Method of virus inactivation for immunoglobulin production

FIELD: medicine.

SUBSTANCE: invention refers to virus inactivation in production of immunoglobulins, particularly of G fraction. The method virus inactivation for production of G fraction immunoglobulin includes purification of dissolved immunoglobulin recovered by Kohn's spirit fractionation. Purified dissolved immunoglobulin is prepared with a solvent detergent mixture which is acetate buffer solution 0.05 M at pH 5.5, containing 1 wt % tri-n-butylphosphate and 1 wt % polysorbate 80. Preparation represents stirring of the mixture within 12-16 hours followed with dilution with acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. Prepared immunoglobulin is immobilised on sulphoprolylcation sorbent with grain size 50 mcm and sorption capacity 55 mg/cm3, and two-stage washed by column chromatography followed with elution. The first stage of washing implies acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. The second stage of washing applies acetate buffer solution 0.05 M at pH 5.5. At the first stage the product is washed with volume related to 5 volumes of sorbent, while at the second stage washing is performed with volume related to three volumes of sorbent.

EFFECT: invention provides high-effective virus inactivation that improves viral safety of immunoglobulin preparations.

3 cl, 7 ex

 

The invention relates to medicine, namely the inactivation of viruses in the production of globulins, which are proteins in blood plasma that participate in immune reactions, and can be used in the manufacture of pharmaceutical preparations which contain immunoglobulin fraction G.

A significant problem with the use of medicines derived from biological material, is the risk of contamination by viruses. Known methods of inactivation of viruses, in particular heat treatment, unacceptable to such biological raw materials, as globular proteins in blood plasma, as they lead to their destruction and loss of biological activity.

The closest is a method of inactivating viruses upon receipt of immunoglobulin, including cleaning solution immunoglobulin, selected alcohol fractionation (RU, patent No. 2261112, CL AC 39/395, publ. 27.09.2005). Purification by a known method provides a processing solution which contains diluted 12 volumes of water for injection alcohol extract of sediment B (III fraction of Koh) and sodium chloride, chloroform, which is added to 3 wt.%, at pH 4.0...5,0. The mixture was incubated for 14 hours at 0...3°C, centrifuged, the precipitate is removed and conduct brightening filtering.

The disadvantage of this method is the low level is the pressure/inactivation of viruses during the course of performing such processing, that leaves him viruscapsid. In this regard, the known method provides for additional inactivation of viruses at room temperature by exposure for 20...25 days.

Object of the invention is the improvement of the method of inactivation of viruses upon receipt of immunoglobulin, which due to the proposed treatment and conditionality increases the efficiency of removal/inactivation of viruses that provides wirosoetisno immunoglobulin for drugs. The proposed method is feasible and easy to control.

The problem is solved by the proposed method inactivation of viruses upon receipt of immunoglobulin, which includes cleaning solution immunoglobulin, selected alcohol fractionation, in which when cleaning solution immunoglobulin pre-treated solvent-detergent mixture, treated with immunoglobulin immobilized on alphapapillomavirus sorbent and is carried out in two stages lavage using column chromatography, followed by elution. In the first stage wash using acetate buffer solution, which contains octanoate sodium, sodium chloride and propylene glycol, for example, 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M chloride n the sodium and propylene glycol at a concentration of 0.2 g/l, and washing are displacement, corresponding to the five volumes of sorbent. In the second stage wash using acetate buffer solution, for example, 0.05 M acetate buffer solution at pH 5.5, and washing are displacement, corresponding to the three volumes of sorbent. Better on the first and second stages rinse hold with a speed of 3 cm/min

As the solvent-detergent mixture using 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The treatment is carried out by mixing the solvent-detergent mixture and solution of immunoglobulin within 12...16 hours followed by dilution with 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L.

For immobilization of immunoglobulin use alphapapillomavirus sorbent, which has a grain size of 50 μm, and the sorption capacity of 55 mg/cm3. The elution of the treated and washed immunoglobulin perform 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride.

Experimentally it was found that the preliminary solvent-detergent treatment solution of immunoglobulin, a dedicated alcohol fractionation, followed by its immobilization on alphapapillomavirus sorbent and washing is definitely acetate buffer solution to increase the efficiency of removal/inactivation of viruses, that provides wirosoetisno of immunoglobulin products.

The method is thus.

Immunoglobulin allocated the alcohol fractionation method con (liquid sludge "In"), dissolved in acetate buffer solution. 3%solution of immunoglobulin treated with a solvent-detergent mixture. This stage of the process involves the exposure of the immunoglobulin solution that contains a solvent and a detergent. As the solvent used, for example, tri-n-butylphosphate as detergent, octoxynol 10, Polysorbate 80, β-propiolactone and other surfactants. Mixing is carried out in the reactor. For example, 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80, is mixed with a solution of immunoglobulin. The mixture is stirred for 12...16 hours at 25°C. the mixture is diluted with 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. thus Treated immunoglobulin using column chromatography with eluent linear velocity of 1 cm/min immobilizer on alphapapillomavirus sorbent, which has a sorption capacity of 55 mg/cm3and grain size of 50 microns.

After the adsorption is carried out in two stages n is the washing of the pretreated immunoglobulin, immobilized on alphapapillomavirus sorbent, eluent linear velocity of 3 cm/min In the first stage wash using acetate buffer solution of the same composition for dilution of the mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/l Washing lead displacement, corresponding to the five volumes of sorbent.

In the second stage wash using 0.05 M acetate buffer solution at pH 5.5. Washing lead displacement, corresponding to the three volumes of sorbent.

The elution of immunoglobulin perform 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride.

The eluate contains immunoglobulin G at a concentration of 3.5...4%.

The following are examples demonstrating the effectiveness of the method of inactivation of viruses upon receipt of immunoglobulin, but not limiting it. According to the GMP requirements, the deliberate introduction of any virus in industrial premises is not permitted. Therefore, the validation was conducted in a separate laboratory, properly equipped, using the model of the production process (examples 1-6).

Example 1.

To demonstrate the effectiveness of the removal/inactivation of viruses proposed method used the model viruses viral diarrhea VRH(BVDV).

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed 6,45 log10TCLD50/cm3virus BVDV. In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus BVDV, added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 12 hours at a speed of rotation of the agitator 80 rpm Later in capacity add 90 ml of solution, which contains: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this pass through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.1 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. The solution is passed at flow rate 5 ml/min (corresponds to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The infectivity titer of the virus BVDV after inactivation - <1,0 log10TCLD50/cm3.

Example 2.

To demonstrate the effectiveness of the removal/inactivation of viruses proposed method used the model viruses pseudoleskeella (PRV).

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed ≥7,33 log10TCLD50/cm3virus PRV. In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus PRV, added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 14 hours at a rotation speed of the agitator 80 rpm Later in capacity add 90 ml of solution, which sod is RIT: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this pass through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Solution is passed at flow rate 5 ml/min (corresponding to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The infectivity titer of the virus PRV after inactivation was <1,0 log10TCLD50/cm3.

Example 3.

To demonstrate the effectiveness of the removal/inactivation of viruses proposed with the special used model enteroviruses pigs (PTV-1).

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed ≥ 7,9 log10TCLD50/cm3virus PTV-1. In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus PTV-1 was added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 16 hours at a speed of rotation of the agitator 80 rpm Later in capacity add 90 ml of solution, which contains: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this purpose, passed through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium ,15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. The solution is passed at flow rate 5 ml/min (corresponding to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The infectivity titer of the virus PTV-1 after inactivation was 3.3 log10TCLD50/cm3.

Example 4.

To demonstrate the effectiveness of the removal/inactivation of viruses proposed method used a model strain of adenovirus type 4 (Ad4).

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed ≥7,0 log10TCLD50/cm3virus Ad4.

In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus Ad4, added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 12 hours at a speed of rotation of the agitator 80 rpm Later in capacity add 90 ml of p is the target, which contains: 0.05 M acetate buffer solution at pH 5.5, 1% wt. octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this pass through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Solution is passed at flow rate 5 ml/min (corresponding to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The infectivity titer of the virus Ad4 after inactivation was ≤ 5,77 log10TCLD50/cm3.

Example 5.

For demonstratieproject removal/inactivation of viruses proposed method used the model hepatitis ducklings (DHV-1).

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed to 3.0×106LD50/cm3virus DHV-1.

In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus DHV-1 was added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 12 hours at a speed of rotation of the agitator 80 rpm Later in capacity add 90 ml of solution, which contains: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this pass through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium ,15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. The solution is passed at flow rate 5 ml/min (corresponding to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The infectivity titer of the virus DHV-1 after inactivation was ≤1,0×103LD50/cm3.

Example 6.

To demonstrate the effectiveness of the removal/inactivation of viruses proposed method used the model viruses HIV-1.

3 g of sediment "In" immunoglobulin, selected alcohol fractionation method Kona, was dissolved in 15 ml of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

A 3%solution of immunoglobulin contributed to 5.5 log10TCLD50/cm3virus HIV-1.

In a container containing 15 ml of 3%aqueous solution of immunoglobulin with the virus HIV-1, was added 15 ml of a solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 12 hours at a speed of rotation of the agitator 80 rpm Later in capacity add 90 ml of solution, of which the first contains: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 15 mm, containing 23 cm3selfoperating sorbent, at a volumetric flow rate of 1.7 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 70 minutes Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this pass through the column 110 ml of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Solution is passed at flow rate 5 ml/min (corresponding to a linear velocity of 3 cm/min) for 20 minutes. Then washed with 70 ml of 0.05 M acetate buffer solution at pH 5.5 for 15 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 50 minutes. The eluate 34 ml collected in a sterile vessel and conducted ultrafiltration.

The titer of infectivity of HIV-1 after inactivation - <1,0 log10TCLD50/cm3of the virus.

Example 7.

3 kg sludge "In" immunoglobulin, selected alcohol fractionation the method of Kona, dissolved in 15 l of 0.05 M acetate buffer solution at pH 5.5 and a temperature of 4°C.

Into the reactor containing 15 l of 3%aqueous solution of immunoglobulin, with the help of vacuum serves 15 liters of solvent-detergent mixture: 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80. The mixture is stirred at 25°C for 12 hours at a speed of rotation of the agitator 80 rpm Next to the reactor with the help of vacuum serves 90 l of a solution that contains: 0.05 M acetate buffer solution at pH 5.5, 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Mixture is stirred and passed through a pre-equilibrated chromatographic column with a diameter of 400 mm, containing 23 l selfoperating sorbent, at a volumetric flow rate of 1200 ml/min (eluent linear velocity of 1 cm/min). Sorption spend about 10 hours. Immobilizovannyi on alphapapillomavirus sorbent pre-treated immunoglobulin subjected to washing in two stages. For this purpose, passed through the column 110 l of 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/L. the Solution is passed at flow rate of 3600 ml/min (corresponding to a linear velocity of 3 cm/min) for 37 minutes. Then the industry is up 70 l of 0.05 M acetate buffer solution at pH 5.5 for 23 minutes.

The elution of immunoglobulin was performed 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride. The elution process lasted about 60 minutes. The eluate 34 l was collected in a sterile 20-l glass bottles and held ultrafiltration.

The titer of the virus after inactivation of the viruses. Thus, the proposed method provides high efficiency in removing viruses, which increases wirosoetisno immunoglobulin drugs, is technologically advanced and can be used in industrial scale.

1. The method of inactivation of viruses upon receipt of the immunoglobulin fraction G, includes cleaning solution immunoglobulin, selected alcohol fractionation method Kona, characterized in that the cleaning solution of the immunoglobulin pre-treated solvent-detergent mixture, which is using 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% tri-n-butylphosphate and 1 wt.% Polysorbate 80, and the treatment is carried out by stirring the mixture for 12 to 16 h followed by dilution with 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/l, treated with immunoglobulin immobilizer on alphapapillomavirus sorbent, with C the RNA size of 50 μm and the sorption capacity of 55 mg/cm 3and carry out the washing in two stages using column chromatography, followed by elution, and in the first stage wash using 0.05 M acetate buffer solution at pH 5.5, containing 1 wt.% octanoate sodium, 0.15 M sodium chloride and propylene glycol at a concentration of 0.2 g/l, in the second stage wash using 0.05 M acetate buffer solution at pH 5.5, while in the first stage washing lead volume corresponding to 5 volumes of sorbent in the second stage washing lead displacement, corresponding to the three volumes of sorbent.

2. The method according to claim 1, characterized in that the first and second stages are washing with a speed of 3 cm/min

3. The method according to claim 1, characterized in that the elution of conduct 0.05 M acetate buffer solution at pH 5.5, containing 0.2 M sodium chloride.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to manufacturing of medical products. There is disclosed method for producing immunoglobulin G that involves ethanol fractionation of donor plasma to make sediment B and purification of prepared immunoglobulin. The sediment B is dissolved in 0.9% saline pH 5.15 and mixed with acetate buffer solution 2 M and 53% ethanol, and centrifugated. Centrifugate is mixed with sodium hydrocarbonate at solution pH 5.5. After centrifugation, the centrifugate is purified, mixed with acetate buffer solution at pH 5.4, 96% (vol/vol) ethanol and sodium bicarbonate at pH 7.2, centrifugated at minus (10-12)°C. The recovered sediment is dissolved in acetate buffer solution 0.05 M at pH 5.5, added with solvent detergent mixture containing acetate buffer solution 0.05 M at pH 5.5, and 1 wt % tri-n-butylphosphate and 1 wt % polysorbate 80. The mixture is stirred, then diluted with acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. Immunoglobulin processed in such a way is immobilised and two-stage washed on sulphoprolylcation sorbent by column chromatography followed with elution, ultrafiltration and sterilisation filtration.

EFFECT: invention provides high-degree purification of immunoglobulin G, absence of viruses and stability of ready preparation.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and biotechnology, and method of obtaining immunoglobulin medicines. Method of obtaining immunoglobulin medicines involves a sequential processing of alcohol sediment B (Cohn III fraction) by tenfold volume of 0.9% sodium chloride solution and by chloroform in 0.1%-3.0% end concentration, centrifugation, addition of copper sulfate to centrifugate at pH 6-8, sediment removal by centrifugation, and further extraction of target product by ultrafiltration method in the presence of complex-forming compounds, product stabilisation, clearing and stabilising filtration. Copper sulfate is taken in 0.003-0.03% concentration.

EFFECT: increased content of separated immunoglobulin A fraction, improved product quality.

3 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns medicine, in particular, to immunology, and can be used for immunotherapy and immunologic prophylaxis of bacteriemic and virus diseases. The base for immunobiological preparations is offered; it contains IgG, IgM and IgA, and also subclasses IgG, IgG1, IgG2, TgG3 and IgG4, thus the content of immunoglobulins makes: IgG 70-80%, IgM 10-15% and IgA 10-15%. The way of reception of immunoglobulin marker base for immunobiological preparations is offered. Suppositories and ointment for prevention and therapy of bacteriemic and virus diseases are offered.

EFFECT: development of an economic and effective way of processing of a deposit A (II+III according Cohn), allowing to receive highly effective paste for immunoglobulin marker preparations.

4 cl, 5 ex, 2 tbl, 2 dwg

FIELD: technological processes; pharmacology.

SUBSTANCE: method is implemented in the following manner: pH of initial solution is brought to 4.6-4.95, and caprilat- and/or heptanoate-ions are added to it, while pH is maintained at the level of 4.8-4.95. Supernatant solution is incubated under conditions, at which concentration of caprilat- and/or heptanoate-ions makes 10-30 mM. Filtered solution is applied on anion-exchanging resin, under conditions that provide binding of contaminating admixtures with resin in the absence of considerable binding of antibodies with resin.

EFFECT: method makes it possible to produce preparation of antibody that is purified, virologically safe and inactivated from virological point of view.

22 cl, 2 dwg, 2 tbl, 3 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: biochemical engineering; medicine.

SUBSTANCE: method involves producing immunoglobulin preparation by concentrating III Kohn spirit method fractions by means of ultrafiltration under рН 3.5-5.0 condition, washing from ethanol and carrying out final solution concentration process. The subsequent restoration hydrate immunoglobulin envelopes is carried out by means of molecular filtration method by creating concentration gradient by introducing low-molecular substance in concentration of 0.1-2.5% by weight into immunoglobulin solution, with water of рН 3.8-4.5 being used, cleared from dialyzing solution. Then stabilizing agent is added and рН value is fixed at 3.5-5.0. Sterilizing filtration is carried out and the produced preparation is dried by subliming when needed at the final stage of refinement process. The so produced immunoglobulin preparation for making intravenous injections has solution transparency index less than 0.01, and contains monomer immunoglobulin IgG more than 97% under full absence of polymeric forms, proteins 4.5-11% and sodium chloride no more than 0.3%.

EFFECT: reduced risk of side reactions when administering the immunoglobulin preparation; mechanically aided and automated process of preparation production; prolonged storage period.

5 cl, 1 dwg, 2 tbl

FIELD: medicine, preparative immunology.

SUBSTANCE: invention relates to a method for preparing IgG (immunoglobulin G) from blood plasma for medicinal using. Method involves the following steps: (1') removal of albumin with preparing IgG fraction followed by carrying out step (I) wherein IgG fraction prepared at stage (1') is concentrated; (2') this IgG fraction is purified on anion-exchange resin and unbound IgG fraction is collected; (3') this unbound IgG fraction is purified on cation-exchange resin and bound IgG fraction is collected and step (II) is carried out wherein pH value of IgG fraction eluted from cation-exchange resin at step (3') is brought about to 4 ± 0.1 and during all steps of the method pH value is maintained less 6.0; (4') virus in IgG fraction prepared from IgG fraction collected at step (3') is inactivated by using chemical reagents at temperature 30° ± 2°C for at least 4 h. Method provides preparing IgG that possesses anti-complementary activity less 1 titer value of complement providing 50% hemolysis (CH50)/mg of immunoglobulin.

EFFECT: improved preparing method.

7 cl, 1 dwg, 2 ex

The invention relates to veterinary medicine, in particular to methods for treating dyspepsia newborn calves

The invention relates to a method of purification of immunoglobulin G from containing the immunoglobulin protein fractions of crude plasma

The invention relates to medicine, in particular to immunology, and can be used for neutralization of TNFy patient in need of such neutralization

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to manufacturing of medical products. There is disclosed method for producing immunoglobulin G that involves ethanol fractionation of donor plasma to make sediment B and purification of prepared immunoglobulin. The sediment B is dissolved in 0.9% saline pH 5.15 and mixed with acetate buffer solution 2 M and 53% ethanol, and centrifugated. Centrifugate is mixed with sodium hydrocarbonate at solution pH 5.5. After centrifugation, the centrifugate is purified, mixed with acetate buffer solution at pH 5.4, 96% (vol/vol) ethanol and sodium bicarbonate at pH 7.2, centrifugated at minus (10-12)°C. The recovered sediment is dissolved in acetate buffer solution 0.05 M at pH 5.5, added with solvent detergent mixture containing acetate buffer solution 0.05 M at pH 5.5, and 1 wt % tri-n-butylphosphate and 1 wt % polysorbate 80. The mixture is stirred, then diluted with acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. Immunoglobulin processed in such a way is immobilised and two-stage washed on sulphoprolylcation sorbent by column chromatography followed with elution, ultrafiltration and sterilisation filtration.

EFFECT: invention provides high-degree purification of immunoglobulin G, absence of viruses and stability of ready preparation.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: there is provided DNA that codes protein able to transform a compound of formula (II) specified in description of invention into a compound of formula (III) specified in description of invention with an electron transport system containing an electron donor. Protein is able to metabolise herbicides.

EFFECT: introduction of DNA to plants with an expression of the specified protein provides herbicide resistance thereto.

26 cl, 66 dwg, 35 tbl, 75 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely oncology and can be used in treatment of patients with colorectal cancer with multiple liver metastases. The first stage of treatment involves neoadjuvant oxalyplatine chemotherapy; in addition Tomudex is introduced in a dose 3 mg/m2 as a 24 hour continuous infusion. After termination of chemotherapy, biotherapy involves intra-arterial injection of Avastin in a dose 10 mg/kg of body weight within 2 hours, neoadjuvant intra-arterial chemotherapy and biotherapy totals in 4-6 courses. At the second stage 4 weeks after chemotherapy surgery is performed. The third stage involves 4-6 courses of adjuvant intra-arterial chemotherapy and biotherapy, as at the first stage.

EFFECT: invention allows increasing survival rate and life expectancy of the patients owing to improved respectability of metastases and lower toxicity of complex neoadjuvant and adjuvant intra-arterial chemotherapy and biotherapy.

1 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and biotechnology, and method of obtaining immunoglobulin medicines. Method of obtaining immunoglobulin medicines involves a sequential processing of alcohol sediment B (Cohn III fraction) by tenfold volume of 0.9% sodium chloride solution and by chloroform in 0.1%-3.0% end concentration, centrifugation, addition of copper sulfate to centrifugate at pH 6-8, sediment removal by centrifugation, and further extraction of target product by ultrafiltration method in the presence of complex-forming compounds, product stabilisation, clearing and stabilising filtration. Copper sulfate is taken in 0.003-0.03% concentration.

EFFECT: increased content of separated immunoglobulin A fraction, improved product quality.

3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and method of obtaining immunoglobulin medicine. Method involves alcohol sediment B (Cohn III fraction) extraction stage by saline solution, removal ballast admixtures by copper sulfate in 0.003-0.03% concentration at pH 4.9-5.1 and temperature of 0-5°C, and extraction of target product by ultrafiltration method in the presence of complex-forming compounds.

EFFECT: toxic reagent excluded from process cycle; enhanced output of extracted immunoglobulin and medicine purity grade.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly oncology, and can be applied in treatment of tumours expressing G250 antigen. Manufacturing of medicine for supporting therapy in case of non-metastatic disease after dissection of primary tumour expressing G250 antigen involves G-250-specific antibody and/or antibody fragment.

EFFECT: reduced risk of tumour recurrence due to cell cytotoxicity dependent on given antibody.

18 cl, 4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: penetrating keratoplasty is performed, preoperative preparation is made and post-surgical treatment is performed using medicine blocking angiogenesis - bevacizumab by the suggested procedure.

EFFECT: method provides regress of the existing newly performed vessels, prevents cornea neovascularisation development in the early postoperative period after penetrating keratoplasty.

2 ex

FIELD: veterinary science.

SUBSTANCE: method pertains to laboratory diagnostic methods of anaplasmosis and may be used in veterinary. Method consists in injecting of antigen and levamisole immunostimulant to the rabbits. Whereat as an antigen an antigen derived by alternating freezing and thawing of the grown on the Vero cells in the growth supporting nutrient medium "Igla" MEM with added embryonic serum, amino acids and antibiotics of vial culture Anaplasma sp. Omsk after the eight days of incubation.

EFFECT: method enables getting anaplasmosis serum with high antibodies diagnostic titer.

5 tbl

FIELD: medicine.

SUBSTANCE: there are disclosed polypeptide variants containing Fc-areas IgG, having amino acid modifications providing changed effector functions Fc in specified polypeptides. There is disclosed composition for antibody targeting on antigen, containing the specified polypeptide. There is described method for preparing the specified polypeptide. Also, there are disclosed the methods for treating V-cell tumour or a malignant disease characterised by V-cell expression of CD20, treating chronic lymphocytic leukosis, relieving the symptoms of the V-cell controlled autoimmune disease, treating a angiogenesis-associated disorder, treating HER2-expressing cancer, treating LFA-1-mediated involvement, treating IgE-mediated involvement wherein specified methods imply introduction to the patient of the therapeutically effective amount of said polypeptide.

EFFECT: higher clinical effectiveness.

63 cl, 6 ex, 13 dwg, 10 tbl

FIELD: medicine.

SUBSTANCE: invention represents a pharmaceutical composition for treatment of rheumatoid arthritis, containing interleukine-6 receptor antibody and methotrexate.

EFFECT: synergetic effect ensured by combined use of components of the composition, extended range of products for treatment of rheumatoid arthritis.

57 cl, 4 ex, 2 tbl

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

Up!