JP6657650B2 - Additive for promoting the production of useful substances - Google Patents

Description

  The present invention relates to an additive for promoting the production of a useful substance in a cell containing the polysaccharide represented by the formula (1); a cell culture medium used for producing a useful substance containing the additive; a cell culture medium containing the additive And a method for producing a useful substance, which comprises a step of culturing cells using the method.

Proteins and peptides are artificially produced using genetic recombination technology. Particularly in the field of medicine, proteins useful for medicine (for example, humanized antibodies and human antibodies) are produced using cultured cells.
As a medium used for culturing cells, a serum medium supplemented with bovine serum has been mainly used for a long time. However, in the production of pharmaceuticals, a serum-free medium containing no serum due to the risk of contamination with pathogens is required. However, serum contains various growth factors involved in cell growth, and simply lacking the serum significantly reduces the proliferation of cells and often renders it impossible to culture. Therefore, in many cases, insulin, transferrin, sodium selenite, and the like are used as serum substitutes. However, even when these serum substitutes are used, a serum-free medium, especially a medium composed only of substances having a clear chemical composition (Chemically defined medium), has a lower cell growth and protein productivity than a conventional serum medium. May be inferior. For the improvement, a yeast, a soybean hydrolyzate, or the like may be used together. It is known that the addition of such a protein hydrolyzate to a culture medium effectively works for improving cell growth and productivity of a target protein (Patent Document 1). However, since protein hydrolyzate is a composite material having many components of unknown composition, it is not preferable from the viewpoint of safety as a raw material used in the production of pharmaceuticals. From the viewpoint of assurance of safety, it is desired to use only a medium component having a clear chemical composition, and a cell growth improving agent and a protein production promoter using a substance having a clear composition are required.

JP-T-2002-520014

  The present invention relates to an additive for promoting the production of a useful substance in a cell containing the polysaccharide represented by the formula (1); a cell culture medium used for producing a useful substance containing the additive; a cell culture medium containing the additive And a method for producing a useful substance, which comprises a step of culturing cells using the method.

（式中、ｎは重量平均分子量が１０，０００〜３，０００，０００であるような値を示す整数である）
で表される多糖またはその誘導体が細胞においてタンパク質などの有用物質の生産量を増大させることを見出し、これに基づき本発明を完成させた。
本発明は、一つの側面として、上記式（１）で表される多糖を含む細胞における有用物質の産生促進用添加剤を提供する。
本発明は、一つの側面として、上記式（１）で表される多糖を含む添加剤を含有する有用物質生産に用いる細胞培養用培地を提供する。
本発明は、一つの側面として、上記式（１）で表される多糖を含む添加剤を含有する細胞培養用培地を用いて細胞を培養する工程を含む、有用物質の製造方法を提供する。 The inventor of the present application has calculated the formula (1):

(In the formula, n is an integer indicating a value such that the weight average molecular weight is 10,000 to 3,000,000.)
Have been found to increase the production of useful substances such as proteins in cells, and the present invention has been completed based on this.
The present invention provides, as one aspect, an additive for promoting production of a useful substance in a cell containing the polysaccharide represented by the above formula (1).
The present invention provides, as one aspect, a cell culture medium used for producing a useful substance containing an additive containing the polysaccharide represented by the above formula (1).
The present invention provides, as one aspect, a method for producing a useful substance, comprising a step of culturing cells using a cell culture medium containing an additive containing the polysaccharide represented by the above formula (1).

  According to the present invention, it is possible to provide an inexpensive and highly safe medium for animal cells, comprising the polysaccharide represented by the above formula (1) as an active ingredient. The culture medium for cell culture containing the polysaccharide represented by the above formula (1) of the present invention increases the production amount of a useful substance regardless of the cell morphology and type, the presence or absence of transformation, and the like. That is, the additive for promoting the production of a useful substance provided by the present invention, a culture medium for cell culture containing the additive, and a method for producing a useful substance using the medium can increase the production of a useful substance in cells. It can be applied in fields such as clinical diagnostic drug production using antibodies, antibody drug production, regenerative medicine, and cell therapy.

FIG. 7 is a graph showing the change over time in the IgG concentration in the culture solution of Example 2. FIG. 7 is a graph showing the specific antibody production rates on days 3 to 7 of culture in Example 2. It is a figure which shows the time-dependent change of the number of viable cells at the time of the CHO cell culture of Example 3. FIG. 9 is a graph showing the specific antibody production rate per cell on days 0 to 4 of culture in Example 3. FIG. 7 is a diagram showing a time-dependent change in IgG antibody concentration in a culture solution during CHO cell culture of Example 3. It is a figure which shows the time-dependent change of the number of viable cells at the time of the CHO cell culture of Example 4. FIG. 7 is a graph showing the specific antibody production rate per cell on days 0 to 3 of culture in Example 4. FIG. 9 is a diagram showing a time-dependent change in the concentration of IgG antibody in the culture solution during CHO cell culture of Example 4.

  The present invention relates to an additive for promoting the production of a useful substance in a cell containing the polysaccharide represented by the above formula (1); a cell culture medium used for producing a useful substance containing the additive; and a cell culture medium containing the additive. Provided is a method for producing a useful substance, including a step of culturing cells using a medium.

（式中、ｎは重量平均分子量が１０，０００〜３，０００，０００であり、好ましくは５０，０００〜２，５００，０００であり、より好ましくは１００，０００〜２，０００，０００であり得るような整数である）また、式中のヒドロキシル基は、その全部または一部が硫酸基で修飾されていてもよい。なお、本発明では、上記式（１）で表される多糖およびその誘導体のいずれかのみを用いてもよいし、両方を用いてもよいが、少なくとも、上記式（１）で表される多糖を用いることが好ましい。 The polysaccharide that can be used in the present invention is, for example, a polysaccharide represented by the following formula (1) or a derivative thereof.
Equation (1):

(Where n is a weight average molecular weight of 10,000 to 3,000,000, preferably 50,000 to 2,500,000, more preferably 100,000 to 2,000,000. The hydroxyl group in the formula may be entirely or partially modified with a sulfate group. In the present invention, either the polysaccharide represented by the above formula (1) or a derivative thereof may be used alone, or both may be used, but at least the polysaccharide represented by the above formula (1) may be used. It is preferable to use

  The polysaccharide represented by the above formula (1) includes glucose, mannose, rhamnose and the like as constituent sugars. The polysaccharide represented by the above formula (1) has a structure in which carboxyethyl rhamnose is bonded at the 3-position position in a glucose molecule to a main chain in which a glucose residue and mannose are 1,4 bonded. In addition, the structure determination of the polysaccharide of the formula (1) can be analyzed by a method used for determining the structure of a normal chemical substance. For example, a product obtained by hydrolyzing a polysaccharide with an acid or the like is derivatized (for example, methylated alditol acetate derivatization), and the derivative is subjected to structural analysis using GC-MS, 1H-NMR or 13C-NMR using NMR. -NMR, structural analysis using 1H-COSY, C, H-COSY, or structural analysis using CI-MS.

  The polysaccharide represented by the above formula (1) can be obtained by culturing a bacterium belonging to the genus Pseudomonas (for example, Pseudomonas stutzeri) and purifying the polysaccharide from the culture. The polysaccharide can be produced by, for example, purifying the polysaccharide from the obtained culture solution by the method described in Japanese Patent No. 3057221. As a method for purifying a polysaccharide from a culture solution, a polysaccharide can be purified without any particular problem as long as it is a commonly used method for purifying a polysaccharide from a culture solution. Examples of the purification method include a dilution method and an ethanol precipitation method. The weight average molecular weight of the polysaccharide can be adjusted by the culture time. For example, when producing a polysaccharide having a large weight average molecular weight, the culture time may be increased. The polysaccharide can also be synthesized by a chemical synthesis method or an enzymatic method.

  The dilution method is to obtain a polysaccharide by adding distilled water or the like to a culture solution obtained by culturing, diluting the solution, centrifuging, dialyzing the supernatant with distilled water, and freeze-drying the dialysate. Can be. The ethanol precipitation method can be obtained by adding ethanol to a culture solution, precipitating a polysaccharide contained in the culture solution, and collecting the precipitated polysaccharide by a method such as centrifugation. Before adding ethanol, the cells may be removed from the culture solution by centrifugation or filtration. Furthermore, the polysaccharide can be further purified by redissolving the recovered polysaccharide in water and performing ethanol precipitation again. In addition, if necessary, the polysaccharide obtained by the above method can be further purified using chromatography or the like. The amount of reagent to be added and various conditions for purification may be appropriately determined depending on the properties of the culture solution used for purification, the reagent, and the like.

Examples of the polysaccharide derivative represented by the above formula (1) include salts, esters, ethers, amides and the like.
Examples of salts include acid addition salts and base addition salts. Examples of acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, phosphoric and nitric acids, and p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, Examples thereof include those derived from organic acids such as oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and trifluoroacetic acid. Examples of base addition salts include those derived from ammonium, potassium, sodium, and calcium, and those derived from quaternary ammonium hydroxides, such as, for example, tetramethylammonium hydroxide.

  Examples of esters include aliphatic esters (eg, methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, t-butyl ester, pentyl ester, 1-cyclopropylethyl ester, and propylene glycol ester) And organic acid esters (eg, acetic acid ester, sulfonic acid ester, phosphoric acid ester, malic acid ester), and inorganic acid esters (eg, sulfuric acid ester).

  Examples of ethers include lower alkyl ethers such as methyl ether and ethyl ether (C1-6 alkyl ether).

  Examples of the amide include lower alkyl amides such as methyl amide and ethyl amide (C1-6 alkyl amide).

  Further, the weight average molecular weight of the polysaccharide represented by the above formula (1) that can be used in the present invention can be measured by a method generally used for a method for measuring the weight average molecular weight of a polysaccharide. For example, the weight average molecular weight of the polysaccharide can be measured by gel permeation chromatography using HPLC. The column used for the measurement may be appropriately selected depending on the physical properties of the polysaccharide to be measured, and a commercially available column for measuring the molecular weight may be used.The measurement can be performed by a usual method, and is particularly limited. It is not something to be done.

  As the polysaccharide represented by the above formula (1), those subjected to a molecular weight reduction treatment by a known method can also be used. For example, any known polysaccharide hydrolysis reaction can be used as a method for reducing the molecular weight. For example, it is known that a polysaccharide is hydrolyzed by heating under pressure in the presence of an acid, and this can be used to reduce the molecular weight of the polysaccharide. It is also possible to reduce the molecular weight by using a hydrolysis reaction by an enzyme. Furthermore, it is also possible to reduce the molecular weight by performing a physical treatment such as an ultrasonic treatment.

  The polysaccharide represented by the above formula (1) can be used for promoting production of useful substances in cells. Preferably, the use of the polysaccharide represented by the above formula (1) is provided for promoting production when producing useful substances using cells in vitro. Regarding production promotion, regardless of the mechanism, the production amount of a useful substance may be increased within a certain period of time, for example, the production of a useful substance may be increased by promoting cell proliferation, The production of a useful substance may be increased by increasing the expression level of the substance, or both.

  The polysaccharide represented by the above formula (1) can be used for promoting production of useful substances by adding it to a cell culture medium. In a preferred embodiment, the production of a useful substance in the absence of serum is promoted by adding the polysaccharide represented by the above formula (1) to a cell culture medium. Therefore, the polysaccharide represented by the above formula (1) can be used alone as an additive for promoting production of a useful substance, which is a factor added to a medium. In addition, it can be used together with substances (eg, proteins such as transferrin and insulin) and / or low molecular weight compounds (eg, glucose, phosphate, selenite) that contribute to promoting the production of other useful substances. That is, the additive for promoting useful substance production is not particularly limited as long as it contains the polysaccharide represented by the above formula (1), and may or may not contain other substances.

  The additive provided by the present invention (for example, an additive for promoting the production of a useful substance) is a polysaccharide represented by the above formula (1) at a final concentration of 0.01 μg / ml or more when added to a medium. 0.1 μg / ml or more is preferable, 0.5 μg / ml or more is more preferable, 1 μg / ml or more is more preferable, and 10,000 μg / ml or less is sufficient. It is preferably at most 5,000 μg / ml, more preferably at most 2,000 μg / ml, even more preferably at most 1,000 μg / ml. The final concentration of the polysaccharide in the medium is, for example, 0.01 to 10,000 μg / ml, 0.01 to 5,000 μg / ml, 0.01 to 2,000 μg / ml, 0.01 to 1,000 μg / ml. ml, 0.1-10,000 μg / ml, 0.1-5,000 μg / ml, 0.1-2,000 μg / ml, 0.1-1,000 μg / ml, 0.5 μg / ml-10, 000 μg / ml, 0.5 μg / ml to 5,000 μg / ml, 0.5 μg / ml to 2,000 μg / ml, 0.5 μg / ml to 1,000 μg / ml, 1 to 10,000 μg / ml, 1 to Any form may be used so long as it is included in the range of 5,000 μg / ml, 1 to 2,000 μg / ml, 1 μg / ml to 1,000 μg / ml, and the like. Additives may be liquid or solid at room temperature. For example, the polysaccharide represented by the above formula (1) is added to a buffer such as a phosphate buffered saline, a phosphate buffer, an acetate buffer, a citrate buffer, a Tris buffer, a HEPES buffer, and a MOPS buffer. The polysaccharide concentration in the medium can be adjusted within the above range by dissolving as an additive provided by the present invention.

The medium to which the polysaccharide represented by the above formula (1) is added may be, for example, MEM, DMEM, HamF12, RPMI1640, Fisher's medium, or a mixture thereof. By adding the polysaccharide represented by the above formula (1) to these basal media, a serum-free medium useful for producing a useful substance can be prepared. Further, it can also be used by adding to a serum-free medium, a protein-free medium or a chemically defined medium, which is presumed to be used without serum by adding a growth factor or a serum substitute. Examples of these media include EX-CELL 302, EX-CELL 325-PF, EX-CELL CD CHO manufactured by SAFC Biosciences, SFM II, CHO-III-PFM, CD CHO manufactured by Life Technologies, and the like. And IS-CHO CD and BalanCD Growth A Medium manufactured by Irvine Scientific, but are not limited thereto. Further, such a serum-free medium, a protein-free medium, or a mixed medium obtained by mixing two or more kinds of a chemically defined medium at an arbitrary ratio can be similarly used. The content of the polysaccharide represented by the above formula (1) in the medium is not particularly limited, but may be, for example, 0.01 to 10,000 μg / ml, 0.01 to 5,000 μg / ml, 0.01 to 2 μg / ml. 2,000 μg / ml, 0.01-1,000 μg / ml, 0.1-10,000 μg / ml, 0.1-5,000 μg / ml, 0.1-2,000 μg / ml, 0.1-1 2,000 μg / ml, 0.5 μg / ml to 10,000 μg / ml, 0.5 μg / ml to 5,000 μg / ml, 0.5 μg / ml to 2,000 μg / ml, 0.5 μg / ml to 1,000 μg / ml, 1 to 10,000 μg / ml, 1 to 5,000 μg / ml, 1 to 2,000 μg / ml, 1 μg / ml to 1,000 μg / ml. The medium containing the polysaccharide represented by the above formula (1) includes, in addition to the polysaccharide, for example, inorganic salts (eg, sodium salt, potassium salt, calcium salt), carbohydrates (eg, sugars such as glucose), amino acids ( For example, glutamine and isoleucine), vitamins (eg, riboflavin, thiamine), fatty acids / lipids (eg, steroids such as cholesterol), proteins / peptides (eg, albumin, transferrin, dipeptides such as L-alanyl-L-glutamine), It may include substances selected from trace elements (eg, magnesium, copper, iron) and combinations thereof. In one embodiment, the liquid medium containing the polysaccharide represented by the above formula (1) contains 0.1 to 4.5 g / L glucose and 0.1 to 0.5 g / L CaCl in addition to the polysaccharide. ₂ , 1 to 10 g / L NaCl, 0.001 to 0.3 g / L L-arginine.HCl, 0.001 to 0.3 g / L L-cysteine.2HCl, 0.001 to 0.3 g / L L of L- histidine _· HCl _· H 2 O, the 0.001～0.3G / L L- isoleucine, the 0.001～0.3G / L L- leucine, the 0.001～0.3G / L L-lysine / HCl, 0.001-0.3 g / L L-methionine, 0.001-0.3 g / L L-phenylalanine, 0.001-0.3 g / L L-threonine, 001-0.3 g / L L-tryptophan, 0.001-0.3 g / L of L- tyrosine · 2Na · _2H 2 O, and a L- valine 0.001~0.3g / L. In one embodiment, the medium may be a powdered medium that has the above composition when dissolved in water. Such a medium containing the polysaccharide represented by the above formula (1) described above can be used for producing a useful substance.
Specifically, a method comprising culturing cells producing a useful substance in a medium containing the polysaccharide represented by the above formula (1), and isolating the useful substance produced from the cells. Can produce a useful substance. For example, when producing an antibody as a useful substance, an antibody can be produced by a method comprising culturing antibody-producing cells in a medium containing the polysaccharide represented by the above formula (1) and purifying the antibody. The antibody purification step includes, for example, a method using affinity column chromatography. In the above-described culturing step, so-called Feed culturing, in which a medium component is added during culturing, may be performed. In this case, the medium to be added may contain the polysaccharide represented by the above formula (1) or may not contain the polysaccharide, but preferably contains the polysaccharide.

  In the present specification, the useful substance is not particularly limited as long as it is a substance useful for medicines, agricultural chemicals, foods, and other chemical industries. Preferably, physiologically active proteins such as antibodies, enzymes (such as urokinase), hormones (such as insulin), cytokines (such as interferon, interleukin, erythropoietin, G-CSF, and GM-CSF), and peptides are exemplified. The antibody is, for example, a mouse monoclonal antibody, a humanized monoclonal antibody or a human monoclonal antibody. The class of the immunoglobulin is not particularly limited, and is, for example, IgG (eg, IgG1, IgG2). The useful substance may be a recombinant protein that is an expression product of a foreign gene.

  In the present specification, the cells are not particularly limited as long as they can be used for producing useful substances such as recombinant proteins. CHO cells, BHK cells, HepG2 cells, rodent myeloma cells (eg, SP2 / O cells, NSO Mammalian cells such as mouse myeloma cells), animal cells such as silkworm cells and insect cells such as Drosophila cells; plant cells such as Arabidopsis cells; and transformed cells obtained by introducing foreign genes into these cells. It is listed as. When producing an antibody as a useful substance, a hybridoma obtained by cell fusion of an animal cell (preferably a mammalian cell) such as a CHO cell, SP2 / O cell, or NSO cell may be used as the antibody-producing cell. it can. Among them, animal cells are preferred, mammalian cells are more preferred, and CHO cells are even more preferred.

  Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

Method for Measuring Weight Average Molecular Weight of Polysaccharide In the method for measuring the weight average molecular weight of the polysaccharide used in the experiment, the weight average molecular weight was measured by the method described below. Note that column fitted with a TOSOH Guard COLUMN, was measured by connecting two of TSK-GEL GMPW _XL in series.
Equipment: High performance liquid chromatography (Nippon Waters)
Column: TSK-GEL GMPW _XL (column size 7.5 mm.D x 30 cm) (Tosoh Corporation)
Detector: 2414 differential refractometer (RI detector) (manufactured by Nippon Waters)
Column temperature: 40 ° C
Mobile phase: 100 mM phosphate (potassium) buffer (pH 2.8)
Flow rate: 1.0 ml / min
Inflow volume: 100 μl
Sample concentration: 0.20% (2 mg / ml)
Pretreatment: 0.45 μm membrane filter filtration (Minisart RC 4)
Molecular weight marker: pullulan (weight-average molecular weight: 2.56, 1.33, 78.8, 404,000, 212,000, 112,000, 473,000, 22.28, 118,000, 0.6000) (Showa Denko KK)

Method for Measuring IgG Concentration Using a human IgG ELISA measurement kit (Human IgG ELISA Quantitation Set, ELISA Starter Accession Kit) manufactured by Bethyl Laboratories, Inc., the concentration of G was determined by the method described in the attached cell culture medium using the G method described in the attached instruction manual. .

Method for measuring the number of viable cells The number of viable cells was measured by a method described in the instruction manual using a Counts Automated Cell Counter manufactured by Life technologies.
(Example 1) Examination by the amount of polysaccharide added

  Pseudomonas stutzeri BL58 strain (FERM P-16025) is cultured by the method described in the patent document (Japanese Patent No. 3057221), and a culture solution containing the polysaccharide represented by the above formula (1) is cultured. Was prepared. 35% hydrochloric acid was added to the culture solution (liquid volume 500 ml) to adjust the pH to 4. After adding pure water to the culture solution and diluting it about twice, the cells were removed from the culture solution by a diatomaceous earth filtration method, followed by centrifugation (14,000 × g, 10 min). Thereafter, the centrifuged supernatant was dialyzed against pure water using a dialysis membrane (Spectra / Pro 4, MWCO 12 to 14,000 Da) (the pure water was replaced three times in total). The solution containing the polysaccharide after dialysis was freeze-dried for 2 days using EYELA FDU-1200 and EYELA DRY CHAMBER DRC-1N (manufactured by Tokyo Rikakiki) to obtain a polysaccharide powder. The obtained polysaccharide powder was dissolved in a phosphate buffered saline, and a polysaccharide solution (polysaccharide solution 1) was adjusted to have a pH of 7.0 ± 0.2, and was used in subsequent experiments. In addition, the obtained polysaccharide was subjected to structural analysis by the above-described structural analysis method, and it was confirmed that the obtained polysaccharide had the structure of formula (1). The weight average molecular weight of the obtained polysaccharide was about 445,000.

Using a CHO cell line (ATCC CRL-12445) into which an IgG gene was introduced and secreting and producing an IgG antibody, cells adapted to BalanCD Growth A Medium (Irvine Scientific) and suspended were used for experiments. 7 ml of BalanCD Growth A Medium was added to a 70 ml T flask, and the adjusted suspended cells were seeded at 2 × 10 ⁵ cells / ml. The above-prepared polysaccharide solutions were added to the respective media so that the final concentrations were 0.064, 0.32, 1.6, 8, 40, 200, and 1000 μg / ml. In addition, as a control, the same culture was performed using a medium in which only a phosphate buffered saline was added to the medium instead of the polysaccharide solution. After static culturing for 8 days at 37 ° C. in a 5% CO ₂ environment, the IgG concentration in the culture solution was measured. Table 1 shows the measurement results of the IgG concentration.

表中カッコ内の値は、多糖濃度０の場合のＩｇＧ産生量に対する相対値（％）を示す。

The values in parentheses in the table indicate relative values (%) to the amount of IgG produced when the polysaccharide concentration is 0.

Increased IgG production was observed in all polysaccharide concentration ranges tested (0.064-1000 μg / ml). The above results indicate that the polysaccharide of the present invention has an effect of promoting the production of IgG, which is a recombinant protein.
(Example 2) Examination by shaking culture

30 ml of BalanCD Growth A Medium was added to a 125 ml Erlenmeyer flask, and the same suspended cells as those used in Example 1 were seeded at 1.5 × 10 ⁵ cells / ml. Further, the polysaccharide solution 1 adjusted in Example 1 so that the final concentration of the polysaccharide was 160 μg / ml was added to the medium. Further, as a control, a medium to which a phosphate buffered saline equivalent to the polysaccharide solution was added was prepared and cultured under the same conditions as those to which the polysaccharide was added. The culture was performed at 37 ° C. in a 5% CO ₂ environment with shaking at 100 rpm for 10 days. The culture solution was sampled on the third, seventh, and tenth days of the culture, and the IgG concentration and the number of viable cells in the culture solution were measured. FIG. 1 shows the measurement results of the IgG concentration. FIG. 2 shows the antibody specific production rates on the 3rd to 7th days of culture calculated from the IgG concentration and the number of living cells.

As shown in FIG. 1, the addition of polysaccharide to the medium increased the IgG production by 48-86% compared to the case where no polysaccharide was added. In addition, as shown in FIG. 2, the addition rate of the polysaccharide to the medium improved the antibody specific production rate per cell about 1.5 times. In addition, the number of viable cells did not differ regardless of the presence or absence of polysaccharide addition.
(Example 3) Influence of weight average molecular weight of polysaccharide

  A culture solution containing a polysaccharide was prepared in the same manner as in Example 1. 35% hydrochloric acid was added to the culture solution (liquid volume 500 ml) to adjust the pH to 4. After adding pure water to the culture solution and diluting it about twice, the cells were removed from the culture solution by a diatomaceous earth filtration method, followed by centrifugation (14,000 × g, 10 min). Thereafter, the centrifuged supernatant was dialyzed using a hollow fiber membrane (MWCO 50,000 Da, MiniKros module, M25S-030-01N, SPECTRUM). The solution containing the polysaccharide after dialysis was freeze-dried for 2 days using EYELA FDU-1200 and EYELA DRY CHAMBER DRC-1N (manufactured by Tokyo Rikakiki) to obtain a polysaccharide powder. The obtained polysaccharide powder was dissolved in phosphate buffered saline, and a polysaccharide solution (polysaccharide solution 2) was adjusted to have a pH of 7.0 ± 0.2, and used in subsequent experiments. In addition, the obtained polysaccharide was subjected to structural analysis by the above-described structural analysis method, and it was confirmed that the obtained polysaccharide had the structure of the formula (1). The weight average molecular weight of the obtained polysaccharide was about 711,000.

Using a CHO cell line (ATCC CRL-12445) into which an IgG gene was introduced and secreting and producing an IgG antibody, cells adapted to BalanCD Growth A Medium (Irvine Scientific) and suspended were used for experiments. 30 ml of BalanCD Growth A Medium was added to a 125 ml Erlenmeyer flask, and the above prepared suspended cells were seeded at 1.5 × 10 ⁵ cells / ml. The polysaccharide solution 2 prepared above was added to the medium to a final concentration of 160 μg / ml. In addition, as a control, the same culture was performed using a medium in which only a phosphate buffered saline was added to the medium instead of the polysaccharide solution. Shaking culture was performed at 37 ° C. in a 5% CO ₂ environment with stirring at 100 rpm. The culture solution on days 4, 7, and 10 of the culture was sampled, and the number of viable cells and the IgG antibody concentration were measured. FIG. 3 shows a time-dependent change in the number of living cells during the culture of CHO cells. FIG. 4 shows the antibody specific production rate on the 0th to 4th days of culture calculated from the IgG concentration and the number of viable cells, and FIG. 5 shows the time course of the IgG antibody concentration in the culture solution during CHO cell culture.

As shown in FIG. 3, no growth inhibition was observed even when the polysaccharide was added to the medium. In addition, as shown in FIG. 4, the addition of polysaccharide to the medium increased the specific antibody production rate per cell by about 1.7 times. Furthermore, as shown in FIG. 5, when the polysaccharide was added, the total IgG production increased 1.8-fold.
(Example 4) Examination by Feed culture

Using a CHO cell line (ATCC CRL-12445) into which an IgG gene was introduced and secreting and producing an IgG antibody, cells adapted to BalanCD Growth A Medium (Irvine Scientific) and suspended were used for experiments. 30 ml of BalanCD Growth A Medium was added to a 125 ml Erlenmeyer flask, and the above prepared suspended cells were seeded at 1.5 × 10 ⁵ cells / ml. In addition, the polysaccharide solution 2 prepared above was added to the medium to a final concentration of 160 μg / ml (B). In addition, as a control, the same culture was performed using the medium (A) in which only the phosphate buffered saline was added to the medium instead of the polysaccharide solution. Shaking culture was performed at 37 ° C. in a 5% CO ₂ environment with stirring at 100 rpm. As shown in Table 2, on the first, third and fifth days of culture, PBS and Feed medium (A), polysaccharide solution and Feed medium (B) (Feed medium (BalanCD CHO FEEDI; manufactured by Irvine Scientific)) were added. The culture solution was sampled on days 3 and 8 of culture, and the number of viable cells and the concentration of IgG antibody were measured. Fig. 6 shows the time-dependent changes of the antibody concentration in the culture solution during the CHO cell culture. The changes are shown in FIG.

ＰＢＳ；リン酸緩衝生理食塩水
Ｆｅｅｄ；ＢａｌａｎＣＤ ＣＨＯ ＦＥＥＤＩ（追加培地）
多糖；多糖溶液２
Ａ；１、３、５日目にＰＢＳとＦｅｅｄ培地を添加。
Ｂ；１、３、５日目に多糖とＦｅｅｄ培地を添加。

PBS; Phosphate Buffered Saline Feed; BalanCD CHO FEEDI (Additional Medium)
Polysaccharide; polysaccharide solution 2
A: PBS and Feed medium were added on days 1, 3, and 5.
B: Polysaccharide and Feed medium were added on days 1, 3, and 5.

  As shown in FIG. 6, the addition of the polysaccharide to the medium did not affect the number of viable cells. As shown in FIG. 7, the addition rate of the polysaccharide and the Feed medium to the medium increased the antibody specific production rate per cell by about 1.8 times. Further, as shown in FIG. 8, when the polysaccharide and the Feed medium were added, the total IgG production was improved by 1.8 times.

The additive, medium and production method provided by the present invention can increase the production amount of useful substances in cells, and can be used in the fields of clinical diagnostic drug production using antibodies, antibody drug production, regenerative medicine, cell therapy, and the like. Application is possible.

Claims (5)

Equation (1):

(In the formula, n is an integer indicating a value such that the weight average molecular weight is 10,000 to 3,000,000.)
Including a polysaccharide or a derivative thereof represented by
A culture medium for CHO cell culture used for antibody production wherein the concentration of the polysaccharide or a derivative thereof is 1 µg / ml to 1000 µg / ml. Antibody is an IgG antibody, according to claim 1 Symbol placement of culture areas. CHO cells are transformed cells, culture locations according to claim 1 or 2. Comprising culturing the CHO cells using culture locations according to any one of claims 1 to 3, the method of manufacturing the antibody. 5. The method according to claim 4 , wherein the antibody is an IgG antibody and / or the CHO cells are transformed cells.

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