The method of producing mielopid

 

(57) Abstract:

The invention relates to medicine, namely to methods of producing biologically active preparations from bone marrow of animals. The method includes preparing a suspension of bone marrow cells, their incubation in a nutrient medium, separating the supernatant and the subsequent allocation of a target product, for suspension and incubation of the cells using an empty environment, and the selection of the target product is carried out by passing the supernatant through a biospecific sorbent Adam and extraction of adsorbed material 50-80% solution of ethanol in 0.1% aqueous acetic acid solution. The composition of the preferred option nutrient medium. The method allows to increase the yield of the target product while simplifying the manufacturing processes and reduce labor costs. 1 C.p. f-crystals, 1 table.

The invention relates to a technology for obtaining an immunomodulating drug from the bone marrow of animals.

A known method of producing Mielopid, comprising preparing a suspension of bone marrow cells into a colored 199 environment, their incubation in the same environment, the separation of the supernatant and the allocation of money is eriley water, sterile filtration and gel chromatographic separation on Sephadex G-25 using phosphate buffer (EN 2041716 C1, 20.08.1995).

The disadvantages of this method are the high complexity and the complexity of the technology associated with the need for purification of the target product from the one used suspendirovanie and incubation of the cells of the dye phenol red, part 199 medium, and low yield of the target product, forming 250-300 doses of 1·10¹⁰cells.

The technical result of the invention is to reduce labor costs, the simplification of technology and the increase of the yield of the target product.

This result is achieved in that in the method of producing Mielopid, comprising preparing a suspension of bone marrow cells, their incubation in a nutrient medium, separating the supernatant and the subsequent allocation of a target product, according to the invention for the suspension and incubation of the cells using an empty environment, and the selection of the target product is carried out by passing the supernatant through a biospecific sorbent Adam and extraction of adsorbed material with an aqueous solution of ethyl alcohol into acetic acid.

Priya and incubation environment, containing the following components (mg/DM³:

CaCl₂200

Fe(NO₃)₂·9H₂O 0,1

KCl 400

MgSO₄·7H₂O 200

NaCl 6800

NaHCO₃2200

NaH₂PO₄·H₂O 140

Adenine 10

Sodium salt of denitrificate 10

Denisova acid 0,2

Cholesterol 0,2

2-deoxyribose 0,5

Glucose 1000

Glutathione 0,05

Guanine chloride 0,3

Gipoksantin 0,3

Ribose 0,5

Sodium acetate 50

Thymine 0,3

Twin 80 20

Uracil 0,3

Xanthine 0,3

DL-alanine 50

L-arginine chloride 70

DL-aspartic acid 50

L-cysteine chloride of 0.1

L-cystein 20

L-glutamine 100

DL-glutamic acid·H₂O 150

Glycine 50

L-histidine chloride 20

L-hydroxyproline 10

DL-isoleucine 40

DL-leucine 120

L-lysine chloride 70

DL-phenylalanine 50

DL-methionine 50

L-Proline 40

DL-serine 50

DL-threonine 60

DL-tryptophan 20

L-tyrosine 40

DL-valine 50

p is

Choline chloride 0,5

Folic acid is 0.01

i-Inositol 0,05

Menadione 0,01

Nicotinamide 0,025

Nicotinic acid 0,025

Sodium salt of Pantothenic acid 0.01

Pyridoxal chloride 0,025

Pyridoxine chloride 0,025

Riboflavin 0,01

Thiamine chloride in 0.01

p-tocopherolacetate sodium 0,01

Vitamin a IS 0.1

Another preferred option is provided the use of 50-80% ethanol in 0.1% aqueous acetic acid solution.

The method is implemented as follows.

Bone marrow cells extracted from crushed animal bones by suspension in a liquid unpainted environment with subsequent filtering, centrifugation and dilution unpainted nutrient medium containing a mixture of vitamins, amino acids and components of nucleic acids in salt Hanks solution, preferably in the above mixing ratio. Then carry out incubation of cells with the traditional parameters of the process and separate the supernatant by centrifugation. From the supernatant produce the target product by its adsorption on biospecific sorbent Adam (IDT with the concentration of ethyl alcohol 50-80% by volume in 0.1% by volume aqueous solution of acetic acid. Using the above composition of the extractant minimizes its consumption, subsequent labor and energy costs on the selection of the target product and the loss of its active components, which increases the yield of the target product.

Example 1.

8 kg tipped rib bones of pigs crushed in sterile conditions. They added 3 l of sterile liquid nutrient medium of the above composition. The suspension is shaken on a shaker at C for 1 hour to flush the bone marrow cells. Cells separated by filtration and washed by centrifugation in the same nutrient medium at 4C. The obtained cells are diluted with the same medium, bringing their concentration up to 1·10⁹cells/DM³and incubated for 18-20 hours at S. Next, separate the supernatant by centrifugation at 5000 rpm and a temperature of 4C. The supernatant is passed through a column Packed with biospecific sorbent Adam. The adsorbed material is extracted with 6S, using as extractant 50-80% solution of ethanol in 0.1% aqueous acetic acid solution. At concentrations above this extraction does not occur. At concentrations below this dramatically increases the flow of extractant that when posleduyu activity of the drug. The product yield factor stimulation 1,8 ranges from about 40,000 doses of the drug with the boundary values of the concentration of the extractant to 50,000 at a concentration of ethyl alcohol in the extractant 60% by volume.

The biological effect of the target product is estimated by the stimulation of antibody productions at the peak of the secondary immune response to sheep erythrocytes (Jerne, N. K., Nordin A. A.// Science, 1983, v. 140, p. 405), by adding the test material in the cell culture of lymph nodes obtained from mice on the fourth day after the second immunization. The stimulation coefficient calculated as the ratio of the number of antibody productive cells (AFC) in the culture with the test sample to the amount of the KLA in control. The results are given in the table.

Thus, when simplified technology and reduced labor costs, the proposed method allows to increase the output of Mielopid.

Example 2.

The product prepared according to example 1, but using a nutrient medium containing the following components (mg/DM³:

CaCl₂200

Fe(NO₃)₃·N₂O 0,1

KCl 400

MgSO₄·7H₂O 200

NaCl 5400

Panso₃3700

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L-arginine chloride 84

L-cystein 48

L-glutamine 780

Glycine 30

L-histidine chloride water 42

L-isoleucine 105

L-leucine 105

L-lysine chloride 146

L-methionine 30

L-phenylalanine 86

L-serine 42

L-threonine 85

L-tryptophan 18

L-tyrosine 72

L-valine 94

Choline chloride 4

Folic acid 4

i-Inositol 7,2

Nicotinamide 4

Sodium salt of Pantothenic acid 4

Pyridoxal chloride 4

Riboflavin 0,4

Thiamine chloride 4

The product yield factor stimulation 1,8 ranges from 20000 to 32000 doses of the drug at various concentrations of ethanol in the extractant.

Example 3.

The product prepared according to example 1, but using a nutrient medium containing the following components (mg/DM³:

CA(NO₃)₂·4H₂O 100

KCl 400

gSO₄·7H₂O 100

NaCl 6000

NaHCO₃2000

Na₂HPO₄801

Glucose 2000

Glutathione 1

Garamycin 15

L-arginine chloride 342

L-asparagine 50

Glycine 10

L-histidine chloride 18,2

L-hydroxyproline 20

L-isoleucine 50

L-leucine 50

L-lysine chloride 50

L-methionine 15

L-phenylalanine 15

L-Proline 20

L-serine 30

L-threonine 25

L-tryptophan 5

L-tyrosine 20

L-valine 20

p-aminobenzoic acid 1

Biotin 0,2

Choline chloride 3

folic acid 1

i-Inositol 36

Nicotinamide 1

Sodium salt of Pantothenic acid 0,75

Pyridoxal chloride 1

Riboflavin 0,2

Thiamine chloride 1

Vitamin B₁₂0,008

The product yield factor stimulation 1,8 ranges from 12000 to 25000 doses of the drug at various concentrations of ethanol in the extractant.

1. The method of producing Mielopid, comprising preparing a suspension of bone marrow cells, their incubation in a nutrient medium, separating the supernatant and the subsequent allocation of a target product, characterized in that the suspension and incubation of the cells using an empty environment, and the selection of the target product is carried out by passing the supernatant is alcohol in 0.1% aqueous solution of acetic acid.

2. The method according to p. 1, characterized in that the suspension and incubation of bone marrow cells using a medium containing the following components (mg/DM³:

l₂200

Fe(NO₃)₂·9H₂O 0,1

KCl 400

MgSO₄·7H₂O 200

NaCl 6800

NaHCO₃2200

NaH₂PO₄·H₂O 140

Adenine 10

Sodium salt of denitrificate 10

Denisova acid 0,2

Cholesterol 0,2

2-Deoxyribose 0,5

Glucose 1000

Glutathione 0,05

Guanine chloride 0,3

Gipoksantin 0,3

Ribose 0,5

Sodium acetate 50

Thymine 0,3

Twin 80 20

Uracil 0,3

Xanthine 0,3

DL-alanine 50

L-arginine chloride 70

DL-aspartic acid 50

L-cysteine chloride of 0.1

L-cystein 20

L-glutamine 100

DL-glutamic acid·N₂About 150

Glycine 50

L-histidine chloride 20

L-hydroxyproline 10

DL-isoleucine 40

DL-leucine 120

L-lysine chloride 70

DL-phenylalanine 50

DL-methionine 50

L-Proline 40

p-Aminobenzoic acid 0,05

Ascorbic acid is 0.05

Biotin 0,01

Calciferol 0,1

Choline chloride 0,5

Folic acid is 0.01

i-Inositol 0,05

Menadione 0,01

Nicotinamide 0,025

Nicotinic acid 0,025

Sodium salt of Pantothenic acid 0.01

Pyridoxal chloride 0,025

Pyridoxine chloride 0,025

Riboflavin 0,01

Thiamine chloride in 0.01

p-Tocopherolacetate sodium 0,01

Vitamin A Is 0.1