AU559677C - Homogeneous human interleukin 2 - Google Patents

Description

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fractions exhibiting human interleukin 2 activity; and

B. passing the pooled active fractions from step A above through a reverse phase high performance column containing diphenyl groups covalently bonded to silica

556 7 TENT OR A STANDARD PATENT OF ADDITION Full name(s) of 1We -l, F.Hoffmann-La Roche Co. Aktiengesellschaft" Applicant(s)

of 124-184 Grenzacherstrasse, Basle, Switzerland

hereby apply for the grant of a stnadpatent for an invention entitled

Title of Invention "Homogeneous Human Interleukin 2 and Process theref or"

which is described in the accompanying complete specification. DETAILS OF BASIC APPLICATIONeS4 Number(-s) of Basic Applicationes) 418,927 I Name(-) of Convention Countrv(ies) in which tas~qs:, Application~t) was/were tiled Cl

United States of America I ve.

Date~s- of biasic Appicationms September 16, 1982 APPL!CATiON ACCEPTED AND AMENDMENTS (r-espeieWy)

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FIEE TO VAM 0

MAIL OKICER I -MyL/Our address for service is: SPRUSON FERGUSON LODGED AT SUE-OFF1 CE PATENT ATTORNEYS CCC C Ce C C IC BA CENTRE, 60 MARGARET ST. Ge I SYDNEY. NEW SOUTH WALES. I CSCS o C AUSTRALIA. 'Dated this 4th day of August, 19 83.

F. HOFFMANN-LA ROCHE CO. AKTI1!NG ESE LLSCH ANFT

Theodor Bjn Martii Vice-Dij.'zx-,to. Mandatory

To: The Commissioner of Patents 1K 1 I ~~1

N .7 1 9 596 V COMMONWEALTH OF AUSTRALIA PATEN7S ACT 1952 F

COMPLETE SPECIFICATION (ORIGINAL)

COMMONWEALTH OF AUSTRALIA AUS TRALIA Spruson Fergusi ~'mi CON ETION THE PATENTS ACT 1952 STANDARD PETTY PATENT DECLARATION IN SUPPORT OF A DECLARATION 1916 0183In supportCONVENTION APPLICATION FOR A PATENT of thle Convention Application made for a SFP4

patent f'or an invention entitled: Title of Invention "Homogeneous Human Interleukin 2 and Process therefor"

I/.We Fridolin Klausner Full name(s) and address(es) of DecI arant(s) of' 187 Baselmattweg, Allschwil, Switzerland

do solemnly and sincerely declare as follows: Full name(s) of Applicant(s) (or, in the case ot'an application bi' a bodi' corporate) I. I arn/WG-ap authorlsed by F.Hoffmarin-La Roche Co. Aktiengesellschaft

thle applicantH-) f-or the patent to make this declaration on its/tlieit-behalf. 2. Thle basic application(s-) as defined by Section 141 of the Act was! were mnade

Basic Country(jes) in United States of Am~erica

Priority Date(s) onl September 16, 1982 Rp'sic Applicant(s) by Alvin S. Stern

3. 1'ul- name(s) and address(es) of invi~ntor(s) (or iw;here a person otlher thian the inv'entor is the applicant)

3. Alvin S. Stern

of 110 Kensington Terrace, Passaic Park, USA

(rgefiyedy3- is/a're the actual inventor(s of' thle invention and the facts upon -Sul out how Applicant(s) which the applicant(-s) is/mrr entitled to make the application are derije title from actual inventer9s) e.g. The as follows: Applicant(s) is/are the Hoffmann-La Roche Inc., Nutley, USA, is the a. signee(,! "4the ento, fixm the assignee of' the said Alvin S. Stern, and F.Hoffmann-La R~oche Co. Aktiengesellschaft, Basle, Su, tzerland, is the assignee of the said Hoffmann-La Roche Inc.

4. The basic application(s) referred to in paragraph 2 of this Declaration was/wer-e the first application(s) made in a Convention country in respect of' the invention thle subject of thle app~ication. Declared at Basle, this 4th day of August, 1983.

SFP4 To: The Commissioner of Patents SF~~~~SgnrTo:Th Cmmssonr f atnt o4 1/8 11/81 t<itzr ti

(12) AUSTRALIAN PATENT ABRIDGMENT (19) AU (11) AU-B-19160/83

(54) HOMOGENEOUS HUMAN INTERLEUKIN 2 (71) F. HOFFMANN-LA ROCHE CO. AKTIENGESELLSCHAFT (21) 19160/83 559677 (2; 2) 15.9.83 (24) 16.9.82 (31) 418927 (32) 16.9.82 (33) US (43) 22.3.84 (44) 19.3.87 (51)3 C12P 21/02 C07C 103/52 A61K 37/02 CO7G 7/00 (72) ALVIN S. STERN (74) (56) 14460/83 CO7G 7/00, C 1 2P 1/00 US 4390623

(57) Claim 1. Human interleukin 2 as a homogeneous protein charac- terized in javing a specific activity of about 1.4 x U/mg, a pi of about 5.68 and a composite amino acid com- position as follows:

Asp 12 Ala 7 Tyr Thr 10 Cys 4-5 Phe Ser 8 Val 6 His Glu 16-17 Met 4 Lys Pro 8-9 Ile 7 Arg Gly 8 Leu

A process for preparing human interleukin 2 in homogeneous form which process comprises in combination:

A. passing a so..ution of crude human interleukin 2 through a reverse phase high performance column containing methyl or octyl groups covalently bonded to silica gel whereby said human interleukin 2 is retained by said column, eluting said column under HPLC conditions with a buffered n-propanol 0-60% gradient and pooling ./2 CI

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fractions exhibiting human interleukin 2 activity; and

B. passing the pooled active fractions from step A above through a reverse phase high performance column containing diphenyl groups covalently bonded to silica gel whereby said human interleukin 2 is retained by said column, eluting said column under HPLC conditions with a buffered n-propanol 20-60% gradient and pooling fractions exhibiting human interleukin 2 activity and optionally repeating step A and/or B. 7. A pharmaceutical composition containing as active agent homogeneous human interleukin 2 as defined by claim 1, and further containing a pharmaceutically acceptable diliment or carrier. 8. A method of modulating lymphocyte reactivity comprising administering homogeneous human interleukin 2, as defined by claim 1, to a patient requiring such modulation.

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xi COMMONWEALTH OF AUSTRALIA PATEN."S ACT 1952 Ff 1

COMPLETE SPECIFICATION (ORIGINAL) FOR OFFICE USE

TIhis docwncent LOniin 111Thih amcnduncnlai L In~t. Class 83(2)by Scc~io heSupor- Application Number Lodged and is cu rnxt fo: jl,,

Complete Specification Ludged Accepted Published risT ducuflleft contains~ ztu arneadndfts m~ade under Priority ',,ction 49.

and is corTect for printing. Related Art

I Name of Applicant (si: F.HOFFMANN-LA ROCHE CO. AKTIENGESELLSCHAFT

Address of Apphicant~s1: 124-184 Grenzacherstrasse, Basle, Switzerland

Actual Inventor ALVIN S. STERN L

Address for Service Spruson Ferguson, Patent Attorneys, CBA Centre Margaret Street, Sydney, New South Wales, 2000 Australia

Complete Specification for the invention entitled

"HOMOGENEOUS HUMAN INTERLEUKIN 2 AND PROCESS THEREFOR"

The following statement is a full description of this invention, including the best method of performing it known to-me/us:

SFP17 1in'#0

RAN 4105/69

Interleukin 2 (IL-2) is a soluble protein which is capable of modulating lymphocyte reactivity and, in the past, has been produced by stimulating mouse, rat or human lymphocyte cells with a mitogen, such as phytohemagglu- tinin (PHA) and concanavalin A (Con see e.g. Gillis et al. [Nature 268, 154 (1977); J. Immunol. 120, 2027 (1978); ibid. 124, 1954 (1980); ibid. 125, 2570 (1980)].

IL-2 purified from mouse, rat and human normal T- lymphocytes, has been found to retain different types of biological activity, including: marked enhancement of thymocyte mitogenesis; promotion of long term in vitro proliferation of antigen specific helper or killer T cell lines; and induction of cytotoxic T lymphocyte (CTL) reactivity and plaque-forming cell responses in cultures of nude mouse spleen cells. Accordingly, these identified biological activities of IL-2 indicate that IL-2 is useful in elevating immune responses and restoring immune defi- cient T cell populations (nude mouse spleen cells) to normal levels of cell and humoral immunity. Furthermore, these results suggest that IL-2 production and response are important parameters of immunological functions which may be useful in clinical diagnosis of aberrant immunity. Moreover, the fact that human IL-2 makes possible the in 4. vitro proliferation of antigen specific human, mouse and rat killer T cells emphasizes the importance of human IL-2 i as a research reagent. a

However, although production of human IL-2 from lectin stimulated human splenic and peripheral blood lymphocyte conditioned :edia has been described by several authors, these production sources and techniques result in weak

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'M concentrations of IL-2, with purification of IL-2 requiring fractionation of large volumes of conditioned media con- taining IL-2 in order to obtain only very small quantities of human IL-2 activity. As a consequence, sufficient quan- tities of concentrated human IL-2 have not been available for in vivo experiments, nor to study effectively the final molecular characterization thereof.

Gillis and Watson Exp. Med. 152, 1709-19 (1980)] describe the production of human IL-2 by induction of a malignant neoplastic cell line such as the Jurkat-FHCRC line with a T-cell mitogen such as PHA optionally in the presence of a phorbol ester such as phorbol myristic acetate (PMA). Partial purification of the IL-2 was achieved by a procedure involving ammonium sulfate preci- pitation/dialysis, gel filtration chromatography (Sephadex G-100), ion exchange chromatography (DEAE cellulose), flat bed isoelectric focusing and analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The majority of IL-2 biological activity was electrophoretically i eluted from the protein band (one of nine to sixteen 1 4 separate bands observed on the gel) having a molecular weight of approximately 14,000 daltons. See also Frank et al., J. Immunol. 127, 2361 (1981), and Watson et al., I

*1 4,

Lymphokines 6, 95 (1982), for a corresponding disclosure. t I

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I I't C Mier and Gallo Immunol. 128, 1122 (1982); Lympho- kines 6, 137 (1982)] have reported on the purification of I iIt 1C I IL-2 from normal lymphocytes to provide "a nearly homo- geneous material" using preparative SDS gel electrophoresis as the last step after a sequence of anion exchange chroma- I I Ic I tography and gel filtration procedures. Their product had C C a molecular weight of 13,000 on SDS-PAGE and 20-25,000 on gel filtration and an isolectric point of 6.8. This material was very unstable even at -70°C and required addition of bovine serum albumin (BSA) or polyethylene glycol to retain 11 activity.

I

Stadler and Oppenheim [Lymphokines 6, 117 (1982)] describe IL-2 purified from peripheral blood mononuclear cells, tonsil and spleen cells which exhibited charge heterogeneity after purification on column chromatography, gel filtration and electrofocusing. Three charge species having pIs of 6.5, 7.2 and 8.2 were found and the hetero- genecity possibly attributed to differences in the degree of glycosylation.

Finally, Robb and Smith [Mol. Immunol. 18, 1087-94 (1981)] have isolated TCGF IL-2) from the human T- leukemia cell line JURKAT which was uniform in charge and size. The same result has also been reported by Robb et al. in J. Exp. Med. 184, 1455-74 (1981).

The present invention relates to human IL-2 derived from induced human malignant cells which is purified to homogeneity using multiple reverse phase high performance liquid chromatography steps. The procedure utilized to produce the homogeneous IL-2 product of the present inven- tion involves passing a crude IL-2 preparation through a reverse phase methyl or octyl bonded silica HPLC column, eluting protein with a n-propanol gradient in buffer, pooling active fractions as determined by assay and passing the pooled active fractions through a reverse phase di- phenyl bonded silica HPLC column. In those instances where the crude preparation used is of relatively low titer it may be necessary to repeat one or more of the HPLC steps in S< 1 order to achieve purification to homogeneity.

Crude IL-2 preparations are readily prepared by cul- turing malignant human neoplastic cells, such as human leukemia and lymphoma cells in vitro in a serum containing medium supplemented with various additives. The culture is stimulated by a T cell mitogen such as PHA thereby produ- cing a.supernate which contains IL-2. After a period of time, approximately 24 hours after incubation the i supernate is collected and processed to purify tht IL-2 r

4

into a more concentrated form. It is also desirable to utilize a phorbol ester such as PMA as an inducing agent either alone or in combination with a T-cell mitogen such as PHA or Con A to enhance the production of IL-2. For further details see Gillis et al., J. Exp. Med. 152, 1709 (1980).

Cell lines which can be employed in the production of crude human IL-2 include various T and B cell lines as well as various T lymphoma cell lines. The cell lines were produced by either spontaneous occurrence, viral infection or by chemical carcinogen. A preferred cell line for this purpose is identified by Gillis et al. as the Jurkat-FHCRC leukemic human T cell line and most preferably a sub-clone of this line designated H33HJ-JAI is used. The process of the invention can also be used to purify IL-2 derived from peripheral blood lymphocytes.

The culture medium used to produce crude human IL-2 in conjunction with the aforesaid cell lines may consist of commercially available media, such as Dulbecco's Modi- fied Eagle Medium, RPMI medium and Click's medium. Addi- tives which may be added to the culture medium individually or in combination include penicillin, streptomycin, genta- mycin, fresh L-glutamine, HEPES buffer, NaHCO 3 fetal calf serum (FCS) or normal human serum. The initial cell density of cells, particularly when Jurkat-FHCRC cells are employed, should be in the range of from 5 x 105 cells/ml ''It

SI I I S to 1 x 10 7 cells/ml, most preferably about 1 x 10 6 cells/ml.

Culture conditions include a temperature in the range *14 S 4 of about 35 to 380 C, a pH in the range of 7.0 to 7.4 and a humidified atmosphere of from approximately 5 to 10% carbon 4 5

dioxide in air. A PHA mitogen concentration in the range of 0.5% to 2.0% by volume, preferably 1% by volume may be used.

The quantity of IL-2 produced by stimulating malignant Ii y~ human cells with a plant mitogen varies with time. Peak

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levels of IL-2 are reached at approximately sixteen to twenty-four hours after stimulation by PHA. Assay of IL-2 for monitoring the cell culture production procedure or to monitor purification procedures involves assessing the capacity of the sample to induce T cell line proliferation. T cell proliferation is determined by measuring the incor- poration of tritiated thymidine. One unit of activity is defined as the number of microliters present in a T cell culture well which induced 50% of maximal thymidine incor- poration. Specific details of the assay procedure are set forth in Immunol. 120, 2027 (1978), Gillis et al..

Preparation of a suitable starting material for carrying out the HPLC purification steps of the present invention involves, precipitating IL-2 containing super- nates derived by centrifuging harvested induced cells by addition of ammonium sulfate to 85% saturation. Such addition is accomplished by the gradual addition of dry ammonium sulfate to the supernatant with gentle stirring. Addition of ammonium sulfate for precipitation is done over an extended period of time, e.g. a 12 hours period. Upon reaching 85% saturation the gentle stirring is con- tinued for an additional period in the cold. The precipi- tated protein is pelleted by centrifugation and the pellets resuspended in sterile double distilled water.

In one alternate process aspect the resuspended crude IL-2 is subjected to ion exchange chromatography. A suitable column for this purpose is CM Biogel A resin (LKB-Produkter, Bromma, Sweden). Preferably the column is pretreated with a fetal calf serum solution to block non-specific i t <binding sites on the resin before application of the IL-2 i" containing sample. Elution from the column is accomplished with a buffered salt gradient. A suitable gradient for this purpose is 50 mM 0.5 M NaCl in HEPES, pH 5.5. A final wash with 0.5M NaCl-HEPES, pH 5.5, is used to ensure that all IL-2 activity is eluted.

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Pooled active fractions from the above ion exchange chromatography step provides a preferred starting material for the HPLC procedures which form the process aspect of the present invention. In an alternate embodiment it is possible where sufficiently high titer supernatant from the ammonium sulfate step is available to use this material directly in the first HPLC column step. However, in such aspect it may be necessary to repeat one or both of the HPLC steps in order to achieve essential homogeneity in the product IL-2.

The HPLC steps employed in the instant process use a reverse phase, methyl, octyl or diphenyl bonded silica Spre'e/'e ro. ly column having a pore sizelof at least about 150A. Suitable reverse phase HPLC columns for use in the practice of the invention are articles of commerce. Particularly preferred for this purpose are the Protesil line of columns commer- cially available from Whatman Separations Inc., Clifton, USA.

Thus, for example, Whatman Protesil 300 Methyl is a column consisting of trimethylsilyl groups covalently bonded by means of a silicon-oxygen-silicon bond to the surface of a 300 A pore diameter silica gel which has been classified to a mean particle size of 8 microns. In similar fashion the Whatman Protesil Magnum 9 Octyl column has octyl groups covalently bonded to 150 R pore diameter silica gel while the Whatman Protesil Diphenyl column has diphenyl groups covalently bonded to the surface of a 300 A silica t gel.

The elution of proteins from the HPLC columns can be carried out in a manner known per se in the art. A suitable elution procedure for removing bound proteins from the methyl or octyl columns used in the first HPLC step of the present process involves the use of a gradient of increasing concentration of a water miscible lower alkanol, preferably ,i n-propanol. A preferred gradient foi this purpose is a I 1-:

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0-60% vol/vol gradient in pyridine-acetic acid, pH

Similar elution conditions may be employed in con- junction with the diphe-yl column used in the second step with the exception that the alkanol gradient is 20-60% vol/vol. Eluted protein can conveniently be moni- tored with detection systems known in the art such as, for example, by use of an automated fluorescence detection system described by Kenny et al., Methods Enzymol. 78, 435 (1981).

After carrying out the HPLC process steps, either in two steps or where low titer crude is used, with one or more repeats of the first and/or second steps, IL-2 is obtained, purified to homogeneity as a single symmetrical peak of bioactivity in good yield.

The ability to prepare homogeneous IL-2 allows for 'Wa the first time the determination of the amino acid corn- position of this molecule. This information, in turn, may result in the determination of the amino acid sequence which again is important in assisting in the cloning of the human IL-2 gene and the ultimate production of large amounts of pure recombinant IL-2 for clinical trials and ultimately for widespread medical use of this substance. Moreover, the availability of homogeneous IL-2 will allow a a accurate biological studies of its activity free from the

contamination of the numerous lymphokine species which are known to be co-produced with IL-2 zuring the induction procedure. While the prior art has claimed to prepare highly purified or nearly homogeneous IL-2 preparations, experience has shown that when material indicated to be single band material on SDS-PAGE analytical electrophoresis

is used as starting material in the instant HPLC process on a methyl or octyl column, the band is resolved into a large number of protein peaks which are unassociated with the IL-2 activity. Based on such observations it is believed that such preparations are no more than 10% pure and in some instances may even be less than 1% pure.

Tests with partially purified IL-2 preparations deri- ved from Jurkat-FHCRC cell line have shown that the par- tially purified lymphokine does not cause fresh peri- pheral blood lymphocytes to proliferate; but does induce proliferation in T-lymphocyte populations previously stimulated with lectins or specific antigens. Moreover, partially purified human IL-2 was found to maintain the proliferation of not only human antigen-specific effector T-cells, but also of murine antigen-specific effector T cells. Thus, Jurkat-FHCRC produced human IL-2, even par- tially purified, has been shown to be a useful reagent for use in the growing of clonal human and murine T-cells with various antigens and effector specifications, and in fact is now an article of commerce for such uses. Homogeneous IL-2 of the present invention can, of course, be employed in similar manner.

20 The process and product of the present invention are further illustrated by the following Example

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IL-2 Production H33HJ-JAI cells (ATCC No. CRL-8163, deposited August 26, 1982), a clone of the parent Jurkat-FHCRC cell line, were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 U/ml.penicillin, 50 ug/ml streptomycin, ug/ml gentamycin and 300 ug/ml fresh L-glutamine, at 37°C in a humidified atmosphere of 5% CO 2 in air. Cells at saturation density (8-10 x 10 cells/ml) were harvested, centrifuged and resuspended in fresh RPMI 1640 medium without serum but containing 50 U/ml penicillin, 50 ig/ml streptomycin, 50 ug/ml gentamycin and 300 jig/ml fresh L-glutamine. Cells were resuspended to a concentration of 2 x 10 cells/ml and were stimulated by the addition of 1% PHA by volume and 10 ng/ml phorbol myristic acetate (PMA). Following 24 hours incubation at 37°C in a humidified ^atmosphere of 5% CO 2 in air, induced cells were harvested, centrifuged and the supernates retained as crude starting S 20 material.

Pooled supernatants from three cell culture runs (nos. 1-3) comprising 900 ml, 900 ml and 1050 ml each or a total of 2850 ml of supernatant, the pooled material having a total activity as determined by the assay of i Gillis et al., J. Immunol. 120, 2027 (1978), of 7.125 x 10 units, were treated by the gradual addition of dry ammonium sulfate to 85% saturation with gentle stirring. i Addition of ammonium sulfate for precipitation was done iI over a 12 hours period. Once the solution reached saturation gentle stirring was continued at 4°C for an additional 24 hours. Protein present in the supernate was pelleted by a 30 minutes centrifugation at 10000 x G. Supernates were decanted and discarded. The pellet was resuspended to 40 ml with double distilled sterile water and the resulting solution contained 205000 U/ml of IL-2 aof total 8.2 x 10 6or units. or a total of 8.2 x 10 units.

10

Additional runs were carried out as summarized below in Table I.

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Supernatant Pooled Total Resuspension Activity Total Activity 6 Run No. volume [ml Activity [U/ml]' Units x 10 volume [ml EU/ml]I [U X-10 6

1200 6500 9.425 200000 11

1250

6500 12.35 163840 13.107

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12

Ion Exchange Chromatography on CM Biogel A Column

CM Biogel A was equilibrated in 0.05 M NaCl-HEPES, pH The column (100 ml) was poured and 100 ml of 0.005M NaCl-HEPES,pH 5.5, containing 10% FCS was then applied to the column. This was done to block non-specific binding sites which could tie up human IL-2 activity. Serum pro- teins which bound to the column were then eluted off by the addition of 500 ml of 0.5 M NaCl-HEPES,pH 5.5. The column was then re-equilibrated with 1000 ml of 0.05 M NaCl.

Combined resuspensions of ammonium sulfate precipi- tated protein comprising runs 1-5 as described above was applied to the CM column. The column was then washed with 200 ml of 0.05 M NaCl-HEPES,pH 5.5. A 500 ml salt gradient running from 50 mM to 0.5 M NaCl-HEPES,pH 5.5,was then applied to the column. After gradient application the column was washed with 200 ml of 0.5 M NaCl-HEPES,pH to ensure that all bound IL-2 had indeed been eluted from the column. Fractions of 5 ml each were obtained. Every third fraction was assayed for IL-2 activity. Peak acti- vity was determined and the active fractions pooled. There was thus obtained a pool of 35 ml having an activity of 983040 U/ml or a total activity of 35.39 x 106 units.

A second CM column run with the resuspended ammonium 2 sulfate precipitates from pooled runs 6 and 7 as described It i above provided pooled fractions having a volume of 215 ml, an activity of 0000 U/ml and a total activity of 17.2 x 106 units. A 130 ml portion of this run was combined with II a CII C the 36 ml derived from the first run thus providing 166 ml 1444CC 4 of crude IL-2 having a total of 10.4 x 10 units when assayed on preparation. i -i-

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Reverse Phase HPLC with Octyl Column

Lot A obtained as described above was pumped in toto directly onto a 9.4 x 250 mm Protesil Magnum 9 Octyl Column (Whatman Separations Inc., Clifton, The column was then washed with 50 ml of 0.9 M acetic acid/ 0.2 M pyridine pH 4.0, buffer. Elution of the proteins was accomplished with a gradient of n-propanol 0-60% (v/v) over 8 hours in the 0.9 M acetic acid/0.2 M pyridine pH 4 buffer. An automated fluorescence detection system using fluorescamine monitored the protein in the column effluents. The recovery of biological activity was 7.13 x 106 units (approx.

Reverse Phase HPLC with Diphenyl Column

The fractions containing the major peaks of activity obtained from the octyl column were pooled, diluted 1:1 with 0.9 M acetic acid/0.2 M pyridine,pH pumped onto a 4.6 x 250 mm Whatman Protesil Diphenyl column. Proteins were eluted with a 20-60% n-propanol gra- dient in 0.9 M acetic acid/0.2 M pyridine pH 4.0 buffer over 6 1/2 hours. This procedure yielded a symmetrical peak coinciding with IL-2 activity. Recovery of activity S. 25 in this step was greater than 60%. Essential homogeneity of this material was ccufirmed by analytical SDS-PAGE electrophoresis and two dimensional gel electrophoresis which yielded a single band and a single spot respectively. i The homogeneous human IL-2 thus obtained had a specific activity of about 1.4 x 10 U/mg and a pi of 5.68.

Samples of this material were subject to amino acid analysis performed with fluorescamine detection (see Stein et al., Arch. Biochem. Biophys. 155, 203 [1973]). The polypeptide was hydrolyzed for 24 and 48 hours at 104°C in constant boiling HC1 containing 0.1% thioglycolic acid. The results are summarized in Table 2 below:

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Table 2

Amino Acid Composition 24 hours 24 hours 48 hours of IL-2

Asp 12.3 11.8 11.8 12 Thr 8.6 9.6 7.4 Ser 7.5 5.2 7.3 8 Glu 18.3 15.4 16.0 16-17 Pro 9.7 10.7 8-9

/O Gly 7.5 6.3 10.8 8

Ala 7.5 6.0 8.4 7 Cys Val 5.0 5.5 5.2 6

Met 4.3 3.3 3.1 4

Ile 5.0 7.5 6.3 7

Leu 13.7 19.4 16.0 16

Tyr 2.4 1.7 4.2 3 Phe 4.6 4.1 6.4 His 4.4 3.8 4.9 4

o2O Lys 7.9 8.6 7.9 8

Arg 3.9 3.9 7.5 4 Total 130-133

24 hour hydrolysis on separate sample

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Sequence analysis yielded the following internal partial

amino acid sequence:

Ile-Leu-Asn-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr. This

sequence is consistent with positions 44-57 of the putative

human 11-2 sequence proposed by Taniguchi et al., Nature 302,

305-310 (1983), from cloned cDNA.

71

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Claims (2)

1. The claims defining the invention are as follows: 1. A process for preparing human interleukin 2 in homogeneous form which process comprises in combination: A. passing the solution of crude human interleukin 2 through a reverse phase high performance column containing methyl or octyl groups covalently bonded to silica gel whereby said human irterleukin 2 is retained by said column, eluting said column under HPLC conditions with a buffered n-propanol 0-60% gradient and pooling fractions exhibiting human interleukin 2 activity; and B. passing the pooled active fractions from step A above through a reverse phase high performance column containing diphenyl groups covalently bonded to silica gel whereby said human interleukin 2 is retained by said column, eluting said column under HPLC conditions with a buffered n-propanol 20-60% gradient and pooling fractions exhibiting human interleukin 2 activity and optionally repeating step A and/or B. 2. The process of claim 1 wherein said column in step A is an octyl column. 3. The process of claim 2 wherein said silica gel in said octyl column has a pore size of about 150 8 and in said diphenyl column has a pore size of about 300 A. 4. The process of claim 3 wherein said buffer in steps A and B is S 0.9 M acetic acid/0.
2. 2 M pyridine, pH The process of any one of claims 1 to 4 wherein said crude human interleukin 2 is derived from induced, cultured human malignant cells. 6. A process for preparing homogeneous human interleukin 2 substantially as hereinbefore described with reference to the example.

   DATED this TNENTY-NINTH day of DECEMBER 1989 F. Hoffmann-La Roche Co., Aktiengesellschaft

   Patent Attorneys for the Applicant SPRUSON FERGUSON

   Sec.

   TMR/4214W

   L UUIEI 'El