DK174066B1 - Process for the preparation of a factor VIII preparation - Google Patents

Abstract

The process allows a pasteurised preparation of blood clotting factor VIII, which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen, to be obtained. A preparation of this type can be used to treat disturbances of blood clotting.

Description

in DK 174066 B1

The invention relates to a process for the preparation of a factor VIII preparation which is characterized by the characterizing part of claim 1, and the invention also relates to such a method which is characteristic of the characterizing part of claim 2. Preferred embodiments of these methods are set forth in claims 3 and 4.

The present composition can be used to treat blood clotting disorders.

10 Blood coagulation is a complex process that runs over several reaction steps, involving at least 13 coagulation factors (F), denoted by Roman numerals. The coagulation factors are predominantly proteins and especially those which are equipped with the properties of proteases or accelerators.

The only active substance is fibrinogen, which, by damage to thrombin, is converted to its insoluble form, the fibrin which forms the primary wound closure. When one of the 13 coagulation factors is missing, the formation of 20 thrombin and fibrin is absent and the result is bleeding. Such a case is present in haemophilia A, the most common bleeding disorder characterized by a deficiency of factor VIII. Hemophilia A and B (F IX deficiency) can only be treated by effective therapy by substitution of the missing factor.

25 The extraction of factor F VIII from human plasma with good yield and high purity, as this is especially necessary for self-treatment of patients, still represents a problem that has not yet been resolved optimally.

This is especially true for pasteurized FVIII concentrates, which have increasingly displaced the usual commodity because it could thereby eliminate the risk of hepatitis transmission.

The isolation of a highly purified F VIII in good yield is hampered primarily by the fact that F VIII is concentrated in the cryoprecipitate but together with fibrinogen and fibronectin, which are two relatively high molecular weight and highly soluble proteins with similar physicochemical properties. such as those with which F VIII is associated.

A factor VIII concentrate should be as pure as possible, ie free of unwanted other proteins 5 and especially free of immunoglobulins, including the isoagglutins, since it is stated that the supply of nonspecific proteins leads to excessive requirements for the reticulocyte. endothelial system (RES), and to an impairment of the immune system characterized by a change in the composition of the lymphocyte population and the immunoglobulins. This is of increasing importance in relation to the fact that haemophiles must be treated with such F VIII concentrates throughout their lifespan. From this comes the requirement for a native, highly purified, pasteurized F III concentrate, ie. a product which excludes a transfer of hepatitis viruses and other infectious material and an sensitization to alloantigens.

A method of preparing such a composition which precludes the transmission of hepatitis and is isoagglutinin-free is the subject of the present invention.

It has been found that an F VIII-containing solution which is practically free of factors of the prothrombin complex (F II, VII, IX and X) can be recovered by addition of stabilizers to protect against thermal inactivation, pasteurize and treat the heated solution at a pH of 5.5 with an anion exchanger, wash the absorbed F VIII free of companion proteins, especially fibrinogen, fibronectin and immunoglobulins, including isoagglutinins , elute with a concentrated solution of Na, K or Ca salts with a halogen and e.g. upon precipitation isolates from the eluate.

Ecteola cellulose and a QAE (Quaternaty Amino Ethyl) group-bearing anion exchanger have already been used for the F VIII purification, cf. S. van Creveld et al., Thromb.

Diath. Haem., VI, No. 2/3, 282 (1961), and R. Baugh et al., Bioch. Biophys. Acta, 371, 360 (1974). However, a transfer to technical or production scale was not possible.

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Only for high purification of pre-fractionated material could ion exchange chromatography be used, cf. J. Fay et al.,

Proc. Natl. Acad. Sci., 79, 7200 (1982).

The higher specificity adsorption takes place only at a pH of about 5.5. This, however, already precipitates most of the proteins contained in the cryoprecipitate, primarily the human fibrinogen, and especially upon contact with the adsorbent in the column. Furthermore, according to the literature cited, relatively large quantities of adsorbent are required. This, in turn, seems to have had a huge impact on the yield, which was minimal, obviously conditioned by a nonspecific adsorption of F VIII - its physiological task is to adhere to an unphysiological surface - on the large amounts of adsorbent. It has also been found that on a QAE exchanger, highly purified material rapidly loses activity, and therefore anion exchangers have not been used for F VIII production.

However, according to the invention, it has now surprisingly been found that a chromatography on basic ion exchangers 20 is particularly suitable for the production of F VHI preparations.

Surprisingly, a highly purified F VHI preparation can be recovered from a pasteurized cryoprecipitate in one step by means of anion exchange chromatography.

The invention thus relates to a process for the preparation of a factor VIII preparation, as set forth in the claims, cf. above.

As a starting material comes a cryoprecipitate, Cohn fraction I, cf. Pool et al., Nature, 203, 312 (1964). GR Minot et al., J. Clin. Invest., 2j4, 704 (1945), Plasma, F VIII: C-containing cell culture medium as well as considering the recovered F VIII-containing bifractions. However, direct cryoprecipitate or Cohn I fraction is preferably used.

As stabilizers to protect against thermal inactivation of F VIII during pasteurization, carbohydrates and amino acids, preferably 35-60 grams, may be used. 100 grams of solution and 1-3 moles of glycine per day. liters of solution and possibly calcium ions. For example, is progressed in accordance with German Publication Nos. 2,916,711 and 3,237,512.

As anion exchangers for the adsorption of F VIII, e.g. DEAE, QAE or Ecteola group-bearing ion exchangers, and thus especially those based on cellulose, Sephadex or Sepharose, are considered, but preferably DEAE-Sepharose.

However, unlike van Creveld and Baugh, cf. the previous 10, these ion exchangers are considered for the F VIII purification, but under conditions not previously known and which will be described in more detail below.

The adsorption conditions have been found to be significant. Surprisingly, it has also been found that, in physiological saline environment, preferably at a pH of 5.5, F VIII is selectively bound to DEAE-Sepharose, while companion proteins such as the fibrinogen and fibronectin remain in the supernatant (by the batch method). ) or 2o a pillar passes. Finally, it has also been surprisingly found that a pasteurized solution of the cryoglobulins can be chromatographed at all at a pH of 5.5 in physiological saline environment, since under these conditions certain cryoglobulins are precipitated, primarily fibrillated. The adsorption, and especially the specificity with which F VIII binds to the anion exchangers, decreases sharply to the neutral point. However, under normal chromatography conditions, DEAE is first charged at a pH greater than 7.0. However, the precipitation fails in the process described here because the carbohydrates present in the cryoprotection from the pasteurization keep the fibrinogen and Cig in solution in slightly acidic medium.

An advantage of the disclosed process is that in the anion exchange adsorption of pasteurized cryoglobulin, fibrinogen and fibronectin remain in the above liquid or in the flow and can be recovered therefrom as pasteurized products. Fibronectin, for example, is extracted according to German Publication No. 2,848,529.

The chromatography on e.g. DEAE or QAE ion exchangers occur in slightly acidic environment (pH = 5.5) and on ion exchangers 5 which are equilibrated correspondingly, e.g. with 0.1 mole of Na-acetate buffer per ml. liter containing 0.1 mole of lysine per liter. liter.

With this buffer, the pasteurized F VIII-containing solution can also be diluted to double the volume before being treated with the ion exchanger by the batch or column method. The adsorption according to the batch method, preferably used, has the advantage that during adsorption one can follow the binding of F VIII to the ion exchanger via the functional determination of F VIII in the above liquid and after the disappearance of the activity the non-adsorbed factors can be separated by 15 sedimentation or centrifugation from the ion exchanger and immediately thereafter can begin washing the F VIII ion exchanger loaded. Preferably, while washing is to be performed batchwise, e.g. on a Nutsch filter, it is advisable for the elution to transfer the ion exchanger to a 20 column, since the column chromatographic elution has the advantage that F VIII appears relatively concentrated under the test conditions in the range of 50 to 100 IU F VIII per liter. ml.

Particularly suitable for the washing of the F VIII loaded ion ions are such buffers which do not partly release F VIII but which are partly suitable for the quantitative separation of nonspecific proteins as far as possible, such as the immunoglobulins and thus also the isoagglutinins. As a corresponding buffer, surprisingly, a buffer was found in which the starting material can be dissolved for the adsorption and containing 0.1 mole of Na-acetate per liter. liter, 0.1 mole of lysine per liter. liter and 1 gram of NaCl per liter. liter at a pH of 5.5.

With this buffer, the ion exchange loaded with F VIII is washed until the eluate is free of isoagglutinins. For test 35, the highly sensitive Coombs test, which also shows incomplete antibodies, is used.

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For the desorption of F VIII from the anion exchangers, concentrated salt solutions are considered, e.g. such containing NaCl.

However, other salts of halogens having Na, K or Ca, e.g. KBr, NaBr

ISLAND

and CaCl2 · They have the advantage that F VIII elutes as a relatively sharp peak with a high activity per minute. volume, an advantage that is significant to the felling.

The concentrations are in the range of 0.05 mol 10 per ml. liter to the saturation limit.

Finally, the yield also depends quite substantially on the elution rate, which must be of an order of magnitude of from 1 to 10 ml per ml. cm per per hour, preferably 2 from 5 to 7 ml per hour. cm per hour.

The F Viil-containing eluate can be concentrated using the usual methods, namely by precipitation with neutral salts, e.g. ammonium sulphate or NaCl, advantageously with NaCl, which, unlike ammonium sulphate, can be more rapidly dialyzed and not necessarily completely removed.

The further processing of the F VHI-containing precipitate takes place in such a way that the precipitation residue e.g. dissolve in a buffer of pH 6.9 containing Na citrate (0.02 mol per liter), NaCl (0.06 mol per liter), 25 glycine (20 grams per liter) and albumin (5 grams per liter) (dialysis buffer), and dialyzed against the same buffer without albumin until equilibrium is reached. The dialyzed solution is adjusted by dilution with the albumin-containing dialysis buffer to an activity of 25-30 IU F VIII per ml. ml and 30 are drained after sterile filtration, and optionally freeze-dried. The final preparation is characterized as a white lyophilizate which dissolves in less than 1 minute, has an F Vin activity of 5-10 IU per day. mg of protein, is pasteurized and isoagglutinin-free. Relative to products 35 of the state of the art, it has the advantage of being free of unwanted proteins and especially those which, such as alloantigens, could lead to a sensitization. Blood group incompatibilities are not induced by use of the preparation. A transmission of viral diseases, especially the various forms of hepatitis, seems excluded. One of the advantages of the 2 method is that it is relatively simple and therefore can be easily transferred to an industrial scale and also consists of a few working steps. The small number of work steps is documented in good yield, with experience every cleaning step being associated with a greater or lesser activity loss.

According to the invention, a pasteurized, highly purified factor VIII preparation can be obtained which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen and is characterized by a specific clotting (C) activity (F VIII: C) of ca. 100 U / mg protein and a ratio of F VIII: C to F VIII R: Ag (Related Antigen) greater than 1.

F VIII can be determined by the following procedure: 1 part, e.g. 0.1 ml partial thromboplastin, e.g.

20 prepared according to German Publication No. 2,316,430, is mixed with 1 part F VIII deficiency plasma and 1 part diluted normal plasma. This mixture is kept at 37 ° C for 6 minutes. After addition of 1 part to 37 ° C, 0.025 molar calcium chloride solution is pre-determined, the time taken from the addition of the calcium chloride solution to the appearance of a coagulate. For quantitative determination, a measurement or calibration curve obtained with a normal plasma dilution series is used.

1 international unit (= 1 IU) of F VIII 30 corresponds to the F VIII activity of 1 ml of normal plasma as a substandard to the 3rd International WHO Standard.

The following example illustrates the process of recovering a pasteurized and isoagglutin-free F VIIl preparation.

35 8 DK 174066 B1 o

Example 1. Starting material.

250 grams of crude cryoprecipitate are dissolved in 750 ml of a NaCl solution (0.08 mol per liter) containing 5 glycine (0.25 mol per liter) and heparin (1.25 USP units per ml) , heated to 30-37 ° C. This yields 1000 ml of solution with a concentration of 0.06 moles of NaCl per ml. liter, 0.2 mole glycine per liter. liter and approx. 1 USP unit of heparin per ml. The pH of the solution is adjusted to 6.5 10 with 1 N HCl.

2. Aluminum hydroxide adsorption.

To 1000 ml of solution from 1. add 80 ml of a suspension containing 10 grams of aluminum hydroxide per ml. liter (Behringwerke, Marburg) and stir for 15 minutes (temperature about 30 ° C). Then, centrifuge for 15 minutes at 3000 g, discard the residue and pasteurize the poured liquid after addition of stabilizers.

3. Pasteurization.

20 ml of poured liquid from 2, the following stabilizers are added in this order: 5 ml of CaCl2 solution, 1 mol per ml. 1000 grams (5 mmol per liter) 1000 grams sucrose (500 grams per kg solution) 150 grams glycine (2 mol to 1 liter solution).

The pH is adjusted to 7.3 with 2N NaOH. In the additions, the volume is increased to 1700 ml. The solution is kept for 10 hours at 60 ° C in a water bath.

4. Ion exchange treatment.

Dilute 1700 ml of solution 3 with 1700 ml of a solution containing 0.2 mole of Na-acetate per ml.

per liter, pH = 5.5, and 0.2 moles of lysine per liter. liter. Adjust the pH to 5.5 with dilute acetic acid and add 70 ml with a solution containing Na acetate (0.1 mole per liter), pH = 5.5, lysine (0.1 mole per liter) ) and NaCl 35 (1 gram per liter), equilibrated DEAE-Sepharose 6B Cl. Stir for 2-3 hours at room temperature, and the binding of the activity is determined by determining the activity of the above liquid. When F VIII activity has decreased from 4 IU to 0.1 IU per ml, the adsorbent is separated by centrifugation.

The above liquid is poured off and the adsorbent is separated from the rest of the above liquid. By washing with a solution containing 0.1 mole of Na-acetate per ml. liter, pH = 5.5, 0.1 moles of lysine per liter. liter and 1 gram of NaCl per liter. per liter, the Sepharose is frozen for entrapped protein and 10 is then slurried with the same buffer in a column (dimensions: 10 cm x 3 cm).

In the column, the ion exchanger is washed so long that no light absorption can be measured at 280 nm and the isoagglutinin values at Coombstest are at the detection limit.

5. Elution.

The ion exchanger from 4. is eluted with a solution, pH = 5.5, containing 0.1 mole of Na-acetate per ml. liter, 0.1 mole of lysine per liter. per liter and 0.3 moles of CaCl 2 per liter. liter. This results in a measurable peak at a wavelength of 280 nm.

The corresponding fractions are pooled and a volume of 180 ml is obtained at 40 IU F VIII per ml. ml.

6. Felling of F VIII.

180 ml of eluate from 5. is added 2.2 moles of glycine 25 per ml. per liter and 150 grams of NaCl per liter. liter and stir for 30 minutes at room temperature.

The precipitate can be recognized by a clear haze. The precipitate is separated by 30 minutes centrifugation at 30,000 G in an ultracentrifuge and kept after 30 hours of pouring the supernatant overnight at 4 ° C.

7. Reprocessing.

The precipitate from 6. is taken up in 145 ml buffer pH = 7.0 containing 0.02 moles of tri-Na citrate per ml. liter, 0.06 moles of NaCl per liter. 10 g of glycine per liter. per liter and 5 g of 35 human albumin (solution buffer) per liter. liter. For the solution, an activity of 40 IU F VIII: C is determined. ml. The solution is dialyzed for 3 hours at room temperature against the aforementioned buffer containing no albumin and the dialysate is heated to 30 ° C and centrifuged for 30 minutes at 30,000 G and 25 ° C. Following a P VHI assay that gives a result of 36 IU per the solution is adjusted using the solution buffer of 30 IU per ml. and the solution is heated to 37 ° C and sterile filtered on a membrane filter.

Fill small flasks, freeze and lyophilize.

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Claims (4)

A process for preparing a factor VIII preparation, characterized in that a pasteurized solution containing factor VIII from a human plasma cryoprecipitate 5 in the presence of a carbohydrate and an amino acid is treated with an ion exchanger bearing DEAE, QAE or Ecteola groups, based on cellulose, Sephadex® or Sepharose® at a pH of 5.5, wash the ion exchanger and elute factor VIII with chaotropic solutions, preferably 10 with a solution of CaCl Process for preparing a factor VIII preparation, characterized in that the cryoprecipitate is dissolved with the addition of sucrose and glycine, preferably 35-60 grams sucrose per day. 100 ml and 1-3 moles of glycine per ml. per liter, 15 calcium ions are added, preferably 1-20 mmol per liter. and, where appropriate, pasteurized, cooled and diluted, and F VIII: C is absorbed at a pH of 5.5 on an ion exchanger bearing DEAE, QAE or Ecteola groups, based on cellulose, Sephardex® or Sepharose® and the absorbance 20 are washed until free of foreign proteins, especially isoagglutinins, and F VIII: C is eluted by eluting with a buffered concentrated chaotropic saline solution, preferably with a solution of CaCl 2, dialyzed and lyophilized. Process according to claim 1 or 2, characterized in that the adsorbent is washed with a buffered dilute brine. Process according to claim 1 or 2, characterized in that DEAE-Sepharose® is used as the ion exchanger.