Title of Invention	PROCESS FOR PREPARATION OF PYROGLU-MCP-1, AN N-TERMINALLY MODIFIED CHEMOTACTIC FACTOR
Abstract	The invention relates to a method for producing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1. According to the inventive method, Gln-MCP-1 is incubated in a buffer solution having a salt concentration ranging between 10 mM and 160 mM and a pH value ranging between 2 and 7.5 at a temperature ranging between 30 DEG C and 80 DEG C until at least 90 percent of the MCP-1 are provided in the form of pyroGlu-MCP-1.

Title of Invention

PROCESS FOR PREPARATION OF PYROGLU-MCP-1, AN N-TERMINALLY MODIFIED CHEMOTACTIC FACTOR

Abstract

The invention relates to a method for producing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1. According to the inventive method, Gln-MCP-1 is incubated in a buffer solution having a salt concentration ranging between 10 mM and 160 mM and a pH value ranging between 2 and 7.5 at a temperature ranging between 30 DEG C and 80 DEG C until at least 90 percent of the MCP-1 are provided in the form of pyroGlu-MCP-1.

Full Text	Boehringer Ingelheim International GmbH Method for the production of an N-terminally modified chemotactic factor 5The invention relates to a process for preparing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1 and compositions containing pyroGlu- MCP-1 preparations. MCP-1 (monocyte chemoattractant protein-1; also known by the names: l0monocyte chemotactic and activating factor 'MCAF', macrophage chemotactic factor 'MCF' tumour necrosis factor stimulated gene-8 'TSG-8', 'HC-11', smooth muscle cell chemotactic factor 'SMC-CF', lymphocyte derived chemotactic factor 'LDCF' as well as glioma derived chemotactic factor 'GDCF') is a member of the CC-chemokine family. Human MCP-1 protein was 15originally described in US Patent No. 5,714,578. It is synthesised under natural conditions in the body (natively) as a precursor protein 99 amino acids long, which is then processed to form a peptide with 76 amino acid groups. Mature human MCP-1 (hMCP-1) is a glycoprotein with a molecular weight of 14 kDa and is secreted by many types of cells, e.g. smooth vascular muscle 20cells and endothelial cells (Leonard and Yoshimura (1990), Immunology Today 11, 97 -101). It contains two intramolecular disulphide bridges and is Oglycosylated and sialylated when expressed natively (J. Yan Ling et al. (1990), Journal of Biological Chemistry, 265, 18318 -18321). 25The protein is a product of the JE gene of the chromosomal location 17q11.2- q21.1. This gene locus is also known as SCY A2 (small inducible cytokine A2). The human gene was first described in US Patent No. 5,212,073. The expression of this gene may be induced by a number of cytokines, such as e.g. tumour necrosis factor alpha, but also by immunoglobulin G, for example. 30The gene sequence and further information on the gene and the gene product are available in the NCBI Data bank under Accession Number M37719 (see Table 1). Case 1-1492-FF 2 Boehringer Ingelheim International GmbH et al. 5 LOCUS HUMMCHEMP 2776 bp DNA linear PRI 13-MAY-1994 DEFINITION Human monocyte chemotactic protein gene, complete cds. ACCESSION M37719 VERSION M37719.1 Gl:187447 5KEYWORDS monocyte chemotactic protein. SOURCE Homo sapiens (human) ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo, PREFERENCE 1 (bases 1 to 2776) AUTHORS Shyy, Y.J., Li, Y.S. and Kolattukudy, P.E. TITLE Structure of human monocyte chemotactic protein gene and its regulation by TPA JOURNAL Biochem. Biophys. Res. Commun. 169 (2), 346-351 (1990) 15MEDLINE 90290466 COMMENT Original source text: Human DNA. FEATURES Location/Qualifiers source 1..2776 /organism="homo sapiens" 20 /db_xref="taxon:9606" gene 598..2080 /gene="SCYA2" CDS join(598..673,1472..1589,1975..2080) /gene="SCYA2" 25 /note="monocyte chemotactic protein" /codon_start=1 /protein_id="AAA18102.1" /db_xref="GI:487124" translation= "MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLA 30 SYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT" exon /gene="SCYA2" /note="monocyte chemotactic protein" /number=1 35 exon 598..673 /gene="SCYA2" /note="monocyte chemotactic protein" /number=1 intron 674..1471 40 /gene="SCYA2" /note="monocyte chemotactic protein intron A" exon 1472.. 1589 Case 1-1492-FF 3 Boehringer Ingelheim International GmbH et al. /gene="SCYA2" /note="monocyte chemotactic protein" /numbeF=2 intron 1590..1974 5 /gene="SCYA2" /note="monocyte chemotactic protein intron B" exon 1975..2080 /gene="SCYA2" /note="monocyte chemotactic protein" 10 /number=3 exon 1975..>2080 /gene="SCYA2" /note="monocyte chemotactic protein" /number=3 15BASE COUNT 700 a 727 c 565 g 781 t 3 others ORIGIN 1 cagttcaatg tttacacaat cctacagttc tgctaggctt ctatgatgct actattctgc 61 atttgaatga gcaaatggat ttaatgcatt gtcagggagc cggccaaagc ttgagagctc 121 cttcctggct gggaggcccc ttggaatgtg gcctgaaggt aagctggcag cgagcctgac 20181 atgctttcat ctagtttcct cgcttccttc cttttcctgc agttttcgct tcagagaaag 241 cagaatcctt aaaaataacc ctcttagttc acatctgtgg tcagtctggg cttaatggca 301 ccccatcctc cccatttgcg tcatttggtc tcagcagtga atggaaaaaa gtgctcgtcc 361 tcacccccct gcttcccttt cctacttcct ggaaatccac aggatgctgc atttgctcag 421 cagatttaac agcccactta tcactcatgg aagatccctc ctcctgcttg actccgccct 25481 ctctccctct gcccgctttc aataagaggc agagacagca gccagaggaa ccgagaggct 541 gagactaacc cagaaacatc caattctcaa actgaagctc gcactctcgc ctccagcatg 601 aaagtctctg ccgcccttct gtgcctgctg ctcatagcag ccaccttcat tccccaaggg 661 ctcgctcagc caggtaaggc cccctcttct tctccttgaa ccacattgtc ttctctctga 721 gttatcatgg accatccaag cagacgtggt acccacagtc ttgctttaac gctacttttc 30781 caagataagg tgactcagaa aaggacaagg ggtgagcccc aaccacacag ctgctgctcg 841 gcagagcctg aactagaatt ccagctgtga acccaaatcc agctccttcc aggattcagg 901 atccagctct gggaacacac tcagcagtta ctcccccagc tgcttccagc agagtttggg 961 gatcagggta atcaaagaga agggtgggtg tgtaggctgt ttccagacac gctggagacc 1021 cagaatctgg tctgtgcttc attcacctta gcttccagag accggtgact ctgcaggtaa 351081 tgagtatcag ggaaactcat gaccaggcat agctattcag agtctaaaag gaggctcata 1141 gtggggctcc cagctgatct tccctggtgc tgatcatctg gattattggt ccgtcttaat 1201 gacacttgta ggcattatct agctttaaca gctcctcctt ctctctgtcc attatcaatg 1261 ttatataccc cattttacag cataggaaac tgagtcattg ggtcaaagat cacattctag 1321 ctctgaggta taggcagaag cactgggatt taatgagctc tttctcttct cctgcctgcc 401381 ttttgttttt tcctcatgac tcttttctgc tcttaagatc agaataatcc agttcatcct 1441 aaaatgcttt tctttgtggt ttattttcca gatgcaatca atgccccagt cacctgctgc 1501 tataacttca ccaataggaa gatctcagtg cagaggctcg cgagctatag aagaatcacc Case 1-1492-FF 4 Boehringer Ingelheim International GmbH et al. 1561 agcagcaagt gtcccaaaga agctgtgatg tgagttcagc acaccaacct tccctggcct 1621 gaagttcttc cttgtggagc aagggacaag cctcataaac ctagagtcag agagtgcact 1681 atttaactta atgtacaaag gttcccaatg ggaaaactga ggcaccaagg gaaaaagtga 1741 accccaacat cactctccac ctgggtgcct attcagaaca ccccaatttc tttagcttga 51801 agtcaggatg gctccacctg gacacctata ggagcagttt gccctgggtt ccctccttcc 1861 acctgcgtcc tcctagtctc catggcagct cgcttttggt gcagaatggg ctgcacttct 1921 agaccaaaac tgcaaaggaa cttcatctaa ctctgtctcc tcccttcccc acagcttcaa 1981 gaccattgtg gccaaggaga tctgtgctga ccccaagcag aagtgggttc aggattccat 2041 ggaccacctg gacaagcaaa cccaaactcc gaagacttga acactcactc cacaacccaa 102101 gaatctgcag ctaacttatt ttcccctagc tttccccaga caccctgttt tattttatta 2161 taatgaattt tgtttgttga tgtgaaacat tatgccttaa gtaatgttaa ttcttattta 2221 agttattgat gttttaagtt tatctttcat ggtactagtg ttttttagat acagagactt 2281 ggggaaattg cttttcctct tgaaccacag ttctacccct gggatgtttt gagggtcttt 2341 gcaagaatca ttaatacaaa gaattttttt taacattcca atgcattgct aaaatattat 152401 tgtggaaatg aatattttgt aactattaca ccaaataaat atatttttgt acaaaacctg 2461 acttccagtg ttttcttgaa ggaaattaca aagctgagag tatgagcttg gtggtgacaa 2521 aggaacatga tttcagaggg tggggcttac attttgaagg aatgggaaag tggattggcc 2581 cnntntcttc ctccactggg tggtctcctc tgagtctccg gtagaagaat ctttatggca 2641 ggccagttag gcattaaagc accacccttc cagtcttcaa cataagcagc ccagagtcca 202701 atgaccctgg tcacccattt gcaagagccc acccccattt cttttgctct cacgaccctg 2761 accctgcatg caattt 25lt has already been explained in the above-mentioned US 5,714,578 that in the case of an MCP-1 protein isolated from a native source the N terminus is blocked. Only later was it discovered that this is due to a post-translational modification in which the glutamine exposed after the cleaving of the leader sequence at the N terminus of the mature protein is cyclised, losing an NH3 30molecule, to form a pyroglutamate group. The identification of the open reading frame coding for MCP-1 in US 5,212,073 allowed recombinant expression of the protein. An MCP-1 produced by the recombinant method, e.g. in E. coli, does not have the 35typical N- or O-glycosylation pattern of the native MCP-1. An MCP-1 preparation prepared in this way also does not oppose Edman decomposition in the same way as a preparation obtained from native material, i.e. it contains unblocked N termini (glutamines). In cellular assays based on the 10Case 1-1492-FF 5 Boehringer Ingelheim International GmbH et al. chemotactic effect on macrophages, originally no difference could be found between the biological activity of native MCP-1 preparations and those obtained by the recombinant method. 5Under physiological conditions MCP-1 acts as an agonist to the beta- chemokine receptors CCR2 and CCR4, both of which are expressed mainly on monocytes and are also found both on basophiles and on T- and B- lymphocytes. MCP-1 induces monocyte chemotaxis even at subnanomolar concentrations. The receptors CCR2 and CCR4 are G-protein-coupled seven- lOtransmembrane domain receptors which lead to the activation of monocytes and increased adhesion of integrins. This process ultimately results in the docking of monocytes to endothelial cells and the subsequent departure of the monocytes from the vascular system. 15WO 98/44953 discloses the influence of MCP-1 on arteriogenesis, i.e. the growth of collateral arteries and/or other arteries from existing arteriolar connections. On the basis of this finding it was proposed in the above- mentioned International Patent Application to use MCP-1 for therapeutic purposes, namely for the treatment of vascular occlusive diseases which 20may be alleviated by stimulating the formation of new blood vessels. Such vascular occlusive diseases are particularly coronary artery disease (CAD), peripheral arterial occlusive disease (PAOD), cerebral and mesenterial arterial occlusive diseases, etc. The application also proposed the use of MCP-1-neutralising agents such as e.g. anti-MCP-1-antibody for preventing 25new vascular formation in order to combat tumour growth, in particular, which is reliant on a sufficient blood supply and hence adequate vascularisation of the tumour tissue. In order to be able to use the MCP-1 protein as explained above in a therapeutic approach for promoting the vascularisation of tissue, large 30enough quantities of this protein have to be made available with sufficient purity for pharmaceutical purposes. Human MCP-1 was originally obtained in native form from cultures of the human glioma cell line U105MG or from human mononuciear leukocytes of peripheral blood. Protein intended for therapeutic purposes cannot be isolated from these sources because the Case 1 -1492-FF 6 Boehringer Ingelheim International GmbH et al. production of large enough amounts would only be possible at unacceptably high cost in terms of labour and/or materials: it would not be possible to obtain human leukocytes in the required quantity. The glioma cells could indeed be replicated in virtually unrestricted amounts, but are unsuitable for 5cultivation in biotechnological fermenters and additionally require foetal calf serum for their cultivation, for example, with all the attendant problems. Instead of the native expression of MCP-1 it is therefore an inviting prospect to prepare MCP-1 by the recombinant method. In fact recombinant human 10MCP-1 is already commercially available, particularly from Messrs R&D Systems (Catalogue no. 279-MC) and Peprotech (Catalogue no. 300-04; see Table 2; the catalogue numbers quoted are those applicable in January 2003). According to the product specification of the commercially available recombinant MCP-1 preparations the MCP-1 protein present therein contains 15the amino acid glutamine (Q) at the N terminus. The product description also indicates that this protein preparation is highly sensitive, particularly in the reconstituted liquid form (recommended max storage : 1 week at 4°C). In fact, experiments by the inventor (internal prior art) showed that when the conventional MCP-1 protein preparations are stored in dissolved form the - 20biologically active - protein preparation appears to show signs of contamination by breakdown products very rapidly, particularly at temperatures above 4 °C (appearance of secondary bands in analytical HPLC chromatographs). Table 2 (extract from the Internet Website of Messrs Peprotech 25 http://www.peprotech.com/content/details.htm?results=1&prod= Recombinant Human MCAF (Human MCP-1) Case 1-1492-FF 7 Boehringer Ingelheim International GmbH et al. 15 30A conventional method of recombinantly producing human MCP-1 protein is by the temperature-induced expression of the protein as a fusion protein, purification of the inclusion bodies formed subsequently, dissolving and refolding of the protein and subsequent enzymatic release of the hMCP-1 protein from the fusion protein. 35 In conventional processes for the recombinant preparation of human MCP-1 protein the problem arises that a protein which is N-terminally shortened by one or more amino acids is often obtained as a by-product. These by- products may behave as antagonists to MCP-1 receptors in biological 40systems. In Van Coiliie et al. (1998), Biochemistry 37: 12672, 12673 a.E. a process is described in connection with experiments on the influence of N-terminal modifications on the biological activity of MCP-2, wherein an MCP-2 45preparation obtained by the recombinant method is incubated in 0.01 M Na2HPO4, pH 8.0, for 24 hours at 37 °C. MCP-2 has 62% homology with MCP-1 at the level of the amino acid sequence and also differs from it to the extent that N-terminally unmodified MCP-1 is biologically active, unlike MCP-2 (cf. the line "Biological Activity" in Table 2). The authors did not determine the 50extent of the conversion of the N-terminal glutamine groups into a Case 1-1492-FF 8 Boehringer Ingelheim International GmbH et al. pyroglutamate group which takes place under the conditions mentioned above. The inventors have found (internal prior art) that the application of comparable conditions to MCP-1 in any case leads to only partial cyclisation of the N-terminal amino acid (cf. Table 3, Comparison test 2). 5 One aim of the invention therefore, in the light of the foregoing discussion, is to provide a process by which an MCP-1-preparation (MCP-1 composition) may be prepared, which (a) is suitable for therapeutic purposes in view of its biological activity and (b) has advantages in terms of the drug licensing lOprocedures, which in the case of biopharmaceuticals contain requirements which are extremely difficult to comply with, e.g. with regard to the purity and reproducibility of production of the pharmaceutical composition, and in particular (c) has an excellent shelf life. Another related aim is to provide compositions or preparations which contain MCP-1 produced by the 15recombinant method and are suitable for the purposes mentioned above or have the aforementioned advantages. These aims are achieved by the processes and compositions containing MCP-1 recited in the claims. 20 Thus according to the invention a process for preparing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1 is provided wherein Gln-MCP-1 is incubated at a temperature in the range from 30°C and 80°C, preferably in the range from 30°C and 70°C and more preferably in the range from 35 °C 25to 60°C, in a buffer solution which has a salt concentration in the range from 10 mM to 160 mM, preferably in the range from 10 mM to 100 mM, and which has a pH in the range from 2 to 7.5, preferably 3.5 to 7.5, more preferably 3.5 to 6.5, more preferably 5 to 6.5, more preferably 5.5 to 6.5 and more preferably has a pH of about 6. The incubation is carried out until at least 3090%, preferably at least 95 % and more preferably at least 96 % of the MCP- 1 contained in the incubating buffer solution is present in the form of the pyroGlu-MCP-1. Case I-I492-FF 9 Boehringer Ingelheim International GmbH et al. By "MCP-1" is meant, within the scope of this disclosure, the MCP-1 protein (without preprosequence), with an N-terminai glutamine group ("Gln-MCP-1"; cf. the amino acid sequence shown in Table 1 and Table 2) or with an N terminus which has already been converted/cyclised into the pyroglutamate 5group ("pyroGlu-MCP-1"), depending on the context. It is clear, however, that modifications of the MCP-1-protein which do not affect its biological function (esp. the monocyte-attractant activity or the effect on the CCR receptor) and do not alter its structure so that the reaction parameters described above no longer produce the desired result (i.e. the protein can no l0longer be converted into a pyroGlu-Variant by the process according to the invention), do not depart from the scope of protection. Thus, the process according to the invention is naturally also applicable to an MCP-1 in which a conservative amino acid exchange, e.g. serine to threonine or leucine to isoleucine, has taken place, provided that this affects neither the biological 15function or activity of the resulting protein nor the convertibility of the N- terminal glutamine into pyroglutamate according to the above process. The process according to the invention can thus also be applied to MCP-1 proteins of other mammals such as e.g. rats, mice, guinea pigs, rabbits and ferrets (and several others). 20 The buffer solution in which the process described above is carried out is preferably a phosphate-buffered (sodium and/or potassium phosphate- buffered) aqueous solution of low or physiological molarity, namely in the range from 10 to 160 mM, 10 to 80 mM, 10 to 50 mM, 20 to 40 mM or around 2520 or 40 mM. If longer incubation times are accepted, according to one particular embodiment of the invention the work may also be done at physiological molarities, such as e.g. a saline concentration of around 150 mM, this molarity preferably being achieved by the use of a phosphate- buffered saline solution or "PBS". The disadvantage of the longer incubation 30period - and the attendant risk of increasing amounts of breakdown, secondary or oxidation products - is made up for in this particular embodiment by the advantage of being able to obtain the protein preparation straight away in the form of a solution with a physiological salt concentration. 20Case 1-1492-FF 10 Boehringer Ingelheim International GmbH et al. The buffer solution in which the modification step is carried out may also contain, for example, a (mild) detergent, an antioxidant, a preservative, a complexing agent, stabilisers, antimicrobial reagents, etc. 5The incubation temperature is selected in the range from 30 °C to 80 °C, i.e. above ambient temperature. At lower temperatures the conversion step proceeds very slowly. In order to achieve virtually total conversion the incubation period would have to be increased substantially to more than a week. This would lead to stand times which are unacceptable in the l0 biotechnological process and would also substantially increase the risk of other forms of contamination of the protein solution (with breakdown or oxidation products, bacteria, viruses or other pathogens). On the other hand, although increasing the incubation temperature to above 80 °C does indeed greatly speed up the reaction of conversion, it also results in an increased 15formation of undesirable by-products and breakdown products (cf. Examples 1 and 2). If stand times of up to 6 days are acceptable, an incubation temperature of 35 to 40 °C is particularly preferred. In cases where incubation should be significantly shorter if possible, the incubation may also be carried out in the range from e.g. 50 to 60, 70 or 80 °C. Temperatures between 40 20and 50 °C will naturally also produce the desired results. The pH of the incubating buffer solution should be in the range from 2 and 7.5, i.e. in the neutral to acidic range. It is preferably in the range from 3.5 to 7.5, more preferably in the range from 3.5 to 6.5, more preferably in the range 25from 5 to 6.5, more preferably in the range from 5.5 to 6.5 and more preferably a pH of about 6 is selected. With a suitable choice of the above parameters, e.g. as described in the Examples, the pyroGlu-MCP-1 variant may be obtained with a purity of 90% 30in any case and possibly higher, e.g. 95%, 96% or 98%, this percentage indicating the amount of pyroGlu-MCP-1 (determined e.g. as the area under the curve in an HPLC elution profile) based on the amount of total MCP-1 present in the solution (i.e. including any remaining amount of Gln-MCP-1 and possible oxidation products and other by-products or breakdown products). Case I-1492-FF 11 Boehringer Ingelheim International GmbH et al. According to another embodiment of the invention a process for preparing a pyroGlu-MCP-1 preparation is provided which comprises at least the following steps: 5- preparing a Gln-MCP-1 preparation by the recombinant method by expression of a gene construct coding for MCP-1 in a host cell, - optionally concentrating and/or purifying the Gln-MCP-1 contained in the Gln-MCP-1 preparation, and finally - converting the Gln-MCP-1 which is contained in the Gln-MCP-1 preparation l0 or which was contained therein before the concentrating and/or purifying, into a pyroGlu-MCP-1 preparation which contains the protein molecule species pyroGlu-MCP-1, this step being carried out according to the "Process for preparing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1" as described above. 15This process may optionally also include, for example, buffering the pyroGlu- MCP-1 preparation or further purification, e.g. by a subsequent step of column chromatography, dialysis, ultrafiltration, etc. Methods of producing recombinant proteins by biotechnology are known. 20They comprise, in particular, fermentation, purification, concentration, and other steps. The step of conversion described above may be included at various points in a "multi-step process", i.e. with an as yet largely unpurified or wholly purified MCP-1 protein solution as the starting solution. It is also conceivable, in particular, to have a process in which not yet (totally) 25converted Gln-MCP-1 in more, less or practically wholly purified form is lyophilised in order to improve its shelf life in as yet unconverted form and is in due course put back into solution and then converted into pyroGlu-MCP-1 according to the above process. Naturally, the protein solution obtained after conversion into pyroGlu-MCP-1 may also be lyophilised. 30 The product of the process, namely the pyroGlu-MCP-1 preparation subjected to N-terminal modification (conversion), contains according to the invention an amount of pyroGlu-MCP-1 of at least 90 %, preferably at least 95 % and more Case I-1492-FF 12 Boehringer Ingelheim International GmbH et al. 25 preferably at least 96 %, based on the total content of MCP-1 (converted plus unconverted protein plus by-products formed by oxidation, for example). The term (Gin- or pyroGlu-MCP-1-) preparation means that in the various 5stages of the process the MCP-1 protein is present in dissolved form in a (buffer) solution and hence other components may be present in addition to the MCP-1, i.e. in any case the ions of the buffer salt used and possibly also other salts, antioxidants, stabilisers, antimicrobial reagents, detergents, preservatives, complexing agents, etc. 10 According to a further aspect of the invention a composition is provided which contains pyroGlu-MCP-1 or a pyroGlu-MCP-1 preparation obtained by the processes described above, in which at least 90 % of the MCP-1 contained in the composition (converted plus unconverted protein plus by-products; i.e. the 15"area under the curve" in an HPLC elution chromatograph, for example) are present in the form of the pyroGIu-MCP-1. Thus, the invention also relates to an MCPO-1 preparation produced by the recombinant method particularly in prokaryotes and particularly preferably in 20E. coli, wherein at least 90 % of the MCP-1 protein is present as pyroGlu- MCP-1. When prepared in common expression cells the protein will frequently not exhibit the natural glycosyfation pattern - unlike in native production. When prepared in prokaryotes such as e.g. E coli, in particular, the pyroGlu- MCP-1 obtained or the pyroGlu-MCP-1 preparation obtained unlike the form 25which occurs in native expression in the human body will not be glycosylated and/or sialylated and will thus differ from a native protein of this kind or from a protein preparation obtained from a native source. A composition of this kind according to the invention may be, in particular, a 30medicament or a pharmaceutical composition which contains, in addition to an amount of pyroGlu-MCP-1 which makes up at least 90 %, 95% or 96% of the total MCP-1 content (converted plus unconverted protein plus by- products), conventional excipients and carriers, salts, antioxidants, stabilisers, antimicrobial reagents, detergents, preservatives, complexing agents, etc. Case 1-1492-FF 13 Boehringer Ingelheim International GmbH et al. The composition according to the invention may be used to treat patients suffering from an arterial occlusive disease such as in particular PAOD or CAD. The treatment comprises administering such a composition in a therapeutically effective amount by a suitable route, e.g. by intraarterial 5infusion or in the form of a periarterially deposited gel, from which the MCP-1 protein is released over a fairly long period. Surprisingly it has also been found that, contrary to expectations, the incubation of a recombinantly prepared MCP-1 protein preparation in lOaqueous solution and at elevated temperature does not lead to its decomposition and biological inactivation; on the contrary, with a suitable choice of a number of parameters, this step, which is very unusual from a biotechnological point of view and is usually inherently undesirable, results in a surprisingly homogeneous, biologically fully active pyroGlu-MCP-1 15preparation, which retains its homogeneity even during lengthy storage, i.e. is more stable than the Gln-MCP-1 (starting) preparation. Bodies such as the Food and Drug Administration in the USA or the EMEA in Europe make the granting of marketing approval for a drug dependent on 20meeting numerous conditions which are imposed on the drugs manufacturers with the ultimate aim of protecting the patient. Thus, the manufacturer in question has to supply proofs which demonstrate, for example, the purity of the pharmaceutical product, its shelf life and its reproducibility of production. These three aspects are only a small selection, but play a central role 25precisely in the licensing of biopharmaceuticals, i.e. drugs which contain macromolecular active substances consisting of natural materials or derived therefrom (proteins, nucleic acids, proteoglycans, polysaccharides, etc.). Often, the requirements imposed on the (bio)pharmaceutical within the framework of this approval are very difficult to meet. For example, active 30substances based on a protein have to be subjected to intensive and complicated multi-stage purification after their production in a more or less complex cellular organism and must not be uncontrollably altered in structure, e.g. by oxidation or other spontaneous chemical reactions. Moreover, the end product should have a reasonable shelf life so that there is no need for Case 1-1492-FF 14 Boehringer Ingelheim International GmbH et al. complicated and expensive supply networks between the manufacturer and the prescribing doctor or patient or the clinic using the product - a requirement which is generally only met with great difficulty, particularly in the case of proteins, where it is important to maintain the correct secondary 5structure (folding). In the case of the MCP-1 protein the quality of commercially obtainable preparations is frequently adequate in many respects for in vitro testing, for example. However, the inventors have found in the course of their work that l0the same preparations are by no means suitable for producing a drug which would be eligible for approval. Being left to stand in solution at ambient temperature even for a short time before being administered to the patient, which could not be ruled out in doctors' surgeries or hospitals, for example, leads to the occurrence of "breakdown" products in the MCP-1 preparations 15which were originally viewed as contamination, in the experience of the inventors. More intense purification of the Gln-MCP-1 originally provided as active substance briefly restored a high level of purity, but even this preparation was again unstable when briefly stored in solution and at ambient temperature. Operating a drug approval procedure on the basis of such an 20unstable active substance preparation is beset by tremendous problems, if not altogether hopeless. Surprisingly, these problems can be overcome by the teaching of the invention, wherein an unusual and at first sight highly counter-productive step, 25namely incubation at elevated temperature (which was previously seen to positively promote inhomogeneity) was included in the actual preparation and purification process. In fact, when carried out under the conditions analysed in detail and perfected by the inventors, such incubation may bring about virtually quantitative conversion of the original active substance Gln-MCP-1 30into the pyroGlu-MCP-1 form which was originally regarded as a contaminant. This latter form is then astonishingly stable against protein-denaturing influences such as incubation at elevated temperature and the like. Thus, an MCP-1 preparation is obtained which, by virtue of its high purity and homogeneity, exceptionally reproducible manufacture and stability on 30Case 1-1492-FF 15 Boehringer Ingelheim International GmbH et al. storage, is suitable for use as a pharmaceutical active substance which has good prospects of complying with the strict requirements in corresponding licensing procedures referred to earlier, and thus makes it possible to implement new therapeutic processes based on the activity of MCP-1. 5 According to a partial aspect of the invention a pyroGlu-MCP-1 preparation prepared by the process described above and in the claims is used - for the therapeutic treatment of vascular occlusive diseases such as, in particular, coronary artery disease (CAD), peripheral arterial occlusive lOdisease (PAOD), cerebral and mesenterial arterial occlusive diseases, or - for preparing a pharmaceutical composition for the treatment of vascular occlusive diseases such as, in particular, coronary artery disease (CAD), peripheral arterial occlusive disease (PAOD), cerebral and mesenterial arterial occlusive diseases. 15 The invention thus also makes it possible to carry out a process for treating a patient suffering from the above-mentioned vascular occlusive diseases, wherein the pyroGlu-MCP-1 preparation prepared by conversion or the pharmaceutical composition prepared using a pyroGlu-MCP-1 preparation of 20this kind is administered by injection or infusion, for example. With regard to the individual optimised process parameters it should also be noted that the biotechnologist will not easily be convinced that he or she should incubate a protein preparation fora fairly long period of, in some 25cases, several days at elevated temperatures of e.g. 60°C. Regarding the neutral to acidic pH of the incubating solution which was previously disclosed as optimum this is contrary to chemical logic: the conversion of glutamine to cyclic pyroglutamate is a nucleophilic substitution reaction in which a pair of free electrons acts on the attacking nitrogen atom of the C-alpha-amino group 30of the glutamate at the C atom of the C-gamma-amide group. An acidic pH would inherently lead to increased protonation of the C-alpha-amino group and would therefore have a negative effect on the speed of the reaction. However, this is precisely not the case, according to the inventors' findings. One possible explanation - which should not be interpreted restrictiveiy - Case 1-1492-FF 16 Boehringer Ingelheim International GmbH et al. might be that the folded MCP-1 protein forms a micro-environment for the N terminus, in which in spite of the acidic pH of the surrounding aqueous solution the amino group is present in unprotonated form, i.e. a pair of free electrons is available for the nucleophilic attack. Case 1-1492-FF 17 Boehringer Ingelheim International GmbH et al. 35 Examples Example 1: Production of a pvroGlu-MCP-1 preparation by the process Saccordina to the invention Fermentation: The fermentation was carried out with a strain of Escherichia coli K12 (W3110). The strain was transformed with a ColE1 plasmid (pBR322 lOderivative) containing the following elements: the genomic sequence of hMCP-1 under the control of an all-purpose promoter (phosphatase, pPhoA), a ColE1-replication origin and the resistance gene for tetracycline. For fermentation the production strain was precultured in the shaking flask in LB medium containing tetracycline. Incubation was carried out at 37°C until an 15OD of 1 was achieved. The preculture was transferred into the fermenter and further cultivated with stirring and with a supply of air at 37°C. The medium contained glucose, various salts, trace elements, yeast extract, amino acids and tetracycline. The pH was maintained at 6.8 with ammonia. As soon as the glucose put in had been used up the dissolved oxygen (pO2) was kept 20constant at the intended level of 40% by the supply of glucose. The induction of the phosphatase promoter took place automatically as soon as the phosphate in the medium had been exhausted. After 39 hours the biomass was harvested with a tube centrifuge (CEPA) and stored at -70°C. 25Cell lysis and protein purification steps: The frozen cell pellet was resuspended in four times as much lysis buffer (200 mM Tris, 55 mM NaCI, 5mM EDTA, pH 7.5) using an Ultraturrax. The cell lysis was carried out by two passages with a homogeniser at 460 bar. Cell fragments were removed with a CEPA centrifuge. The supernatant was 30optionally filtered with Polysep II (1.2 rn) filters (Millipore), and then loaded onto a column combination consisting of a Q sepharose FF and an SP sepharose FF. The columns were equilibrated with lysis buffer. Case 1-1492-FF 18 Boehringer Ingelheim International GmbH et al. After the loading was complete the column combination was washed with lysis buffer and the Q sepharose column was clamped off. The SP sepharose was washed with lysis buffer, then with 5 M urea (in lysis buffer) and with lysis buffer again. The product was eluted in a linear NaCI gradient. 5 The eluate was salted with ammonium sulphate to a final concentration of 1.3 mol/L and centrifuged for 15 min at 3200 g. The supernatant was loaded onto a phenyl sepharose column which was equilibrated with 1.3 M ammonium sulphate (in lysis buffer). 10 The flow of phenyl sepharose was applied to a Source 30 RPC column which was equilibrated with lysis buffer. Washing was then carried out with lysis buffer, water and buffer A (5% EtOH, 0,1% TFA). The product was eluted in a linear gradient from 20% buffer B (95% EtOH, 0.1% TFA) to 70% buffer B 15in 10 column volumes. Conversion step: The eluate was diluted 1:10 in different buffers, e.g. phosphate buffer, 10 - 40 mM, pH 6.0 - 7.4. According to a preferred embodiment a value of 20between 6.0 and 6.5 was sought as the final pH of the conversion solution. The protein concentration was between 0.2 and 1.0 mg/mL. The solution was filter sterilised and incubated at temperatures of 35 - 80°C (in different batches) with gentle agitation (60 rpm). The progress of the reaction was monitored by HPLC analysis. 25 Final purification steps: As soon as the end of the reaction was reached, the conversion solution was loaded onto an SP sepharose HP column which was equilibrated in buffer A (40 mM phosphate, pH 6.0). Elution was carried out with a linear NaCI 30gradient. The NaCI concentration in the eluate was about 350 mmol/L, and the protein concentration was about 3 mg/mL. The eluate was diluted with buffer A to a conductivity corresponding to 250 mM NaCI. Then it was further diluted with 40 mM phosphate, pH 6.0, 250 mM NaCI until the protein Case 1-1492-FF 19 Boehringer Ingelheim International GmbH et al. concentration was 1 mg/mL The bulk solution thus formed was filter sterilised and stored at4°C or-70°C until ready to be formulated. Analysis: 5The progress of the conversion step discussed above was monitored by RP- HPLC. Chromatographs of the elution profiles were taken before the start of the conversion reaction (to) and at times t = 24 h, t= 48 h and t = 120 h. As can be seen from the four chromatographs shown in Fig. 1, after 48 h approx. 90 % of the protein had been converted into pyroGlu-MCP-1 at an incubation lOtemperature of 35 °C. HPLC analyses showed that the two disulphide bridges had formed properly. Fig. 2 shows the kinetics of the reaction of Gln-MCP-1 to pyroGlu-MCP-1 under the conditions described. It shows that as the duration of the reaction 15increases an impurity appears, which is referred to as "variant 2" in Fig. 2 and not further characterised. Similar reaction patterns were observed when carrying out the conversion step in 0.1 M sodium acetate buffer, pH 5.5, or 0.1 M sodium citrate buffer, 20pH 3.5 (data not shown). The reaction described above was repeated except that temperatures of 24° C, 60 °C and 80 °C were used instead of a reaction temperature of 35°C. As is clear from the data assembled in Table 3, no satisfactory yields of pyroGlu- 25MCP-1 were obtained at 24°C within a reasonable time. At 60°C and 80°C, on the other hand, significantly higher reaction rates are observed. The reaction rate at 80°C was approximately 50 times the rate at ambient temperature. 40Case 1-1492-FF 20 Boehringer Ingelheim International GmbH et al. n.d. = not determined, the end of the reaction had not been reached after the specified time 5Certainly, a higher reaction temperature favours the formation of by-products or breakdown products, reducing the purity level - particularly when the test is carried out at 80 °C (Table 3). Fig. 3 shows in high resolution the chromatographs of the elution profile of l0pyroGlu-MCP-1 preparations which were obtained under the conditions summarised in Table 3 (20 mM phosphate buffer, 35°C, 60°C or 80°C). As can be seen from this, significantly more impurities occur with MCP-1 by- products or breakdown products at high temperatures, even with a short incubation period (e.g. 3 hours at 80°C), than during five days' incubation at 1535°C. The content of (e.g. 5 %) ethanol (EtOH) in certain embodiments of the process resulting from the production method did not have a negative effect on the reaction rates or purity of the product, under otherwise constant test 20conditions (20 mM phosphate buffer, pH 6.0). Example 2: Production of a pyroGlu-MCP-1 preparation by an alternative process according to the invention Case 1-1492-FF 21 Boehringer Ingelheim International GmbH et al. The fermentation, cell lysis and protein purification steps were carried out as explained in Example 1. 5Conversion step: The eluate of the Source 30 RPC was diluted 1:10 with PBS, 0.02% Tween 20. The protein concentration was then between about 0.2 and 0.5 mg/mL, the pH was about 7.4. In addition to the buffer components the solution contained about 3.5% EtOH and 0.01% TFA, which originated from the eluate 1 Oof the previous Source 30 RPC step. The solution was filter sterilised into polypropylene flasks with a Millipak 20 (0.2 n, Millipore) and incubated at 35°C with gentle agitation. The progress of the reaction was monitored by HPLC analysis. As soon as the end of the reaction was reached the conversion solution was ultradiafiltered with a Pellikon 2 UDF membrane (3k, 15Millipore) against PBS, 0.02% Tween 20, and the protein concentration was adjusted to 0.1 mg/mL. The preparation was decanted in 1mL batches into glass containers through a Millipore GV filter (0.m) under sterile conditions. 20Analysis: The progress of the conversion step was monitored by RP-HPLC. As can be seen in Fig. 4, after 5 days more than 90 % of the protein had been converted into pyroGlu-MCP-1, while surprisingly in spite of the long incubation period a very high purity of 95 % was obtained (Table 3). As is apparent from a 25comparison of mixtures containing 20 mM phosphate buffer and those containing 20 mM phosphate buffer plus 150 mM salt (Example 1, Table 3, line 3 to line 6), the relatively low speed constant in mixtures containing PBS as incubation solution may be put down to the higher salt concentration, ionic strength or osmolarity. 30 Example 3: Preparation of a pharmaceutical composition based on the MCP- 1 preparation produced according to Example 1 or Example 2 Case 1-1492-FF 22 Boehringer Ingelheim International GmbH et al. 45 The pyroGlu-MCP-1 preparation obtained in Example 1 or Example 2 was diluted to a concentration of 0.1 mg/mL in PBS (sodium chloride, disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate; pH 7.0), also containing 0.02 % Tween 20, and transferred into glass 5containers in volumes of 1 mL The finished pharmaceutical solution was clear, colourless and odourless and can be administered by injection or infusion. Example 4: Comparison of the biological activity of pyroGlu-MCP-1 and Gln- 10MCP-1 Measuring principle: MCP-1 binds to and activates the MCP-1 receptor CCR2b. The activation of 15the receptor leads to an influx of calcium into the cytosol. This can be measured using a Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices). To do this, the inactive fluorescent dye ester Fluo-4 AM is sluiced into cells which express hCCR2b on their surface, this ester then being cleaved by intracellular esterases. In this form the fluorescent dye binds Ca2+ 20ions. On excitation with a wavelength of 488 nm there is an emission with a peak at 528 nm. The intensity of the emission is dependent on the concentration of calcium in the cytosol. The change in intensity of the emitted light thus correlates with the concentration of calcium in the cytosol which is in turn dependent on the state of activation of the receptor. 25 A characteristic time-triggered measuring signal after the addition of MCP-1 is shown in Fig. 5. It is apparent from this that after the addition of MCP-1 there is a rapid release of calcium which is linked with a sharp rise in fluorescence. The peak (b) is reached just a few seconds after the application (a). The 30interval between the application (a) and maximum stimulation (b) is roughly 20-30 sec. In the tests that follow, the evaluation was carried out using the maximum values as they are less affected by secondary effects such as e.g. a calcium- 35induced calcium influx, and therefore more precise measurement is possible. Case 1-1492-FF 23 Boehringer Ingelheim International GmbH et al. Preparation of stably expressing CHO/hCCR2B-K1 cells: The coding region of the human CCR2b receptor (Gene bank Accession No: 5D29984) was amplified by PCR. Then the 1.08 kb BamHI-Xbal fragment was cloned into an expression vector. CHO-K1 cells were transfected with this plasmid acting as an expression vector. The cells were cultivated in a culture medium based on Ham's F12 medium lOand regularly passaged. On the day before the measurement 5000 cells were plated out in 384-well assay plates (Corning Costar) with 40 pi of culture medium and left to adhere overnight (approx. 24 h) at 37 °C, 5 % CO2, 95 % relative humidity. In all the assays the measurements were carried out four times. 15 On the day of the test, first of all the substance plates were prepared. Hanks buffer with 0.1 % BSA (protease-free) was used as the diluting buffer for the various MCP-1 preparations. Hanks buffer with 0.1 % BSA was used as the blank control. Then 40 l / well of Fluo-4 dye medium were added to the cells 20and the preparations were incubated for 45 min. at 37 °C, 5 % CO2, 95% relative humidity. The stained cells were then washed four times with 60 ul washing buffer, leaving a residue of 25 l of washing buffer in each well. The cells were then incubated with washing buffer for a further 5 min at ambient temperature. 25 To measure the fluorescence the FLIPR measuring device was adjusted so that stained and unstained wells differed by at least a factor 1:5 and the stained wells had approx. 11,000 fluorescence counts. The other FLIPR settings were: 30 Excitation: 488 nm Emission: 510 - 570 nm (band pass) Negative correction: Hanks/BSA (= comparison with blank control) Case 1-1492-FF 24 Boehringer Ingelheim International GmbH et al. Bias subtraction: 6 (= levelling all the wells to 0 before adding the substance) Presoak of the tips: with 25 ul of the relevant substance solution from the substance plate (= minimising an adhesion 5 artefact) The measuring process comprised the following steps: 6 intervals in a 5 sec. cycle 15 ul substance added 10 60 intervals in a 1 sec. cycle 18 intervals in a 5 sec cycle For evaluation the maximum signal of the 78 intervals after the addition of the substance was used. The control mixtures used were: 15 blank control: for negative correction positive control (ATP): for checking the staining reference control (standard): -monitoring the receptor expression on the cells -basis of calculation for determining the 20 activity of unknown samples The samples to be tested were pipetted parallel with the reference control in identical dilution steps. The concentrations used in the tests were selected so as to be in the EC50 region of the reference control. 25 50Case 1-1492-FF 25 Boehringer Ingelheim International GmbH et al. Comparison of MCP-1 preparations according to the invention and not according to the invention: Two pyroGlu-MCP-1 preparations ("Batch 0711OOkh" and "Batch 0141921") 5prepared by the process according to the invention and an MCP-1 preparation prepared by the recombinant method, whose N-terminal glutamine group had not been converted into a pyroglutamate group in the process step according to the invention as described above (MCP-1 preparations obtainable from Peprotech) were tested and compared using the lOtest system described previously. The EC50 was determined from the measurement curves recorded as described above (see Fig. 6). Virtually identical EC50 values were obtained for the two pyroGlu-MCP-1 preparations, while the values for the MCP-1 preparation not according to the 15invention and not N-terminally modified differed significantly (Fig. 6). The EC50 value of the latter preparation proved to be worse by a factor 2 to 3 (pyroGlu- MCP-1: EC50 = 7.82 nM; MCP-1 preparation from Peprotech: EC50 = 20.76 nM). 2QThe biological activity of the two preparations was calculated as follows: First, a positive correction is made to the pyroGlu MCP-1 at the concentration 1e-8 M, i.e. the value obtained at a concentration of 10 nM pyroGlu-MCP-1 was set at 100 %. This is in the almost linear part of the ascent of the curve. 25Using this percentage signal the activity of other preparations, such as e.g. the Peprotech MCP-1 is calculated according to the formula % activity (sample) = RFU(Sampleat 1e-8M) X 1 00 % / RFU(standard at 1e-8M) 30ln the above Example, as shown in Table 4, this yields a value of 48 % activity for the non-N-terminally modified MCP-1 preparation based on the pyroGlu-MCP-1 preparation. Case 1-1492-FF 26 Boehringer Ingelheim International GmbH et al. 5Example 5: Temperature sensitivity of pyroGlu-MCP1 pyroGlu-MCP-1 was incubated for 1 or 2 hours at 56°C or 95°C. The biological activity still present thereafter was then measured using the test system in Example 4. 10 It was found that no denaturing occurs when a pyroGlu-MCP-1 preparation is incubated at 56 °C for either one hour or two hours. However, as shown in Fig. 7, incubation at 95°C results in a very marked denaturing effect. 15Example 6: Treatment of patients suffering from an arterial occlusive disease: For treating PAOD patients a pyroGlu-MCP-1 preparation as prepared above is adjusted to a concentration of between 1.2 and 120 g/ml. immediately 20before use this solution is adjusted to a final concentration of between 0.1 and 10 g/ml and infused into the patient at a flow rate of 2 to 12 ml/min over a period of 1 to 6 hours by intraarterial route close to the region affected by the vascular occlusion. The infusion may be repeated after 1 to 7 days. 25 Case 1-1492-FF 27 Boehringer Ingelheim International GmbH et al. 55 Claims 1. Process for preparing pyroGlu-MCP-1 from recombinantly produced Gln- MCP-1, wherein Gln-MCP-1 is incubated 5 - at a temperature in the range from 30°C and 80°C - in a buffer solution, with -- a salt concentration in the range from 10 mM to 160 mM and - at a pH in the range from 2 to 7.5 until at least 90 % of the MCP-1 is present in the form of the pyroGlu- 10 MCP-1. 2. Process according to claim 1, wherein the buffer solution is a phosphate buffer with a concentration in the range from 20 mM to 50 mM and with a pH in the range from 3.5 to 6.5. 15 3. Process according to one of Claims 1 or 2, wherein the buffer solution additionally contains a detergent, an antioxidant, a preservative, a stabiliser, an antimicrobial reagent and/or a complexing agent. 204. Process for preparing a pyroGlu-MCP-1 preparation, comprising at least the steps of - preparing a Gln-MCP-1 preparation by expression of a gene construct coding for MCP-1 in a host cell, - optionally concentrating and/or purifying the Gln-MCP-1 contained in 25 the Gln-MCP-1 preparation, - converting the Gln-MCP-1 of the Gln-MCP-1 preparation into a pyroGlu-MCP-1 preparation which contains pyroGlu-MCP-1, according to one of processes 1 to 3, and - optionally buffering and/or further purifying the pyroGlu-MCP-1 30 preparation, the proportion of pyroGlu-MCP-1 based on the total content of MCP-1 in the resulting pyroGlu-MCP-1 preparation being at least 90%. Case 1-1492-FF 28 Boehringer Ingelheim International GmbH et al. 5. Composition containing a pyroGlu-MCP-1 preparation prepared according to claim 4, wherein at least 90 % of the MCP-1 contained in the pyroGlu-MCP-1 preparation is present in the form of the pyroGlu- -?( MCP-1. 5 6. Process for preparing a pharmaceutical composition containing pyroGlu- MCP-1, wherein a pyroGlu-MCP-1 preparation prepared according to claim 4 is used. 107. Medicament or pharmaceutical composition containing a pyroGlu-MCP-1 preparation prepared according to claim 4, wherein at least 90 % of the MCP-1 contained therein is in the form of the pyroGlu-MCP-1. 8. Medicament or pharmaceutical composition according to claim 7, 15 containing pyroGlu-MCP-1 in a phosphate buffer with sodium chloride and optionally a detergent as additives. 9. Use of pyroGlu-MCP-1 or a pyroGlu-MCP-1 preparation prepared according to claim 4 for preparing a pharmaceutical composition for the 20 treatment of vascular occlusive diseases such as, in particular, coronary artery disease (CAD), peripheral arterial occlusive disease (PAOD), cerebral and mesenterial arterial occlusive diseases. 10. Recombinant MCP-1 preparation produced by the process according to 25 claim 4, the biological activity of which, with respect to a recombinantly produced MCP-1 preparation which has not been subjected to a process according to claim 1 (N-terminally unmodified MCP-1 preparation), is in the ratio 100 : 48 or higher. 3011. Recombinantly produced MCP-1 preparation, wherein at least 90 % of the MCP-1 protein is present as pyroGlu-MCP-1. 12. MCP-1 preparation according to claim 11, wherein the pyroGlu-MCP-1 is present in non-glycosylated form. Case 1-1492-FF 29 Boehringer Ingelheim International GmbH et al. 13. Process for inducing a biological or physiological reaction which substitutes for or potentiates the biological or physiological activity of endogenous native MCP-1-protein, characterised in that a composition 5 according to claim 5 or a pharmaceutical composition according to claim 7 or a preparation according to at least one of Claims 10 to 12 is added to cells or tissues or organs in an amount which is suitable for evoking the biological or physiological activity. 10 14. Process according to claim 13, wherein the cells or tissues or organs comprise CCR-2 and/or CCR-4-receptors. 15. Process according to claim 14, wherein the cells are mammalian cells which natively or recombinantly express the MCP-1 receptor subtype 15 CCR2, particularly CCR2b. The invention relates to a method for producing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1. According to the inventive method, Gln-MCP-1 is incubated in a buffer solution having a salt concentration ranging between 10 mM and 160 mM and a pH value ranging between 2 and 7.5 at a temperature ranging between 30 DEG C and 80 DEG C until at least 90 percent of the MCP-1 are provided in the form of pyroGlu-MCP-1.

Full Text

Boehringer Ingelheim International GmbH
Method for the production of an N-terminally modified
chemotactic factor
5The invention relates to a process for preparing pyroGlu-MCP-1 from
recombinantly produced Gln-MCP-1 and compositions containing pyroGlu-
MCP-1 preparations.
MCP-1 (monocyte chemoattractant protein-1; also known by the names:
l0monocyte chemotactic and activating factor 'MCAF', macrophage chemotactic
factor 'MCF' tumour necrosis factor stimulated gene-8 'TSG-8', 'HC-11',
smooth muscle cell chemotactic factor 'SMC-CF', lymphocyte derived
chemotactic factor 'LDCF' as well as glioma derived chemotactic factor
'GDCF') is a member of the CC-chemokine family. Human MCP-1 protein was
15originally described in US Patent No. 5,714,578. It is synthesised under
natural conditions in the body (natively) as a precursor protein 99 amino acids
long, which is then processed to form a peptide with 76 amino acid groups.
Mature human MCP-1 (hMCP-1) is a glycoprotein with a molecular weight of
14 kDa and is secreted by many types of cells, e.g. smooth vascular muscle
20cells and endothelial cells (Leonard and Yoshimura (1990), Immunology
Today 11, 97 -101). It contains two intramolecular disulphide bridges and is
Oglycosylated and sialylated when expressed natively (J. Yan Ling et al.
(1990), Journal of Biological Chemistry, 265, 18318 -18321).
25The protein is a product of the JE gene of the chromosomal location 17q11.2-
q21.1. This gene locus is also known as SCY A2 (small inducible cytokine
A2). The human gene was first described in US Patent No. 5,212,073. The
expression of this gene may be induced by a number of cytokines, such as
e.g. tumour necrosis factor alpha, but also by immunoglobulin G, for example.
30The gene sequence and further information on the gene and the gene
product are available in the NCBI Data bank under Accession Number
M37719 (see Table 1).

Case 1-1492-FF 2 Boehringer Ingelheim International GmbH et al.
5
LOCUS HUMMCHEMP 2776 bp DNA linear PRI 13-MAY-1994
DEFINITION Human monocyte chemotactic protein gene, complete cds.
ACCESSION M37719
VERSION M37719.1 Gl:187447
5KEYWORDS monocyte chemotactic protein.
SOURCE Homo sapiens (human)
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo,
PREFERENCE 1 (bases 1 to 2776)
AUTHORS Shyy, Y.J., Li, Y.S. and Kolattukudy, P.E.
TITLE Structure of human monocyte chemotactic protein gene and its
regulation by TPA
JOURNAL Biochem. Biophys. Res. Commun. 169 (2), 346-351 (1990)
15MEDLINE 90290466
COMMENT Original source text: Human DNA.
FEATURES Location/Qualifiers
source 1..2776
/organism="homo sapiens"
20 /db_xref="taxon:9606"
gene 598..2080
/gene="SCYA2"
CDS join(598..673,1472..1589,1975..2080)
/gene="SCYA2"
25 /note="monocyte chemotactic protein"
/codon_start=1
/protein_id="AAA18102.1"
/db_xref="GI:487124"
translation= "MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLA
30 SYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT"
exon /gene="SCYA2"
/note="monocyte chemotactic protein"
/number=1
35 exon 598..673
/gene="SCYA2"
/note="monocyte chemotactic protein"
/number=1
intron 674..1471
40 /gene="SCYA2"
/note="monocyte chemotactic protein intron A"
exon 1472.. 1589

Case 1-1492-FF 3 Boehringer Ingelheim International GmbH et al.
/gene="SCYA2"
/note="monocyte chemotactic protein"
/numbeF=2
intron 1590..1974
5 /gene="SCYA2"
/note="monocyte chemotactic protein intron B"
exon 1975..2080
/gene="SCYA2"
/note="monocyte chemotactic protein"
10 /number=3
exon 1975..>2080
/gene="SCYA2"
/note="monocyte chemotactic protein"
/number=3
15BASE COUNT 700 a 727 c 565 g 781 t 3 others
ORIGIN
1 cagttcaatg tttacacaat cctacagttc tgctaggctt ctatgatgct actattctgc
61 atttgaatga gcaaatggat ttaatgcatt gtcagggagc cggccaaagc ttgagagctc
121 cttcctggct gggaggcccc ttggaatgtg gcctgaaggt aagctggcag cgagcctgac
20181 atgctttcat ctagtttcct cgcttccttc cttttcctgc agttttcgct tcagagaaag
241 cagaatcctt aaaaataacc ctcttagttc acatctgtgg tcagtctggg cttaatggca
301 ccccatcctc cccatttgcg tcatttggtc tcagcagtga atggaaaaaa gtgctcgtcc
361 tcacccccct gcttcccttt cctacttcct ggaaatccac aggatgctgc atttgctcag
421 cagatttaac agcccactta tcactcatgg aagatccctc ctcctgcttg actccgccct
25481 ctctccctct gcccgctttc aataagaggc agagacagca gccagaggaa ccgagaggct
541 gagactaacc cagaaacatc caattctcaa actgaagctc gcactctcgc ctccagcatg
601 aaagtctctg ccgcccttct gtgcctgctg ctcatagcag ccaccttcat tccccaaggg
661 ctcgctcagc caggtaaggc cccctcttct tctccttgaa ccacattgtc ttctctctga
721 gttatcatgg accatccaag cagacgtggt acccacagtc ttgctttaac gctacttttc
30781 caagataagg tgactcagaa aaggacaagg ggtgagcccc aaccacacag ctgctgctcg
841 gcagagcctg aactagaatt ccagctgtga acccaaatcc agctccttcc aggattcagg
901 atccagctct gggaacacac tcagcagtta ctcccccagc tgcttccagc agagtttggg
961 gatcagggta atcaaagaga agggtgggtg tgtaggctgt ttccagacac gctggagacc
1021 cagaatctgg tctgtgcttc attcacctta gcttccagag accggtgact ctgcaggtaa
351081 tgagtatcag ggaaactcat gaccaggcat agctattcag agtctaaaag gaggctcata
1141 gtggggctcc cagctgatct tccctggtgc tgatcatctg gattattggt ccgtcttaat
1201 gacacttgta ggcattatct agctttaaca gctcctcctt ctctctgtcc attatcaatg
1261 ttatataccc cattttacag cataggaaac tgagtcattg ggtcaaagat cacattctag
1321 ctctgaggta taggcagaag cactgggatt taatgagctc tttctcttct cctgcctgcc
401381 ttttgttttt tcctcatgac tcttttctgc tcttaagatc agaataatcc agttcatcct
1441 aaaatgcttt tctttgtggt ttattttcca gatgcaatca atgccccagt cacctgctgc
1501 tataacttca ccaataggaa gatctcagtg cagaggctcg cgagctatag aagaatcacc

Case 1-1492-FF 4 Boehringer Ingelheim International GmbH et al.
1561 agcagcaagt gtcccaaaga agctgtgatg tgagttcagc acaccaacct tccctggcct
1621 gaagttcttc cttgtggagc aagggacaag cctcataaac ctagagtcag agagtgcact
1681 atttaactta atgtacaaag gttcccaatg ggaaaactga ggcaccaagg gaaaaagtga
1741 accccaacat cactctccac ctgggtgcct attcagaaca ccccaatttc tttagcttga
51801 agtcaggatg gctccacctg gacacctata ggagcagttt gccctgggtt ccctccttcc
1861 acctgcgtcc tcctagtctc catggcagct cgcttttggt gcagaatggg ctgcacttct
1921 agaccaaaac tgcaaaggaa cttcatctaa ctctgtctcc tcccttcccc acagcttcaa
1981 gaccattgtg gccaaggaga tctgtgctga ccccaagcag aagtgggttc aggattccat
2041 ggaccacctg gacaagcaaa cccaaactcc gaagacttga acactcactc cacaacccaa
102101 gaatctgcag ctaacttatt ttcccctagc tttccccaga caccctgttt tattttatta
2161 taatgaattt tgtttgttga tgtgaaacat tatgccttaa gtaatgttaa ttcttattta
2221 agttattgat gttttaagtt tatctttcat ggtactagtg ttttttagat acagagactt
2281 ggggaaattg cttttcctct tgaaccacag ttctacccct gggatgtttt gagggtcttt
2341 gcaagaatca ttaatacaaa gaattttttt taacattcca atgcattgct aaaatattat
152401 tgtggaaatg aatattttgt aactattaca ccaaataaat atatttttgt acaaaacctg
2461 acttccagtg ttttcttgaa ggaaattaca aagctgagag tatgagcttg gtggtgacaa
2521 aggaacatga tttcagaggg tggggcttac attttgaagg aatgggaaag tggattggcc
2581 cnntntcttc ctccactggg tggtctcctc tgagtctccg gtagaagaat ctttatggca
2641 ggccagttag gcattaaagc accacccttc cagtcttcaa cataagcagc ccagagtcca
202701 atgaccctgg tcacccattt gcaagagccc acccccattt cttttgctct cacgaccctg
2761 accctgcatg caattt
25lt has already been explained in the above-mentioned US 5,714,578 that in
the case of an MCP-1 protein isolated from a native source the N terminus is
blocked. Only later was it discovered that this is due to a post-translational
modification in which the glutamine exposed after the cleaving of the leader
sequence at the N terminus of the mature protein is cyclised, losing an NH3
30molecule, to form a pyroglutamate group.
The identification of the open reading frame coding for MCP-1 in US
5,212,073 allowed recombinant expression of the protein. An MCP-1
produced by the recombinant method, e.g. in E. coli, does not have the
35typical N- or O-glycosylation pattern of the native MCP-1. An MCP-1
preparation prepared in this way also does not oppose Edman decomposition
in the same way as a preparation obtained from native material, i.e. it
contains unblocked N termini (glutamines). In cellular assays based on the

10Case 1-1492-FF 5 Boehringer Ingelheim International GmbH et al.
chemotactic effect on macrophages, originally no difference could be found
between the biological activity of native MCP-1 preparations and those
obtained by the recombinant method.
5Under physiological conditions MCP-1 acts as an agonist to the beta-
chemokine receptors CCR2 and CCR4, both of which are expressed mainly
on monocytes and are also found both on basophiles and on T- and B-
lymphocytes. MCP-1 induces monocyte chemotaxis even at subnanomolar
concentrations. The receptors CCR2 and CCR4 are G-protein-coupled seven-
lOtransmembrane domain receptors which lead to the activation of monocytes
and increased adhesion of integrins. This process ultimately results in the
docking of monocytes to endothelial cells and the subsequent departure of
the monocytes from the vascular system.
15WO 98/44953 discloses the influence of MCP-1 on arteriogenesis, i.e. the
growth of collateral arteries and/or other arteries from existing arteriolar
connections. On the basis of this finding it was proposed in the above-
mentioned International Patent Application to use MCP-1 for therapeutic
purposes, namely for the treatment of vascular occlusive diseases which
20may be alleviated by stimulating the formation of new blood vessels. Such
vascular occlusive diseases are particularly coronary artery disease (CAD),
peripheral arterial occlusive disease (PAOD), cerebral and mesenterial
arterial occlusive diseases, etc. The application also proposed the use of
MCP-1-neutralising agents such as e.g. anti-MCP-1-antibody for preventing
25new vascular formation in order to combat tumour growth, in particular, which
is reliant on a sufficient blood supply and hence adequate vascularisation of
the tumour tissue.
In order to be able to use the MCP-1 protein as explained above in a
therapeutic approach for promoting the vascularisation of tissue, large
30enough quantities of this protein have to be made available with sufficient
purity for pharmaceutical purposes. Human MCP-1 was originally obtained in
native form from cultures of the human glioma cell line U105MG or from
human mononuciear leukocytes of peripheral blood. Protein intended for
therapeutic purposes cannot be isolated from these sources because the

Case 1 -1492-FF 6 Boehringer Ingelheim International GmbH et al.
production of large enough amounts would only be possible at unacceptably
high cost in terms of labour and/or materials: it would not be possible to
obtain human leukocytes in the required quantity. The glioma cells could
indeed be replicated in virtually unrestricted amounts, but are unsuitable for
5cultivation in biotechnological fermenters and additionally require foetal calf
serum for their cultivation, for example, with all the attendant problems.
Instead of the native expression of MCP-1 it is therefore an inviting prospect
to prepare MCP-1 by the recombinant method. In fact recombinant human
10MCP-1 is already commercially available, particularly from Messrs R&D
Systems (Catalogue no. 279-MC) and Peprotech (Catalogue no. 300-04; see
Table 2; the catalogue numbers quoted are those applicable in January
2003). According to the product specification of the commercially available
recombinant MCP-1 preparations the MCP-1 protein present therein contains
15the amino acid glutamine (Q) at the N terminus. The product description also
indicates that this protein preparation is highly sensitive, particularly in the
reconstituted liquid form (recommended max storage : 1 week at 4°C). In fact,
experiments by the inventor (internal prior art) showed that when the
conventional MCP-1 protein preparations are stored in dissolved form the -
20biologically active - protein preparation appears to show signs of
contamination by breakdown products very rapidly, particularly at
temperatures above 4 °C (appearance of secondary bands in analytical HPLC
chromatographs).
Table 2 (extract from the Internet Website of Messrs Peprotech
25 http://www.peprotech.com/content/details.htm?results=1&prod=
Recombinant Human MCAF (Human MCP-1)

Case 1-1492-FF 7 Boehringer Ingelheim International GmbH et al.
15
30A conventional method of recombinantly producing human MCP-1 protein is
by the temperature-induced expression of the protein as a fusion protein,
purification of the inclusion bodies formed subsequently, dissolving and
refolding of the protein and subsequent enzymatic release of the hMCP-1
protein from the fusion protein.
35
In conventional processes for the recombinant preparation of human MCP-1
protein the problem arises that a protein which is N-terminally shortened by
one or more amino acids is often obtained as a by-product. These by-
products may behave as antagonists to MCP-1 receptors in biological
40systems.
In Van Coiliie et al. (1998), Biochemistry 37: 12672, 12673 a.E. a process is
described in connection with experiments on the influence of N-terminal
modifications on the biological activity of MCP-2, wherein an MCP-2
45preparation obtained by the recombinant method is incubated in 0.01 M
Na2HPO4, pH 8.0, for 24 hours at 37 °C. MCP-2 has 62% homology with
MCP-1 at the level of the amino acid sequence and also differs from it to the
extent that N-terminally unmodified MCP-1 is biologically active, unlike MCP-2
(cf. the line "Biological Activity" in Table 2). The authors did not determine the
50extent of the conversion of the N-terminal glutamine groups into a

Case 1-1492-FF 8 Boehringer Ingelheim International GmbH et al.
pyroglutamate group which takes place under the conditions mentioned
above. The inventors have found (internal prior art) that the application of
comparable conditions to MCP-1 in any case leads to only partial cyclisation
of the N-terminal amino acid (cf. Table 3, Comparison test 2).
5
One aim of the invention therefore, in the light of the foregoing discussion, is
to provide a process by which an MCP-1-preparation (MCP-1 composition)
may be prepared, which (a) is suitable for therapeutic purposes in view of its
biological activity and (b) has advantages in terms of the drug licensing
lOprocedures, which in the case of biopharmaceuticals contain requirements
which are extremely difficult to comply with, e.g. with regard to the purity and
reproducibility of production of the pharmaceutical composition, and in
particular (c) has an excellent shelf life. Another related aim is to provide
compositions or preparations which contain MCP-1 produced by the
15recombinant method and are suitable for the purposes mentioned above or
have the aforementioned advantages.
These aims are achieved by the processes and compositions containing
MCP-1 recited in the claims.
20
Thus according to the invention a process for preparing pyroGlu-MCP-1 from
recombinantly produced Gln-MCP-1 is provided wherein Gln-MCP-1 is
incubated at a temperature in the range from 30°C and 80°C, preferably in
the range from 30°C and 70°C and more preferably in the range from 35 °C
25to 60°C, in a buffer solution which has a salt concentration in the range from
10 mM to 160 mM, preferably in the range from 10 mM to 100 mM, and which
has a pH in the range from 2 to 7.5, preferably 3.5 to 7.5, more preferably 3.5
to 6.5, more preferably 5 to 6.5, more preferably 5.5 to 6.5 and more
preferably has a pH of about 6. The incubation is carried out until at least
3090%, preferably at least 95 % and more preferably at least 96 % of the MCP-
1 contained in the incubating buffer solution is present in the form of the
pyroGlu-MCP-1.

Case I-I492-FF 9 Boehringer Ingelheim International GmbH et al.
By "MCP-1" is meant, within the scope of this disclosure, the MCP-1 protein
(without preprosequence), with an N-terminai glutamine group ("Gln-MCP-1";
cf. the amino acid sequence shown in Table 1 and Table 2) or with an N
terminus which has already been converted/cyclised into the pyroglutamate
5group ("pyroGlu-MCP-1"), depending on the context. It is clear, however,
that modifications of the MCP-1-protein which do not affect its biological
function (esp. the monocyte-attractant activity or the effect on the CCR
receptor) and do not alter its structure so that the reaction parameters
described above no longer produce the desired result (i.e. the protein can no
l0longer be converted into a pyroGlu-Variant by the process according to the
invention), do not depart from the scope of protection. Thus, the process
according to the invention is naturally also applicable to an MCP-1 in which a
conservative amino acid exchange, e.g. serine to threonine or leucine to
isoleucine, has taken place, provided that this affects neither the biological
15function or activity of the resulting protein nor the convertibility of the N-
terminal glutamine into pyroglutamate according to the above process. The
process according to the invention can thus also be applied to MCP-1
proteins of other mammals such as e.g. rats, mice, guinea pigs, rabbits and
ferrets (and several others).
20
The buffer solution in which the process described above is carried out is
preferably a phosphate-buffered (sodium and/or potassium phosphate-
buffered) aqueous solution of low or physiological molarity, namely in the
range from 10 to 160 mM, 10 to 80 mM, 10 to 50 mM, 20 to 40 mM or around
2520 or 40 mM. If longer incubation times are accepted, according to one
particular embodiment of the invention the work may also be done at
physiological molarities, such as e.g. a saline concentration of around 150
mM, this molarity preferably being achieved by the use of a phosphate-
buffered saline solution or "PBS". The disadvantage of the longer incubation
30period - and the attendant risk of increasing amounts of breakdown,
secondary or oxidation products - is made up for in this particular
embodiment by the advantage of being able to obtain the protein preparation
straight away in the form of a solution with a physiological salt concentration.

20Case 1-1492-FF 10 Boehringer Ingelheim International GmbH et al.
The buffer solution in which the modification step is carried out may also
contain, for example, a (mild) detergent, an antioxidant, a preservative, a
complexing agent, stabilisers, antimicrobial reagents, etc.
5The incubation temperature is selected in the range from 30 °C to 80 °C, i.e.
above ambient temperature. At lower temperatures the conversion step
proceeds very slowly. In order to achieve virtually total conversion the
incubation period would have to be increased substantially to more than a
week. This would lead to stand times which are unacceptable in the
l0 biotechnological process and would also substantially increase the risk of
other forms of contamination of the protein solution (with breakdown or
oxidation products, bacteria, viruses or other pathogens). On the other hand,
although increasing the incubation temperature to above 80 °C does indeed
greatly speed up the reaction of conversion, it also results in an increased
15formation of undesirable by-products and breakdown products (cf. Examples
1 and 2). If stand times of up to 6 days are acceptable, an incubation
temperature of 35 to 40 °C is particularly preferred. In cases where incubation
should be significantly shorter if possible, the incubation may also be carried
out in the range from e.g. 50 to 60, 70 or 80 °C. Temperatures between 40
20and 50 °C will naturally also produce the desired results.
The pH of the incubating buffer solution should be in the range from 2 and
7.5, i.e. in the neutral to acidic range. It is preferably in the range from 3.5 to
7.5, more preferably in the range from 3.5 to 6.5, more preferably in the range
25from 5 to 6.5, more preferably in the range from 5.5 to 6.5 and more
preferably a pH of about 6 is selected.
With a suitable choice of the above parameters, e.g. as described in the
Examples, the pyroGlu-MCP-1 variant may be obtained with a purity of 90%
30in any case and possibly higher, e.g. 95%, 96% or 98%, this percentage
indicating the amount of pyroGlu-MCP-1 (determined e.g. as the area under
the curve in an HPLC elution profile) based on the amount of total MCP-1
present in the solution (i.e. including any remaining amount of Gln-MCP-1 and
possible oxidation products and other by-products or breakdown products).

Case I-1492-FF 11 Boehringer Ingelheim International GmbH et al.
According to another embodiment of the invention a process for preparing a
pyroGlu-MCP-1 preparation is provided which comprises at least the following
steps:
5- preparing a Gln-MCP-1 preparation by the recombinant method by
expression of a gene construct coding for MCP-1 in a host cell,
- optionally concentrating and/or purifying the Gln-MCP-1 contained in the
Gln-MCP-1 preparation, and finally
- converting the Gln-MCP-1 which is contained in the Gln-MCP-1 preparation
l0 or which was contained therein before the concentrating and/or purifying, into
a pyroGlu-MCP-1 preparation which contains the protein molecule species
pyroGlu-MCP-1, this step being carried out according to the "Process for
preparing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1" as
described above.
15This process may optionally also include, for example, buffering the pyroGlu-
MCP-1 preparation or further purification, e.g. by a subsequent step of
column chromatography, dialysis, ultrafiltration, etc.
Methods of producing recombinant proteins by biotechnology are known.
20They comprise, in particular, fermentation, purification, concentration, and
other steps. The step of conversion described above may be included at
various points in a "multi-step process", i.e. with an as yet largely unpurified or
wholly purified MCP-1 protein solution as the starting solution. It is also
conceivable, in particular, to have a process in which not yet (totally)
25converted Gln-MCP-1 in more, less or practically wholly purified form is
lyophilised in order to improve its shelf life in as yet unconverted form and is
in due course put back into solution and then converted into pyroGlu-MCP-1
according to the above process. Naturally, the protein solution obtained after
conversion into pyroGlu-MCP-1 may also be lyophilised.
30
The product of the process, namely the pyroGlu-MCP-1 preparation subjected
to N-terminal modification (conversion), contains according to the invention an
amount of pyroGlu-MCP-1 of at least 90 %, preferably at least 95 % and more

Case I-1492-FF 12 Boehringer Ingelheim International GmbH et al.
25
preferably at least 96 %, based on the total content of MCP-1 (converted plus
unconverted protein plus by-products formed by oxidation, for example).
The term (Gin- or pyroGlu-MCP-1-) preparation means that in the various
5stages of the process the MCP-1 protein is present in dissolved form in a
(buffer) solution and hence other components may be present in addition to
the MCP-1, i.e. in any case the ions of the buffer salt used and possibly also
other salts, antioxidants, stabilisers, antimicrobial reagents, detergents,
preservatives, complexing agents, etc.
10
According to a further aspect of the invention a composition is provided which
contains pyroGlu-MCP-1 or a pyroGlu-MCP-1 preparation obtained by the
processes described above, in which at least 90 % of the MCP-1 contained in
the composition (converted plus unconverted protein plus by-products; i.e. the
15"area under the curve" in an HPLC elution chromatograph, for example) are
present in the form of the pyroGIu-MCP-1.
Thus, the invention also relates to an MCPO-1 preparation produced by the
recombinant method particularly in prokaryotes and particularly preferably in
20E. coli, wherein at least 90 % of the MCP-1 protein is present as pyroGlu-
MCP-1. When prepared in common expression cells the protein will frequently
not exhibit the natural glycosyfation pattern - unlike in native production.
When prepared in prokaryotes such as e.g. E coli, in particular, the pyroGlu-
MCP-1 obtained or the pyroGlu-MCP-1 preparation obtained unlike the form
25which occurs in native expression in the human body will not be glycosylated
and/or sialylated and will thus differ from a native protein of this kind or from a
protein preparation obtained from a native source.
A composition of this kind according to the invention may be, in particular, a
30medicament or a pharmaceutical composition which contains, in addition to
an amount of pyroGlu-MCP-1 which makes up at least 90 %, 95% or 96% of
the total MCP-1 content (converted plus unconverted protein plus by-
products), conventional excipients and carriers, salts, antioxidants, stabilisers,
antimicrobial reagents, detergents, preservatives, complexing agents, etc.

Case 1-1492-FF 13 Boehringer Ingelheim International GmbH et al.
The composition according to the invention may be used to treat patients
suffering from an arterial occlusive disease such as in particular PAOD or
CAD. The treatment comprises administering such a composition in a
therapeutically effective amount by a suitable route, e.g. by intraarterial
5infusion or in the form of a periarterially deposited gel, from which the MCP-1
protein is released over a fairly long period.
Surprisingly it has also been found that, contrary to expectations, the
incubation of a recombinantly prepared MCP-1 protein preparation in
lOaqueous solution and at elevated temperature does not lead to its
decomposition and biological inactivation; on the contrary, with a suitable
choice of a number of parameters, this step, which is very unusual from a
biotechnological point of view and is usually inherently undesirable, results in
a surprisingly homogeneous, biologically fully active pyroGlu-MCP-1
15preparation, which retains its homogeneity even during lengthy storage, i.e. is
more stable than the Gln-MCP-1 (starting) preparation.
Bodies such as the Food and Drug Administration in the USA or the EMEA in
Europe make the granting of marketing approval for a drug dependent on
20meeting numerous conditions which are imposed on the drugs manufacturers
with the ultimate aim of protecting the patient. Thus, the manufacturer in
question has to supply proofs which demonstrate, for example, the purity of
the pharmaceutical product, its shelf life and its reproducibility of production.
These three aspects are only a small selection, but play a central role
25precisely in the licensing of biopharmaceuticals, i.e. drugs which contain
macromolecular active substances consisting of natural materials or derived
therefrom (proteins, nucleic acids, proteoglycans, polysaccharides, etc.).
Often, the requirements imposed on the (bio)pharmaceutical within the
framework of this approval are very difficult to meet. For example, active
30substances based on a protein have to be subjected to intensive and
complicated multi-stage purification after their production in a more or less
complex cellular organism and must not be uncontrollably altered in structure,
e.g. by oxidation or other spontaneous chemical reactions. Moreover, the
end product should have a reasonable shelf life so that there is no need for

Case 1-1492-FF 14 Boehringer Ingelheim International GmbH et al.
complicated and expensive supply networks between the manufacturer and
the prescribing doctor or patient or the clinic using the product - a
requirement which is generally only met with great difficulty, particularly in the
case of proteins, where it is important to maintain the correct secondary
5structure (folding).
In the case of the MCP-1 protein the quality of commercially obtainable
preparations is frequently adequate in many respects for in vitro testing, for
example. However, the inventors have found in the course of their work that
l0the same preparations are by no means suitable for producing a drug which
would be eligible for approval. Being left to stand in solution at ambient
temperature even for a short time before being administered to the patient,
which could not be ruled out in doctors' surgeries or hospitals, for example,
leads to the occurrence of "breakdown" products in the MCP-1 preparations
15which were originally viewed as contamination, in the experience of the
inventors. More intense purification of the Gln-MCP-1 originally provided as
active substance briefly restored a high level of purity, but even this
preparation was again unstable when briefly stored in solution and at ambient
temperature. Operating a drug approval procedure on the basis of such an
20unstable active substance preparation is beset by tremendous problems, if
not altogether hopeless.
Surprisingly, these problems can be overcome by the teaching of the
invention, wherein an unusual and at first sight highly counter-productive step,
25namely incubation at elevated temperature (which was previously seen to
positively promote inhomogeneity) was included in the actual preparation and
purification process. In fact, when carried out under the conditions analysed
in detail and perfected by the inventors, such incubation may bring about
virtually quantitative conversion of the original active substance Gln-MCP-1
30into the pyroGlu-MCP-1 form which was originally regarded as a contaminant.
This latter form is then astonishingly stable against protein-denaturing
influences such as incubation at elevated temperature and the like. Thus, an
MCP-1 preparation is obtained which, by virtue of its high purity and
homogeneity, exceptionally reproducible manufacture and stability on

30Case 1-1492-FF 15 Boehringer Ingelheim International GmbH et al.
storage, is suitable for use as a pharmaceutical active substance which has
good prospects of complying with the strict requirements in corresponding
licensing procedures referred to earlier, and thus makes it possible to
implement new therapeutic processes based on the activity of MCP-1.
5
According to a partial aspect of the invention a pyroGlu-MCP-1 preparation
prepared by the process described above and in the claims is used
- for the therapeutic treatment of vascular occlusive diseases such as, in
particular, coronary artery disease (CAD), peripheral arterial occlusive
lOdisease (PAOD), cerebral and mesenterial arterial occlusive diseases, or
- for preparing a pharmaceutical composition for the treatment of vascular
occlusive diseases such as, in particular, coronary artery disease (CAD),
peripheral arterial occlusive disease (PAOD), cerebral and mesenterial
arterial occlusive diseases.
15
The invention thus also makes it possible to carry out a process for treating a
patient suffering from the above-mentioned vascular occlusive diseases,
wherein the pyroGlu-MCP-1 preparation prepared by conversion or the
pharmaceutical composition prepared using a pyroGlu-MCP-1 preparation of
20this kind is administered by injection or infusion, for example.
With regard to the individual optimised process parameters it should also be
noted that the biotechnologist will not easily be convinced that he or she
should incubate a protein preparation fora fairly long period of, in some
25cases, several days at elevated temperatures of e.g. 60°C. Regarding the
neutral to acidic pH of the incubating solution which was previously disclosed
as optimum this is contrary to chemical logic: the conversion of glutamine to
cyclic pyroglutamate is a nucleophilic substitution reaction in which a pair of
free electrons acts on the attacking nitrogen atom of the C-alpha-amino group
30of the glutamate at the C atom of the C-gamma-amide group. An acidic pH
would inherently lead to increased protonation of the C-alpha-amino group
and would therefore have a negative effect on the speed of the reaction.
However, this is precisely not the case, according to the inventors' findings.
One possible explanation - which should not be interpreted restrictiveiy -

Case 1-1492-FF 16 Boehringer Ingelheim International GmbH et al.
might be that the folded MCP-1 protein forms a micro-environment for the N
terminus, in which in spite of the acidic pH of the surrounding aqueous
solution the amino group is present in unprotonated form, i.e. a pair of free
electrons is available for the nucleophilic attack.

Case 1-1492-FF 17 Boehringer Ingelheim International GmbH et al.
35
Examples
Example 1: Production of a pvroGlu-MCP-1 preparation by the process
Saccordina to the invention
Fermentation:
The fermentation was carried out with a strain of Escherichia coli K12
(W3110). The strain was transformed with a ColE1 plasmid (pBR322
lOderivative) containing the following elements: the genomic sequence of
hMCP-1 under the control of an all-purpose promoter (phosphatase, pPhoA),
a ColE1-replication origin and the resistance gene for tetracycline. For
fermentation the production strain was precultured in the shaking flask in LB
medium containing tetracycline. Incubation was carried out at 37°C until an
15OD of 1 was achieved. The preculture was transferred into the fermenter and
further cultivated with stirring and with a supply of air at 37°C. The medium
contained glucose, various salts, trace elements, yeast extract, amino acids
and tetracycline. The pH was maintained at 6.8 with ammonia. As soon as
the glucose put in had been used up the dissolved oxygen (pO2) was kept
20constant at the intended level of 40% by the supply of glucose. The induction
of the phosphatase promoter took place automatically as soon as the
phosphate in the medium had been exhausted. After 39 hours the biomass
was harvested with a tube centrifuge (CEPA) and stored at -70°C.
25Cell lysis and protein purification steps:
The frozen cell pellet was resuspended in four times as much lysis buffer
(200 mM Tris, 55 mM NaCI, 5mM EDTA, pH 7.5) using an Ultraturrax. The
cell lysis was carried out by two passages with a homogeniser at 460 bar. Cell
fragments were removed with a CEPA centrifuge. The supernatant was
30optionally filtered with Polysep II (1.2 rn) filters (Millipore), and then loaded
onto a column combination consisting of a Q sepharose FF and an SP
sepharose FF. The columns were equilibrated with lysis buffer.

Case 1-1492-FF 18 Boehringer Ingelheim International GmbH et al.
After the loading was complete the column combination was washed with
lysis buffer and the Q sepharose column was clamped off. The SP sepharose
was washed with lysis buffer, then with 5 M urea (in lysis buffer) and with lysis
buffer again. The product was eluted in a linear NaCI gradient.
5
The eluate was salted with ammonium sulphate to a final concentration of 1.3
mol/L and centrifuged for 15 min at 3200 g. The supernatant was loaded onto
a phenyl sepharose column which was equilibrated with 1.3 M ammonium
sulphate (in lysis buffer).
10
The flow of phenyl sepharose was applied to a Source 30 RPC column which
was equilibrated with lysis buffer. Washing was then carried out with lysis
buffer, water and buffer A (5% EtOH, 0,1% TFA). The product was eluted in
a linear gradient from 20% buffer B (95% EtOH, 0.1% TFA) to 70% buffer B
15in 10 column volumes.
Conversion step:
The eluate was diluted 1:10 in different buffers, e.g. phosphate buffer, 10 -
40 mM, pH 6.0 - 7.4. According to a preferred embodiment a value of
20between 6.0 and 6.5 was sought as the final pH of the conversion solution.
The protein concentration was between 0.2 and 1.0 mg/mL.
The solution was filter sterilised and incubated at temperatures of 35 - 80°C
(in different batches) with gentle agitation (60 rpm). The progress of the
reaction was monitored by HPLC analysis.
25
Final purification steps:
As soon as the end of the reaction was reached, the conversion solution was
loaded onto an SP sepharose HP column which was equilibrated in buffer A
(40 mM phosphate, pH 6.0). Elution was carried out with a linear NaCI
30gradient. The NaCI concentration in the eluate was about 350 mmol/L, and
the protein concentration was about 3 mg/mL. The eluate was diluted with
buffer A to a conductivity corresponding to 250 mM NaCI. Then it was further
diluted with 40 mM phosphate, pH 6.0, 250 mM NaCI until the protein

Case 1-1492-FF 19 Boehringer Ingelheim International GmbH et al.
concentration was 1 mg/mL The bulk solution thus formed was filter sterilised
and stored at4°C or-70°C until ready to be formulated.
Analysis:
5The progress of the conversion step discussed above was monitored by RP-
HPLC. Chromatographs of the elution profiles were taken before the start of
the conversion reaction (to) and at times t = 24 h, t= 48 h and t = 120 h. As
can be seen from the four chromatographs shown in Fig. 1, after 48 h approx.
90 % of the protein had been converted into pyroGlu-MCP-1 at an incubation
lOtemperature of 35 °C. HPLC analyses showed that the two disulphide
bridges had formed properly.
Fig. 2 shows the kinetics of the reaction of Gln-MCP-1 to pyroGlu-MCP-1
under the conditions described. It shows that as the duration of the reaction
15increases an impurity appears, which is referred to as "variant 2" in Fig. 2 and
not further characterised.
Similar reaction patterns were observed when carrying out the conversion
step in 0.1 M sodium acetate buffer, pH 5.5, or 0.1 M sodium citrate buffer,
20pH 3.5 (data not shown).
The reaction described above was repeated except that temperatures of 24°
C, 60 °C and 80 °C were used instead of a reaction temperature of 35°C. As
is clear from the data assembled in Table 3, no satisfactory yields of pyroGlu-
25MCP-1 were obtained at 24°C within a reasonable time. At 60°C and 80°C,
on the other hand, significantly higher reaction rates are observed. The
reaction rate at 80°C was approximately 50 times the rate at ambient
temperature.

40Case 1-1492-FF 20 Boehringer Ingelheim International GmbH et al.

n.d. = not determined, the end of the reaction had not been reached after the specified time
5Certainly, a higher reaction temperature favours the formation of by-products
or breakdown products, reducing the purity level - particularly when the test is
carried out at 80 °C (Table 3).
Fig. 3 shows in high resolution the chromatographs of the elution profile of
l0pyroGlu-MCP-1 preparations which were obtained under the conditions
summarised in Table 3 (20 mM phosphate buffer, 35°C, 60°C or 80°C). As
can be seen from this, significantly more impurities occur with MCP-1 by-
products or breakdown products at high temperatures, even with a short
incubation period (e.g. 3 hours at 80°C), than during five days' incubation at
1535°C.
The content of (e.g. 5 %) ethanol (EtOH) in certain embodiments of the
process resulting from the production method did not have a negative effect
on the reaction rates or purity of the product, under otherwise constant test
20conditions (20 mM phosphate buffer, pH 6.0).
Example 2: Production of a pyroGlu-MCP-1 preparation by an alternative
process according to the invention

Case 1-1492-FF 21 Boehringer Ingelheim International GmbH et al.
The fermentation, cell lysis and protein purification steps were carried out as
explained in Example 1.
5Conversion step:
The eluate of the Source 30 RPC was diluted 1:10 with PBS, 0.02% Tween
20. The protein concentration was then between about 0.2 and 0.5 mg/mL,
the pH was about 7.4. In addition to the buffer components the solution
contained about 3.5% EtOH and 0.01% TFA, which originated from the eluate
1 Oof the previous Source 30 RPC step. The solution was filter sterilised into
polypropylene flasks with a Millipak 20 (0.2 n, Millipore) and incubated at
35°C with gentle agitation. The progress of the reaction was monitored by
HPLC analysis. As soon as the end of the reaction was reached the
conversion solution was ultradiafiltered with a Pellikon 2 UDF membrane (3k,
15Millipore) against PBS, 0.02% Tween 20, and the protein concentration was
adjusted to 0.1 mg/mL. The preparation was decanted in 1mL batches into
glass containers through a Millipore GV filter (0.m) under sterile
conditions.
20Analysis:
The progress of the conversion step was monitored by RP-HPLC. As can be
seen in Fig. 4, after 5 days more than 90 % of the protein had been converted
into pyroGlu-MCP-1, while surprisingly in spite of the long incubation period a
very high purity of 95 % was obtained (Table 3). As is apparent from a
25comparison of mixtures containing 20 mM phosphate buffer and those
containing 20 mM phosphate buffer plus 150 mM salt (Example 1, Table 3,
line 3 to line 6), the relatively low speed constant in mixtures containing PBS
as incubation solution may be put down to the higher salt concentration, ionic
strength or osmolarity.
30
Example 3: Preparation of a pharmaceutical composition based on the MCP-
1 preparation produced according to Example 1 or Example 2

Case 1-1492-FF 22 Boehringer Ingelheim International GmbH et al.
45
The pyroGlu-MCP-1 preparation obtained in Example 1 or Example 2 was
diluted to a concentration of 0.1 mg/mL in PBS (sodium chloride, disodium
hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate;
pH 7.0), also containing 0.02 % Tween 20, and transferred into glass
5containers in volumes of 1 mL The finished pharmaceutical solution was
clear, colourless and odourless and can be administered by injection or
infusion.
Example 4: Comparison of the biological activity of pyroGlu-MCP-1 and Gln-
10MCP-1
Measuring principle:
MCP-1 binds to and activates the MCP-1 receptor CCR2b. The activation of
15the receptor leads to an influx of calcium into the cytosol. This can be
measured using a Fluorescence Imaging Plate Reader (FLIPR; Molecular
Devices). To do this, the inactive fluorescent dye ester Fluo-4 AM is sluiced
into cells which express hCCR2b on their surface, this ester then being
cleaved by intracellular esterases. In this form the fluorescent dye binds Ca2+
20ions. On excitation with a wavelength of 488 nm there is an emission with a
peak at 528 nm. The intensity of the emission is dependent on the
concentration of calcium in the cytosol. The change in intensity of the emitted
light thus correlates with the concentration of calcium in the cytosol which is in
turn dependent on the state of activation of the receptor.
25
A characteristic time-triggered measuring signal after the addition of MCP-1 is
shown in Fig. 5. It is apparent from this that after the addition of MCP-1 there
is a rapid release of calcium which is linked with a sharp rise in fluorescence.
The peak (b) is reached just a few seconds after the application (a). The
30interval between the application (a) and maximum stimulation (b) is roughly
20-30 sec.
In the tests that follow, the evaluation was carried out using the maximum
values as they are less affected by secondary effects such as e.g. a calcium-
35induced calcium influx, and therefore more precise measurement is possible.

Case 1-1492-FF 23 Boehringer Ingelheim International GmbH et al.
Preparation of stably expressing CHO/hCCR2B-K1 cells:
The coding region of the human CCR2b receptor (Gene bank Accession No:
5D29984) was amplified by PCR. Then the 1.08 kb BamHI-Xbal fragment was
cloned into an expression vector. CHO-K1 cells were transfected with this
plasmid acting as an expression vector.
The cells were cultivated in a culture medium based on Ham's F12 medium
lOand regularly passaged. On the day before the measurement 5000 cells
were plated out in 384-well assay plates (Corning Costar) with 40 pi of culture
medium and left to adhere overnight (approx. 24 h) at 37 °C, 5 % CO2, 95 %
relative humidity. In all the assays the measurements were carried out four
times.
15
On the day of the test, first of all the substance plates were prepared. Hanks
buffer with 0.1 % BSA (protease-free) was used as the diluting buffer for the
various MCP-1 preparations. Hanks buffer with 0.1 % BSA was used as the
blank control. Then 40 l / well of Fluo-4 dye medium were added to the cells
20and the preparations were incubated for 45 min. at 37 °C, 5 % CO2, 95%
relative humidity. The stained cells were then washed four times with 60 ul
washing buffer, leaving a residue of 25 l of washing buffer in each well. The
cells were then incubated with washing buffer for a further 5 min at ambient
temperature.
25
To measure the fluorescence the FLIPR measuring device was adjusted so
that stained and unstained wells differed by at least a factor 1:5 and the
stained wells had approx. 11,000 fluorescence counts. The other FLIPR
settings were:
30 Excitation: 488 nm
Emission: 510 - 570 nm (band pass)
Negative correction: Hanks/BSA (= comparison with blank
control)

Case 1-1492-FF 24 Boehringer Ingelheim International GmbH et al.
Bias subtraction: 6 (= levelling all the wells to 0 before adding the
substance)
Presoak of the tips: with 25 ul of the relevant substance solution from
the substance plate (= minimising an adhesion
5 artefact)
The measuring process comprised the following steps:
6 intervals in a 5 sec. cycle
15 ul substance added
10 60 intervals in a 1 sec. cycle
18 intervals in a 5 sec cycle
For evaluation the maximum signal of the 78 intervals after the addition of the
substance was used. The control mixtures used were:
15 blank control: for negative correction
positive control (ATP): for checking the staining
reference control (standard): -monitoring the receptor expression on
the cells
-basis of calculation for determining the
20 activity of unknown samples
The samples to be tested were pipetted parallel with the reference control in
identical dilution steps. The concentrations used in the tests were selected
so as to be in the EC50 region of the reference control.
25

50Case 1-1492-FF 25 Boehringer Ingelheim International GmbH et al.
Comparison of MCP-1 preparations according to the invention and not
according to the invention:
Two pyroGlu-MCP-1 preparations ("Batch 0711OOkh" and "Batch 0141921")
5prepared by the process according to the invention and an MCP-1
preparation prepared by the recombinant method, whose N-terminal
glutamine group had not been converted into a pyroglutamate group in the
process step according to the invention as described above (MCP-1
preparations obtainable from Peprotech) were tested and compared using the
lOtest system described previously. The EC50 was determined from the
measurement curves recorded as described above (see Fig. 6).
Virtually identical EC50 values were obtained for the two pyroGlu-MCP-1
preparations, while the values for the MCP-1 preparation not according to the
15invention and not N-terminally modified differed significantly (Fig. 6). The EC50
value of the latter preparation proved to be worse by a factor 2 to 3 (pyroGlu-
MCP-1: EC50 = 7.82 nM; MCP-1 preparation from Peprotech: EC50 = 20.76
nM).
2QThe biological activity of the two preparations was calculated as follows:
First, a positive correction is made to the pyroGlu MCP-1 at the concentration
1e-8 M, i.e. the value obtained at a concentration of 10 nM pyroGlu-MCP-1
was set at 100 %. This is in the almost linear part of the ascent of the curve.
25Using this percentage signal the activity of other preparations, such as e.g.
the Peprotech MCP-1 is calculated according to the formula
% activity (sample) = RFU(Sampleat 1e-8M) X 1 00 % / RFU(standard at 1e-8M)
30ln the above Example, as shown in Table 4, this yields a value of 48 %
activity for the non-N-terminally modified MCP-1 preparation based on the
pyroGlu-MCP-1 preparation.

Case 1-1492-FF 26 Boehringer Ingelheim International GmbH et al.

5Example 5: Temperature sensitivity of pyroGlu-MCP1
pyroGlu-MCP-1 was incubated for 1 or 2 hours at 56°C or 95°C. The
biological activity still present thereafter was then measured using the test
system in Example 4.
10
It was found that no denaturing occurs when a pyroGlu-MCP-1 preparation is
incubated at 56 °C for either one hour or two hours. However, as shown in
Fig. 7, incubation at 95°C results in a very marked denaturing effect.
15Example 6: Treatment of patients suffering from an arterial occlusive
disease:
For treating PAOD patients a pyroGlu-MCP-1 preparation as prepared above
is adjusted to a concentration of between 1.2 and 120 g/ml. immediately
20before use this solution is adjusted to a final concentration of between 0.1
and 10 g/ml and infused into the patient at a flow rate of 2 to 12 ml/min over
a period of 1 to 6 hours by intraarterial route close to the region affected by
the vascular occlusion. The infusion may be repeated after 1 to 7 days.
25

Case 1-1492-FF 27 Boehringer Ingelheim International GmbH et al.
55
Claims
1. Process for preparing pyroGlu-MCP-1 from recombinantly produced Gln-
MCP-1, wherein Gln-MCP-1 is incubated
5 - at a temperature in the range from 30°C and 80°C
- in a buffer solution, with
-- a salt concentration in the range from 10 mM to 160 mM and
- at a pH in the range from 2 to 7.5
until at least 90 % of the MCP-1 is present in the form of the pyroGlu-
10 MCP-1.
2. Process according to claim 1, wherein the buffer solution is a phosphate
buffer with a concentration in the range from 20 mM to 50 mM and with
a pH in the range from 3.5 to 6.5.
15
3. Process according to one of Claims 1 or 2, wherein the buffer solution
additionally contains a detergent, an antioxidant, a preservative, a
stabiliser, an antimicrobial reagent and/or a complexing agent.
204. Process for preparing a pyroGlu-MCP-1 preparation, comprising at least
the steps of
- preparing a Gln-MCP-1 preparation by expression of a gene construct
coding for MCP-1 in a host cell,
- optionally concentrating and/or purifying the Gln-MCP-1 contained in
25 the Gln-MCP-1 preparation,
- converting the Gln-MCP-1 of the Gln-MCP-1 preparation into a
pyroGlu-MCP-1 preparation which contains pyroGlu-MCP-1, according
to one of processes 1 to 3, and
- optionally buffering and/or further purifying the pyroGlu-MCP-1
30 preparation,
the proportion of pyroGlu-MCP-1 based on the total content of MCP-1 in
the resulting pyroGlu-MCP-1 preparation being at least 90%.

Case 1-1492-FF 28 Boehringer Ingelheim International GmbH et al.
5. Composition containing a pyroGlu-MCP-1 preparation prepared
according to claim 4, wherein at least 90 % of the MCP-1 contained in
the pyroGlu-MCP-1 preparation is present in the form of the pyroGlu- -?(
MCP-1.
5
6. Process for preparing a pharmaceutical composition containing pyroGlu-
MCP-1, wherein a pyroGlu-MCP-1 preparation prepared according to
claim 4 is used.
107. Medicament or pharmaceutical composition containing a pyroGlu-MCP-1
preparation prepared according to claim 4, wherein at least 90 % of the
MCP-1 contained therein is in the form of the pyroGlu-MCP-1.
8. Medicament or pharmaceutical composition according to claim 7,
15 containing pyroGlu-MCP-1 in a phosphate buffer with sodium chloride
and optionally a detergent as additives.
9. Use of pyroGlu-MCP-1 or a pyroGlu-MCP-1 preparation prepared
according to claim 4 for preparing a pharmaceutical composition for the
20 treatment of vascular occlusive diseases such as, in particular, coronary
artery disease (CAD), peripheral arterial occlusive disease (PAOD),
cerebral and mesenterial arterial occlusive diseases.
10. Recombinant MCP-1 preparation produced by the process according to
25 claim 4, the biological activity of which, with respect to a recombinantly
produced MCP-1 preparation which has not been subjected to a process
according to claim 1 (N-terminally unmodified MCP-1 preparation), is in
the ratio 100 : 48 or higher.
3011. Recombinantly produced MCP-1 preparation, wherein at least 90 % of
the MCP-1 protein is present as pyroGlu-MCP-1.
12. MCP-1 preparation according to claim 11, wherein the pyroGlu-MCP-1 is
present in non-glycosylated form.

Case 1-1492-FF 29 Boehringer Ingelheim International GmbH et al.
13. Process for inducing a biological or physiological reaction which
substitutes for or potentiates the biological or physiological activity of
endogenous native MCP-1-protein, characterised in that a composition
5 according to claim 5 or a pharmaceutical composition according to claim
7 or a preparation according to at least one of Claims 10 to 12 is added
to cells or tissues or organs in an amount which is suitable for evoking
the biological or physiological activity.
10 14. Process according to claim 13, wherein the cells or tissues or organs
comprise CCR-2 and/or CCR-4-receptors.
15. Process according to claim 14, wherein the cells are mammalian cells
which natively or recombinantly express the MCP-1 receptor subtype
15 CCR2, particularly CCR2b.

The invention relates to a method for producing pyroGlu-MCP-1 from recombinantly produced Gln-MCP-1. According to the inventive method, Gln-MCP-1 is incubated in a buffer solution having a salt concentration ranging between 10 mM and 160 mM and a pH value ranging between 2 and 7.5 at a temperature ranging between 30 DEG C and 80 DEG C until at least 90 percent of the MCP-1 are provided in the form of pyroGlu-MCP-1.

Documents:

02415-kolnp-2005-abstract.pdf

02415-kolnp-2005-claims.pdf

02415-kolnp-2005-description complete.pdf

02415-kolnp-2005-drawings.pdf

02415-kolnp-2005-form 1.pdf

02415-kolnp-2005-form 3.pdf

02415-kolnp-2005-form 5.pdf

02415-kolnp-2005-international publication.pdf

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Patent Number

235029

Indian Patent Application Number

2415/KOLNP/2005

PG Journal Number

26/2009

Publication Date

26-Jun-2009

Grant Date

24-Jun-2009

Date of Filing

29-Nov-2005

Name of Patentee

BOEHRINGER INGELHEIM INTERNATIONAL GMBH

Applicant Address

BINGER STRASSE 173, 55216 INGELHEIM

Inventors:

#	Inventor's Name	Inventor's Address
1	WANDL ROBERT	LANDSTRASSE HAUPTSTRASSE 107/101, A-1030 WIEN
2	NECINA ROMAN	SCHILLGASSE 32/18, A-1210 WINE
3	SEIDLER RANDOLPH	4 LESTER ROAD, SANDY HOOK, CT 06482,
4	LENTER MARTIN	PROMENADE 23, 89073 ULM
5	DOODS HENRI	FREIHERR-VON-KOENIG-STRASSE 6, 88447 WARTHAUSEN

PCT International Classification Number

C07K 14/52

PCT International Application Number

PCT/EP2004/003856

PCT International Filing date

2004-04-13

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	03010014.3	2003-05-02	EUROPEAN UNION