A method for industrial production of recombinant granulocyte - macrophage colony stimulating factor human

 

(57) Abstract:

The invention relates to biotechnology and can be used in the production of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on an industrial scale. Recombinant Escherichia coli strain SG 20050/R DM grown in standard LB-medium at a temperature of 25-37°C. the Cultivation is carried out in the presence of ampicillin and tetracycline. The concentration of dissolved oxygen 10-40% of saturation. The cultivation can be carried out at a temperature of 29+1°C. the Cultivation may be carried out in three stages, with successive increase in the volume of the nutrient medium from 50 ml to 100 L. the Invention allows for the maximum accumulation of glycoprotein GM-CSF in bacterial cells. 2 C.p. f-crystals, 3 tables.

The invention relates to biotechnology, specifically to the production of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on an industrial scale.

GM-CSF stimulates the growth of myeloid cells predecessors, but also enhances the effector function of Mature monocytes and neuropile. His sources are T-lymphocytes, endothelial cells and fibroblasts.

and eosinophilic leukocytes and macrophages. He is also involved in many biological and immunological functions, such as changing the morphology of effector cells, their mobility, the cytotoxic activity and phagocytosis. The ability of GM-CSF to stimulate the growth of granulocytes and macrophages makes possible its clinical use to reduce the side effects of the beam - and chemotherapy, enhancing resilience to infections, therapy side effects of bone marrow transplantation. The use of recombinant GM-CSF is promising to speed up the recovery of hematopoiesis after chemotherapy, which have significant damaging effect on the bone marrow. It also shows that the introduction of recombinant GM-CSF cancer patients leads to activation of endogenous monocytes and macrophages.

A method of obtaining another polypeptide - granulocyte colony stimulating factor using E. coli strain [1]. The method involves fermentation procuring master in the presence of >1 amino acids in amounts that increase the accumulation of polypeptide. You can add in a nutrient medium threonine and/or leucine at a concentration of 1.25 - 5 g/l Threonine and/or leucine in these quantities inhibits Rho the article of owner - the hydrolysate coseine or isoleucine. The accumulation of the polypeptide is 20 to 40 g/L.

A method of obtaining GM-CSF using E. coli strain SG963 [2, prototype]. Transformed E. coli cells SG963 the one grown in the amounts of about 3 to 5 liters at a temperature of 28oC in LB medium with antibiotics. The content of ampicillin in the environment of 50 mg/ml kanamycin (10%) 200 mg/ml, the Cells are grown to an optical density of 30 units, at a wavelength of 650 nm. After temperature shock at 42oC and the introduction of additional protection cells grow another three hours.

Analysis on productivity in GM-CSF is conducted in the following periods of time: at the time of seeding, 4 hours later, an hour after the induction of high temperature (42oC) and after 3 h after induction. The analyses show that the accumulation of target glycolysated in this way is 8-9% of the total protein.

In this paper does not specify the amount of the nutrient medium, the type of fermenter. From the process conditions specified temperature is not specified the amount of the medium fed after thermal shock.

The disadvantages of this method are

- the presence of stage induction;

- the presence of additional operations in the form of making pKa: 8-9% compared to 15% of the total protein of the proposed method.

The objective of the invention is the development and the search for optimal conditions of industrial cultivation, allowing maximum accumulation of glycoprotein GM-CSF in bacterial cells.

The problem is solved in that in the method for industrial preparation of GM-CSF by submerged cultivation of recombinant producer strain E. coli on standard LB-medium at a temperature of 25 - 37oC, according to the invention as a producer strain using recombinant Escherichia coli strain SG 200500/p280 GM. Cultivation conduct in-depth method in the presence of ampicillin and tetracycline in a nutrient medium containing casein hydrolysate, Baker's yeast extract and sodium chloride at an initial pH of 7.0 to 7.2. The optimal cultivation temperature is 29 1oC and the concentration of dissolved oxygen not less than 5.0% and not more than 50% of saturation. The cultivation is carried out in three stages, with successive increase in the volume of the nutrient medium from 50 ml to 100 l and achieving an optical density of biomass at each stage of not less than 1 OE at a wavelength of 540 nm and the length of kivity 1 see three-stage cultivation is performed to obtain the optimal seeding concentration and allocation of m is about 15% of the total cellular protein.

When using E. coli strain SG 20050/p280 GM there is no need of applying thermal shock to induce glycoprotein GM-CSF.

The strain developed by the all-Union scientific research Institute of molecular biology NGOs Vector (SRC VB "Vector") and deposited in the all-Union collection of industrial microorganisms Institute "Vniigenetika" under number B-6613.

The method is as follows.

Prepare a nutrient medium with antibiotics in tap water, purified from mechanical and organic impurities by ultrafiltration for the separation apparatus with a pore diameter of 5 microns (e.g., ultrafiltration apparatus Spa "Khimvolokno").

Sterilized nutrient medium known method of sterilizing filtration (for example, using cartridge filters firm "Millipor" or "Bladisat").

Laboratory fermenter with a volume of 5 l of sterile nutrient medium with antibiotics inoculated with E. coli cells SG 20050/p280 GM. The volume of the nutrient medium of 3.0 liters. For inoculation of the fermenter using colonies grown on agar nutrient medium, suspended in 50 ml of culture medium. Cultivation of conduct for 16-24 h at constant PE is possible. The initial pH of the medium to 7.0 to 7.2. During cultivation, the pH value does not regulate. The cultivation of finish upon reaching an optical density of not less than 1 OE at a wavelength of 540 nm and the length of the cuvette 1 cm

Grown inoculum (optical density, measured on a spectrophotometer at less than 1 OE at a wavelength of 540 nm and the length of the cuvette 1 cm) sowing industrial fermenter with a capacity of 250 l and cultured under the following conditions: the volume of nutrient medium (100 l; temperature 25 - 37oC; initial pH 7,0 - 7,2; constant stirring and aeration, providing the concentration of dissolved oxygen not less than 10 and not more than 30% of saturation.

Immediately after planting and then every 4 hours take a sample of the culture fluid and analyzed for the following parameters: the optical density, the sterility of the suspension, pH.

The process is complete when you reach a total optical density of not less than 1 OE, at a wavelength of 540 nm.

The accumulation of glycoprotein GM-CSF is thus 20-70 mg per 1 liter of nutrient medium.

The essential features of the invention are:

- the use of dissolved oxygen concentration in the range of 10 - 40% of nasasatellite on the compliance of the claimed invention, the criterion of "novelty" and "inventive step".

The method is illustrated by the following examples of specific performance.

Example 1. The search for the optimum dissolved oxygen concentration.

Cultivation in a fermenter with a capacity of 250 l is carried out at constant temperature and different values of dissolved oxygen concentration.

The volume of nutrient medium (100 l), the temperature of cultivation 291oC, initial pH of 7.0 to 7.2. The results of the experiments are shown in table 1.

From table 1 it is evident that the maximum accumulation of the target glycoprotein is when the dissolved oxygen concentration from 10 to 30% of saturation.

Moving beyond the specified range leads to a significant reduction of the yield of the target product.

Example 2. The choice of the optimal cultivation temperature.

Cultivation in a fermenter with a capacity of 250 l is carried out at various temperatures and at a constant value of dissolved oxygen concentration.

The volume of nutrient medium (100 l), dissolved oxygen concentration of 10 to 40% of saturation, the initial pH of 7.0 to 7.2.

The results of the experiments are shown in table 2.

Table 2 shows that the maximum accumulated oC leads to a significant increase in time of cultivation and declining productivity, increasing the temperature up to 37, 1oC leads to a sharp decrease in the productivity of the culture of the target protein.

From table 3 it can be seen that an industrial method to produce GM-CSF person using Escherichia coli SG 20050/p280 GM allows to obtain a higher yield of the target protein with less material and energy costs than in the cultivation of recombinant E. coli bacteria SG963.

References

1. UK application N 2241505 "Fermentation method of producing polypeptides", C 12 P 21/00, published RJ ISM N 3, 93.

2. The international application N 87/02060 Human granulocyte-macrophage colony-stimulating factor-like polypeptides and process for producing them in high yields in microbial cells", C 12 N 15/00, published RJ ISM N 1, 88.2

1. A method for industrial production of recombinant granulocyte-macrophage colony stimulating factor human by submerged cultivation of recombinant producer strain of Escherichia coli on standard LB-medium at a temperature of 25 - 37oC, characterized in that as a producer strain using recombinant Escherichia coli strain SG 20050/R GM, cultivation is carried out in the presence of ampicillin and tetrazine fact, the cultivation is carried out at a temperature of 29 1oC.

3. The method according to p. 1, wherein the cultivation is carried out in three stages, with successive increase in the volume of the nutrient medium from 50 ml to 100 l and achieving an optical density of biomass at each stage for at least 1 SECOND at a wavelength of 540 nm and length of 1 cm cuvette

 

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