IE57914B1 - Purification of recombinant interleukin-2 - Google Patents

Abstract

Homogeneous recombinant human interleukin-2 is prepared by extraction of IL-2 from the cell membrane components of transformed microorganisms after lysis and purification of the extract by chromatography. <IMAGE>

Description

Description Human interleukin-2 (IL-2), formerly called T-cell growth factor, is a soluble protein which can be synthesized by lectin or antigen-activated T-cells and which is capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific T-effector lymphocytes. As has been found, IL-2 also enhances thymocyte mitogenesis and induces cytotoxic T-lymphocyte reactivity. Accordingly, this lymphocyte-regulating substance is useful in enhancing humoral and cellular immune responses and in restoring a normal humoral and cellular immunity. These identified immunological activities indicate that IL-2 is useful for medical immunotherapy of disorders of the immune system, including neoplastic disorders, bacterial and viral infections, immune deficient disorders, autoimmune disorder etc. (Papermaster, B. et al., Adv. Immunopharm., 507. [1980]). A recently published review article in the Journal of The American Medical Association, Vol. 249, No. 2 (14 January 1983) discusses at pages 166-171 the clinical applications of various lymphokines with particular reference to human IL-2.

The purification of homogeneous human IL-2 from induced human malignant cells up to homogeneity has recently been achieved by a multi-stage high performance liquid chromatography (HPLC) process. The resulting homogeneous human IL-2 was characterized by a specific activity of about 1.4 x 109 units/mg (see e.g. European Patent Application No. 83.109202.8, Publication No. 106 179).

Taniguchi et al. reported on the cloning and sequencing of the IL-2 gene and expression of immature human IL-2 at the Third Annual Recombinant DNA Congress in Philadelphia, Pennsylvania on the 9th February 1983 (Nature, 302. 305-310 [1983]). The cDNA from which the amino acid sequence of the IL-2 protein was derived was isolated from mRNA of Jurkat cells which had been induced with Concanavalin A. The full length cDNA clone is 800 base pairs long and codes for a protein having 153 amino acids. See also European Patent Application, Publication No. 91539.

Other groups have also reported the cloning and sequencing of the IL-2 gene. See Devos et al., Molecular Cloning of Human lnterleukin-2 cDNA and Its Expression in E. coli, Nucleic Acids Research, 11. 4307-4323 (1983). See also European Patent Application, Publication No. 118 977.

Although the expression of IL-2 in transformed host cells, especially in E. coli, is described in various literature references, no purification procedures are disclosed which would permit a person skilled in the art to obtain mature recombinant human IL-2 in a homogeneous or substantially pure form.

The present invention relates to a process for the purification of recombinant, especially mature, human IL-2. It provides a process for the purification of recombinant mature human IL-2 to homogeneity which is superior to prior art methods and which is characterized by (a) cultivating microorganisms transformed with a DNA sequence which codes for mature human IL-2; (b) causing the transformed microorganisms to express and accumulate mature human IL-2; (c) lysing the microorganisms to give a cell lysate; (d) separating the cell membrane components present in the cell lysate; (e) extracting IL-2 from the isolated cell membrane components using a wash solution which contains about 4 to 7M guanidine HCl; and (f) chromatographically purifying the IL-2 from the wash solution.

EP-A-0 145 390 describes a process for the preparation of recombinant human IL-2 with the aid of 7M guanidine HCl and chromatography, but in this process a prior lysis of the microorganisms and the isolation of membrane components are not carried out. The specific activity of the IL-2 obtained is lower than that of the IL-2 obtained according to the process described in the present Application.

Brief Description of the Drawings Figure 1 represents the DNA nucleotide sequence of plL2-2B and the corresponding amino acid sequence of mature human IL-2. The amino terminus of mature IL-2 is denoted by an arrow.

Figure 2 schematically illustrates the construction of the plasmid pRC2 starting from pBR322.

Figure 3 schematically illustrates the construction of the plasmid pRC23 having a Pl promoter.

Figure 4 described the construction of the IL-2 structural gene which expresses Ser-IL-2.

Figure 5 schematically illustrates the construction of the plasmid pRC201/IL2 which was used for the construction of the plasmid pRC233/IL2 (see Figure 6).

Figure 6 shows schematically the construction of an expression vector having a Pl promoter system which was used for the expression of mature IL-2.

The process of the present invention is based on the surprising finding that the IL-2 protein expressed by a microbe tends to associate with the membrane fractions of the host microbe, principally with the inner membrane of the microbe (which is that membrane whose lipid bi-layer is often associated with hydrophobic proteins). Therefore, the separation of the membranes during the purification process of IL-2 from transformed microorganisms leads to high yields and an end product of high purity.

Although different methods of cell lysis, e.g. enzymatic or chemical lysis, can be used in connection with the present invention, lysis by sonification is the preferred method. The inner and outer membranes are then separated from the other cellular components by known methods such as e.g. centrifugation.

After separation of the cell membranes from the cell lysate, these are washed with extraction solutions, preferably salt and detergent solutions, to give a solution containing at least about 50% IL-2. In a preferred embodiment, the cell membranes are washed in 4 separate steps with salt and detergent solutions. The first step is characterized in that the cell membranes are preferably washed with a salt solution, especially with 1M NaCl. In the second step the cell membranes are washed with a detergent solution, preferably 1% Triton X100. In the third step the cell membranes are washed with another salt solution, preferably 1.75-2M guanidine HCl. The final wash is also effected with a salt solution, preferably about 4-7M guanidine HCl. The wash solution which results from the fourth and last wash then contains approximately about 50% IL-2.

The last IL-2 wash solution is finally purified by chromatography, preferably using reverse phase high performance liquid chromatography (HPLC). This HPLC purification step leads to active IL-2 which is present in almost 100% pure or homogeneous form. It is also forseeable that antibody-affinity chromatography columns with polyclonal or monoclonal antibodies against IL-2 could be used in place of HPLC. Other chromatographic purification procedures such as dye-affinity chromatography (e.g. with Procion red agarose as described in European Patent Application No. 83103582.9, published under Publication No. 92163 on 26th October 1983) or Sephacryl S200 columns can also be utilized. In another preferred embodiment of this invention the chromatography is carried out in multiple steps, i.e. HPLC followed subsequently by dye-affinity chromatography.

The IL-2 purified in accordance with the present invention can be used in the same manner as the other known substances having immunomodulating activity, e.g. as a means for treating immunosuppressive conditions. It may be administered in the form of pharmaceutical preparations orally, by injection or locally. Dosage and dose frequency may be established on the basis of experience with the clinical application of known immuno- modulating substances, typically 1-200 χ 106 units daily. The medicaments resulting from this invention contain the said IL-2 together with a physiologically compatible carrier material. Any conventional carrier material can be used. The io carrier material can be an inert, organic or inorganic material which is suitable for enteral, percutaneous or parenteral administration. Suitable carriers are water, gelatin, gum arabic, lactose, corn starch, stearic acid, talc, vegetable oils, polyalkylene glycols, especially polyethylene glycols, natural Vaseline and the like. Furthermore, the pharmaceutical preparations may contain other pharma- ceutically active substances. Additional additives such as flavouring agents, coating agents, stabilizers, emulsifiers, buffers and the like may be added according to conventional galenical practice.

The pharmaceutical preparations can be made up in any administration form, i.e. a) in solid form, e.g. in the form of tablets, capsules, powders, granules and the like for oral administration; b) in liquid form e.g. in the form of solutions, syrups, suspensions, elixirs and the like for oral administration; c) as preparations for parenteral administration, e.g. as sterile solutions, suspensions or emulsions; and d) as preparations for local use in the form of solutions, suspensions, ointments, creams, gels, micronized powders, aerosols and the like. The pharmaceutical preparations may be sterilized and/or may contain adjuvants such as coating agents, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.

Infusion or injection solutions, which can be injected intravenously or intramuscularly, come into consideration as parenteral dosage forms.

The construction of IL-2 expression vectors and of transformants capable of expressing IL-2 is described in detail hereinafter.

The amino acid sequence of mature IL-2 protein is given in Figure 1. The protein may be expressed in its mature form with methionine as the amino-terminal amino acid provided that the gene initiation codon is ATG. The methionine is in certain circumstances cleaved off by the host cell after the IL-2 expression.

The expression vectors used in this invention are pBR322 derivatives containing the Pl promoter isolated from bacteriophage lambda DNA. Pl is the promoter of choice, since it is a very strong promoter which is effectively controlled by the lambda cl repressor, the gene for which is located on the chromosome of the microorganism, the vector which contains the Pl promoter or another compatible vector. The gene which codes for the repressor carries a gits mutation which renders the repressor temperature-sensitive. Such a vector which may be used in this invention and which carries the gl mutation is pRK 248 gits, which is known in the art and which has been described by Kahn et al., Methods in Enzymology, 68. 268 (1979). At a temperature of 30°C the repressor functions normally, but at temperatures from about 37°-42°C it is inactivated. Thus, the Pl promotor is repressed (turned-off) at 30°C and derepressed (turned-on) at 37°-42°C. This method of Pl promoter control permits one to grow the culture at about 30°C - 36°C without expressing the desired gene product and at an optimum point in time, to raise the temperature to about 42°C, at which the desired mature human IL-2 product is expressed.

The preferred vector used in this invention has an additional EcoRI restriction site distal (downstream in 3' direction) to the SD sequence. The following description serves to illustrate the construction of a preferred vector, the vector pRC23, into which the IL-2 gene is introduced. However, other vectors may also be used.

In accordance with the procedure described in Figures 2 and 3, 20 micrograms of pBR322 DNA were digested with EcoRI and subsequently used in two different reactions: 1. for treatment with SI nuclease in order to remove the 5' overhang, and 2. for treatment with DNA polymerase I klenow fragment in order to fill-in the cohesive termini. Both reactions were terminated by phenol extraction and subsequent ethanol extraction. The DNA from each reaction batch was ligated with synthetic Bg1 II linker, digested with Bg1 II and Pst I and thereafter separated electro- phoretically in 1% agarose. The fragments containing 3600 bp (base pairs) and 760 bp from both reaction batches were isolated from the gel. For the construction of pRC2, the 3600 bpcon- taining fragment from the Klenow reaction was ligated to the 760 bp-containing fragment from the SI reaction. E. coli strain RR1 was transformed with this DNA combination and transformants were selected in a medium containing 50 pg/ml of ampicillin. Transformants having the expected plasmid combination, i.e. a plasmid with a Bg1 II restriction site in the proximity of the EcoRI restriction site, were identified by restriction analysis of the isolated plasmid DNA. Plasmid pRC23 was constructed by ligating a synthetic oligonucleotide which codes for a consensus RBS (computer-generated ribosomebinding site) [Scherer et al. Nucleic Acids Research, & 3895 (1980)] to a 250 bp-containing Bg1 II - Hae III fragment containing the lambda Pl promoter, and inserting into the plasmid pRC2.

In order to isolate the 250 bp-containing DNA fragment, which contains the lambda Pl promoter, 1 microgram of a 450 bpcontaining Bg1 II - Hpa I DNA fragment (from bp # 35260 tp 35710 of the lambda phage DNA sequence) was digested with Hae III and the resulting products were isolated with the aid of preparative gel electrophoresis on 5% polyacrylamide. Approximately 200 ng of the 250 bp-containing Bg1 II - Hae III fragment were ligated to 60 pM each of the synthetic oligonucleotides shown in Figure 3, which for the most part code for a consensus RBS sequence, the sequence of which has been developed by means of computer analysis as described by Scherer et al. The resulting molecules were then digested with Bg1 II and EcoRI (in order to eliminate oligomers), purified by gel electrophoresis and ligated with pRC2 DNA which also had been treated with Bg1 II and EcoRI. The transformation of E. coli RR1 (pRK 248 cJts) was carried out according to standard methods and the transformants were selected in a medium containing 50 pg/ml of ampicillin at 30°C. 50 transformants were obtained of which 8 were selected for further analysis. The plasmid DNA of these 8 transformants was isolated and analyzed by means of Hinc II cleavage. 6 of these 8 showed the expected restriction pattern and also Maxam-Gilbert nucleotide sequence analysis of one of these 6 confirmed the expected construction (designated pRC23).

Construction of a plasmidic expression vector containing DNA coding for mature human IL-2 (1) Isolation of mRNA coding for human IL-2 mRNA was isolated from H33HJ-JAI cells (ATCC No. CRL8163, deposited on 26th August 1982), a clone of Jurkat cell line FHCRC, after induction with PHA and PMA. 12000 ml of # H33HJ-JAI clone cell cultures (106 cells/ml) were grown in RPMI 1640 tissue culture medium supplemented with 10% foetal bovine serum, 50 U/ml penicillin, 50 pg/ml streptomycin, 50 pg/ml gentamycin and 300 pg/ml fresh L-glutamine. These cells were collected by centrifugation and resuspended in 6 I of the above-detailed medium, lacking serum, but supplemented with 1% PHA and 10 ng/ml PMA. 6 I aliquots of resuspended ceils were dispensed into sterile glass roller bottles and placed on a roller mill (10 rpm at 37°C). hours later the cells were collected by centrifugation and mRNA was extracted by a standard phenol-chloroform procedure. Thereafter, the ethanol-precipitated RNA was sedimented by high speed centrifugation and taken up in a 0.5M salt solution. Oligo-(dT)-cellulose chromatography was used to separate the mRNA with poly(A) tails from the total RNA. The ethanol precipitated mRNA was taken up in water in a concentration of 500 pg/ml. 30 ng of RNA were then microinjected into Xenopus oocytes. After 24 hours of incubation in sterile Barth's solution, 4 eggs were transferred into a sterile 1.5 ml Eppendorf tube and fed with 500-1500 microliters of fresh sterile Barth’s solution. After 48 hours, 200 μΙ of incubation medium were harvested and assayed for IL-2 activity using a standard CTLL Cell 3H-Tdr incorporation assay (Gillis et al., J. Immunol. 120. 2027 [1978]). mRNA probes which after translation in the ooctye test showed significant IL-2 activity, were pooled, separated according to size by standard sucrose density gradient, centrifugation and, after ethanol precipitation, used for the production of a cDNA library. 2) cDNA Synthesis 3.5 pg of purified mRNA (with a sediment coefficient of 10S in sucrose gradients) were used to produce double-stranded complementary DNA (ds cDNA) according to the following method (Gubler and Hoffmann, Gene 2£, 263-269 [1983]). (a) First strand cDNA synthesis: The mRNA was incubated in 17.5 μΙ Tris HCl pH 8.3, 10 mM MgCl2, 10 mM DTT, 4 mM Na pyrophosphate, 1.25 mM dATP, 1.25 mM dGTP, 1.25 mM dTTP, 0.5 mM dCTP, 100 pg/ml of oligo(dT)i2-ie and 10 Ci of 32P-dCTP (Amersham 3000 Ci/mMol). After incubation for 5 minutes at 43°C, 3000 units of AMV reverse transcriptase/ml were added to the incubation mixture and this was incubated for a further 30 minutes at 43°C. The reaction was stopped by the addition of EDTA to 20 mM, the cDNAmRNA hybrid was extracted with phenol-cresol and concentrated by ethanol precipitation. The yield of the first strand synthesis was 58.6 ng (1.7%) as assayed by TCA-insoluble radioactivity. (b) Second strand cDNA synthesis; The cDNA-mRNA hybrid was taken up in 5.8 μΙ of H2O. To this solution were added 7.7 μΙ of the second strand synthesis mix to give a solution containing 20 mM Tris HCl, pH 7.5, 5 mM MgCl2, 10 mM (NH4)2SO4, 100mM KCI, 0.15 mM betaNAD, 50 pg/ml BSA, 40 mM dNTPs, 8.5 units/ml of E. coli RNase H (Bethesda Research Labs), 230 Units/ml DNA polymerase I, 10 units/ml E.coli DNA ligase. The mixture was incubated for 60 minutes at 12°C and then for 60 minutes at 22°C. The reaction was stopped by the addition of EDTA to 20 mM and the ds cDNA was extracted with phenol-cresol. A Sephadex G-50 fine filled column equilibrated with 10 mM TEAB was used to separate the ds cDNA from the free nucleotides. The yield from this reaction was 107 ng of dsc DNA (91%). (c) Annealing and transformation ng of ds cDNA were tailed with deoxyguanosine.(dG) residues according to standard methods, The vector, pBR 322, was firstly digested with EcoRV and subsequently tailed with deoxycytidine (dc) residues. 100 ng of tailed pBR 322 DNA and 1.25 ng of tailed cDNA fragments (ratio 80:1) were annealed in 250 μΙ of 0.01 M Tris, pH 7.5, 1 mM EDTA, 0.15 M NaCl for 90 minutes at 58°C and used for the transformation with com- potent E.coli RRI cells. The transformed cells were plated on LB plates containing 100pg/ml of ampicillin and incubated at 37<>C for 12 hours. 3200 colonies were obtained for the IL-2 cDNA library. 1 (3) Screening of cDNA library for IL-2 gene sequences In order to detect cDNA copies with the complete IL-2 gene sequence, a synthetic deoxy-oligonucleotide probe was used which corresponds to nucleotides 45-65 of the human IL-2 cDNA sequence published by Taniguchi et al. (Nature 202., 305-310 [1983]). This probe [ACAATGTACAGGATGCAACTC] was synthesized according to the solid-phase phosphodiester method, purified by HPLC and labelled using 32PATP (ICN) Pharmaceuticals, 7000 Ci/mM) and polynucleotide kinase. Colonies of the cDNA library were transferred to nitrocellulose filters according to the method of Grunstein and Hogness (Methods in Enzymology 68. 379 [1979]). After hybridization at 30°C for 16 hours, the filters were washed in 4 x SSC buffer at 45°C for 45 minutes, dried and used for autoradiography. A single positive colony, which contained the complete IL-2 gene sequence, was obtained. From this colony, designated plL-2-2B, there was prepared plasmid DNA which was used for nucleotide sequence analysis and expression in E.coli. The nucleotide sequence was identical to that of the sequence published by Taniguchi et al. with two differences. These differences are: an insert which begins at nucleotide 17 of the Taniguchi sequence and a substitution of G in position 503. (4) Production of an E.coli plasmid vector which contains DNA coding for Ser-lL-2 An E.coli expression vector which contains DNA coding for Ser-IL-2 was produced by combining three segments (see Figure 4): (1) the vector pRC23 with the lambda Pl promoter, (2) a synthetic adapter and (3) a HgiAl - Aha III fragment isolated from II-2 cDNA. pRC23 vector DNA (50 ng) was digested with EcoRi and EcoRV, extracted with phenol and then precipitated with ethanol The synthetic adapter was prepared by hybridizing two complementary synthetic deoxynucleotide sequences A and B: (A) 5’ AATTCAATTATGAGTGCA 3' 2 (Β) 3' GTTAATACTC 5' This double-stranded adapter can anneal to an EcoRI binding site at its 5' end and to a HgiAl binding site at its 3' end.

The 1 kb IL-2 cDNA insert was isolated by gel purification from plL-2-2B DNA after treatment with the restriction enzyme Bam HI. The thus-obtained DNA fragment was further treated with HgiAl and Aha III, extracted with phenol and precipitated with ethanol. HgiAl cuts between the alanine and the proline codon at the beginning of the sequence coding for mature IL-2. In the cloning for the expression of the corresponding IL-2 gene there is used the restriction site adjacent to the Pl promoter in the pRC23 vector. The synthetic adapter can join this EcoRI restriction site with the HgiAl restriction site at the start of the IL-2 cDNA fragment. By ligating the adapter with the cDNA there are simultaneously produced the methionine codon required for translation initation and the codons for serine and alanine, the first amino acids of the mature IL-2 protein. The cDNA is joined to the pRC23 vector DNA with the blunt Ahalll end via the blunt EcoRV end.

The ligation of the three DNA segments was carried out in 10 μΙ of reaction mixture containing 65 mM Tris, pH 7.6, mM MgCl2, 0.5 mM ATP, 15 mM beta-mercaptoethanol and 0.5 pM each of vector DNA, synthetic adapter and cDNA inert. The ligation mixture was firstly heated at 65°C for 5 minutes, then cooled to 4°C and, after the addition of 200 units of T4 DNA ligase, incubated at 4°C for 16 hours. The T4 DNA ligase was inactivated by heating the reaction mixture at 65°C for 5 minutes. Subsequently, the ligated DNA fragments were treated with EcoRV to remove undigested vector DNA. The mixture was then used for transformation with E.coli RR1 (pRK 248 cits). These cells were grown at 30°C for 15 hours.

The colonies were transferred to nitrocellulose filters as described previously, dried and hybridized with the 32P13 labelled deoxynucleotide sequence A. One positive colony, designated pRC 23/IL-2 # 4-1, was chosen for further analysis. This clone synthesized more that 200 000 units/ml of IL-2 after the Pl promoter had been induced by a temperature increase to 42°C. The IL-2 activity was detected by biological testing on CTLL cells, an indicator cell line for IL-2 (described in Gillis et al. J. Immunol. 120. 2027 [1978]). (5) Production of an E.coli expression vector which synthesizes mature IL-2 (with alanine at the amino terminus) In order to express mature IL-2 in E.coli, the following strategy was chosen. As shown in Figure 5, a HgiAl restriction site is located at the Ser-Ala peptide bond, which is simultaneously the cleavage site for the removal of the signal peptide (Robb et al., PNAS, 80. 5990-5994 [1983]). After treatment with HgiAl, the cohesive terminal was converted into a blunt end by treatment with T4 DNA polymerase. The resulting fragment was then ligated via its blunt end to a 108 bp Bg1ll-Hae III fragment isolated from phage lambda, thereafter treated with Bg1 II and Xba I and inserted between Bg1 II and Xba I restriction sites in the vector pRC23/IL-2, # 4-1. This intermediate cloning step was effected to ensure that the T4 DNA polymerase treatment had functioned as expected, which was confirmed by the creation of a new Stu I restriction site from the joining of the Hae III terminus to the blunt HgiAl terminus. Re-treatment with Stu I conveniently regenerates at this site the blunt end beginning with the proline codon CCT.

This intermediate plasmid construction was designated pRC 201/IL-2. 2 synthetic deoxyoligonucleotides were prepared which incorporate an ATG-translation initiation codon, restore the condon for the amino acid alanine and create an EcoRI terminus. These oligomers were ligated to the expression vector pRC23 via the EcoRI termini. The thusobtained product was treated with Pst I so that there resulted a 1025 bp-containing fragment which was isolated by gel electrophoresis. The 1025 bp-containing fragment (with a Pst I and a blunt end) was inserted between the Pst I restriction site and the newly created Stu I restriction site in the plasmid pRC201/IL-2. In order to identify transformants having the desired construction, the plasmid DNAs were isolated and analyzed by restriction enzyme analysis. The confirmed plasmid construction was designated pRC233/IL-2 (see Figure 6).

Preferred host organisms for transformation with a vector containing the IL-2 gene in accordance with the invention are bacteria such as e.g. E. coli; Bacillaceae such as e.g. Bacillus subtilis and the like. Yeasts from a further group of preferred microorganisms for transformation.

An especially preferred microorganism which is used as the recipient in the transformations is Escherichia coli K-12 strain 294 as described in British Patent Application No. 2055382A. This strain was deposited at the American Type Culture Collection on 28th October 1978, under ATCC No. 446. Moreover, other E. coli strains such as e.g. E. coli RR1, ATCC Accession No. 31 343, or other microorganisms many of which have been deposited in and are generally available from a depository such as the American Type Culture Collection can also be used as host organisms. In the performance of this invention in practice overnight cultures of the transformed E. coli cells are grown in LB medium at 30°C. One liter of the overnight culture is preferably diluted to 10 liters with M-9 minimal medium supplemented with casamino acids. After the logarithmic growth phase has been reached, the growth temperature of the culture is increased from 30°C to 42°C to induce IL-2 production. After incubation at 42°C for 2-3 hours, the bacteria are harvested by centrifugation. The entire procedure was carried out while observing the guidelines of the National Institute of Health. The purification steps are described in detail in the following Examples.

Example 1 g of transformed E. coli cells which produce IL-2 (having serine at the amino terminus of the protein) were resuspended in 30 mM Tris HCl, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulphonyl fluoride and lysed by sonification. The lysate was centrifuged at 14000 x g for 15 minutes. The membrane components of the centrifuged lysate were separated from the other components of the lysate. The membrane components were then subjected to 4 washing or IL-2 extraction steps, the results of which are compiled in Table 1. 5 ml of wash solution were used per gram of cells. The bulk of the IL-2 activity was extracted in the final wash as detailed in Table 1. This wash solution, which contained the bulk of the IL-2 activity and at least 50% pure IL-2 measured with the aid of SDS-PAGE, was subjected to reverse phase HPLC (RP-C8). The yield was 40-60% of almost 100% pure IL-2 (with serine at the amino terminus of the protein. The IL-2 bioactivity lay at 1 x 108 U/mg.

Table 1 Extraction of IL-2 from E, coli membranes Extraction Total protein Ima) Total IL-2 Activity (units) Specific Activity lUZma) 1M NaCl wash 0.4 mg 0.03 x 106 0.1 x 108 1% Triton wash 1.75M guanidine 2 mg 0.75 x 108 0.4 x 108 HCl wash 7M guanidine 1 mg 1.5 x 108 1.5 x 108 HCl wash 4.7 mg 250 x 108 (99%) 53 x 108 252.3 x 108 Example 2 This Example serves to demonstrate the tendency of IL-2 to become associated with the membrane components of the transformed E. coli host cells.

In order to demonstrate this, 1 g of transformed E. coli cells which produced IL-2 with serine at the amino terminus were taken up in 10 ml of 20 percent sucrose, 30 mM Tris HCl, pH 8.0, and lysed using lysozyme-EDTA in order to separate proteins which are localized in the periplasmic space. The remainder of the lysate was subsequently subjected to sonification. The membranes (inner and outer membranes) were then separated from the other cellular components by sucrose gradient centrifugation and the inner and outer membranes were separated. Table 2 shows that the majority of biological IL-2 activity is to be found in the inner cellular membrane.

Table 2 Localization of IL-2 in E, coli cells Fraction Total protein (mo) Total IL-2 Activity (units) Yield of IL-2 ca Soluble fraction 37 mg 2 χ 108 0.3 Periplasmic fraction 2 mg 10 x 106 1.5 Outer membrane 6 mg 8 x 106 1.2 Inner membrane 15 mg 6.6 χ 108 97 Example 3 g of transformed E. coli cells producing mature IL-2 was resuspended in 30 mM Tris HCl, pH 8, 5 mM EDTA, 1 mM phenylmethylsulphonyl fluoride and lysed by sonification. The lysate was centrifuged at 14000 x g for 15 minutes. The membrane components of the centrifuged lysate were separated from the other components of the lysate. The membrane components were then subjected to 4 washing or IL-2 extraction steps, the results of which are compiled in Table 3. 5 ml of wash solution were used per gram of cells. The bulk of the IL-2 activity was extracted in the final wash as detailed in Table 3. This wash solution, which contained the bulk of the IL-2 activity and at least 50% pure IL-2 measured with the aid of SDS-PAGE, was subjected directly to reverse phase HPLC (RP-C8). The yield was approximately 60% of almost 100% pure IL-2 protein. The IL-2 bioactivity lay at 4 χ 108 U/mg.

Table 3 E xtraction of mature IL-2 from E, coli membrane Extraction Total protein (ma) Total IL-2 Activity (units) Specific Activity (U/mq) 1M NaCl wash 0.5 mg 0.3 χ 108 0.6 χ 108 1% Triton wash 3.3 mg 5.5 χ 108 1.7 χ 108 1.75M guanidine HCl wash 2.5 mg 10.0 x 108 4.0 χ 108 7M guanidine HCl wash 10.1 779 χ 108 77.1 χ 108 (98%) 794.8 χ 108 Example 4 The following Example describes the purification of mature IL-2 from a paste of transformed E. coli cells by Procion red agarose chromatography and HPLC.

Cell membrane extraction Frozen E. coli cells (containing a plasmid coding for human IL-2) are thawed and 1 g of these cells is taken up in 5 ml of Buffer A (0.03M Tris HCl, pH 8.0, 0.005M EDTA). The resulting cell suspension is then stirred for 15 minutes and thereafter the cells are collected by centrifugation in a Sorval SS-34 rotor (10000 rpm for 10 minutes). The supernatant is discarded and the cells are resuspended in 5 ml of Buffer A. The resuspended cells are broken with a sonicator (6 x 30 seconds). The broken cells are centrifuged (10000 rpm for 10 minutes) and the supernatant containing the soluble proteins is discarded. The residue is washed once with 5 ml of Buffer A and centrifuged (10000 rpm for 10 minutes). The residue, which corresponds to the membrane fraction, is dissolved in Buffer Β (1M NaCl, 0.03M Tris HCl, pH 8.0, 0.005M EDTA) with a tissue homogenizer and stirred for 10 minutes. The membrane fraction is removed by centrifugation in a Sorval SS-34 rotor (15000 rpm for 10 minutes). The residue is dissolved in 5 ml of Buffer C (1% Triton X-100, 0.03M Tris HCl, pH 8.0), homogenized, stirred and again centrifuged (15000 rpm for 10 minutes). The residue is dissolved in 5 ml of 1.75M guanidine HCl, homogenized, stirred and centrifuged (15000 rpm for 10 minutes). The resulting residue is washed once with 5 ml of Buffer A and centrifuged. The membrane fraction (residue) is extracted with 5 ml of 7M guanidine HCl. After centrifugation, the extract containing the IL-2 is saved and the residue is extracted a second time with 5 ml of 7M guanidine HCl. This extract is also saved.

Procion red agarose chromatography The column is filled at room temperature, washed with two column volumes of 7M guanidine HCl and then equilibrated with equilibration buffer (0.01 M Tris HCl, pH 7.9, 0.035M NaCl) at 4°C. At least 1 ml of Procion red agarose should be used per 100 pg of IL-2 protein expected. The flow rate is 2 bed volumes per hour. The 7M guanidine HCl extract containing the IL-2 is diluted 40fold with equilibration buffer and the precipitate is removed by centrifugation. The column is loaded with the resulting supernatant. The column is washed with two column volumes of equilibration buffer. IL-2 appears as a peak after 1.5-2 volumes of elution buffer (0.01 M Tris HCl, pH 7.9, 1.035M NaCl). After elution of the IL-2, the column is freed from extraneous materials by washing with 6M guanidine HCl.

HPLC RP-P (C-18) chromatography The pH of the Procion red eluate is adjusted to 7 by the slow addition of 1.0M acetic acid. Aliquots of 0.1 ml each are removed for activity determination before and after the pH adjustment. The neutralized eluate (60 ml) is pumped at a flow rate of 1 ml/minute on to a 0.31 x 25 cm RP-18 column and equilibrated with a buffer mixture consisting of 5% HPLC-Buffer II and 95% HPLC-Buffer I. The column flowthrough is collected and assayed for its protein content and its IL-2 bioactivity. The column is eluted at room temperature according to the gradient profile described in Table 4. The effluent is monitored at wavelength 220 nm and 1.5 ml fractions are collected. Their protein content and their IL-2 bioactivity are determined. The peak with maximum IL-2 activity elutes at about 74% HPLCBuffer II (59% acetonitrile). The fractions are stored at 4-8°C and are pooled according to their specific activity.

Table 4 Conditions for the HPLC (RP-18 column of a typical Procion-red eluate.

Sample amount: Column dimensions HPLC Buffer I: HPLC-Buffer II: Flow rate: ml 0.41 x 25 cm 0.01 M phosphoric acid 0.05M lithium chloride 0.01 M phosphoric acid 0.05M lithium chloride, 80% acetonitrile 1 ml/min.

Equilibration % HPLC-Buffer II 5 Duration (min) Sample amount Wash Step 1, gradient Step 2, gradient Step 3, constant Step 4, constant to 35 35 to 85 All chromatographic steps are carried out at room temperature.

Claims (14)

Claims

1. A process for the production of homogeneous mature recombinant human interleukin-2 (IL-2), characterized by a) cultivating microorganisms transformed with a DNA sequence which codes for mature human IL-2; (b) causing the transformed microorganisms to express and accumulate mature human IL-2; (c) lysing the microorganisms to give a cell lysate; (d) separating the cell membrane components present in the cell lysate; (e) extracting IL-2 from the isolated cell membrane components using a wash solution which contains about 4 to 7M guanidine HCl; and (f) chromatographically purifying the IL-2 from the wash solution.

2. A process according to claim 1, characterized in that the transformed microorganisms are lysed by sonification.

3. A process according to claim 1 or 2, characterized in that the cell membrane components are separated from the cell lysate by centrifugation.

4. A process according to any one of claims 1-3, characterized in that the isolated cell membrane components are washed with a wash solution containing about 4 to 7M guanidine HCl, with salt and detergent solutions prior to the extraction of the IL-2.

5. A process according to claim 4, characterized in that the washings with the salt and detergent solutions are carried out in 3 sequential, separate steps.

6. A process according to claim 5, characterized in that the cell membrane components are washed in the first step with a NaCl solution, in the second step with a detergent solution and in the third step with a guanidine HCl solution having a molarity of approximately 1.75 to 2.0.

7. A process according to claim 6, characterized in that the extraction of the IL-2 from the isolated cell membrane components is carried out using a wash solution which contains approximately 7M guanidine HCl.

8. A process according to claim 1, characterized in that the chromatographic purification is carried out by high performance liquid chromatography (HPLC).

9. A process according to claim 1, characterized in that the chromatographic purification is carried out by affinity chromatography.

10. A process according to claim 9, characterized in that the affinity chromatography is dye affinity chromatography.

11. A process according to claim 1, characterized in that the chromatographic purification is a multiple step procedure in which high performance liquid chromatography and affinity chromatography are used.

12. A pharmaceutical preparation containing as active ingredient homogeneous, mature, recombinant human interleukin2 (IL-2) having a specific activity of at least 53 x 10 6 U/mg, obtainable in accordance with the process according to any one of claims 1 to 11, and a physiologically compatible carrier material.

13. A process according to claim 1, substantially as hereinbefore described and exemplified.

14. Homogeneous mature recombinant human interleukin-2 whenever produced by a process claimed in a preceding claim.