NO166727B - PROCEDURE FOR MANUFACTURING RECOMBINANT ALFA INTERFERON - Google Patents

Abstract

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

Description

The object of the present invention is a method for the production of recombinant, homogeneous, pure, non-immunogenic, antiviral and immunoregulatory active α-interferon. Reference is also made to the accompanying claim 1.

Interferons are body-specific proteins that can be detected in the most diverse species. The antiviral and immunoregulatory properties that are special to them have led to them already appearing to be suitable for the most diverse areas of application. Studies have shown that there are different interferon classes. In addition to the interferons a, 3 and y, interferon-u> and its structure were recently discovered.

The high expectations attached to the interferons as an effective agent against viral diseases and against cancer have already led early on to investigations with interferon preparations of natural material, whereby serious side effects have, however, occurred. The preparations used in these investigations contained, even after very thorough purification, complex mixtures of different interferons and in many cases also other proteins. The reason lies in the fact that some of the interferons have sub-types that differ more or less from each other. Thus, for example, more than 20 different types of interferon-a are known to date.

It was only when the interferons were produced by genetic engineering methods that the types of interferon preparations could be investigated.

These also include the recombinant interferons-o<2 (also called a A) used in the clinical tests.

In the production of human proteins using micro-organisms, purification of the proteins is of decisive importance. Any admixture originating from the host organism would, when the product is used on humans, lead to immune rejection reactions which could be life-threatening. Separation of admixtures of this kind hardly presents any problems today, and the extremely sensitive analytical methods allow endotoxins to be detected in the smallest concentrations. Also in the area of interferon research, purification methods have been developed that make it possible to provide interferon preparations that contain virtually no endotoxins. For example, the work by Staehelin et al, J. Biol can be mentioned here. Chem., 256, 9750 (1981).

All right. α-interferons used for clinical tests are practically endotoxin-free.

All the more surprising, therefore, was the occurrence of side effects that even necessitated the interruption of the interferon treatment. Someone straight. α-interferons surprisingly proved to be immunogenic. Antibodies against the interferon had been stimulated.

(Quesada et al., J. Nat.. Cancer Inst., 70, No. 6, 1041-1046 [1983]; Protzman et al., J. Immunol. Methods 75, 317-323

[1984] ).

These antibodies can lead to dangerous results if they influence the action of the interferon. They then affect not only the rack. the a-interferon, but then rec. The a-interferon is identical to the body's own interferon, also to the same extent as the body's own interferon.

In this connection, it is a concern that these antibodies also continue to work after the end of the interferon treatment and can worsen the course of the disease, weaken the body's own defense against viral infections and thus make the organism more susceptible to further infections.

These effects were already confirmed in animal experiments. For

to be able to have maximum drug safety, one must therefore demand that rec.-interferon-a^ is pure, practically endotoxin-free and, last but not least, non-immunogenic.

The task for the present invention was thus to develop

le a method for producing a non-immunogenic, antiviral and immunoregulatory active strain. α-interferon.

The reason for the immunogenicity of the mentioned α-interferons is. not known. It can only be ruled out that endotoxic additives are responsible for this.

The preparations used for testing differ primarily by very small variations in their amino acid sequence.

In addition to the structural differences in the primary structure of the clinically used proteins, it is known that genetically engineered alpha interferons are always a mixture of monomeric, low molecular weight, reduced and oligomeric forms of the interferon (see for example EPA 108 585 , 110,302 and 118,808). Some of these forms show similar in vitro, others, however, also reduced effects, and some are said not to possess possible immunogenic properties (see EPA 108 585 and 110 302).

In the aforementioned patent applications, methods are described for separating these interferon forms.

In EPA 108 585, a method for separating a "slow moving monomer" and oligomers is described, in which the interferon sample is incubated at a pH value of 3-5 for a certain time at a temperature of 28-40°C.

EPA 110 302 describes a method by which the monomer is formed from the oligomers by reduction with a redox system.

In EPA 118,808, recombinant interferon-α is purified from the oligomeric forms by means of metal chelate resins.

The pherons obtained according to these methods should contain monomeric interferon in almost quantitative form; however, immunogenicity tests are not available.

In-depth own analytical investigations have shown that recombinantly produced α-interferons are a mixture of different interferon forms in varying concentrations.

These include oligomers, tetramers, trimers and dimers of the interferon, methionine interferon, reduced forms and fragments of the interferon and surprisingly also various monomeric forms of the interferon.

The oligomers are α-interferons with a molecular weight of > 70,000, the reduced α-interferons are the protein with free SH groups and the methionine interferon is an α-interferon which at the N-terminal end in additionally carries a methionine (conditional on the microbiological production of the α-interferon).

The different monomeric forms constitute different S-S isomers of α-interferon, as shown by analysis.

In order to distinguish between these isomers linguistically, in the following the expression non-native monomer is used for the isomers of the predominantly occurring α-interferon monomer and this is designated as native monomeric α-interferon. With this, however, it should not be ruled out that the non-native monomers can likewise also occur in the "natural" material.

In an E. coli fermentation batch for the production of interferon-a2, for example, it was possible to detect at least 7 different interferon components (K 1 - K 7).

The analysis showed that these are oligomers, di-/trimers, methionine interferon, reduced interferons and an S-S isomer of the native monomeric interferon.-α^, each of which exhibits a disulfide bridge between the amino acids in positions 1 and 98 and 29 and 138 (see also Wetzel et al., J. Interferon. Res., Vol. 1, No. 3, 381-391 (1981).

As already stated above, the reason for the immunogenicity of rec»a-interferon is not known. However, it is also likely that all forms of a-interferon act immunogenically, which differs from the body's own. These also include the low molecular weight, dimers and oligomers and also the non-native monomers which contain differently linked disulphide bridges.

As long as the reasons for immunogenicity have not yet been clarified, the method for the production of shrimp. α-interferons are not used in any conditions that could require the formation of these, in their effect unknown forms. This means that purification of the interferon must also be carried out under the most gentle conditions, which do not endanger the nativity. Elevated temperature and reducing agents do not belong to these conditions.

Of course, at all cleaning stages, it must be ensured that no foreign substances are introduced, for example when using metal chelate resins or similar problematic reagents.

The subject of the invention is a method for the production of recombinant α-interferon, which is characterized by the characteristics specified in claim 1. The method is suitable for the production and purification of α-interferons of different species, for example human or animal α-interferons. The host organism used for the preparation can be a prokaryote or eukaryote, for example E. coli or Saccharomyces cerevisiae, preferably E. coli. The cultivation conditions for the various host organisms are well known to those skilled in the art.

It was surprisingly found that the growth time not only has an influence on the yield of a-interferon, but above all is also partly responsible to a decisive extent for how the interferon mixture is composed. Thus, for example, the composition of the interferon mixture changed when using E. coli with regard to the amount of methionine interferon according to the duration of the growth.

The fermentation rate is therefore advantageously controlled at short time intervals regarding the formation of α-interferon derivatives which are induced by the host organism, as an indicator of the best growing time. Methionine interferon may serve as one such indicator. In case of timely interruption, for example after the formation of less than 20%, preferably less than 5%, particularly preferably less than 1%, methionine interferon, one therefore already obtains a particularly pure interferon with ideal conditions for the subsequent purification process according to to the invention.

The method for producing acid-stable α-interferon is particularly suitable. In this connection, for example, the method according to DE 34 12 196.9, in which the cells are destroyed at pH 2 in a homogenizer, can be used.

When using a tandem chromatography, i.e. with successively coupled chromatographic steps with different adsorptive agents, with suitable washing and elution solutions, a large part of the contaminants can surprisingly be separated. Preferably, one combination of a cellulose-for-column is used which is directly connected to an affinity column. Particularly preferred is a DE-52 cellulose with a monoclonal anti-interferon IgG antibody linked to a carrier, for example Sepharose, such as the EBI 1 antibody described in BRD official publication 33 06 060.

For example, a TRIS/NaCl buffer pH 7.5 has proven to be a suitable washing solution, but there are also suitable washing solutions which do not influence the binding of the interferon to the antibody, but which nevertheless wash out the contaminants and allow them to be -constituents that have a negative influence on the properties of the antibody column remain bound to the pre-column.

A suitable eluent for the interferon is, for example, a buffer solution of 0.1 M citric acid in 25% ethylene glycol, but other eluents are also suitable which have similar properties. In general, the eluent must be matched to the α-interferon to be purified.

Some of the impurities in the "tandem eluate" were surprisingly already separated by debuffering the pH value to 4.0-4.8, particularly advantageously to pH 4.5. The pH value should be chosen so that there is as little monomeric interferon as possible in the precipitate.

Final purification of the interferon was achieved by chromatography on a cation exchanger, preferably with a MONO-S, type HR 10/10 cation exchanger. For elution of the highly purified α-interferon, a flat step gradient was used with a volatile buffer, for example an ammonium acetate buffer, where the pH value was constant and the concentration was varied (concentration gradient). Just as well, the concentration was also allowed to remain constant and the pH value to vary (pH gradient). It is crucial that the eluent is capable of separating interferon admixtures, particularly S-S isomers from the predominantly occurring monomers. Particularly well suited for this purpose is a flat concentration gradient of an ammonium acetate buffer, prepared from 0.1-1.0 M, preferably from 0.1-0.5 M ammonium acetate in a pH range of 4.0-5.0, preferably at pH 4.5. This buffer can also be removed by lyophilization, so that the highly purified α-interferons can be obtained for the first time in solid form, free of buffer salts and precipitating agents.

The method according to the invention is particularly suitable for the production of α-interferons of different species, which after use in the corresponding species do not stimulate any antibodies.

The procedure according to. the invention relates to the production of a recombinant, homogeneous, pure and non-immunogenic α-interferon which is free of reduced forms and fragments of the interferon, which exhibits less than 0.2% oligomers and less than 2% dimers/trimers/tetramers and less than 5% methionine interferon and in which more than 90% of the monomer content consists of native monomeric α-interferon.

As particularly advantageous, the method according to the invention can be used for the production of an α-interferon as described above, in which the host organism contains the gene that codes for the human α-interferon in accordance with the amino acid sequence:

and the native monomeric α-interferon in each case exhibits a disulfide bridge between the cysteines at positions 1 and 98 and 29 and 138.

Conditional on the choice of host organism and the species of

the fermentation conditions, recombinantly produced α-interferons can, in addition to the methionine interferon, also contain di-, tri-, tetra- and oligomers, reduced forms and fragments and S-S-isomers of the mainly occurring monomeric α-interferon in varying amounts, which, as described , for the first time can be suppressed, respectively removed, according to the present invention.

Recombinant α-interferons produced by the invention preferably contain no oligo-, tetra-, tri- and dimers, preferably contain more than 90% native monomer, and preferably are completely free of S-S isomers of the native monomer α-interferon .

The invention thus produces recombinant α-interferons of different species, which after use in the relevant species do not stimulate any antibodies, likewise fixed interferons.

Preferred is a recombinant human-α interferon with the above-described properties, preferably a recombinant human-α interferon according to the amino acid sequence

Particularly preferred is a recombinant, non-immunogenic, solid human α-interferon, which corresponds to the amino acid sequence

and is present in homogeneous, pure form, with less than 5% methionine interferon, which is free from reduced forms and fragments of the interferon, with less than 0.2% oligomers and less than 2% dimers/trimers/tetramers and at which more than .95% of the monomer content consists of the native-monomeric α-interferon with in each case a disulfide bridge between the cysteines in position 1 and 98 and 29 and 138. The method according to the invention enables separation of the impurities and interferon mixtures under the most gentle conditions. It will be explained in more detail by using interferon-α2Arg as an example according to the amino acid sequence

but also other α-interferons can be produced and purified by the method according to the invention, if necessary with small modifications that do not form part of the invention.

The acid-precipitated deep-frozen biomass was thawed by stirring

in 1 percent acetic acid. These and all subsequent manipulations were carried out at approx. 5°C.

The extraction of the protein from the cells took place, as described in detail in DRB publication 34 32 196.9, by breaking up the bacterial cells in a homogenizer, adding a precipitation aid, for example polyethyleneimine PEI-600 in a concentration range of 0 .1-0.25%, adjusting the pH value with NaOH to 7.5-10.0' and stirring the suspension for several hours. The pH value was then adjusted to 7.5, the crude extract clarified in a gentle manner and samples taken for protein determination and interferon testing.

Despite the known sensitivity of the polypeptides, such as interferon in relation to the shearing forces that occur during mechanical influences (Proe. Soc. Exp. Biol. Med., 146, 249-253

(1974)) this method at these pH conditions could surprisingly give high yields of crude interferon.

Control of different fermentation rates showed that the composition of the mixture changed depending on the fermentation conditions.

Especially the share; of component 1, methionine-interferon-a, a barely separable component, changed with the duration of fermentation: methionine-interferon-a was only formed after 8-9 h of fermentation duration.

If the fermentation is interrupted in time, it is also possible

to obtain interferon preparations which do not contain any methionine interferon.

Solid ammonium sulfate was added to the clarified crude solution with stirring until 65% saturation. After complete dissolution of the ammonium sulfate, the batch was kept refrigerated overnight, the precipitate that had formed was separated and stored at -20°C for further use.

From the clear; of the remaining material, samples were again taken for the interferon test, to check the precipitation of the interferon. No more than 5% of the interferon should remain in the above.

The ammonium sulfate pellet was dissolved in 0.01 M NaCl, the pH was adjusted to 7.5 with NaOH and the solution stirred for 2 hours. The insoluble portion' was separated and optionally extracted again with 0.01 M NaCl.

They combined? clear solutions were dialyzed using a sterile, pyrogen-free. dialysis cartridge against 0.01 M NaCl. The osmolarity of the interferon solution should be approx. 390-430 mOsmol/1 after 2-3 times of application. Aliquots were taken from the clarified solution for the interferon test.

For further purification "tandem chromatography" was used; a combination of a cellulose pre-column and a post-coupled affinity chromatography with highly specific, monoclonal antibodies. The pre-column, an ion-exchange column, served to keep sparingly soluble sample constituents away from the antibody column.

For the pre-column, DE-52 cellulose (Fa. Whatman) was stirred well with TRIS/NaCl buffer, pH 7.5 and filled into a chromatography column. The absorbent was washed with the buffer until the eluate no longer showed any pH and osmolarity changes. For the pre-column, 0.5-1.0 g DE 52-Cellulosei 0.025 M TRIS/HC1 + 0.2 M NaCl was used per grams of biomass; it was freshly prepared for each cleansing.

For the antibody column, monoclonal anti-interferon IgG recovered from mouse ascites and purified was coupled to BrCN-activated Sepharose 4B as a carrier. The finished co-

pocket material was stored in phosphate-buffered saline (PBS) with sodium azide in a cold room. Before first use or after long storage, the antibody column was washed with 0.1 M citric acid in 25% ethylene glycol, to remove any soluble parts, and finally washed neutral with PBS. Per gram of biomass, a column volume of 0.2-1.0 ml was required in the antibody column; this column could be used several times. The dialyzed interferon solution was first pumped over both columns (pre-column and antibody column) and the eluate checked by measuring the extinction at 280 nm. After application of the interferon solution, it was washed for a long time with TRIS/NaCl buffer pH 7.5

that the amount of protein in the eluate fell to 1/20 of the plateau value. To check that the interferon had been bound to the antibody column, the eluate was tested for interferon content. The anti-substance f column was then separated from the pre-column and washed alone with TRIS/NaCl buffer pH 7.5, until no protein could be detected in the eluate.

The elution of the interferon bound to the antibody took place with 0.1 M citric acid in 25% ethylene glycol, whereby the extinction of the eluate was measured at 280 nm. The protein peak containing the interferon was collected. For the final purification, the interferon pool was stored at -20°C. Interferon test, protein determination and the reverse-phase HPLC analysis showed that this purification yielded 60-90% pure IFN-α. In addition to oligomeric forms, this interferon pool contained reduced forms with free SH groups, dimers, trimers, tetramers and the non-native monomer. These components are all biologically and immunologically characterized as IFN. Surprisingly, part of the dis-

see components themselves; by precipitation at pH 4.5 (with ammonia) remove. In particular, the proportions of the components with reduced sulfur bridges were thereby reduced, also the forms with free SH groups (components 5 and 6). The analysis of this precipitate shows that the monomeric interferon was only entrained in small amounts.

For final purification, an FPLC apparatus from the company Pharmacia was used with a MONO-S column, type HR 10/10 (cation exchange), which could be filled with up to 60 mg of protein. This column material constitutes a high-efficiency ion exchanger with an excellent separation effect and the great advantage that despite the relatively large amount of protein to be purified, the final purification lasted only a few hours, the buffer solutions could be used sterile filtered and work could be done at room temperature.

The clear supernatant material obtained after the precipitation was applied to the column. Of particular importance was the buffer used in the FPLC separation. It had to elute the interferon components so that they could be clearly distinguished from each other and afterwards could be completely removed. The ammonium acetate buffer used with which the interferon was eluted in a gradient program had these properties. Interferon was eluted as a sharp peak with a weak shoulder. Both the "shoulder" fraction (K 3) and the later eluted fractions (K 5 - K 7) were separated from the main peak of the pure interferon. The peak of the pure interferon was collected and aliquoted. this for HPLC analysis, SDS gel electrophoresis, protein determination, interferon test and endotoxin determination.

By means of this chromatography, practically all components were separated from the main peak and a homogeneous interferon was obtained which, by gel permeation HPLC, showed a monomer content of over 99%. Reverse-phase HPLC showed only approx. 1% of the non-native monomer, the chromatofocusing a proportion of 2.5% non-native monomer.

Before reuse, the MONO-S column was washed with 0.5 M NaCl + 0.1 M Na- phosphate, pH 8.0, to remove adsorbed contaminants; it was stored in 25% ethanol.

The volatile puff could be completely removed by lyophilization. For this, the IFN pot was filled in portions of up to 2 ml in autoclaved lyo ampoules (volume 8 ml); this corresponded to ampoule an amount of 1 to approx. 8 mg pure interferon. The ampoules were then provided with pre-washed and autoclaved lyo-stoppers and cooled to at least 20°C. The lyophilization took place at -10°C and a vacuum of less than 1 Torr. After removal of the buffer solution, the temperature was raised to 25°C and lyophilized further for at least 1 hour. The vacuum was lifted and the stoppers were immediately pressed firmly. Fitted with aluminum covers, the ampoules were then stored in a cold room or at -20°C.

As was shown, with the help of good fermentation management (relatively early harvesting) together with the method according to the invention, it has become possible for the first time to produce an interferon-a which, not only with regard to its interferon content, is above 98% purity, but also as far as homogeneity is concerned, calculated on the different interferon components, consists to a degree of more than 95% of native monomeric interferon-a.

This high degree of purity and homogeneity also made it possible for interferon to be obtained for the first time, free of salts and buffer components, in solid form. It is therefore for the first time possible to store a-interferon for months without stabilizers, which offers significant advantages, in terms of storage, shipping and not least for the galenic developments, compared to the interferons that have always been stabilized with albumin. Even after 11 months of storage at 4°C, no loss of content could be determined.

The crystalline human leukocyte interferon described in EPA 83 734 involves crystals of polyethylene glycol as a precipitating agent with interferon, but not pure, homogeneous and crystalline interferon as the title might lead one to believe.

The interferon-a produced according to the invention was, as is known, dissolved by the addition of human serum albumin, sterile filtered and distributed aseptically in bottles; according to application in suitable concentrations.

In the clinical examinations, the interferon-a produced by means of the invention proved to be non-immunogenic and excellently tolerable.

In all, up to January 1985, 75 patients were treated with the non-immunogenic interferon-a: 58 patients with tumor indications and 17 with viral indications.

In not a single patient were antibodies stimulated during the therapy, whereby the duration of the treatment was partly 15 weeks

and more, up to 35' weeks.

The method according to the invention made it possible for the first time to provide a highly pure, homogenous, solid, solid, free of salts and buffer components based on the native monomeric interferon and; non-immunogenic interferon-c^ •

The amino acid sequence analysis according to known methods gave the following amino acid sequence:

The following examples shall illustrate the invention in more detail, without limiting it in any way.

Above all with regard to the fermentation rate, the method according to the invention is unrestrictedly applicable within wide limits.

Thus, it is also possible to use biomass from other host organisms, which after mechanical digestion give comparable IFN yields and with interferons, which similarly react specifically with the EBI-1 monoclonal antibody, for example IFN-a^. Other interferons with weak homology to interferon-a can also be purified by the method according to the invention using a corresponding highly specific monoclonal antibody.

Subject matter: the present invention is in detail methods for the production of recombinant α-interferon which are characterized in that the host organism containing the interferon gene is grown under normal conditions, the cells are killed after the normal growth time and harvested, the expressed interferon is separated in the usual way, cell residues separated in a weakly alkaline environment, the interferon is concentrated and pre-purified using a tandem chromatography, the eluate is adjusted to pH 4.0-4.8 to separate impurities, the interferon is finally purified by chromatography on a cation exchange column with a volatile buffer as eluent and then lyophilized .

The host organism used in the method is preferably E. coli; the cells are killed after a growth period in which no more than 20%, preferably no more than 5%, particularly preferably no more than 1% methionine interferon is formed.

The cells are destroyed in a homogenizer at pH 2 and the interferon is separated.

In one embodiment, the tandem chromatography consists of a cellulose pre-column and an affinity chromatography, and the substance to be purified over both columns is washed with a suitable washing solution, and then the α-interferon is eluted with a suitable eluent from the affinity column.

In one embodiment, the pre-column is loaded with DE-52 cellulose, the affinity column with a monoclonal anti-interferon-IgG antibody coupled to a carrier, and the substance to be purified is washed with a TRIS-NaCl buffer, pH 7.5 over both columns, and afterwards the α-interferon is eluted with 0.1 M citric acid in 25% ethylene glycol from the affinity column.

The monoclonal antibody EBI 1 is preferably used.

It is preferable that the eluate from the tandem chromatography is adjusted to pH 4.5 for separation of impurities.

For final cleaning, a MONO-S, type HR 10/10 cation exchanger is preferably used.

For elution, a flat concentration gradient prepared from 0.1-1.0 M, preferably from 0.1-0.5 M, ammonium acetate buffer is preferably used at pH values of 4.0-5.0, preferably at pH 4, 5.

One embodiment of the invention relates to a method for producing a solid α-interferon.

Another embodiment of the invention relates to a method for producing a non-immunogenic α-interferon.

Another method according to the invention works out

to produce a recombinant, homogeneous, pure α-interferon which exhibits less than 5% methionine interferon, which is free from reduced forms and fragments of interferon, which contains less than 0.2% oligomers and less than 2% dimer/trimer /tetramer and in which more than 90% of the monomer content consists of native monomeric interferon.

A further embodiment of the invention relates to a method for producing a recombinant, homogeneous,

pure human-α interferon of the amino acid sequence:

which exhibits less than 5% methionine interferon, which is free from reduced forms and fragments of α-interferon, which contains less than: than 0.2% oligomer and less than 2% dimer/trimer/tetramer and in which the native monomeric α-interferon in each case exhibits a disulfide bridge between the cysteines in positions 1 and 98 and 29 and 138.

The recombinant human α-interferon produced according to the invention preferably has the following amino acid sequence:

It exists in homogeneous, pure form, has less than 5% methionine interferon, less than 0.2% oligomers and less than 2% dimers/trimers/tetramers, it is free of reduced forms and fragments of the α-interferon and more than 90% of the monomer content of the native monomeric α-interferon consists in each case of a disulfide bridge between the cysteines in position 1 and 98 and 29 and 138.

The α-interferon produced according to the invention is applicable in the therapeutic treatment of viral and tumoral diseases.

Pharmaceutical agents for therapeutic treatment will contain an α-interferon produced according to the invention and one or more pharmaceutical, inert auxiliary and/or carrier substances.

Example 1

(E. coli; IFN-α2Arg; 28°C)

a) 251 g of acid field biomass, which was stored at -20°C, was

taken up in 2500 ml of 1 percent acetic acid, stirred for 1/2 h in an ice bath and homogenized 2x1 min. using Ultraturax type 45/6. Polymin P was added to a final concentration of 0.25%, pH adjusted to 10.0 with 5 N NaOH and stirred for 2 h in an ice bath, and finally the pH was adjusted back to 7.50 with 5 N HCl. Centrifugation for 1 hour in a Christ Cryofuge 6-6 S at 4°C and 3000 rpm gave 2540 ml of clear crude extract with an interferon content of 17.1 x 10<9> I.E. (=100%) and a protein content of 5330 mg, from which a specific activity of 3.21 x 10^ I.E./mg protein could be calculated.

b) Ammonium sulfate was added to 65 percent saturation (430 g/liter extract). The mixture was kept overnight in wood

4-8°C and the precipitate that had formed, centrifuged within half an hour in a Beckmann J 2-21 Highspeed centrifuge,

Rotor JA 10 at 4°C, 10,000 rpm. The clear supernatant, 3120 ml, contained 0.7% of the interferon contained in the crude extract (120 x 10<6> I.E.).

The pellet was taken up in 0.01 M NaCl and stirred for 2 h at 4-8°C. The pH was adjusted to 7.50 with 5 N NaOH and the solution centrifuged clear, as described above. The clear solution was dialyzed against 0.01 M NaCl at 390 mOsmol/1 using a dialysis cartridge (Nephross Allegro, company Organon Technika). The interferon content was 13.3 x 10<9>I.E. 77.6%).

c) The dialysate was then chromatographed (tandem chromatography). For the pre-column, 125 g of DE 52 cellulose powder from

company Whatman in TRIS/NaCl buffer pH 7.5 (0.025 M TRIS/HCl + 0.2 M NaCl); this corresponded to 0.5 g column material/g biomass. For the affinity column, monoclonal anti-interferon-IgG (EBI 1) was used which was coupled to Br-CN-activated Sepharose 4 B (Pharmacia). The finished column material was stored in phosphate-buffered saline (PBS) with sodium azide at 4-8°C. Before use, the antibody column was washed with 0.1 M citric acid in 25% ethylene glycol and then rinsed neutrally with PBS. For each gram of biomass, a column volume of 0.2-1.0 ml was required for the antibody column. The dialyzed interferon solution was first pumped over both columns (pre-column and antibody column) and the eluate checked by measuring the extinction at 280 nm. After application of the interferon solution, it was washed with TRIS/NaCl buffer pH 7.5, until the amount of protein in the eluate had fallen to 1/20 of the plateau value. The antibody column was then separated from the pre-column and washed alone with TRIS/NaCl buffer pH 7.5 until no more protein could be detected in the eluate.

The elution of the interferon bound to the antibody took place with 0.1 M citric acid in 25% ethylene glycol, whereby the extinction of the eluate was again examined at 280 nm. The protein peak containing the interferon was collected. 16.8 ml of eluate was obtained with an interferon content of 12.3 x lo<9>I.E. (=' 71.9%). The total amount of protein was 54.4 mg, of which one could calculate

ne a specific activity of 226 x 10^ I.E./mg protein.

d) For further purification, the eluate was adjusted to pH 4.5 with ammonia and the precipitate separated. The clear supernatant (18.3 ml) contained 46.3 mg of protein and an interferon proportion of 11.8 x 10 Q I.E. calculated on the crude protein. This corresponded to a yield of 65% (255 x 10<6> I.E./mg protein). e) For final purification, an FPLC apparatus from the company Pharmacia with a MONO-S column, type HR 10/10 (cation exchange) was used.

The clear supernatant obtained after the precipitation was applied to the column pre-washed with 0.1 M ammonium acetate buffer, pH 4.5-5.0 and washed until the extinction at 280 nm had returned to the initial value. The elution of the absorbed interferon took place with a flat salt gradient by mixing 0.5 M ammonium acetate buffer, pH 4.5-5.0. Interferon was eluted as a sharp peak. Both the "shoulder" fraction (K 3 ) and the later eluted fractions (K5 - K7) were separated from the main peak of the pure interferon. The peak of the pure interferon was collected, and aliquots were taken from this for HPLC analysis, SDS-gel electrophoresis, protein determination, interferon test and endotoxin determination. In the "shoulder" fraction (9.1 ml) a total of 4.1 mg of protein was determined; the interferon content was 1.3 3 x 10q I.E. (7.7%). From this, one could conclude that 324 x IO<6> I.E./ mg protein.

The main pool of 9.8 ml contained 5.18 x 10<9>I.E. (30.3%)

of the purest interferon and a total of 16.1 mg of protein. This resulted in a specific activity of 322 x 10^ I.E./mg protein.

f;a) The IFN pool was filled in portions of up to 2 ml in autoclaved Lyo ampoules (volume 8 ml), which per ampoule corresponded to a quantity of 1 and up to approx. 8 mg pure interferon. The vials were then fitted with pre-washed and autoclaved Lyo stoppers and cooled to at least -20°C. The lyophilization took place at -10°C and a vacuum of less than 1 Torr. After removing the buffer solution, the temperature was increased to 25°C, and it was further lyophilized for at least 1 hour. The vacuum was lifted and the stoppers were immediately pressed firmly. Fitted with aluminum covers, the ampoules were then stored in a cold room or at -20°C.

/3) To investigate the stability, four different fermentation batches were lyophilized, independently from example 1a-f;a but purified in an analogous way.

The lyophilisates were dissolved in IRMA dilution buffer and analyzed using NK2-IRMA for human IFN-alpha (company Celltech

UK).

The lyophilisation therefore produced no losses. After 11 months of storage of the lyophilisate; at approx. At 4°C (in a refrigerator) it was dissolved in 0.1 M ammonium acetate and checked for purity (by gel permeation-HPLC) and content (by NK2-IRMA tests).

Example 2

To check the influence of the fermentation time on the composition of the interferon components, samples were taken from a fermentation batch (E. coli HB 101; 28°C) after 8, 9, 10 or 11 hours, according to the usual technique at pH 2 precipitated with acid and worked up and? analyzed according to the methodology according to the invention. The following table shows the contents of the samples of Kl, K 2 and K 3 (K. 1: Met - I FN, K 2: native IFN; K 3: non-native IFN). The values were obtained using chromatofocusing.

Example 3

Binding of EBI-1 antibody to CNBr-activated

Sepharose 4 B (see DE official document 33 06 060)

EBI-1 antibody was first dissolved with 0.5 M NaCl/0.2 M NaHCO-j, pH 8.4 (in the smallest possible buffer) and dialyzed against the buffer until no more sulfate ions could be detected in the external solution with barium chloride. Careful removal of the ammonium sulfate was absolutely necessary, as ammonium ions interfered with the subsequent coupling to the support. The protein concentration was then adjusted to 5 mg/ml with buffer. For coupling, CNBr-activated Sepharose 4 B was used as carrier. This was in

adhere to the manufacturer's instructions (delivery note) pre-washed. For every 25 mg of EBI-1 antibody, 1 g of activated Sepharose was used. The coupling took place in the above-mentioned buffer, pH 8.4 at room temperature for 2 hours. The EBI-l-Sepharose was then decanted and washed according to the instructions on the package insert. No more than 5% of the EBI-1 antibody used must remain in the filtrate. The finished EBI-1-Sepharose was stored in PBS/azide in a cold room.

PBS/azide:

PBS: 7.30 g, sodium chloride p.a. (Merck 6404)

3.00 g Na2HP04 x 2 H20 p.a. (Merck 6580)

1.15 g NaH2P0^ x H20 p.a. (Merck 6346) dissolved and

filled up to 1000 ml; pH 7.0

Azide: 1.0 g/l sodium azide of the purest type (Merck 6688) was added to PBS.

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

Interferon antibody assay (neutralization assay)

Material

Cells

Human lung carcinoma cells "A-549" ATGC CCL 185.

Virus

Encephalomyocarditis virus (EMC), ATCC VR 129.

Interferon standard

HS-11 (1 ampoule "HS-11" = lyophilized Hu IFN ra-A, taken up

in 1.2 ml H20>,i gives 12,000 iU/ml)

Tissue culture plates

96 bowls with. lid, flat bottom, Corning, New York, No. 25,860, dish diameter: 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. 33468, quality A

FCS = fetal calf serum Boehringer Mannheim

HUMAN SERUM ALBUMIN Behring Inst., 20%, for infusion antibiotic, Tlamulin hydrogen fumarate Biochemie Kundl/Tirol, Austria (Sandoz C.)

Growth medium: DMEM = 10% FCS/inactivated 30 min/56°C

Assay medium: As growth medium, but 5% FCS instead

10% and addition of 5 ug Tiamulin/ml

Dilution medium: Growth medium without serum, with

5 ug/ml Tiamulin

Virus medium: growth medium without serum; with 5 ug/ml Tiamulin and with 3.5 mg/ml human serum albumin.

The cells were treated as a permanent cell line. It was propagated by trypsinization and dilution in the growth medium. To carry out the assay, the cells were poured into a hemocytometer and suspended in assay medium so that a seeding solution of 4 - 5 x 10^ cells per ml per toast; this was distributed among the plates. The incubation was carried out in an atmosphere of 5% CO2 and 80% humidity at 37°C.

After 8-24 h the monolayer was usually complete. At this point, the interferon and serum dilutions were prepared in separate test tubes.

For the control plate, HS-11 dilutions of 1 : 1000,

1 : 2000 ..... to 1 : 32,000 incubated at 37°C for 1 h.

For the sample plate, the serum samples were diluted to 1:4,1:8

..... to 1 : 64 with a dilution medium containing so much HS-11 that a final concentration of 10 iU HS-11/ml was obtained in

each glass, and this was likewise incubated for 1 h at 37°C.

The plates were decanted and each dish was filled with 100 ml of the dilution medium (rows 2, 3, 10 and 11) or with 100 ml of the dilutions (rows 4 - 9). The plates were incubated as above for 4 h at 37 ° C. The plates were then overlaid with 100 µl of the virus medium (without virus) per dish and 50 µl of the virus dilution (rows 3, 11, 4 - 9) to achieve a cytopathic effect of approximately 90% during 36 h and incubated again After 24 h and microscopic control, the cells were stained with methyl violet.

The results are shown in figures 12a-12f, see the attached form.

METHODOLOGY

The following methods were used for 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 determination of the top surfaces measured at 214 nm,

which was recorded by gel permeation HPLC. The conversion took place using a factor of the calibration substances bovine serum albumin, ovalbumin, trypsinogen and lysozyme. Above all, this determination was made additionally or exclusively on the preparations after the purification step tandem chromatograph, pH 4.5 precipitation and FPLC by MONO-S.

Interferon determination

The from the company CELLTECH (U.K.) available "NK2-IRMA2

for human interferon-a was used. As a standard, a laboratory standard "HS-11" served, which by means of a biological assay (plaque reduction test WISH cells and vesicular stomatitis virus) was adjusted to the international standard B 69/19.

SDS gel electrophoresis

The method of LAEMMLI (Nature 227, 680, 1980) was used. The dye used for staining the proteins was Coomassie Brilliant Blue. In the purity checks of the interferon preparations, 20 µg was used in each case.

Chromatofocusing

The method of Bodo and Adolf was used ("Separation and Characterization of Human IFN-alpha Subtype" in "The Biology of the Interferon System", pp. 113-118, Elsevier 1983, ed. E. DeMaeyer and H. Schellekens), whereby a MONO-P chromatofocuser column HR 5/20 (Pharmacia) was used in a pH range of 4-7. The buffers contained, instead of the indicated addition of 25% 1,2-propanediol, 25% acetonitrile in order to increase the flow rate. The recording of the protein concentration took place at 280 nm, the pH recording took place automatically. The samples to be analyzed were lyophilized, dissolved in water to 1 mg/ml and then diluted with 5 volumes of buffer A (pH 7.1). 0.2-1.0 mg of interferon was used per analysis.

Gel Permeation HPLC (High Performance Liquid Chromatography)

Stationary phase:

WATERS 1-125, 2 x (300mm x 7.8mm);

10 pm particle diameter

Mobile phase:

0.5 M NaOSO.

2 4

0.02 M NaH 2 PO 4 , adjusted to pH 7.0 with NaOH

0.04% Tween 20

25% propylene glycol

S flow rate:

0.5 ml/min

Detection:

UV absorption at 214 mm

Molecular weight calibration:

Reverse phase HPLC (high pressure liquid chromatography)

Stationary phase:

Bakerbond WP C 18; 250mm x 4.6mm; 5 nm particle diameter;

30 nm pore diameter

Mobile phase:.

A: 0.1% trifluoroacetic acid in water, pH 2.2

B: 0-1% trifluoroacetic acid in acetonitrile Gradient program:

0 - 2 min: 45% B

2-32 min: 45-53% B 32 - 40 min: 53% B 40 - 50 min: 45% B

S flow rate:

1 ml/min

Detection:;

UV absorption at 214 nm

Explanation of the figures

Figure 1: Chromatogram for reverse-phase HPLC of the acidic eluate after tandem chromatography; presentation of the components K 1 - K 7. Figure 2: Chromatogram of gel permeation HPLC for the acidic

eluate after tandem chromatography.

Figure 3: Chromatogram for reverse-phase HPLC after precipitation at pH

4.5;; showing the components Kl, K2, K 3 and X 6. Figure 4: Chromatogram of gel permeation HPLC after precipitation at pH 4.5. Figure 5: Chromatogram of FPLC on M0N0-S at pH 4.5 using

an ammonium acetate gradient of 0.1-0.5 M

Figure 6: Chromatogram of reverse-phase HPLC to "shoulder" fraction en" to the MONO-S peak. Figure 7: Gel permeation HPLC chromatogram to

the "shoulder fraction" of the MONO-S peak.

Figure 8: Reverse-phase HPLC chromatogram of the "main fraction"

to the MøNO-S peak.

Figure 9: Chromatogram of gel permeation HPLC for "main- fraction" of the MONO-S peak. Figure 10: Chromatogram for the chromatofocusing of the "main fraction" of the MONO-S peak. Figure 11: Photo of the gel electrophoresis of the acidic eluate after tandem chromatography as well as reverse-phase HPLC - separate components K 1 - K 7.

Figure 12a - 12f:

Results of anti-IFN-α antibodies;

investigations for different indications

Figure 13: Form for anti-interferon-a antibody assay Table 1: Overview of purification

Table 2: Assessment of the cleanliness using

by HPLC

Claims (3)

1. Process for the production of recombinant, homogeneous, pure, non-immunogenic, antiviral and immunoregulatory active α-interferon which exhibits less than 5% methionine interferon, which is free from reduced forms and fragments of the interferon, which contains less than 0 .2% oligomers and less than 2% dimers/trimers/tetramers and in which more than 90% of the monomer content consists of native monomeric interferon, by fermentation of a host organism, preferably E. coli, which contains the interferon gene, preferably that gene which codes for the human α-interferon with the amino acid sequence characterized in that the transformed microorganism is grown under normal conditions, the cells are lysed after a growth period;,,, depending on the microorganism used, in which no more than 20%, preferably no more than 5%, particularly preferably not more than 1%, methionine interferon is formed, whereby preferably the cells are destroyed in a homogenizer at pH 2 and harvested, cell debris is separated in a weak alkaline environment, the interferon is concentrated and pre-purified by means of a tandem chromatography, consisting of a cellulose pre-column and an affinity chromatography, the eluate is adjusted to pH 4.0-4.8 to separate impurities, the interferon is finally purified by chromatography on a cation exchange column, preferably with a MONO-S, type HR 10/10 cation exchanger with a volatile buffer which eluent, preferably with a flat concentration gradient, prepared from 0.1-1.0 M, preferably from 0.1-0.5 M, ammonium acetate buffer at pH values of 4.0-5.0, preferably at pH 4 ,5, and then lyophilized. 2. Procedure as stated in claim 1, characterized in that the tandem chromatography consists of a cellulose pre-column with DE-52 cellulose, and an affinity chromatography with a monoclonal anti-interferon IgG antibody which is linked to a carrier, particularly preferably with the monoclonal antibody EBI 1, and that the substance to be is cleaned, washed over both columns with a suitable washing solution, preferably with a TRIS-NaCl buffer pH 7.5, and that the a-interferon is then eluted with a suitable eluent, preferably with 0.1 M citric acid in 25% ethylene glycol, from the affinity column. 3. Method as stated in one of the preceding claims, characterized in that the eluate in the tandem chromatography is adjusted to pH 4.5 for separation of contaminants.