GB1601744A

GB1601744A - Processes for the purification of interferon certain new and useful intermediates formed therein the purified interferon thus produced and pharmaceutical compositions containing

Info

Publication number: GB1601744A
Application number: GB17225/78A
Authority: GB
Original assignee: Agence National de Valorisation de la Recherche ANVAR
Current assignee: Bpifrance Financement SA
Priority date: 1977-05-02
Filing date: 1978-05-02
Publication date: 1981-11-04
Also published as: FR2389380B1; JPS53139714A; DE2819297A1; FR2389380A1; CH633716A5

Abstract

The separation of products having interferon activity from preparations containing them is accomplished by bringing these preparations into contact with a phase comprising at least one polynucleotide compound. Purified products having interferon activity, which may be used as medicaments, can be prepared in this way.

Description

(54) PROCESSES FOR THE PURIFICATION OF INTERFERON, CERTAIN NEW AND USEFUL INTERMEDIATES FORMED THEREIN, THE PURIFIED INTERFERON THUS PRODUCED, AND PHARMACEUTICAL COMPOSITIONS CONTAINING IT (71) We, AGENCE NATIONALE DE VALORISATION DE LA RECHERCHE (ANVAR), a Public Corporation organized under the Laws of the Republic of France, of 13, rue Madeleine Michelis, 92522 Neuilly-sur-Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following- statement:- The subject of the invention is a process for separating products having interferon-type activity and preparations containing them and more particularly aprocess for purifying such products.

The subject of the invention is also the corresponding purified preparations and the use of these as active principle in medicaments.

It is known that interferon is a therapeutic product of great value especially because of its remarkable antiviral and immunodepressive properties.

According to the usual methods of synthesis in vitro this product is obtained by the action of viruses or of chemical substances upon tissue cultures. However, the resulting preparations also contain contaminating substances. In particular these preparations contain proteins which have pyrogenic activity and, in addition, have the disadvantage of being capable of inducing specific sensitisation in the recipient.

The crude preparations of interferon cannot, because of this be used directly, in particular for the applications considered above, and must be purified.

Various purification methods have been proposed, especially by means of filtration on gel or by means of ion exchangers.

However, use on a large scale of such techniques for the purification of interferon preparations has come up against difficulties especially because of very small yields.

Affinity chromatography processes have also been proposed.

It is known that the latter are based upon a selective affinity between a given product and an adsorbent phase containing a ligand fixed in a covalent manner to a solid support. The passage over the adsorbent phase, of a preparation containing the product which it is desired to separate enables the product in question to be retained selectively on the ligand and thus to separate it from the preparation containing it. In order to recover the fixed product it is then necessary to have available a desorption agent.

According to techniques based upon the principle of affinity chromatography there are used as ligand anti-interferon antibodies. However, the results obtained up till now still do not give satisfaction especially because of the production of antisera which is difficult, long and costly and this afortiori if an attempt is made to develop it on the industrial scale.

Affinity chromatography processes are also known in which the ligand employed is selected because of the hydrophobic properties of interferon. These processes, therefore, call for organic solvents for desorption.

Now, with a view to the use in therapy of interferon, it is necessary to remove all traces of organic solvents without destroying the interferon activity by transferring it to an aqueous medium. These processes thus involve extremely complex handling operations.

In other processes of this type the ligand used is constituted by a derivative having a polycyclic structure of the type of that of the blue chromophore such as that sold under the brand "Bleu Cibacron F3GA".

The latter processes have the advantage of being capable of being employed under conditions in which work can be carried out in aqueous medium, which removes the disadvantages encountered in the previous techniques in the face of the use in therapy of purified preparations of interferon.

These processes, in addition, enable a large part of the contaminating proteins to be removed.

Now, the inventors have at present established that it is possible to improve the results even more by using, to fix and separate the products having interferon activity from preparations containing them, new ligand agents. In the following description and in the claims it is understood that the term "preparation" denotes a composition containing the products having interferon activity both in non-bonded and in bonded form and especially in the form of a complex This invention, therefore, is based upon the establishment of a remarkable affinity of the products having interferon activity for a certain type of structure.

Thus it is possible, by using this particular affinity, to separate, in an extremely specific manner, products having interferon activity from preparations or the like containing them and to obtain, easily, preparations of interferon which are more satisfactory than those proposed up till now especially as much from the point of view of their interferon titre as of their purity.

The process according to the invention for purifying preparations containing products having interferon activity by selectively separating said products from the contaminating substances of the preparations is characterised by the fact that the said preparations are put into contact with a phase containing at least one compound of the polynucleotide type capable of acting like a ligand to bind the products having interferon activity.

The remar & able specific affinity between the polynucleotide and the products having interferon activity enables the latter to be separated, with very great selectivity, from preparations containing them.

The results obtained give complete satisfaction with preparations of interferon both of human and animal origin and constituted especially by the supernatant liquids of tissue cultures, of fresh explantation (primary or secondary cultures) or of continuous lineage, where the cells have been induced to produce interferon, advantageously in a culture medium devoid of serum, by a virus or a synthetic nucleic acid such as poly I.C (polymer of inosinic acid and cytidylic acid).

The interferons of the preparations employed can, moreover be of fibroblast or leucocyte origin. Preparations obtained by conversion into a cellular medium, especially according to the techniques reported in "Molecular Mechanism of Prot.

Biol. Synthesis" edited by H. Weissbach and S. Pestka, Acad. Press 1977, pages 574 to 582, can also be used in the process of the invention.

According to one arrangement of the invention a preparation to be purified, of products having interferon activity is put into contact with an adsorbent put into equilibrium by a buffer of which the concentration in salts is isomolar or preferably hypomolar by comparison with that of blood, the said adsorbent containing, for the one part, a solid support forming a gel, of which the size of the lattices is such that it permits the passage of macromolecules and, for the other part, a compound of the polynucleotide type, fixed upon the said support, playing the part of ligand with respect to the products which it is desired to separate selectively, the amount of adsorbent employed and the duration of contact between the ligand and the preparation to be purified being sufficient to permit the desired fixing of the products having interferon activity.

In order to obtain purified preparations having interferon activity an additional stage is resorted to according to which the desorption of the fixed products is carried out using a buffer of which the concentration in salts is hypermolar by comparison with that of blood.

It is thus possible to have available extremely pure products having interferon activity, with a high titre in interferon, which can be used directly in therapy.

It will be noted that the interferon can be recovered easily from the coupling product which it forms with the ligand and this can be done practically totally and without it being altered. Interest in this process has increased even more from the fact that the adsorption capacity of the adsorbent used is practically undamaged.

The same adsorbent can then be used for numerous purification operations and consequently is convenient from a point of view of economy.

According to another arrangement of the invention a composition containing a complex containing products having interferon activity fixed upon a ligand is put into contact with a phase containing at least one compound of the polynucleotide type, capable of competing with the ligand for the fixing of the products having interferon activity and of detaching the latter, the composition and phase in question being placed in equilibrium using a buffer of which the concentration in salts is isomolar or hypomolar by comparison with that of blood, the amount of polynucleotides in the above-mentioned phase and the duration of contact between the polynucleotides and the products having interferon activity fixed upon the ligand being sufficient to permit desorption of the products having interferon activity.

It will be noted that when employing this arrangement the products having interferon activity are fixed upon a ligand for which they have less affinity than for the polynucleotides. Given the very important nature of the affinity of interferon for the polynucleotides, competition to fix the interferon takes place to the advantage of the latter.

In addition, it will be observed that the result of this is additional purification for the products having interferon activity. In fact, the contaminating substances, in particular the proteins which are present in the crude preparations of interferon and which have a tendency to become fixed with the interferon on the adsorbent under consideration will, in practice not become detached by the polynucleotides.

This arrangement of the invention constitutes a very selective purification process.

In addition, this process offers the advantage of conferring upon interferon considerable protection against thermal denaturing and consequently great stability.

According to a preferred method of carrying out the above-mentioned first arrangement of the invention products having interferon activity are separated from crude preparations containing them by putting these preparations into contact with an adsorbent phase constituted by at least one derivative of polynucleotide type coupled to a solid support.

The derivative of polynucleotide type is a polyribonucleotide or a polydesoxyribonucleotide in which one or more members in the constituent motifs can be modified without this modification impairing the specific recognition phenomenon which interferon has in this respect.

Advantageously polynucleotides are employed containing about 10 to 500 nucleotide motifs. These derivatives, which are suitable by their configuration and their constitution for fixing products having interferon activity, can be both synthesised and of natural origin.

Amongst the derivatives of the first type of polynucleotide such as polyriboinosinic acid or poly I, polyriboadenosic acid or poly A and polyribouridylic acid or poly U have proved to be particularly suitable. Generally, products having a molecular weight of the order of 50,000 to about 250,000 are used.

As polynucleotides of natural origin suitable for carrying out the process of the invention, advantageously the cellular ribonucleic acids and more particularly the transfer ribonucleic acids or t-RNA are used.

All the t-RNA fractions extracted from the cells as well as a part of these fractions could be concerned, or even specific fractions with respect to the synthesis of a given amino acid.

It is known that these t-RNA contain about 70 to 85 nucleotide residues and have a molecular weight of the order of 25,000.

Different sources of t-RNA can be used, especially amongst the eucaryotes or the procaryotes and bacteria can be used such as those belonging to the genus E.

Coli or even yeasts such as those belonging to the type of beer yeast.

The solid support of the adsorbent forming a gel includes a polysaccharide such as agarose or a derivative of this, especially a modified agarose in which the polysaccharide chains are cross-linked into a three dimensional lattice in particular that sold under the brand Sepharose (registered Trade Mark) or even a polyacrylamide or an acrylic resin.

For physiological reasons which are still unexplained it has been proved that the products having interferon activity have very great affinity for the abovementioned polynucleotides. This property can, therefore, be used from a practical point of view to purify interferon.

By simple contact, according to the conditions of iso- or hypo-molarity set out above, advantageously established using a buffer having a molarity of about 0.01 to 0.05 M, the said products are adsorbed selectively whilst the major part of the undesirable substances present in the preparations are not retained.

It is particularly convenient to contact the adsorbent and the preparation to be purified in a chromatography column, passing through this column the preparation to be purified. The column filled with the adsorbent is placed in equilibrium with the above-mentioned buffer of which the pH is of the order of 3 to 9, preferably close to neutrality.

Crude preparations of interferon are used of which the interferon titre is about 104 to 106 international units per 0.1 to 0.2 g of proteins, which gives a specific activity (International Units of Interferon or U.I.F. per mg of proteins) lying between 105 and 107. These preparations can be used as they are in isomolar conditions for passage through the column or they can be subjected first to a dialysis against the buffer which served to equilibrate the said column. Operating under conditions isomolar with a tissue culture medium, the yield of interferon is about 80 to 90%, whereas it is 100% under hypomolar conditions. In the latter event a particularly suitable buffer is constituted by 10 mM Tris-HC1, pH 7.5 (it is recalled that Tris is the abbreviation currently adopted to denote tris-hydroxy methylaminomethane).

The stage of separating the products having interferon activity from preparations containing them comes, therefore, within the scope of the invention.

In addition, the invention aims, as new industrial products, at complexes formed by the products having interferon activity and fixed on the adsorbent phases described above containing at least one derivative of polynucleotide type.

As already indicated, the origin of these interferons can be very varied. These complexes are the result of the interaction between the products having interferon activity and the polynucleotides. The method of interaction is not clarified at present but the work carried out leads one to think that it is a question of non covalent bonds and that Van der Waals forces, hydrogen bonds, hydrophobic bonds, weak ionic forces, etc. can exist.

The fixed products can be recovered by increasing the salt concentration of the buffer-especially by the addition of acetates, phosphates, chlorides or other salts-so as to obtain a salt concentration which is hypermolar by comparison with that of blood and of at least 0.5 M and preferably 1 M.

This recovery of the products having interferon activity is carried out advantageously by eluting in a column. By recovering the eluted fractions one has available purified interferon preparations with a high content of interferon and practically completely devoid of contaminating proteins.

In addition, by carrying out the desorption phase with an output not exceeding 0.2 mVcm2/mn the major part of the products having interferon activity is recovered in an eluate fraction of small volume, in particular when the adsorption phase has been carried out under hypomolar conditions, which shows the advantage of providing preparations having a high concentration of interferon by comparison with the concentration in the crude preparations.

By use of the process of the invention with an almost total yield and in a single stage interferon preparations of very great purity can be obtained.

In addition, it will be noted that these preparations are advantageously in aqueous solution and can, therefore, be used as they are in therapeutic applications as indicated hereinafter: Thus, as far as interferon from mice, for example, is concerned, for which the method was perfected the first time, preparations are obtained capable of reaching 109 units per mg of proteins.

As indicated above it is also possible, due to the remarkable affinity, made evident by the inventors, between the products having interferon activity and the polyribonucleotides, to separate the latter from complexes or compositions in which they are bound up and to form a new complex of polynucleotide-typeproducts having interferon activity.

According to a preferred method of carrying out this arrangement of the invention, for the one part of composition containing products having interferon activity fixed upon an adsorbent and, for the other part, a phase containing at least one derivative of polynucleotide type are put in contact.

The said adsorbent contains a solid support forming a gel of which the size of the lattices is such that it permits the passage of macromolecules and on the other hand a compound fixed to the said support, playing the part of ligand with respect to the products which it is desired to separate selectively and which has a polycyclic structure of the type of that of blue chromophore, sold under the brand Bleu Cibacron F3GA, (Cibacron is a Registered Trade Mark) of formula

or an equivalent product of which the configuration enables the interferon to be retained selectively.

As solid support of this type advantageously a support forming a gel is used such as that employed to form a coupling product with a polynucleotide according to the above-mentioned first arrangement of the invention.

The ligand coupled to the solid support is advantageously constituted by Bleu Cibacron F3GA itself or the product sold under the brand Bleu Cibacron 3GA (which differs essentially from the previous one by the presence of a hydrogen atom in place of the group -SO2ONa on the phenyl radical at the end of the chain), or also in an equivalent product. By equivalent product there is understood, in the description and the claims, a product which, even if it differs from the blue chromophores indicated above by the nature and/or the position of the substitutions, nevertheless permits the desired selective fixing of the interferon to be obtained.

The cyclic compound used as ligand can be attached, with no disadvantage, to a polysaccharide, especially to a polysaccharide of high molecular weight, of the order of 2 millions, called Dextran 2000. In this case it is the polysaccharide which is fixed to the solid support.

A preferred compound of this kind, that is to say constituted by a cyclic compound fixed to the Dextran 2000, is the product known under the brand name Bleu Dextran 2000 which corresponds to the above-mentioned Bleu Cibacron F3GA fixed to the Dextran 2000. This product is advantageously fixed by means of the Dextran 2000 to a solid support constituted by agarose, in particular Sepharose, which corresponds to the product sold under the brand name Bleu Dextran Sepharose.

The adsorbent containing the products having interferon activity bonded to the said solid support is placed in equilibrium by a buffer of which the salt concentration is isomolar or preferably hypomolar by comparison with that of blood. These conditions are advantageously established using a buffer having a molarity of less than 50mM and especially with the Tris buffer used in the phase of adsorbing products having interferon activity on to a phase containing polynucleotides, as described above.

As far as the phase containing at least one derivative of the polynucleotide type is concerned it contains these polynucleotides in solution in a buffer of which the salt concentration is advantageously hypomolar by comparison with that of blood.

The polynucleotides in question are those mentioned above. Thus, the polyribonucleotides containing about 10 to 500 nucleotide motifs constitute remarkable desorption agents. Amongst the suitable polyribonucleotides there may be mentioned polyriboguanilic acid or poly G, poly I and poly U.

Ribonucleic acids, in particular t-RNA's can also be used. The latter can be extracted from bacteria such as those of the genus E. Coli. They can also come from yeast such as beer yeast.

Advantageously ribonucleic acids of animal or human origin are also used, more particularly coming from animal organs.

Amongst the latter those extracts of the very cells which produced the interferon which it is desired to purify are especially preferred. Thus, to purify preparations containing products having mouse interferon activity, advantageously the cells of mice of the cell lineage called C 243 are advantageously used.

Preferably the polynucleotides used as desorption agents are put into solution in a buffer at the rate of 10 to 100uglml, preferably from 30 to 50 ,ug.

A buffer suitable for this purpose is constituted by 10 mM tris-HC1, pH 7.5.

From a practical point of view it is advantageous for the contacting on the one hand of the complexes containing the products having interferon activity with, on the other hand the polynucleotides, to correspond to a desorption operation in an affinity chromatography column.

This operation consists, then, in recovering, using polynucleotides, the products having interferon activity fixed on to the adsorbent filling the column.

For this purpose a solution of polynucleotides containing from 10 to 100,ug/ml and preferably from 30 to 50 ,vg'ml of an elution buffer constituted by 10 mM Tris HCI, pH 7.5 is passed through a column containing an adsorbent gel constituted by Bleu Dextran Sepharose to which are fixed products having interferon activity.

The passage of this solution is advantageously carried out at the rate of about 0.2 mVcm2/mn.

To facilitate desorption on a small volume of eluant, especially in the case in which the affinity between the polynucleotides and the products having interferon activity is not clearly marked, the solution of polynucleotides is advantageously left in contact with the adsorbent gel before proceeding with the elution. Generally a contact period of about half an hour to 18 hours proves to be sufficient.

Experimentation has shown that the desorption phase is advantageously carried out with a volume of eluant corresponding to approximately from 2 to 4 times the total volume of adsorbent gel.

Due to the remarkable specificity of this process products having interferon activity of very great purity are recovered.

The interferon is fixed to the polynucleotides and the resulting coupling products also come within the scope of the invention.

As indicated above, the polynucleotides ensure, advantageous protection of the products having interferon activity against thermal denaturing. Thus, it is established, by heating to about 60"C a coupling product according to the invention, that a considerable part especially of the antiviral activity is maintained whereas more than 95% of this activity disappeared with the control preparations.

The purified preparations of products having interferon activity and which contain the products having interferon activity recovered by desorption from complexes which they form with polynucleotides fixed to a solid support, as well as the complexes which they form according to another arrangement of the invention with the said polynucleotides, possess remarkable therapeutic properties to which is added especially the advantage of antiviral activity of great interest. This activity was made evident especially in patients suffering from chronic hepatitis B, proceeding according to the techniques described by Desmyter et al in The Lancet ii 645--647 1976 and Greenberg et al, New England in J. Med. 295-517-522, 1976.

These interferon preparations can also be used to cure cases of dendritic keratitis, as shown by tests carried out according to the techniques described by Sundmacher et al in Albrecht v. Graefes Arch. Klin. exp. Ophtal 201:39--45, 1976.

These preparations are also shown to be effective for preventing recurrences of herpetic keratitis, as made evident by the tests carried out according to Kaufman et al, J. Infect. Dis. 133 (suppt) A 165-168, 1976. In addition, the interferon preparations can have a favourable effect in patients suffering from cancerous tumours. On the one hand these preparations reduce the frequency and the duration of viral complications, especially due to herpes Zoster, as is apparrent by carrying out tests according to Joran et al, J. Infect. Dis. 130:56-62, 1974. On the other hand they seem to promote, by their anti-tumour properties, the survival of patients suffering from osteosarcoma. These experiments can be made evident by operating according to Strander et al. Acta Orthop. Scand. 45:958-959, 1974.

All these tests have enabled the remarkable innocuousness (in terms of side effects) of the preparations having interferon activity according to the invention to be established.

As shown by numerous works, interferon does not, moreover, cause phenomena of obvious toxicity. Reference can be made, regarding this subject, to the results given by J. Desmyter et al in The Lancet, September 25, 1976, pages 645 to 647 and to the references indicated in this article relating to the absence of toxicity of these products, namely the article by H. Strander et al in Natn. Cancer Inst. 1973, 51, 733, by G. W. Jordan et al. in Infect. Dis. 1974, 130, 56, and by G.

Emodi et al. in Natn. Cancer Inst. 1975, 54, 1045.

Interferon being specific to the species, it is, therefore, necessary to use, for example for therapeutic applications in man, interferon from human cells.

The medicaments according to the invention which contain the purified preparations of the invention as active principles can be administered to man especially by intramuscular route and by sub-cutaneous route in the form of a solution. By way of example a dose of fibroblast interferon of 107 units was administered every two days for two weeks in the case of chronic hepatitis or 3x 106 units of leucocyte interferon 3 times per week in the case of osteosarcoma.

Example 1 The preparation of an affinity column of polynucleotides with a view to the separation of products having interferon activity from preparations containing them was carried out as follows: First of all an activated Sepharose gel is formed which will serve as solid support for the polynucleotides then, in a second stage, fixing of the polynucleotides to the activated Sepharose is carried out, proceeding according to a method derived from that described by D.L. Robberson and N. Davidson in Biochem., II, 4, 1973, 533.

a) Preparation of the activated Sepharose gel Agarose of the Sepharose 4 B type is employed, such as that sold by Pharmacia (Uppsala, Sweden). This product is in the form of small balls. The latter are washed with water by filtration through fritted glass, then put back into suspension in water at a proportion of 5 ml of initial Sepharose per 10 ml of water.

The Sepharose is then activated with cyanogen bromide CNBr. To each 10 ml of Sepharose suspension, the latter being maintained in suspension by slight magnetic agitation, one adds gradually 15 ml of a 6.5%7% aqueous solution of CNBr.

The temperature of the suspension is maintained at 40C by an ice bath; the pH, controlled by a pH meter, is adjusted to 10 by the addition of NaOH. Once the pH has been stabilised and a temperature of about 20"C is reached the thusactivated Sepharose is filtered through a fritted-glass filter and it is washed with a 0.1 M bicarbonate buffer pH 9, previously cooled by using one volume of buffer corresponding to about 10 times the volume of Sepharose gel. The gel is then put back into suspension in the same bicarbonate buffer, using 8.5 ml per 10 ml of initial suspension. The thus-activated Sepharose is coupled to the methyl ester of amino-caproic acid. Each 8.5 ml fraction is made up to 10 ml with a 30% solution of the ester in the bicarbonate buffer at pH 9. The pH of the mixture is adjusted to 9 using NaOH and the mixture is left at 40C for 24 hours with slow rotatory agitation.

The Sepharose gel is then washed with 10 volumes of iced water and put back into suspension in water until the initial volume is reached. 0.7 ml of a 98% solution of hydrazine hydrate is then added, then this mixture is taken to 700C for 15 minutes then it is cooled.

The gel is again filtered and washed copiously with iced water and then put back into su

a-treatment with an alkaline phosphatase Before carrying out this fixing, the polynucleotides are subjected to an oxidation reaction with sodium periodate. To increase the yield of the reaction the polynucleotides are systematically treated previously with the alkaline phosphatase to remove any possible presence of a PO4 group at the end 3' of the sugar portion of the polynucleotides.

stage of oxidising the polynucleotides The oxidation of the periodate is carried out according to the method of Hunt (Bioch. J., 1965, 95, 542). The polynucleotides, at a concentration of 1-2 mg/ml in an acetate buffer of pH 5.2 are oxidised by the addition of one-tenth volume of a freshly-prepared 0.2 M solution of Nail4. The reaction is carried out away from the light for 45 minutes. The excess periodate is removed by the addition of onetenth volume of methylene glycol. The polynucleotides thus oxidised are recovered by precipitation with ethanol then they are put into solution in a buffer of 0.1 M sodium acetate, pH 5, and dialysis of this solution is carried out against the same buffer of 0.1 M acetate, which enables formaldehyde formed previously to be eliminated.

coupling of the polynucleotides to the activated Sepharose gel About 50 to 60 A 260 units of polynucleotides per ml of activated Sepharose gel blocked by hydrazine as indicated above (volume measured as deposit), are added. This mixture is put into suspension in 5--6 ml and left under gentle agitation at 4"C for 12-15 hours. The polynucleotides not fixed are removed by successive washing with a 0.1 M acetate buffer of pH 5.2 then a 0.1 M bicarbonate buffer of pH 9.

The Sepharose gel bearing the polynucleotide chains is then put back into suspension in the desired buffer. To form a complex of activated Sepharose and polynucleotides such as poly A and poly C work is advantageously carried out in buffer medium at pH 6 according to the process described by Wagner et al. in BBRC. (1971) 45 184.

To a solution of polynucleotides (at a concentration of 2W mg/ml in 0.2 M 4morpholino 2-ethane-sulphonic acid, of pH 6.0) is added a suspension of activated Sepharose gel obtained as described above in a proportion of 1/3 (V/V). The mixture is agitated gently at 40C for 12-15 hours. The polynucleotides which have not reacted with the Sepharose are removed by filtration, then by successive washing, with the 4 - morpholino - ethane - sulphonic acid buffer then with water.

In this case the polynucleotides are assumed to be fixed by intermediate of the PO4 group which is at the end of the group at position 5' in the sugar portion of the polynucleotides.

To fix the poly U on to the activated Sepharose work is carried out as indicated above in a buffer having a neutral pH such as Tris, at weak concentrations of 1020 mM.

c) Preparation of the affinity chromatography column The polynucleotides-Sepharose gel is poured into a chromatography column of suitable dimensions. It is estimated that one ml of gel is capable of fixing about 8 million International units of interferon.

Before the very first use of the column it is rinsed with 20 times its (dead) volume of 10 mM Tris-HC1 buffer, pH 7.5, to which has been added 0.02?(' of sodium azide.

Given that the sodium azide is toxic to the cells, it is necessary to pass through the column at least three dead column volumes of Tris-HC1 buffer without azide before proceeding with fixing the interferon.

Example 2 The purification of a preparation of mouse interferon by affinity chromatography through a column according to Example 1 of poly U-Sepharose was carried out as follows: The crude preparation of interferon consists of a supernatant liquid of tissue culture derived from C 243 cells induced to produce interferon by the Newcastle disease virus. After inactivation of the virus at pH 2 the supernatant liquid is dialysed against 10 mM Tris-HC1, pH 7.5, for about 24 hours at a temperature of about 4"C.

The interferon titre of the crude preparation is 1.3x 106 international units, its protein content is 0.1 mg per ml and the specific activity is 1.3x107 international units per mg of proteins.

The chromatogram of the purification operation is represented on Figure 1 of the accompanying drawings.

10 ml of the preparation to be purified are passed through a column 0.9 cm in diameter, containing 2 ml of poly-U Sepharose 4B gel (product obtained commercially from Pharmacia, Uppsala, Sweden).

During the sorption phase as well as during the subsequent washing and desorption phases fractions of a volume of 2 ml are recovered.

After the interferon preparation has penetrated the gel (fractions 1 to 4 of the chromatogram) washing of the column is carried out with 6 ml of 10 mM Tris-HC1 buffer, pH 7.5, to ensure the removal of the non-adsorbed contaminating substances (fractions 5 to 7).

To recover the interferon one proceeds to the desorption phase, passing through the column 10 ml of 10 mM Tris-HC1, pH 7.5, to which has been added 1 M Na Cl (fractions 8 to 13 on the chromatogram of Figure 1); The interferon titre is measured in each fraction recovered during the operation, washing and desorption phases. It is then established that the interferon contained in the starting crude preparation is recovered in toto and is collected almost totally in 2 tubes, namely tubes number 8 and 9 (that is 4 ml) during the desorption phase.

The interferon titre (IUF/ml) in these tubes is, respectively 2.5x103 (tube 8) and 5x 106 (tube 9) The recovery of the interferon is, therefore, total.

The protein content in tube number 9 is less than 2.5 yg per ml; the specific activity is, therefore, above 2x 109 which corresponds to a purification greater than 150 times.

Example 3 The purification of a preparation of human interferon by affinity chromatography through a column according to Example 1 of poly 1-Sepharose was carried out as follows: The crude preparation of interferon is a supernatant liquid of a culture of human fibroblasts induced to produce interferon by poly I and poly C.

The interferon titre is 4096 I.U. of interferon per ml and the protein content is 660 ,ug per ml which gives a specific activity of 6.2x103 I.U. per mg of protein.

5 ml (20,480 I.U.) of the preparation, not previously dialysed, are passed, at the rate of 1 ml per minute, through a column 0.9 cm in diameter, containing 2 ml of Sepharose-poly I gel according to Example 1, this column being placed in equilibrium with 10 mM Tris-HC1 buffer, pH 7.5.

As soon as all the preparation has penetrated the gel one proceeds with washing as indicated in Example 2.

The desorption of the interferon is then carried out, by passing through the column with a flow of about 0.2 ml/mn, 10 ml of 10 mM Tris-HC1 of pH 7.5, enriched with 1 M of Na Cl.

During these different adsorption, rinsing, washing and desorption phases 2 ml fractions are recovered. By determining the interferon titre in these different fractions it is established that 1024 units are recovered in the first three tubes (2 ml fractions) that is in toto 6,144 units, which is equivalent to 30% of the interferon passed through the column. No interferon activity can be detected in the washing tubes.

At the desorption it is established that the products having interferon activity are principally recovered in the fractions in tubes 10 and 11 (tube 10 containing a fraction of 1 ml only). The interferon titre in these tubes is, respectively 2,048 (tube 10) and 16,384 (tube 11) which corresponds to a total of 18,432 I.U. of interferon/ml.

Given that the crude preparation which was purified through the column containing initially 20,480 I. U., 90 Ó of interferon has, therefore, been recovered.

Example 4 The preparation of an affinity column of Bleu Dextran Sepharose with a view to the purification of interferon of animal origin on the one hand and interferon of human origin on the other hand, with the use, as desorption agents, of polynucleotides was carried out as follows: 1. Preparation of the Bleu Dextran Sepharose gel The process of manufacturing Bleu Dextran Sepharose gels has been described by L.D. Ryan and C.S. Vestling (Arch. Biochem. Biophys., 160, 279-284, 1974). Since this publication it is no longer necessary to continue to activate the Sepharose with cyanogen bromide according to Cuatrecasas (Cuatrecasas P., J.

BioL Chem., 3059-3065, 1970) since there are lyophilised commercial preparations, ready for use, of already activated Sepharose.

Sepharose 4B activated with cyanogen bromide and Bleu Dextran 2000, such as those sold by Pharmacia (Uppsala, Sweden) are used.

a) Preparation of the Sepharose (CNBr Sepharose 4B) gel In order to swell the gel and to remove the bactericidal agents present in the lyophilised preparation the desired amount of CNBr-Sepharose, knowing that one gram of dry product gives about 3.5 ml of gel, is washed on a fritted-glass filter with a I mM solution of HCI for about a quarter of an hour. For this purpose 200 ml of 1 mM HCI is used per gram dry weight of CNBr-Sepharose. The gel thus formed is immediately coupled with Bleu Dextran 2000.

b) Coupling of the Sepharose gel with the Bleu Dextran 2000 The amount of Bleu Dextran necessary for coupling, is determined, bearing in mind that one gram dry weight of Sepharose is capable of fixing from 80 to 100 mg of Bleu Dextran.

This amount is dissolved in an alkaline buffer of a pH of the order of from 8 to 10, in particular a 0.4 M carbonate buffer of pH 10 (20 mg of Bleu Dextran 2000 are dissolved easily in one ml of carbonate buffer).

The solution of Bleu Dextran is poured on to the Sepharose gel, previously rinsed with 3 or 4 times its volume of 0.4 M carbonate buffer, pH 10. The mixture of gel and of Bleu Dextran is poured into a dark glass flask and is subjected to slight agitation, for example wit a revolving wheel, for about 18 to 24 hours, at a temperature of the order of 4"C to promote coupling After the coupling phase the Bleu Dextran Sepharose product obtained is rinsed, on a fritted-glass filter, with an excess of 0.4 M carbonate buffer, pH 10, so as to remove the Bleu Dextran not fixed, until the buffer remains colourless (optical density less than or equal to 0.02).

To eliminate the active positions which might not have been fixed with Bleu Dextran the product of the coupling is then put unto contact with an alkanolamine solution, in particular 1M ethanolamine at pH 8, for about 2 hours, at a temperature of +40 C. For this operation preferably the product of the coupling is transferred from the flask into another receptacle.

To make the gel resistant to variations in pH, in molarity or other variations, it is then subjected to three consecutive rinsing cycles with an acid solution constituted by a 0.1 M acetate buffer of pH 4.0 to which has been added I M NaCI, and an alkaline solution constituted by a 0.1 M borate buffer of pH 8.5 to which has been added 1M NaCI.

After these rinsing cycles the gel is equilibrated with a 10 mM Tris-HC1 buffer, pH 7.5, to which has been added a bactericidal and antiseptic agent, in particular 0.02% sodium azide.

2. Preparation of the affinity chromatography column The Bleu Dextran Sepharose is poured into a chromatography column of appropriate dimensions (bearing in mind that one ml of gel is capable of fixing about 8 million international units of interferon).

Before the very first use of the column, it is rinsed with 20 times its (dead) volume of Tris HCI 10 mM buffer, pH 7.5. to which has been added 0.02% of sodium azide.

To eliminate molecules of Bleu Dextran which might have a tendency to become detached, the elution buffer, made up from Tris-HC1 10 mM, pH 7.5, to which has been added 1 M NaCI is passed through until the optical densitv at 254 nm remains at 0. (In the case of columns of large dimensions, it is recommended to undertake several successive rinsing cycles with Tris buffer both with and without NaCI in order to increase the adaptation of the column to variations in pH, in molarity or other variations.

A Bleu Dextran Sepharose column can be employed over several months and for numerous interferon purification cycles, provided that it is not contaminated with bacteria. When not in use it is therefore desirable to protect it with sodium azide and to keep it cold, at about +4"C.

However, since sodium azide is toxic for the cells, before undertaking the fixing of the interferon it is necessary to pass through at least three dead column volumes of the Tris-HC1 buffer without azide in order to eliminate every trace of the antiseptic.

Example 5 The purification of a preparation of mouse interferon, on the one hand, and of the preparation of human interferon, on the other hand, by chromatography in a Bleu Dextran Sepharose column according to Example 4, using polynucleotides as desorption agents was carried out as follows: a) Preparations of interferon As regards the preparation of mouse interferon, it consists of the supernatant liquid from a tissue culture (C 243 cells) which has been induced to produce interferon by the virus of Newcastle disease. After deactivation of the virus at pH 2, the supernatant liquid is dialysed against Tris-HC1 10 mM, pH 7.5, for about 24 hours at a temperature of about 4"C.

The titre of interferon, the content of proteins in the preparation and the specific activity correspond to those of the preparation employed in Example 2.

So far as concerns the preparation of human interferon to be purified, it is constituted by a supernatant liquid from a culture of fibroblasts such as that in Example 3. This preparation is employed as it is, without preliminary dialysis.

b) Adsorption phase From 3 to 8 ml of the preparation of mouse interferon indicated above (being 3.8x106 to lox10' I.U. of interferon) are made to pass through a Bleu Dextran Sepharose column of from 1.5 to 4 ml according to the experiment, at a rate of 0.5 mVmn. In the case of the purification of human interferon, one puts on the column 7 ml of the preparation described above, titring 2,560 interferon units per ml, which corresponds to a total of 17,920 interferon units.

This adsorption phase is carried out as described in Examples 2 and 3.

c) Desorption phase After penetration of the preparations and rinsing of the column, as in Examples 2 and 3, desorption of the interferon is carried out using polyffucleotides and operating as follows: An eluant comprising 0.1 g of polynucleotides per ml of a Tris-HC1 10 mM buffer of pH 7.5 is made to pass through the column, at a rate of 0.5 ml/mn. The total volume of eluant employed corresponds to two volumes of gel (in the case of poly U a quantity corresponding to 4 volumes of gel has also been employed). The polynucleotides employed were synthetic polyribonucleotides, namely poly G, poly I and poly U such as those sold by Choay laboratories.

Polynucleotides of natural origin were equally employed, such as the t-RNA (total fraction) of beer yeasts, those of Escherichia Coli and those of mouse cells of C-243 type. These products are sold under their chemical names, polyinosinic acid being sold for example under the name poly I.

After the desorption stage described above, a second washing is undertaken, and the residue of the products with interferon activity still fixed upon the column is eluted by means of NaCI I M in Tris 10 mM buffer, pH 7.5.

Fractions each corresponding to a third of the total volume of the Bleu Dextran Sepharose (BDS) column were recovered. The table given below records the percentage of interferon recovered with two total volumes of eluant in terms of the polynucleotide employed. (The values have been corrected in such a way that the percentage of interferon desorbed by the polynucleotides and the percentage of interferon subsequently desorbed by the hyper-molar buffer are together equal to 100).

Percentage of desorption obtained according to the type of polynucleotides employed Type of polynucleotide Percentage of interferon desorbed in the column of BDS.

I-Mouse interferon poly G 17 poly I 86 poly U 43 (if the elution is continued 73% is obtained) t-RNA (total fraction extracted from beer yeasts) 90 t-RNA (total fraction extracted from E. Coli) 23 t-RNA (total fraction extracted from C-243 cells) 30 Il-Human interferon poly I 94 In another series of experiments the degree of purification obtained was studied by carrying out the desorption of interferon fixed upon a Bleu Dextran Sepharose column by means of polynucleotides.

The results obtained with preparations of mouse interferon and of human interferon are respectively recorded in Examples 6 and 7 which follow.

Example 6 A preparation of mouse interferon (C 243 cells) induced to produce interferon by the virus of Newcastle disease was used, such as that employed in Example 2.

The titre of interferon in the crude preparation was 2.6x 106 I.U./ml. Its protein content was 100 ,ug/ml and its specific activity was 2.6x 107 interferon units/mg of proteins.

130 Ml of such a preparation (which thus corresponds in toto to 3.3x103 interferon units) was passed through a Bleu Dextran Sepharose column, as in Example 4, having a total volume of 9 ml.

The adsorption rinsing and washing stages were carried out as previously described. The desorption was effected according to the procedure described in Example 5, employing 18 ml of an elution solution containing poly I as desorbent agent at a rate of 100ylml of buffer.

The interferon was recovered in two fractions a and h, of 7 and 5 ml respectively. Their titres in interferon were 4.1x107 and 5.1 x 106.

3.16x108 I.U. in toto were recovered, which corresponds to a percentage of recovery of 96%.

In fraction a, 19y proteins were found to be present, which gives a specific activity of 2.1x109.

The degree of purification is thus about 80 (specific activity at the start/specific activity at the finish: 2.1x108/2.6x107).

Example 7 The preparation of human interferon to be purified was constituted by a supernatant liquid from a culture of human fibroblasts induced to produce interferon by poly IC.

The titre of this preparation was 2,560 I.U./ml, its protein content was 410 g/ml and its specific activity was 6.2x103.

7 Ml of this preparation (which corresponds to 17,920 units were passed through a Bleu Dextran Sepharose column of 5 ml.

The adsorption, rinsing and washing stages were carried out according to the operative methods described in the previous Examples The desorption of the products with interferon activity was affected by 10 ml of poly I in solution in a Tris-HC1 10 mM buffer, pH 7.5, at a rate of 100 FLg/ml.

The products with interferon activity were recovered in 5 fractions of 2 ml.

The titre of each of these fractions is recorded hereinbelow.

Fraction Titre

i 656 2 5120 3 1280 > x2=16960 units in toto 4 1280 5 160J The percentage of recovery is therefore: 16960 =95% 17920 Fraction 2 contains 10 yg/ml of proteins. The specific activity of this fraction was 5.1x105.

The degree of purification is therefore: 82 times (5.1x105/6.2x10-3).

Example ides coupled 8 As indicated previously the polynucleotides coupled to the products with interferon activity confer upon them a protection against thermal denaturisation which is of great importance. To illustrate this effect there are attached some graphs showing the percentage of activity of the products of this coupling as a function of the period of incubation at 600 C.

In Figure 2, there are shown the results obtained with the products of coupling between on the one hand interferon from mouse cells C 243 induced to produce interferon by the virus of Newcastle disease and on the other hand respectively poly U, poly I, poly C, poly I-poly C and poly A, in comparison with control preparations of interferon containing no polynucleotides. The concentration of polynucleotides in the preparation is 10 yg/ml.

Figure 3 records the results obtained, working with the products of coupling between interferon and respectively t-RNA (valine), t-RNA (tryptophane), t-RNA (total fraction of beer yeasts t-RNA (tyrosine), t-RNA (total fraction of wheat germ) and t-RNA (lysine).

According to these investigations, the antiviral activity of interferon preparations is measured by a microtest quantitative analysis using the following method: appropriate dilutions of the samples to be estimated are made in the plates sold by Falcon for microanalysis (microtiter plates-Falcon Plastic Company). At each site on the plate, 100,al of a suspension of cells derived from the same species as the interferon to be measured are then added, and the plates are allowed to incubate for 18 hours at 370C in a CO2 incubator. The indicator virus is then added (in the Examples here given, the virus of vesicular stomatitis was used at a titre of 100 infectious units per site).

The plates are then replaced in the CO2 incubator, and 24 hours later, the cellular destruction caused by the virus is read off under the microscope.

The titre of interferon corresponds to the inverse of the dilution at which 50% of the cells, at least, are protected against the destructive effect of the virus. Thus if at a dilution of 1/20000 there are 50% of the cells still intact, the titre is 20000.

The microtest units used in the laboratory have been converted into international reference units (1 microtest unit/0.2 ml corresponds to 10 international units/ml).

In the test whose results are recorded in Figure 2, the crude preparation of mouse interferon was dialysed in Tris-HC1 10 mM, pH 7.5. Into that polynucleotides were incorporated at a concentration of 10 yg per ml. Aliquots of 0.3 ml (being 3.9x 105 interferon units and 3,ag of polynucleotide) were incubated on a water-bath at 60"C. At the times indicated in the Figure, samples of 20,al were removed. These were diluted 100 times in a culture medium containing 3% of calf serum, and immediately cooled in an ice bath. Titring of the interferon was carried out at the end of the incubation.

In the investigations whose results are recorded in Figure 2, the procedure was carried out under the same conditions as those indicated above for the investigation of the thermal denaturisation in the presence of polynucleotides, but in their case t-RNA was used.

Examination of these graphs shows that the products with interferon activity when coupled with the polynucleotides retain a significant therapeutic activity even after more than 30 minutes of thermal treatment at 60"C although this activity decreases rapidly in the controls. Thanks to this protective effect of polynucleotides upon products with interferon activity, it is possible to store the interferon preparations without problems, and to subject them in the course of various investigations to variations in temperature without causing any denaturisation of their properties.

Example 9 The purification of a mouse interferon preparation by chromatography in a Bleu Dextran Sepharose column by means of t-RNA as desorption agents was carried out as follows: The mouse interferon preparation to be purified was similar to that employed in Example 5. Proceeding as described in that Example 5 after dialysis of the preparation, it107 units of interferon were put into a Bleu Dextran Sepharose column. The column was rinsed with Tris buffer (4 volumes in total), then desorption of the interferon was effected by putting 3 to 4 ml of t-RNA into the column, at a concentration of 100 ,ug per ml, in the said Tris-HC1 10 mM buffer.

A second stage of rinsing was carried out with the buffer in question, then desorption of the remaining products with interferon activity was carried out by means of Tris-HC1 10 mM buffer, pH 7.5, to which NaCI 1M have been added.

The percentages of desorption of mouse interferon by means of total fractions of t-RNA of beer yeasts, of E. coli, of C 243 mouse cells and of rat liver were respectively 91, 23, 54 and 10.

WHAT WE CLAIM IS: 1. Process for purifying preparations containing products with interferon activity by selectively separating said products from the contaminating substances of the preparations, characterised by the fact that the said preparations are contacted with a phase containing at least one compound of the polynucleotide type capable of acting like a ligand to bind the products with interferon activity.

2. Process according to claim 1, characterised by the fact that a preparation containing products with interferon activity, which is to be purified, is contacted with an adsorbent equilibrated with a buffer whose salt concentration is isomolar or hypomolar relative to that of blood, the said adsorbent comprising a gel forming solid support whose lattice size is such that it permits passage of macromolecules and a said polynucleotide-type compound fixed upon the said support.

3. Process according to claim 2, for obtaining purified preparations possessing interferon activity, characterised by a supplementary stage in which the desorption of the fixed products having interferon activity is achieved by means of a buffer whose salt concentration is hypermolar relative to that of blood.

4. Process according to claim 1, characterised in that a composition including a complex containing products with interferon activity fixed upon a ligand is contacted with a phase containing at least one compound of polynucleotide type, capable of competing with the ligand for the products with interferon activity and detaching them, the composition and phase in question being equilibrated by means of a buffer whose salt concentration is isomolar or hypomolar relative to that of blood.

5. Process according to any one of claims 1 to 4. characterised in that the polynucleotide derivative employed is constituted by at least one polyribonucleotide and/or one polydesoxyribonucleotide of synthetic or natural origin.

6. Process according to claim 5, characterised in that a polyribonucleotide is used containing from 10 to 300 nucleotide residues.

7. Process according to claim 6, characterised in that the polyribonucleotide is selected from the group consisting of polyriboinosinic acid, polyriboadenosic acid, polyribouridylic acid and polyriboguanylic acid.

8. Process according to claim 6, characterised in that the polyribonucleotide is constituted by a t-RNA.

9. Process according to claim 8, characterized in that it is the t

Claims

**WARNING** start of CLMS field may overlap end of DESC **. even after more than 30 minutes of thermal treatment at 60"C although this activity decreases rapidly in the controls. Thanks to this protective effect of polynucleotides upon products with interferon activity, it is possible to store the interferon preparations without problems, and to subject them in the course of various investigations to variations in temperature without causing any denaturisation of their properties. Example 9 The purification of a mouse interferon preparation by chromatography in a Bleu Dextran Sepharose column by means of t-RNA as desorption agents was carried out as follows: The mouse interferon preparation to be purified was similar to that employed in Example 5. Proceeding as described in that Example 5 after dialysis of the preparation, it107 units of interferon were put into a Bleu Dextran Sepharose column. The column was rinsed with Tris buffer (4 volumes in total), then desorption of the interferon was effected by putting 3 to 4 ml of t-RNA into the column, at a concentration of 100 ,ug per ml, in the said Tris-HC1 10 mM buffer. A second stage of rinsing was carried out with the buffer in question, then desorption of the remaining products with interferon activity was carried out by means of Tris-HC1 10 mM buffer, pH 7.5, to which NaCI 1M have been added. The percentages of desorption of mouse interferon by means of total fractions of t-RNA of beer yeasts, of E. coli, of C 243 mouse cells and of rat liver were respectively 91, 23, 54 and 10. WHAT WE CLAIM IS:

1. Process for purifying preparations containing products with interferon activity by selectively separating said products from the contaminating substances of the preparations, characterised by the fact that the said preparations are contacted with a phase containing at least one compound of the polynucleotide type capable of acting like a ligand to bind the products with interferon activity.

9. Process according to claim 8, characterized in that it is the total fraction oft- RNA deriving from bacteria, yeasts or mammalian organs.

10. Process according to claim 9, characterized in that the said t-RNA derives from bacteria of the genus Escherichia Coli, yeasts of the genus of beer yeasts, or cells of animal or human origin, specifically cells from the same animal or human origin as those which have produced the interferon which is to be purified.

11. Process according to any one of claims 1 to 3 and 5 to 10, characterized in

that as the solid support for the gel forming adsorbent there is used a polysaccharide such as agarose or a derivative thereof, specifically a modified agarose in which the polysaccharide chains are cross-linked into a threedimensional lattice, or alternatively a polyacrylamide or an acrylic resin.

12. Process according to any one of claims 1 to 3 and 5 to 11, characterized in that the salt concentration of the buffer employed to equilibrate the adsorbent is less than or equal to 0.15 M.

13. Process according to any one of claims 3 to 12, characterized in that the salt concentration of the elution buffer is at least 0.5 M.

14. Products with interferon activity fixed upon an adsorbent phase displaying the properties and characteristics of those obtained by carrying out the process according to any one of claims 1 to 3 and 5 to 13.

15. Process according to any one of claims 4 to 10, characterized in that the composition containing products with interferon activity not fixed upon a ligand is an adsorbent composition comprising a gel forming solid support the size of whose lattices is such as to permit the passage of macro-molecules and a compound fixed upon the said support, playing the role of ligand relative to the products which are to be separated in a selective manner and which possesses a polycyclic structure similar to that of the blue chromophore of the formula

or an equivalent product whose structure enables interferon to be retained in a selective manner.

16. Process according to claim 15, characterized in that the ligand coupled to the solid support is constituted by the blue chromophore having the formula given in claim 15 or a compound having that formula with a hydrogen atom in place of the -SO2ONa group on the phenyl group at end of the chain.

17. Process according to claim 16, characterised in that the said chromophore is bound to a polysaccharide, and in particular one having a molecular weight of about 2,000,000.

18. Process according to claim 17, characterised in that an adsorbent composition is used containing a ligand comprising the blue chromophore according to claim 15 or claim 16 coupled to a solid support according to claim 17 and bound to a polysaccharide according to claim 11.

19. Process according to any one of claims 14 to 18, characterised in that the salt concentration of the buffer employed to equilibrate the adsorbent is less than or equal to 0.5 M.

20. Process according to any one of claims 14 to 19, characterised in that the phase containing at least one derivative of polynucleotide type comprises these polynucleotides in solution in a buffer whose salt concentration is hypomolar as compared with that of blood.

21. Process according to claim 20, characterised in that the said polyribonucleotides are used in solution in a buffer at a rate of 10 to 100 ,ug/ml, preferably from 30 to 50 ssg/ml.

22. Products with interferon activity when fixed upon polynucleotides.

23. Products with interferon activity fixed upon nucleotides such as are obtained by operation of the process according to any one of claims 4 to 10 and 14 to 21.

24. Purified preparations with interferon activity displaying the properties and characteristics of those obtained by operating the process according to any one of claims I to 21.

25. Medicaments containing the preparations according to claim 23 as the active principle.