CS220725B1 - A method for producing immunoglobulins for intravenous use from human blood plasma - Google Patents

Abstract

The invention solves a simplified method of producing Immunoglobulins from human blood plasma, taking into account the possibility of using it for intravenous administration. The essence of the invention is that human blood plasma is converted by heat into serum, from which the majority of serum proteins except immunoglobulins are removed by adsorption on an ion exchanger. The remaining filtrate is dried by lyophilization and the obtained immunoglobulin solid is dissolved in a stabilized solution of human albumin. The solution is sterile filtered and heated for at least 10 hours to at least 60 °C. The invention can be used in healthcare in the production of medicinal products from blood plasma

Description

(54) A method for producing immunoglobulins for intravenous use from human blood plasma

2

The invention solves a simplified method for the production of immunoglobulins from human blood plasma with regard to the possibility of use for intravenous administration.

It is an object of the invention that human blood plasma is converted by heat into serum, from which most of the serum proteins except immunoglobulins are removed by adsorption to an ion exchanger. The remaining filtrate is dried by freeze-drying and the obtained immunoglobulin solids are dissolved in a stabilized solution of human albumin. The solution is sterile filtered and heated to a minimum of 60 ° C for at least 10 hours.

The invention may be used in the medical field in the manufacture of medicaments from blood plasma.

SUMMARY OF THE INVENTION The present invention provides a process for the production of immunoglobulins for intravenous use from human blood plasma, which is converted by heat into serum, from which most of the serum proteins except immunoglobulins are removed by adsorption to an ion exchanger.

The main prerequisite for the possibility of administering immunoglobulins for intravenous use is the reduction of anticomplementary activity, which could cause adverse clinical reactions. Most of the existing methods for the preparation of intravenous immunoglobulins use as the basis immunoglobulin isolated by one of the classical fractionation methods, which is further adapted for intravenous use by enzymatic cleavage (pepsin, plasmin), low pH incubation, ultracentrifugation, etc.

The disadvantage of these methods is the low yield of the final product, moreover, in enzymatic cleavage, the preparations contain components with a lower sedimentation constant than the uncleaved immunoglobilin and consequently are more rapidly excreted from the body.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process which allows the preparation of immunoglobulins for intravenous use from human blood plasma with a substantial reduction in these disadvantages. This object is achieved by the process according to the invention by which immunoglobulins can be prepared for intravenous use from human blood plasma. It is an object of the invention that human blood plasma is converted by heat into serum, from which most of the serum proteins except immunoglobulins are removed by adsorption to an ion exchanger. The remaining filtrate is freeze-dried, the obtained immunoglobulin solids are dissolved in a solution containing stabilized human albumin at a concentration of 50 + 5 g / l, and aminoacetic acid at a concentration of 22.5 + +7.5 g / l, the amount of solution used to Dissolution of dry matter corresponds to the original volume of serum obtained by thermal conversion of plasma, the solution is sterile filtered and heated for at least 10 hours to at least 60 degrees Celsius.

The object of the invention is to achieve a higher effect by virtue of the fact that the isolation method has practically no negative effects on the immunoglobulin molecule, which results in a significant effect in yield, with high electrophoretic purity of the final product. In addition, the method has the advantage of maintaining the full biological activity of the immunoglobulin molecule without undesirable aggregation, which makes this preparation suitable for intravenous use. The significance of the method is also that the final product, after appropriate stabilization, is heated to a minimum of 60 ° C for at least 10 hours, which are generally accepted parameters necessary to inactivate hepatitis virus and thereby prevent eventual transmission of viral hepatitis by human blood plasma preparations.

An example of a process for making immunoglobilin for intravenous use with a high content of anti-Rho (D) antibodies of the invention.

Hyperimmune plasma with a high content of specific anti-Rh ₀ (D) antibodies with a titre of 512 to 10124 in the indirect antiglobulin assay is heated at 56 ° C for 40 minutes to convert the plasma to serum fibrin. The fiber is removed by centrifugation at 1500 g for 20 minutes. The current acidity is adjusted to pH = 6.5. Next, an ion exchanger cycled as follows is added to the serum:

100 g of dry ion exchanger (for example DEAE Sephadex A50) are swollen in 5000 ml of 0.1 M phosphate buffer at pH = 6.5 with occasional mixing. After swelling (at least 2 hours), the gel is agitated and after 15 minutes the supernatant liquid is aspirated to remove fine particles. This wash is repeated using 0.01 mol / L phosphate buffer also at pH-6.5. Excess buffer was removed by filtration. The ion exchanger is further washed with 1000 ml of 0.01 mol / l phosphate buffer at pH = 6.5 by pouring over the ion exchanger, stirring, leaving to stand for 15 minutes and then filtering off. The operation is repeated until the wash buffer is used up. Finally, the ion exchanger is washed with water for injection, sucked dry to the extent possible and optionally autoclaved.

The cyclized ion exchanger is added in an amount corresponding to 33 g dry ion exchanger per 1000 ml of serum. The mixture was stirred at room temperature for 20 minutes. The ion exchanger with adsorbed ballast proteins is removed by filtration. For the filtrate obtained, ion-exchange purification is repeated twice in the same manner. The final filtrate is lyophilized after filling and freezing. The anti-Rho (D) immunoglobulin dry matter is dissolved in a solution containing stabilized human albumin at a concentration of 50 ± 5 g / l, followed by aminoacetic acid at a concentration of 22,5 ± 7,5 g / l, the amount of solution used to dissolve the dry matter corresponds to the original serum volume obtained by thermal conversion of the plasma. Next, the solution is sterile filtered. The filtrate is heated to a minimum of 60 ° C for at least 10 hours. After determining the microgram content of specific anti-Rh ₀ (D) antibodies, the final product is filled in an amount corresponding to 100 micrograms of active anti-Rh ₀ (D) antibody per package.

In addition to the chemical composition, the finished product must comply with the biological requirements, ie tests of harmlessness, pyrogenicity, sterility and, in particular, the content of anticomplementary activity required as an assay to detect the presence of undesirable aggregates in immunoglobulin preparations intended for intravenous use.

The process object of the present invention can be extended to the preparation of other specific immunoglobulins for intravenous use, where hyperimmune plasma with a high content of specific antibodies is available as a feedstock.

Claims (1)

Subject Method for producing immunoglobulins for intravenous use from human blood plasma, which is converted by heat to serum, from which most of the serum proteins other than immunoglobulins are removed by adsorption to an ion exchanger, characterized in that the remaining filtrate is freeze dried; containing the invention stabilized human albumin at a concentration of 50 + 5 g / l, and aminoacetic acid at a concentration of 22.5 + 7.5 g / l, the amount of solution used to dissolve the dry matter corresponding to the original serum volume obtained by thermal plasma conversion; filtered and heated for at least 10 hours at least 60 ^Q C.