RU2324495C1 - Method of making preparation of human blood coagulaton factor viii - Google Patents

Abstract

FIELD: technological processes.

SUBSTANCE: preparation of human blood coagulation factor VIII is made by producing cryoprecipitate out of blood plasma, virus inactivating treatment, purification of cryoprecipitate solution, concentration, sterilizing filtration, bottling and drying. At the same time cryoprecipitate is produced with the help of running water with the temperature of (4±2)°C during unfreezing of fresh frozen plasma, virus inactivating treatment is carried out by means of solvent-detergent method, chromatographic purification of virus inactivated cryoprecipitate solution is carried out with application of sorbent with fractionation interval up to 20,000 kilodaltons, at the flow rate of (20±15) cm/hr, with further concentration by means of ultrafiltration on hollow fibers. Stabilizers are added, such as albumin in final concentration of (5±3) g/l, sugars and amino acids up to final concentration of (10±3) g/l. During drying initial preparation temperature is (25±15)°C below zero, final temperature is (25±8)°C, total duration of preparation drying process is (45±7) hours.

EFFECT: coagulation factor yield stability is increased and technological production design is simplified.

2 ex

Description

The invention relates to biotechnology and relates to methods for producing preparations of factor VIII coagulation system of human blood.

The present method allows to obtain a drug, which is a factor VIII of coagulation of human blood, isolated by cryofraction followed by chromatographic purification from plasma for fractionation (FS 42-0091-02) and used as a medicine.

A known method for producing purified preparations of factor VIII coagulation system from blood plasma by obtaining cryoprecipitate from blood plasma, virusinactivating treatment of a solution of cryoprecipitate, chromatographic purification of a solution of cryoprecipitate by ion exchange chromatography, sterilizing filtration, bottling, drying by sublimation (E.P. Ivanov, V.N. Gapanovich , A.V. Litvinchuk, I.A. Lisova; Research work of the I quarter of 1996 - the VI quarter of 1997; New in transfusiology, issue 26, Dereza T.L., Fetisova L.V., Azhigirova M. BUT.).

However, this method has the following disadvantages.

1. The output of factor VIII is unstable with respect to the content of factor VIII in the initial cryoprecipitate (65-100%).

2. For chromatographic purification, an expensive sorbent is used.

The objective of the invention is to increase the stability of the output of factor VIII, simplify the technological scheme of production, use a cheaper chromatographic sorbent, increase the profitability of the production of the drug.

The problem is solved by developing a new method, including obtaining cryoprecipitate from blood plasma, purification of a solution of cryoprecipitate, concentration, sterilizing filtration, bottling and drying.

New in the proposed method is that cryoprecipitate is obtained using running water when defrosting freshly frozen plasma, purification is carried out by gel chromatography, followed by concentration by ultrafiltration on hollow fibers, stabilizers are added, which are used as albumin, sugars and amino acids, drying of a sterile eluate concentrate, containing factor VIII, is carried out by freezing the product to a temperature of minus (25 ± 15) ° С and then increasing the temperature by (5 ± 2) ° С every hour to the pace temperature (25 ± 8) ° С.

The essence of the method is as follows.

Freshly frozen plasma is thawed in running water with a temperature of (4 ± 2) ° С. After the complete disappearance of ice and the formation of a precipitate in containers with plasma, the containers are immediately opened and the plasma with sediment is pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air (0.022 ± 0.002) MPa enters the flow centrifuges. After centrifugation, the precipitate is removed from supercentrifuge rotors and immediately frozen at minus (70 ± 10) ° С. The resulting precipitate, hereinafter referred to as cryoprecipitate, consists of blood plasma proteins insoluble at a plasma temperature of (4 ± 2) ° С. From one liter of plasma, 9 to 11 g of cryoprecipitate are obtained.

A centrifugate, which is a cryo-depleted blood plasma, is collected in a container and subsequently used for the manufacture of other blood products. The yield from this stage is 38-43% of the content of factor VIII in the original plasma. Then the cryoprecipitate solution is purified as follows: (40 ± 20) mM citrate buffer, pH 6.8-7.5, is added to the container with frozen cryoprecipitate. Then cryoprecipitate with citrate buffer is thawed in a water bath at a temperature of (25 ± 1) ° С. The thawed cryoprecipitate solution is centrifuged, the supernatant of the cryoprecipitate solution is separated and transferred for clarification filtration. Clarifying filtration is carried out on a filter filled with membranes with a pore diameter of 3.0-10 microns. From the tank, the supernatant of the cryoprecipitate solution is filtered under pressure (0.055 ± 0.005) MPa. In a container with a clarified supernatant of a cryoprecipitate solution, add a heparin solution in the ratio of 0.3 kg of cryoprecipitate / 7000-10000 U of heparin and mix. Then, virus-inactivating treatment is carried out using the solvent-detergent method. In this case, the supernatant of the cryoprecipitate solution is treated with tri-n-butyl phosphate and Tween 80 at final concentrations of 0.3% and 1%, respectively, for (6 ± 2) hours at a temperature of (30 ± 2) ° С with constant stirring. Also, virus-inactivating treatment is carried out using copper phenanthroline buffer. In this case, the supernatant of the cryoprecipitate solution is treated with a solution of copper phenanthroline buffer in the ratio of 1 part of copper phenanthroline buffer / 40 parts of the supernatant of the cryoprecipitate solution. Copper phenanthroline buffer (CuPH) is prepared by mixing 10 ml of 0.1 M histidine, 8 ml of 0.01 M 5-aqueous copper sulfate and 8 ml of 0.5 M 1,10-phenanthroline. The volume was adjusted to 200 ml with water for injection. Then chromatographic purification of the virus-inactivated cryoprecipitate solution is carried out using a sorbent with a fractionation interval of up to 20,000 kDa at a flow rate of 20 ± 15 cm / h.

After chromatographic purification, the eluate containing factor VIII is collected in sterile vials and albumin is added to a final concentration of (5 ± 3) g / l, alanine, or arginine, or glycine, or histidine to a final concentration of (10 ± 3) g / l , maltose, or glucose, or sorbitol or sucrose to a final concentration of (10 ± 3) g / l and freeze at a temperature of minus (60 ± 30) ° С.

Next, the eluate containing factor VIII is thawed at a temperature of (20 ± 1) ° C and poured into a container. Then, clarifying filtration is carried out through membranes with a pore diameter of 3.0-10.0 μm under a pressure of (0.055 ± 0.005) MPa. The yield from this stage is 80-85% of the content of factor VIII in the initial cryoprecipitate. Then, concentration is carried out.

Concentration is carried out by ultrafiltration on a membrane filtration unit using membranes in which the cut-off threshold is not more than 200 kDa. For this, the ultrafiltration unit is filled with an eluate containing factor VIII, and the concentration process is started. The eluate containing factor VIII, entering the plant from the tank, circulates in a closed loop, creating a tangential flow along the fibers along their inner part. Part of the eluate containing factor VIII leaves without filtration and returns to the bottle. This part of the solution is called a concentrate and contains all the material that lingers in accordance with the pore size. With each transmission cycle of the eluate containing factor VIII, part passes through the membrane and is sent to a separate container. This part is called permeate, which is a waste product and contains water with salts dissolved in it. Permeate is periodically (at least after 15 minutes) checked for protein content. To do this, equal volumes of permeate and a 20% trichloroacetic acid solution are mixed in vitro. The formation of sediment in the test tube indicates a violation of the integrity of the membranes. The eluate containing factor VIII is concentrated to values from 10 to 15 ME of factor VIII in 1 ml of eluate, or from 20 or more ME of factor VIII in 1 ml of eluate. The yield from this stage is 95-99% of the content of factor VIII in the initial eluate containing factor VIII. Then sterilizing filtration is carried out simultaneously with the process of filling the drug into bottles. The filtration system consists of a container, a filter and a capsule. Installation of the system is carried out in compliance with aseptic rules. After that, the eluate concentrate containing factor VIII from the container under pressure (0.055 ± 0.005) MPa is filtered through a filter filled with membranes with a pore size of 3.0-10.0 μm, and then through a filter filled with membranes with a pore size of 0.45 μm, 0.22 μm. A sterile solution of the eluate concentrate is poured into vials.

The yield from this stage is 83-85% of the content of factor VIII in the initial concentrate of the eluate containing factor VIII.

Drying of the sterile concentrate of the eluate containing factor VIII begins with freezing the product at a temperature of minus (25 ± 5) ° С. Then the temperature is increased by (5 ± 2) ° C every hour to a temperature of (25 ± 8) ° C. The final temperature of the product should be (25 ± 8) ° C. The total duration of the drying process of the drug is 45 ± 7 hours. The residual moisture of the drug should be 2.2 ± 0.2%.

The yield from this stage is 80-90% of the content of factor VIII in a sterile eluate concentrate containing factor VIII.

Example 1

Freshly frozen plasma was thawed in running water with a temperature of 2 ° C. After the ice disappeared completely and a precipitate formed in the containers with plasma, the containers were immediately opened and the plasma with sediment was pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air of 0.020 MPa was supplied to flowing centrifuges. After centrifugation, the precipitate was removed from supercentrifuge rotors and immediately frozen at minus 60 ° C.

The centrifugate was collected in a container and subsequently used for the manufacture of immunoglobulin and albumin preparations.

The yield from this stage was 38% of the content of factor VIII in the original plasma. Then, the cryoprecipitate solution was purified as follows: 20 mM citrate buffer, pH 6.8, was added to the container with frozen cryoprecipitate. Then cryoprecipitate with citrate buffer was thawed in a water bath at a temperature of 24 ° C. The thawed cryoprecipitate solution was centrifuged, the supernatant of the cryoprecipitate solution was separated and transferred for clarification filtration. Clarifying filtration was carried out on a filter filled with membranes with a pore diameter of 3.0 μm. From the tank, the supernatant of the cryoprecipitate solution was filtered under a pressure of 0.05 MPa into a sterile container. In a container with a clarified supernatant of a cryoprecipitate solution, heparin solution was added in the ratio of 0.3 kg of cryoprecipitate / 7000 units of heparin and mixed. Then virusinactivation treatment was carried out using the solvent-detergent method. In this case, the supernatant of the cryoprecipitate solution was treated with tri-n-butylphosphate and Tween 80 at final concentrations of 0.3% and 1%, respectively, for (6 ± 2) hours at a temperature of (3 ± 2) ° С and constant slight stirring. Then, a virus-inactivated cryoprecipitate solution was chromatographically purified using a sorbent with a fractionation interval of up to 20,000 kDa at a flow rate of 5 cm / h.

After chromatographic purification, the eluate containing factor VIII was collected in sterile vials and albumin was added to a final concentration of 2 g / l, glycine to a final concentration of 7 g / l, sucrose to a final concentration of 7 g / l and frozen at a temperature of minus 40 ° С . Next, the eluate containing factor VIII was thawed at a temperature of 19 ° C and poured into a container. Then, clarifying filtration was performed through membranes with a pore diameter of 3.0 μm under a pressure of 0.05 MPa into a sterile container. The yield from this stage was 80% of the content of factor VIII in the initial cryoprecipitate. Then, concentration was carried out.

Concentration was carried out by ultrafiltration on a membrane filtration unit using membranes with a cutoff threshold of not more than 200 kDa. For this, the ultrafiltration unit was filled with an eluate containing factor VIII, and the concentration process was started. The eluate containing factor VIII, entering the plant from the tank, circulated in a closed loop, creating a tangential flow along the fibers along their inner part. Permeate was checked for protein content every 15 minutes. For this, equal volumes of permeate and a 20% trichloroacetic acid solution were mixed in vitro. The eluate containing factor VIII was concentrated to 13 IU of factor VIII in 1 ml. The yield from this stage was 99% of the content of factor VIII in the initial eluate containing factor VIII. Then sterilizing filtration was carried out simultaneously with the process of filling the drug into bottles. The filtration system consisted of a container, a filter and a capsule. Installation of the system was carried out in compliance with aseptic rules. After that, the eluate concentrate containing factor VIII was filtered from a container under a pressure of 0.05 MPa through a filter filled with membranes with a pore size of 3.0 μm, and then through a filter filled with membranes with a pore size of 0.45 μm, 0.22 μm . A sterile solution of the eluate concentrate was poured into vials.

The yield from this stage was 83% of the factor VIII content in the initial eluate concentrate containing factor VIII.

Drying of the sterile concentrate of the eluate containing factor VIII began with freezing of the product at a temperature of minus 10 ° C. Then the temperature was increased by 3 ° C every hour to a temperature of 17 ° C. The final temperature of the product was 17 ° C. The total duration of the drying process of the drug was 38 hours. The residual moisture of the drug was 2.4%.

The yield from this stage was 80% of the factor VIII content in a sterile eluate concentrate containing factor VIII.

Example 2

Freshly frozen plasma was thawed in running water with a temperature of 6 ° C. After the ice disappeared completely and a precipitate formed in the containers with plasma, the containers were immediately opened and the plasma with sediment was pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air of 0.024 MPa was supplied to flowing centrifuges. After centrifugation, the precipitate was removed from supercentrifuge rotors and immediately frozen at minus 80 ° C.

The centrifugate was collected in a container and subsequently used for the manufacture of immunoglobulin and albumin preparations.

The yield from this stage was 43% of the content of factor VIII in the initial plasma. Then, the cryoprecipitate solution was purified as follows: 60 mM citrate buffer, pH 7.5, was added to the container with frozen cryoprecipitate. Then cryoprecipitate with citrate buffer was thawed in a water bath at a temperature of 26 ° C. The thawed cryoprecipitate solution was centrifuged, the supernatant of the cryoprecipitate solution was separated and transferred for clarification filtration. Clarification filtration was carried out on a filter filled with membranes with a pore diameter of 10.0 μm. From the tank, the supernatant of the cryoprecipitate solution was filtered under a pressure of 0.06 MPa into a sterile container. In a container with a clarified supernatant of a cryoprecipitate solution, heparin solution was added in the ratio of 0.3 kg of cryoprecipitate / 10000 U of heparin and mixed. Then virusinactivation treatment was performed using copper phenanthroline buffer. The supernatant of the cryoprecipitate solution was treated with a solution of copper phenanthroline buffer in the ratio of 1 part of copper phenanthroline buffer / 40 parts of the supernatant of the cryoprecipitate solution. Copper phenanthroline buffer (CuPH) was prepared by mixing 10 ml of 0.1 M histidine, 8 ml of 0.01 M 5-aqueous copper sulfate and 8 ml of 0.5 M 1,10-phenanthroline. The volume was adjusted to 200 ml with water for injection. Then, a virus-inactivated cryoprecipitate solution was chromatographically purified using a sorbent with a fractionation interval of up to 20,000 kDa at a flow rate of 35 cm / h.

After chromatographic purification, the eluate containing factor VIII was collected in sterile vials and albumin was added to a final concentration of 8 g / l, glycine to a final concentration of 13 g / l, sucrose to a final concentration of 13 g / l and frozen at a temperature of minus 80 ° С . Next, the eluate containing factor VIII was thawed at a temperature of 21 ° C and poured into a container. Then, clarifying filtration was performed through membranes with a pore diameter of 10.0 μm under a pressure of 0.06 MPa into a sterile container. The yield from this stage was 85% of the content of factor VIII in the initial cryoprecipitate. Then, concentration was carried out.

Concentration was carried out by ultrafiltration on a membrane filtration unit using membranes with a cutoff threshold of not more than 200 kDa. For this, the ultrafiltration unit was filled with an eluate containing factor VIII, and the concentration process was started. The eluate containing factor VIII, entering the plant from the tank, circulated in a closed loop, creating a tangential flow along the fibers along their inner part. Permeate was checked for protein content every 15 minutes. For this, equal volumes of permeate and a 20% trichloroacetic acid solution were mixed in vitro. The eluate containing factor VIII was concentrated to 22 IU in 1 ml of eluate. The yield from this stage was 95% of the content of factor VIII in the initial eluate containing factor VIII. Then sterilizing filtration was carried out simultaneously with the process of filling the drug into bottles. The filtration system consisted of a container, a filter and a capsule. Installation of the system was carried out in compliance with aseptic rules. After that, the eluate concentrate containing factor VIII was filtered from a container under a pressure of 0.06 MPa through a filter filled with membranes with a pore size of 10.0 μm, and then through a filter filled with membranes with a pore size of 0.45 μm, 0.22 μm . A sterile solution of the eluate concentrate was poured into vials.

The yield from this stage was 85% of the factor VIII content in the initial eluate concentrate containing factor VIII.

Drying of a sterile concentrate of the eluate containing factor VIII began with freezing of the product at a temperature of minus 40 ° C. Then the temperature was increased by 7 ° C every hour to a temperature of 33 ° C. The final product temperature was 33 ° C. The total duration of the drying process of the drug was 52 hours. The residual moisture of the drug was 2%.

The yield from this stage was 90% of the factor VIII content in a sterile eluate concentrate containing factor VIII.

The rationale for the execution modes of the proposed method

Defrosting freshly frozen plasma in running water with a temperature of (4 ± 2) ° C provides a yield of 38-43% of the content of factor VIII in the initial plasma. The use of running water with a temperature below 2 ° C when defrosting freshly frozen plasma leads to a prolongation of the defrosting process and, as a result, to a decrease in the yield of less than 38% of the content of factor VIII in the initial plasma. This leads to a decrease in the profitability of production, since the yield of factor VIII from a liter of freshly frozen plasma is reduced, as well as the complexity of the technological scheme, since an additional concentrated eluate containing factor VIII is required.

The use of running water with a temperature above 6 ° C during defrosting of freshly frozen plasma leads to disruption of the precipitation of coagulation factor VIII, resulting in its cryoprecipitate yield of less than 38% of the content of factor VIII in the initial plasma. This leads to a decrease in the profitability of production.

When cleaning cryoprecipitate, a heparin solution in the ratio of 0.3 kg of cryoprecipitate / 7000-10000 U of heparin is added to the container with the clarified supernatant of the cryoprecipitate solution and mixed. When a heparin solution is added to a final concentration of less than 7000 Units per 0.3 kg of cryoprecipitate, the yield from this stage is less than 80% of the content of factor VIII in the initial cryoprecipitate, which reduces the profitability of the preparation. When a heparin solution is added to a final concentration of more than 10,000 Units per 0.3 kg of cryoprecipitate, the activity of factor VIII in the eluate containing factor VIII decreases, which complicates the technological scheme, since an additional concentrated eluate containing factor VIII is required.

When cleaning a virus-inactivated cryoprecipitate solution by gel chromatography using a sorbent with a fractionation interval of up to 20,000 kDa at a flow rate of 20 ± 5 cm / h, a yield of 80-85% of the content of factor VIII in the initial cryoprecipitate is ensured. When using a sorbent with a higher fractionation interval, the interaction of factor VIII molecules with the sorbent is enhanced. As a result, the yield of factor VIII decreases and is less than 80% of its content in the initial cryoprecipitate. At a flow rate of less than 5 cm / h, the time of chromatographic purification is increased, which complicates the technological scheme and reduces the profitability of the preparation. At a flow rate of more than 35 cm / h, the quality of the chromatographic purification of the eluate containing factor VIII decreases, which complicates the technological scheme due to the need for additional purification steps.

When concentrating an eluate containing factor VIII by ultrafiltration on a membrane filtration unit using membranes with a cut-off threshold of not more than 200 kDa, the yield is 95-99% of the content of factor VIII in the initial eluate containing factor VIII. When concentrating the eluate containing factor VIII using membranes with a cut-off threshold of more than 200 kDa, some of the molecules of factor VIII pass through the membranes and enter the permeate, which is a waste product. This leads to a decrease in the yield of factor VIII of less than 95% of the content in the initial eluate containing factor VIII.

The addition of albumin to a final concentration of (5 ± 3) g / l in the eluate containing factor VIII stabilizes the factor VIII molecule, increasing the stability of the yield of factor VIII after concentration, sterilizing filtration and drying. Adding albumin to a final concentration of less than 2 g / l causes a decrease in the stability of factor VIII molecules, resulting in a decrease in the yield of factor VIII after concentration, sterilizing filtration and drying. Thus, the stability of the yield of coagulation factor VIII during production of the drug is reduced. The addition of albumin to a final concentration of more than 8 g / l does not increase the stability of factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

Adding alanine, or arginine, or glycine, or histidine to a final concentration of (10 ± 3) g / l in the eluate containing factor VIII stabilizes the factor VIII molecule, increasing the stability of the yield of factor VIII after concentration, sterilizing filtration and drying. The addition of alanine, or arginine, or glycine, or histidine to a final concentration of less than 7 g / l causes a decrease in the stability of factor VIII molecules, resulting in a decrease in the yield of factor VIII after concentration, sterilizing filtration and drying. Thus, the stability of the yield of coagulation factor VIII during the production of the drug is reduced. The addition of alanine, or arginine, or glycine, or histidine to a final concentration of more than 13 g / l does not increase the stability of factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

The addition of maltose, or glucose, or sorbitol or sucrose to a final concentration of (10 ± 3) g / l in the eluate containing factor VIII stabilizes the factor VIII molecule, increasing the stability of the yield of factor VIII after concentration, sterilizing filtration and drying. The addition of maltose, or glucose, or sorbitol or sucrose to a final concentration of less than 7 g / l causes a decrease in the stability of factor VIII molecules, resulting in a decrease in the yield of factor VIII after concentration, sterilizing filtration and drying. Thus, the stability of the yield of coagulation factor VIII during the production of the drug is reduced. The addition of maltose, or glucose, or sorbitol, or sucrose to a final concentration of more than 13 g / l does not increase the stability of factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

When carrying out drying, including freezing, at a temperature of minus (25 ± 15) ° С, a temperature increase of (5 ± 2) ° С per hour to a final temperature of (25 ± 8) ° С and a total duration of the drying process of the preparation of 45 ± 7 h , the yield is 80-90% of the content of factor VIII in a sterile eluate concentrate containing factor VIII. Freezing at a temperature of less than 10 ° C causes a decrease in the yield of less than 80% of the content of factor VIII in a sterile concentrate of the eluate containing factor VIII. Freezing at temperatures above 40 ° C does not increase the yield of factor VIII, but increases the cost of the drug, therefore, reduces the profitability of the drug. When the temperature rises by less than 3 ° C per hour or more than 7 ° C per hour, the yield decreases and amounts to less than 80% of the factor VIII content in a sterile eluate concentrate containing factor VIII. When the drying process lasts less than 38 hours, the drug has a residual moisture content of more than 2.4% and does not meet the requirements of the FSF for this drug. When the drying process takes more than 52 hours, the yield of coagulation factor VIII decreases and is less than 80% of the content of factor VIII in the sterile concentrate of the eluate containing factor VIII.

Obtained by the claimed method, the preparation of coagulation factor VIII human blood provides an increase in the content of factor VIII in blood plasma and temporarily eliminates the coagulation defect in patients with hemophilia A (hereditary bleeding, characterized by insufficient activity of a specific plasma protein, factor VIII blood coagulation) and is intended for the treatment and prevention of bleeding in patients with hemophilia A, including during emergency or planned surgical intervention.

Claims (1)

A method of obtaining a preparation of coagulation factor VIII of human blood, which consists in obtaining cryoprecipitate from blood plasma, virus-inactivating treatment, purification of a solution of cryoprecipitate, concentration, sterilizing filtration, bottling and drying, characterized in that cryoprecipitate is obtained using running water with a temperature of 4 ± 2 ° C when defrosting freshly frozen plasma, virus-inactivating treatment is carried out using the solvent-detergent method before cleaning the cryoprecipitate solution, chromatogram An aphic purification of a virus-inactivated cryoprecipitate solution is carried out using a sorbent with a fractionation interval of up to 20,000 kDa, at a flow rate of 20 ± 15 cm / h, followed by concentration by ultrafiltration on hollow fibers, the addition of stabilizers, which use albumin at a final concentration of 5 ± 3 g / l, sugar and amino acids to a final concentration of 10 ± 3 g / l, upon drying, the initial temperature of the drug is minus 25 ± 15 ° C, the final temperature is 25 ± 8 ° C, the total duration of the process is drying of the drug is 45 ± 7 hours