DD250336A5 - Process for the production of alpha interferon - Google Patents

Abstract

The invention relates to a process for the production of α-interferon for the treatment of viral and tumoral diseases. The aim of the invention is an improved process for the production of highly pure, non-immunogenic, antiviral and immunoregulatory effective, homogeneous α-interferon. According to the invention, the cells containing the interferon gene, preferably the gene coding for the human a-interferon, preferably E. coli, are cultivated under usual conditions, the cells after usual growth time, preferably, when not more than 1% methionine interferon has been formed , killed, wherein the cells are destroyed and harvested in a homogenizer at p H 2, the experimentally separated interferon in the usual manner, cell truncated in weakly alkaline medium separated, the interferon concentrated and pre-purified by tandem chromatography, the eluate to remove impurities adjusted to p H 4.0-4.8 and the interferon on a cation exchange column of MONO-S, type HR 10/10 at p H 4.0-5.0 final purified and lyophilized. Fig. 1

Description

For this 18 pages drawings and 2 pages tables

Field of application of the invention

The present invention is a process for producing a high-purity, non-immunogenic, antiviral and immunoregulatory effective, homogeneous α-interferon, the protein itself and its use. The interferon prepared according to the invention is used as a medicament, for example for the treatment of viral and tumoral diseases.

Characteristic of the known technical solutions

Interferons are endogenous proteins that can be detected in a wide variety of species. Their own antiviral and immunoregulatory properties have made them appear early on as suitable for a wide variety of applications. Studies have shown that there are different classes of interferon. In addition to the interferons α, β and y , the interferon-ω was recently found and its structure elucidated.

The high expectations placed in the interferons as an effective remedy against viral diseases and against cancer have led early to investigations with interferon preparations from natural material, but in which serious side effects occurred. The preparations used in these studies contained complex mixtures of different interferons, and in many cases also other proteins, even after extensive purification. The reason is that some of the interferons have subtypes that are more or less different from each other; For example, more than 20 different types of interferon-α are known.

Only by the production of the Interferone by gen-technological procedures could be examined pure interferon preparations.

These include the recombinant interferons used in clinical trials (also called aA ).

Crucial importance in the production of human proteins by microorganisms is the purification of the proteins. Any addition from the host organism would lead to immune defenses in the use of the product in humans, which can be life-threatening. The separation of admixtures of this type, however, presents little problem today and the now extremely sensitive analytical methods can detect endotoxins in the lowest concentrations.

Also in the field of interferon research, purification methods have been developed which make it possible to obtain interferon preparations which contain virtually no endotoxins. As an example, the work of Staehelin et al. J. Biol.

Chem. 256,9750 (1981).

All rek. a-interferons are practically endotoxin-free.

All the more surprising was the occurrence of side effects that even forced the discontinuation of interferon treatment. Some rec. a-interferons surprisingly proved to be immunogenic. Antibodies to the 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 can cause threatening effects if they affect the effect of interferon. They do not only work on the rek. α-interferon, but because the rek. α-interferon is identical to the body's interferon, equally to the body's interferon.

What is disastrous is that these antibodies can continue to function after the end of interferon treatment, worsen the course of the disease, weaken the body's defense against viral infections and thus make the organism more susceptible to further infections. These effects have already been confirmed in animal experiments. In the interests of maximum drug safety, it is therefore necessary to demand that rec. Interferon-a _{2 must be} type-pure, practically endotoxin-free and not least non-immunogenic.

The object of the present invention was therefore to provide a method for producing a non-immunogenic, antiviral and immunoregulatory effective rec. to develop α-interferons.

The cause of the immunogenicity of said a-interferons is not known. It can only be excluded that endotoxic admixtures are responsible for this.

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

Amino acid 23 Amino acid 34 Preparation I: lysine histidine Preparation II: arginine histidine Preparation III: arginine arginine

In addition to the structural differences in the primary structure of the clinically used proteins, it is known that genetically engineered α-interferons are always a mixture of monomeric, low molecular weight, reduced and oligomeric forms of interferon (see, e.g., EPO 108585, 110302 and 118808). Some of these forms have the same in vitro, but other diminished effects and some are said to have possible immunogenic properties (s.

EPO 108585 and 110302).

Described in these patent applications are methods to separate these interferon forms.

In EPA 108 585 a process for separating a "slow moving monomer" and oligomers is described in which the interferon sample is incubated at a pH of 3 to 5, some time at a temperature of 28-40 ⁰ C.

EPA 110302 describes a process in which the monomer is formed from the oligomers by reduction with a redox system.

Finally, in EPA 118808, recombinant interferon-α is purified from the oligomeric forms by means of metal chelating resins.

The interferons obtained by these methods should contain monomeric interferon in almost quantitative form; Immunogenicity tests are not available.

Detailed in-house analytical investigations have shown that recombinantly produced a-interferons are a mixture of different forms of interferon in varying concentrations.

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

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

The different monomeric forms have been shown to be different S-S isomers of α-interferon.

To linguistically distinguish these isomers, the term non-native monomer is used hereinafter for the isomers of the predominantly occurring a-interferon monomer, and this is referred to as native monomeric α-interferon. However, this does not mean that the non-native monomers are also present in the "natural" material

In an E. coli fermentation batch for the production of interferons, for example, at least 7 different interferon components could be detected (K 1-K7).

The analysis revealed that these are oligomers, di- / trimers, methionine interferon, reduced interferons and an SS isomer of native monomeric interferons, each having a disulfide bond between the amino acids at positions 1 and 98 and 29 and 138 (See also Wetzel et al., J. Interferon Res., Vol. 1, No.3,381-391 [1981].

As stated above, the cause of the immunogenicity of the rek. α-interferons not known. However, it is also obvious that all the forms of α-interferon have an immunogenic effect, which is different from the body's own. These include the low molecular weight dimers and oligomers as well as the non - native monomers containing differently linked disulfide bridges.

As long as the causes of immunogenicity have not been clarified, the procedures for producing rek. In interferons, no conditions are used which could promote the formation of these forms, which are unknown in their impact. This means that also the cleaning of the interferon should be carried out under the most gentle conditions, which do not endanger the nativity. Elevated temperature and reducing agent are not among these conditions.

Of course, in all cleaning steps to ensure that no foreign substances are introduced, for example, when using metal chelating the similar problematic reagents.

Object of the invention

The aim of the invention is to provide an improved process for the production of α-interferon, with which a very pure product can be obtained in a simple and economical manner.

Explanation of the essence of the invention

The invention has for its object to carry out the production under mild conditions to prevent the formation of interfering forms of α-interferon, such as. To avoid oligomers, tetramers, trimers and dimers, methionine interferon, reduced forms and fragments of interferon.

According to the invention, a method for the production of recombinant α-interferon is provided, which is characterized in that the interferon gene, preferably that for the human α-interferon of the amino acid sequence Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin GIuGIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr Me Pro Vai Leu His GIu Met Gin Gin He Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Giu LeuTyrGln GIn Leu Asn Asp Leu GIu AIa Cys Vai Gin GIy Vai GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser Me Leu Ala VaI Arg Lys Tyr Phe Gin Arg le Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys Ala Trp GIu VaI VaI Arg Ala GIuIIe Met Arg Ser Phe Ser Leu Ser Thr Asn Leu GInGIu Ser Leu Arg Ser Lys GIu

coding gene containing host organism, preferably E. coli, is cultivated under customary conditions,

the cells after the usual growth time, preferably after a growth time in which not more than 20%, preferably not more than 5%, more preferably not more than 1% methionine interferon is formed, killed, wherein preferably destroys the cells in a homogenizer at pH2 and to be harvested,

the experimented interferon is separated in a conventional manner, cell debris are separated in the weakly alkaline, the interferon is concentrated and pre-purified by tandem chromatography, the eluate is adjusted to pH4.0-4.8 to remove impurities, the interferon by chromatography a cation exchange column, preferably with a MONO-S, type HR10 / 10 cation exchanger with a volatile buffer as eluent, preferably with a shallow concentration gradient prepared from 0.1-1.0M, preferably from 0.1-0.5Μ ammonium acetate buffer at pH Values of 4.0-5.0, preferably at pH4.5, and then lyophilized.

The process is suitable for the preparation and purification of α-interferons from various species, e.g. human or animal a-interferons. The host organism used for the preparation may be a prokaryote or eukaryote, for example E. coli Saccharomyces cerevisiae, preferably E. coli. The cultivation conditions 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 has an influence on the yield of α-interferon, but above all is also decisively responsible for how the interferon mixture is composed. For example, the composition of the interferon mixture using E. coli changed with respect to the amount of methionine interferon depending on the duration of growth.

Advantageously, therefore, the fermentation batch is checked at short intervals for the formation of α-interferon derivatives produced by the host organism as an indicator of the best growth time. Methionine interferon may serve as such indicator. By timely termination, for example, after the formation of less than 20%, preferably less than 5%, more preferably less than 1% methionine interferon, therefore, a particularly pure interferon with ideal conditions for the subsequent purification process according to the invention is obtained. Particularly suitable is the process for the preparation of acid-stable α-interferon. Here, for example, the method according to DE 3432196.9, in which the cells are destroyed at pH 2 in a homogenizer, are used. By using tandem chromatography, i. H. with successive chromatographic steps on various adsorbents with suitable washing and elution solutions, surprisingly, a large part of the impurities can be separated off. Preferably, a combination of a cellulose precolumn directly connected to an affinity column is used here. Particularly preferred is a DE-52 cellulose having a monoclonal anti-interferon IgG antibody coupled to a carrier, such as Sepharose, such as the EBH antibody described in DEOS 3306060.

For example, a TRIS / NaCl buffer pH7.5 has been found to be a suitable wash solution, but wash solutions are also suitable which do not affect the binding of interferon to the antibody, but which wash out the impurities, and the components which adversely affect the properties of the antibody column. leave tied to the guard column. For example, a suitable eluent for the interferon is a buffer solution of 0.1 M citric acid in 24% ethylene glycol, but other eluents having similar properties are also suitable. In general, the eluent must be adapted to the particular α-interferon to be purified.

Surprisingly, a portion of the impurities of the "tandem eluate" could already be separated by buffering off the pH to pH 4.0-4.8, more preferably to pH 4.5 Precipitation finds as little monomeric α-interferon as possible.

The final purification of the α-interferon was achieved by chromatography on a cation exchanger, preferably with a MONO-S type HR10 / 10 cation exchanger. For the elution of the highly purified α-interferon, a flat step gradient with a volatile buffer, for example an ammonium acetate buffer, was used in which the pH was constant and the concentration was varied (concentration gradient). Equally, the concentration could be kept constant and the pH varied (pH gradient). It is crucial that the eluent is able to separate interferon additions, in particular S-S-isomers of the predominantly occurring monomer. Particularly suitable for this purpose is a low 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, 5th This buffer can also be removed by lyophilization, so that the highly purified a-interferons can be obtained for the first time in solid form, free from buffer salts and precipitants.

The process according to the invention is particularly suitable for the production of α-interferons of various species which do not stimulate antibodies after application in the corresponding species.

The process according to the invention has proven to be particularly advantageous in the preparation of a recombinant, homogeneous, pure solid and non-immunogenic α-interferon which is free of reduced forms and fragments of interferon containing 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. The process according to the invention can be used to particular advantage in the production of an α-interferon as described above, in which the host organism has the human α-interferon corresponding 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 He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin GIu GIu Phe GIy Asn Gin Phe Gin Lys Ala GIu TIiV lie Pro Vai Leu Giu Met Gin Gin lie Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala AlaTrp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr GIu LeuTyrGln Gin Leu Asn Asp Leu GIu Ala Cys Vai Gin GIyVaI GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser lie Leu Ala VaI Arg Lys Tyr Phe GIn Arg Ile Le Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu VaI VaI Arg Ala Glu He Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys GIu

contains coding gene and the native monomeric α-interferon each having a disulfide bridge between the cysteines at position 1 and 98 and 29 and 138.

The present invention also provides recombinant α-interferons which can be prepared by the process according to the invention, preferably recombinant α-interferons in homogeneous, pure form which contain less than 20%, preferably less than 5%, particularly preferably less than 1%, methionine. Interferon included.

Due to the choice of the host organism and the nature of the fermentation conditions, recombinantly produced a-interferons may contain not only methionine interferon but also di-, tri-, tetra- and oligomers, reduced forms and fragments and SS isomers of the predominantly occurring monomeric α-interferon contained in varying amounts, which, as described, for the first time suppressed by the novel process, or can be eliminated. Another object of the invention are therefore recombinant inferferons in homogeneous, pure form, containing less than 20%, preferably less than 5%, more preferably less than 1% methionine interferon, free from reduced forms and fragments of the α-interferon which contain less than 0.2% oligomers and less than 2% dimers / trimers / tetramers, preferably no oligo-, tetra-, tri- and dimers, which preferably contain more than 90% of native monomer, and preferably completely free from SS isomers of native monomeric α-interferon.

The invention further relates to the described recombinant a-interferons of various species which do not stimulate antibodies after administration in respective species, as well as solid interferons. Preferred is a recombinant human a-interferon having the properties described above, preferably a recombinant human a-interferon according to the amino acid sequence.

Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr lie Pro VaI Leu His GIu Met NeGInGIn He PheAsn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr GIu Leu Tyr Gin GIn Leu Asn Asp Leu GIu AIa Cys VaI He Gin GIy VaI GIuVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe GIn Arg IleThr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIu Ne Met Arg Ser Phe Ser Leu Ser Thr Asn Leu GInGIu Ser Leu Arg Ser Lys GIu.

Particularly preferred is a recombinant, non-immunogenic, solid human α-interferon having 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 He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro GInGIuGIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thrlie Pro VaI Leu His GIu Met HeGInGInle PheAsn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin GIn Leu Asn Asp Leu GIu AIa Cys VaI He Gin GIy VaI GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser Me Leu Ala VaI Arg Lys Tyr Phe Gin Arg le Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys Ala Trp GIu VaI VaI Arg Ala GIu He Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu

is present in homogeneous, pure form, with less than 5% methionine interferon, which is free of reduced forms and fragments of interferon, with less than 0.2% oligomers and less than 2% dimers / trimers / tetramers and in which more than 95% of the monomer content consists of the native monomeric α-interferon, each having a disulphide bridge between the cysteines at positions 1 and 98 and 29 and 138.

The inventive method allows the separation of the impurities and the interferon admixtures under the gentlest conditions. The example of the interferon-a ₂ Arg according to the amino acid sequence Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thrlie Pro VaI Leu His GIu Met Gin Gin He PheAsn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr Giu Leu Tyr Gin GIn Leu Asn Asp Leu GIu AIa Cys Vai Gin GIy VaI GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe Gin Argulle Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIuIIe Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu

However, other a-interferons, if necessary with minor non-inventive modifications, are to be prepared and purified by the process according to the invention.

The acid-precipitated frozen biomass was thawed by stirring in 1% acetic acid. These and all subsequent manipulations were performed at about 5 ° C.

The extraction of the protein from the cells was carried out, as described in detail in the patent application DE 3432196.9, by disrupting the bacterial cells in a homogenizer, adding a precipitation aid such as polyethyleneimine PEI-600 in a concentration range of 0.1-0.25%, adjusting the pH with NaOH to 7.5-10.0 and stir the suspension for several hours. The pH was then adjusted to 7.5, the crude extract was clarified in a gentle manner and samples were taken for protein determination and the interferon test.

Despite the known sensitivity of the polypeptides, such as interferon, to the shear forces occurring under mechanical influences (Proc. Soc. Exp. Biol. Med. 146, 249-253 [1974]), surprisingly high yields of crude interferon could be achieved under these pH conditions ,

Examination of various fermentation approaches showed that the composition of the mixture changed depending on the fermentation conditions.

In particular, the proportion of the component 1, the methionine-interferon-a, a hardly separable component, changed with the duration of the fermentation: methionine-interferon-a was formed only after 8-9 hours of fermentation. By timely termination of fermentation so can be obtained interferon preparations containing no methionine interferon.

Into the clarified crude solution was added with stirring solid ammonium sulfate up to 65% saturation. After complete dissolution of the ammonium sulfate, the batch was kept cooled overnight, the resulting precipitate was separated and stored at -20 ° C until further use.

From the clear supernatant samples were again taken for the interferon assay to control the precipitation of 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 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.

The combined clear solutions were dialyzed against 0.01 M NaCl with a sterile, pyrogen-free dialysis cartridge. The osmolarity of the interferon solution should be about 390-430 mOsmol / l after 2-3 times throughput. Aliquots were taken from the clarified solution for the interferon assay.

For further purification "tandem chromatography" was used, a combination of a cellulose precolumn and a downstream affinity chromatography with highly specific monoclonal antibodies The precolumn, an ion exchange column, was used to keep poorly soluble sample components away from the antibody column: for the precolumn

DE-52 cellulose was well stirred with TRIS / NaCl buffer, pH 7.5, and filled into a chromatography column. The adsorbent was washed with the buffer until the eluate no longer showed pH and osmolarity changes. For the precolumn, 0.5-1.0 g of DE-52 cellulose in 0.025 MTRIS / HCl + 0.2 M NaCl per gram of biomass was used; she was reconditioned for every clean.

For the antibody column, purified mouse monoclonal anti-interferon IgG was coupled to BrCN-activated Sepharose 4B as vehicle. The finished column material was stored in phosphate buffered saline (PBS) with sodium azide in the refrigerator. Prior to first use or after prolonged storage, the antibody column was washed with 0.1 M citric acid in 25% ethylene glycol to remove any soluble content and then washed neutral with PBS. Per gram of biomass, the antibody column required a column volume of 0.2 to 1.0 ml; this column could be used several times. Diedialysierte interferon solution was first pumped over both columns (precolumn and antibody column) and the eluate by measuring the absorbance at 280nm controlled. 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 dropped to V20 of the plateau value. To control that the interferon was bound to the antibody column, the eluate was tested for interferon content. The antibody column was then separated from the precolumn and washed alone with TRIS / NaCl buffer pH 7.5 until no protein was detectable in the eluate.

Elution of the interferon bound to the antibody was done with 0.1 M citric acid in 25% ethylene glycol, monitoring the absorbance of the eluate at 280 nm. The interferon-containing protein peak was collected. Until final cleaning, the interferon pool was stored at -20 ° C. Interferon test, protein determination and Reserve 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 characterizable as IFN. Surprisingly, some of these components were removed by precipitation at pH 4.5 (with ammonia). In particular, the proportions of the components with reduced sulfur bridges, ie the forms with free SH groups (components 5 and 6), were reduced. The analysis of this precipitate shows that the monomeric interferon was entrained only in small amounts.

For final purification, a FPLC apparatus with a MONO-S column, type HR10 / 10 (cation exchanger) was used, which could be loaded with up to 60 mg protein. This column material is a high performance ion exchanger, with excellent separation efficiency 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 sterilized by filtration and worked at room temperature.

The clear supernatant obtained after the precipitation was applied to the column. Of particular importance was the buffer used in the FPLC separation. He had to elute the interferon components clearly distinguishable and subsequently be completely removable. These properties possessed the ammonium acetate buffer used with which the interferon was eluted by a gradient program. Interferon was eluted as a sharp peak with a weak shoulder. Both the "shoulder" fraction (K3) and the later eluted portions (K5-K7) were separated from the main peak of pure interferon, and the pure interferon peak was collected from this aliquot for HPLC analysis, SDS Gel electrophoresis, protein determination, interferon test and endotoxin determination.

By means of this chromatography, virtually all components were separated from the main peak and a homogeneous interferon was obtained, which showed a monomer content of more than 99% in gel permeation HPLC. The reverse-phase HPLC showed only about 1% of the non-native monomer, the chromatofocusing accounted for 2.5% non-native monomer.

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

The volatile buffer could be completely removed by lyophilization. For this purpose, the IFN-pool was filled in portions up to a maximum of 2 ml each in autoclaved lyo-ampoules (volume 8 ml); this corresponded to an amount of 1 to about 8 mg of pure interferon per ampule. The vials were then provided with pre-washed and autoclaved lyo-stoppers and cooled to at least -20 ° C. The lyophilization was carried out at -10 ⁰ C and a vacuum of less than 1 Torr. After removal of the buffer solution, the temperature was raised to 25 ° C and further lyophilized for at least 1 hour. The vacuum was released and the plugs immediately pressed firmly. Provided with aluminum closures, the ampoules were then stored in the refrigerator or at -20 ⁰ C.

As shown, it has become possible for the first time to produce an interferon-α by a clever fermentation regime (relatively early harvesting) together with the purity of more than 98% not only in terms of its interferon content, but also in terms of homogeneity to the various interferon components to more than 95% of native monomeric interferon-α consists.

This high degree of purity and homogeneity also made it possible for the first time to obtain the interferon in solid form, free from salts and buffer components. It is therefore possible for the first time to store α-interferon for months without stabilizers, which offers significant advantages for storage, shipping and, not least, for galenic developments compared to albumin-stabilized interferons. Even after 11 months storage at 4 ° C, no loss of salary could be detected.

The crystalline human leukocyte interferon described in EPA 83734 are crystals of polyethylene glycol as a precipitating agent with interferon, but not pure, homogeneous and crystalline interferon, as the title suggests.

The interferon-α prepared according to the invention was dissolved, as known, by addition of human serum albumin, sterile filtered and aseptically distributed on vials; depending on the application in suitable concentrations.

In clinical investigations, the interferon-α prepared according to the invention proved to be non-immunogenic and highly compatible.

In total, 75 patients were treated with non-immunogenic interferon-α until January 1985: 58 patients with

Tumor indications and 17 with viral indications. _Λ

Antibodies were not stimulated during therapy in any single patient, with treatment durations partially 15 and more, up to 35 weeks.

The process according to the invention made it possible for the first time to provide a high-purity, solid, free from salts and buffer constituents, based on the native, monomeric interferon, and non-immunogenic interferon-a ₂ .

Amino acid sequence analysis by known methods yielded the following amino acid sequence: Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg Me Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr Me Pro Vai Leu His GIu Met Gin Gin He PheAsn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr Giu LeuTyrGln GIn Leu Asn Asp Leu GIu AIa Cys VaI Me Gin GIy VaI GIyVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser Me Leu Ala VaI Arg Lys Tyr Phe GIn Arg HeThr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIu Me Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu

embodiment

The following examples are intended to illustrate the invention without limiting it in any way.

Especially with regard to the fermentation approach, the process according to the invention can be used without restriction within wide limits. Thus, it is also possible to use biomass from other host organisms which yield comparable IFN yields after mechanical disruption and to interferons which react similarly with the EBI-I monoclonal antibody, e.g. B. IFN-O ₁ . Other interferons with less homology to interferon-alpha can also be purified using a corresponding highly specific monoclonal antibody according to the method of the invention.

The present invention relates in detail to a process for the production of recombinant alpha interferon; characterized in that

the host organism containing the interferon gene is cultured under normal conditions, the cells are killed and harvested after the usual growth time,

the expressed interferon is separated in the usual way,

Cell debris can be separated in weakly alkaline

the interferon is concentrated and pre-purified by tandem chromatography, the eluate is adjusted to pH 4.0-4.8 to remove impurities, the interferon is finally purified by chromatography on a cation exchange column with a volatile buffer as eluent and then lyophilized;

characterized in that the host organism is E. coli;

characterized in that the cells are killed after a growing time in which not more than 20%, preferably not more than 5%, more preferably not more than 1% methionine interferon is formed; characterized in that the cells are destroyed in a homogenizer at pH 2 and the interferon is separated; characterized in that the tandem chromatography consists of a cellulose precolumn and an affinity chromatography and that the substance to be purified is washed over both columns with a suitable washing solution and the α- interferon is subsequently eluted from the affinity column with a suitable eluent; characterized in that the precolumn is charged with DE-52 cellulose, the affinity column is loaded with a monoclonal anti-interferon IgG antibody coupled to a support and that the substance to be purified is washed with TRIS-NaCl buffer, pH 7.5, over both columns and the α-interferon is then eluted from the affinity column with 0.1 M citric acid in 25% ethylene glycol;

characterized in that the monoclonal antibody EB11 is used; characterized in that the eluate of tandem chromatography is adjusted to pH 4.5 to remove impurities;

characterized in that a MONO-S type HR10 / 10 cation exchanger is used for the final cleaning; characterized in that, for elution, a low 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 is used.

Process for the preparation of a solid interferon.

Process for the preparation of a non-immunogenic α-interferon.

A process for producing a recombinant, homogeneous, pure α-interferon having less than 5% methionine interferon free of reduced forms and fragments of interferon, containing less than 0.2% oligomers and less than 2% dimers / trimers Contains more than 90% of the monomer content of native monomeric interferon.

A method, characterized in that the host organism that for the human a-interferon of the amino acid sequence Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg Me Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr He Pro VaI Leu His GIu Met He Gin Gin He Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr GIu Leu Tyr GInG Leu Asn Asp Leu GIu AIa Cys Vai Gin GIyVaI GIyVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe GIn Arg HeThr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys Ala Trp Glu VaI VaI Arg Ala Glu Met Arg Ser Phe Ser Leu Ser Thr Asn Leu GInGIu Ser Leu Arg Ser Lys GIu

contains coding gene.

A method for producing a recombinant, homogeneous, pure human a-interferon of the amino acid sequence Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg lie Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro GInGIuGIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thrlie Pro VaI Leu His GIu Met Gin Gin He PheAsn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr GIu LeuTyrGln GIn Leu Asn Asp Leu GIu AIa Cys Vai Gin GIyVaI GIyVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser Me Leu Ala VaI Arg Lys Tyr Phe GIn Arg He Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIu He Met Arg Ser Phe Ser Leu Thr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu

containing less than 5% methionine interferon,

which is free from reduced forms and fragments of the α-interferon containing less than 0.2% oligomers and less than 2% dimers / trimers / tetramers, and wherein the negative monomeric α-interferon each has a disulfide bridge between the cysteines at position 1 and 98 and 29 and 138.

In addition, recombinant interferons, which can be prepared by one of the methods according to the invention, are the subject matter of the present invention. characterized in that it is present in homogeneous, pure form, with less than 20%, preferably less than 5%, more preferably less than 1% methionine interferon; characterized in that it is in homogeneous, pure form, free from reduced shapes and fragments and contains less than 0.2% oligomers and 2% dimers / trimers / tetramers;

free of oligomers and dimers / trimers / tetramers;

wherein more than 90% of the monomer content is native monomeric α-interferon; free from non-monomeric interferon;

characterized in that it does not stimulate antibodies;

characterized in that it is in solid form.

Human α-interferon according to the invention.

α-interferon, characterized in that it is in the amino acid sequence

Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr He Pro VaI Leu His GIu Met HeGInGInIIe Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr GIu LeuTyrGln GIn Leu Asn Asp Leu GIu AIa Cys VaI He Gin GIy VaI GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe GIn Arg He Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIu He Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu

equivalent.

Recombinant human a-interferon, the amino acid sequence

Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr He Pro VaI Leu His GIu Met He Gin Gin He Phe Asn Leu Phe Ser Thr Lys AspSer Ser Ala Ala Trp Asp GIu Thr Leu Leu Asp Lys Phe Tyr Thr Giu LeuTyrGln GIn Leu Asn Asp Leu GIu AIa Cys VaI He Gin GIyVaI GIyVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe GIn Arg He Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIuIIe Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Giu Ser Leu Arg Ser Lys Giu

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

Use of a interferon according to the invention for the therapeutic treatment of viral and tumoral diseases.

Pharmaceutical agent for therapeutic treatment, characterized in that it contains an α-interferon according to the invention and one or more pharmaceutical inert excipients and / or carriers.

Example 1 (batch E. coli; IFN-α ₂ Arg; 28 ^° C.)

a) 251g acid precipitated biomass, which had been stored at -20 ⁰ C, was added to 2 500 ml of 1% acetic acid, stirred for V2h in an ice bath and homogenized 2 times 1 minute using the Ultraturax type 45/6. Polymin P was added to a final concentration of 0.25%, the pH was adjusted to 10.0 with 5 N NaOH and stirred in an ice bath for 2 h, finally the pH was returned to 7.50 with 5N HCl.

Centrifugation for 1 hour in a Christ Cryofuge 6-6S at 4 ° C and 3000 rpm gave a clear crude extract of 2540 ml with an interferon content of 17.1 χ 10 ⁹ LE. (A 100%) and a protein content of 5330 mg, resulting in a specific activity of 3.21 χ 10 ⁶ 1.E./mg protein.

b) Ammonium sulfate was added to 65% saturation (430 g / liter extract). The batch was stored overnight at 4-8 ⁰ C and the precipitate formed in a Beckman J 2-21 high-speed centrifuge rotor JA 10 at 4 ° C, 10000 rpm spun within 1 hour. The clear supernatant, 3120 ml, contained 0.7% of the interferon contained in the crude extract (120 χ 10 ⁶ IU).

The pellet was taken up in 0.01 M NaCl and stirred at 4-8 ⁰ C for 2 h. The pH was adjusted to 7.50 with 5 N NaOH and the solution was clear-centrifuged as described above. The clear solution was dialyzed against 0.01 M NaCl to 390 mOsmol / l with the aid of a dialysis cartridge (Nephross Allegro, Organon Technika). The interferon content was 13.3 χ 10 ⁹ IU (A 77.6%)

c) The dialysate was then chromatographed (tandem chromatography). For the precolumn, 125g DE-52 cellulose powder from Whatman was used in TRIS / NaCl buffer pH7.5 (0.025 M TRIS / HCl + 0.2 M NaCl); this corresponded to 0.5 g of column material / g of biomass. For the affinity column, monoclonal anti-interferon IgG (EB11) coupled to Br-CN activated Sepharose 4B (Pharmacia) was used. 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 neutral with PBS. Per gram of biomass, column volume of 0.2-1, OmI was required in the antibody column. The dialyzed interferon solution was first pumped over both columns (guard columns and antibody column) and the eluate was monitored by measuring the absorbance at 280 nm. After application of the interferon solution was washed with TRIS / NaCl buffer pH 7.5 until the amount of protein in the eluate had fallen to V20 of the plateau value. The antibody column was then separated from the precolumn and washed alone with TRIS / NaCl buffer pH 7.5 until no protein was detectable in the eluate.

The elution of the interferon bound to the antibody was carried out with 0.1 M citric acid in 25% ethylene glycol, the extinction of the eluate was again monitored at 280 nm. The interferon-containing protein peak was collected. 16.8 ml of eluate with an interferon content of 12.3 × 10 ⁹ IU (A 71.9%) were obtained. The total amount of protein was 54.4 mg, resulting in a specific activity of 226 × 10 ⁶ IU / mg protein.

d) For further purification, the eluate was adjusted to pH 4.5 with ammonia and the precipitate formed was separated off. The clear supernatant (18.3 ml) contained 46.3 mg of protein and an interferon content of 11.8 × 10 ⁹ IU, based on the crude protein. This corresponded to a yield of 69% (255 × 10 ⁶ IU / mg protein)

e) For final cleaning, an FPLC apparatus with a MONO-S column, type HR10 / 10 (cation exchanger) was used.

The clear supernatant obtained after the precipitation was applied to the column prewashed with 0.1 M ammonium acetate buffer pH 4.5-5.0 and washed until the absorbance at 280 nm returned to baseline was. The elution of adsorbed interferon was done with a shallow salt gradient by admixing 0.5 M ammonium acetate buffer, pH 4.5-5.0. Interferon was eluted as a sharp peak. Both the "shoulder" fraction (K3) and the later eluted portions (K5-K7) were separated from the main peak of pure interferon, and the pure interferon peak was collected and this aliquot for HPLC analysis, SDS In the "shoulder" fraction (9.1 ml) a total of 4.1 mg of protein was detected; the interferon content was 1.33 χ 10 ^s IU (7.7%). This resulted in 324 × 10 ⁶ IU / mg protein.

The 9.8 ml main pool contained 5.18 × 10 ⁹ IU (30.3%) purest interferon and a total of 16.1 mg protein; this resulted in a specific activity of 322 χ 10 ⁶ IU / mg protein.

f; a) The lFN-pool was filled in portions up to a maximum of 2 ml each in autoclaved lyo-ampoules (volume 8 ml), which corresponded per ampoule to an amount of 1 up to about 8 mg of pure interferon. The vials were then provided with pre-washed and autoclaved lyo-stoppers and cooled to at least -20 ° C. The lyophilization was carried out at -10 ⁰ C and a vacuum of less than 1 Torr. After removal of the buffer solution, the temperature was raised to 25 ⁰ C and continued for at least 1 hour. The vacuum was released and the plugs immediately pressed firmly. Provided with aluminum closures, the ampoules were then stored in the refrigerator or at -2O ⁰ C.

ß) To test the stability, four different fermentation mixtures were used, independently of Example 1 a-f; a, but similarly purified, lyophilized.

The lyophilisates were dissolved in IRMA dilution buffer and analyzed by NK2-IRMA for human IFN alpha. Charge IFN-Titervor IFN-Titernach ±%

Lyophilization lyophilization

A 720x10 ⁶ 754x10 ⁶ + 5 B 1337X10 ⁶ 1526x10 ⁶ + 14 C 981x10 ⁶ 852x10 ° -13 D 1230X10 ⁶ 1 149x10 ^e -7

The lyophilization therefore gave no losses. After 11 months of storage of the lyophilisate at about 4 ⁰ C (refrigerator) was dissolved in 0.1 M ammonium acetate and both purity checked (by gel permeation-HPLC) as well as to content (by the NK2-IRMA assay).

before lyophilization after 11 months storage

alsLyophilisat (4 ° C)

Purity (gel-HPLC) 98.5% 98.7

IFN titer 1510x10 ^e unit / ml 1464x 10 ⁶ units / ml

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 HB101, 28 ° C.) after 8.9, 10 or 11 hours, filled with acid at pH 2 according to the usual Tecknik method and after processed and analyzed methodology according to the invention. The following table shows the content of the samples at K1, K2 and K3 (K1: Met-IFN, K2: native IFN, K3: non-native IFN). The values were determined by means of chromatofocusing

Harvest time after hours JJ 9 10 11

g wet biomass per liter of culture 14 15 18 21 mg IFN / g biomass (measured in crude extract) 0.28 0.21 0.16 0.12 mg IFN / I culture volume 3.9 3.2 2.9 2.5 K1 pl = 5.78 0.7% n.d. * 10.7% 19.4% K 2 pl = 5.64 96.2% 97.5% 86.1% 78.5%

K 3 pl = 5.49 3.1% 2.5% 3.3% 2.1%

* N.D.. = not detected = not detectable

Example 3 Coupling of the EB11 antibody to CNBr-activated Sepharose 4B (cf DE-OS 3306060)

The EBI-1 antibody was first dissolved with 0.5 M NaCl / 0.2 M NaHCO ₃ , pH 8.4 (in as little buffer as possible) and dialyzed against the buffer until no sulfate was present in the external solution with barium chloride. I could show more ions. Careful removal of the ammonium sulfate was absolutely necessary because ammonium ions interfere with subsequent coupling to the carrier. The protein concentration was then adjusted to 5 mg / ml with buffer. For coupling, CNBr-activated Sepharose 4B was used as support. This was prewashed according to the manufacturer's instructions. For every 25 mg of EBI-I antibody, 1 g of activated Sepharose was used. The coupling was carried out in the above buffer, pH 8.4 at room temperature for 2 hours. Afterwards, the EBI-I Sepharose was filtered by suction and washed. The filtrate should not contain more than 5% of the EBI-I antibody used. The finished EBI-I Sepharose was stored in PBS / Azide in the refrigerator PBS / Azide:

PBS: 7.30 g sodium chloride pA (Merck 6404) '3.00 g Na ₂ HPO ₄ x 2 H ₂ O pA (Merck 6580)

1.15 g NaH ₂ PO ₄ × H ₂ O pA (Merck 6346) and made up to 1 000 ml; pH 7.0 azide: In addition, 1.0 g / l of sodium azide in pure form (Merck 6688) were added to PBS. The final solution was sterile filtered (0.2 micron pore size) and stored in the refrigerator.

Interferon antibody assay (neutralization assay) material

cell

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

Encephalomyocarditis virus (EMC), ATCC VR129.

Interferon standard

HS-11 (1 ampoule "HS-11" = lyophilized Hu IFN ra-A, taken in 1.2 ml H ₂ O, gives 12,000 iU / ml)

Tissue culture plates

96 wells with lid, flat bottom, cup diameter 6.4mm, tissue culture treated.

media

DMEM = Dulbecco's modified Eagle medium, with glutamine, without sodium carbonate, FlowKat. No. 10-331-24 (IF-017D) HEPES Sigma no. H-3375

TRICINE Calbiochem. No. 33468, A grade

FCS = fetal calf serum

HUMANSERUM ALBUMIN 20%, for infusion

Antibiotic, tiamulin hydrogen fumarate

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

+ 13mM HEPES + 6mM TRICINE + 1.6g / l NaHCO ₃ without antibiotics pH 7.2-7.4

Assay medium: as growth medium, but 5% FCS instead of 10% and adding 5 / xgTiamulin / ml dilution medium: growth medium without serum, containing 5 / xg / ml tiamulin virus medium: growth medium without serum containing 5yu.g / ml tiamulin and with 3.5mg / ml human serum albumin

Methyl violet stock solution:

Methyl violet 6g

Ethanol 100ml

Dissolve and filter at approx. 50 ^° C. Methyl violet working solution

Stock solution 50ml

Water (pH neutral) 950 ml

The cells were treated as a permanent cell line. Propagated by trypsinization and dilution in the growth medium. For the assay, the cells were counted in a hemacytometer and suspended in assay medium to give a challenge of 4-5χ10 ⁴ cells per ml per well; this was distributed on the plates. It was incubated in an atmosphere of 5% CO ₂ and 80% humidity at 37 ° C.

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

1000,1: For the control plate HS-11 dilutions of 1 was added 2000 ... up to 1: 32000 incubated at 37 ⁰ C. 1

For the sample plate, the serum samples were set at 1: 2.1: 4.1: 8 ... to 1:64 with a dilution medium containing as much HS-11 to a final concentration of 10μU HS-11 / ml in each jar obtained, diluted and incubated at 37 ⁰ C for 1 h as well.

The plates were decanted and each well was filled with 100 ml of the dilution medium (pure 2,3,10 and 11) or with 100 μl of the dilutions (series 4-9). The plates were incubated at 37 ° C. for ⁴ hours as above.

Thereafter, the plates were overlaid (without virus) per well and 50 μ \ of virus dilution (series 3.11, 4-9) with 100μΙ of the virus medium to reach a cytopathic effect of approximately 90% within 36 hours, and again incubated. After 24 h and microscopic control, the cells were stained with methyl violet.

The results are shown in Figures 12a-12f: the scheme is attached.

METHODOLOGY

The following methods were used for the analysis:

protein determination

BIORAD PROTEIN ASSAY: This assay uses the Coomassie Brilliant Blue dye and measures the protein-dye complex at 595 nm. The standard used is bovine serum albumin.

Planimetric determination of 214nm peak areas recorded on gel permeation HPLC. The conversion was carried out using a factor from the calibration substances bovine serum albumin, ovalbumin, trypsinogen and lysozyme. This determination was made especially for the preparations after the purification step tandem chromatography, pH4.5 precipitation and FPLC on MONO-S additionally or exclusively.

interferon determination

The commercially available "NK ₂ -IRMA" for human interferon alpha was used as standard, a laboratory standard "HS 11", which with the aid of a biological assay (plaque reduction test WISH cells and vesiculate stomatitis virus) to the International Standard B 69/19 was set.

SDS gel electrophoresis

The method was used by LAEMMLI (Nature 227,680, [1980]). The dye used to stain the proteins was Coomassie Brilliant Blue. In the purity controls of the: interferon preparations were used in each case 20 micrograms.

chromatofocusing

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. in which a MONO-P Chromatofokussiersäule HR 5/20 (Pharmacia) in a pH range of 4-7 was used.The buffers contained instead of the indicated addition of 25% 1,2-propanediol 25% acetonitrile to increase the flow rate The protein concentration was recorded at 280 nm, pH registration was automatic, the samples to be analyzed were lyophilized, dissolved in water at 1 mg / ml, and then diluted with 5 volumes of Buffer A (pH 7.1). 2-1.0 mg of interferon per analysis were used.

Gel permeation - HPLC {high pressure liquid chromatography) Stationary phase:

WATERS 1-125; 2 χ (300mm χ 7.8mm);

ΙΟμ, ιτι particle diameter mobile phase:

0.5MNa ₂ SO ₄

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

0.04% Tween 20

25% propylene glycol flow rate:

0.5ml / min detection:

UV absorption at 214 nm molecular weight calibration:

Bovine serum albumine M 66 000

Ovalbumin M 45 000

Trypsinogen Μ 24 000 '

  Lysozyme M 14 300

Reverse Phase HPCL (High Pressure Liquid Chromatography)

Stationary phase::

Bakerbond WP C18; 250mm χ 4.6mm; '~

5 / xm particle diameter; 30nm pore diameter MobilePhase:

A: 0.1% trifluoroacetic acid in water, pH 2.2 B: 0.1% trifluoroacetic acid in acetonitrile Gradient program:

0-2min: 45% B 2-32min: 45-53% B 32-40min: 53% B 40-50min: 45% B Flow Rate:

1 ml / min N,

detection:

UV adsorption at 214nm

Lecende to the pictures

Fig. 1 Chromatogram of the reverse phase HPLC of the acidic eluate after tandem chromatography: Representation of components K1-K7;

Fig. 2: chromatogram of the gel permeation HPLC of the acidic eluate after tandem chromatography; Fig. 3: Chromatogram of reverse phase HPLC after precipitation at pH 4.5; Representation of the components K1, K 2, K3 and K6;

Fig. 4: Chromatogram of gel permeation HPLC after precipitation at pH 4.5; FIG. 5: Chromatogram of the FPLC on MONO-S at pH 4.5 with the aid of an ammonium acetate gradient of 0.1-0.5 M; Fig. 6: Chromatogram of reverse phase HPLC of the "shoulder fraction" of the MONO S peak Fig. 7: Chromatogram of gel permeation HPLC of the "shoulder fraction" of the MONO S peak; Fig. 8: Chromatogram of the reverse phase HPLC of the "main fraction" of the MONO S peak; Fig. 9: Chromatogram of the gel permeation HPLC of the "main fraction" of the MONO S peak;

10: chromatogram of the chromatofocusing of the "main fraction" of the MONO S peak, Fig. 11: Photo of the gel electrophoresis of the acid eluate after tandem chromatography and the components K1-K7 separated by reverse phase HPLC.

Fig. 12 a Results of anti-IFN-α antibodies; Investigations in various indications bis12f:

Test Type: Neutralization Assay (10iU / ml IFN-a / A-599 / EMC)

Total number of patients: 75_

Number of patients with tumor indications:. 58

Number of patients without stimulated antibodies 58

Number of patients with virus Indications: 17

Number of patients without stimulated antibodies 17

Fig. 13: Scheme for the anti-interferon a-antibody assay; Table 1: Overview of the cleaning; Table 2: Evaluation of purity by HPLC.

table

cleaning stage

VoI CmI) mg of protein

IFN _A (IE)

I.E.IFN / mg protein% yield

crude extract

2540 17,1 χ 10 ⁹ 3,21 χ ΙΟ ⁶

100

Arnmonsul fat supernatant

Pellet after dissolution + dialysis

3120

282

o, 12 χ 10

nb. 13.3 χ 10 ⁹

0.7

77.6

Eluate of tandem chromatography

12.3-x 10 '

χ IO

71.9

CO CIJ

survived the pH 4.5 precipitation

11.8 χ 10 '

x 10

; 6

69.0

FPLC to Mono-S

a) "shoulder" fraction

b) Main fraction

4.1 1.33 χ 10 "16.1 5.18 χ 10 ^S

χ 10 χ ΙΟ ⁶

7.7

30.3

n.d. = not determined

T. E. = International Units

table

Gelperinetations HPLC

IFN (mg / ml) purity of MW> 70,000 monomers (MW 19,000)

Acid eluate after tandem chromato- 2,14 traphy

Supernatant after

Precipitation at pH 4.5 2.0 after HPLC on MONO-S

a) Shoulder Frakt. 0.45 b) Main fract. 1.64

74 %

79 %

99 % 99 %

On G '

, Z / u

U, Z , o

Reverse phase HPLC **

K 2 + 1 K3

'K4

.. 100 7.0 n.n.

4.0 n.n

K5

l <6

1.6 22.8

<1 5.4

100 16 ο. η • η η .η η. η η .η 100 1, 2 η .η η .η η. η η .η

* not detectable ** Relative peak heights (214 ran), K 2 * 1 = 100 %

Claims (7)

1. A process for the production of recombinant α-interferon, characterized in that the interferon gene, preferably that for the human a-interferon of the amino acid sequence. Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg Me Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr Ne Pro VaI Leu His GIu Met 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 GIu Ala Cys VaI lie Gin GIy VaI GIy VaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser lie Leu Ala VaI Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Lys GIu Lys Lys Tyr Ser Pro Cys Ala Trp GIu VaI VaI Arg Ala GIu He Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu-encoding gene containing host organism, preferably E. coli, is cultured under normal conditions, the cells after usual growth time, preferably after a growth time in which not more than 20% , preferably not more than 5%, more preferably not more than 1% methionine interferon is formed, killed preferably the cells are destroyed and harvested in a homogenizer at pH 2, the interferon is separated in the usual way, cell debris is separated in the weakly alkaline, the interferon is concentrated and pre-purified by tandem chromatography, the eluate is removed to remove impurities to pH 4.0-4.8, the interferon is purified by chromatography on a cation exchange column, preferably with a MONO-S type HR10 / 10 cation exchanger with a volatile buffer as eluent, preferably with a shallow concentration gradient prepared from 0.1-1, OM , 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. The method according to item 1, characterized in that the tandem chromatography of a cellulose precolumn, preferably with DE-52 cellulose and an affinity chromatography, preferably with a monoclonal anit interferon IgG antibody coupled to a carrier, particularly preferably with the monoclonal Antibody EB11 and that the substance to be purified is washed over both columns with a suitable washing solution, preferably with a TRIS-NaCl buffer pH 7.5 and the α-interferon then with a suitable eluent, preferably with 0.1 M citric acid in 25 % ethylene glycol is eluted from the affinity column. 3. The method according to any one of the preceding points, characterized in that the eluate of the tandem chromatography for the separation of impurities is adjusted to pH 4.5. 4. Method according to one of the preceding points, characterized in that a recombinant, homogeneous, pure α-interferon is produced, which has less than 5% methionine interferon, which is free of reduced forms and fragments of the interferon, which is less than 0 , 2% oligomers and less than 2% dimers / trimers / tetramers, and wherein more than 90% of the monomer content is native monomeric interferon. 5. The method according to any one of the preceding points, characterized in that a recombinant, homogeneous, pure human a-interferon of the amino acid sequence Cys Asp Leu Pro GIn Thr His Ser Leu GIy Ser Arg Arg Thr Leu Met Leu Leu Ala GIn Met Arg Arg He Ser Lei Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe GIy Phe Pro Gin GIu GIu Phe GIy Asn GIn Phe GIn Lys Ala GIu Thr He Pro Vai Leu His GIu Met Gin Gin He Phe Asn Leu Phe SerThr Lys Asp Ser Ser Ala Ala Trp Asp Giu Thr Leu Leu Asp Lys Phe Tyr Giu Leu Tyr GIn GIn Leu Asn Asp Leu GIu AIa Cys Vai Gin GIyVaI GIyVaI Thr GIu Thr Pro Leu Met Lys GIu Asp Ser He Leu Ala VaI Arg Lys Tyr Phe GIn Arg He Thr Leu Tyr Leu Lys GIu Lys Lys Thr Ser Pro Cys AIa Trp GIu VaI VaI Arg Ala GIu He Met Arg Ser Phe Ser Leu SerThr Asn Leu Gin GIu Ser Leu Arg Ser Lys GIu which has less than 5% methionine interferon, which is free of reduced forms and fragments of the alpha interferon, the less contains 0.2% oligomers and less than 2% dimers / trimers / tetramers, and wherein the native monomeric α-interferon each has a disulfide bridge between the cysteines at positions 1 and 98 and 29 and 138. 6. The method according to any one of the preceding points, characterized in that a solid a- interferon is produced. 7. The method according to any one of the preceding points, characterized in that a non-immunogenic α-interferon is produced.