IE61444B1 - Process for preparing and purifying interferon - Google Patents

Abstract

The process yields a highly pure, non-immunogenic, homogeneous alpha -interferon which has antiviral and immunoregulatory activity.

Description

PROCESS FOR PREPARING AND PURIFYING INTERFERON _ 1 _ The present invention relates to a process ® for preparing a very pure, non-inununogenic, homogeneous a-interferon having antiviral and immunoregulatory * activity, the protein itself and the use thereof.

Interferons are proteins naturally occurring in the body and which have been detected in a great variety of species. Their inherent antiviral and immunoregulatory properties indicated at an early stage that they might be suitable for a wide variety of 'applications. Tests have shown that ther® are different classes of interferon., In addition to c-., 6 and b interferons,a? -interferon has recently been discovered and its structure clarified.

The high expectations placed on the interferons as an effective agent against viral diseases and cancer have already led fco trials with interferon preparations obtained from natural material, but serious side effects occurred- The preparations used in these trials, even after laborious purification, contained complex mixtures of different interferons and, in many cases, other proteins. The reason for this is that some of the interferons have subtypes differing from one another to a greater or lesser extent; thus, for example, more than 20 different types of a-interferon are known.

Only by producing interferons by genetic engineering has it been possible to conduct trials with pure types of interferon preparations. These include the recombinant interferons used in the clinical trials (also known as c A). The purification - of any human proteins produced by microorganisms is of critical importance. Any contamination originating from the host organism would lead to immune defence reactions if the product were to be used in humans and these could be life-threatening. The removal of contaminants of this kind is now possible and extremely sensitive analytical methods can detect endotoxins in very 614 4 4 tiny concentrations. In the field of interferon research methods of purification have been developed % with which interferon preparations containing virtually no endotoxins can be obtained. Mention may be * made, for example, of th® work of Staehelin et al. J. Biol. Chem. 256, @750 (1981).

All rec. a-interferons used in clinical trials are virtually free from endotoxins and it was therefore surprising that side effects which were severe enough to cause the interferon treatment to be discontinued should occur. Sven some rec» e-interferons were found to be immunogenic and antibodies against interferon had been stimulated. (Quesada et al.

J. Natl. Cancer Inst. 70, No. 6, 1041-1046 (1983); Protzman et al. J. Immunol. Methods 75, 317 - 323 (1984)).

These antibodies may lead to serious effects if they influence the action of the interferon.

This is because, in this case, they act not only on the rec. α-interferon but, since the rec. einterferon Is identical to the body’s own interferon,.· on the body’s own interferon as well.

The disastrous aspect of this Is that these antibodies go on acting even after the interferon treatment has ended. They may cause a deterioration in the course of the disease, weaken the body’s own defences against virus infections and thus make the organism even more susceptible to other infections.

These effects have already been confirmed in tests on animals. Therefore, with a view to maximum safety of drug treatment, it is essential that the rec. a., interferon must be of a pure type, virtually free from endotoxins and, not least, non-immunogen ic.

One object of this invention was therefore to develop a process for preparing a non-immunogenic rec.. α-interferon with antiviral and immunoregulatory activities.

The cause of the immunogenicity of the abovementioned a interferons is not known. The only fact that can be ruled out is that endotoxic contaminants are responsible.

The preparations used for trials differ primarily by slight variations in their amino acid sequences.

Preparation I: Preparation II: Preparation III: Amino acid 23 Lysine Arginine Arginine Amino acid 34 Sistidine Histidine Arginine η.

Apart from the structural differences in the primary structure of the proteins used in clinical trials, it is known that the a-interferons produced by genetic engineering always consist of a mixture of monomeric, shortened-molecular, reduced and oligomeric forms of interferon (see for example SPA 108 585, 110 302 and 118 808). Some of these forms show the same activities in vitro but others show reduced activities and some are reputed to have immunogenic properties (see EPA 108 585 and 110 302).

These patent applications describe processes for separating these forms of interferon...

EPA 108 585 describes a process for separating a ’slow moving monomer" and oligomers wherein the interferon probe is incubated for some time at a temperature of 28~40°C at a pH of 3 to 5.

EPA 110 302 describes a process wherein fche monomer is formed from the oligomers by reduction with a redox system.

Finally, in EPA 118 808, recombinant e-interferon is purified with the aid of metal chelate resins from the oligomeric forms. _ 4 The interferons obtained by these methods are supposed to contain monomeric interferon in « virtually quantitative form; however, there are no tests of immunogenicity.

Our own detailed analytical investigations have shown that α-interferons prepared by recombinant methods are a mixture of different forms of interferon in fluctuating concentrations. These include oligomers„ tetramers, trimers and dimers of interferon,t methionine interferon, reduced forms and fragments of interferon and, surprisingly, various monomeric forms of interferon as well.

The oligomers are e-interferons with a molecular weight >· 70,000, the reduced c-interferons are the protein with free SH groups and the methionine interferon is an e-interferon which additionally carries a methionine at the N-terminal end (caused by the microbiological method of preparation of the e-interferon). Analysis has shown that the various monomeric forms are different S-S isomers of e-interferon.

In order to distinguish between these isomers linguistically, the term non-native monomer will be used hereinafter for the isomers of the predominantly occurring e-interferon monomer and the latter will be referred to as native monomeric e-interferon.

However, this should not exclude the possibility that the non-native monomers may also exist in Knatural" material.

In an Ξ. coli fermentation mixture for preparing c2 interferon, for example, 7 different interferon components could be detected (K 1 - K 7). Analysis showed that these were oligomers, dimers and trimers, methionine interferon, reduced Interferons and an S-S Isomer of native monomeric ©2 interferon which had a disulphide bridge between the amino acids at positions 1 and 98 and 29 and 138 (see also Wetzel et al. J. Interferon Res., Vol. 1, Mo. 3, 381 - 391 (1981).

As already stated, the cause of the immunogenicity of rec. a -interferon is not known. However, it is also obvious that all the forms of a -interferon which differ from the body’s own interferon have an immunogenic activity. These forms also include the shortened molecules, the dimers and oligomers and also the non-active monomers which contain differently linked disulphide bridges, While the causes of immunogenicity remain unclear, conditions which might promote the formation of these forms with their unknown effects should not be used in processes for preparing rec, a-interferons This means that even the purification of interferon must be carried out under the mildest possible conditions which would not endanger nativity. Elevated temperature and reducing agents are not among these conditions.

Obviously, efforts must be made during all steps of the purification to ensure that no foreign substances are introduced, for example as a result of the use of metal chelate resins or similar Problematic reagents.

According to one feature of the present invention there is provided a process for the preparation of a recombinant interferon characterised in that the host organism containing the interferon gene is cultivated under conventional conditions, after a growth period in 25which not more than 20% methionine interferon is formed the cells are killed off and harvested, the expressed interferon is removed in conventional manner, the cell debris is removed in a slightly alkaline medium, the interferon is concentrated and subjected to preliminary purfication by tandem chromatography, the eluate is adjusted to pH 4.0-4.8 to remove any impurities, the interferon is finally purified by chromatography on a cation exchanger column with a volatile buffer as eluant at pH levels from 4-0 to 5.0 and is then lyophilised.

The process according to the invention is suitable for the preparAtlon aNd purification of interferons* especially a-interferons* from different species* such as,, for example* human or animal ©-interferons. The host organism used for the preparation may be a prokaryote or eukaryote* such as. e.g. Ξ. coli or Saccharomyces cerevisiae* preferably E. coli. The conditions of cultivation for the various host organisms are well known to those skilled in the art.

Surprisingly* it has been found that the growth time not only affects the yield of ©-interferon but is also a crucial factor in determining the composition of the interferon mixture. Thus* if S. coli is used* the composition of the interferon mixture changes with regard to the quantity of methionine interferon depending on the duration of growth.

Advantageously* therefore* the fermentation mixture is checked at short intervals for the formation of ©-interferon derivatives produced by the host organism* as an indicator of the best growth time. Methionine interferon may be used as an indicator of this kind. Therefore* by discontinuing the process at the. appropriate time* for example after the formation of less than 20%* preferably less than 5%* more particularly less than. 1% of methionine interferon* a particularly pure interferon is obtained with ideal prerequisites for the subsequent purification process according to the invention.

The process is particularly suitable for the preparation of acid-stable α-interferon» For example* the process according to SPA-173924 wherein the cells are disrupted in a homogeniser at a pH of 2 may be used» The majority of the impurities can surprisingly be removed by tandem chromatography* i»e» with successive chromatographic stages using different -7adsorption agents with suitable washing and eluting solutions. Preferably, the tandem chromatography consists of a preliminary cellulose column and affinity chromatography and the substance to be purified is washed through both columns with a suitable washing S solution and the a-interferon is subsequently eluted from the affinity column with a suitable eluant. It is particularly preferred to use a DE-52 cel lulose mth a monoclonal .anti-, interferon IgG-antibodys such as/for example the EBI 1 antibody described in DE-OS 33 06 069coupled to a carrier such as Sepharose.

A TRIS/NaCl buffer pH 7.5 has proved suitable a§ a washing solutions but is is also possible to use washing solutions which do not affect the bidding of the interferon to the antibody but wash out the contaminants and leave those constituents which have a negative effect on the properties of the antibody column bound to the pre! imi nary column.

A suitable eluant for interferon is, for example, a buffer solution consisting of 0.1 ¢4 citric acid in 25% ethyleneglycol, but other eluants having similar properties are also suitable. In general, the eluant must be matched to the particular interferon which is to be purified.

Surprisingly, some of the impurities in the "tandem eluate" could be removed by buffering the pH value, preferably to pH 4.0 -· 4.8, more particularly pH 4.5. The pH value should be selected so that there is as little monomeric e-interferon as possible in the precipitate.

Final purification of the interferon was achieved by chromatography using a cation exchanger, preferably a MONO-S, type HR 10/10 (Messrs. Pharmacia) cation exchanger. A flat graduated gradient with a volatile buffer such as an ammonium acetate buffer in which the pH was kept constant and the concentration was varied (concentration gradient) was used to elute the highly purified β-interferon. It would be equally possible to keep the concentreeion constant and vary the pH (pH gradient) . The crucial point is that the eluant should be capable of removing any interferon contaminants, particularly S-S isomers of the main monomer which occurs. A linear concentration gradient of an ammonium acetate buffer produced from 0,1 to 1.0 fcb preferably 0.1 to 0,5 " ammonium acetate in a pH range of from 4.0 to 5,.0,, preferably pH 4.5/ is particularly suitable for this purpose.

This buffer can moreover be removed by lyophilisation so that the highly purified c-xnterferons can be obtained for the first time in solid form’ free from buffer salts and precipitation agents.

The process according to the invention is particularly suitable for the preparation of cinterferons of various species which do not stimulate any antibodies when administered to the corresponding species.

The process according to the Invention has proved particularly advantageous in the preparation of a recombinant^ homogeneousr pure a-interferon which contains 0 to 5% methionine and which is free from reduced forms and fragments of interferon3 which contains 0 to 0.2% oligomer and 0 to 2% dimer/trimer/tetramer and in which 90 to 100% of the monomer contents-consists of the a-interferon with disulphide bridges between the cysteines at positions 1 arid 98 and 29 and 138.

The process according to the invention may be used particularly advantageously for the preparation of ah a-interferon as described above wherein the host organism contains the gene which codes for human a-interferon according to the amino acid sequence Cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu Me t Leu Leu Ala Gl n Met Arg Arg ile -Se r Leu phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pr o Gin Glu Glu Phe Gly Asr. Gin Phe Gin Lys Ala Glu Thr lie Pro Va 1 Leu His Glu Me c lie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Se r Ser Ala Ala Tr p Asp Glu Thr Leu Leu Asp Lys Phe Tyr Th r Glu Leu Tv «ί ** Gin Gin Leu Asn Asp Leu Glu Ala Cys val lie Gin Gly Val Gly Val Thr Glu Th r Pro Leu Met Lys Gl u Asp Se r I A € Leu Ale Va 1 A£ Ci Lvs Tyr Phe Glirs Arg J X The Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Cys Ale ¢=¾ β & Glu vel Val Ar g Ala Glu n -» A *" Me t Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu which contains 0 to 5% methionine interferon which is free from 15 reduced forms and fragments of «-interferon, which contains 0 - 0.2% oligomer and 0 to 2% dimer/trimer/tetramer and wherein the ;·.« -interferon contains disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

This invention also relates to recombinant Depending on the choice of host organism and the nature of the fermentation conditions, a-interferons prepared by the recombinant method may contain, in addition to methionine interferon, di-, tri-e tetra- and oligomers, reduced forms and fragments and SS isomers of the predominantly occurring monomeric «-interferon in fluctuating quantities which can be suppressed or eliminated for the first time using the process according to the invention.

The exceptionally pure recombinant α-interferon which may be prepared by the process of our invention is believed to be novel per se. since we know of no prior art process capable of yielding an equivalent product. A further feature of the present invention therefore provides recombinant α-interferons in substantially pure homogeneous form which contain less than 20%, preferably less than 5%, more particularly less than 1% methionine interferon, which are substantially free from reduced forms and fragments of a-interferon, which contain less than 0,2% oligomer and less than 2% dimer/trimer/tetramer, preferably no oligomers, tetramers, trimers or dimers, and which preferably contain more than 90% of the native monomer and preferably are substantially entirely free from S-S isomers of native monomeric e-interferon.

A still further feature of the present invention provides recombinant e-interferons of various species described which, when administered to particular species, do not stimulate any antibodies.

The interferons of our invention are preferably in solid form.

A recombinant human c-interferon with the properties described above, preferably a recombinant human α-interferon according to the amino acid sequence Cys Asp Leu Pro Gin Thr His Ser Leu Gly ter Arg Arg Thr Le u Mer Leu Leu Ala Gin Mer Arg Arg lie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu The ile Pro Va 1 Leu His Glu Met Tie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Th r Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys val lie Gin Gly val Gly val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser lie Leu Ala val Ar c Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Sec Pro Cys Ala Tr p Glu Val Val Arg Ala Glu lie Met Ar g Se r Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu. is preferred.

A recombinant, non-immunogenic, solid human c-interferon which corresponds to the amino acid sequence - η Cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Ar g Arg Xie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin Gl u Glu Phe Gly Asn Cln Phe Gin Lys Ala Glu Thr Xie Pro Val Leu His Glu Met Xie Cln Gin Xie Phe Asn Leu Phe Sec Thr Lys Asp Ser Ser Ala Ala Tr p Asp Glu Thr Leu Leu ASO Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Xie Gin Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Xie Leu Ala Val Ar g Lys Tyr Phe Gin Arg Xie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Tr p Glu Val Val Arg Ala Glu Xie Met Ar g Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu and is present in pure homogeneous form with 0 to 5% methionine interferon» which is free from reduced forms and fragments of α-interferon» with 0 to 0-2% oligomer and 0 to 2% dimer/trimer/tetramer and wherein 90 to 100% of the monomer contents consists of the α-interferon with disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138» is particularly preferred.

The process according to the invention enables impurities and . interferon contaminants to be removed 5 under very mild conditions. This will be explained more fully using the example of the c^Arg interferon according to the amino acid sequence Cys Asp Leu Pro Gin Thr His Ser Leu Giv Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Me t Arg Arg Tie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg ASp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Gl u Thr Tie Pre Va 1 Leu His Glu Met Tie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys val Tie Gin Gly Val Gly Val Th r Glu Th r Pro Leu Met Lys Gl u Asp Ser Tie Leu Ala Val Ax g Lys Tyr Phe Gin Arg Tie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Tr p Glu Va 1 Val Ar g Ala Gl u Tie Met Ar g Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Ar g Ser Lys Glu but other e-interferons may also be prepared and purified using the process according to the invention, if necessary with slight non-inventive modifications.

The acid-precipitated deep frozen biomass was thawed and taken up in in 1% acetic acid.

This and all subsequent operations were carried out at about 5°C, The protein was extracted from the cells, as described in. detail in EPA.-173924, by breaking up the bacterial cells in a homogeniser, adding a precipitation adjuvant such as polyethyleneimine ΡΞΙ-600 in a concentration ranging from 0.1 to 0.25%, adjusting the pH to 7.5 - 10.0 with the aid of NaOH and stirring the suspension for several hours. The pH was then adjusted to 7.5, the crude extract was clarified under mild conditions and samples were taken for determining the protein and for the Interferon test.

In spite of the known vulnerability of polypeptides such as interferon to the shearing forces which occur under mechanical influences (Proc. Soc. Exp.

Biol. Med. 146, 249 - 253 (1974)) surprisingly high yields of crude interferon could be achieved using this process under these pH conditions.

Examination of different fermentation batches showed that the composition of the mixture varied as a function of the fermentation conditions.

In particular the proportion of component 1,, namely methionine ©-interferon* which is a verydifficult component to remove* varied with the duration of fermentations methionine ©-interferon was only formed after 8-9 hours of fermentation.

By stopping fermentation in good time* therefore* it was possible to obtain interferon preparations which contained no methionine interferon.

Solid ammonium sulphate was added to the clarified crude solution up to 65% saturation.

After all the ammonium sulphate had dissolved the mixture was kept cool overnight* the precipitate formed was separated off and stored at. -2Q®C until required.

Samples for the interferon test were again taken from the clear supernatant in order to monitor the precipitation of the Interferon. Not more than 5% of the interferon should remain in the supernatant.

The ammonium sulphate pellet was dissolved in 0.01 M NaCl, the pH was adjusted to 7.5 with NaOH and the solution was stirred for 2 hours.

The insoluble fraction was removed and possibly extracted once more with 0.01 M NaCl.

The combined clear solutions were dialysed with 0.01 M NaCl using a sterile* pyrogen-free dialysis cartridge. The osmolarity of the interferon solution should be about 390 - 430 mOsmol/1 after running through two to three times. Aliquots for the interferon test were taken from the clarified solution.

’’Tandem chromatography" was used for the further purifications a combination of a cellulose preliminary column and subsequent affinity chromatography with highly specific monoclonal antibodies. The preliminary column, an ion exchanger columnf was used to keep any difficultly soluble sample constituents away from the antibody column. For the preliminary column, DE-52 cellulose (Messrs. Whatman) was stirred thoroughly with TRIS/NaCl buffer, pH 7.5, and introduced into a chromatography column. The adsorbent was washed with the buffer until the eluate showed. no further changes in pH and osmolarity. ' For the preliminary column, 0.5 - 1.0 g of DE 52 cellulose, 0.025 M TRIS/BC1 + 0.2 M NaCl were used per gram of biomass; it was freshly prepared for each purification.

For the antibody column, purified monoclonal anti-interferon-IgG obtained from mouse ascites was coupled to BrCN activated Sepharose 43 (Messrs. Pharmacia) as the carrier. The finished column material was stored in phosphate-buffered saline solution (PBS) with sodium azide in a cold store.

Before being used for the first time or after a lengthy storage period, the antibody column was washed with 0.1 M citric acid in 25% ethyleneglycol in order to eliminate any soluble components and then washed with PBS until neutral. A column volume of from 0.2 - 1.0 ml was required for each gram of biomass in the antibody column; this column could be used several times. The dialysed interferon solution was first pumped through both columns (preliminary column and antibody column) and the eluate was monitored by measuring the extinction at 280 nm. After the Interferon solution had been applied, it was washed with TRIS/NaCl buffer, pH 7.5, until the quantity of protein In the eluate had fallen to 1/20th of the plateau value. In order to check that the interferon had bound to the antibody column., the eluate was tested for its interferon content. The antibody column was then separated from the preliminary column and washed on its own with TRIS/NaCl buffer, pH 7.5, until no further protein could be detected in the eluate.

Flution of the interferon bound to the antibody was carried out using 0.1 M citric acid in 25% aqueous ethyleneglycol, and the extinction of the eluate at 280 nm was monitored» The protein peak containing the interferon was collected. The interferon pool was stored at ~20®C until final purification.

An interferon test, protein determination and reverse phase HPLC analysis showed that 60 - 90% pure IFN-c was obtained after this purification. In addition to oligomeric forms this interferon pool contained reduced forms with free SH groups, dimers, trimers, tetraraers and the non-native monomer. These components are all biologically and immunologically characterisable as XFN. Surprisingly, some of these components could be removed by precipitation at pH 4.5 (with ammonia). The fractions of the components with reduced sulphur bridges, i.e. the forms with free SH groups (components 5 and 6), in particular, were thus reduced. Analysis of the precipitate shows that only small amounts of the monomeric interferon were carried down.

Final purification was carried out using an FPLC apparatus made by Messrs. Pharmacia with a MONO-S column. Type HR 10/10 (Messrs. Pharmacia) cation exchanger, which could be charged with up to 60 mg of protein.

This column material constitutes a high performance ion exchanger with an exceptional separating action and the great advantage that final purification takes only a few hours, in spite of the relatively large quantity of protein to be purified, the buffer solutions could be used after being filtered sterile and the work could be done at ambient temperature.

The clear supernatant obtained after precipitation was applied to the column. The buffer used in the FPLC separation was particularly important.

It had to be capable of eluting the interferon components so chat they could be clearly distinguished and then it had to be completely removable. The ammonium acetate buffer with which the interferon was eluted through a series of gradients had these properties. Interferon was eluted as a sharp peak with a weak shoulder. Both the *shoulder" fraction (K 3) and also the fractions eluted subsequently (K 5 - X 7) were separated from the main peak of the pure interferon. The peak of pure interferon was collected and aliquots were taken from It for the HPLC analysis* SDS gel electrophoresis* protein determination* interferon testing and endotoxin determination.

By this chromatography* virtually all the components were separated from the main peak and homogeneous interferon was obtained showing a monomer content of over 99% in gel permeation .HPLC. Reverse phase HPLC showed only about 1% of non-native monomer and chromatofocussing showed a proportion of 2.5% of non-native monomer.

The MONO-S column was washed before re-use with 0.5 M NAC1 + 0.1 M Na-phosphate* pH 8.0* In order to eliminate any adsorbed impurities; it was stored in 25% ethanol.

The volatile buffer could be totally removed by lyophilisation. For this* the IFM pool was transferred into autoclaved lyo-ampoules (capacity 8 ml) in batches of not more than 2 ml? this corresponded to a quantity of from 1 to about 8 mg of pure interferon per ampoule. The ampoules were then sealed with pre-washed and autoclaved lyo-stoppers and cooled to at least -20®C„ Lyophilisation was carried out at -10®C under, a vacuum of less than 1 torr.

After removal of the buffer solution the temperature was increased to 25°C and lyophilisation was continued for at least 1 hour. The vacuum was released and the stoppers were immediately pressed in. After being sealed with aluminium closures, the ampoules were then stored in the refrigerator or at -20°C.

As has been shown, careful guidance of fermentation (relatively early harvesting) together with the process according to the invention has made it possible for the first time to prepare an e-interferon which not only has a degree of purity of over 98% with regard to its interferon content but also consists of more than 95% native monomeric e-interferon with regard to its homogeneity based on the various Interferon components.

This high degree of purity and homogeneity has also made it possible for the first time to obtain Interferon in solid form free from salts and buffer constituents. It Is therefore possible for the first time to store e-interferon for months without the use of stabilisers; this has significant advantages in terms of storage, dispatch and, not least, galenic developments, over the Interferons which have hitherto always been stabilised with albumin. Even after 11 months’ storage at 4°C, no loss of contents could be detected.

The crystalline human leucocyte interferon described in EPA 83 734 consists of crystals of polyethyleneglycol as the precipitating agent with Interferon, but not a pure, homogeneous and crystalline interferon as the title would have one believe.

The e-interferon prepared according to the invention was, as is already known, dissolved by the addition of human serum albumin, filtered until sterile and transferred into vials under aseptic conditions, In suitable concentrations depending on th© particular application.

In clinical trials, the e-interferon prepared according to the invention proved to be non-immunogenic and exceptionally well tolerated.

In all, up to January 1985, 75 patients have 5 been treated with the non-immunogenic a-interferon: patients with tumour indications and 17 with viral indications.

Antibodies were not stimulated in a single patient throughout the therapy, the period of treatment being 15 or more weeks in some cases and up to 35 weeks.

The process according to the invention has made it possible for the first time to prepare a highly pure c^-interferon which is homogeneous in terras of the native monomeric interferon, solid, free from salts and buffer constituents and nonimmunogen ic„ Amino acid sequence analysis by known methods yielded the following amino acid sequences Cys Asp Leu Pro Gin Thr •i: s Ser Leu Gly Ser Ar c /-. Γ G Thr Leu Met Leu Leu Ala Gin Met Arg Arg lie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr lie pro val Leu His Glu Met Xie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Gl u Leu Tyr Gin Gin Leu Asn A£p Leu Glu Ala Cys val lie Gin Gly val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Tr p Glu Val val Arg Ala Glu lie Met Ar g Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Sec Leu Arg Sec Lys Glu With regard to the fermentation mixture, In particular, the process according to the invention can be used without restriction, within wide limits. Thus, it is also possible to use biomasses of other host organisms which give comparable IFN yields after mechanical decomposition and interferons which react in a similarly specific manner with the '331-1 monoclonal antibody, e.g. IFN-α-ρ Other Interferons with a lesser homology to interferon a. may also be purified using the method according to the invention if a corresponding highly specific monoclonal antibody is used.

Salient features of a preferred embodiment of our process are as follows: the host organism containing the interferon gene is cultivated under conventional conditions, after the conventional growth period the cells are killed off and harvested, the expressed Interferon is removed in conventional manner, the cell debris Is removed in a slightly alkaline medium, the interferon. Is concenerated and subjected to preliminary purification by tandem chromatography, the eluate is adjusted to pH 4.0-4.8 to remove any impuri ties, the Interferon Is finally purified by chromatography on a cation exchanger column with a volatile buffer as eluant and is then lyophilised.

Preferably the tandem chromatography consists of a preliminary cellulose column and affinity chromatography and the substance to be purified is washed through both columns with a suitable - ZO washing solution and the e-interferon is subsequently eluted from the affinity column with a suitable eluant.

More preferably the preliminary column is charged with DE-52 cellulose, the affinity column is charged with a monoclonal anti-interferon IgG antibody, e.g. S8I, coupled to a carrier and the substance to be purified is washed through both columns with a TRIS-NaCX buffer, about pH 7.5, and the c-interferon is subsequently eluted from the affinity column with about 0.1 M citric acid in about 25% aqueous ethyleneglycol? The eluate from the tandem chromatography is preferably adjusted to about pH 4.5 to remove impuritiesj Preferably the buffer eluant for the .final purification has a concentration gradient prepared from 0.1-1.0 ¢4 ammonium acetate buffer and/or a pH gradient in the range 4.0-5-0.

The a-interferon according to the invention may be used for the therapeutic treatment of viral diseases and tumours. For such purposes it may be formulated into pharmaceutical compositions containing one or more Inert pharmaceutical excipients and/or carriers.

The following Examples are given by way of Illustration only. The Examples should be read in conjunction with the accompanying drawings, whose significance is explained in more detail hereinafter.

Example 1 (E. coli for example Ξ. col1 K12 HB101; IFN-a2Arg; 28°C) a) 251 g of acid-precipitated biomass which had been stored at "20**C were taken up in 2500 ml of 1% acetic acid, stirred for half an hour in an ice bath and homogenised twice for 1 minute using the Ultraturax Type 45/5,. Polymin P was added to give a final concentration of 0.25%, the pH was adjusted to 10.0 using 5 N NaOH and the mixture was stirred for 2 hours over an ice bath and finally the pH was adjusted to 7.50 using 5 N HCl.

Centrifuging for 1 hour in a Christ Crvofuge 5 - 6 S at 4°C and 3000 rpm yielded a clear crude extract of 2540 ml with an interferon content of 17.1 x 10 I.U- (=100%) and a protein content of 5330 mg, from which a specific activity of 3.21 x 10® I.U./mg of protein can be calculated. fo) Ammonium sulphate was added until 55% saturation was reached (430 g/litre of extract). The mixture was stored overnight at 4 - 8°C and the precipitate formed was removed by centrifuging in a Beckmann J 2-21 highspeed centrifuge, Rotor JA 10 at 4eC, 10,000 rpm within 1 hour. The clear supernatant, 3120 ml, contained 0.7% of the Interferon contained in the crude extract (120 x 10® I.U.).

The pellet was taken up in 0.01 M NaCl and stirred for 2 hours at 4 - 8°C. The pH was adjusted to 7.50 using 5 N NaOH and the solution was clarified by centrifuging as described above.

The clear solution was dialysed with 0.01 >1 NaCl using a dialysis cartridge (Nephross Allegro, Messrs. Organon Technika) to give 390 mOsmol/1.

Q The interferon content was 13.3 x 10ϊ.ϋ. (= 77.6%). c) The dialysed material was then chromatographed (Tandem chromatography). For the preliminary column* 125 g of DE 52 cellulose powder made by Messrs. Whatman was used in TRIS/NaCl buffer,, pH 7.5 (0.025 M TRIS/HC1 + 0.2 M NaCl); this corresponded to 0.5 g of column material per g of biomass. For the affinity column* monoclonal anti-interferon IgG (EBI 1) coupled to' Br-CNactivated sepharose 4 3 (Messrs. Pharmacia) was used. The finished column material was stored in phosphate-buffered saline solution (PBS) with sodium azide at 4 - 8®C. Before use* the antibody column was washed with 0.1 M citric acid in 25% ethyleneglycol and then rinsed with PBS until neutral. A column volume of from 0.2 to 1.0 ml was required for each gram of biomass in the antibody column. The dialysed interferon solution was first pumped through both columns (preliminary column and antibody column) and the eluate was monitored by measuring the extinction at 280 nm. After the interferon solution had been applied* it was washed with TRIS/NaCl buffer* pH 7.5* until the quantity of protein in the eluate had fallen to 1/20th of the plateau value. The antibody column was then separated from the preliminary column and washed on its own with TRIS/NaCl buffer* pH 7.5* until no further protein could be detected in the eluate.

Elution of the interferon bound to the antibody was carried out using 0.X M citric acid in 25% ethyleneglycol* and again extinction of the eluate at 280 nm was monitored. The protein peak containing the interferon was collected. 16.8 ml of eluate were obtained with an interferon content of 12.3 x 10 I.U. (= 71.9%). The total quantity of protein was 54.4 mg, from which a specific activity of 226 x 10® I.U./mg of protein can be calculated. d) For further purification, the eluate was adjusted to pH 4.5 with ammonia and the precipitate formed was removed. The clear supernatant (18.3 ml) contained 46.3 mg of protein and had ah interferon Q content of 11.8 x 10 I.U. based on the crude protein. This corresponded to a yield of 69% (255 x 10® I.U./mg of protein). e) Final purification was carried out with an FPLC apparatus made by Messrs. Pharmacia with a MONO-S column, Type HR 10/10 (Messrs. Pharmacia) cation exchanger.

The clear supernatant obtained after precipitation was applied to the column, which had previously been washed with 0.1 M ammonium acetate buffer, pH 4.5 - 5.0, and this column was then washed until the extinction at 280 nm had reverted to the original value. Biution of the adsorbed interferon was carried out with a planar salt gradient by admixing 0.5 M ammonium acetate buffer, pH 4.5 to 5.0. Interferon was eluted as a sharp peak. Both the shoulder fraction (K 3) and also the fractions eluted later (K 5 - K 7) were separated from the main peak of pure interferon. The peak of pure interferon was collected and from it aliquots were taken for HPLC analysis, SDA gel electrophoresis, protein determination, interferon testing and endotoxin determination.

A total of 4.1 mg of protein were found in the shoulder fraction (9.1 ml)? the interferon G content was 1.33 χ 10 I.U. (7.7%). This gave 324 x 10^ ϊ.ϋ./mg of protein.

Q The main pool of 9.8 ml contained 5.18 χ ΙΟΙ.ΰ. (30.3%) of very pure interferon and a total of 16.1 mg of protein; this gave a specific activity of 322 x 10° Ι.ϋ./mg of protein. f;c) The 1FN pool was transferred into autoclaved lyo-ampoules (capacity 8 ml) in batches of up to a maximum of 2 ml, corresponding to' a quantity of from 1 to about 8 mg of pure Interferon per ampoule. The ampoules were then sealed with prewashed and autoclaved lyo-stoppers and cooled to at least -20®C. Lyophilisation was carried out at -10®C under a vacuum of less than 1 torr. After elimination of the buffer solution, the temperature was increased to 25°C and lyophilisation was continued for at least 1 hour. The vacuum was released and the stoppers were immediately pressed firmly in. After being capped with aluminium seals, the ampoules were then stored in a refrigerator or at -20®C.

S) To check stability, four different fermentation mixtures were lyophilised after being purified separately from Examples la - f; a but in a similar manner.

The lyophilised mixtures were dissolved in IRMA dilution buffer and analysed with the aid of NK2-IRMA for human XPN alpha (Messrs. Celltech U.K.).

Batch IFN titre before XFN titre after + % lyophilisation_lyophilisation A 720 X 10® 754 X 10® + 5 B 1337 X 10® 1526 X 10® 4- 14 C 981 X 106 852 X 10® - 13 D 1230 X 10® 1149 X 10® — 7 Thus, lyophilisation did not cause any losses.

After 11 months storage of the lyophilised material at about 4®C (refrigerator) it was dissolved in 0.1 M ammonium acetate and checked for both purity (by gel permeation HPLC) and also for content (by the NK2-IKMA test). before lyophilisation after 11 months storage in lyophilised form Purity (gel-HPLC 98.5% IFN titre 1510 x 10® units/ml 98.7 1464 x 10® units/ml Example 2 To test the effect of the fermentation time on the composition of the interferon components, samples were taken from a fermentation mixture (F, coli S3 101; 28°C) after 8, 9, 10 or 11 hours, precipitated with acid at pH 2 by the usual method and worked up and analysed using the method according to the Invention. The following Table shows the content of K 1, K 2 and K 3 (K 1: Met - IFN, K 2: native IFN; K 3: non-native IFN J in the samples.

The values were determined by chromatofocussing. - 26 Harvest time after hours 8 9 10 11 g moist biomass per litre of culture 14 15 18 21 mg IFN/<3 biomass (measured in the crude extract) 0.28 0.21 0.16 0.12 mg IFN/1 of culture volume 3.9 3.2 2.9 2.5 K 1 pi « 5.78 0.7% n. d. * 10.7% 19.4% K 2 pi = 5.64 96.2% 97.5% 86.1% 78.5% K 3 pi = 5.49 3.1% 2.5% 3.3% 2.1% * n.d. = not detected.

Example 3 Coupling of the EBI 1 antibody to CNBr-activated Sepharose 4 B (cf. DS-OS 33 06 OSO) The EBI-l antibody was first dissolved with 0.5 ¢4 NaCl/0.2 M NaHCO^, pH 8.4 (in as little buffer as possible) and dialysed with the buffer until no further sulphate ions could be detected in the external solution with barium chloride. Careful removal of the ammonium sulphate was absolutely essential since ammonium ions disrupt the subsequent coupling to the carrier. The protein concentration was then adjusted to 5 mg/ml with buffer. For the coupling, CNBr-activated Sepharose 4 B (Pharmacia) was used as carrier. It was first given a preliminary wash in accordance with the manufacturer's instructions (leaflet enclosed with the package). 1 g of activated sepharose was used for every 25 mg of SBI-1 antibodyCoupling was carried out in the above buffer at pH 8.4 for 2 hours at ambient temperature. Then - 27 the EBI-1 Sepharose was removed by suction filtering and washed in accordance with the instruction leaflet. Not more than 5% of the EBI-1 antibody used should remain in the filtrate. The finished EBI-1 Sepharose was stored in PBS/aside in a cold store.

PBS/azide: .'PBS: 7.30 g sodium chloride p.A. (Merck 6404) 3.00 g Na2HPO4 x 2 H2O p.A. (Merck 6580) 1.15 g NaB^PO^ x 02O p.A. (Merck 6346) dissolved and made up to 1000 mlj pH 7.0 Azide: 1.0 g/1 of very pure sodium azide (Merck 6688) were added to the PBS.

The finished solution was filtered sterile (0.2 micron pore size) and stored in a cold store.

Interferon antibody assay (neutralisation assay) Material Cells Human lung carcinoma cells A-549 ATCC CCL 185.

Virus Encephalomyocarditis virus (EMC)* ATCC VR 129.

Interferon standard HS-11 (1 ampoule '3S-X1~ = lyophilised Hu IFN rc-A taken up in 1.2 ml HjO? yielding 12000 iu/ml) Tissue culture plates wells with lid* flat bottom* Corning* New York* No. 25850 diameter of well 6.4 mm* tissue culture treated.

Media DMEM - Dulbecco's modified eagle medium* with glutamine* without sodium bicarbonate* flow cat. no. 10-331-24 (1F-017D) HEPES Sigma No. H-3375 TRICINE Calbiochem No. 33458., A grade FCS " foetal calf serum Boehringer Mannheim HUMANSERUM-ALBDMIN Behring Inst.* 20%* for infusion Antibiotic* tiamulin hydrogen fumarate* Biochemie Kundl/Tirol* Austria (Sandoz C.) Growth medium: DMEM + 10% FCS/deactivated 30 min/56eC + 13 mM HEPES + 6 mM TRICINE + l.S g/1 NaHCO2 without antibiotics pH 7.2 - 7.4 Assay mediums same as growth medium but 5% FCS instead of 10% and with the addition of 5 ug of tiamulin/ml Dilution medium: growth medium with no serum but with 5 ug/ml of tiamulin Virus medium: growth medium with no serum but with 5 ug/ml of tiamulin and 3.5 mg/ml of human serum albumin Methyl violet storage solutionί methyl violet Merck No. 1402 ........... 6 g ethanol ................................TOO ml dissolved and filtered at about 50°C methyl violet solution for use storage solution ....................... 50 ml water (pH neutral) ...................... 950 ml The cells were treated as a permanent cell line. They were propagated by trypsinisation and dilution in growth medium. For the assay, the cells were counted in a haemoeytomefcer and suspended In assay medium In order to obtain an inoculation solution of 4 - 5 x 10 * cells per ml per well? these were distributed over the dishes. Incubation was carried out in an atmosphere consisting of 5% CO? and 80% relative humidity at 37°C. After 8 - 24 hours the mono-layer was usually complete.

At this time the interferon and the serum dilutions were prepared in. separate test tubes.

For the control dish, OS-11 dilutions of 1:1000, 1:2000 up to 1:32,000 were Incubated for 1 hour at 3/°C.

For the test dish, the serum samples were diluted to 1:2, 1:4, 1:8 and so on up to 1:64 with a dilution medium which contained sufficient HS-11 to give a final concentration of 10 10 HS-ll/ml in each g-lass and then incubated for 1 hour at 37°C.

The dishes were decanted and each well was filled with 100 ml of the dilution medium (series 2, 3, 10 and 11) or with 100 ul of the dilutions (series 4-9). The dishes were incubated at 37°C for 4 hours as above. Then the dishes were given a coating of 100 ul of the virus medium (without the virus) for each well and 50 ul of the virus dilution (series 3, 11, 4-9) in order to achieve a cytopathic effect of approximately 90% within 36 hours and then incubated again. After 24 hours and microscopic monitoring the cells were stained with methyl violet.

The results are'shown in Figures 12a - 12f; the diagram appears in the Appendix.

METHOD The following methods were used for the analysis? Protein determination BIORAD PROTEIN ASSAY? This assay uses the dye Coomassie brilliant blue and measures the protein/dye complex at 595 nm. The standard used is bovine serum albumin.

Planimetric determinations Of the peak surfaces measured at 214 nm which were recorded by'gel permeation HPLC. The results are converted with the aid of a factor from the calibrating substances bovine serum albumin, ovalbumin, trypsinogen and lysozyme.

This measurement was carried out particularly on the preparations after the Tandem chromatography purification stage, pH 4.5, precipitation and FPLC on MONO-S, additionally or exclusively.

Interferon determination The NHj-IHMA" for human alpha Interferon, commercially available from Messrs. CELLTECH (U„K„), was used. The standard used was a laboratory standard HS 11’ which was adjusted to International Standard B 59/19 by biological assay (plaque reduction test WISH cells and vesicular stomatitis virus)„ SDS-gal electrophoresis The method of LAEMMLI (Nature 227, 680, (1980)) was used. The dye used to stain the proteins was Coomassie brilliant blue. 20 micrograms of the Interferon preparations were used in the purity checks.

Chromatofocussing The method of Bodo and Adolf (Separation and Characterization of Human IFN-alpha Subtypes, in The Biology of the Interferon System, pages 113-118, Elsevier 1983, Edts.E.DeMaeyer and H.Schellekens) was used, with a MONO-·? chromatofocussing column HR 5/20 (Pharmacia) in a pH range of 4 - 7. The buffers contained 25% acetonitrile instead of the specified 25% 1,2-propandiol in order to increase the flow rate. The protein concentration was recorded at 280 nm and the pH was recorded automatically.

The samples to be analysed were lyophilised, dissolved in water in amounts of 1 mg/ml and then diluted with 5 volumes of buffer A (pH 7.1). 0.2 - 1.0 mg of interferon were used for each analysis.

Gel permeation HPLC (high pressure liquid chromatography) Stationary phases WATERS 1-125; 2 χ (300 mm x 7.8 mm); pm particle diameter Mobile phase: 0.5 M ^a2SO4 0.02 M NaHjPO^, adjusted to pH 7.0 with NaOH 0.04% Tween 20 % propyleneglycol Flow speeds 0.5 ml/min Detections UV absorption at 214 nm Molecular weight calibrations Bovine serum albumin M 66,000 Ovalbumin M 4.5,000 Trvpsinogen M 24,000 Lysozyme M 14,300 Reverse phase HPLC (high pressure liquid chromatography) Stationary phases Bakerbond WP C 18y 250 mm x 4.6 ram; pm particle diameter nm pore diameter Mobile phases As 0,1% trifluoroacetic acid in water, pH 2.2 Bs 0.1% trifluoroacetic acid in acetonitrile Gradient programme: 0 2 mins 45% B 2-32 mins 45 - - 53% 3 32 - 40 mins 53% B 15 40 - 50 mins 45% B Flow speed 1 ml/min Detections ϋν absorption at 214 nm Legend for the figures Figure 1: Chromatograph of reverse phase HPLC of the acid eluate after Tandem chromatography; representation of components K 1 - K 7. Figure 2 s Chromatograph of gel permeation HPLC of the acid eluate after Tandem chromatography. Figure 3s Chromatograph of reverse phase HPLC after precipitation at pH 4.5; representation of components K 1, K 2f K 3 and K 6. Figure 4: Chromatograph of the gel permeation HPLC after precipitation at pH 4.5. Figure 5: Chromatograph of the FPLC on MONO-S at pH 4.5 with an ammonium acetate gradient from 0.1-0.5M. Figure 6s Chromatograph of reverse phase HPLC of the "shoulder fraction of the MONO S-peak. Figure 7s Chromatograph of the gel permeation HPLC of the shoulder fraction of the MONO S-peak, Figure 8s Chromatograph of the reverse phase HPLC of the main fraction of the MONO S-peak. Figure 9s Chromatograph of the gel permeation HPLC of the main fraction of the MONO S-peak.

Figure 10s Chromatograph of chromatofocussing of the ’’main fraction" of the MONO S-peak.

Figure 11: Photo of gel electrophoresis of the 5 acid eluate after Tandem chromatography and the components K 1 - K 7 separated by reverse phase HPLC.

Figures 12 - 12.f: Results of the anti-IFN-a antibodies; tests for various indications Type of test: neutralisation assay (10 iU/ml IFN ©/A-599/EMC) Total number of patients: 75 Number of patients with tumour indications: 58 Number of patients without stimulated antibodies 58 Number of patients with virus indications: 17 Number of patients without stimulated antibodies 17 Figure 13: Diagram of the anti interferon-α antibody assay Table 1: Survey of purification Table 2i Assessment of purity by HPLC

Claims (12)

1. Process for the preparation of recombinant a-interferon, characterised in that the host organism containing the interferon gene is cultivated under conventional conditions, after a growth period in which not more than 20% methionine interferon is formed the cells are killed off and harvested, the expressed interferon is removed in conventional manner, the cell debris is removed in a slightly alkaline medium, the interferon is concentrated and subjected to preliminary purification by tandem chromatography, the eluate is adjusted to pH 4.0 - 4.8 to remove any impurities, the interferon is finally purified by chromatography on a cation exchanger column with a volatile buffer as eluant at pH levels from 4.0 to 5.0 and is then lyophilised.

2. » Process according to claim 1, characterised Mn that the tandem chromatography consists of a preliminary cellulose column and affinity chromatography and in that the substance to be purified is washed through both columns with a suitable washing solution and the a-interferon is subsequently eluted from the affinity column with a suitable eluant.

3. Process according to one of the preceding claims, characterised in that the eluate from the tandem chromatography is adjusted to pH 4.5 to remove any impurities.

4. Process according to one of the preceding claims for the preparation of a recombinant, homogeneous, pure a -interferon which contains 0 to 5% methionine interferon, which is free from reduced forms and fragments of interferon, which contains 0 to 0.2% oligomer - 37 and 0 ΐο 2% dimer/trimer/tetramer and in which 90 to 100% of the monomer content consists of the «-interferon with disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

5. Process according to one of the preceding claims for the preparation of a recombinant, homogeneous, pure, human a-interferon with the amino acid sequence cys Asp Leu Pro Gin v** His Ser Leu Gly Ser Arg Arg Thr Leu het Leu Leu Ala Gin Met Arg Arg lie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr lie Pro Val Leu His Glu Met lie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala cys Val Ile Gin Gly Val Gly Val Thr G lu Thr Pro Leu Met Lys Glu Asp Ser lie Leu Ala Val Arg Lys Tvr »3 Phe Gin Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Xie Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu which contains 0 to 5% methionine-interferon, which is free from reduced forms and fragments of α-interferon, which contains 0 - 0.2% oligomer and 0 to 2% dimer/trimer/tetramer and wherein the a-interferon contains disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

6. Process according to one of the preceding claims for preparing a solid «-interferon.

7. Process according to one of the preceding claims for preparing a non-immunogenic «-interferon. 38

8. Recombinant α-interferon, characterised in that it corresponds to the amino acid sequence cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala GXh Met Arg Arg Xie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe' Pro Gin Glu Glu Phe Gly Asn G In Phe Gin Lys Ala Glu Thr lie Pro Val Leu His Glu lie Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu TV'S Gin Gin Leu Asn Asp Leu Glu Ala cys Val Xie Gin Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg Xie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser LVS Glu Λ 20

9. - Recombinant a-interferon, which corresponds to the amino acid sequence cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg Arg lie Ser Leu Phe Ser Cys Leu Lvs Asp Arg Arg Asp Phe Gly Phe Pro Gin Glu GlU Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Ile Gin Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu is present in a homogeneous, pure form, with 0-5% methionine interferon, 0 - 0.2% oligomers and 0-2% dimers/trimers/tetramers, is - 39 free from reduced forms and fragments of the a-interferon and wherein 90 - 100% of the monomer content consists of the a-interferon with disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

10. , Human α-interferon according to one of claims 8 and 9.

11. , a-Interferon according to one of claims 8 to 10 for use in the therapeutic treatment of viral and tumoral diseases,

12. , Pharmaceutical composition for therapeutic treatment, characterised in that it contains an α-interferon according to one of claims 8 to 10 and one or more pharmaceutical, inert excipients and/or carriers.