RU2731988C2 - Composition for preventing aggregation and higher homogeneity of culture, increasing productivity of recombinant proteins producing cell lines - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to compositions for improving characteristics of antibody-producing cell culture.

EFFECT: addition of the composition obtained according to the present invention to a serum-free basic medium is capable of preventing aggregate formation, as well as promotes higher homogeneity of medium during cultivation of CHO cell lines (Chinese hamster ovary cells), producing monomer and dimeric forms of recombinant monoclonal IgG and IgA antibodies.

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Description

Technology area

The invention relates to biotechnology, in particular to a composition for preventing aggregation and increasing the homogeneity of the culture, increasing productivity, and can be used for suspension cultivation of mammalian cells, as well as for producing recombinant antibodies of IgG and IgA classes during expression of mammalian cells.

State of the art

Currently, several serum-free media have been developed and successfully used for the cultivation of various eukaryotic cells, and in particular CHO cells. However, it should be noted that different cell lines have specific metabolism and specific nutrient utilization. It is known that at a high cell density, the production of recombinant antibodies can decrease due to the aggregation of CHO cells and, as a result, a deterioration in the supply of cells with oxygen and nutrients. However, for the purposes of the Pharmaceutical Industry, it is necessary to optimize the level of expression of immunoglobulins in CHO cells. Some authors also report an increase in the production of therapeutic proteins and improved cell growth when supplements are added to the serum-free medium, which are combinations of vitamins and hormones, as well as hormones and inorganic salts (Morris A.E., Schmid J. "Effects of insulin and LongR3 on serum-free Chinese Hamster Ovary cell cultures expressing two recombinant proteins ", Biotechnol. Progr. 2000. Vol. 16. P. 693-697), which allowed to develop an approach to substitution of components of animal origin (Kim DY, Lee J.C., Chang YN, Duk J.O. "Effects of supplementation of various medium components on Chinese hamster ovary cell cultures producing recombinant antibody", Cytotechnology. 2005. Vol. 47. P. 37-49).

According to the literature, fibroblast growth factor affects the growth of corneal cells and stem cells (deVere JK, White RW "Fibroblast growth factor 2: its structure and property, paracrine function tumor angiogenesis, and prostate-related mitogenic and oncogenic functions", Urology, 2000 Vol. 55. P. 800-806). Numerous studies confirm the ability of FGF-2 to stimulate the proliferation of corneal epithelial cells, stromal fibroblasts and endothelial cells in vitro, as well as in cell culture and systems for growing organs in culture (Giguere L., Cheng J, Gospodarowicz D. "Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium ", J. Cell Physiol. 1982. Vol. 110. P. 72-80., Sun P., Shen L., Zhang C., Du L., Wu X. "Promoting the expansion and function of human corneal endothelial cells with an orbital adipose-derived stem cell-conditioned", Stem Cell Res. Ther. 2017. Vol. 8. N. 1. P. 287-296. Chmielowiec J. , Borowiak M. "In vitro differentiation and expansion of human pluripotent stem cell-derived pancreatic progenitors" Rev. Diabet Stud. 2014. Vol. 11. N. 1. P. 19-34).

It is known from the literature that the addition of zinc sulfate can increase the production of IgG antibodies when added to the culture medium. Several studies have investigated the effect of microadditives on serum-free media. Trace minerals often replace the use of serum in serum-free media. Particularly important is the presence in the media of such elements as Zn, Cu, Se, as well as tricarboxylic acids necessary for normal cell growth, in particular for maintaining the functioning of enzymes (Arora M. Cell Culture Media: A Review. Mater Metods. 2018. Vol. 3. N175).

The composition of the nutrient medium is known, including succinic acid (RU 2558253 "Nutrient medium for suspension cultivation of mammalian cells", 2014-02-10 or RU 2014104467 A, 2015-08-20). However, this medium contains glutamine, and the content of succinic acid does not exceed 0.06 mM, which does not provide the proper cultivation time and productivity. In addition, the invention shows the use of the composition only for the cultivation of BHK-21 (Syrian hamster kidney cells).

Also known is a composition for the cultivation of mammalian cells, using iron chelate, zinc chelate and dextran sulfate in the composition of protein-free media (EP 1482031 A1 "Serum-free mammalian cell culture medium, and uses thereof", 1996-08-30, 2004-12-01 ). The described method is taken as a prototype of the invention. A common feature is the use of iron and zinc ions in the composition. The disadvantage is the presence of iron and zinc in the composition of chelates and dextran sulfate in a multicomponent medium, which do not prevent cell aggregation.

Thus, the known compositions (formulations) do not provide the possibility of obtaining a homogeneous suspension culture. The use of the components did not prevent the formation of cell aggregates in the cultivation medium, and as a consequence did not contribute to the improvement of the growth and density of cell cultures.

Brief Description of Drawings

The invention is illustrated by the following graphic materials:

FIG. 1 Influence of fibroblast growth factor (FGF-2) on the viability and concentration of cells of a stable cell line producing recombinant monoclonal antibodies of the dimeric form of the IgA2m1 isotype.

FIG. 2 Influence of adding a composition of fibroblast growth factor and zinc sulfate to the culture medium on the productivity of a stable cell line producing recombinant monoclonal antibodies of the dimeric form of the IgA2m1 isotype.

FIG. 3 Comparison of the effect of the composition of dextran sulfate and iron citrate on the growth and productivity of stable cell lines of IgG1 and IgA1 isotypes.

Disclosure of invention

The technical problem solved by the invention is the increase in productivity during the cultivation of cell lines producing antibodies based on CHO cells in basic media.

The technical result of the claimed invention is to prevent aggregation of culture cells and obtain homogeneous suspension cell cultures, which makes it possible to use them for efficient expression of stable cell lines producing both IgG and IgA. This is especially true for cell lines producing dimeric forms of IgA, which, due to the peculiarities of their structure and glycosylation, are prone to the formation of cell aggregates, which leads to a decrease in the density and viability of cells, and, as a result, to a decrease in productivity. This makes it possible to replace expensive nutrient media and additives for the cultivation of cell lines producing both IgG and IgA.

The invention includes the developed multicomponent additives to base serum-free media IMDM (Iskov's medium IMDM - modification of Dulbecco's medium) or DMEM, which include: an aqueous solution of fibroblast growth factor-2 (FGF-2), zinc salts, iron citrate and dextran sulfate. This invention increases the homogeneity of the suspension culture, increases the density of the culture, increases the length of time of cultivation and increases the level of expression of recombinant antibodies of the IgG and IgA classes.

This solution was found using compositions to the base environment. As a basic medium, we used IMDM medium (Gibco, USA); Biolot, RF and PanEco, RF.

The claimed composition for preventing aggregation, increasing the density and uniformity of the culture, increasing productivity includes aqueous solutions of fibroblast growth factor-2 with a concentration of 5.6-11.2 ng / ml and zinc sulfate with a concentration of up to 8.3 μM / ml, while additionally using dextran sulfate and iron citrate taken at a concentration of 0.15-0.33 mM / ml and dextran sulfate at a concentration of up to 50 μg / ml.

The best result is achieved when using a composition containing fibroblast growth factor (FGF-2), zinc sulfate, dextran sulfate and iron citrate, while the FGF-2 solution is contained in a concentration of 11.2-22.4 ng / ml, iron citrate - 0.15-0.33 mM / ml, zinc sulfate - up to 8.3 μM / ml, dextran sulfate - up to 50 μg / ml.

For the first time, it was found that the use of the proposed multicomponent additive to the base medium makes it possible to increase the density, cultivation time and productivity of cell lines producing recombinant antibodies of cells during their suspension cultivation.

The well-known nutrient media and growth additives offered for the cultivation of CHO cells by the manufacturers Lonza and Invitrogen are costly in terms of finance and delivery time, since they are manufactured abroad and imported into the Russian Federation. For example, CD OptiCHO medium (Invitrogen, USA) is intended for cultivation of CHO DG44 cells. However, biotechnological manufacturing requires a large amount of media and additives, which increases the cost of antibody-based drugs.

The invention makes it possible to obtain a high-quality and cost-effective nutrient medium.

Implementation of the invention

The invention is illustrated by examples of specific implementation, which describe in more detail the possibility of implementing the claimed invention with the achievement of the claimed technical result.

Example 1. Increasing the concentration, viability of cells and productivity of a stable cell line producing recombinant monoclonal antibodies of the dimeric form of the IgA2m1 isotype by adding fibroblast growth factor (FGF-2) to the culture medium.

The experiment uses a suspension stable cell line - a producer of recombinant antibodies of the IgA2m1 isotype, based on the CHO DG44 (dhfr-) cell line (Chinese hamster ovary cells). Cell cultures are grown in serum-free DMEM / F12 basic medium (PanEco, RF). To determine the effect on the productivity, density and viability of cell cultures of cultivation, the experiment is carried out in the format of static cultures in 6-well plates under standard growing conditions at 37 ° C and 8% CO2 and 96% humidity with continuous stirring at a speed of 130 rpm.

To compare the effect of influencing the growth of cell cultures-producers of IgA2m1-isotype antibodies as a growth stimulant, fibroblast growth factor (FGF-2), obtained in the laboratory of protein engineering of the Institute of Bioorganic Chemistry, RAS and the addition of insulin-transferrin-sodium selenite (ITS) from Gibco ". The standard ITS supplement is usually used to increase culture density and stimulate growth, but is not cheap and increases the cost of producing therapeutic immunoglobulins.

To study the effect on the growth of CHO cells, fibroblast growth factor FGF-2 is added to the medium for cultivation of DMEM / F12 at different concentrations: at a final concentration of 5.6 ng / ml and 11.2 ng / ml.

A cell culture cultured in a medium supplemented with insulin-transferrin-selenite (ITS) was used as a control. The increase in the density of the cell culture occurs on the 5th day of cultivation, and the increase in growth depends on the concentration of fibroblast growth factor in the culture. FIG. 1A shows the increase in cell culture density upon addition of FGF-2 to DMEM / F12 base medium. When a solution of fibroblast growth factor was added to the medium at a concentration of 5.6 ng / ml, the culture density increased by 6 times, and at a FGF-2 concentration of 11.2 ng / ml, by 6.7 times, compared with the control sample.

The use of fibroblast growth factor as a growth supplement also shows its prolonged effect on maintaining cell culture density and growth time for 9 days in the base medium, in contrast to the ITS supplement. When ITS is added to the medium on the 9-10th day of cultivation, there is a significant decrease in cell density and a virtual death of the cell culture. (Fig. 1A)

FIG. 1B demonstrates the effect of fibroblast growth factor on cell viability. After 9 days of cultivation, all samples that are cultured on a medium using fibroblast growth factor have a high degree of viability - over 85%, the highest degree of viability of about 94% remains only for the sample to which the FGF-2 preparation is added at a concentration of 11.2 ng / ml ... (Fig. 1B)

Measurement of the concentration of cells and their viability is carried out using a dye of 0.4% trypan blue solution in a Goryaev chamber.

Example 2. Influence of the composition of a solution of fibroblast growth factor and zinc sulfate on an increase in the productivity of a stable cell line expressing recombinant monoclonal antibodies of the dimeric form of the IgA2m1 isotype.

The experiment uses a suspension stable cell line producing recombinant antibodies of the IgA2m1 isotype, based on the CHO DG44 (dhfr-) cell line (Chinese hamster ovary cells). Cell cultures are grown on a serum-free base IMDM medium (PanEco, Russia). To determine the effect on the productivity, density and viability of cell cultures of cultivation, the experiment is carried out in the format of static cultures in 6-well plates under standard growing conditions at 37 ° C and 8% CO2 and 96% humidity with continuous stirring at a speed of 130 rpm.

To determine the effect on the production of IgA antibodies, a solution of zinc sulfate, as well as a combination of fibroblast growth factor and zinc sulfate, are added to the PanEco serum-free base medium IMDM. A sterile zinc sulfate solution was added at a concentration of 8.3 μM / ml, and a FGF-2 solution at a concentration of 11.2 ng / ml. As a control sample, a cell culture grown in a basic IMDM medium without additives is used.

Productivity is determined by ELISA on the 6th and 10th days of cultivation. FIG. 2 shows the effect of the composition on the increase in productivity in comparison with the control sample. When using the composition of fibroblast growth factor and zinc sulfate on the 10th day of growth, the increase in the concentration of antibodies compared to the control was 30%.

In this case, the addition of only a solution of zinc sulfate increases productivity on the 6th day of cultivation compared to the control sample and the sample cultivated on the medium with the composition. However, on the 10th day, the concentration of antibodies in the medium becomes lower compared to the data of the control sample.

The use of fibroblast growth factor promotes both an increase in cell density and the duration of their cultivation and, as a consequence, an increase in productivity.

Example 3. Influence of the composition of dextran sulfate with iron citrate on the proliferative activity and productivity of stable cell lines of two immunoglobulin isotypes: IgG1 monomer and IgA1 dimeric form.

The effect of the composition on the homogeneity of the suspension culture is demonstrated on stable cell lines producing different classes of immunoglobulins: IgG1 and IgA1. To determine the effect on both productivity and culture time, the experiment is carried out in static culture format in 6-well plates under the standard growing conditions mentioned above. Unsupplemented IMDM medium was used as a culture control. The anti-clumping reagent from Gibco is added to the base medium as a reference.

As a composition for the base medium, the sodium salt of dextran sulfate at a final concentration of 50 μg / ml and iron citrate at 2 concentrations are used: 0.33 mM / ml or 0.15 mM / ml. The formation of aggregates in cell cultures and the duration of their cultivation are influenced by the composition of the sodium salt of dextran sulfate at a final concentration of 50 μg / ml and iron citrate at two final concentrations: 0.33 mM / ml and 0.15 mM / ml. In the control cell culture sample growing on the basic IMDM medium without additives, and the cell culture sample growing on the IMDM basic medium with "Anti-clumping reagent", cell aggregates were formed on the 7th day of culture, while the samples growing on the base medium with the investigated additives have a homogeneous format. However, differences should be noted for the cell lines producing antibodies of IgG1 and IgA1 isotypes.

FIG. 3, A and B shows a diagram of the growth and vitality of crops.

For a cell line producing antibodies of the IgG1 isotype, the highest density of 3.9 × 10 ⁶ cells / ml is shown by samples with the addition of sodium salt of dextran sulfate at a final concentration of 50 μg / ml, which is a high indicator for cultivation in the medium (Fig. 3, A).

The results obtained by culturing a cell line producing antibodies of the IgA1 isotype demonstrate that the cell density both with and without the addition of the compositions have similar values. However, the culture samples to which the combinations of sodium dextran sulfate at a final concentration of 50 μg / ml and iron citrate in 2 concentration variants are added continue to grow on the 11th day of cultivation, while the viability of other samples decreases below 40%. Thus, the addition of sodium salt of dextran sulfate and iron citrate increases the uniformity of the culture and increases the time of cultivation of the cell line producing IgA1 antibodies (Fig. 3, B)

FIG. 4, A and B shows a diagram of productivity for cell lines producing IgG1 and IgA1 isotypes.

The productivity of a cell line producing IgA1 when cultured in a medium supplemented with sodium salt of dextran sulfate at a final concentration of 50 μg / ml and iron citrate at a concentration of 0.33 mM exceeds the values for a control culture sample growing in a medium without additives and a sample cultured in a medium with by adding "Anti-clumping reagent", samples with the addition of dextran sulfate or with a composition of dextran sulfate with iron citrate at a concentration of 0.33 mM / ml - by 62%, 33%), 90% and 123%, respectively (Fig. 4, B) ...

The productivity results for the cell line producing antibodies of the IgG1 isotype demonstrate that the productivity of the culture when using the composition with the addition of sodium dextran sulfate at a concentration of iron citrate of 0.33 mM exceeds the control and other samples (Fig. 4, A).

Claims (8)

1. Composition for introduction into a serum-free basic growth medium to prevent aggregation and increase the productivity of CHO cell lines - producers of monomeric and dimeric forms of recombinant monoclonal antibodies of IgG and IgA classes, based on a solution of fibroblast growth factor-2 (FGF-2) and zinc sulfate at the following ratio of components: aqueous solutions of fibroblast growth factor-2 - from 5.6 to 11.2 ng / ml and zinc sulfate with a concentration of up to 8.3 μM / ml. 2. A composition according to claim 1, characterized in that IMDM or DMEM is used as a serum-free base medium. 3. Composition for introduction into a serum-free basic growth medium to prevent aggregation and increase the productivity of CHO cell lines - producers of monomeric and dimeric forms of recombinant monoclonal antibodies of IgG and IgA classes, based on a solution of dextran sulfate and ferrous citrate (Fe ²⁺ ) with the following ratio components: dextran sulfate - up to 50 μg / ml and ferrous citrate (Fe ²⁺ ) - from 0.15 to 0.33 mM / ml. 4. A composition according to claim 3, characterized in that IMDM or DMEM is used as a serum-free base medium.

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