Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1998-06-11
2000-09-05
Kemmerer, Elizabeth
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530414, 530416, 530418, 435 701, 435 711, C07K 114, C07K 116, C07K 118, C07K 1400
Patent
active
061145106
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for the purification of recombinant human interleukin-8 that has been obtained in various ways.
SUMMARY OF THE INVENTION
Processes for the purification of recombinant human interleukin-8 are already known. Various study groups have developed different purification processes. The purification differs essentially in the chromatography steps chosen, the following methods having been described: Mono S with subsequent reversed phase HPLC (Lindley, I.; Aschauer, H.; Seifert, J.-M.; Lam, C.; Brunowski, W.; Kownatzki, E.; Thelen, M.; Peveri, P.; Dewald, B.; von Tscharner, V.; Walz, A.; Baggiolini, M. (1988): Synthesis and expression in E. coli of the gene encoding monocyte-derived neutrophil-activating factor: Biological equivalence between natural and recombinant neutrophil-activating factor. Proceedings of the National Academy of Science, USA, 85), DEAE Sephacel throughflow for heparin Sepharose followed by Sephacryl S-200 and CM-3SW column (Matsushima, K.; Oppenheimer, J. J. (1989): Interleukin-8 and MCAF: novel inflammatory cytokines inducible by IL 1 and TNF. Cytokine 1), CM-Sepharose CL-6B with subsequent Toyopearl HW-55 (Furuta, R.; Yamagishi, J.; Kotani, H.; Sakamoto, F.; Fukui, T.; Matsui, Y.; Sohmura, Y.; Yamada, M.; Yoshimura, T.: Larsen, C. G.; Oppenheim, J. J.; Matsushima, K. (1989): Production and characterization of recombinant human neutrophil chemotactic factor. Journal of Biochemistry, Vol. 106, No. 3) and batch adsorption with silicate acid followed by heparin Sepharose CL-6B and Utrogel AcA 54 (Van Damme, J.; Van Beeumen, J.; Conings, R.; Decock, B.; Billau, A. (1989): Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of .beta.-thromboglobulin, European Journal of Biochemistry 181). Downstream processing for the production of interleukin-8 under GMP conditions on a commercial scale is not known.
Disadvantages of the known processes are low yield and the lack of purity of interleukin-8 with, at the same time, high material costs. A further disadvantage of the known processes is their use only for research purposes, that it to say their inability to be translated to production methods conforming to GMP.
A process for the purification of recombinant interleukin-8 has now been developed that overcomes the disadvantages of the known processes.
The process of the invention for the purification of interleukin-8 is characterised in that by repeated cross-flow ultrafiltration in such a manner that accompanying components and high-resolution purification by means of cation-exchange chromatography, wherein the highly positively charged surface of the interleukin-8 molecule is used in a form such that the application and elution pH value is selected to be sufficiently high that the interleukin-8 can just still bind, then is exchanged by gel filtration, dialysis or ultrafiltration and finally
The cell lysis according to process step a) is necessary only in the case of intracellularly formed protein, with enzymatic (for example by means of lysozyme), chemical (for example by means of organic solvents) and other physical methods of cells lysis (for example by means of ultrasound) also being possible. For commercial cell lysis physical methods are generally preferred (Schewedes and Bunge, 1988 Mechanische Zellaufschlu.beta.verfahren, Jahrbuch der Biotechnologie, VCH Verlagsgesellschaft mhB, Weinheim, Germany). Of the physical methods, apart from the ball mill, especially high-pressure homogenisation (Agerkvist and Enfors, 1990, Sauer et al., 1988) may be mentioned as an established and well described method for cell lysis.
The various cell lysis methods mentioned above may be used for the purification process of the invention.
Preferably, high-pressure homogenisation is chosen for process step a) of the process of the invention. Lysis of the cells is effected at a pressure of from 2000 to 15,000 psi and in from 1 to 6 cycles, preferably at a pressure of from 5000 to 7000 p
REFERENCES:
patent: 4599176 (1986-07-01), Wittenberger
patent: 5302384 (1994-04-01), Gimbrone, Jr. et al.
Furuta et al., J. Biochem., vol. 106, pp. 436-441 (1989).
Ricketts et al., American Chemical Society, Washington, vol. 271, pp. 21-49 (1985).
Van Damme et al., Journal of Experimental Medicine, vol. 167, No. 4, pp. 1364-1376 (1988).
Tutunjian, Biotechnology, vol. 3, pp. 615-626 (1985).
Boidol Werner
Daum Joachim
Donner Peter
Koltermann Andre
Scholz Peter
Basi Nirmal S.
Kemmerer Elizabeth
Schering Aktiengesellschaft
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