CH633716A5 - Method for separating and purifying products having interferon activity, purified preparations having interferon activity and use of the preparations - Google Patents

Description

The subject of the invention is a process for selectively separating, by eliminating contaminating substances, products with interferon activity from preparations containing them, and more particularly relates to a process for the purification of such products.

It also relates to the corresponding purified preparations and the use of the latter as active principle of medicament.

We know that interferon is a therapeutic product of great value, in particular because of its remarkable antiviral and immunosuppressive properties.

According to the usual in vitro synthesis methods, this product is obtained by the action of viruses or chemicals on tissue cultures. However, the resulting preparations also contain contaminating substances. In particular, these preparations contain proteins which have a pyrogenic action and also have the disadvantage of being able to induce specific sensitization in the recipient.

Crude preparations of interferon cannot therefore be used directly, in particular for the applications envisaged above, and must be purified.

Various purification methods have been proposed, in particular methods by gel filtration or by ion exchangers.

However, the large-scale use of such techniques for the purification of interferon preparations has encountered difficulties, notably due to very low yields.

Affinity chromatography methods have also been proposed.

We know that these are based on a selective affinity between a given product and an adsorbent phase comprising a ligand covalently attached to a solid support. The passage, over the adsorbent phase, of a preparation containing the product which it is desired to separate makes it possible to selectively retain the product in question on the ligand, and thus to separate it from the preparation containing it. To recover the fixed product, it is then necessary to have a desorption agent.

According to techniques based on the principle of affinity chromatography, anti-interferon antibodies are used as ligand. However, the results obtained to date are not yet satisfactory, in particular because of the production of antisera which is difficult, long and costly, all the more so if we try to develop it on an industrial scale.

Also known are affinity chromatography methods in which the ligand used is chosen because of the hydrophobic properties of interferon. These processes then use organic solvents for desorption.

However, with a view to the therapeutic use of interferon, it is necessary to eliminate all traces of organic solvent, without destroying the interferon activity by transferring it to an aqueous medium. These methods therefore involve extremely complex manipulations.

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.

These latter methods have the advantage of being able to be implemented under conditions in which it is possible to work in an aqueous medium, which eliminates the drawbacks encountered in the previous techniques in the case of the use in therapy of purified preparations of interferon.

These methods also make it possible to remove a large part of the contaminating proteins.

However, the inventors have now established that it is possible to further improve these results by using, to separate and fix the products with interferon activity of preparations containing them, new ligand agents. In the following description and in the claims, it is understood that the term “preparation” denotes both a composition containing the products with interferon activity in unbound and bound form, and in particular in the form of a complex.

This invention is therefore based on the observation of a remarkable affinity of products with interferon activity for a certain type of structure.

It is thus possible, using this particular affinity, to separate in an extremely specific manner products with interferon activity from preparations, or the like, containing them, and to obtain, easily, interferon preparations more satisfactory than those proposed. to date, especially both from the point of view of their interferon titer and their purity.

The method according to the invention, for selectively separating, by eliminating the contaminating substances, products with interferon activity from preparations containing them is characterized in that these preparations are brought into contact with a phase comprising at least one polynucleotide, playing on specifically the role of ligand vis-à-vis products with interferon activity.

The remarkable specific affinity between the polynucleotides and the products with interferon activity makes it possible to separate the latter, with very high selectivity, from preparations containing them.

The results obtained are entirely satisfactory with interferon preparations of both human and animal origin and in particular constituted by supernatants from tissue cultures, fresh explantation (primary or secondary cultures) or continuous lines, where the cells have been induced to produce interferon, preferably in a serum-free culture medium, by a virus or a synthetic nucleic acid such as poly-IC The interferons of the preparations used can moreover be of fibroblastic or leukocytic origin. Preparations obtained by translation in cell-free medium, in particular according to the techniques reported in “Molecular Mechanism of Prot. Biol. Synthesis ”edited by H. Weissbach and S. Pestka, Acad. Press 1977, pp. 574 to 582, can also be used in the process of the invention.

According to a provision of the invention, a preparation to be purified of products with interferon activity is brought into contact with an adsorbent balanced by a buffer whose salt concentration is isomolar or preferably hypomolar relative to that of blood, said adsorbent comprising, on the one hand, a solid support forming a gel, the mesh size of which is such that it allows the passage of macromolecules and, on the other hand, a compound of the polynucleotide type, fixed on said support, playing the role of screw ligand with regard to the products which it is desired to separate selectively, the quantity of adsorbent used and the duration of contact between the ligand and the preparation to be purified being sufficient to allow the desired fixation of the products with interferon activity.

To obtain purified preparations with interferon activity, an additional step is used according to which the desorption of the fixed products is carried out using a buffer whose salt concentration is hypermolar compared to that of blood.

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

It will be noted that the interferon is easily recoverable from the coupling product which it forms with the ligand, and this in an almost total manner and without it being denatured. The advantage of this process is further increased by the fact that the adsorption capacity of the adsorbent used is practically not damaged. The same adsorbent can then be used for many purification operations, and therefore is advantageous from an economic point of view.

According to another arrangement of the invention, a composition comprising a complex containing products with interferon activity attached to a ligand is brought into contact with a phase comprising at least one compound of the polynucleotide type, capable of competing with the ligand for the fixing of the products with interferon activity and detaching the latter, the composition and phase in question being balanced using a buffer whose salt concentration is isomolar or hypomolar relative to that of blood, the amount of polynucleotides in the above phase and the duration of contact between the polynucleotides and the products with interferon activity fixed on the ligand being sufficient to allow the desorption of the products with interferon activity.

It will be noted that, in the implementation of this arrangement, the products with interferon activity are found attached to a ligand for which they have less affinity than for polynucleotides. Given the very important character of the interferon affinity for polynucleotides, the competition for the fixation of interferon is to the benefit of the latter.

It will further be observed that this results in additional purification for the products with interferon activity. Indeed, the contaminating substances, in particular the proteins which are present in the crude interferon preparations and which tend to bind with the interferon on the adsorbent considered will not be practically detached by the polynucleotides. This arrangement of the invention constitutes a very selective purification process.

In addition, this method offers the advantage of giving interferon significant protection against thermal denaturation and, consequently, great stability.

According to a preferred embodiment of the aforesaid first provision of the invention, the products with interferon activity are separated from crude preparations containing them by bringing these preparations into contact with an adsorbent phase consisting of at least one derivative of polynucleotide type coupled to solid support.

The polynucleotide type derivative is a polyribonucleotide or a polydeoxyribonucleotide in which one or more elements in the constituent units is possibly modified, without this modification altering the phenomenon of specific recognition which the interferon presents towards it.

Advantageously, the polynucleotides are used, comprising from approximately 10 to 500 nucleotide units. These own derivatives, by their configuration and their constitution, to fix the products with interferon activity can be as well of synthesis as of natural origin.

Among the derivatives of the first type of polynucleotides such as polyribo-inosinic acid or poly-I, polyniboadenosic acid or poly-A and polyribo-uridylic acid or poly-U, are particularly suitable. Generally, products having a molecular weight in the range of 50,000 to about 250,000 are used.

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

It can be either all of the t-RNA fractions extracted from the cells as part of these fractions, or alternatively specific fractions with respect to the synthesis of a given amino acid.

It is known that these t-RNAs comprise approximately 70 to 85 nucleotide units and have a molecular weight of the order of 25,000.

Different sources of tRNA can be used, in particular among eukaryotes or prokaryotes, it is possible to use bacteria, such as those belonging to the genus E. coli, or yeasts, such as that belonging to the type of yeast.

The solid support of the gel-forming adsorbent comprises a Polysaccharide such as agarose or a derivative thereof, in particular a modified agarose in which the Polysaccharide chains are crosslinked in a three-dimensional network, in particular that sold under the trademark Sepharose, or a Polyacrylamide or an acrylic resin.

For physiological reasons still unexplained, it turns out that the products with interferon activity have a very great affinity for the above-mentioned polynucleotides. This property can therefore be used from a practical point of view to purify interferon.

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

It is particularly convenient to bring the adsorbent into contact with the preparation to be purified in a chromatography column, by passing the preparation to be purified through this column. The column loaded with the adsorbent is balanced with the above buffer, the pH of which is around 3 to 9, preferably close to neutral.

Crude interferon preparations are used, the interferon titer of which is approximately 10+ to 10® international units per 0.1 to 0.2 g of protein, which gives specific activity (international interferon units or UIF , per milligram of protein) lying between 105 and 107. These preparations can be used as such under isomolarity conditions, for passage through a column, or undergo prior dialysis against the buffer which served to balance said column. By operating under the conditions of isomolarity of a tissue culture medium, the yield of interferon is approximately 80 to 90%, while it is 100% under hypo-larity conditions. In the latter eventuality, a particularly suitable buffer consists of tris-HCl 10 mM pH 7.5 (it is recalled that tris is the abbreviation commonly adopted to denote tris- (hydroxymethylaminomethane).

The step of separating products with interferon activity from preparations containing them therefore falls within the scope of the invention.

The invention further relates, as new industrial products, to the complexes formed by the products with interferon activity attached to the adsorbent phases described above comprising at least one derivative of polynucleotide type.

Such complexes result from the interaction between products with interferon activity and polynucleotides. The mode of interaction has not been clarified to date, but the work carried out suggests that these are noncovalent bonds and that there may be Van der Waals forces, hydrogen bonds, hydrophobic bonds , weak ionic forces ...

The fixed products can be recovered by increasing the salt concentration of the buffer - in particular by adding acetates, phosphates, chlorides or other salts - so as to obtain a hypermolar salt concentration relative to that of blood and at least 0 , 5 M, and preferably 1 M.

This recovery of products with interferon activity is advantageously carried out by column elution. By collecting the eluted fractions, there are purified interferon preparations, with a high interferon content and practically completely free of contaminating proteins.

In addition, by carrying out the desorption phase with a flow rate not exceeding 0.2 ml / cm 2 / min, the majority of the products with interferon activity are collected in a fraction of eluate of low volume, in particular when the phase adsorption was conducted

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under hypomolar conditions, which has the advantage of providing preparations having a high concentration of interferon compared to the concentration in crude preparations.

By using the process of the invention, with almost total yield and in a single step, very high purity interferon preparations can be obtained.

It should also be noted that these preparations are advantageously in aqueous solution and can therefore be used as such in therapeutic applications as indicated below. 10

Thus, as regards for example the mouse interferon for which the method was developed the first time, preparations are obtained which can reach 10® units per milligram of protein.

As indicated above, it is also possible, thanks to the remarkable affinity, demonstrated by the inventors, between the products with interferon activity and the polyribonucleotides, to separate the latter from complexes or compositions in which they are found bound and to form a new complex of poly-nucleotide type / products with interferon activity.

According to a preferred embodiment of this arrangement of the invention, contact is made, on the one hand, with a composition comprising products with interferon activity fixed on an adsorbent and, on the other hand, a phase containing at least one derivative polynucleotide type.

Said adsorbent comprises a solid gel-forming support, the mesh size of which is such that it allows the passage of macromolecules and, on the other hand, a compound fixed on said support, playing the role of ligand with respect to the products. which one wishes to selectively separate and which has a polycyclic structure of the type of that of the blue chromophore, marketed under the brand Bleu Cibacron F3GA, of formula

S020Na

SOgONa or an equivalent product, the configuration of which allows interferon to be selectively retained.

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

The ligand coupled to the solid support advantageously consists of the Bleu Cibacron F3GA itself, or the product sold under the brand Bleu Cibacron 3GA (which essentially differs from the previous one by the presence of a hydrogen atom in place of the group - S020Na on the phenyl radical at the chain end), or an equivalent product. By equivalent product is meant,

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 makes it possible to obtain the desired selective fixation of the interferon.

The cyclic compound used as ligand can be attached without disadvantage to a Polysaccharide, in particular to a Polysaccharide of high molecular weight, of the order of 2 million, called Dextran 2000. In this case, it is the Polysaccharide which is attached to the support. solid.

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

The adsorbent comprising the products with interferon activity linked to the aforesaid solid support is balanced by a buffer whose concentra60

tion in salts is isomolar or preferably hypomolar compared to that of blood. These conditions are advantageously established using a buffer having a molarity of less than 50 mM and in particular with the tris buffer used in the adsorption phase of products with interferon activity on a phase comprising polynucleotides as described above. .

As regards the phase containing at least one derivative of the polynucleotide type, it comprises these polynucleotides in solution in a buffer whose salt concentration is advantageously hypomolar compared to that of blood.

The polynucleotides in question are as mentioned above. Thus, polyribonucleotides comprising from approximately 10 to 500 nucleotide units constitute remarkable desorption agents. Among the suitable polyribonucleotides, mention may be made of poly-riboguanylic or poly-G acid, poly-I, poly-U.

Ribonucleic acids, in particular t-RNAs, can also be used. These can be extracted from bacteria such as those of the genus E. coli. They can also come from yeasts such as brewer's yeast. Ribonucleic acids of animal or human origin are also advantageously used, more particularly originating from animal organs.

Among these, those extracted from the same cells which produced the interferon which it is desired to purify are very particularly preferred. Thus, to purify preparations containing products possessing the activity of mouse interferon, mouse cells of the so-called C 243 cell line are advantageously used.

Preferably, the polynucleotides used as desorption agents are dissolved in a buffer at a rate of 10 to 100 ng / ml, preferably from 30 to 50 µg.

A buffer suitable for this purpose consists of 10 mM tris-HCl, pH 7.5.

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

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

To this end, a polynucleotide solution containing 10 to 100 ng / ml and preferably 30 to 50 (ig / ml of an elution buffer consisting of 10 mM tris-HCl, pH 7.5, is passed through. on a column containing an adsorbent gel constituted by Blue Dextran Sepharose to which products with interferon activity are attached.

The passage of this solution is advantageously carried out at a rate of approximately 0.2 ml / cm 2 / min.

To facilitate desorption on a small volume of eluent, in particular in the case where the affinity between the polynucleotides and the products with interferon activity is not clearly marked, the solution of polynucleotides is advantageously left in contact with the adsorbent gel before to proceed with the elution. Generally speaking, a contact time of between about 1 a.m. and 6 p.m. is sufficient.

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

Thanks to the remarkable specificity of this process, products with very high purity interferon activity are collected.

Interferon is attached to the polynucleotides and the resulting coupling products also fall within the scope of the invention.

As indicated above, the polynucleotides advantageously provide protection for products with interferon activity against thermal denaturation. Thus, it is found, by heating to about 60 ° C a coupling product according to the invention, that a significant part in particular of the antiviral activity is retained, while more than 95% of this activity disappears in the control preparations.

Purified preparations of products with interferon activity and which include products with interferon activity recovered by desorption from complexes which they form with polynucleotides fixed on a solid support, as well as the complexes which they form according to another provision of l he invention with said polynucleotides have remarkable therapeutic properties, and there is added in particular the advantage of an antiviral action of great interest. This activity has been demonstrated in particular in patients suffering from chronic hepatitis B, using 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 the 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 in preventing recurrences of herpetic keratitis, as demonstrated by the tests carried out according to Kaufman et al., "J. Infect. Dis. ”, 133 (suppt) A 165-168,1976. In addition, interferon preparations can have a favorable effect in patients with cancerous tumors. On the one hand, these preparations decrease the frequency and the duration of viral complications, in particular due to herpes zoster, as it appears by carrying out tests according to Jordan et al., “J. Infect. Dis. ”, 130: 56-62.1974. On the other hand, they seem to favor, by their anti-tumor properties, the survival of patients suffering from osteosarcoma. These experiences can be highlighted by operating according to Strander et al., “Acta Orthop. Scand. ", 45: 958-959, 1974.

All these tests made it possible to note a remarkable harmlessness of the preparations with interferon activity according to the invention.

As numerous studies have shown, interferon does not cause obvious toxicity phenomena. We can refer to this subject to the results given by J. Desmyter et al. in

"The Lancet", September 25,1976, pp. 645 to 647, and to the references indicated in this article concerning the absence of toxicity of such 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. Emödi et al. in "Natn. Cancer Inst. ”, 1975, 54, 1045.

Since interferon is specific to the species, interferon from human cells must therefore be available, for example for therapeutic applications in humans.

The medicaments according to the invention, which contain the purified preparations of the invention as active principles, can be administered to humans, in particular by the intramuscular route and by the subcutaneous route in the form of a solution. For example, a dose of fibroblastic interferon of 107 units was administered every

2 d, for two weeks, in cases of chronic hepatitis or

3 x 10fi units of leukocyte interferon, 3 times a week in cases of osteosarcoma.

Example 1:

Preparation of an affinity column of polynucleotides for the separation of products with interferon activity from preparations containing them.

First of all, an activated Sepharose gel is formed which will serve as a solid support for the polynucleotides, then, in a second step, the polynucleotides are fixed to the activated Sepharose by proceeding according to a method derived from that described by DL Robberson and N. Davidson. in "Biochem. ", II, 4,1973,533.

a) Preparation of activated Sepharose gel

Sepharose 4 B type agarose is used, such as that sold by Pharmacia (Uppsala, Sweden). This product is in the form of beads. The latter are washed with water by filtration on sintered glass, then resuspended in water in a proportion of 5 ml of initial Sepharose per 10 ml of water.

Sepharose is then activated by cyanogen bromide CNBr. Each 10 ml of Sepharose suspension, the latter being kept in suspension by slight magnetic stirring, 15 ml of a 6.5-7% aqueous CNBr solution are gradually added.

The temperature of the suspension is maintained at 4 ° C by an ice bath; the pH, controlled by a pH meter, is adjusted to 10 by addition of NaOH. Once the pH has stabilized and a temperature of approximately 20 ° C. has been reached, the Sepharose thus activated is filtered on a sintered glass filter and washed with a 0.1M bicarbonate buffer, pH 9, previously cooled using a volume of buffer corresponding to approximately 10 times the volume of Sepharose gel. The gel is then resuspended in the same bicarbonate buffer, using 8.5 ml per 10 ml of initial suspension. The Sepharose thus activated is coupled to the methyl ester of s-amino-caproic acid. Each fraction of 8.5 to 10 ml is made up with a solution of the 30% ester in the bicarbonate buffer at pH 9. The pH of the mixture is adjusted to 9 using NaOH and the mixture is left to 4 ° C for 24 h with slow rotary stirring.

The Sepharose gel is then washed with 10 volumes of ice water and resuspended in water to the initial volume. 0.7 ml of a 98% hydrazine hydrate solution is then added, then this mixture is brought to 70 ° C. for 15 min and cooled.

The gel is again filtered and washed thoroughly with ice water and then resuspended in water to the initial volume.

To block the carboxyl groups of Sepharose, 9 g of glycinamide hydrochloride are added per 100 ml of Sepharose / hydrazide suspension. The pH is adjusted to 4.75 and then 1.12 g of carbodiimide (per 100 ml of suspension) are added to the mixture. The mixture is left for 2 h, while maintaining the pH at 4.75 by addition of IN HCl.

After washing with ice-cold water by filtration, the Sepharose gel is suspended in a double volume of a 5 × 10_4M EDTA solution, pH7.

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The Sepharose thus activated can be stored at 4 ° C.

b) Attachment of Sepharose to the Polynucleotides

Several methods have been proposed for forming a complex of activated polynucleotides and agarose such as the above-mentioned Sepharose and they can make it possible to obtain the desired fixation, in particular at the end of the P04 group in position 3 ′ in the ose portion of the polynucleotides or still at the end of group P04 in position 5 '.

To fix Sepharose activated by the end of the P04 group in position 3 'of the dare portion of polynucleotides such as poly-I, poly-U or t-RNA, we will advantageously proceed according to a method based on that described by DL Robberson and N. Davidson in the article mentioned above. This method comprises the treatment of the polynucleotides with an alkaline phosphatase, then a step of oxidation of the polynucleotides and their coupling with the activated Sepharose gel.

a-Treatment with alkaline phosphatase

Before carrying out this fixation, the polynucleotides are subjected to an oxidation reaction with sodium periodate. To increase the yield of the reaction, the polynucleotides are systematically treated beforehand with alkaline phosphatase to remove any possible presence of a PO 4 group at the 3 ′ end of the dare portion of the polynucleotides.

ß-Polynucleotide oxidation step

Periodate oxidation is carried out according to the Hunt method ("Bioch. J.", 1965,95,542).

The polynucleotides, at a concentration of 1-2 mg / ml in an acetate buffer of pH 5.2, are oxidized by adding a tenth of volume of a freshly prepared 0.2 M NaI04 solution. The reaction is carried out protected from light for 45 min. The excess periodate is removed by adding a tenth of a volume of ethylene glycol. The polynucleotides thus oxidized are recovered by precipitation with ethanol, then it is dissolved in a 0.1 M sodium acetate buffer, pH 5, and a dialysis of this solution is carried out against the same aetate buffer. 0.1M, which allows the elimination of the formaldehyde previously formed.

y-Coupling of polynucleotides to activated Sepharose gel

About 50 to 60 units of A 260 of polynucleotides are added per milliliter of activated Sepharose gel, blocked by hydrazine as indicated above (volume measured on deposit). This mixture is suspended in 5-6 ml and left in gentle stirring at 4 ° C for 12-15 h. The unbound polynucleotides are eliminated by successive washings with an 0.1 M acetate buffer of pH 5.2, then a 0.1 M bicarbonate buffer of pH 9.

The Sepharose gel carrying the polynucleotide chains is then resuspended in the desired buffer. To form a complex of activated Sepharose and of polynucleotides such as poly-A and poly-C, one advantageously operates in a buffer medium at pH 6 according to the method described by Wagner et al. in "BBRC" (1971) 45,

184.

To a solution of polynucleotides (at a concentration of 2-4 mg / ml in 0.2M 4-morpholinoethanesulfonic acid, pH 6.0), a suspension of activated Sepharose gel obtained as described above is added in a proportion of 1/3 (v / v).

The mixture is gently stirred at 4 ° C for 12-15 h. The polynucleotides which have not reacted with Sepharose are eliminated by filtration then, by successive washings, with the buffer of 4-morpholinoethanesulfonic acid, then with water.

In this case, the polynucleotides are assumed to be fixed via the group PO4 which is located at the end of the group in position 5 'in the dare portion of the polynucleotides.

To fix poly-U on activated Sepharose, the procedure is carried out as indicated above in a buffer at neutral pH such as tris, at low concentrations of 10-20 mM.

c) Preparation of the affinity chromatography column

The Sepharose polynucleotide gel is poured into a chromatography column of suitable dimensions. It is estimated that 1 ml of gel is capable of fixing approximately 8 million international units of interferon.

Before the very first use of the column, a rinsing is carried out with 20 times the dead volume of 10 mM tris-HCl buffer, pH 7.5, added with 0.02% of sodium azide.

Since sodium hydroxide is toxic to cells, at least three dead volumes of azide-free tris-HCl buffer must be passed through the column before binding the interferon.

Example 2:

Purification of a mouse interferon preparation by affinity chromatography on a column according to Example 1 of poly-U / Sepharose.

The crude preparation of interferon consists of a tissue culture supernatant derived from C 243 cells induced to produce interferon by the Newcastle disease virus. After inactivation of the virus at pH 2, the supernatant is dialyzed against 10 mM tris-HCl, pH 7.5, for approximately 24 h at a temperature of approximately 4 ° C.

The crude preparation interferon titer is 1.3 x 10® international units, its protein content is 0.1 mg / ml, and the specific activity is 1.3 x 107 interferon units per milligram protein.

The chromatogram of the purification operation is shown on the fig. 1.

10 ml of the preparation to be purified are passed through a 0.9 cm diameter column containing 2 ml of poly-U / Sepha-pink gel 4 B (product obtained commercially from Pharmacia,

Uppsala, Sweden).

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

After penetration of the interferon preparation into the gel (fractions 1 to 4 of the chromatogram), the column is washed with 6 ml of 10 mM tris-HCl buffer, pH 7.5, to ensure removal non-adsorbed contaminating substances (fractions 5 to 7).

To recover the interferon, the desorption phase is carried out by passing through the column 10 ml of 10 mM tris-HCl, pH 7.5, supplemented with 1M NaCl (fractions 8 to 13 on the chromatogram of fig 1).

The interferon titer is measured in each fraction collected during the sorption, washing and desorption phases. It is then found that the interferon contained in the raw starting preparation is recovered in full and is collected almost completely in 2 tubes, namely tubes Nos 8 and 9 (ie 4 ml), during the desorption phase.

The interferon titer (UIF / ml) in these tubes is respectively

2.5 x 10th (tube 8), and 5 x 10® (tube 9)

Interferon recovery is therefore total.

The protein content in tube No. 9 is less than 2.5 (ig / ml; the specific activity is therefore above 2 × 10 9, which corresponds to a purification greater than 150 times.

Example 3:

Purification of a human interferon preparation by affinity chromatography on a column according to Example 1 of poly-I / Sepharose.

The crude interferon preparation is a supernatant from a culture of human fibroblasts induced to produce interferon by poly-I / poly-C.

The interferon titer is 4096 IU of interferon per milliliter and

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the protein content of 660 (ig / ml, which gives a specific activity of 6.2 x 10 3 IU per milligram of protein.

5 ml (20,480 IU) of the preparation, not dialyzed beforehand, are passed at a rate of 1 ml / min through a column of

0.9 cm in diameter containing 2 ml of Sepharose / poly-I gel according to Example 1, this column being equilibrated with 10 mM tris-HCl buffer, pH 7.5.

As soon as all the preparation has entered the gel, the washing is carried out as indicated in Example 2.

The desorption of the interferon is then carried out by passing through the column, with a flow rate of approximately 0.2 ml / min, 10 ml of 10 mM tris-HCl of pH 7.5 enriched with 1M NaCl.

During these various adsorption, rinsing, washing and desorption phases, 2 ml fractions are collected. By determining the interferon titer in these different fractions, we note that 1024 units are collected in the first 3 tubes (fractions of 2 ml), that is to say 6144 units, which is equivalent to 30% of the interferon passed over the column. No interferon activity is detectable in the wash tubes.

Upon desorption, it is found that the products with interferon activity are mainly collected in the fractions of tubes 10 and 11 (tube 10 containing a fraction of only 1 ml). The titer of interferon in these tubes is respectively

2048 (tube 10), and 16 384 (tube 11),

which corresponds to a total of 18,432 IU of interferon / ml.

Since the crude preparation which was purified on the column initially contained 20480 IU, 90% of interferon was therefore recovered.

Example 4:

Preparation of an Affinity Column of Bleu Dextran Sepharose for Purification of Interferon of Animal Origin, on the One Hand, and of Interferon of Human Origin, on the Other Hand, with Use, as desorption, of polynucleotides.

1. Preparation of Dextran Sepharose Blue Gel

The manufacturing process for 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 activate Sepharose with cyanogen bromide according to Cuatrecasas (Cuatrecasas P., "J. Biol. Chem.", 3059-3065, 1970) since there are ready-to-use lyophilized commercial preparations of Sepharose already activated.

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

a) Preparation of Sepharose gel (CNBr Sepharose 4B)

To swell the gel and remove the bactericidal agents present in the lyophilized preparation, the desired quantity of CNBr-Sepharose is washed on a sintered glass filter with a HCl ImM solution for approximately V * h, knowing that 1 g of dry product gives about 3.5 ml of gel. 200 ml of HCl ImM / g dry weight of CNBr-Sepharose are used for this purpose. The gel thus formed is immediately coupled to Bleu Dextran 2000.

b) Coupling of Sepharose gel with Bleu Dextran 2000

The quantity of Bleu Dextran necessary for coupling is determined, considering that 1 g of dry weight of Sepharose is capable of fixing 80 to 100 mg of Bleu Dextran.

This quantity is dissolved in an alkaline buffer with a pH of the order of 8 to 10, in particular a 0.4M carbonate buffer of pH 10 (20 mg of Blue Dextran 2000 are easily dissolved in 1 ml of carbonate buffer).

The Blue Dextran solution is poured onto the Sepharose gel, rinsed beforehand with 3 or 4 times its volume of 0.4M carbonate buffer,

pH 10. The mixture of gel and Blue Dextran is poured into a dark glass bottle and is subjected to slight stirring, for example with a rotating wheel, for approximately 18 to 24 h, 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 sintered glass filter with an excess of 0.4M carbonate buffer, pH 10, in order to remove the unfixed Bleu Dextran, until the buffer remains colorless (optical density less than or equal to 0.02).

To remove the active sites which would not have been fixed with Bleu Dextran, the coupling product is then brought into contact with a solution of alkanolamine, in particular 1M ethanolamine at pH 8, for approximately 2 h, at a temperature + 4 ° C. For this operation, the coupling product is preferably transferred from the bottle to another container.

To make the gel resistant to pH, molarity or other variations, it is then subjected to three alternating rinsing cycles with an acid solution constituted by an 0.1 M acetate buffer of pH 4.0 added with 1 M NaCl, and a aslcaline solution consisting of a 0.1 M borate buffer, pH 8.5, added with 1 M NaCl.

After these rinsing cycles, the gel is equilibrated with a 10 mM tris-HCl buffer, pH 7.5, added with a bactericidal and antiseptic agent, in particular with 0.02% sodium azide.

2. Preparation of the affinity chromatography column

The Blue Dextran Sepharose is poured into a chromatography column of appropriate dimensions (it is estimated that 1 ml of gel is capable of fixing approximately 8 million international units of interferon).

Before the very first use of the column, rinsing is carried out with 20 times the dead volume of the 10 mM tris-HCl buffer, pH 7.5, supplemented with 0.02% sodium azide.

To eliminate the molecules of Blue Dextran which would tend to detach, the elution buffer is passed through, consisting of 10 mM tris-HCl, pH 7.5, added with 1M NaCl, until the optical density at 254 nm remains at 0. (In the case of large columns, it is recommended to carry out several successive rinsing cycles with sorted buffer with and without NaCl to reinforce the adaptation of the columns to variations in pH, molarity, or others.)

A column of Blue Dextran Sepharose can be used for several months and for many cycles of purification of interferon, provided that it is not contaminated with bacteria. It should therefore be kept under sodium hydroxide and cold, at around + 4 ° C outside of periods of use.

However, since sodium hydroxide is toxic to cells, before fixing the interferon, at least three dead volumes of azide-free tris-HCl buffer must be passed to remove any trace of antiseptic.

Example 5:

Purification of a preparation of mouse interferon, on the one hand, and of a preparation of human interferon, on the other hand, by chromatography on a column of Blue Dextran Sepharose according to Example 4, using of polynucleotides as desorption agents.

a) Interferon preparations

As regards the preparation of mouse interferon, it consists of a tissue culture supernatant (C 243 cells) induced to produce interferon by the Newcastle disease virus. After inactivation of the virus at pH 2, the supernatant is dialyzed against 10 mM tris-HCl, pH 7.5, for approximately 24 h at a temperature of approximately 4 ° C.

The interferon titer, the protein content of the preparation and the specific activity correspond to those of the preparation used in Example 2.

As for the preparation of human interferon to be purified, it consists of a supernatant from a culture of fibroblasts

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such as that of Example 3. This preparation is used as it is without prior dialysis.

b) Adsorption phase

Passing from 3 to 8 ml of the preparation of mouse interferon mentioned above (i.e. 3.8 x 10® to 1 x 107 IU of interferon) on a column of Blue Dextran Sepharose from 1.5 to 4 ml according to experience, at a rate of 0.5 ml / min. In the case of the purification of human interferon, 7 ml of the preparation described above, titrated 2560 interferon units per milliliter, are deposited on the column, which corresponds to 17 920 interferon units in total.

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, according to Examples 2 and 3, the desorption of the interferon is carried out using polynucleotides using the following procedure:

An eluent comprising 0.1 g of polynucleotides per milliliter of a 10 mM tris-HCl buffer of pH 7.5 is passed through the column, at a rate of 0.5 ml / min. The total volume of eluent used corresponds to two volumes of gel (we also used, in the case of poly-U, an amount corresponding to 4 volumes of gel). As polynucleotides, synthetic polyribonucleotides are used, namely poly-G, poly-I and poly-U such as those marketed by Choay laboratories.

Polynucleotides of natural origin are also used, such as t-RNA (total fraction) from brewer's yeast, those from Escherichia coli and those from mouse C 243 cells. These products are sold under their chemical name, polynosinic acid being sold for example under the name poly-I.

After the desorption phase described above, a second washing is carried out, and the rest of the products with interferon activity still fixed on the column are eluted using 1M NaCl in 10 mM tris buffer, pH 7.5.

Fractions are each collected corresponding to one third of the total volume of the Blue Dextran Sepharose (BDS) column. In the table given below, the percentages of interferon collected are reported with two total volumes of eluent according to the polynucleotide used. (The values have been corrected so that the percentage of interferon desorbed by the polynucleotides and the percentage of interferon subsequently desorbed by the hypermolar buffer are equal to 100.)

Percentage of desorption obtained according to the type of polynucleotides used

Percentage

Desorbed interferon polynucleotide type

from the BDS column

I. Mouse interferon

poly-G

17

poly-I

86

poly-U

43 (if we continue the elution,

we get 73%)

T-RNA (total fraction

extracted from yeast

beer)

90

T-RNA (total fraction

extracted from £. coli)

23

T-RNA (total fraction

extracted from C cells 243)

30

II. Human interferon

poly-I

94

In another series of experiments, the degree of purification obtained by desorption by polynucleotides of interferon fixed on a column of Blue Dextran Sepharose is studied.

The results obtained with preparations of mouse interferon and human interferon are reported in Examples 6 and 7, respectively.

Example 6:

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

The crude preparation interferon titer is 2.6 x 10 6 IU / ml. Its protein content is 100 ng / ml and its specific activity of 2.6 x 107 interferon units / mg of proteins.

130 ml of such a preparation is passed (which therefore corresponds to a total of 3.3 x 108 units of interferon) over a column of Blue Dextran Sepharose according to Example 4, having a total volume of 9 ml.

The adsorption, rinsing and washing phases are carried out as described above. Desorption is carried out according to the method described in Example 5, using 18 ml of an elution solution containing, as desorbing agent, poly-I at the rate of 100 μl / ml of buffer.

Interferon is collected in two fractions a and b of 7 and 5 ml respectively. Their interferon titer is 4.1 x 107 and 5.1 x 106.

In total, 3.16 × 10 5 IU is collected, which corresponds to a recovery percentage of 96%.

In fraction a, the presence of 19 y of proteins is observed, which gives a specific activity of 2.1 × 10 9.

The degree of purification is therefore 80 (specific activity at the start / specific activity at the end: 2.1 × 10 9 / 2.6 × 10 7).

Example 7:

The preparation of human interferon to be purified consists of a supernatant of a culture of human fibroblasts induced by the poly-IC to produce interferon.

The title of this preparation is 2560 IU / ml, its protein content is 410 ng / ml and the specific activity of 6.2 x 103.

7 ml of this preparation (which corresponds to 17,920 units) are passed over a 5 ml column of Blue Dextran Sepharose.

The adsorption, rinsing and washing phases are carried out according to the procedures described in the preceding examples.

Desorption of products with interferon activity is carried out with 10 ml of poly-I in solution in a 10 mM tris-HCl buffer, pH 7.5. at a rate of 100 (i / ml.

The products with interferon activity are collected in 5 fractions of 2 ml. The title of each of these fractions is reported below.

Fraction

Headline

1

650

2

5120

3

1280 Ì

x 2 = in total 16,960 units

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1280 V

5

160 J

The recovery percentage is therefore:

17920

Fraction 2 contains 10 Jig / ml of protein. The specific activity of this fraction is 5.1 x 105.

The degree of purification is therefore:

82 times (5.1 x 105 / 6.2 x 103).

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Example 8:

As indicated above, the polynucleotides coupled to the products with interferon activity give them protection of great interest with respect to thermal denaturation. To illustrate this effect, curves are shown in the appendix representing, as a function of the incubation time at 60 ° C. of the coupling products in question, the percentage of activity of the latter.

In fig. 2, the results obtained from the coupling products, on the one hand, of mouse interferon of C 243 cells induced to produce interferon by the Newcastle disease virus with, on the other hand, have been indicated. poly-U, poly-I, poly-C, poly-I / poly-C, poly-A compared to control interferon preparations not containing polynucleotides. The concentration of polynucleotides is 10 ng / ml of preparation.

Fig. 3 represents the results obtained, by operating with products of coupling of interferon with t-RNA (valine); Ftryptophone t-RNA); T-RNA (total fraction of brewer's yeast); T-RNA (tyrosine); T-RNA (total fraction of wheat germ), and t-RNA (lysine).

According to these tests, the antiviral activity of the interferon preparations is measured by a micro test assay according to the following method: appropriate dilutions of the samples to be measured are made in plates sold by Falcon for microdoses (microtiter plates - Falcon Plastic Company). To each well of the plate, 100 μl of a suspension of cells derived from the same species as the interferon to be assayed is then added, and the plates are left to incubate for 18 h at 37 ° C. in a CO 2 incubator. The revealing virus is then added (in the examples cited, the vesicular stomatitis virus was used as 100 infectious units per scoop).

The plates are then returned to the CO 2 incubator and, 24 h later, the cell destruction caused by the virus is read under a microscope.

The titer of interferon corresponds to the reverse of the dilution at which 50% of cells, at least, are protected against the destructive effect of the virus. Thus, if at the dilution of 1/20000 we have 50% of the cells still intact, the titer is 20000.

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

In the test, the results of which are reported in FIG. 2, the crude preparation of mouse interferon was dialyzed in tris-

LOmM HCl, pH 7.5. Polynucleotides were incorporated therein at a concentration of 10 μg / ml. 0.3 ml aliquots (ie 3.9 × 10 5 units of interferon and 3 μg of polynucleotide) were incubated in a water bath at 60 ° C. At the times indicated in the figure, samples were taken. 20 days 1. The latter were diluted 100 times in culture medium containing 3% calf serum and immediately cooled in an ice bath. Interferon titrations were performed at the end of the incubation.

In the tests, the results of which are reported in FIG. 2, one proceeds under the same conditions as those indicated above for the study of thermal denaturation in the presence of polynucleotides but, instead of the latter, t-RNAs are used.

Examination of these curves shows that the products with interferon activity coupled with the polynucleotides retain significant therapeutic activity, even after more than 30 min of heat treatment at 60 ° C., while this activity decreases rapidly in the controls; thanks to this protective effect of the polynucleotides with respect to products with interferon activity, it is possible to store the interferon preparations without problems and to subject them, during various tests, to temperature variations without it results from denaturation of their properties.

Example 9:

Purification of a mouse interferon preparation by chromatography on a column of Blue Dextran Sepharose using t-RNA as desorption agents.

The preparation of mouse interferon to be purified is similar to that used in Example 5. By proceeding as described in this Example 5, 1 × 107 is deposited on a column of Blue Dextran Sepharose, after dialysis of the preparation interferon units. The column is rinsed with tris buffer (4 total volumes), then the interferon is desorbed by depositing on the column 3 to 4 ml of t-RNA, at a concentration of 100 ng / ml, in the above 10 mM tris-HCl buffer.

A second rinsing phase is carried out with the buffer in question then, using a 10 mM tris-HCl buffer, pH 7.5, added with 1M NaCl, the desorption of the products with remaining interferon activity is carried out.

The percentages of desorption of mouse interferon using total fractions of brewer's yeast t-RNA, E. coli, mouse C 243 cells and rat liver are 91.23, 54 and 10, respectively.

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1 drawing sheet

Claims (23)

633716 2 1. Method for selectively separating, by eliminating contaminating substances, products with interferon activity from preparations containing them, characterized in that these preparations are brought into contact with a phase comprising at least one polynucleotide, playing in a specific manner the role of ligand vis-à-vis products with interferon activity. 2. Method according to claim 1, characterized in that the polynucleotide derivative used consists of at least one polyribonucleotide and / or a polydeoxyribonucleotide of synthetic or natural origin. 3. Method according to claim 2, characterized in that one implements a polyribonucleotide comprising from 10 to 300 nucleotide units. 4. Method according to claim 3, characterized in that said polyribonucleotide is chosen from the group comprising polyribo-inosinic acid, polyriboadenosine acid, polyribo-uridylic acid and polyriboguanilic acid. 5. Method according to claim 3, characterized in that said polyribonucleotide consists of a transfer ribonucleic acid. 6. Method according to claim 5, characterized in that it is the total fraction of transfer ribonucleic acids from bacteria, yeast or mammalian organs. 7. Method according to claim 6, characterized in that the above-mentioned transfer ribonucleic acids come from bacteria of the genus Escherichia coli, yeasts from the genus of brewer's yeast or from cells of animal or human origin, in particular cells themselves who produced the interferon that we want to purify. 8. Method according to one of claims 1 to 7, characterized in that a preparation to be purified of products with interferon activity is brought into contact with an adsorbent balanced by a buffer whose salt concentration is isomolar or preferably hypomolar compared to that of blood, said adsorbent comprising, on the one hand, a solid gel-forming support, the mesh size of which is such that it allows the passage of macromolecules and, on the other hand, a polynucleotide, fixed on said support, playing the role of ligand with respect to the products which it is desired to separate selectively, the quantity of adsorbent used and the duration of contact between the ligand and the preparation to be purified being sufficient to allow fixation desired products with interferon activity. 9. Method according to claim 8, characterized in that, to constitute the solid support of the gel-forming adsorbent, use is made of a polysaccharide such as agarose or a derivative thereof, in particular a modified agarose in which the Polysaccharide chains are crosslinked in a three-dimensional network, for example a polyacrylamide or an acrylic resin. 10. Method according to one of claims 8 or 9, characterized in that the salt concentration of the buffer used to balance the adsorbent is less than or equal to 0.15 M. 11. Method according to one of claims 8 to 10, for obtaining purified preparations having an interferon activity, characterized by an additional step according to which the 15 desorption of products with interferon activity fixed using a buffer whose salt concentration is hypermolar compared to that of blood. 12. Method according to claim 11, characterized in that the salt concentration of the elution buffer is at least 0.5 M. 13. Method according to one of claims 1 to 7, characterized in that a preparation comprising a complex comprising products with interferon activity attached to a first ligand is brought into contact with a phase comprising at least one polynucleotide, capable to compete with the first ligand for the fixation of the products with interferon activity and to detach the latter, the preparations and phases in question being balanced using a buffer whose salt concentration is isomolar or hypomolar by compared with that of blood, the quantity of polynucleotides in the abovementioned phase and the duration of contact between the polynucleotides and the products with interferon activity fixed on the first ligand being sufficient to allow the desorption of the products with interferon activity. 14. Method according to claim 13, characterized in that the preparation comprising products with interferon activity to be brought into contact with a phase comprising at least one polynucleotide comprises a solid support forming a gel, the mesh size of which is such that it allows the passage of macromolecules and, on the other hand, a compound fixed on said support, playing the role of ligand with respect to the products which it is desired to selectively separate and which has the polycyclic structure of the blue chromophore of formula S020Na SOgONa whose configuration allows selective retention of interferon. 15. The method of claim 14, characterized in that the ligand coupled to the solid support consists of the blue chromophore or the corresponding product in which the group - SO20Na under the phenyl radical at the chain end of the formula given in claim 14 is replaced by a hydrogen atom. 16. The method of claim 15, characterized in that said chromophore is linked to a Polysaccharide, which has a molecular weight of about 2,000,000. 17. The method of claim 16, characterized in that said chromophore linked to the Polysaccharide is fixed on a support 65 consisting of agarose. 18. Method according to one of claims 14 to 17, characterized in that the salt concentration of the buffer used to balance the adsorbent is less than or equal to 0.5 M. 3 633716 19. Method according to one of claims 14 to 18, characterized in that the phase comprising at least one polynucleotide comprises these polynucleotides in solution in a buffer whose salt concentration is hypomolar with respect to that of blood. 20. The method of claim 19, characterized in that the above polyribonucleotides are used in solution in a buffer at a rate of 10 to 100 ng / ml, preferably from 30 to 50 ng / ml. 21. Purified preparations with interferon activity obtained by implementing the method according to claim 1. 22. Preparations according to claim 21, obtained by implementing the method according to one of claims 2 to 12. 23. Preparations according to claim 21, composed of products with interferon activity fixed on nucleotides and obtained by implementing the method according to one of claims 13 to 20. 24. Use of the preparations according to claim 21 as active principle of medicaments.