RU2353386C1 - Method for making human coagulation factor ix concentrate - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and pharmaceutical industry area and concerns methods for making frozen-dried high-purity blood plasma factor IX preparation. In the present invention supernatant made of plasma after solvent-detergent processing is applied on an anion-exchange column to concentrate factor IX and remove viral inactivation components. Then salt fractionation follows with ammonium sulphate. This reasonably priced and effective stage allows eliminating the majority of prothrombin-complex impurity factors with keeping those components well separated from end protein at the following stage. The last stage involves final purification by metalchelate chromatography. The problem of the invention is to simplify blood plasma factor IX technology enabling for high-active product to be effective in clinical practice.

EFFECT: invention aims at simplifying the method for making coagulation factor IX of high specific activity of protein 150-200 ME/mg.

2 cl, 2 ex

Description

The invention relates to the field of biotechnology and the pharmaceutical industry and relates to methods for producing a lyophilized highly purified factor IX preparation from blood plasma.

Factor IX is a protein that plays a key role in the process of blood coagulation and is intended for replacement therapy for hemophilia B.

Factor IX containing drugs are concentrates enriched with this protein. Their main components are factors II, VII, IX and X, known as the prothrombin complex. Obviously, with prolonged transfusion of such concentrates, complications associated with the activation of impurity proteins are possible. Therefore, one of the leading tasks in creating medicines containing factor IX is to minimize the amount of impurities.

Since the specific concentration of factor IX in plasma is small and amounts to about 0.01 IU / mg of total protein, all known methods are reduced at the first stage to the concentration of the target product. Most often, ion-exchange chromatography is used for this. However, the non-selectivity of this method allows one to concentrate other proteins, including the components of the prothrombin complex. The specific activity in this case increases to 10-20 IU / mg of total protein. There are preparations where the method for producing the concentrate is limited to only one type of chromatography (for example, RF patent 2163140 dated February 20, 2001). Naturally, this is not enough, and additional purification is necessary to obtain a long-term drug. In a two-stage technology for the preparation of factor IX concentrate, the second stage of purification is most often chromatography (affinity, hydrophobic, etc.) based on the use of other properties of this protein.

For example, a method is known combining two sequential chromatographies: using an ion exchanger on which the prothrombin complex is obtained and affinity chromatography on heparin sepharose (US Pat. No. 5,457,181 of October 10, 1995). The specific activity of factor IX in the final preparation was about 130 IU / mg protein.

However, two steps are not enough to obtain a highly purified protein. Therefore, the search for additional methods for purification of factor IX is currently an important task.

For example, a method is known combining ion exchange chromatography, hydrophobic chromatography and chromatography on heparin sepharose (US patent 5919909 dated July 6, 1999), allowing to obtain the final preparation with specific activity of more than 100 IU / mg protein.

This example shows that the decisive moment is the selection of a combination of the second and third stages of purification of factor IX.

The closest in technical essence and the achieved result is a method for the isolation and purification of factor IX from blood plasma, which includes multi-stage sequential chromatography on various sorbents (ion-exchange resins, for example DEAE-sepharose, affinity heparin-containing resins, for example, heparin-sepharose, hydroxyapatite resins, for example, Ceramic-Hydroxyapatite, and affinity sorbents with immobilized metals) with specially selected buffers for elution of the target protein, the result is a product with high specific activity (240 V / mg) (EP 1571208, 09/07/2005).

The disadvantage of this method is its complexity and complexity of each stage, the use of specific expensive types of sorbents.

The objective of the invention is to simplify the technology for the preparation of factor IX drug from blood plasma with the achievement of a sufficiently high activity of the resulting product, which will allow its successful application in clinical practice. The method of obtaining it is based on a simpler technique and the use of more affordable means. Thus, the technical result achieved by the implementation of this invention is to simplify the method of obtaining factor IX blood coagulation with a high level of activity of the drug.

In the present invention, the supernatant obtained from plasma and subjected to solvent detergent treatment is applied to a column with an anion exchanger to concentrate factor IX and remove the components of viral inactivation. Then salting out is carried out using ammonium sulfate. This inexpensive and effective stage allows you to discard most of the impurity factors of the prothrombin complex and leave those components that are well separated from the target protein in the next stage. At the last stage, the final purification is carried out using metal chelator chromatography. This allows to increase the specific activity of factor IX to 150-200 IU / mg protein.

A method for producing concentrate IX of coagulation factor from human blood plasma is proposed, including ion exchange chromatography on DEAE-Sepharose, precipitation with ammonium sulfate and metal chelating chromatography followed by dialysis against a buffer, characterized in that the plasma is centrifuged beforehand, the supernatant is treated with 0.5-2% Tween 80 and 0.1-1% tri-n-butylphosphate, chromatography on DEAE-Sepharose uses 15-15 mm Tris-HCl buffer, pH 7.5, containing 40-60 mm sodium citrate, 30-60 mm sodium chloride, with the same elution with a buffer containing 100-200 mm sodium chloride, the eluate is treated with ammonium sulfate to a degree of saturation of 25-35%, followed by centrifugation, the precipitate is discarded, the supernatant is diafiltered and chromatographed on a Cu-chelate sepharose column using 5-15 mm Tris HCl buffer containing 30-60 mm sodium citrate, pH 7.0-7.8, carry out the removal of ballast proteins with a buffer containing an additional 50 mm sodium chloride, and the target protein is eluted with 5-15 mm Tris HCl buffer, pH 7.2, containing 30-60 mm sodium citrate, 90-140 mm glycine and 100-200 mm chloride sodium, which is dialyzed against 5-15 mm Tris HCl buffer containing 30-60 mm sodium citrate, 30-60 mm glycine, pH 7.2.

The implementation of the method is illustrated by the following examples.

Example 1

100 l of plasma containing 80080 IU of factor IX were thawed and centrifuged. After separating the precipitate, tween 80 was added to 95 l of the supernatant to a final concentration of 1% and tri-n-butyl phosphate to a final concentration of 0.3%, and then incubated for 16 hours. The mixture was applied to a column filled with DEAE-Sepharose. Then it was washed first with a balancing buffer containing 10 mM Tris-HCl and 50 mM sodium citrate, then with the same buffer, but containing an additional 50 mM sodium chloride. Elution was performed with the same buffer, but containing 150 mM sodium chloride. The resulting 4.5 L of eluate containing 62635 ME of Factor IX was diafiltered against equilibration buffer and applied to a Cu-chelate Sepharose column. After washing with equilibration buffer (10 mM Tris HCl, 50 mM sodium citrate, pH 7.5), ballast proteins were removed with a buffer containing an additional 50 mM sodium chloride. The desired product was eluted with 10 mM Tris HCl buffer, pH 7.2, containing 50 mM sodium citrate, 120 mM glycine and 150 mM sodium chloride. The 3.5 L eluate contained 56372 ME of factor IX activity. After dialysis against formulation buffer (10 mM Tris HCl, pH 7.2, 50 mM sodium citrate, 50 mM glycine), the solution was sterilized and lyophilized. The specific activity of factor IX in the preparation was 118 IU / mg protein.

Example 2

As in example 1, but the eluate obtained after the column with DEAE-Sepharose was cooled to + 6 ° C, ammonium sulfate was added to a final concentration of 30% and left for 16 hours at the same temperature. After this, the suspension was centrifuged, the precipitate was discarded, and the supernatant was diafiltered and applied to a Cu-chelate Sepharose column. After washing with equilibration buffer (10 mM Tris HCl, 50 mM sodium citrate, pH 7.5), ballast proteins were removed with a buffer containing an additional 50 mM sodium chloride. The desired product was eluted with 10 mM Tris HCl buffer, pH 7.2, containing 50 mM sodium citrate, 120 mM glycine and 150 mM sodium chloride. The eluate contained 51175 ME of factor IX activity in 3.5 L. After dialysis against formulation buffer (10 mM Tris HCl, pH 7.2, 50 mM sodium citrate, 50 mM glycine), the solution was sterilized and lyophilized. The specific activity of factor IX in the preparation was 287 IU / mg protein.

Claims (2)

1. A method of obtaining a concentrate of IX coagulation factor from human blood plasma, including ion exchange chromatography on DEAE-Sepharose, diafiltration and metal chelating chromatography followed by dialysis against a buffer, characterized in that the plasma is centrifuged beforehand, the supernatant is treated with 0.5-2% with stirring Tween 80 and 0.1-1% tri-n-butyl phosphate, using chromatography on DEAE-Sepharose, use 5-15 mm Tris-HCl buffer containing 40-60 mm sodium citrate, 30-60 mm sodium chloride, pH 7.0 -7.9 with elution with the same buffer containing 100-200 M sodium chloride, the eluate is treated with ammonium sulfate to a degree of saturation of 25-35%, followed by centrifugation, the precipitate is discarded, the supernatant is diafiltered and chromatographed on a Cu-chelate Sepharose column using 5-15 mM Tris HCl buffer containing 30-60 mM sodium citrate, pH 7.0-7.8, carry out the removal of ballast proteins with a buffer containing an additional 50 mm sodium chloride and the target protein is eluted with 5-15 mm Tris HCl buffer, pH 7.0-7.8 containing 30-60 mm sodium citrate, 90-140 mm glycine and 100-200 mm sodium chloride, which dialysis t against 5-15 mM Tris HCl buffer containing 30-60 mM sodium citrate, 30-60 mM glycine, pH 7.2, and having a specific activity of at least 150-287 IU / mg protein. 2. The method according to claim 1, characterized in that, if necessary, the product is subjected to freeze drying.