Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-05-09
2003-06-03
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C435S069100, C435S069500, C435S071100, C514S002600, C530S412000, C530S350000, C530S351000
Reexamination Certificate
active
06573366
ABSTRACT:
The present invention refers to a new purification process of the human Interleukin-1 receptor-antagonist (IL-1ra) obtained by fermenting a strain of recombinant
E. coli.
As known, the members of the interieukin-1 (IL-1) family are important mediators of inflammatory, and immune responses. Among these Interleukin-1&agr; and &bgr; (IL-1&agr; and IL-1&bgr;) behave as agonists on the IL-1 receptors, being responsible for the stimulation of a number of cellular activities related to inflammatory and immune responses.
IL-1ra (the third member of the IL-1 family) is a protein that, like IL-1&agr; and IL-1&bgr;, binds both the IL-1receptor type I (IL-1R type I) on the T-cells (see Cannon J. H. et al., J. Infect. Dis., 1990, 161, 79 and Hannum C. H. et al., Nature, 1990, 343, 336) and the IL-1R type II on polymorphonuclear leukocytes and and B-cells (see Grarowitz E. V. et al., J. Biol. Chem., 991, 266, 14147 and Dripps D. J. et al., J. Biol. Chem., 991, 266, 20311). Nevertheless, differently from IL-1&agr; and IL-1&bgr;, IL-1ra acts as a pure antagonist, blocking the binding of the IL-1&agr; and IL-1&bgr; proteins to the said receptor and thereby providing a relevant control of on the whole IL-1 system.
Although extremely important for mediating the immunological response against pathogens, overproduction of IL-1(i.e. IL-1&agr; and IL-1&bgr;) may in some cases lead to undesired pathogenic conditions. This is, for instance, the case of septic shock, graft versus host disease or autoimmune diseases, such as rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis and certain types of leukemia.
As showed by a number of authors, the inhibition of the IL-1 activity by IL-1ra in said pathogenic conditions could have therapeutic effects (see for instance, Dinarello C. A., Adv. Pharmacol., 1994, 25, p. 21 and Dower K. S. et al., Therap. Immunol., 1994, 1, p. 113). It appears however that relatively high amounts of protein are required in order to achieve significant clinical results (Fisher C. J. et al., JAMA, 1994, 271, 1836).
Because of the evident difficulties of providing high amounts of the human IL-1ra for further studies, the preparation of recombinant IL-1ra proteins is highly desirable.
The IL-1ra protein produced by recombinant
E. coli
according to the present fermentation process is an unglycosylated protein of about 17.5 kDa which has an affinity for the IL-1R type I similar to the one of the natural occurring glycosylated protein (Molecular weight of about 22 kDa), as disclosed by Schreuder et al., Eur. Jour. Biochem., 227, 838-847, (1995). Furthermore, it has been shown that said recombinant IL-1ra inhibits a variety of IL-1 dependent processes both in vitro (see Eisenberg S. P. et al., Nature, 1990, 343, 341; Arend W. P. et al., J. Clin. Invest., 1990, 85, 1694 and McIntyre K. W. et al., J. Exp. Med., 1991, 73, 931) and in vivo (see Rambaldi A. Et al., Blood, 1990, 76, 114; Cominelli F. et al., J. Clin. Invest., 1991, 86, 972 and Alexander H. R. et al., J. Exp. Med., 1991, 173, 1029).
The expression of the recombinant IL-1ra and its purification from the fermentation broth are disclosed by Schreuder et al., Eur. Jour. Biochem., 227, 839-847 (1995). Briefly, a IL-1ra producing recombinant Escherichia coli is prepared by inserting the human IL-1ra gene (from BBL—British Biotechnology Labs) into the TAC-Bsp vector by cutting the BBL IL-1ra vector with HindIII and BamHI, isolating the HindIII-BamHI fragment and inserting this latter into a BspMI/BamHI-digested TAC-Bsp vector with the addition of two linker oligonucleotides to bridge the restriction site and to put the gene in frame.
The so obtained recombinant
E. coli
is grown overnight at 37° C. in Luria broth containing 50 &mgr;g/ml of ampicillin; bacterial cells are then harvested by centrifugation and stored at −80° C. until use.
According to the recovery procedure disclosed in the above document, bacterial cells are then lysed by sonication, centrifuged and clarified to remove cell debris.
For purification purposes, the clarified suspension is first passed through a hydrophobic interaction matrix (Phenyl-sepharose Fast-Flow, Pharmacia) using a salt-gradient elution; fractions containing the IL-1ra are pooled, dialyzed and passed through an anionic matrix (MonoQ with FPLC system, Pharmacia), then ammonium sulfate is added to the fractions containing the IL-1ra which are again passed through the hydrophobic interaction matrix and finally purified by gel filtration.
According to other purification, procedures, the recombinant IL-1ra protein contained in the clarified solution may also be purified by using two anionic exchange matrices and a size exclusion chromatography, as disclosed by D. B. Carter et al., Nature, 344 (19901), pp. 633-638, or by employing an anionic exchange chromatography, an hydrophobic interaction chromatography and a size exclusion chromatography, as disclosed by O. M. P. Singh et al., Spec. Publ.—R. Soc. Chem. (1994), 158 (Separations for Biotechnology 3), pp. 474-481.
It has now been found that the purification of IL-1ra protein can be easily achieved by using a simple two-step purification process which employs a cation exchange matrix and an anion exchange matrix.
In particular, the first elution is achieved by increasing the pH of the eluent with respect to the one of the loading mixture. The eluates containing the IL-1ra protein from the first chromatographic column are then directly loaded onto the second chromatographic column, without any intermediate operation such as diafiltration or dialysis; the second elution is then achieved by increasing the ionic strength of the eluent with respect to the one of the mixture eluted from the first chromatographic step.
As the skilled man will appreciate, the present purification method can be applied, using a conventional chromatographic system, to the clarified suspension obtained by the fermentation process described above, after treatment of the recombinant
E. coli
fermentation mass.
However, according to a preferred embodiment, the first purification step onto the cationic exchange matrix is preferably performed under the conditions of the so-called “expanded bed adsorption technique”. This technique, based upon fluidization, relies on the use of particular columns and ion exchangers, which allow to feed the column with a crude feed, without the need of preliminary operations for cell debris removal, such as concentration and/or clarification. Whilst the desired protein is adsorbed onto the matrix, the cell debris passes through the column unhindered and is then discarded (together with the unbound proteins). Accordingly, the homogenate obtained after cell disruption does not need to be clarified and filtered, as disclosed in the above prior art documents for eliminating the cell debris, but may directly be loaded onto the first cationic exchange matrix (see Hjorth et al., Bioseparation, 5(4), 217-223, 1995).
By coupling the expanded bed adsorption technique with the improved two-step chromatographic purification, a new simple procedure for the purification of IL-1ra from recombinant
E. coli
is thus provided; as a further advantage, in view of the low number of operations, the present purification process is also easily scalable for an industrial production of the IT-1ra recombinant protein.
As a general procedure, the cationic exchange matrix is first swallen by addition, of a suitable aqueous buffered solution (buffer A) having a pH lower than 6.2, preferably from about 5.0 to about 6.2, particularly preferred being a pH of about 6.0. For instance, buffered solutions containing MES (4-morpholineethanesulfonic acid), BIS-TRIS (2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propandiol) or citrate may be employed. If desired, further compounds may be added to the buffering solution, for instance protease inhibitors such as EDTA (ethylendiamino-tetraacetic acid) or PMSF (benzenemethanesulfonyl fluoride). A suitable buffering solution may be prepared, for instance, with 20 mM MES and 1 mM EDTA (pH about 6.0). The colum
Sarubbi Edoardo Giacomo
Soffientini Adolfo
Zanette Dino
Brannock Michael
Eyler Yvonne
Gruppo Lepetit S.p.A.
Wang George G.
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