CA1340281C - Process for preparing and purifying alpha-interferon - Google Patents

Abstract

This invention relates to a process for the preparation of recombinant interferon wherein the host organism containing the interferon gene is cultivated, the cells are harvested and disrupted, the cell debris is removed, the interferon is concentrated and subjected to preliminary purification 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 exchanger column with a volatile buffer as eluant, and is then lyophilised whereby a highly pure, non-immunogenic, homogeneous .alpha.-interferon having anti-viral and immunoregulatory activities is obtained, to the proteins produced by the process and to the use thereof.

Description

13 1q2~1 BE IT KNOWN THAT GERHARD BODO of Belhofergasse 27/5 A-1120 Wein, Austria; INGRID MAURER-FOGY, Mariennengasse 24/2/20, A-1090 Wein, Austria; EDGAR FALKNER, Strohberggasse 9, A-1120 Wein, Austria; SILVIA JUTTA LINDNER, Sossenstrasse 6, A-2380 Perchtoldsdorf, Austria, all citizens of Austria, having made an invention entitled "Process for preparing and purifying interferon"
the following disclosure contains a correct and full description of the invention and of the best mode known to the inventors for taking advantage of the same.

~ ~0~ 8 1 The present invention relates to a process for preparing a very pure, non-immunogenic, homogeneous ~-interferon having antiviral and immunoregulatory activity, the protein itself and the use thereof.
Interferons are proteins naturally occurring in the body and which have been detected in a great variety of species. Their inherent antiviral and immunoregulatory properties indicated at an early stage that they might be suitable for a wide variety of applications. Tests have shown that there are different classes of interferon. In addition to ~, B and ~ interferons,~ -interferon has recently been discovered and its structure clarified.
The high expectations placed on the interferons as an effective agent against viral diseases and cancer have already led to trials with interferon preparations obtained from natural material, but serious side effects occurred. The preparations used in these trials, even after laborious purification, contained complex mixtures of different interferons and, in many cases, other proteins. The reason for this is that some of the interferons have subtypes differing from one another to a greater or lesser extent; thus, for example, more than 20 different types of ~-interferon are known.
Only by producing interferons by genetic engineering has it been possible to conduct trials with pure types of interferon preparations. These include the recombinant ~2 interferons used in the clinical trials (also known as ~ A). The purification of any human proteins produced by microorganisms is of critical importance. Any contamination originating from the host organism would lead to immune defence reactions if the product were to be used in humans and these could be life-threatening. The removal of contaminants of this kind is now possible and extremely sensitive analytical methods can detect endotoxins in very tiny concentrations. In the field of interferon research methods of purification have been developed with which interferon preparations containing virtually no endotoxins can be obtained. Mention may be made, for example, of the work of Staehelin et al. J. Biol. Chem. 256, 9750 (1981).
All rec. ~-interferons used in clinical trials are virtually free from endotoxins and it was therefore surprising that side effects which were severe enough to cause the interferon treatment to be discontinued should occur. Even some rec. ~-interferons were found to be immunogenic and antibodies against interferon had been stimulated. (Quesada et al.
J. Natl. Cancer Inst. 70, No. 6, 1041-1046 (1983);
Protzman et al. J. Immunol. Methods 75, 317 - 323 (1984)).
These antibodies may lead to serious effects if they influence the action of the interferon.
This is because, in this case, they act not only on the rec. ~-interferon but, since the rec. ~-interferon is identical to the body's own interferon, on the body's own interferon as well.
The disastrous aspect of this is that these antibodies go on acting even after the interferon treatment has ended. They may cause a deterioration in the course of the disease, weaken the body's own defences against virus infections and thus make the organism even more susceptible to other infections.
These effects have already been confirmed in tests on animals. Therefore, with a view to maximum safety of drug treatment, it is essential that the rec. ~2 interferon must be of a pure type, virtually free from endotoxins and, not least, non-immunogenic.
One object of this invention was therefore to develop a process for preparing a non-immunogenic rec. ~-interferon with antiviral and immunoregulatory activities.
The caus~e of the immunogenicity of the abovementioned ~-interferons is not known. The only fact that can be ruled out is that endotoxic contaminants are responsible.
The preparations used for trials differ primarily by slight variations in their amino acid sequences.
Amino acid 23 Amino acid 34 Preparation I: Lysine Histidine Preparation II: Arginine Histidine Preparation III: Arginine Arginine Apart from the structural differences in the primary structure of the proteins used in clinical trials, it is known that the ~-interferons produced by genetic engineering always consist of a mixture of monomeric, shortened-molecular, reduced and oligomeric forms of interferon (see for example EPA 108 585 (published May 16th, 1984), 110 302 (published June 13th, 1984) and 118 808) (published September l9th, 1984). Some of these forms show the same activities in vitro but others show reduced activit-ies and some are reputed to have immunogenic properties (see EPA
108 585 and 110 302).
These patent applications describe processes for separat-ing these forms of interferon.
EPA 108 585 describes a process for separating a "slow moving monomer" and oligomers wherein the interferon probe is incubated for some time at a temperature of 28-40~C at a pH of 3 to 5.

02~1 EPA 110 302 describes a process wherein the monomer is formed from the oligomers by reduction with a redox system.
Finally, in EPA 118 808, recombinant ~-interferon is purified with the aid of metal chelate resins from the oligomeric forms.

- 4a -The interferons obtained by these methods are supposed to contain monomeric interferon in virtually quantitative form; however, there are no tests of immunogenicity.
Our own detailed analytical investigations have shown that ~-interferons prepared by recombinant methods are a mixture of different forms of interferon in fluctuating concentrations. These include oligomers, tetramers, trimers and dimers of interferon, methionine interferon, reduced forms and fragments of interferon and, surprisingly, various monomeric forms of interferon as well.
The oligomers are ~-interferons with a molecular weight ~ 70,000, the reduced ~-interferons are the protein with free SH groups and the methionine interferon is an ~-interferon which additionally carries a methionine at the N-terminal end (caused by the microbiological method of preparation of the ~-interferon). Analysis has shown that the various monomeric forms are different S-S isomers of ~-interferon.
In order to distinguish between these isomers linguistically, the term non-native monomer will be used hereinafter for the isomers of the predominantly occurring ~-interferon monomer and the latter will be referred to as native monomeric ~-interferon.
However, this should not exclude the possibility that the non-native monomers may also exist in "natural n material.
In an E. coli fermentation mixture for preparing ~2 interferon, for example, 7 different interferon components could be detected (K 1 - K 7). Analysis showed that these were oligomers, dimers and trimers, methionine interferon, reduced interferons and an S-S isomer of native monomeric ~2 interferon which had a disulphide bridge between the amino acids at positions 1 and 98 and 29 and 138 (see . . .

6 ~L34Q281 also Wetzel et al. J. Interferon Res., Vol 1 No. 3,381 - 391 (1981).
As already stated, the cause of the immunogenicity of rec.
~-interferon is not known. However, it is also obvious that all the forms of ~-interferon which differ from the body's own interferon have an immunogenic activity. These forms also include the shortened molecules, the dimers and oligomers and also the non-native monomers which contain differently linked disulphide bridges.
While the causes of immunogenicity remain unclear conditions which might promote the formation of these forms with their unknown effects should not be used in processes for preparing rec.
~-interferons. This means that even the purification of interferon must be carried out under the mildest possible conditions which would not endanger nativity. Elevated temperature and reducing agents are not among these conditions.
Obviously, efforts must be made during all steps of the purification to ensure that no foreign substances are introduced, for example as a result of the use of metal chelate resins or similar problematic reagents.
According to one feature of the present invention there is provided a process for the preparation of recombinant ~-interferon wherein the host organism containing the interferon gene is cultivated, the cells are harvested and disrupted, the cell debris is removed, the interferon is concentrated and subjected to preliminary purification by tandem chromatography comprising a combination of cellulose preliminary column and an affinity column ~, .. .. , .. . ... . . ,, .. .. , . . ~

6a 1~ 2~1 the elute is adjusted, if required to pH 4.0-4.8 to remove impurities, the interferon is finally purified by chromatography on a cation exchanger column with a volatile buffer as eluant, and is then lyophilised.
The process according to the invention is . . , ~ . _ .

iO2,~1 suitable for the preparation and purification of interferons, especially ~nterferons, from different species, such as, for example, human or animal ~-interferons. The host organism used for the preparation may be a prokaryote or eukaryote, such as, e.g. E. coli or Saccharomyces cerevisiae, preferably E. coli. The conditions of cultivation for the various host organisms are well known to those skilled in the art.
Surprisingly, it has been found that the growth time not only affects the yeild of ~-interferon but is also a crucial fact-or in determining the composition of the interferon mixture. Thus,if E. coli is used, the composition of the interferon mixture changes with regard to the quantity of methionine interferon depending on the duration of growth.
Advantageously, therefore, the fermentation mixture is checked at short intervals for the formation of ~-interferon derivatives produced by the host organism, as an indicator of the best growth time. Methionine interferon may be used as an indic-ator of this kind. Therefore, by discontinuing the process at the appropriate time, for example after the formation of less than 20%, preferably less than 5%, more particularly less than 1% of methion-ine interferon, a particularly pure interferon is obtained with ideal prerequisites for the subsequent purification process accord-ing to the invention.
The process is particularly suitable for the preparation of acid-stable ~-interferon. For example, the process according to EPA-173924 (published on March 12, 1986) wherein the cells are disrupted in a homogenizer at a pH of 2 may be used.

, . . . . . . .

~0~1 Therefore, in one aspect this invention provides a process for preparing a substantially non-immunogenic interferon, the improvement which comprises purifying said interferon by tandem chromatography to yield an eluate containing said interferon having less than 20% methionine, said eluate being adjusted, if required, to a pH of 4.0 to 4.8 to remove impurities, said tandem chromatography comprising a combination of a diethylaminoethyl cellulose preliminary column and an affinity column consisting of a monoclonal anti-interferon IgG coupled to Sepharose*.
The majority of the impurities can surprisingly be removed by tandem chromatography, i.e. with successive chromatographic stages using different adsorption agents with suitable washing and eluting solutions. Preferably, a combination of a cellulose preliminary column directly connected to an affinity column is used. It is particularly preferred to use a DE-52* cellulose with a monoclonal anti-interferon IgG-antibody, such as, for example the EBI 1 antibody described in DE-OS 33 06 060 published on August 23rd, 1984, coupled to a carrier such as Sepharose*.
The invention further provides a process for preparing recombinant interferon which process comprises (a) cultivating cells of a host organism containing an interferon gene to synthesise interferon, (b) harvesting and disrupting said cells, removing cell debris and subsequently concentrating the interferon obtained thereby, *Trade-mark .

. .

8a (c) purifying said interferon by tandem chromatography consisting of a diethylaminoethyl cellulose column and a monoclonal anti-interferon IgG coupled to a Sepharose* column and obtaining said interferon having less than 20% methionine in an eluate, (d) adjusting the pH of the eluate to 4.0 to 4.8 to remove impurities, (e) further purifying the interferon in a cation exchanger MONO-S* column using an ammonium acetate buffer as an eluant, and (f) lyophilising the eluant to yield said recombinant interferon.
Preferably the cation exchanger comprises hydrophilic polymer beads with an extremely narrow particle size distribution.
A TRIS/NaCl buffer pH 7.5 has proved suitable as a washing solution, but it is possible to use washing solutions which do not affect the binding of the interferon to the antibody but wash out the contaminants and leave those constituents which have a negative effect on the properties of the antibody column bound to the preliminary column.
A suitable eluant for interferon is, for example, a buffer solution consisting of 0.1 M citric acid in 25%
ethyleneglycol, but other eluants having similar properties are also suitable. In general, the eluant must be matched to the particular a-interferon which is to be purified.
Surprisingly, some of the impurities in the "tandem eluate" could be removed by buffering the pH value, preferably to pH 4.0 - 4.8, more particularly pH 4.5. The pH value should be *Trade-mark ,, 8b selected so that there is as little monomeric a-interferon as possible in the precipitate.
Final purification of the interferon was achieved by chromatography using a cation exchanger, preferably a MONO-S*, type HR 10/10 (Messrs. Pharmacia) cation exchanger. A flat graduated gradient with a volatile buffer such as an ammonium acetate buffer in which the pH was kept constant and the concentration was varied (concentration gradient) was used to elute the highly purified a-interferon. It would be equally possible to keep the concentration constant *Trade-mark 2 ~ 1 g and vary the pH (pH gradient). The crucial point is that the eluant should be capable of removing any interferon contaminants, particularly S-S isomers of the main monomer which occurs. A linear concentration gradient of an ammonium acetate buffer produced from 0.1 to 1.0 M, preferably 0.1 to 0.5 M ammonium acetate in a pH range of from 4.0 to 5.0, preferably pH 4.5, is particularly suitable for this purpose.
This buffer can moreover be removed by lyophilisation so that the highly purified ~-interferons can be obtained for the first time in solid form free from buffer salts and precipitation agents.
The process according to the invention is particularly suitable for the preparation of ~-interferons of various species which do not stimulateany antibodies when administered to the corresponding species.
The process according to the invention has proved particularly advantageous in the preparation of a recombinant, homogeneous, pure, solid and non-immunogenic ~-interferon which is_free from reduced forms and fragments of interferon, which contains less than 0.2% oligomer and less than 2~ dimer/trimer/tetramer and less than 5% methionine interferon and wherein more than 90% of the monomer content consists of native monomeric ~-interferon.
The process according to the invention may be used particularly advantageously for the preparation of an ~-interferon as described above wherein the host organism contains the gene which codes for human ~-interferon according to the amino acid sequence Cys Asp Leu Pro Gln Thc His ~er Leu Glv Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arq Ar9 lle.Ser Leu ?he Ser Cys Leu Lvs ~sp Arg Aca Asp ?he Gly Phe Pro Gln Glu Glu Phe Glv Asr.
Gln ?he Gln Lys Ala Glu Thc Jle Pro Val Leù His Glu Me~ lle Gln Gln Ile Phe ~sn Leu ?he Se~ Thr 'vs ,~sp Ser Ser Ala Ala Trp Asp Glu T~. Leu Leu ~52 Lys Ph~ ,vr Thr Glu Leu Ty~ Gln Gln Leu Asn ~sp Leu Glu Ala Cvs Val lle Gln Glv Val Gly Val ,.~r Glu Thr ?ro Leu .~e~ Lys Gi~ ~s Ser ~le Leu ~ 1 2 Val ~rg Lvs Tyr Phe Gln ~r9 lle T~r Leu Tyr Leu r vs Glu Lvs Lys Tyr Ser Pco Cys Ala Trp Glu Val Val .~9 ~la Glu Ile Met Acg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Ar~ Ser Lys Glu and the native monomeric ~-interferon contains disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.
This invention also relates to recombinant ~-interferons which can be prepared using the process according to the invention, preferably recombinant ~-interferons in homogeneous pure form which contain less than 20%, preferably less than 5%, more particularly less than 1% of methionine interferon.
Depending on the choice of host organism and the nature of the fermentation conditions, ~-interferons prepared by the recombinant method may contain, in addition to methionine interferon, di-, tri-, tetra- and oligomers, reduced forms and fragments and S-S isomers of the predominantly occurring monomeric ~-interferon in fluctuating quantities which can be suppressed or eliminated for the first time using the process according to the invention.
The exceptionally pure recombinant ~-interferon which may be prepared by the process of our invention is believed to be novel per se, since we know of no prior art process capable of yielding an equivalent product. A further feature of the present invention therefore provldes recomblnant a- lnterferons in substantlally pure homogeneous form whlch contaln less than 20%, preferably less than 5%, more partlcularly less than 1% methlonlne lnterferon, whlch are substantlally free from reduced forms and fragments of a-lnterferon, whlch contaln less than 0.2%
ollgomer and less than 2% dlmer/trlmer~tetramer, preferably no ollgomers, tetramers, trlmers or dlmers, and whlch preferably contaln more than 90% of the natlve monomer and preferably are substantlally entlrely free from S-S lsomers of natlve monomerlc a-lnterferon.
A stlll further feature of the present lnventlon provldes recomblnant a-lnterferons of varlous specles described whlch, when admlnlstered to partlcular specles, do not stlmulate any antlbodles.
The lnterferons of our lnventlon are preferably ln solld form.
A recomblnant human a-lnterferon wlth the propertles descrlbed above, preferably a recomblnant human a-lnterferon accordlng to the amlno acld sequence Cys Asp Leu Pro Gln Thr Hls Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu Hls Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg X

~3 1~2,~1 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu.
ls preferred.
A recombinant, non-lmmunogenlc, solld human a-lnterferon whlch corresponds to the amlno acld sequence Cys Asp Leu Pro Gln Thr Hls Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu Hls Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu and ls present ln pure homogeneous form contalnlng less than 5% methlonlne lnterferon, whlch ls free from reduced forms and fragments of lnterferon, wlth less than 0.2% ollgomer and less than 2% dlmer/trlmer/tetramer and whereln more than 95% of the monomer content conslsts of the natlve monomerlc a-lnterferon wlth dlsulphlde brldges between the cystelnes at posltlons 1 and 98 and 29 and 138, ls partlcularly preferred.
The process accordlng to the lnventlon enables lmpurltles and lnterferon contamlnants to be removed under X

, . . . . , ~ ..... . ,. . . _ - 13 ~ 281 very mlld condltlons. Thls wlll be explalned more fully uslng the example of the a2Arg lnterferon accordlng to the amlno acld sequence Cys Asp Leu Pro Gln Thr Hls Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu Hls Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Set Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu but other a-lnterferons may also be prepared and purlfled uslng the process accordlng to the lnventlon, lf necessary wlth sllght non-lnventlve modlflcatlons.
The acld-preclpltated deep frozen blomass was thawed and taken up ln ln 1% acetlc acld. Thls and all subsequent operatlons were carrled out at 5~C.
The proteln was extracted from the cells, as descrlbed ln detall ln EPA-173924, by breaklng up the bacterlal cells ln a homogenlser, addlng a preclpltatlon ad~uvant such as polyethylenelmlne PEI-600 ln a concentratlon ranglng from 0.1 to 0.25%, ad~ustlng the pH to 7.5 - 10.0 wlth the ald of NaOH and stlrrlng the suspenslon for several hours.

- 13a - ~3~
The pH was then ad~usted to 7.5, the crude extract was clarlfied under mlld condltlons and samples were taken for determlning the proteln and for the lnterferon test.
In splte of the known vulnerablllty of polypeptldes such as lnterferon to the shearlng forces whlch occur under mechanlcal lnfluences (Proc. Soc. Esp. Blol. Med. 146, 249 -253 (1974)) surprlslngly X

. . .

- 14 - ~ 2~1 high yields of crude interferon could be achieved using this process under these pH conditions.
Examination of different fermentation batches showed that the composition of the mixture varied as a function of the fermentation conditions.
In particular the proportion of component 1, namely methionine ~-interferon, which is a very difficult component to remove, varied with the duration of fermentation: methionine ~-interferon was only formed after 8 - 9 hours of fermentation.
By stopping fermentation in good time, therefore, it was possible to obtain interferon preparations which contained no methionine interferon.
Solid ammonium sulphate was added to the clarified crude solution up to 65% saturation.
After all the ammonium sulphate had dissolved the mixture was kept cool overnight, the precipitate formed was separated off and stored at -20~C until required.
Samples for the interferon test were again taken from the clear supernatant in order to monitor the precipitation of the interferon. Not more than 5% of the interferon should remain in the supernatant.
The ammonium sulphate pellet was dissolved in 0.01 M NaCl, the pH was adjusted to 7.5 with NaOH and the solution was stirred for 2 hours.
The insoluble fraction was removed and possibly extracted once more with 0.01 M NaCl.
The combined clear solutions were dialysed with 0.01 M NaCl using a sterile, pyrogen-free dialysis cartridge. The osmolarity of the interferon solution should be about 390 - 430 mOsmol/l after running through two to three times. Aliquots for the interferon test were taken from the clarified solution.
"Tandem chromatography" was used for the further purification: a combination of a cellulose preliminary column and subsequent affinity chromatography with highly specific monoclonal antibodies. The preliminary column, an ion exchanger column, was used to keep any difficultly soluble sample constituents away from the antibody column. For the preliminary column, DE-52 cellulose (Messrs. Whatman) was stirred thoroughly with TRIS/NaCl buffer, pH 7.5, and introduced into a chromatography column. The adsorbent was washed with the buffer until the eluate showed no further changes in pH and osmolarity. For the preliminary column, 0.5 - 1.0 g of DE 52 cellulose, 0.025 M TRIS/HCl + 0.2 M NaCl were used per gram of biomass; it was freshly prepared for each purification.
For the antibody column, purified monoclonal anti-interferon-IgG obtained from mouse ascites was coupled to BrCN activated Sepharose 4B (Messrs.
Pharmacia) as the carrier. The finished column material was stored in phosphate-buffered saline solution (PBS) with sodium azide in a cold store.
Before being used for the first time or after a lengthy storage period, the antibody column was washed with 0.1 M citric acid in 25% ethyleneglycol in order to eliminate any soluble components and then washed with PBS until neutral. A column volume of from 0.2 - 1.0 ml was required for each gram of biomass in the antibody column; this column could be used several times. The dialysed interferon solution was first pumped through both columns (preliminary column and antibody column) and the eluate was monitored by measuring the extinction at 280 nm. After the interferon solution had been applied, it was washed with TRIS/NaCl buffer, pH
7.5, until the quantity of protein in the eluate had fallen to 1/20th of the plateau value. In order to check that the interferon had bound to the antibody column, the eluate was tested for , . . . . . . . .

- 16 - ~31~32~1 its interferon content. The antibody column was then separated from the preliminary column and washed on its own with TRIS/NaCl buffer, pH 7.5, until no further protein could be detected in the eluate.
Elution of the interferon bound to the antibody was carried out using 0.1 M citric acid in 25%
aqueous ethyleneglycol, and the extinction of the eluate at 280 nm was monitored. The protein peak containing the interferon was collected. The interferon pool was stored at -20~C until final purification.
An interferon test, protein determination and reverse phase HPLC analysis showed that 60 - 90% pure IFN-~
was obtained after this purification. 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 characterisable as IFN. Surprisingly, some of these components could be removed by precipitation at pH 4.5 (with ammonia). The fractions of the components with reduced sulphur bridges, i.e. the forms with free SH groups (components 5 and 6), in particular, were thus reduced. Analysis of the precipitate shows that only small amounts of the monomeric interferon were carried down.
Final purification was carried out using an FPLC
apparatus made by Messrs. Pharmacia with a MONO-S
column, Type HR 10/10 (Messrs. Pharmacia) cation exchanger, which could be charged with up to 60 mg of protein.
This column material constitutes a high performance ion exchanger with an exceptional separating action and the great advantage that final purification takes only a few hours, in spite of the relatively large quantity of protein to be purified, the buffer solutions could be used after being filtered sterile and the work could be done at ambient temperature.

.. ., . . . . .. , ~ .................................. . .

1 ~ 0~1 The clear supernatant obtained after precipitation was applied to the column. The buffer used in the FPLC separation was particularly important.
It had to be capable of eluting the interferon S components so that they could be clearly distinguished and then it had to be completely removable. The ammonium acetate buffer with which the interferon was eluted through a series of gradients had these properties. Interferon was eluted as a sharp peak with a weak shoulder. Both the "shoulder" fraction (K 3) and also the fractions eluted subsequently (K 5 - K 7) were separated from the main peak of the pure interferon. The peak of pure interferon was collected and aliquots were taken from it for the HPLC analysis, SDS gel electrophoresis, protein determination, interferon testing and endotoxin determination.
By this chromatography, virtually all the components were separated from the main peak and homogeneous interferon was obtained showing a monomer content of over 99% in gel permeation HPLC. Reverse phase HPLC showed only about 1% of non-native monomer and chromatofocussing showed a proportion of 2.5%
of non-native monomer.
The ~ONO-S column was washed before re-use with 0.5 M NACl + 0.1 M Na-phosphate, pH 8.0, in order to eliminate any adsorbed impurities; it was stored in 25% ethanol.
The volatile buffer could be totally removed by lyophilisation. For this, the IFN pool was transferred into autoclaved lyo-ampoules (capacity 8 ml) in batches of not more than 2 ml; this corresponded to a quantity of from 1 to about 8 mg of pure interferon per ampoule. The ampoules were then sealed with pre-washed and autoclaved lyo-stoppers and cooled to at least -20~C. Lyophilisation was carried out at -10~C under a vacuum of less than 1 torr.

After removal of the buffer solution the temperature was increased to 25~C and lyophilisation was continued for at least 1 hour. The vacuum was released and the stoppers were immediately pressed in.
After being sealed with aluminium closures, the ampoules were then stored in the refrigerator or at -20~C.
As has been shown, careful guidance of fermentation (relatively early harvesting) together with the process according to the invention has made it possible for the first time to pre-pare an ~-interferon which not only has a degree of purity of over 98~ with regard to its interferon content but also consists of more than 95~ native monomeric ~ -interferon with regard to its homogeneity based on the various interferon components.
This high degree of purity and homogeneity has also made it possible for the first time to obtain interferon in solid form free from salts and buffer constituents. It is therefore possible for the first time to store ~-interferon for months without the use of stabilizers; this has significant advantages in terms of storage, dispatch and, not least, galenic developments, over the interferons which have hitherto always been stabilized with albumin. Even after 11 months' storage at 4~Cj no loss of contents could be detected.
The crystalline human leucocyte interferon described in EPA 83 734 (published on July 20th, 1983) consists of crystals of polyethyleneglycol as the precipitating agent with interferon, but not a pure, homogeneous and crystalline interferon as the title would have one believe.

.

. .

Q 2 ~ 1 The ~-interferon prepared according to the invention was, as is already known, dissolved by the addition of human serum albumin, filtered until sterile and transferred into vials under aseptic conditions, in suitable concentrations depending on the particular application.

- 18a -~5 xn2~1 In cllnlcal trlals, the a-lnterferon prepared accordlng to the lnventlon proved to be non-lmmunogenlc and exceptlonally well tolerated.
In all, up to January 1985, 75 patlents have been treated wlth the non-lmmunogenlc a-lnterferon: 58 patlents wlth tumour lndlcatlons and 17 wlth vlral lndlcatlons.
Antlbodles were not stlmulated ln a slngle patlent throughout the therapy, the perlod of treatment belng 15 or more weeks ln some cases and up to 35 weeks.
The process accordlng to the lnventlon has made lt posslble for the flrst tlme to prepare a hlghly pure a2-lnterferon whlch ls homogeneous ln terms of the natlve monomerlc lnterferon, solld, free from salts and buffer constltuents and non-lmmunogenlc.
Amlno acld sequence analysls by known methods ylelded the followlng amlno acld sequence:

Cys Asp Leu Pro Gln Thr Hls Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu Hls Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser . , , , , _ ~ . .. ...

- l9a -Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Wlth regard to the fermentation mlxture, ln partlcular, the process accordlng to the lnventlon can be used wlthout restrlctlon, wlthln wlde llmlts. Thus, lt ls also posslble to use blomasses of other .... . ~ . .

~028 L

host organisms which give comparable IFN yields after mechanical decomposition and interferons which react in a similarly specific manner with the EBI-l monoclonal antibody, e.g. IFN-~l. Other interferons with a lesser homology to interferon may also be purified using the method according to the invention if a corresponding highly specific monoclonal antibody is used.
Salient features of a preferred embodiment of our process are as follows:

the host organism containing the interferon gene is cultivated under conventional conditions, after the conventional growth period the cells are killed off and harvested, the expressed interferon is removed in conventional manner, the cell debris is removed in a slightly alkaline medium, the interferon is concentrated and subjected to preliminary purification by tandem chromatography, the eluate is adjusted to pH 4.0-4.8 to remove any impurities, the interferon is finally purified by chromatography on a cation exchanger column with a volatile buffer as eluant and is then lyophilised.

Preferably the tandem chromatography consists of a preliminary cellulose column and affinity chromatography and the substance to be purified is washed through both columns with a suitable .. ..

washing solution and the ~-interferon is subsequently eluted from the affinity column with a suitable eluant.
More preferably the preliminary column is charged with DE-52 cellulose, the affinity column is charged with a monoclonal anti-interferon IgG
antibody, e.g. EBI, coupled to a carrier and the substance to be purified is washed through both columns with a TRIS-NaCl buffer, about pH 7.5, and the ~-interferon is subsequently eluted from the affinity column with about 0.1 M citric acid in about 25% aqueous ethyleneglycol;
The eluate from the tandem chromatography is preferably adjusted to about pH 4.5 to remove impurities;
Preferably the buffer eluant for the final purification has a concentration gradient prepared from 0.1-1.0 M ammonium acetate buffer and/or a pH gradient in the range 4.0-5Ø
The ~-interferon according to the invention may be used for the therapeutic treatment of viral diseases and tumours. For such purposes it may be formulated into pharmaceutical compositions containing one or more inert pharmaceutical excipients and/or carriers.
The following Examples are given by way of illustration only. The Examples should be read in conjunction with the accompanying drawings, whose significance is explained in more detail hereinafter.

.. . ...

~ 14~

In drawin~s ~hich illu~trate various embodiments of the invention;

Figure 1: Chromatograph of reverse phase HPLC
of the acid eluate after Tandem chromatography;
representation of components K 1 - K 7.

Figure 2: Chromatograph of gel permeation HPLC
of the acid eluate after Tandem chromatography.

Figure 3: Chromatograph of reverse phase HPLC
after precipitation at pH 4.5; representation of components K 1, K 2, K 3 and K 6.

Figure 4: Chromatograph of the gel permeation HPLC after precipitation at pH 4.5.

Figure 5: Chromatograph of the FPLC on MONO-S
at pH 4.5 with an ammonium acetate gradient from 0.1-0.SM.
Figure 6: Chromatograph of reverse phase HPLC
of the "shoulder fraction" of the MONO
S-peak.

Figure 7: Chromatograph of the gel permeation HPLC of the "shoulder fraction" of the MONO S-peak.

Figure 8: Chromatograph of the reverse phase HPLC of the "main fraction" of the MONO S-peak.

Figure 9: Chromatograph of the gel permeation HPLC of the "main fraction" of the MONO S-peak.

Figure 10: Chromatograph of chromatofocussing - 21a -.

., , . , . , _ .

~3la2sl of the "main fraction" of the MONO
S-peak.

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

Figures 12 - 12f:
Results of the anti-IFN-~ antibodies;
tests for various indications Type of test: neutralisation assay (10 iU/ml IFN ~/A-599/EMC) Total number of patients: 75 Number of patients with tumour indications: 58 Number of patients without stimulated antibodies 58 Number of patients with virus indications: 17 Number of patients without stimulated antibodies 17 Figure 13: Diagram of the anti interferon-~ antibody assay ~' - 21b -J~

... . . .... ~ .. . . . .

22 1'3~
Example 2 (E. coli for example E. coli K12 HB101 containing plasmid pBR322 (Pst) lF7 coding for IFN-a2Arg, described in European Patent No. 0,095,702 and which is deposited under DSM
No. 2362; 28~C) a) 251 g of acid-precipitated biomass which had been stored at -20 C were taken up in 2500 ml of 1% acetic acid, stirred for half an hour in an ice bath and homogenised twice for 1 minute using the Ultraturax Type 45/6*. Polymin P was added to give a final concentration of 0.25%, the pH was adjusted to 10.0 using 5 N NaOH and the mixture was stirred for 2 hours over an ice bath and finally the pH was adjusted to 7.50 using 5 N HCl.

Centrifuging for 1 hour in a Christ Cryofuge* 6 - 6 S at 4~C
and 300 rpm yielded a clear crude extract of 2540 ml with an interferon content of 17.1 x 10 I.U. (=100%) and a protein content of 5330 mg, from which a specific activity of 3.21 x 106 I.U./mg of protein can be calculated.

b) Ammonium sulphate was added until 65% saturation was reached t430 g/litre of extract). The mixture was stored overnight at 4 - 8 C and the precipitate formed was removed by centrifuging in a Beckmann* J 2-21 highspeed centrifuge, Rotor JA 10 at 4 C, 10,000 rpm within 1 hour. The clear supernatant, 3120 ml, contained 0.7% of the interferon contained in the crude extract (120 x 10 I.U.).

*Trade-mark -22a '~ 2 R l The pellet was taken up in 0.01 M NaCl and stirred for 2 hours at 4 - 8 C. The pH was adjusted to 7.50 using 5 N
NaOH and the solution was clarified by centrifuging as described above. The clear solution was dialysed with 0.01 M
NaCl using a dialysis cartridge (Nephross Allegro*, Messrs.
Organon Technika) to give 390 mOsmol/l.

*Trade-mark . .

0 2 ~ 1 The interferon content was 13.3 x lO9I.U. (-77.6%).

c) The dialysed material was then chromatographed (Tandem chromatography). For the preliminary column, 125 g of DE 52 cellulose powder made by Messrs. Whatman was used in TRIS/NaCl buffer, pH 7.5 (0.025 M TRIS/HCl + 0.2 M NaCl); this corresponded to 0.5 g of column material per g of biomass. For the affinity column, monoclonal anti-interferon IgG (EBI 1) coupled to Br-CN-activated sepharose 4 B (Messrs. Pharmacia) was used. The finished column material was stored in phosphate-buffered saline solution (PBS) with sodium azide at 4 - 8~C. Before use, the antibody column was washed with 0.1 M
citric acid in 25% ethyleneglycol and then rinsed with PBS until neutral. A column volume of from 0.2 to 1.0 ml was required for each gram of biomass in the antibody column. The dialysed interferon solution was first pumped through both columns (preliminary column and antibody column) and the eluate was monitored by measuring the extinction at 280 nm. After the interferon solution had been applied, it was washed with TRIS/NaC1 buffer, pH 7.5, until the quantity of protein in the eluate had fallen to l/20th of the plateau value. The antibody column was then separated from the preliminary column and washed on its own with TRIS/NaCl buffer, pH
7.5, until no further protein could be detected in the eluate.

Elution of the interferon bound to the antibody was carried out using 0.1 M citric acid in 25%
ethyleneglycol, and again extinction of the eluate at 280 nm was monitored. The protein -~d~3281 peak containing the interferon was collected.
16.8 ml of eluate were obtained with an interferon content of 12.3 x 109 I.U. (= 71.9%). The total quantity of protein was 54.4 mg, from which a specific activity of 226 x 106 I.U./mg of protein can be calculated.

d) For further purification, the eluate was adjusted to pH 4.5 with ammonia and the precipitate formed was removed. The clear supernatant (18.3 ml) contained 46.3 mg of protein and had an interferon content of 11.8 x 109 I.U. based on the crude protein. This corresponded to a yield of 69%
(255 x 10~ I.U./mg of protein).
e) Final purification was carried out with an FPLC
apparatus made by Messrs. Pharmacia with a MONO-S
column, Type HR 10/10 (Messrs. Pharmacia) cation exchanger.
The clear supernatant obtained after precipitation was applied to the column, which had previously been washed with 0.1 M ammonium acetate buffer, pH 4.5 - 5.0, and this column was then washed until the extinction at 280 nm had reverted to the original value. Elution of the adsorbed interferon was carried out with a planar salt gradient by admixing 0.5 M ammonium acetate buffer, pH 4.5 to 5Ø Interferon was eluted as a sharp peak. Both the "shoulder" fraction (K 3) and also the fractions eluted later (K 5 - K 7) were separated from the main peak of pure interferon.
The peak of pure interferon was collected and from it aliquots were taken for HPLC analysis, SDA gel electrophoresis, protein determination, interferon testing and endotoxin determination.
A total of 4.1 mg of protein were found in the "shoulder" fraction (9.1 ml); the interferon ,, . . ~ .

- ~. 3,i,~2~1 content was 1.33 x 109 I.U. (7.7%). This gave 324 x 106 I.U./mg of protein.

The main pool of 9.8 ml contained 5.18 x lO9I.U.
(30.3%) of very pure interferon and a total of 16.1 mg of protein; this gave a specific activity of 322 x 106 I.U./mg of protein.

f;~) The IFN pool was transferred into autoclaved lyo-ampoules (capacity 8 ml) in batches of up to a maximum of 2 ml, corresponding to a quantity of from 1 to about 8 mg of pure interferon per ampoule. The ampoules were then sealed with prewashed and autoclaved lyo-stoppers and cooled to at least -20~C. Lyophilisation was carried out at -10~C under a vacuum of less than 1 torr.
After elimination of the buffer solution, the temperature was increased to 25~C and lyophilisation was continued for at least 1 hour. The vacuum was released and the stoppers were immediately pressed firmly in. After being capped with aluminium seals, the ampoules were then stored in a refrigerator or at -20~C.

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

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

*Trade Mark 1~'10281 BatchIFN titre before IFN titre after + %
lyophilisation lyophilisation A 720 x 106 754 x 106 + 5 B 1337 x 106 1526 x 106 + 14 C 981 x 106 852 x 106 - 13 D 1230 x 106 1149 x 10 - 7 Thus, lyophilisation did not cause any losses.
After 11 months storage of the lyophilised material at about 4~C (refrigerator) it was dissolved in 0.1 M ammonium acetate and checked for both purity (by gel permeation HPLC) and also for content (by the NK2-IRMA test).

before lyophilisation after 11 months storage in lyophil-ised form Purity (gel-HPLC 98.5% 98.7 IFN titre 1510 x 106 units/ml 1464 x 106 units/ml Example 2 To test the effect of the fermentation time on the composition of the interferon components, samples were taken from a fermentation mixture (E. coli HB 101; 28~C) after 8, 9, 10 or 11 hours, precipitated with acid at pH 2 by the usual method and worked up and analysed using the method according to the invention. The following Table shows the content of K 1, K 2 and K 3 (K 1: Met - IFN, K 2:
native IFN; K 3: non-native IFN) in the samples.
The values were determined by chromatofocussing.

... -- , ... . . . . . . ~ . .

0~1 Harvest time after hours 8 9 10 11 g moist biomass per 14 15 18 21 5 litre of culture mg IFN/g biomass (measured in the 0.28 0.21 0.16 0.12 crude extract) mg IFN/l of culture volume 3.9 3.2 2.9 2.5 K 1 pI = 5.78 0.7% n.d.* 10.7~ 19.4%
K 2 pI = 5.64 96.2% 97.5% 86.1% 78.5%
K 3 pI = 5.49 3.1% 2.5% 3.3% 2.1%

* n.d. = not detected.

Example 3 Coupling of the EBI 1 antibody to CNBr-activated Sepharose 4B (see DE-OS 33 06 060 or EP-A-0,119,476) The EBI-l antibody was first dissolved with 0.5 M NaCl/0.2 M NaHCO3, pH 8.4 (in as little buffee as possible) and dialysed with the buffer until no further sulphate ions could be detected in the external solution with barium chloride. Careful removal of the ammonium sulphate was absolutely essential since ammonium ions disrupt the subsequent coupling to the carrier. The protein concentration was then adjusted to 5 mg/ml with buffer. For the coupling, CNBr-activated Sepharose 4 B (Pharmacia) was used as carrier. It was first given a preliminary wash in accordance with the manufacturer's instructions (leaflet enclosed with the package). 1 g of activated sepharose was used for every 2~ mg of EBI-l antibody.
Coupling was carried out in the above buffer at pH 8.4 for 2 hours at ambient temperature. Then ,, . . . .. . ., _ ., 2 ~ 1 the EBI-l Sepharose was removed by suction filtering and washed in accordance with the instruction leaflet.
Not more than 5% of the EBI-l antibody used should remain in the filtrate. The finished EBI-l Sepharose was stored in PBS/azide in a cold store.

PBS/azide:
PBS: 7.30 g sodium chloride p.A. (Merck 6404) 3.00 g Na2HPO4 x 2 H2O p.A. (Merck 6580) 1.15 g NaH2PO4 x H2O p.A. (Merck 6346) dissolved and made up to 1000 ml; pH 7.0 Azide: 1.0 g/l of very pure sodium azide (Merck 6688) were added to the PBS.
The finished solution was filtered sterile (0.2 micron pore size) and stored in a cold store.

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

Virus Encephalomyocarditis virus (EMC), ATCC VR 129.

Interferon standard HS-ll (1 ampoule "HS-lln = lyophilised Hu IFN r~-A taken up in 1.2 ml H2O, yielding 12,000 iU/ml) Tissue culture plates 96 wells with lid, flat bottom, Corning, New York, No. 25860, diameter of well 6.4 mm, tissue culture treated.

Media DMEM = Dulbecco's modified eagle medium, with glutamine, without sodium bicarbonate, flow cat. no. 10-331-24 (lF-017D) HEPES Sigma No. H-3375 TRICINE Calbiochem No. 33468, A grade FCS = foetal calf serum Boehringer Mannheim HUMANSERUM-ALBUMIN Behring Inst., 20%, for infusion Antibiotic, tiamulin hydrogen fumarate, Biochemie Kundl/Tirol, Austria (Sandoz C.) Growth medium: DMEM + 10% FCS/deactivated 30 min/56~C
+ 13 mM HEPES
+ 6 mM TRICINE
+ 1.6 g/l NaHCO3 without antibiotics pH 7.2 - 7.4 Assay medium: same as growth medium but 5% FCS

~t 3 f-~ ~) 2 ''$ ~

instead of 10% and with the addition of 5 ug of tiamulin/ml Dilution medium: growth medium with no serum but with 5 ug/ml of tiamulin Virus medium: growth medium with no serum but with 5 ug/ml of tiamulin and 3.5 mg/ml of human serum albumin Methyl violet storage solution:
methyl violet Merck No. 1402 .............. 6 g ethanol ................................. 100 ml dissolved and filtered at about 50~C

methyl violet solution for use storage solution ......................... 50 ml water (pH neutral) ...................... 950 ml The cells were treated as a permanent cell line. They were propagated by trypsinisation and dilution in growth medium. For the assay, the cells were counted in a haemocytometer and suspended in assay medium in order to obtain an inoculation solution of 4 - 5 x 104 cells per ml per well;
these were distributed over the dishes. Incubation was carried out in an atmosphere consisting of 5% C~2 and 80% relative humidity at 37~C. After 8 - 24 hours the mono-layer was usually complete.
At this time the interferon and the serum dilutions were prepared in separate test tubes.
For the control dish, HS-ll dilutions of 1:1000, 1:2000 up to 1:32,000 were incubated for 1 hour at 37~C.
For the test dish, the serum samples were diluted to 1:2, 1:4, 1:8 and so on up to 1:64 with a dilution medium which contained sufficient HS-ll to give a final concentration of 10 iU HS-ll/ml .. . . . . . ...... . .. . ..

z 8 l in each glass and then incubated for 1 hour at 37~C.
The dishes were decanted and each well was filled with 100 ml of the dilution medium (series 2, 3, 10 and 11) or with 100 ul of the dilutions (series 4-9). The dishes were incubated at 37~C
for 4 hours as above. Then the dishes were given a coating of 100 ul of the virus medium (without the virus) for each well and 50 ul of the virus dilution (series 3, 11, 4-9) in order to achieve a cytopathic effect of approximately 90% within 36 hours and then incubated again. After 24 hours and microscopic monitoring the cells were stained with methyl violet.
The results are shown in Figures 12a - 12f;
the diagram appears in the Appendix.

~ ?~ 2 81 METHOD
The following methods were used for the analysis:

Protein determination BIORAD PROTEIN ASSAY: This assay uses the dye Coomassie brilliant blue and measures the protein/dye complex at 595 nm. The standard used is bovine serum albumin.

Planimetric determination: Of the peak surfaces measured at 214 nm which were recorded by gel permeation HPLC. The results are converted with the aid of a factor from the calibrating substances bovine serum albumin, ovalbumin, trypsinogen and lysozyme.
This measurement was carried out particularly on the preparations after the Tandem chromatography purification stage, pH 4.5, precipitation and FPLC
on MONO-S, additionally or exclusively.

Interferon determination The "NH2-IR~" for human alpha interferon, commercially available from Messrs. CELLTECH (U.K.), was used. The standard used was a laboratory standard "HS ll*'lwhich was adjusted to International Standard B 69/19 by biological assay (plaque reduction test WISH cells and vesicular stomatitis virus).

SDS-gel electrophoresis The method of LAEMMLI (Nature 227, 680, (1980)) was used. The dye used to stain the proteins was Coomassie brilliant blue. 20 micrograms of the interferon preparations were used in the purity checks.

Chromatofocussing The method of Bodo and Adolf (Separation and Characterization of Human IFN-alpha Subtypes, *Trade Mark ... .. .... . . . .. . ... . . . ~

in The Biology of the Interferon System, pages 113-118, Elsevier 1983, Edts.E.DeMaeyer and H.Schellekens) was used, with a MONO-P chromatofocussing column HR 5/20 (Pharmacia) in a pH range of 4 - 7. The buffers contained 25% acetonitrile instead of the specified 25% 1,2-propandiol in order to increase the flow rate. The protein concentration was recorded at 280 nm and the pH was recorded automatically.
The samples to be analysed were lyophilised, dissolved in water in amounts of 1 mg/ml and then diluted with 5 volumes of buffer A (pH 7.1). 0.2 - 1.0 mg of interferon were used for each analysis.

Gel permeation HPLC (high pressure liquid chromatography) Stationary phase:
WATERS I-125; 2 x (300 mm x 7.8 mm);
10 ,um particle diameter Mobile phase:
0.5 M Na2SO4 0.02 M NaH2PO4, adjusted to pH 7.0 with NaOH
0.04% Tween~20 25% propyleneglycol Flow speed:
0.5 ml/min Detection:
UV absorption at 214 nm Molecular weight calibration:
Bovine serum albumin M 66,000 Ovalbumin M 45,000 Trypsinogen M 24,000 Lysozyme M 14,300 rG~ Q ~a~

,. ,., . .. ~ . ..

~3 1~ 2~1 Reverse phase HPLC (high pressure liquid chromatography) Stationary phase:
Bakerbond WP C 18; 250 mm x 4.6 mm;
5 ,um 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 programme:
0 - 2 min: 45% B
2 - 32 min: 45 - 53% B
32 - 40 min: 53% B
40 - 50 min: 45% B

Flow speed:
1 ml/min Detection:
UV absorption at 214 nm .. . ..

l~ln2~1 O r~
~ O ' 1-- _I ~ I' o O~o ~ o r~ r~
~o O
~ ~ O O o O
Z ~ ~1 ~1 ~1 ~--IX
H a~ K X X X
. ~_I
N ~D u~ ~r N
~ h ~ NIr) N N
H ~~1 N N ~ ~T~

O O O O O O O
* K X K ~C X X ~C
3 _~ N ~ ~ X ~ CO
Z ~ '--I ' ' ' li:, Hr~ N
H --_I O ~1 O

_I O
0 . , h ~ ~ ~ ' ' O
.
o o ao -~ _I ~ N N
o u~ _I X ~
N ~ N ~1 ~1 ~1 a~ 3 U~ ~
a a, ~ Q. ' ~--I
p ~ ~
al a ~ 0 ~ oa3 c s a ~ L _ O C I :
C V -- L~ + ~ O O ~ V~ J~
~ 0 ~ ~ C a alo c 1-~ ~ O _l ~- ~ O '~ H
~ ~ a~ c ~ ~ a 0 0 X ~_0 ~ ~ o J~
V a~ :5 2~ I ~ a-~ . c c _I~ ~ ~ ~ 0~ ~ o S ~ ~ -a~ c ~a) o ~ a~
o ~ a ~ o~ v ~;--I ta _ C ~L a~ ~ C H
Ei ~ - 0 s .~c u ~ a~ o u. Q~ ~ 0 Q

..... . . . . .

Table 2 Assessment of Purlty By HPLC

Gelpermeatlon-HPLC Reverse-Phase-HPLC**

IFN(mg/ml) Prlorlty of MW > K2 + 1 K3 K4 K5 K6 K7 Monomer 70.000 (MW 19.000) Acld Eluate 2,14 74% 100 7,0 n.d.* 1,6 22,8 ~1 after Tandem-Chromatography Supernatant after Preclpltatlon at pH 4.5 2,0 79% 100 4,0 n.d. <1 5,4 n.d.

after FPLC on MONO-S

a)Shoulder Frac. 0,45 > 99% < 0,2% 100 16,0 n.d. n.d. n.d. n.d.
b)Main Fractlon 1,64 > 99% ~ 0,2% 100 1,2 n.d. n.d. n.d. n.d. ;~
* = not detectable ~_ ** = relatlve peak-helght (214 nm), K 2 + 1 = 100%

Claims (27)

1. In a process for preparing a substantially non-immunogenic .alpha.-interferon, the improvement which comprises purifying said interferon by tandem chromatography to yield an eluate containing said interferon having less than 20% methionine, said eluate being adjusted, if required, to a pH of 4.0 to 4.8 to remove impurities, said tandem chromatography comprising a combination of a diethylaminoethyl cellulose preliminary column and an affinity column consisting of a monoclonal anti-interferon IgG coupled to Sepharose*.

2. The process of claim 1 wherein said interferon is produced by cultivating E. coli cells which genetically code for said interferon.

3. A process for preparing recombinant interferon which process comprises (a) cultivating cells of a host organism containing an interferon gene to synthesise interferon, (b) harvesting and disrupting said cells, removing cell debris and subsequently concentrating the interferon obtained thereby, (c) purifying said interferon by tandem chromatography consisting of a diethylaminoethyl cellulose column and a monoclonal anti-interferon IgG coupled to a Sepharose* column and *Trade-mark obtaining said interferon having less than 20% methionine in an eluate, (d) adjusting the pH of the eluate to 4.0 to 4.8 to remove impurities, (e) further purifying the interferon in a cation exchanger MONO-S* column using an ammonium acetate buffer as an eluant, and (f) lyophilising the eluant to yield said recombinant interferon.

4. A process according to claim 1 or 3 wherein the interferon is synthesised by E. coli cells containing an interferon gene.

5. A process according to claim 3 wherein the interferon gene codes for human .alpha.-interferon.

6. A process according to claim 5 wherein the human .alpha.-interferon gene codes for the amino acid sequence Cys Asp Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr * Trade-mark Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu

7. A process according to claim 3 comprising growing the cells of the host organism in a fermentation mixture until less than 20% methionine interferon is formed.

8. A process according to claim 7 wherein said interferon is an .alpha.-interferon, is substantially non-immunogenic, pure and homogeneous, substantially free from reduced forms and fragments, contains less than 0.2% oligomer and less than 2% dimer/trimer/tetramer.

9. A process according to claims 3, 7 or 8 comprising growing the cells of the host organism in a fermentation mixture until less than 5% methionine interferon is formed.

10. A process according to claims 3, 7 or 8 comprising growing the cells of the host organism in a fermentation mixture until less than 1% methionine interferon is formed.

11. A process according to claim 3 wherein the cells are disrupted in a homogenizer at a pH of about 2.

12. A process according to claim 3 wherein the cation exchanger comprises hydrophilic polymer beads with an extremely narrow particle size distribution.

13. A process according to claim 3 wherein said buffer eluant has a concentration gradient prepared from 0.1-1.0M
ammonium acetate buffer or a pH gradient in the range pH 4.0-5Ø

14. A process according to claim 13 wherein the gradient is 0.1-0.5M ammonium acetate buffer.

15. A process according to claim 13 or claim 14 wherein the ammonium acetate buffer has a pH of about 4.5.

16. A process according to claim 1 or 3 wherein the affinity chromatography column has monoclonal antibody EBI 1 coupled to a carrier.

17. A process according to claim 1 or 3 additionally comprising employing Tris-NaCl buffer of about pH 7.5 as a washing solution.

18. A process according to claim 1 or 3 wherein the interferon is eluted from the affinity column with about 0.1M
citric acid in about 25% aqueous ethyleneglycol.

19. A process according to claim 1, 2 or 3 wherein the eluate from the tandem chromatography is adjusted to about pH 4.5 to remove impurities.

20. A process according to claim 3 for the preparation of a recombinant, substantially non-immunogenic, homogeneous, substantially pure, .alpha.-interferon wherein the product contains less than 5% methionine interferon, is substantially free from reduced forms and fragments of .alpha.-interferon, contains less than 0.2% oligomer and less than 2% dimer/trimer/tetramer and in which more than 90% of the monomer content consists of native monomeric interferon.

21. A process according to claim 3 for the preparation of recombinant, homogeneous, substantially pure, human .alpha.-interferon with the amino acid sequence Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu wherein the product contains less than 5% methionine-interferon, contains less than 0.2% oligomer and less than 2% dimer/trimer/
tetramers, is substantially free from reduced forms and fragments of .alpha.-interferon, and wherein the native monomeric .alpha.-interferon contains disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

22. A process according to claim 1, 2 or 3 wherein the resulting .alpha.-interferon is in solid form.

23. A recombinant .alpha.-interferon that is substantially pure, nonglycosylated and homogeneous, and substantially free from reduced forms and fragments, and contains less than 0.2%
oligomer and less than 2% dimer/trimer/tetramer with less than 20% methionine-interferon which has the amino-acid sequence Cys Asp Leu Pro Gln Thr Hls Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu Hls Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu

24. An interferon as claimed in claim 23 in a homogeneous pure form with less than 5% methionine interferon and in which more than 90% of the monomer content consists of the native monomeric .alpha.-interferon with disulphide bridges between the cysteines at positions 1 and 98 and 29 and 138.

25. An .alpha.-interferon as claimed in claim 23 or 24 which is substantially non-immunogenic.

26. An .alpha.-interferon as claimed in claim 23 or 24 in solid form.

27. An .alpha.-interferon as claimed in claim 23 or 24 of human type.