a water-insoluble magnesium compound than in the case of no treatment.
From these results, it was found that the adsorption treatment with the water-insoluble magnesium compound increased the filtrate volume of the virus-inactivated solution. The reason for this is thought to be that the amount of impurities contained in the virus-inactivated solution before filtration was reduced by the adsorption and removal of impurities by the water-insoluble magnesium compound, and clogging of the filter was reduced.

Claims

A method for producing a useful substance, comprising:
using cells to obtain a liquid sample containing the useful substance;
a step of treating the liquid sample with a water-insoluble inorganic compound containing one or more elements selected from magnesium, calcium, and aluminum to obtain an adsorption-treated liquid sample;
removing the water-insoluble inorganic compound from the adsorption-treated liquid sample;
A method comprising the step of lowering the pH of said adsorption-treated liquid sample from which said water-insoluble inorganic compounds have been removed to 6 or less and then raising it by 0.1 or more.
The method of claim 1, further comprising filtering the adsorption-treated liquid sample after the step of lowering and then raising the pH.
After the step of removing the water-insoluble inorganic compounds from the adsorption-treated liquid sample and before the step of raising after lowering the pH, the method further comprises subjecting the adsorption-treated liquid sample to chromatography. 3. The method according to 1 or 2.
The method according to any one of claims 1 to 3, further comprising a step of subjecting the adsorption-treated liquid sample to chromatography after the step of lowering and then raising the pH.
The OD600nm value of the liquid sample that has undergone the step of lowering and then raising the pH is higher than the OD600nm value of the liquid sample that has undergone the step of lowering and raising the pH without the step of treating with the water-insoluble inorganic compound. is also reduced by 0.01 or more.
The method according to any one of claims 1 to 5, wherein the water-insoluble inorganic compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium phosphate, calcium sulfate, and aluminum oxide.