DE2635894A1 - Prepn. of concentrates contg. anti-haemophilic globulin:A - by adsorption onto di:ethylamino-ethyl-cellulose from plasma etc., then elution with buffer - Google Patents

Abstract

Method for preparing a concentrate contg. antihaemophilia globulin A (I) uses as starting protein soln. an undiluted soln. obtd. conventionally from stabilise plasma, or a diluted factor 8 contg. soln. of protein concn. about 1%. Diethylaminoethylcellulose (II) is stirred into the soln. and when adsorption is complete, (II) is centrifuged off, washed with a buffer, then eluted with a second buffer soln. Pref. the starting material is a plasma stabilised with citrate, heparin or citric acid-dextrose; a cryoprecipitate or Cohn-I fractions. The plasma left after removal of (I) contains no additives; recovery of (I) is higher than in known processes (e.g. 40-50% using cryoprecipitate c.f. not above 20%, usually about 10%) since adsorption onto (II) is nearly quantitative; and (I) is only very slightly contaminated by other proteins.

Description

Process for the production of an antihemophilic globulin A.

containing concentrate The invention relates to a method for Production of a concentrate containing antihemophilic globulin A from protein solutions using diethylaminoethyl cellulose, which is suitable for therapeutic application suitable is.

So far one has for the production of therapeutically applicable concentrates of the antihemophilic globulin (ARG or factor VIII) for the enrichment of the antihemophilic Globulins always used precipitation methods.

The methods described in the literature for purifying ARG modified cellulose to become clinically useful Preparations consistently say that diethylaminoethylcellulose is only unsatisfactory Yields of the desired product are obtained (cf. Blood Coagulation, Haemostasis and Thrombosis, edited by Rosemary Biggs, Blackwell Scientific Publications, pp. 249, 1972). According to the literature, precipitation processes should also be used of alcohol, ether, cold, polyethylene glycol, amino acids or tannic acid products result in which the plasma proteins are changed or denatured so that they often lead to intolerance. Also are those present in the source material Proteins changed or denatured in such a way that they can no longer be used intravenously can be. In all cases, the yield of ARG is only very low (see Blood Coagulation, Haemostasis and Thrombosis, edited by Rosemary Biggs, Blackwell Scientific Publications, pp. 233-248).

That of J. Pool (Pool, J.G., Hershgold, E.J. and Shannon, A.E. Federation Froc. 24 (1965) B. 512) described ery precipitate as a starting material for highly concentrated F VIII preparations. When applied this procedure should be canceled in the best case 50 * of the ARG, normally at 20 - 30 * the initial amount of AHG. The cryoprecipitate is either directly used or serves as a starting material of more highly purified AHG preparations. Again, use the methods for further purifying the ARG from cryoprecipitate Precipitation methods.

The disadvantage of all the methods described for the further purification of the ARG from cryoprecipitate always consists in the large yield losses that occur with this Proceedings occur.

It has now surprisingly been found that under certain Experimental conditions ARG adsorb on diethylaminoethyl cellulose and then elute and in this way produce a therapeutically suitable LHG concentrate can.

The invention relates to a method for producing an antihemophilic Concentrate containing globulin A from protein solutions using diethylaminoethyl cellulose, which is characterized in that it is made as a protein solution in a known manner undiluted protein solutions obtained from stabilized plasma or factor VIII Dilute protein solutions containing a protein concentration of about 1 * used, stir diethylaminoethylcellulose into these solutions, after the end Adsorption centrifuged the cellulose and washes with a buffer solution and then eluted with another buffer solution.

A cellulose such as that from Whatman is preferably used is used in your ion exchanger, for which the following features are known: Makers- Functional Physics- Lowest Protein- Water Degrees Groups of Ions- capacity recovery condition capacity mg / g mEquivalent / g CM 32 CO (Naform) micro- 1.0 1260 3.1 - 4.0 granular (Na + form) CM 52 C000 micro- 1.0 1260 granular, pre-swollen The AHG-containing protein solutions used can be of various types. Plasma (the plasma can be stabilized in various ways, such as B. with citrate, citric acid dextrose, Heparin) cryoprecipitate, ethanol cryoprecipitate, Cohn I fraction, F VIII-containing PEG precipitations and F VIII-containing NaCl precipitations.

The cellulose is stirred into the ARG-containing solutions and remains in it until the ARG is adsorbed. For the various protein solutions containing AHG the optimal adsorption conditions are very different from each other, due to the different protein concentrations of the AHG solutions are conditional. The AHG adsorption takes place from plasma stabilized in a known manner from the undiluted plasma solutions. Are F VIII-containing protein solutions such as z. B. Cryoprecipitate, Xthanol cryoprecipitate, Cohn-I-Braktion, F VIll-Eftrakte from PEG or NaCl precipitations as starting material for the ARG adsorption with the special If cellulose is used, these solutions must have a protein concentration of about 1% diluted. At higher protein concentrations of these solutions takes place only inadequate adsorption of the F VIII y VIII-containing solutions with protein values around 5 * only about 30 70 to 50 * of the present one ARG adsorbed, while almost 100 * of the ARG is adsorbed from these solutions, when the protein concentration of the solutions is adjusted to about 1%.

After the ARG adsorption on the diethylaminoethyl cellulose, it is centrifuged and the cellulose is washed with a suitable buffer system.

Suitable washing buffers are saline-containing citrate buffers or phosphate buffers with low molarity. A 0.03 M phosphate buffer is preferably used with a pH of 7.0. Washing removes excess protein from the Removed diethylaminoethyl cellulose.

The ARG is then separated from the diethylaminoethyl cellulose using a suitable buffer system removed. Suitable eluents are 0.3-0.7 M phosphate buffer. Preferably an 0.4 M phosphate buffer with a pH of 7.0 is used. The other Purification of the ARG from this solution can be done in a known manner. This procedural scheme, in which therapeutically applicable AHG concentrate is obtained has the following advantages compared to the extraction of AHG according to known methods: 1. The plasma freed from MG does not contain any additives, as is the case with various known processes, such as B. Ethanol in the Cohn fractionation or polyethylene glycol in the polyethylene glycol fractionation.

2. The AHG yield is much higher than any other known Process because the ARG can be adsorbed almost quantitatively on the cellulose.

3. The ARG is only minimally contaminated with other proteins.

In the method according to the invention, with reference to the cryoprecipitate used as starting material a yield of ARG of 40 - -50 *, while with the known Usually only 10 so, in the cheapest Trap not more than 20%, based on the plasma used, were recovered.

The invention is illustrated by the following examples.

Example 1 Preparation of antihemophilic globulin A concentrate from Plasma donor venous blood was mixed in a known manner with 3.8% Imatriun citrate solution mixed in a ratio of 9 + 1. The blood was possible within 2 hours after centrifuged after taking blood. The plasma was released within 48 hours, preferably at -40, frozen.

The following were used for the further processing described below Reagents used: Buffer preparations 1. 0.02 M Tris buffer pH 7.0: 2.42 g Tris (hydroxymethyl) aminomethane were distilled in 900 ml. H20 dissolved. Through encores of about 16 ml of 1N HCl, the pH was adjusted to 7.0 and the solution with distilled H20 made up to 1 liter.

2. 0.03 M phosphate buffer pH 7.0: 5.34 g secondary sodium phosphate (Na2HPO4 x 2EI20) were mixed with distilled H20 solved and on 1 Liters filled up. 4.14 g of primary sodium phosphate (NaH2P04 x H20) were diluted with dist. H20 dissolved and made up to 1 liter.

The secondary sodium phosphate solution was initially introduced to adjust the p11 and the pH is adjusted to 7.0 by adding the primary sodium phosphate solution.

3. 0.4 M phosphate buffer pH 7.0: 71.2 g secondary sodium phosphate (Na 2HP04 x 2H20) were dissolved with distilled H20 and made up to 1 liter. 55.2 g of primary sodium phosphate (NaH2P04 x H20) were dissolved with distilled H20 and made up to 1 liter. The pH was adjusted to 7.0 by adding the Solution of the primary sodium phosphate for the solution of the secondary sodium phosphate.

4.1hatman DE 52 cellulose from Vetter KG was treated as follows: 1 kg of the cellulose was stirred with 15 liters of 0.5 N HCl for 30 minutes at room temperature. The cellulose was then centrifuged or filtered to a volume of 16 liters distilled H2O for 5 minutes at room temperature and then twice with 15 liters of 0.5 N DSaOH stirred for 30 minutes at room temperature, with between the NaOH washes followed by a wash with distilled H2O. Subsequently to the NaOH washes were followed by a neutral wash with 1120. The neutral washed cellulose was suspended with physiological NaCl and 20 minutes at 121 ° C with hot air sterilized. Before using the cellulose for adsorption, the cellulose was in 0.03 lm phosphate buffer suspended from pH 7.0. A well pourable suspension contains 100 g dry substance in 150 ml.

5. Regeneration of cellulose: Oellulose has already been used washed again with 0.4 M phosphate buffer from p11 7.0 for 30 minutes at room temperature and then treated as described under 4.

6. F VIII solution buffer: 5.88 g of tri-sodium citrate were in about 800 ml of distilled water dissolved. The pH of this solution was increased by adding 0.1 M citric acid (dissolve 2.1 g citric acid in 100 ml distilled K20) adjusted to a pH of 7.2. This solution was made up with distilled H20 1 liter filled.

1 liter of 0.9% NaCl solution was added to this. Per 1 liter of the A 1: 1 mixture of the two solutions was added to 7.5 g of glycerol.

Procedure: The frozen plasma was thawed at room temperature (the plasma can be thawed at temperatures up to 37 s).

Plasma from multiple donors was pooled. Became the plasma pool the pretreated, sterilized cellulose up to a concentration of 5 to 20 g *, preferably 10 g *, given. The adsorption of the ARG took place in 20 minutes until 2 hours, preferably with stirring in 30 minutes at room temperature. Then it was centrifuged. The supernatant can be used for further plasma protein fractionation be used.

The centrifuged cellulose is 30 minutes at room temperature while stirring with 10 of the initial plasma volume of 0.03 M phosphate buffer with a Washed pH 7.0. After centrifuging off the diethylaminoethyl cellulose the ARG with 0.4 M phosphate buffer with a pH value of 7.0 is removed from the cellulose eluted. Elution conditions: 2.5 l of elution buffer were used per 100 liters of plasma.

Elution occurred within 30 minutes at room temperature. After separating the eluate from the cellulose by centrifugation, a second Elution carried out.

The conditions of the second elution were identical to the conditions for the first elution. The ARG was better known from the combined eluates Weise (loc. Cit., Pages 233-248) further purified, sterile-filtered and freeze-dried. If you dissolve the freeze-dried AHG concentrate in water, it contains about one 25-fold activity of F VIII compared to normal plasma. The concentrate is for Therapy of F VIII deficiency states, for example haemaphilia A, is suitable.

Example 2 The starting material for the production of AHG was from human plasma produced eryoprecipitate.

The thawed eryoprecipitate was washed with 0.02 M Tris buffer from pH 7.0 brought to a protein value of 1 * and extracted for 30 minutes at room temperature. Undissolved constituents were centrifuged off. Thereafter, the solution with 2 * Al (OH) 3 suspension (20 ml of Al (011) 3 suspension per 1 liter of solution) stirred for 5 minutes at room temperature.

Pretreated, sterilized diethylaminoethyl cellulose was added to the F VIII solution given up to a concentration of 10 g%. The AHG adsorption took place under Stir in 30 minutes at room temperature. Then it was centrifuged. the Cellulose centrifuged off was treated further as described in Example 1.

Example 3 As a starting material for EIG production according to Method described in Example 2 contains the F VIII-containing ones listed below Protein solutions processed: 1) Ethanol cryoprecipitate 2) Cohn I fraction 3) AHG-containing polyethylene glycol precipitations 4) AHG-containing common salt precipitations.

The yields obtained are around 40 Cfó.

Claims (7)

Claims: oil)) Process for the production of an antihemophile Concentrate containing globulin A from protein solutions using diethylaminoethyl cellulose, characterized in that as a protein solution in a known manner from stabilized Undiluted protein solutions obtained from plasma or diluted protein solutions containing factor VIII Protein solutions with a protein concentration of about 1 Vo are used in this Stir in solutions of diethylaminoethylcellulose, after adsorption has ended, the cellulose centrifuged off and washed with one buffer solution and then with another buffer solution eluted. 2.) The method according to claim 1, characterized in that as Starting protein solution stabilized with citrate, heparin or citric acid dextrose Plasma or cryoprecipitate or Cohn-I fractions used. 3.) Process according to claims 1 or 2, characterized in that As a detergent, citrate buffers containing sodium chloride or phosphate buffers with lower levels are used Molarity, especially a 0.030 phosphate buffer with a pH of 7.0 is used. 4.) Process according to Claims 1-3, characterized in that one as eluent 0.3-0.7 M phosphate buffer, in particular a 0.4 M phosphate buffer used with a pH of 7.0. 5.) Process according to claims 1-4, characterized in that one the diethylaminoethyl cellulose up to a concentration of 5 - 20, preferably 10 eo, admits. 6.) Process according to claims 1-5, characterized in that the Adsorption from 20 minutes to 2 hours, preferably with stirring at room temperature, is carried out. 7.) Process according to Claims 1-6, characterized in that one to 100 1 plasma 2.5 1 elution buffer is used and preferably at room temperature Eluted for 30 minutes, then centrifuged the eluate from the cellulose and in the same Way, a second elution is carried out, the two eluates obtained are then combined and further cleans and reconditioned in a known manner.