Purified Recombinant Interleukin-2 Compositions

Patentamt Europaisches ||| || 1 1| || || || || ||| || || || ||| || (19) J European Patent Office

Office europeen des brevets (11) EP 0 470 586 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publicationation and mention (51) |nt. CI.6: A61 K 38/20 of the grant of the patent: 15.07.1998 Bulletin 1998/29

(21) Application number: 91113235.5

(22) Date of filing: 27.03.1985

(54) Purified recombinant interleukin-2 compositions Gereinigtes rekombinantes lnterleukin-2 enthaltende Zusammensetzungen Compositions contenant de l'interleukine-2 recombinante purifiee

(84) Designated Contracting States: (74) Representative: BE CH DE FR GB IT LI NL SE Goldin, Douglas Michael et al J.A. KEMP & CO. (30) Priority: 28.03.1984 US 594223 14 South Square Gray's Inn (43) Date of publication of application: London WC1 R 5LX (GB) 12.02.1992 Bulletin 1992/07 (56) References cited: (62) Document number(s) of the earlier application(s) in EP-A- 91 539 EP-A- 92163 accordance with Art. 76 EPC: EP-A- 94 317 EP-A- 147 819 85103649.1 /0156 373 EP-A- 0 145 390 EP-B-0 211 835

(73) Proprietor: CHIRON CORPORATION • JOURNAL OF IMMUNOLOGY, vol. 128, no. 3, Emeryville, California 94608 (US) March 1982, US, pages 1122-1127; J.W MIER et al.: "The purification and properties of human T Inventors: (72) cell growth factor" Kirston Edward • Koths, • CHEMICAL ABSTRACTS, vol. 74, no. 1 0, 8 March CA 94702 Berkeley, (US) 1971, Columbus, Ohio, US; abstract no. 45571, • James William Thomson, NOG AMI, HISASHI et al.: "Decomposition and California 94706 Albany, (US) stabilization of drugs in solution. XVII. Michael • Kunitani, Stabilization mechanism of acylcholine esters in Oakland, California 94611 (US) aqueous solution by sodium lauryl sulfate", • Wilson, Kenneth page 226; & CHEMICAL AND Walnut California 94598 Creek, (US) PHARMACEUTICAL BULLETIN, 1970, 18(11), • Hanisch, Wolfgang Helmut 2297-2302 Oakland, CA 94611 (US)

CO CO CO 10

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in o a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. Q_ 99(1) European Patent Convention). LU Printed by Xerox (UK) Business Services 2.16.3/3.4 1 EP 0 470 586 B1 2

Description non-IL-2 proteins selectively from the cellular material; This invention is in the field of biochemical engi- (c) solubilizing the IL-2 in the solid phase of the neering. More particularly, the invention concerns com- extraction mixture with an aqueous solution of a positions comprising purified interleukin-2 (IL-2) which s solubilizing agent that forms a water soluble com- may be separated or recovered from micro-organisms plex with the IL-2, said solution containing a reduc- that have been transformed to produce IL-2. ing agent; and Native human IL-2 is an antigen-nonspecific, genet- (d) separating the IL-2 from the resulting solution ically unrestricted soluble factor produced by erythro- under reducing conditions. cyte rosette positive T cells stimulated with antigens, 10 mitogens and alloantigens. It is a protein with a reported The present invention provides a purified recom- molecular weight in the approximate range of 13,000 to binant IL-2 product wherein the IL-2 does not include 17,000 daltons (S. Gillis and J. Watson, J Exp Med the initial N-terminal alanine, the product comprising at (1980) 159:1709) and an isoelectric point in the approx- least about 95%, preferably greater than about 98%, IL- imate range of pH 6-8.5. Human IL-2 has a number of in is 2 as determined by reducing SDS-PAGE analysis, and vitro and in vivo effects including enhancing the prolifer- having an endotoxin content of less than about 0.1 nan- ative responses of human peripheral blood mononu- ogram/mg of IL-2 and being substantially free of pyro- clpar cells or murine thymocytes, enhancing the gens as determined by the U.S. P. rabbit pyrogen test at immune response in humans and in animals against a dosage of 3.3 x 105 U/kg. Preferably the IL-2 is des- bacterial, parasitic, fungal, protozoan and viral infec- 20 Ala-Ser125 IL-2. The product may further comprise a tions, and supporting the growth of continuous T cell solubilizing agent, such as SDS. lines. In a particular embodiment, the IL-2 is obtainable IL-2 and IL-2 muteins in which the cysteine residue from the cellular material of the micro-organism that at amino acid 125 has been replaced with serine and/or produced it by a process comprising disrupting the cell the initial alanine has been eliminated have been pro- 25 membrane of the micro-organism, extracting the partic- duced microbially through genetic engineering tech- ulate fraction of the disruptate with an aqueous solution niques. Microbially produced IL-2 is not glycosylated of a chaotropic agent that selectively extracts non-IL-2 and is produced in a reduced state by the microorgan- proteins from the cellular material, solubilizing the IL-2 isms. When purified and oxidized, these microbially pro- in the solid phase of the extraction mixture with an duced IL-2s exhibit activity comparable to native human 30 aqueous solution of a solubilizing agent that forms a IL-2. water soluble complex with the IL-2, said solution con- Procedures for purifying native IL-2 from T cells are taining a reducing agent, and separating the IL-2 from described by Watson, J., et al, J Exp Med (1979) the resulting solution. 150:849-861; Gillis, S., et al, J Immunology (1980) In preferred embodiments of the process which 124:1954-1962; Mochizuki, D. Y, et al, J Immun Meth 35 may be used to obtain the product of the invention the (1980) 39:185-201; Welte, K., et al, J Exp Med (1982) chaotropic agent is urea at a concentration of about 3.5 156:454-464: and European patent applications M to about 4.5 M in the extraction mixture, the solubiliz- 83103582.9 (published October 26, 1983 under no. ing agent is sodium dodecyl sulfate (SDS) or sodium 92163) and 83400938.3 (published November 16, 1983 lauryl sarcosine (sarcosyl), the solubilized IL-2 is further under no. 94317). In general, these procedures involve 40 extracted with 2-butanol or 2-methyl-2-butanol and the precipitating proteins from culture supernatants with final separation is carried out by gel filtration, the result- ammonium sulfate followed by a chromatographic frac- ing sized product is oxidized and the oxidized product is tionation. purified by reverse phase high performance liquid chro- U.S. Patent No 4,450,103 issued May 22, 1984 and matography (RP-HPLC). Derynck, R., et al, Nature (1980) 287:193-197 describe 45 Figure 1 shows a flow diagram of two alternative procedures for recovering IFN-p-producing E.coli. The embodiments of the above process in which gel filtration patent describes a procedure in which IFN-p is chromatography is used as a final purification step. The extracted from cellular material with 2-butanol or 2- embodiment designated Method 1 A uses SDS as a sol- methyl-2-butanol. ubilizing agent; the embodiment designated Method 1 B EP-A 156,373, from which the present application so uses sarcosyl as a solubilizing agent. The figure is divided, describes and claims a process for recover- includes densitometer scans of SDS-polyacrylamide gel ing IL-2 from a transformed micro-organism containing electrophoresis (SDS-PAGE) analyses of the product at the IL-2 comprising:- various steps in the process. Figure 2 is an HPLC chromatogram and SDS- (a) disrupting the cell membrane of the micro- 55 PAGE analysis of the product of Example 3. organism; Figure 3 is an HPLC chromatogram of the product (b) extracting the disruptate at a basic pH with an of Example 9. aqueous solution of a chaotropic agent that extracts Figure 4 is a flow diagram of a preferred procedure

2 3 EP 0 470 586 B1 4 for processing microbially produced IL-2. or other conventional methods. As used herein the term "IL-2" denotes an unglyco- Following concentration the cell membranes of the sylated protein that is (a) produced by a microorganism microorganisms are disrupted. The main purpose of that has been transformed with a human interleukin-2 disruption is to facilitate the following extraction and sol- gene or a modification of the human interleukin-2 gene 5 ubilization steps. Conventional cell disruption tech- that encodes a protein having: (a) an amino acid niques such as homogenization, sonication, or pressure sequence that is at least substantially identical to the cycling may be used in this step of the process. Pre- amino acid sequence of native human interleukin-2 and ferred methods are sonication or homogenization with a (b) has biological activity that is common to native Manton-Gaulin homogenizer. The end point of the dis- human interleukin-2. Substantial identity of amino acid 10 ruption step may be monitored by optical density, with sequences means the sequences are identical or differ the optical density of the suspension typically decreas- by one or more amino acid alterations (deletions, addi- ing about 65% to 85%. In any event, the disruption tions, substitutions) that do not cause an adverse func- should break substantially all of the cells so that sub- tional dissimilarity between the synthetic protein and stantially no intact cells are carried through to the solu- native human interleukin-2. Examples of such proteins 15 bilization step. Before the disruption, the pH of the liquid are the IL-2s described in European patent application phase of the concentrate is adjusted, if necessary, to a 83101035.0 filed February 3, 1983 (published October level that facilitates removal of E. coli proteins in subse- 19, 1983 under publication no. 91539) and European quent steps, while retaining IL-2 protein as an insoluble patent application 82307036.2 filed December 22, 1982 complex in the cellular debris. The pH may be so (published September 14, 1983 under no. 88195), the 20 adjusted by adding suitable buffers. In most instances IL-2s described in European patent application pHs in the range of about 8 to about 8.5 will be used. 83306221.9 filed October 13, 1983 (published May 30, The steps in the recovery process subsequent to 1984 under no. 109748), and the IL-2s described in the the disruption step are primarily designed to separate examples of this application. The product of the inven- the IL-2 from E. coli proteins to a high level of purity (at tion specifically comprises IL-2 which does not include 25 least about 95% and preferably at least about 98%) in an initial N-terminal alanine residue. good yields while maintaining the IL-2 in a reduced As used herein the term "transformed microorgan- state. Simultaneously, these purification processes, in ism" denotes a microorganism that has been genetically combination, also reduce pyrogenic substances in the engineered to produce a protein that possesses native final product to a level believed to be acceptable for human interleukin-2 activity. Examples of transformed 30 parenteral administration to patients. microorganisms are described in said European patent After the cells have been disrupted the particulate publications 88195, 91539, and 109748 and the exam- matter may be separated from the liquid phase of the ples of this application. Bacteria are preferred microor- disruptate and resuspended in an aqueous medium ganisms for producing IL-2. Synthetic IL-2 may also be buffered to the optimal pH for the extraction. The partic- made by suitably transformed yeast and mammalian 35 ulate matter may optionally be washed with buffer at this cells. E. coli is particularly preferred. stage to remove any water soluble E. coli proteins The transformed microorganisms are grown in a therein. In any event, the protein concentration of the suitable growth medium, typically to an optical density cell suspension subjected to the extraction will usually (OD) of at least about 30 at 680 nm, and preferably be in the range of about 5 to about 60 mg/ml, preferably between about 20 and 40 at 680 nm. The composition 40 20 to 40 mg/ml. of the growth medium will depend upon the particular The extraction of E. coli proteins from the particu- microorganism involved. The medium is an aqueous late cellular material may be carried out concurrently medium containing compounds that fulfill the nutritional with the disruption or sequentially following the disrup- requirements of the microorganism. Growth media will tion. It is preferably carried out as a separate step fol- typically contain assimilable sources of carbon and 45 lowing the disruption. The extractant is an aqueous nitrogen, energy sources, magnesium, potassium and solution of a chaotropic agent (i.e., a mild protein denat- sodium ions, and optionally amino acids and purine and urant that dissociates hydrogen bonds and affects the pyrimidine bases. (See Review of Medical Biology. tertiary structure of proteins). The extractant selectively Lange Medical Publications, 14th Ed pp 80-95 (1980.)) removes the bulk of the E. coli proteins from the cellular In expression vectors involving the trp promoter, the so debris leaving at least a substantial portion of the IL-2 tryptophane concentration in the medium is carefully associated (contained in or bound to) with the cellular controlled to become limiting at the time IL-2 expression debris. The selectivity is facilitated by the hydrophobicity is desired. Growth media for E. coli are well known in of the IL-2 and the fact that it is in a reduced, insoluble the art. state at a pH near the isoelectric point of the protein. In After the cells are harvested from the culture, they 55 addition, a substantial portion of the IL-2 may be may be concentrated, if necessary, to about 20 to 150 present in vivo as inclusion bodies of significant mass, mg/ml, preferably 80 to 100 mg/ml (OD 40 to 300, pref- as has been the case with other cloned proteins erably 160 to 200 at 680 nm) by filtration, centrifugation, expressed at high levels in E. coli. Examples of extract-

3 5 EP 0 470 586 B1 6 ants are urea and guanidinium hydrochloride (guanidin- carrying out the organic extraction the protein concen- ium hydrochloride should not be used when SDS is tration of the aqueous solution is preferably adjusted, if used as a solubilizing agent). Urea is preferred. The necessary, to less than about 6 mg/ml, preferably about concentration of the chaotropic agent in the extraction 0.5 to 4 mg/ml. Reducing conditions are maintained by mixture will depend upon the particular agent that is 5 carrying out the extraction in the presence of a reducing used and the amount of cellular material in the extrac- agent (e.g., DTT). The butanol will normally be added to tion mixture. In the case of urea, concentrations (final) the aqueous solution of solubilized IL-2 in volume ratios between about 3.5 M and 4.5 M, preferably about 4 M, in the range of about 1 :1 to about 3:1 (extractant:aque- will be used in batch processes at 25°C. If the extraction ous solution), preferably about 1:1. The extraction may is run on a continuous basis over longer time periods it 10 be carried out in a batch or continuous operation. The may be desirable to use lower concentrations. Temper- temperature will normally be in the range of 20°C to atures in the range of 20°C to 25°C will normally be 100°C and the pH will normally be about 4 to 9, prefera- used in extraction, with room temperature being used bly about 5 to 6. The time of contact between the solu- for convenience. Mixing will typically be used to tion and the butanol is not critical and relatively short enhance contact between the solution and particulate 15 times on the order of a few minutes may be used. After matter and thus decrease the time required to extract the extraction is complete, the aqueous phase and buta- non-IL-2 proteins from the cellular debris. Kinetic analy- nol phase are separated and the IL-2 is separated from sis of the extraction process was performed on the the butanol phase. A preferred procedure for separating supernatants using SDS-PAGE, and the extraction was the IL-2 from the butanol phase is acid precipitation. found to be essentially complete by 15-30 minutes. 20 This is done by adding the butanol phase to aqueous Following the extraction, the mixture is separated buffer, pH 7.5 until the organic phase is dissolved into solid and liquid phases. The IL-2 in the solid phase (approx. 2-3 vol buffer per vol of organic), and then low- is then selectively solubilized by contacting the solid ering the pH to about 5.5 to 7.0, preferably 6.0 to 6.2, to phase with a neutral, aqueous buffer containing a cause the IL-2 to precipitate. reducing agent and a solubilizing agent. Surface active 25 The next step in the process is to separate the IL-2 agents (detergents) that have a suitable hydrophobic- and any E. coli contaminants remaining after the extrac- hydrophilic balance to solubilize the hydrophobic IL-2 tion^) and optimally from the solubilizing agent. Gel fil- may be used. Alkali metal sulfates containing 10 to 14 tration chromatography, RP-HPLC, or a combination of carbon atoms and alkali metal alkyl sarcosinates are gel filtration chromatography and RP-HPLC are used. preferred solubilizing agents, with SDS and sarcosyl 30 The gel filtration chromatography is preferably carried being particularly preferred. out in two stages that remove both pyrogenic compo- The amount of solubilizing agent used in the solubi- nents and protein contaminants having molecular lization will depend upon the particular agent. When weights higher or lower than IL-2. (IL-2 has a molecular SDS or sarcosyl are used, the preferred ratio (w/w) of weight of about 1 5.5K daltons.) Gels that are capable of SDS/sarcosyl to solid phase protein is about 0.5:1 to 35 fractionating the solution to permit separation of the IL- 1 .4:1 . The solubilizing medium also contains a sufficient 2 from these contaminants are commercially available. amount of reducing agent to prevent the solubilized IL-2 Sephacryl S-200 is a preferred gel for removing the from undergoing oxidation to any significant degree. higher molecular weight components and Sephadex G- Protein reduciny agents such as dithiothreitol (DTT) and 25, G-75 or G-100 gels are preferred for removing the 2-mercaptoethanol may be used. The concentration of 40 low molecular weight contaminants. The gel filtrations reducing agent such as DTT in the medium will usually will typically be run in buffered solutions (pH 5.5 to 7.0) range between about 5 to 20 mM. The solubilization will containing about 0.1% to 1.0% solubilizing agent and typically be carried out at temperatures in the range of about 1 to 10 mM reducing agent. The column will be 20°C to 25°C with mixing to facilitate contact between sized to permit suitable resolution of the desired compo- the solid phase and the solubilizing medium. Higher 45 nents. temperatures may solubilize unwanted E. coli proteins. RP-HPLC is an alternative to gel filtration. Also, RP- The solubilization is considered complete when the HPLC is capable of removing molecules from the solu- sample has sat 15 minutes or the solution turns translu- tion that have molecular weights close to IL-2 and can- cent. Insoluble material is separated after completing not, therefore, be removed completely by gel filtration. In the solubilization. so addition, contaminants such as bacterial endotoxin are After the IL-2 is solubilized the IL-2 may optionally also removed effectively by RP-HPLC. Therefore, RP- be extracted from the aqueous solution under reducing HPLC may also be used as a final purification step after conditions with 2-butanol or 2-methyl-2-butanol to gel filtration. Supports (stationary phases) that provide remove additional E. coli proteins, notably including cer- good resolution of proteins may be used in the RP- tain contaminants that have molecular weights very 55 HPLC. C-4, C-8, or C-18 on 300 angstrom pore-size close to the IL-2. Conditions (e.g., ionic strengths in the supports are examples of preferred supports. The sep- range of 0.05 and 0.15) at which the aqueous solution aration is carried out at an acidic pH of less than about and butanol are substantially immiscible are used. In 2.3, usually 2.1 to 2.3, in order to keep the IL-2 in solu-

4 7 EP 0 470 58636 B1 8 tion. In this regard, the pH of the solution from the solu- trate the above process whereby the IL-2 used in the bilization (gel filtration) will preferably be adjusted to this present composition may be obtained. These Examples range. The solution is loaded into the RP-HPLC column are not intended to limit the invention in any manner. and is adsorbed onto the stationary phase. A gradient solvent system comprising an organic acid such as ace- 5 Example 1 tic acid or trifluoroacetic acid and organic solvent such as propanol or acetonitrile is used to elute the IL-2 from IL-2 was recovered from E. coli K-12 strain MM294 the column. Acetic acid-propanol, trifluoroacetic acid- that had been transformed with the plasmid pLW1 propanol, and trifluoroacetic acid-acetonitrile are pre- (deposited at the American Type Culture Collection on ferred solvent systems. IL-2 elutes in the acetic acid- w August 4, 1983 under accession number 39,405) as fol- propanol system at about 40% propanol, in the trifluoro- lows. acetic acid-propanol system at about 50% propanol, The E. coli were grown in a fermenter using the fol- and in the trifluoroacetic acid-acetonitrile system at lowing growth medium. about 62% acetonitrile. For convenience, the organic solvent content of the elutant will usually be increased 75 rapidly to a level somewhat below the solvent concentration at which the IL-2 elutes followed slow by a gradi- (NH4)2S04 150 mM ent change in the range of about 0.1% to 1 .0%/min. mM As soon as the IL-2 is recovered from the chroma- KH2P04 21.6 tography step, it is lyophilized and resuspended in a 20 Ca3 Citrate 1 .5 mM neutral aqueous buffer containing the reducing agent (to ZnS04 • 7H20 30 mM keep the IL-2 in a reduced state) and the solubilizing agent (to keep it in solution). The IL-2 is stable in this MnS04 • H20 30 mM form and be stored for further treatment and formu- may CuS04 • 5H20 1 mM lation before being used. 25 An alternative and preferred procedure is to oxidize pH adjusted to 6.50 with 2.5 N the IL-2 after it has been separated by gel filtration and NaOH autoclaved purify the oxidized product by RP-HPLC or gel filtration Sterile Additions (post autoclave) followed by RP-HPLC. This results in efficient removal of contaminants surviving the gel filtration as well as 30 MgS04 • 7H20 3 mM unwanted oxidation products. A preferred oxidation pro- FeS04 100 nM cedure is to oxidize a fully reduced microbially produced L-tryptophan 1 4 mg/l synthetic protein having an amino acid sequence substantially identical to a useful protein which sequence Thiamine-HCI 20 mg/l includes cysteines which in the useful protein are linked 35 Glucose 5 g/l intramolecularly to form a cystine in a controlled manner so that the cysteines are oxidized selectively to form the Tetracycline 5 mg/l In this the reduced cystine. process fully microbially pro- Ethanol (optional) 2% duced synthetic protein is reacted with o-iodosobenzoate, which oxidizes cysteines selectively in an 40 Casamino acids 2% aqueous medium, at a pH at least about one-half pH unit below the pKa of said cysteines, wherein the concentration of synthetic protein in the reaction mixture is Dow Corning Antifoam B, 20% solution, glucose, less than about 5 mg/ml and the mol ratio of o-iodoso- 50% solution, and KOH, 5N, were added on demand. benzoate to protein is at least stoichiometric, with the 45 The pH of the fermenter was maintained at 6.8 with proviso that the o-iodosobenzoate is in excess in the 5 N KOH. Residual glucose was maintained between 5- terminal portion of the reaction. RP-HPLC purification of 10 g/l, dissolved oxygen at 40%, and temperature at 37 the oxidized product may be carried out under the con- ±1 °C. The casamino acids (20% stock solution) were ditions described above in the absence of a reducing added when the OD680 was about 10. Harvest was agent and presence of a detergent at a concentration so made three hours after the OD680 reached about 20. equal to or less than those used in the above described The harvested material was concentrated by hollow gel filtration. fiber filtration and/or centrifugation. Twenty to forty g The purity of the IL-2 after the chromatography (wet weight) of the concentrate were resuspended in step(s) is at least about 95% and usually at least about 200 ml of 50 mM Tris, 1 mM ethylenediaminetetraacetic 98%. This highly pure material contains less than about 55 acid (EDTA) (pH 8.1-8.5) (Tris/EDTA buffer). The sus- 5 ng endotoxin, usually less than about 0.01 ng endo- pension was centrifuged at 3,000-4,000 x g for 10 min- toxin per 100,000 Units II-2 activity. utes, the supernatant was removed, and the solids were The following Examples further describe and illus- resuspended in 200 ml Tris/EDTA buffer at 4°C. The

5 9 EP 0 470 586 B1 10 suspension was loaded into a sonicator (Heat Systems, 2% sarcosyl instead of 2% SDS as a solubilizing agent Model W-375) and sonicated at 4°C for 45 minutes (end and using sarcosyl in place of SDS in the chromatogra- point = OD680 reduction of about 85%) using large phy columns. Figure 1 shows the densitometer scan for probe, pulsing with 50% duty on power setting "9". An this crude extract using sarcosyl as a solubilizing agent alternative disruption technique is to pass the suspen- 5 (crude extract of Method 1 B). As indicated, the use of sion three times through a Manton-Gaulin homogenizer sarcosyl in place of SDS gave improved purity (58% vs on M-1 setting. Cellular debris was separated from the 37%) at similar IL-2 yield (50% vs 60%). disruptate by centrif uging at 4,500 x g for 1 0 minutes. The cellular debris was resuspended in 60 ml Example 3 Tris/EDTA buffer at room temperature and an equal vol- 10 ume of 8 M urea (Schwarz/Mann ultrapure) in Tris/EDTA The procedure of Example 1 was repeated through buffer was added to the suspension over five mintues the steps preceding urea extraction and was then solu- with rapid stirring (final urea concentration, 4 M). After bilized and clarified as described. continued slow stirring for 15-30 minutes, the suspen- The IL-2 was separated from the solution by RP- sion was centrifuged at 12,000 x g for 15 minutes to 15 HPLC as follows. The solution was diluted 10-fold in recover extracted cellular debris. (If a solid phase does 0.1% trifluoroacetic acid (TFA) and was applied to a 4.6 not form, the supernatant is withdrawn, an equal volume mm I.D. x 5 cm L. Brownlee Aquaport RP-300 column of Tris/EDTA buffer is added and the mixture is recentri- equilibrated in 0.1% TFA. The IL-2 was eluted with a fuged. ) gradient of 30%-60% acetonitrile containing 0.1% TFA The extracted cellular debris is then resuspended in 20 over 45 minutes. The yield of IL-2 activity following 9 ml of 50 mM sodium phosphate (pH 6.8), 1 mM EDTA, HPLC was 80-100%. Figure 2 shows a silver-stained 10 mM DTT at 20°C. One ml of 20% SDS is added to SDS-PAGE analysis of this product. the suspension, and the suspension is mixed vigorously for 5 minutes. The liquid phase is recovered from the Example 4 suspension by centrif uging at 1 2,000 x g for 1 0 minutes 25 at room temperature. The liquid phase was then heated The procedure of Example 1 was repeated through to 40°C for 15 minutes to insure that the IL-2 in the solu- the steps preceding gel filtration chromatography. The tion is fully reduced. A sample of this crude extract was soluble, clarified, reduced material was subjected to G- analyzed by 15% SDS-PAGE. Figure 1 shows a densit- 100 chromatography in 0.1% SDS as described in ometer scan of that analysis product of Method 1 A) indi- 30 Example 1 . The pooled peak fractions of IL-2 were fur- eating the extract contained about 37% IL-2. ther purified by RP-HPLC as described in Example 3. IL-2 was separated from the solution by gel filtration The resulting purified, reduced IL-2 was oxidized and chromatography as follows. The solution was loaded subjected to RP-HPLC as described in Example 3. onto a 2.6 cm x 100 cm S-200 column run in 50 mM sodium phosphate (pH 6.8), 1 mM EDTA, 1 mM DTT, 35 Example 5 1% SDS. The column effluent was collected in 4 ml fractions with samples of the fractions analyzed in 15% The procedure of Example 1 was repeated through SDS-PAGE minigels stained with Coomassie blue. The the steps preceding the G-100 column. The procedure fractions containing the fewest contaminants (minimiz- for Example 3 was repeated using a solvent system of ing contaminants at about 35K daltons, 1 6-1 8K daltons, 40 propanol in 1 M acetic acid. The IL-2 was eluted with a and 12K daltons) were pooled and concentrated to 5-10 gradient of 35% - 60% propanol over 200 minutes. Col- ml by ultrafiltration (Amicon YM5 ultrafilter). The con- umn dimensions were either 10 mm ID x 30 cm L or 48 centrate was loaded onto a 2.6 cm x 100 cm G-100 col- mm ID x 50 cm L, and the column was packed with a umn, run as above except that the SDS concentration bonded phase wide-pore silica gel. The bonded phase was 0.1% rather than 1%. Fractions were analyzed by 45 wide-pore silica used was Vydac TP214. The purity and SDS-PAGE and the purest fractions were pooled. The yield of product was comparable to that of Example 3. drawing shows a densitometer scan of the chromato- graphed product. Analysis indicated the product was Example 6 98% pure and contained 0.5 ng endotoxin/100,000 units of IL-2 activity as measured by the limulus amebocyte so The procedure of Example 3 was repeated using a lysate assay (Associates of Cape Cod, Inc., Woods solvent system of propanol in 0.1% TFA. The IL-2 was Hole, MA). The N-terminal amino acid sequence of this eluted with a gradient of 35% - 60% propanol over 1 20 IL-2 is the same as the native human molecule except minutes. The column and support materials were the that the initial N-terminal alanine is missing. same as in Example 5. The purity and yield of product 55 were comparable to that of Example 3. Example 2

The procedure of Example 1 was repeated using

6 11 EP 0 470 586 B1 12

Example 7 above. Following reduction the solution was adjusted to pH 5.5 with acetic acid. The solution was purified by gel The procedure of Example 1 was repeated except filtration using S-200 and G-25 columns. The resulting that the E_coN-produced IL-2 was one designated des- purified, reduced IL-2 was oxidized, and the oxidized Ala Ser125 IL-2. The amino acid sequence of this IL-2 is s product was purified by G-25 chromatography followed different from that of the native molecule in that the by RP-HPLC as in Example 3. The resulting purified cysteine at position 125 has been changed to serine recombinant IL-2 product has an IL-2 content greater and the initial N-terminal alanine residue is missing. The than about 95% as determined by reducing SDS-PAGE strain of des-Ala Ser125 IL-2 producing E. coli that pro- analysis, an endotoxin content of less than about 0.1 duced this IL-2 was deposited in the American Type 10 nanograms/mg of IL-2, and it is substantially free of Culture Collection on March 6, 1984 under accession pyrogens as determined by the U.S. P. rabbit pyrogen number 39,626. test at a dosage of 3.3 x 105 U/kg. As previously indicated, the endotoxin content is less than about 5 nano- Example 8 grams, and preferably less than 0.01 nanograms 15 endotoxin per 100,000 units IL-2 activity. Typically, the The procedure of Example 1 was repeated except purified recombinant IL-2 products purified by the proc- that the IL-2 was recovered from E. coli K-12 strain that ess of the invention have an IL-2 content greater than had been transformed with the plasmid pLW55 (depos- 98% as determined by reducing SDS-PAGE or RP- ited in the American Type Culture Collection on Novem- HPLC, as shown in Figure 3 in addition to being sub- ber 18, 1983 under accession number 39,516). The 20 stantially free of endotoxins and pyrogens as indicated amino acid sequence of this molecule is different from above. that of the native molecule in that it has an N-terminal A variation of the process described in Example 9, methionine and the cysteine at position 125 has been such as might be used to produce IL-2 on a larger scale, changed to serine. is shown in Figure 4. The process shown in Figure 4 dif- 25 fers from that described in Example 9 as regards (1) Example 9 minor changes in the buffers, (2) use of an acetic acid- propanol (Example 5) solvent system in the RP-HPLC, Des-Ala Ser125 IL-2 producing E. coli were grown, and (3) the inclusion of post-oxidation dilution/diafiltra- the cells disrupted and the cellular debris was recov- tion S-200 gel filtration, and ultrafiltration steps. The ered from the disruptate using the general procedures 30 process as shown in Figure 4 may be modified with var- of Example 1 . The cellular debris was suspended in 50 ious refinements, for example, following the second S- mM Tris, 1 mM EDTA pH 8.5 buffer at a ratio of about 200 column pass, in 1% SDS, the IL-2 solution is diluted 1 :4.5 (w/v). DTT was added to a final concentration of 1 :10 to give a 0.1% SDS concentration and then diafil- 25 mM. 8 M urea in the same buffer was slowly added tered against 10 mM phosphate buffer at a pH of 7.5 with stirring to a final concentration of 4 M and then 35 and 5 ppm SDS. The solution is then concentrated as allowed to mix at room temperature for 30 minutes. After required for appropriate use dosage. 30 minutes, the insoluble material remaining was centrifuged. The resulting paste was resuspended in 50 mM Claims sodium phosphate buffer, 1 mM EDTA pH 7.0. The suspension was then solubilized by addition of solid SDS to 40 1. A purified recombinant interleukin-2 (IL-2) product a final concentration of 5% w/v. wherein the IL-2 does not include the initial N-termi- The 5% SDS solution was diluted to 2% SDS with nal alanine, the product comprising at least about 0.1 M Na2P04, pH 8.0. The protein concentration was 95% IL-2, as determined by reducing SDS-PAGE determined, the pH was adjusted to 8.5, and DTT to 50 analysis, and having an endotoxin content of less mM and EDTA to 2 mM were added. The mixture was 45 than about 0.1 nanogram/mg of IL-2 and being sub- heated to 40°C under N2 to reduce the IL-2. The mixture stantially free of pyrogens as determined by the was then cooled and the pH was adjusted to 5.0. U.S.P. rabbit pyrogen test at a dosage of 3.3 x 105 The solution was then extracted at a 1 :1 ratio (v/v) U/kg. with 2-butanol containing 1 mM DTT at room temperature. Residence time was 2-2.5 minutes. The extraction 50 2. A product as claimed in claim 1 , wherein the IL-2 was carried out in a liquid-liquid phase separator using content is greater than about 98% as determined by a flow rate of 200 ml/min. The organic extract was sep- reducing SDS-PAGE. arated and its pH was adjusted to 8.0 with NaOH. The extract was then added slowly to 0.1% SDS in 10 mM 3. A product according to claim 1 or 2 wherein the IL- Na2P04, 2 mM DTT, pH 6 and stirred for 1 5-20 minutes. 55 2 isdes-Ala-Ser125 IL-2. The resulting precipitate was separated and the resulting paste was resuspended in 5% SDS in PBS. The 4. A product as claimed in claim 1 , 2 or 3 which further solution was clarified by centrifugation and reduced as comprises a solubilizing agent.

7 13 EP 0 470 586 B1 14

5. A product as claimed in claim 4 in which the solubi- durch den U.S.P.-Kaninchen-Pyrogen-Test bei lizing agent is SDS. einer Dosierung von 3,3 x 105 E/kg bestimmt werden. 6. A product as claimed in any one of claims 1 to 5 wherein the IL-2 is obtainable from the cellular 5 2. Produkt nach Anspruch 1, dadurch gekenn- material of the microorganism that produced it by a zeichnet, daB der IL-2-Gehalt bei Bestimmung mit process comprising disrupting the cell membrane reduzierender SDS-PAGE groBer als etwa 98% ist. of the microorganism, extracting the particulate fraction of the disruptate with an aqueous solution 3. Produkt nach Anspruch 1 oder 2, dadurch gekenn- of a chaotropic agent that selectively extracts non- 10 zeichnet, daB das IL-2 des-Ala-Ser125-IL-2 ist. IL-2 proteins from the cellular material, solubilizing the IL-2 in the solid phase of the extraction mixture 4. Produkt nach Anspruch 1, 2 oder 3, dadurch with an aqueous solution of a solubilizing agent that gekennzeichnet, daB es weiterhin ein Solubilisie- forms a water soluble complex with the IL-2, said rungsmittel umfaBt. solution containing a reducing agent, and separat- 15 ing the IL-2 from the resulting solution. 5. Produkt nach Anspruch 4, dadurch gekennzeichnet, daB das Solubilisierungsmittel SDS ist. 7. A product as claimed in claim 6 wherein in extracting IL-2 the chaotropic agent is urea and the solubi- 6. Produkt nach einem der Anspriiche 1 bis 5, lizing agent is sodium lauryl sarcosyl. 20 dadurch gekennzeichnet, daB das IL-2 aus dem cellularen Material des Mikroorganismus, der es 8. A product as claimed in claim 6 or 7, wherein the IL- erzeugt, durch ein Verfahren erhaltlich ist, das das 2 is separated from the resulting solution by gel fil- Zerstoren der Zellmembran des Mikroorganismus, tration or reverse-phase high performance liquid die Extraktion der partikularen Fraktion des Disrupt- chromatography. 25 ats mit einer waBrigen Losung eines chaotropen Mittels, das in selektiver Weise die Nicht-IL-2-Pro- 9. A product as claimed in claim 6 or 7, wherein the IL- teine von dem cellularen Material extrahiert, die 2 is separated from the resulting solution by gel fil- Solubilisierung des IL-2 in der Festphase des tration followed by reverse-phase high performance Extraktionsgemisches mit einer waBrigen Losung liquid chromatography. 30 eines Solubilisierungsmittels, das einen wasserlos- lichen Komplex mit IL-2 bildet, wobei die Losung ein 10. A product as claimed in any one of claims 6 to 9, Reduktionsmittel enthalt, und das Trennen des IL-2 wherein after solubilizing the IL-2 with a solubilizing von der so entstandenen Losung umfaBt. agent in aqueous solution the IL-2 is extracted with 2-butanol or 2-methyl-2-butanol, precipitated with 35 7. Produkt nach Anspruch 6, dadurch gekenn- acid and then purified by gel filtration. zeichnet, daB bei der Extraktion des IL-2 das chao- trope Mittel Harnstoff und das Solubilisierungs- 1 1 . A product as claimed in claim 1 0 wherein the sepa- mittel Natriumlaurylsarcosyl ist. rated IL-2 is selectively oxidized to form intramo- lecular disulphide bonds whilst avoiding unwanted 40 8. Produkt nach Anspruch 6 oder 7, dadurch gekenn- IL-2 oxidation products. zeichnet, daB das IL-2 aus der entstandenen Losung durch Gelfiltration oder reverse 12. A product as claimed in claim 1 1 wherein the selec- Phasenhochleistungsflussigchromatographie tively oxidized product is purified by reverse phase abgetrennt wird. high performance liquid chromatography or gel fil- 45 tration followed by reverse phase high performance 9. Produkt nach Anspruch 6 oder 7, dadurch gekenn- liquid chromatography. zeichnet, daB IL-2 aus dem entstandenen Gemisch durch Gelfiltration, gefolgt von reverser Patentanspruche Phasenhochleistungsflussigchromatographie so getrennt wird. 1. Gereinigtes rekombinantes lnterleukin-2-(IL-2)- Produkt, dadurch gekennzeichnet, daB das IL-2 10. Produkt nach einem der Anspriiche 6 bis 9, das initiale N-terminale Alanin nicht einschlieBt, dadurch gekennzeichnet, daB nach der Solubili- daB das Produkt wenigstens etwa 95% IL-2 bei sierung des IL-2 mit einem Solubilisierungsmittel in Bestimmung durch reduzierende SDS-PAGE-Ana- 55 waBriger Losung das IL-2 mit 2-Butanol oder 2- lyse umfaBt, und daB es einen Endotoxingehalt von Methyl-2-butanol extrahiert wird, mit Saure gefallt weniger als etwa 0,1 Nanogramm/mg IL-2 hat und wird und dann durch Gelfiltration gereinigt wird. daB es im wesentlichen frei von Pyrogenen ist, die

8 15 EP 0 470 586 B1 16

11. Produkt nach Anspruch 10, dadurch gekenn- dans lequel, dans I'extraction de IL-2, I'agent chao- zeichnet, daB das abgetrennte IL-2 in selektiver tropique est I'uree et I'agent solubilisant est le lau- Weise oxidiert wird, so daB intramolekulare Disul- royle sarcosinate de sodium. fidbindungen gebildet werden, wohingegen uner- wtinschte IL-2-Oxidationsprodukte vermieden 5 8. Un produit tel que revendique a la revendication 6 werden. ou 7, dans lequel la IL-2 est separee de la solution resultante par filtration sur gel ou chromatographie 12. Produkt nach Anspruch 11, dadurch gekenn- liquide a haute performance en phase inverse. zeichnet, daB das in selektiver Weise oxidierte Produkt durch reverse Phasenhochleistungsfliis- 10 9. Un produit tel que revendique a la revendication 6 sigchromatographie oder Gelfiltration, gefolgt von ou 7, dans lequel la IL-2 est separee de la solution reverser Phasenhochleistungsflussigchromatogra- resultante par filtration sur gel suivie d'une chroma- phie, gereinigt wird. tographie liquide a haute performance en phase inverse. Revendications 15 10. Un produit tel que revendique dans I'une quelcon- 1. Un produit d'interkeuline-2 (IL-2) recombinant puri- que des revendications 6 a 9, dans lequel apres la f ie dans lequel la IL-2 ne comprend pas I'alanine N- solubilisation de IL-2 avec un agent solubilisant en terminale initiale, le produit comprenant au moins solution aqueuse, la IL-2 est extraite avec du 2- environ 95 % de IL-2, tel que determine par analyse 20 butanol ou du 2-methyl-2-butanol, precipitee avec SDS-PAGE en conditions reductrices, et ayant un de I'acide et ensuite purifiee par filtration sur gel. contenu en endotoxines de moins d'environ 0,1 nanogramme/mg de IL-2 et etant essentiellement 1 1 . Un produit tel que revendique a la revendication 1 0 exempt de pyrogenes tel que determine par le test dans lequel la IL-2 separee est selectivement oxyde pyrogenes sur lapin U.S. P. pour une dose de 3,3 25 dee pour former des liaisons disulfure intramolecu- x105U/kg. laires tout en evitant des produits d'oxydation de IL- 2 non souhaites. 2. Un produit tel que revendique a la revendication 1 , dans lequel le contenu en IL-2 est superieur a envi- 1 2. Un produit tel que revendique a la revendication 1 1 ron 98 % tel que determine par SDS-PAGE reduc- 30 dans lequel le produit oxyde selectivement est puri- trice. f ie par chromatographie liquide a haute performance en phase inverse ou par filtration sur gel 3. Un produit selon la revendication 1 ou 2 dans lequel suivie d'une chromatographie liquide a haute per- la IL-2 estdes-Ala-Ser125 IL-2. formance en phase inverse. 35 4. Un produit tel que revendique a la revendication 1 , 2 ou 3 qui comprend en outre un agent solubilisant.

5. Un produit tel que revendique a la revendication 4 dans lequel I'agent solubilisant est du SDS.

6. Un produit tel que revendique dans I'une quelcon- que des revendications 1 a 5 dans lequel la IL-2 peut etre obtenue a partir du materiel cellulaire du micro-organisme qui la produit par un procede 45 comprenant la rupture de la membrane cellulaire du micro-organisme, I'extraction de la fraction particulate du produit de disruption avec une solution aqueuse d'un agent chaotropique qui extrait selectivement les proteines non IL-2 du materiel cellu- 50 laire, la solubilisation de IL-2 dans la phase solide du melange d'extraction avec une solution aqueuse d'un agent solubilisant qui forme un complexe soluble dans I'eau avec IL-2, ladite solution contenant un agent reducteur, et la separation de IL-2 de la solution resultante.

7. Un produit tel que revendique a la revendication 6

9 EP 0 470 586 B1

FIG. I

IL-2 METHOD 1A c cell dedris / Crude Extract 4M urea extract 37% PURE (~60% YIELD) 2% SDS solubilized

Mb [ HOD 1 B

cell debris Crude Extract 4M urea extract 58% PURE (~50% YIELD) ?% sarcosyl solubilized

Method 1B extract

Final Product S-200 chromatography 98% PURE (—30% YIELD) G-100 chromatography

30K 14. 4K

DENSITOMETRY SCANS OF 15% SDS PAGE

u EP 0 470 586 B1

PYROGEN CONCENTRATION (LAL)

CRUDE IL-2 PURIFIED IL-2 12.000 ng <5 ng

11 EP 0 470 586 B1

FIG. 3

• 15h EP 0 470 586 B1

FERMENTATION

I 4M UREA BATCH EXTRACTION i

FIG 4 2-BUTANOL ■ ■ EXTRACTION w. ~r I 1% SDS, 50mM ACETATE, S-200 COLUMN 2mM DTT, 1mM EDTA, pH 5.5 l _ 0.1% SDS, 50mM ACETATE, G-25nc COLUMN, 1mM EDTA, pH 5.5

OXIDATION IODOSOBENZOIC ACID

G 25 COLUMN 0.1% SDS, 50mM ACETATE, 1mM EDTA, pH 5.5 I

RP-HPLC _ £3&™<>-

^.PiVfffi?-?.^^ 1% SDS- 50mM ACETATE, Dl AFILTR ATION 1mM EDJA pH g g I

S-200 COLUMN 1% SDS- 50mM ACETATE, 1mM EDTA, pH 5.5 I

ULTRAFILTRATION 2mM SODIUM PHOSPHATE, pH 7.5 I

FORMULATION

LYOPHILIZATION