Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1992-12-01
1995-10-24
Fleisher, Mindy B.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
424 857, 530351, C07K 1456, C12P 2100, A61K 3821
Patent
active
054609569
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The human interferon alphas are a family of proteins comprising at least 24 subspecies, Zoon K.C., Interferon 9, 1-12 (1987), Gresser I., ed. Academic Press, New York. They were originally described as agents capable of inducing an antiviral state in cells but are known as pleiotropic lymphokines affecting many functions of the immune system, Opdenakker, et al., Experimentia 45, 513-520 (1989). Apart from their in vitro biological activities the human Interferon alphas are currently used for several important indications, e.g. hairy cell leukemia, Kaposi Sarcoma, veneral warts, and are being investigated for several others Intron A (interferon alpha-2b) Clinical status (1989) Proceedings from a satellite symposium at the 5.sup.th European Conference on Clinical Oncology, London, U.K. September 1989. The demand for highly purified and crystalline forms of interferon alpha, especially the recombinant type alpha-2b is of foremost importance for structure elucidation as well as for formulation of vadous dosage forms.
Two forms of crystalline human interferon alpha-2 have been reported. Miller et al., Science, 215, 689-690 (1982); Kung et al., U.S. Pat. No. 4,672,108; Weissmann, The Cloning of interferon and other Mistakes, In: Interferon 1981, Ion Gresser, ed., Academic Press, New York, 101-134; Weissmann, Phil. Trans. R. Soc. Lond., B299, 7-28 (1982); and Nagabhusban, et al., Characterization of Genetically Engineered Alpha-2 Interferon, In: Interferon: Research Clinical Application and Regulatory Consideration, Zoon, et al., Elsevier, New York 79-88 (1982). These publications describe methods for crystallizing interferon alpha-2 from polyethylene glycol at low temperature or from a phosphate buffer solution by adjusting the pH or temperature. These methods normally provide needle crystals which cannot be well characterized by X-ray diffraction techniques. The Miller et al. article also mentions crystalline interferon alpha-2 in a "prismatic form".
In general, the methods for crystallizing proteins such as interferon have been found to be unpredictable. For example, Kung et al., U.S. Pat. No. 4,672,108 (1987) specifically states in column 1, lines 52-64: proteins, however, no generalized procedure has been discovered, and many proteins remain uncrystallized. Thus, crystallization of proteins is an unpredictable art utilizing tdal and error procedures among many possible alternative methodologies. solution of a crystallizing agent, which is commonly a salt, such as ammonium sulfate or ammonium citrate or an organic solvent, such as ethanol or 2-ethyl-2,4-pentanediol. However, such procedures do not provide a suitable means for producing crystalline human leukocyte interferons." (Emphasis added.)
SUMMARY OF THE INVENTION
We have now surprisingly found that high quality crystalline interferon alpha-2 can be produced efficiently even at room temperature by a method which comprises equilibrating a solution of interferon alpha-2 against a sulfate salt solution that will cause the interferon alpha-2 solution to become more concentrated and form interferon alpha-2 crystals. Preferably, the equilibration is effected by means of ultrafiltration or dialysis, or using drops, e.g., by hanging or sandwiched droplets.
The solution of interferon alpha-2 preferably contains a sulfate salt, and this solution is preferably equilibrated against a more concentrated sulfate salt solution. The sulfate salt is preferably selected from an ammonium, calcium, cadmium, potassium, lithium, magnesium or sodium salt, more preferably it is ammonium sulfate. The sulfate salt is preferably present in the crystalline interferon alpha-2 solution at the time crystals begin to form in a concentration of from about 12% to about 30% saturated, more preferably in a concentration of from about 15% to about 25% saturated ammonium sulfate. As noted below, the concentration of sulfate salt at the start of the equilibration procedure will be lower, i.e., from about 2% to about 18% saturated.
Preferably, the interferon-alph
REFERENCES:
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patent: 4315852 (1982-02-01), Leibowitz et al.
patent: 4672108 (1987-06-01), Kung et al.
T. L. Nagabhushan, et al. Interferon: Research, Clinical Application & Regulatory Consideration, pp. 79-88. (1982).
David L. Miller, et al. Science 215, pp. 689-690, 5 Feb. 1982.
C. Weissmann, et al., Structure and expression of human IFN-a genes Phil. Trans. R. Soc. Lond. B299, 7-28 (1982).
Alexander McPherson, Preparation and Analysis of Protein Crystals. John Wiley & Sons, pp. 102-104. (1982).
Sydney Pestka, et al., Interferons and Their Actions. Ann. Rev. Biochem. 1987. 56:727-77.
Michael Steuli, et al, At Least Three Human Type a Interferons: Structure of a2., Science 209, 19 Sep. 1980.
Senadhi Vijay-Kumar, et al. Crystallization and Preliminary X-ray Investigation of a Recombinant Form of Human .alpha.-Interferon, pp. 4804-4805, (1987).
K. Henco, et al. Structure Relationship of Human Interferon Alpha Genes and Pseudogenes, J. Mol. Bio. (1985) 185, 227-260.
Interferon Y Biotechnologia vol. 5, No. 3 Sep. 1988, Havana-Cuba pp. 281-287, A. Diaz: La Cristalizacion del Interferon Alpha-2 Recombinante Humano.
Hammond Gerald S.
Le Hung V.
Nagabhushan Tattanahalli L.
Reichert Paul
Trotta Paul P.
Carter Philip W.
Dulak Norman C.
Fleisher Mindy B.
Lunn Paul G.
Nelson James R.
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