PRODUCTION OF RECOMBINANT HUMAN INTERLEUKIN-1 INHIBITOR

Abstract: A method for the production of commercial quantities of highly purified interleukin-1 inhibitor (IL-li) from a recombinant host is disclosed. A preferred recombinant E. coli host for sue in this method is also disclosed.

Description: PRODUCTION OF RECOMBINANT HUMAN INTERLEUKIN-1 INHIBITOR BACKGROUND OF THE INVENTION In the recombinant-DNA field, many proteins have been prepared in the laboratory in an amount suitable for research purposes. However, even though techniques to produce research quantities of these proteins have been optimized, these labora production and purification processes are often inadequate to produce commercial quantities of the desired protein which is quality sufficient to be used as a human pharmaceutical. In order to produce commercial quantities of a given prot of an appropriate quality, unique fermentation, isolation, and purification techniques are often required. Moreover, the combination of the techniques and the order in which they are practiced often affect the amount of the protein recovered and purity of the final: roduct^ As previously described in co-pending U.S. patent applications Serial Numbers 199,915, 238,713, 248,521 and 266, filed May 27, 1988, August 31, 1988, September 23, 1988, and November 3, 1988, respectively, a unique protein named human interleu in-1 inhibitor has been isolated. These applications, specifically incorporated herein by reference, also describe methods for producing recombinant human interleukin-1 inhibito hereinafter referred to as "IL-li," in laboratory quantities i transformed organisms useful in laboratory methods. However, these methods did not result in production of commercial quantities of IL-li of a quality suitable for administration to humans. The present inventors have found certain combinations of fermentations, isolation, and purification techniques which a capable of producing commercial quantities of highly purified IL-li. These methods are described in this application. As herein, the term "commercial quantities" is intended to mean least several to tens to hundreds of grams of highly purified product obtained from each 100 liters of fermentation broth. "highly purified product" is meant a material of sufficient p to be administered to humans. In a preferred embodiment, "hi purified product" has less than 5 E.U. per dose of endotoxin less than 0.0025% contamination by E_-. coli protein. SUMMARY OF THE INVENTION It is an object of the present invention to provide a m for the productioβ. of.commercial quantities of recombinant h interleukin-1 inhibitor. This object is achieved by the met described herein. In order to achieve these objects, an improved strain f production of IL-li is described herein. That strain, named SGE90, is capable of producing at least 50 grams of highly purified IL-li per 100 liters of fermentation broth when use the methods described herein. One method which is preferred for production of commerc quantities of the highly purified IL-li described herein inc the following steps: (1) fermentation; (2) cell processing, including: (a) cell recovery, (b) lysis, and (c) clarification of the lysates; (3) a first ion exchange step; (4) a second ion exchange step; (5) the final processing steps including concentration and diafiltration. A third ion exchange step may be optionally added to achieve e greater product purity. In a preferred embodiment, the fermentation step is carri out in microorganisms, particularly Ej. coli, while the first i exchange step is conducted with a column filled with the catio exchange resin S-Sepharose. Also in the preferred embodiment, second ion exchange step is conducted with a column filled wit anion exchange resin, preferably Q-Sepharose. If the optional third ion exchange step is added, a column filled with a catio exchange resin, preferably S-Sepharose, is used. It is to be understood that both the foregoing general description and the following detailed description are exempla and explanatory only, and are not restrictive of the invention claimed. The accompanying drawings, which are incorporated in constitute a part of this specification, illustrates various embodiments of the invention and, together with the descriptio serve to explain the principles of the invention. RRTEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the construction of pRJl and pRJ2 as s forth in Example 1. DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with following examples, serve to explain the principles of the invention. As noted above, the present invention relates to a proc for the production of commercial quantities of IL-li. As us herein, the term "commercial quantities" is intended to mean at least several to tens to hundreds of grams of highly puri product from each 100 liters of fermentation broth are produ As noted previously, one of the preferred methods for production of commercial quantities of IL-li described herei includes the following steps: (1) fermentation of E^. coli comprising a plasmid cont a DNA encoding IL-li; (2) cell processing, including: (a) cell recovery, (b) lysis, and (c) clarification of the lysates; (3) a first ion exchange step; (4) a second ion exchange step; (5) the final processing steps including concentration and diafiltration. An optional third ion exchange step may also be conducted. Su an optional step would be performed immediately after the seco ion exchange step. The fermentation and cell processing steps for use in Εj_ contemplated in this invention include those routinely known t one of ordinary skill in the art. Preferred embodiments of th steps are set forth in the examples which follow. However, an comparable procedures may be inserted in the place of those preferred procedures set forth below. The process for the production of commercial quantities o IL-li utilizes a first ion exchange column. As noted previous the preferred column (described in Example 2), is filled with cationic S-sepharσse rββ±ή. • Other interchangeable resins may be used, including but not limited to resins such as SP-C25 Sephadex, CM Sephadex, CM Sepharose, or CM cellulose. A secon ion exchange column is then used for further purification of t IL-li. As noted above, in the preferred embodiment Q-Sepharos used as an anion exchange resin in this column. In addition, other comparable resins including but not limited to resins su as DEAE-Sepharose, Q-Sephadex, or DEAE-cellulose may be employ In one embodiment of the present invention, a third ion exchange step is included immediately after the second ion exchange step. In this optional third step, a cation exchange column is used. This column preferably contains S-Sepharose resin, however other interchangeable resins may also be used. Such other resins include, but are not limited to, SP-25 Seph CM Sephadex, CM Sepharose, CM cellulose or CM Toyopearl. Following these steps, the final process steps are undertaken. These include a concentration step, if desired, diafiltration of the IL-li. The parameters of these steps a routinely known to those of ordinary skill in the art, in li the teachings found in the examples which follow. Important to the operation of this process is a suitable of quantitative analytical tools to evaluate yield and purit As described in greater detail in the procedures in Exa 4, the assays which have been developed for these purposes i a reverse phase HPLC (RP-HPLC) assay, an ion exchange HPLC (IE-HPLC), an SDS-PAGE assay, a size exclusion assay, a tryp peptide map, and xά. essay for biological activity. When tes the first four of these assays, the highly-purified IL-li pr by the present methods is greater than 90% pure. Preferably tested in the IE-HPLC, the highly purified IL-li is at least pure for both Mono Q and Mono S columns. Preferably, when t in the SDS-PAGE assay, the highly purified IL-li is at least pure, and is at least 98% pure when tested in size exclusion assay. The trypsin peptide map of the highly purified IL-li matches the pattern theoretically expected. The highly puri IL-li demonstrates inhibition of IL-1 in the bioassay. The following examples are provided to illustrate certa preferred embodiment of the present invention. These exampl intended to be illustrative only and are not intended to lim scope of the claims appended hereto. All references provide i these examples are specifically incorporated herein by referenc EXAMPLE 1 1. Transfer of the IL-li cDNA from a lambda phage to a Bluescript plasmid cloninσ vector. The lambda phage GT10-IL-li-2a (ATCC #40488) was digested with EcoRI and the 1.7 kb fragment carrying the IL-li cDNA was purified by gel electrophoresis. This fragment was ligated to EcoRI-digested Bluescript SKM13-(Stratagene) , resulting in the plasmid BS-IL-li#2. 2. Development of an IL-li expression vector using the "T7" system A. Description of pT5T. The T7 expression vector used for IL-li production is call pT5T. It is essentially the same as pJU1003 [Squires, et al., Biol. Chem. (1988) 31:16297-16302], except that there is a shor stretch of DNA between the unique Bgl2 site 5' to the T7 promot and the Clal. site in the tetracycline resistance gene. The sequence of this DNA is: A J_£S&XGATA AGCTGTCAAA CATGAGAATT GAGCTCCCCG GAGATCCTTA G C GAAAG Llαl AGGATTTTTT TTAGATC Bgl2 The vector was linearized with BamHl and Smal restriction enzymes. The plasmid BS-IL-li#2 was digested with PflMl and Se and the 453 bp fragment carrying the sequence coding for a ino acids 4 to 152 of the mature IL-li gene along with the terminat codon and 3 bp of the 3' untranslated region was purified by polyacrylamide gel electrophoresis. Oligonucleotides with the sequences: ~^ 5' GATCCATTGGAGGATGATTAAATGCCGCCCT 3' 3 ' GTAACCTCCTACTAATTTACGGC 5' were synthesized, phosphorylated at their 5' ends and anneal These oligonucleotides contain sequences essential for the translational coupling of the T7 Φ10 gene to the IL-li gene. mixture containing the annealed oligonucleotides, the linear vector fragment and the 453 bp IL-li gene fragment was treat with T4 DNA ligase and then used to transform the Σ^ coli st JM109 (See Figure 1). B. Mutagenesiε of IL-li. Once a plasmid was isolated and shown to have the corre sequence, it was designated pRJl. pRJl carries sequences co for a variant of the IL-li protein. The amino-terminal sequ of this variant is Met-Pro-Pro-Ser-... rather than Arg-Pro-S which is the aminoterminal sequence of the natural human pro The aim here was to express a protein that is as close as po to the natural protein, and that was done by mutagenizing th coding for the IL-li protein such that it codes for Met-Arg-Pro-Ser- . . . , as follows. The gene for IL-li in pRJl removed by digesting the plasmid at the unique BamHl and Pst sites. The 1375 bp fragment was cloned between the BamHl an sites of M13 mp 19 and designated M13-IL-lil. Oligonucleoti site directed mutagenesis was performed on isolated single s DNA of M13IL-1H according to the procedure described in the -<? - BioRad Mutagene mutagenesis kit. The mutagenic oligonucleoti sequence is given below, along with the corresponding amino terminal amino acid sequence of the mutated IL-li: TGATTAAATGCGTCCGTCTGGGAG M R P S G R This mutagenesis produced M13IL-H2 which differs from M13IL- in that the IL-li protein encoded on this plasmid has the des amino-terminal sequence and that the codons for Arg and Pro a those used preferably by E^. coli. C. Expression of IL-li protein. The mutagenized "IL-li gene was then transferred back int pT5T using the same-procedure as described above. This secon expression plasmid is designated pRJ2. pRJ2 was transformed the- E. coli strain BL21 (DE3) for expression. This strain [described in Studier and Moffat J. Mol. Biol. (1986) 189:113 contains the T7 RNA polymerase gene under control of the IPTG inducible lac promoter on a nonexcisable lysogenic lambda bacteriophage. High level expression of rIL-li was achieved growing the cells [BL21(DE3)pRJ2] in Luria broth with 15 μg/m tetracycline up to a cell density corresponding to an Ag-- of IPTG was added to a final concentration of 1.0 mM and the cel were allowed to grow for four hours. The cells were harveste centrifugation and the rIL-li was purified from the soluble ce lysate by standard protein chemistry techniques. 3. Development of an IL-li expression vector using the "tac promoter" system. A. Preparation of pDDl. The plasmid pJU 1003 (Squires, et al.) was cut with Hind BamHl and fused to a synthetic Human Pancreatic Secretory Try Inhibitor (HPSTI) gene whose sequence is: ScnRl GAA77CGA7A 7C7CG77GGA GA7A77CA7G ACGTATTTTG GA7GΛ7AAC3 C77AAGC7ΛT AGAGCAACCT C7A7AAG7AC TGCATAAAAC C7AC7A77GC ____i AGGCGCAAAA AATGAAAAAG ACAGC7A7CG CGΛTCGCAGT GGCAC7GGC7 7CCGCG7777 77AC77777C 7G7CGA7AGC GCTAGCGTCA CCGTGACCGA GG777CGC7A CC37AGCGCA GGC7GAC7C7 CTGGGTCGTG AAGCTAAGTG CCAAAGCGA7 GGCA7CGCG7 CCGACTGAGA GACCCAGCAC 77CGA77CAC CTACAACGAA CTGAACGGT7 GCACTAAAAT CTACAACCCG GTATGTGGTA GATGT7GCTT GACTTGCCAA CGTGATTTTA GATGTTGGGC CATACACCAT CCGACGGTGA CACCTACCCG AACGAATGCG TGCTGTGCTT CGAAAACCGT GGCTGCCAC7 G7GGA7GGGC 7TGC77ACGC ACGACACGAA GCTTT7GGCA AAACG7CAGA CC7CCATCC7 GA7CCAGAAA 7CTGGTCCG7 GCTAAG7CGAC TT7GCAG7C7 GGAGG7AGGA C7AGG7C77T AGACCAGGCA CGAT7CAGC7G Hind 3 CCTGCAGAAG CTT• • • GGACG7C7TC GAA- - • ~ y cutting the HPSTI gene with Pvul and Hind3 and ligating th ? vuI/Hind3 fragment to the BamHI-Hind3 cut plasmid using a do stranded oligonucleotide adaptor with the sequence: 5' GAT CCG ATC TTG GAG GAT GAT TAA ATG AAA AAG ACC GCT A 3' GC TAG AAC CTC CTA CTA ATT TAC TTT TTC TGG CGA T GCC AT 3' CGG 5' /' ****• This synthetic HPSTI gene codes for a protein consisting of t signal (or leader) peptide for the E^. coli o pA protein fused the mature HPSTI protein. Thus, the purpose of this manipula was to incorporate sequences coding for the ompA signal pepti into pJU 1003, for work described below. The resulting plasm pDDl. Plasmid pDDl was digested with BstXl and Hind3. B. Construction of pDD3. Addition of Ej. coli translational signals to the IL-li cDNA. The plasmid pT5T (described above) was cut with BamHl an Smal. The plasmid BS-IL-li#2 was cut with the PflMl and Seal, releasing a fragment 453 bp in length which codes for a porti the IL-li protein (see above). The BamHl/Smal cut pT5T, the 4 bp IL-li fragment, and an oligonucleotide adaptor with the sequence: B?m 5 GA TCC ATC GCA GTG GCA CTG GCT GGT TTC GCT ACC GTA GCG 3 G TAG CGT CAC CGT GAC CGA CCA AAG CGA TGG CAT CGC CAG GCC CGT CCC T 3' GTC CGG GCA G 5' were fused to produce the plasmid pDD2. Plasmid pDD2 was cut BstXl and Hind3, releasing a 499 bp fragment which codes for a of the IL-li protein and a portion of the ompA signal sequence This 499bp fragment was fused to BstXl/Hind3 cut pDDl, resulti in the plasmid pDD3. C. Construction of pT3XI-2. Modification of pKK223-3. The starting plasmid for this construction was plasmid pKK223-3 purchased from Pharmacia. Plasmid pKK223-3 carries a partial gene for tetracycline resistance. This non-functional gene was replaced by a complete tetracycline resistance gene carried on plasmid pBR322. Plasmid pKK223-3 was digested completely with Sphl and partially with BamHl. A 4.4 kilobas pair fragment was gel purified and combined with a synthetic adaptor with the seouenr_e»- 5' G ATCTAGAATTGTCATGTTTGACAGCTTATCAT 3 • 3 • ATCTTAACAGTACAAACTGTCGAATAGTAGC 5 • and a 539 base pair fragment of DNA from a Clal - Sphl digest the tetracycline resistance gene of pBR322 (PL Biochemicals, 27-4891-01). The resulting plasmid was designated pCJl. Next a Xhol linker purchased from New England Biolabs wa inserted into plasmid pCJl's PvuII site to form plasmid pCJX- This insertion disrupts the rop gene which controls plasmid c number. An EcoRI fragment containing the lacl gene was purif from plasmid pMC9 [Calos, et al., Proc. Natl. Acad. Sci. USA (1983), 80:3015-3019] then inserted into the Xhol site with X to EcoRI adaptors having the sequence: 5' TCGAGTCTAGA 3• 3 ' CAGATCTTTAA 5• The polylinker sequence between the EcoRI and Pstl sites plasmid pK 223-3 was next replaced with a polylinker sequence shown here: 5* AATTCCCG G G TACCAGATCT GAGCTCACTA GTCTG C A 3 3 * GGGCCC ATGGTCTAGA CTCGAGTGAT CAG - The plasmid vector so obtained is designated pCJXI-1. Finally, the tetracycline resistance gene was replaced similar gene which had the recognition sites for restriction enzymes Hind3, BamHl, and Sail destroyed by bisulfite mutage The following procedure was used to mutate the tetracycline Resistance gene of pBR322. Plasmid pB322 was cut with Hind3, inutagβnized with sodium bisulfite [Shortle and Nathans, Proc. :flatl. Acad. Sci. USA (1978) 5:2170-2174)]. The mutagenzied DN *as ligated to form circular DNA, then cut with Hind3 to linea any plasmid that escaped mutagenesis. JL. coli JM109 [Yanish-Perron et al.. Gene (1985) 33:103-119] was transformed with the plasmid, then plated on selective media. Plasmids we isolated from tetracycline resistance colonies and checked for loss of the Hind3 site in the tetracycline resistance gene. T successfully mutated plasmid was designated pTl. A similar procedure was followed to mutagenzie the BamHl site in pTl, yielding plasmid pT2. Plasmid pT2 in turn was mutagenized to remove the Sail site, forming plasmid pT3. A Clal-BsmHl fragm of pT3 carrying the mutated tetracycline resistance gene was isolated and used to replace the homologous fragment of pCJXI- form pT3XI-2 v . The mutated tetracycline resistance gene still encodes a functional protein. D. Formation of pT3XI-2-Φ10TC3FGFsyn. Preparing the tac promoter vector for IL-li. Initially a "gene" for basic Fibroblast Growth Factor (FG was synthesized. This "gene" codes for the same sequence as t reported for FGF by Sommer et al. , but uses the codons that are found preferably in highly expressed genes in J . coli. The structure of this is such that the coding portion is preceded b translational coupler sequence (see Squires, et al., 1988) to ensure efficient initiation of translation. The FGF synthetic gene was first inserted into M13mp between the EcoRI and Hind3 sites and sequenced. The str this gene is: AATTCAGGA TCCGATCGTG GAGGATGATT AAATGGGTAC CATGGCTGCT GGCT GTCCT AGGCTAGCAC CTCCTACTAA TTTACCCATG GTACCGACGA CCGA EcoRI BamHl ESS FGFstart Translational Coupler 3 CTACCCTGCC GGCACTGCCG GAAGACGGTG GCTCCGGTGC TTTCCCGCCG GGCC GATGGGACGG CCGTGACGGC CTTCTGCCAC CGAGGCCACG AAAGGGCGGC CCGG AAGACCCGAA ACGTCTGTAC TGTAAAAACG GTGGCTTCTT CCTGCGTATC CACC TTCTGGGCTT TGCAGACATG ACATTTTTGC CACCGAAGAA GGACGCATAG GTGG GTCGTGTCGA CGGCGTACGT GAAAAAAGCG ACCCGCACA TCAAACTGCA GCTGC CAGCACAGCT TGCCGCATGC ACTTTTTTCC TGGGCGTGT AGTTTGACGT CGACG AAGAACGTG GTGTTGTATC TATCAAAGGC GTTTGCGCAA ACCGTTACCT GGCT TTCTTGCAC CACAACATAG ATAGTTTCCG CAAACGCGTT TGGCAATGGA CCGA AAGACGGTC GTCTGCTGGC TAGCAAATGT GTAACTGACG AATGTTTCTT CTTC TTCTGCCAG CAGACGACCG ATCGTTTACA CATTGACTGC TTACAAAGAA GAAG TGGAAAGCA ACAACTACAA CACCTACCGT TCTCGTAAAT ACACTTCTTG GTAC ACCTTTCGT TGTTGATGTT GTGGATGGCA AGAGCATTTA TGTGAAGAAC CATG TGAAACGTA CCGGCCAGTA CAAACTGGGT TCCAAAACTG GCCCGGGTCA GAAA ACTTTGCAT GGCCGGTCAT GTTTGACCCA AGGTTTTGAC CGGGCCCAGT CTTT TGTTCCTGC CGATGAGCGC TAAATCTTAA ACTAGTA ACAAGGACG GCTACTCGCG ATTTAGAATT TGATCAH£S&__ FGFstop HinDIII Relevant features of the gene are highlighted, - is - It was then isolated by digestion with BamHl and Hind3 a inserted into BamHl/Hind3 cut pJU1003 (Squires, et al., 1988) yielding pJU1003-synFGF. This plasmid was cut with Xbal and and the Xbal/Hind3 fragment carrying the FGF gene was isolate This fragment was ligated into pT3Xl-2 cut with EcoRI and Hin using an EcoRI-Xbal linker: 5» pAAT TCC ACA ACG GTT TCC CT 3' 3. P GG TGT TGC CAA AGG GAG ATCp 5 ; The new plasmid is designated pT3XI-2-Φl0TC3FGFsyn. E. Formation of pDD4. Inserting IL-li into a tac promoter vector. pT3XI-2-Φ10TC3FGFsyri was cut with BamHl and Hind3, which resulted in the linearization of the 7.4 kb expression vector the release of the insert DNA. The DNA was then cut with Ncol Smal, which further fragmented the insert DNA. pDD3 was diges with BamHl and Hind3 and the 546 bp IL-li fragment was gel purified and fused with the BamHl/Hind3-cut pT3XI-2-Φl0TC3FGFs 7.4 kb vector DNA fragment, resulting in the plasmid pDD4. F. Formation of pDD5. Use of E. coli preferred codons. The plasmid pDD4 carries DNA coding for the ompA signal sequence and the full length of the IL-li protein as it was derived from the original cDNA. Plasmid pDD4 was cut with Bam and Spel, thus releasing a small fragment (170 bp) carrying th sequences for the ompA signal peptide and the codons for the f 29 amino acid residues of the IL-li protein, and the large (7. kb) vector fragment. The large BamHl/Spel vector fragment was fused to two small fragments of DNA assembled from four synthe oligonucleotides. The sequences of these fragments are: / & 5' GAT CCG ATC TTG GAG GAT GAT TAA ATG CGT CCG AGC GGC CG 3 ' GC TAG AAC CTC CTA CTA ATT TAC GCA GGC TCG CCG GC Sacl AAG AGC TCC AAA AT 3' TTC TCG AGG TTT TAC GTC CG 5' 5' G CAG GCT TTC CGT ATC TGG GAC GTT AAC CAG AAA ACC TTC 3 ■ A AAG GCA TAG ACC CTG CAA TTG GTC TTT TGG AAG CTG CGC AAC AAC CAA 3' GAC GCG TTG TTG GTT GAT C 5 » These fragments carry sequences coding for the first 29 resid of the IL-li protein using JL. coli preferred codons [accordin deBoer and Kastelein in From Gene to Protein: Steps Dictati Maximal Level for Gene Expression (1986) Davis and Reznikoff, pp. 225-283, Butterworths, NY]- and a unique Sacl site after sixth codon of IL-li. The resulting plasmid is called pDD5. G. Formation of pDD6. Changes to remove secondary structure in mRNA. Plasmid pDD5 was digested with BamHl and Sacl. The lar (7.8 kb) Vector fragment resulting from this digestion was l to a synthetic fragment of DNA: 5« G AT CCG ATC TTG GAG GAT GAT TAA ATG CGA CCG TCC GGC CG 3 • GC TAG AAC CTC CTA CTA ATT TAC GCT GGC AGG CCG GC AAG AGC T 3 1 TTC 5' that codes for the first 6 residues of the IL-li protein, bu utilizes codons that prevent the formation of any hairpin lo near the 5' end of the mRNA, especially involving the "Shine-Dalgarno" sequence or the initiation codon for the IL protein. This resulted in the formation of pDD6 which is the expression vector for production of IL-li. Plasmid pDD6 was transformed into JM107 to yield the production strain SGE90. EXAMPLE 2 1. Production of IL-li from E. coli SGE90 Seed Growth. Ampules of a culture of SGE90 are prepared to be used for seed as follows. A culture streak is grown on Luria agar supplemented with 15 mg/1 tetracycline HCl at 37°C. A single colony is picked and grown in Luria broth supplemented with 15 1 tetracycline HCl at 37°C. Growth is monitored by absorbance i|660 nm (henceforth referred to an OD). When the culture reach about 1 OD it is aseptically centrifuged and resuspended in 20 glycerol:Luria broth (1:1). It is then distributed into ampul (1.5 ml per ampule) and stored at -70°C. Working stocks are m from this cell bank by growing one ampule in Luria broth supplemented; with 15 mg/1 tetracycline HCl to about 1 OD, then preparing ampules as above. The fermentor used is prepared by thawing ampules in 40 β C water and inoculating 1 ml from the ampule prep into each of t liter flasks containing 0.5 liter of Seed Media (Formula 1). flasks are incubated for about 8 hours on a shaker at 37°C at 3 rpm. The seed OD reaches about 3-4 by this time. 500 ml of the seed culture is used to inoculate 10 liters Fermentation Media (Formula 2). This seed tank is then grown a 37 β C for 5-6 hours with pH control at 7.0, until the OD reaches approximately 5. The seed tank is then used to inoculate the fermentor. 2. Fermentation. The fermentation is carried out in 1600 liters of Fermentation Media (Formula 2). Temperature is controlled at 37°C. Dissolved oxygen is maintained at 30% (saturation with at 3 psig) . pH is controlled at 7.0 by the addition of HCl a NaOH as required. Growth is monitored by OD. At approximately 10 OD synthe of IL-li is induced by the addition of Isopropyl-B-D-thiogalactoside (IPTG) to a final concentration 150 uM. Fermentation is continued until the culture reaches of about 40. Cell yield is about 150 kg solids per 1600 lite fermentation media. 3. Cell recovery and washing. Cells are recovered using a desludging centrifuge (for example an Alfa Laval BTUX 510) and washed with 150 mM NaCl. Cells are resuspended to approximately 16% solids in 150 mM N and then frozen and stored at -20°C. 4. Cell rupture and debris removal Fourteen kg of resuspended cells (about 2.2 kg solids) a thawed. EDTA is added to 5 mM and the cells lysed with two p through a high pressure homogenizer. The pH is adjusted to 5 using 1 M acetic acid. The lysate is diluted to 20±2 liters water and clarified by centrifugation at 14,000 x G for 20 minutes. 5. First Ion Exchange. (a) Column Specifications. The column used is an Amicon G300x250 filled with 7.5 liter of S-Sepharose resin - (b) Column Specifications. The column used is an lA icon G180x250, filled with 5 liters of Q-Sepharose (Pharmac All solutions are pumped through the columns at 350 ml/min. (c ) Column Operation. The following buffer sequen used for each cycle on the column. Buffer formulas are give Appendix A. Solution Formula Number Equilibration 8 Diafiltrate Equilibration 8 Salt Gradient Elution* 8/9 NaOH Wash 5 Acetic Acid Wash 6 Storage 7 ♦ Salt gradient is run from 0 to 100 mM NaCl. Eluate is collected by following the absorbance at 280 collecting the peak eluting during * _he salt gradient. Recov about 45 g of IL-li in about 10 1 of the pooled fractions fr of Diafiltrate 1. 7. Final Processing. (a) Concentration and Diafiltration. The pooled from the second ion exchange column is concentrated to approximately 6 1 using YM10 membrane (Amicon). The materia then diafiltered against 5 volumes of Diafiltration buffer (Formula 10). Final concentration then takes place to approximately 1-2 1, with a target concentration of 10-30 g/ ■ -$ t - The precipitate which forms at this step is removed by filtrat through 3 uM and 0.22 uM filters. The final concentrate is th filtered through a 0.22 uM filter into sterile, pyrogen free t and stored at -70°C. Recovery is about 80% from the pooled fractions from the second ion exchange column. EXAMPLE 3 1. Removal of the N-terminal Methionine from E. coli produced IL-li IL-li produced in E_j_ coli has a sequence identical to tha IL-li-x from human monocytes with the exception that the N-terminus has an additional methionine residue. This residue be removed by incubating the inhibitor with the exoprotease Aminopeptidase 1 from S. cerevisiae. 10 mg of recombinant IL-li (from the first S-Sepharose or Q-Sepharose step of purification) is incubated with 1 mg of Ye Aminopeptidase 1 purified as described by Change and Smith (J. Biol. Chem. 264, 6979, (1989)), in 50 mM ammonium carbonate pH for 6 hours. The desmethionyl IL-li is purified from the react mixture by further S-Sepharose chromatograph . If wished, this step of the production process for desmethionyl can be avoided by expressing the IL-li in an EL. co which contains the cDNA for yeast Aminopeptidase 1 enzyme in a suitable expression vector. This E^. coli should also be unable express the gene for Aminopeptidase P (Yoshimoto et al. J♦ Bioc (Tokyo) 104 93 (1988) since removal of the N-terminal methionin will otherwise lead to removal of the N-terminal arginine. It will be apparent to those skilled.in the art that vario modifications and variations can be made in the processes of th present invention. Thus, it is intended that the present invention cover the modifications and variations of these processes provided they come within the scope of the appended claims and their equivalents. 'EXAMPLE 4 A. Media and formula recipes Formula U____L_____ Step Name Fermentation Seed Medium All ingredients except tetracycline are mixed and the pH adjusted to 7.5 with sodium hydroxide. Tetracycline is filte .sterilized and added separately. Fermentation Fermentation Media All ingredients through antifoaa are sterilized together. Trace minerals, thiamine and tetracycline are filter sterilized and added separately. * Added as needed. Fermentation λntifoam Macol 19 750 m GE60 an ifoam 250 m le tart Gradient) nd Gradient) mg/ml with water. SμppU.r Cfll. f po . Sigma T-6508 Baker 9017-03 6 ograph Software NaCl 126 tograph Software DETECTOR SETTINGS: 280 nm MOBILE PHASE: A: 25 mM Naλc, pH 5.5 + 1 mM EDTA B: 25 mM NaAc, pH 5.5 + 1 mM EDTA + 500 mM NaCl GRADIENT CONDITIONS: 0% to 60% B in 36 minutes. FLOW RATE: 0.5 ml/min ule 406 aph Software to a final F . RZDUCING SDS PAGE OF IL-li GEL PREPARATION : Follow procedure outlined by Lammli in J. Mol Biol., 80, 575-599 (1973) . SEPARATING GEL: Aery1 mide 15% TRIS pH 8.8 375 mM SDS 0.1% STACKING GEL: Acrylamide pH 6.8 5% SDS 0.1% SAMPLE PREPARATION: Sample is diluted 1:1 with Sample Buffer. The samples are then heated for 15 minute* at 65*C, spun and loaded onto the gel. SAMPLE BUFFER: TRIS pH 6.8 250mM SDS 2.5% 2-Mβrcaptoethano1 5% Gycerol 12.5% SLSCTROPHPRSSSIS CONDITIONS; 50 V until samples have reached the separating gel. 100V until the broaophenol blue runs out of the gel. S AIFire. Stain overnight at room temperature with gentle shaking. Methanol 30.0% Acethic Acid 12. 5% water 57.5% I Olestain overnight or until background is clear at room tjemperature . F ? qrtad MW 2,300 and 3,400 Insulin (A and B) Bovine Trypsin Inhibitor 5,200 14,300 Lysozyme 18,400 β-Lactoglobulin 25,700 Alpha-Chymotrypsin 43,000 Ovalbumin 5 ug of the protein mixture is loaded onto the SDS PAGE gel ; t 4.6 inject onto the HPLC column. 5. Reverse-phase of peptide fragments produced by trypsin digestion. 5.1 HPLC system and column as in Section 2. 5.2 Detector settings: Channel A: 215 mm Channel B: 200 mm Range: 0-0.5 AU 5.3 Mobile phase: A: 0.1% TFA in water B: 0.1% TFA in acetonitrile 5.4 Gradient conditions e 5.5 H. ττ-11 Bioassav. The assay for IL-1 inhibitor is based on an IL-1 assay developed by S. Nakai, . Mizuno, M. aneta and Y. Hirai. (Biochero. Biophvs. Res. Comm. 154:1189-1196. 1988). principle of this assay is that prolonged exposure to IL-1 is eytotoxic to the human melanoma cell line A375. The cytotoxi is mediated via the IL-1 receptor. IL-li antagonizes this cytotoxicity in a dose dependent manner by competing with IL- binding to the IL-1 receptor. The level of toxicity can be quantitated by staining the live cells with crystal violet, extracting the stain from the cells by overnight incubation 100% ethanol, and measuring the optical density of the extra stain with a spectrophotometer. The rationale for the use of A375 melanoma cell bioasβay is that it is a simple and direc method for measuring both IL-1 and IL-li activity. Most oth assays that have been described in the literature depend on ability of IL-1 to activate other products, such as prostagl E2 and lactic acid in fibroblasts, and interleukin-2 in T-ce These secondary products are then measured in order to deter the level of IL-1 present. Although all of these IL-1 activ are receptor mediated, the existence of more than one stage these alternative assays cumbersome and subject to a greater probability of error.

Claims: WHAT IS CLAIMED IS: 1. A method for the production of commercial quantitie highly purified interleukin-1 inhibitor (IL-li) comprising: (1) fermentation of JL. coli comprising a plasmid conta a DNA encoding IL-li; (2) cell processing, including: (a) cell recovery, (b) lysis, and (c) clarification of the lysates; (3) a first ion exchange step; (4) a second ion exchange step; (5) the final processing steps including concentration and diafiltration. 2. The method of claim 1 wherein the plasmid is pDD6. 3. The method of claim 1 wherein the first ion exchange step uses a column packed with a cationic resin. 4. The method of claim 3 wherein the cationic resin is selected from the group consisting of S-Sepharose, SP-C25 Sephadex, CM Sephadex, CM Sepharose, CM cellulose, or CM Toyopearl. 5. The method of claim 4 wherein the cationic resin is S-Sepharose. 6. The method of claim 1 wherein the second ion exchang step uses a column packed with an anionic resin. 7. The method of claim 6 wherein the anionic resin is selected from the group consisting of Q-Sepharose, DEAE-Sephar Q-Sephadex and DEAE cellulose. 8. The method of claim 7 wherein the anionic resin is Q-Sepharose. 9. The method of claim 1 which further comprises a thi exchange ion exchange step conducted immediately prior to the final processing steps. 10. The method of claim 9 wherein the third ion exchang step is conducted using a cationic resin. 11. The method of claim 10 wherein the cationic resin i selected from the group consisting of S-Sepharose, SP-C25 Sephadex, CM Sephadex, CM Sepharose, CM cellulose, or CM Toyopearl. 12. The method of claim 1-1 wherein the cationic resin S-Sepharose. 13. A transformed Ej_ coli host comprising at least one plasmid containing a DNA sequence encoding IL-li which is ca of producing commercial quantities of highly purified IL-li. 14. The plasmid pDD6. 15. Interleukin-1 inhibitor bound to an anionic resin. 16. Interleukin-1 inhibitor bound to a cationic resin.

Inventors: CUDNY HENRYK, DRIPPS DAVID, EISENBERG STEPHEN P, EVANS RONALD, HAGEMAN ROBERT, THOMPSON ROBERT C, CUDNY, HENRYK, DRIPPS, DAVID, EISENBERG, STEPHEN, P., EVANS, RONALD, HAGEMAN, ROBERT, THOMPSON, ROBERT, C.
Applicants: SYNERGEN INC, SYNERGEN, INC.
Application year: 1990
Main classification: C12N1
IPC codes: C07K1, C07K14, C12N1, C12N15, C12P21, C12R1