CS273467B1 - Method of precallicreine's and hageman's factor's from blood plasma standard concentrates preparation - Google Patents

Abstract

The invention concerns a method of preparation of standard concentrates of precallicreine and Hageman factors from blood plasma, particularly from intact citric human blood plasmas, for diagnostic purposes, above all for control of the quality of intravenously applied blood derivatives. The essence of the invention is the method of processing blood plasma into a precallicreine concentrate by sorption with an equilibrated sorbent on a base of diethylaminoethyl derivatives of polysaccharides or hydroxyalkyl methacrylate or glycol methacrylate gel in the free space of an electrical charge and into a Hageman factor fragment, which is achieved by desorption of protein from the sorbent by enrichment of an eluate by proteolysis of the Hageman factor fragment after desalting and subsequent fractionalisation with a cation exchanger on a base of a carboxymethyl derivative of dextran or cellulose. The method simplifies and accelerates the process of preparation of the precallicreine concentrate and Hageman factor fragment concentrate.

Description

The invention relates to methods for preparing standard concentrates of precallikrein and a fragment of Hageman factor from blood plasma, in particular from intact citrate blood plasma, for diagnostic purposes.

Plasma precallikrein serves as the only specific agent for laboratory demonstration of the presence of a fragment of Hageman factor (a precallikrein activator, also HFf) in preparations prepared from blood plasma. HFf is the initiator of processes that lead to the release of kinins from plasma kininogens in a bloodstream by a known mechanism. Thus, contamination with a Hageman factor fragment in intravenously administered blood derivatives represents a threat to the recipient of a sharp drop in blood pressure, leading to circulatory collapse. The prior art manufacturing processes for the preparation of a stable plasma solution, albumin or intravenous gamma-globulin do not prevent the HFf present in the starting material as such or in the form of a precursor from passing into the final formulation as a contaminant. Determination of the level of HFf, whose hypotensive effects are known, may be an indicator of the possible reactivity of blood plasma derivatives in clinical applications.

Demonstration of the Hageman factor fragment in clinically used preparations is based on the fact that the preparation containing this factor is able to activate precallikrein to the enzyme kallikrein, the released amount of which is proportional to the precallikrein activator present and is determined by a specific substrate. Some authors use substrates based on basic amino acid esters (cf. lit. 1, 2), but later more specific chromogenic oligopeptide derivatives of p-nitroaniline are used (cf. lit. 3). In order to standardize the HFf assay in preparations prepared from blood plasma, a standard precallikrein and a standard precallikrein activator must be available.

Methods are known for separating precallikrein from blood plasma by column chromatography on sorbents of the dextran diethylaminoethyl derivative type, which are generally known and commercially available (cf. lit. 4). Since the surface electrical charge of precallikrein is close to the surface electrical charge of immunoglobulin G, the procedures described for the isolation of this fraction from blood plasma can also be used for this purpose (cf. lit. 5). The disadvantage of these column chromatography methods is, in particular, the several-hour separation process, during which the plasma being treated may be activated by precallikrein into the enzyme kallikrein due to activation cofactors present in blood plasma and due to the negative electrical charge of the glass separator surface. There is a known way to prevent undesirable activation of precallikrein: To separate it from plasma, it is necessary to use chromatographic tubes with an intact surface, ie free of electric charge, or glass tubes coated inside with a layer of silicone.

The method according to the invention has succeeded in eliminating the disadvantages of the prior art described above, in particular the long-term separation and considerable demands on the special treatment of the apparatus, and at the same time utilizes the blood plasma residues after precallikrein isolation to prepare the Hageman factor fragment concentrate. It is based on the fact that the starting material with the addition of a neutral electric charge neutralizing agent, such as hexadimethrin bromide, previously equilibrated with a pH buffer of 6.3 to 8.0 and a molarity of 0.0175 to 0.1, is introduced in a surface-free apparatus. charge, in constant contact with an anion exchange resin, in particular based on diethylaminoethyl derivatives of polysaccharides, for example dextran or cellulose, or on the basis of a hydroxyalkyl methacrylate or glycol methacrylate gel, prewashed with equilibration buffer, after adsorption, again in an apparatus free of surface electric charge, then the anion exchange sorbent protein portion containing the Hageman factor fragment precursor is released by elution with a buffer solution having a pH of equilibration buffer and a sodium chloride content of at least 1.0 mol / l, the salt, for example by dialysis, and subjected to proteolysis, for example by kallikrein, and the released HFf is isolated on a cation exchanger, for example based on dextran or cellulose carboxymethylderivative, after equilibrating both the eluate and cation exchanger concentrraCS 273 467 Bl · <£ (!

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or from 0 to 1.0 mol / L sodium chloride.

In carrying out the method of the invention, the basic precaution is to bring the blood plasma into contact with the anion exchange resin in a container having an intact surface, i. free of electrical charge, under precisely defined pH and ionic strength conditions, under which precallikrein, along with the immunoglobulin fraction, does not adsorb to the anion exchange resin. The non-adsorbed portion serves as a precallikrein concentrate. The anion exchange protein fraction containing the precursor of the Hageman factor fragment is released by elution with an increased ionic strength solution and is used to prepare the HFf concentrate. After removal of the salts, the fraction obtained is subjected to proteolytic cleavage to enrich for the Hageman factor fragment. under defined conditions, it is separated by cation exchange column chromatography. The HFf concentrate is processed to a precallikrein activator standard in a manner known per se as illustrated in Example 2.

The method of the invention by applying a batch process in a vessel (or apparatus) free of surface electric charge allowed the preparation of the precallikrein concentrate to be shortened to a maximum of 30 min. thus substantially reducing the risk of autoactivating or proteolytic processes in the plasma. It does not require any special chromatographic apparatus and at the same time utilizes sorbed protein fractions to prepare HFf concentrate. Under the recommended conditions according to the invention, both concentrates are of constant quality.

An advantage of the method according to the invention is that it allows a very simple, fast and economical preparation of the plasma components necessary to control the presence of the Hageman factor fragment in preparations obtained from human blood plasma.

The following examples illustrate the invention but do not limit it in any way.

Example 1

a) Preparation of precallikrein concentrate

To 100 ml of intact human citrate plasma is added 5 mg of hexadimethrin bromide (synthetic heparin antagonist, Polybren) and then the plasma is equilibrated by dialysis at 4 ° C against Tris-HCl buffer (0.1 mol / l, pH 8.0) with hexadimethrin bromide added. at a concentration of 50 mg / l. To the equilibrated plasma, 8 g of dextran diethylaminoethyl derivative having a particle size of 40 to 120 µm or 10 g of cellulose diethylaminoethyl derivative or 8 g of hydroxyethyl methacrylate, previously washed with equilibration buffer, was added and the mixture stirred gently in a polyethylene vessel for 20 minutes. After the sorption is complete, the sorbent is aspirated on a suction cup equipped with fast flowing filter paper and the filtrate is collected in a polyethylene bag or container. The filtrate obtained is precallikrein concentrate. As described above, all precallikrein present in the starting plasma is obtained, together with IgG, constituting 95 to 98% of the protein of the concentrate. The number of international precallikrein units is determined by activation with a standard precallikrein activator.

b) Preparation of the Hageman Factor Fragment Concentrate

The anion exchange resin separated from the liquid fraction containing the protein fraction containing the precursor of the Hageman factor fragment is eluted twice with 150 ml of a high ionic strength buffer, for example Tris-HCl (0.1 mol / l, pH 8), 0) with 2.0 mol / l sodium chloride. The eluate of about 300 ml is dehydrated by dialysis against distilled water (or diafiltration) and concentrated to 50 ml by diafiltration. Addition of 10 ml of active kallikrein enzyme solution (activity of 80-100 U / ml) and incubation for at least 8 hours at 25 ° C enriched in HFf. The mixture was fractionated on a cation exchange column (CM Sephadex, particle size 40-120 / un, column size 1.6 x 40 cm) with a salt gradient of 0 to 1.0 mol / l sodium chloride in acetate buffer (0.1 mol / l pH 5.9). The HFf-containing fractions were combined and, after removal of salts by dialysis against distilled water (or diafiltration), concentrated to a volume such that the obtained Hageman factor fragment concentrate had an activity of at least 90 U / ml. Together with albumin, HFf accounts for more than 98% of the protein.

CS 273 467 Bl

Example 2

Preparation of precallikrein activator standard in albumin

For the preparation of the precallikrein activator standard, commercial albumin is used to remove any kallikrillin system inhibitors present by binding to a glycoprotein affinity sorbent (e.g., Concanavalin lectin-linked sieve agaresis) and free kallikrein as described in Example 1 (a). for separating precallikrein. The sorbed albumin is eluted with the high ionic strength buffer as described in Example 1 (b), the salts are removed in the same manner and the solution is concentrated by ultrafiltration. The Hageman factor fragment concentrate is mixed with the albumin concentrate so that the final protein concentration is approximately 5% and the Hageman factor fragment activity is comparable to the International Precallikrein Activator Standard (National Institute for Biological Standards and Control, London). The osmolality of the solution is adjusted to 300 mOs / kg by addition of sodium chloride.

Claims (1)

SUBJECT OF THE INVENTION Process for the preparation of standard concentrates of precallikrein and of the Hageman factor fragment from blood plasma, in particular from intact citrate human blood plasma using generally known methods and materials such as column chromatography on sorbents of the diethylaminoethyl derivative of polysaccharides or hydroxyalkyl methacrylate or glycol methacrylate gel precallikrein and on carboxymethyl derivative sorbents using elution, desalination and proteolysis for the Hageman factor fragment concentrate, characterized in that the starting material with the addition of a neutral electric charge neutralizing agent, for example hexadimethrin bromide, is previously equilibrated with a buffer of pH 6.3 to 8, 0 with a molarity of 0.0175 to 0.1, states in an area free of surface electric charge, in constant movement in contact with an anion exchange resin, in particular based on diethylaminoethyl derivatives, polysaccharide dextran or cellulose, or based on a hydroxyalkyl methacrylate or glycol methacrylate gel, pre-washed with an equilibration buffer, after the sorption is complete, the solution of the unabsorbed protein containing the precallikrein concentrate is separated, for example by filtration, again in a surface free electric charge space. anion exchange resin adsorbed protein containing a Hageman factor fragment precursor, eluting with a pH buffer of equilibration buffer containing 1.0-2.0 mol / l sodium chloride, desalting the obtained eluate, for example by dialysis, and subjecting to proteolysis, for example, kallikrein and released the Hageman factor fragment is isolated on a cation exchanger, for example based on dextran or cellulose carboxymethylderivatives, after equilibrating both the eluate and the cation exchanger with a pH buffer of 5.5 to 6.0, eluting with a salt gradient, e.g. at a concentration of 0 to 1.0 mol / l chloride sodium.