RU2298409C2 - Method for preparing immune lactoglobulin - Google Patents

Abstract

FIELD: medicine, immunology.

SUBSTANCE: invention relates to a method for preparing immune lactoglobulin. Method involves ultrafiltration that is carried out for two steps in flow circulating under pressure and perpendicularly to filtrate flow. At the first step filtration is carried out on membrane cassette modulus carrying out clearing lactoserum and purifying its from far and casein. The second step is carried out in modulus with hole fibers carrying out washing out of working mixture from low-molecular components with 0.4% sodium chloride solution at pH = 5.0-6.0 followed by protein concentrating in the limits 4.5-5.5%, and prepared filtrate is subjected for sterilization and bottled into flasks in a liquid state. Washing out the lactoglobulin concentrate prepared on the second step from low-molecular components is carried out three times by its filtering on a fiber modulus with addition of three-fold volume of 0.4% sodium chloride solution at pH = 5.0-6.0. Except for, the necessary protein concentration in a lactoglobulin preparation is obtained by measurement of the final volume of lactoglobulin solution. Invention provides simplifying technology in preparing immune lactoglobulin. Invention can be used in manufacturing lactoglobulin for immunotherapy aims.

EFFECT: improved preparing method.

2 tbl, 3 dwg, 1 ex

Description

The present invention relates to medicine, in particular to immunology, and can be used in the production of lactoglobulin for immunotherapy and immunoprophylaxis of bacterial and viral diseases, namely intestinal infectious diseases and intestinal dysbiosis.

A known method of producing lactoglobulin anti-lipid cow for oral use (see USSR autosw. No. 1743025, class A61K 35/20, 06.06.1989), which consists in isolating colostrum lactic acid, fractionating it in the cold with ethanol, separating the precipitate by centrifugation, dilution of the precipitate, clarifying and sterilizing filtration, and after clarifying filtration, Trilon B and sorbitol are added to the lactoglobulin solution to a concentration of 0.05-0.15% and 0.5-1.5%, respectively.

The disadvantage of this method is that at the stage of alcohol deposition using the Kohn method, which adversely affects the final product, and the lyophilization process leads to a decrease in biologically active substances of enzymatic nature, which provide bactericidal properties of lactoglobulins, which reduces its quality.

In addition, the technology for producing lactoglobulin is very laborious, unproductive, requires expensive equipment, refrigeration units and a large amount of alcohol.

For the prototype, a method for producing an immunoglobulin preparation was selected (see RU, Pat. No. 2108794, class A61K 35/20, 04/20/1998), which includes the use of milk or colostrum of immune cows as the initial product, followed by removal of fat and casein by acid coagulation, while the obtained serum is treated with caprylic acid at the rate of 2-10 ml of acid per 100 ml of serum, coagulating non-immunoglobulin proteins and lipids, after which the precipitate is removed.

However, carrying out the precipitation of the components with caprylic acid will also not make it possible to obtain a high-quality preparation because the acid itself has a negative effect on the enzymatic activity of lactoglobulin, in particular lactoperoxidase, which provides a non-specific protective effect of lactoglobulins.

In addition, the known method can be used only in laboratory conditions and requires a large amount of caprylic acid, namely, 100 liters of serum requires 10 liters of acid.

The technical task of the invention was to simplify the production technology of immune lactoglobulin with high quality of the final product.

The problem is achieved in that in the known method for producing immune lactoglobulin, which includes obtaining lactoserum from milk or colostrum from immune cows, followed by clarification of the lactoserum and its concentration by ultrafiltration, the latter is carried out in two stages in a circulating pressure stream perpendicular to the filtrate current, while the first stage of filtration is carried out on a membrane cassette module, producing clarification of the lactoserum, purifying it of residues of fat and casein, and the second stage of on a module with hollow fibers, washing the working mixture from low molecular weight components with a 0.4% sodium chloride solution pH 5.0-6.0 and concentrating the protein in the range 4.5-5.5%, and the resulting filtrate is sterilized and liquid form is poured into bottles.

In this case, the washing of the lactoglobulin concentrate obtained in the second stage from the low molecular weight components is carried out three times, filtering it on a fiber module with the addition of a three-fold volume of a 0.4% sodium chloride solution.

In addition, the required protein concentration in lactoglobulin is obtained from the calculation of the final volume of the lactoglobulin solution according to the formula:

где

Where

V ₀ is the final volume of the lactoglobulin solution, in liters;

x ₁ - protein concentration obtained in solution, in%;

x ₂ - the required amount of protein, in%;

V ₁ - the volume of lactoglobulin concentrate, in liters,

and from the difference in volumes of the final product V ₀ and concentrate V ₁ , the volume of the required solution of 0.4% sodium chloride is obtained.

The essence of the invention is illustrated in the drawing, where

figure 1 shows the installation for implementing the method of obtaining immune lactoglobulin;

figure 2 - diagram of the passage of the working fluid through the cassette module;

figure 3 - diagram of the passage of the working fluid through the module with hollow fibers.

The installation consists (see Fig. 1) of a container 1 for lactoserum 2. Through flexible hoses 3, 4 of silicone rubber, the container 1 is connected respectively to a peristaltic pump 5 and to an ultrafiltration membrane cartridge module 6. The latter is equipped with a hose 7 that interacts with the peristaltic pump 5, and a hose 8 connected to the container 9.

Cassette module 6 is a set of ten membrane cassettes 10, each of which is a plate with pores with a diameter of 0.22 μm (see figure 2) mounted horizontally in the module. Capacity 9 using a flexible hose 11 interacts with the ultrafiltration module 12 with hollow fibers 13 (see figures 1, 3), mounted vertically for the supply of processed lactoserum 2.

Between the tank 9 and the module 12, a second peristaltic pump 14 is placed, which is connected by a hose 15 to the said tank 9, and by a hose 16 to the module 12. At the same time, a tank 17 is installed behind the module 12, which is connected with the hose 18.

To create pressure in the system, hose valves are used (not shown in the drawing), which are installed at the output of ultrafiltration modules 6, 12. The pressure is controlled by pressure gauges. The connection of the hoses with the installation elements is carried out by means of quick-release clamps.

In addition, the process control is carried out from control units located on the front panel of the peristaltic pumps 5, 14.

The proposed method is implemented as follows.

Milk or colostrum from immune cows is separated and pepsinized to produce lactoserum 2. Lactoserum 2 with residues of fat and casein is poured into tank 1 (see Fig. 1) and a peristaltic pump 5 is turned on.

The working mixture (lactoserum 2) flows under pressure into the pores of the membrane cassettes 10 perpendicular to the filtrate current (see FIG. 2) and begins to circulate through the hoses 3, 4, 7 several times in a closed loop. The filtrate leaving the module 10 through the hose 8, after passing through the ultrafiltration module 10 is collected in the tank 9.

Thus, after carrying out the first stage of ultrafiltration, clarified lactoserum with a pH of 5.0-6.0 is obtained without a residue of fat and casein.

The second stage of ultrafiltration is to clean the working mixture from low molecular weight components. To do this, from the tank 9 (see Fig. 1), the working fluid is supplied through the hoses 15, 16 with a peristaltic pump 14 to the module 12. The mixture is supplied under pressure through the hollow fibers 13 (see Fig. 3), while separating the substance according to molecular parameters. Using pressure drops, a driving force is created on both sides of the fibers to transfer matter through the pores of the hollow fibers. The low molecular weight filtrate enters through a hose 18 (see Fig. 1) into a container 17, and the purified concentrate into a container 9.

Washing the working mixture from low molecular weight components is carried out three times with three times the volume of a 0.4% sodium chloride solution, which is added to the tank 9. Moreover, after each washing in the resulting concentrate, the presence of protein is determined, and then the calculation is carried out according to the formula:

где

Where

V ₀ is the final volume of the lactoglobulin solution, in liters;

x ₁ - protein concentration obtained in solution, in%;

x ₂ - the required amount of protein, in%;

V ₁ - the volume of lactoglobulin concentrate, in liters.

Then from the expression:

где

Where

V ₀ is the final volume of the lactoglobulin solution, in liters;

V ₁ - the volume of lactoglobulin concentrate, in liters;

V is the volume of the required sodium chloride solution, in liters,

determine how much to pour in a 0.4% sodium chloride solution to obtain a given concentration of protein in the finished product. The protein content is determined by biuret reagent according to FS 42-3874-99.

The next stage of the technology for the production of immune lactoglobulin is sterilizing filtration and sterile bottling.

Example.

Lactoserum obtained from immune cows in the amount of 15 liters is filtered on a membrane cassette module in a pressure-circulating stream until clarification.

After that, the clarified filtrate (15 liters) is passed through an UPV-6 ultrafiltration fiber module and concentrated to obtain 5 liters of concentrate. Then the concentrate is washed, up to 15 liters of a 0.4% sodium chloride solution pH 5.0-6.0 are added to it and re-filtered on a hollow fiber module.

The concentrate is washed twice more, and after each washing, the amount of protein is determined.

To comply with the amount of protein from 4.5-5.5% (weight / volume), the calculation is carried out according to the formula:

где

Where

V ₀ is the final volume of the lactoglobulin solution, in liters;

x ₁ - protein concentration obtained in solution, in%;

x ₂ - the required amount of protein, in%;

V ₁ - the volume of lactoglobulin concentrate, in liters.

In 5 liters of concentrate, the protein was 10.2%. In order for lactoglobulin to have a protein concentration of 5.0%, it is necessary to carry out the calculation:

Therefore, it is necessary to add (10.2-5) = 5.2 liters of a 0.4% sodium chloride solution to the concentrate.

The resulting solution was sterilized by filtration on low molecular weight plate filters with a pore size of 0.22 μm and sterilely poured into 20 ml vials (2 doses).

The finished drug is controlled by FS 42-3496-98.

As a result of experimental data of a new method for producing immune lactoglobulin by specialists of the Rostov Research Institute of Microbiology and Parasitology, it was revealed (see table 1) that the obtained lactoglobulin in comparison with the previously developed technology provides a high specific immunological activity of the drug in the content of protective antibodies against microorganisms.

The results shown in table 2 indicate that the replacement of the Cohn alcohol fractionation operation with ultrafiltration, the process at pH 5.5 and the exclusion of lyophilization leads to the preservation of the lactoperoxidase activity of lactoglobulin as a nonspecific factor that increases the resistance of the macroorganism against opportunistic bacteria and salmonella .

Using the present invention, due to the double ultrafiltration of lactoserum, first on the membrane cassette module, and then on the fiber module in a pressure-circulating stream perpendicular to the filtrate current, the technology of production of immune lactoglobulin is simplified, since a number of deposition, lyophilization operations are reduced and no additional expensive equipment is required.

In addition, this method allows you to:

- improve the quality of liquid lactoglobulin by increasing stability and preserving non-specific factors of the drug’s protection,

- prevent the formation of a "secondary membrane" on the filter surface, which significantly increases the performance and quality of the filter;

- reduce the cost of obtaining lactoglobulin;

- use a low molecular weight filtrate enriched with the active components of lactoserum for the production of new medicines.

Table 1 name of diagnosticum for strains specific activity of lactoglabulin according to the proposed method, mcg / 10 μl specific activity of lactoglabulin according to the RNIIMP technology (a.s. No. 1743025), μg / 10 μl SOP Ser 10-0404 Salmonella groups B-1,4,12 15.6 31,2 31,2 Salmonella Group D-1,9,12 31,2 15.6 62.5 Proteus mirabilis serogroup 0-35 15.6 15.6 15.6 Proteus vulgaris serogroup 0-43 7.8 15.6 15.6 Klebsiellapneumonia 15.6 31,2 31,2 Pseudomonas aeruginosa 7.8 31,2 31,2

table 2 series of the drug (name of operation) incubation temperature, t degrees C lactoperoxidase activity (arbitrary units of activity) 0 days 7 days 14 days 28 days lactoglabulin (alcohol precipitation) before lyophilization according to the RNIIMP technology (AS No. 1743025) four 4134 + 1.6 2071 + 5.5 1010 + 2.2 490 + 1.2 35 4134 + 1.6 0 0 0 lactoglabulin (alcohol precipitation) after lyophilization according to the RNIIMP technology (AS No. 1743025) four 168 + 4,5 165 + 2.2 154 + 3.1 98 + 6.4 35 168 + 4,5 110 + 1.3 81 + 2,3 59 + 3.1 lactoglabulin according to the proposed method (ultrafiltration operation at pH 7.0) four 4632 + 3.4 2090 + 4.1 1045 + 2.6 540 + 3.2 35 4632 + 3.4 0 0 0 lactoglabulin according to the proposed method (ultrafiltration operation at pH 5.5) four 19904 + 107 18909 + 114 17715 + 105 15753 + 110 35 19904 + 107 15127 + 111 8957 + 51 828 + 13

Claims (3)

1. A method for producing immune lactoglobulin, comprising obtaining lactoserum from milk or colostrum from immune cows, followed by clarification of the lactoserum and its concentration by ultrafiltration, characterized in that ultrafiltration is carried out in two stages in a circulating pressure stream perpendicular to the filtrate current, while in the first stage filtration is carried out on a membrane cassette module, clarifying the lactoserum, cleansing it of the remains of fat and casein, and the second stage is carried out on a module with hollow with fibers, washing the working mixture from low molecular weight components with a 0.4% solution of sodium chloride pH 5.0-6.0 and concentrating the protein in the range of 4.5-5.5%, and the resulting filtrate is sterilized and poured in liquid form into bottles. 2. The method according to claim 1, characterized in that the washing of the lactoglobulin concentrate obtained in the second step is carried out three times from low molecular weight components, filtering it on a fiber module with the addition of a three-fold volume of a 0.4% sodium chloride pH 5.0- 6.0. 3. The method according to claim 1, characterized in that the required concentration of protein in lactoglobulin is obtained from the calculation of the final volume of the lactoglobulin solution according to the formula

, where V ₀ is the final volume of the lactoglobulin solution, l; x ₁ - protein concentration obtained in solution,%; x ₂ - the required amount of protein,%; V ₁ - the volume of lactoglobulin concentrate,%, and from the difference in volumes of the final product V ₀ and concentrate V ₁ , the volume of the required solution of 0.4% sodium chloride is obtained.

Images