Natural human interleukin-2 and production thereof

Claim 1

Human interleukin 2A which has the following physico- chemical properties: (i) Molecular weight: 17 ± 1 kilodaltons (as determined by SDS-polyacrylamide gel electrophoresis), (ii) Amino acid composition: Contains the following amino acids and, in addition, Pro, 1/2 Cys, and Trp: (The amino acid content values given above are expressed in terms of mole percent based on the total number of amino acid residues except Pro, 1/2 Cys, and Trp.) (iii) Amino terminal amino acid sequence: The first and second amino acids from the amino terminal amino acid are from the 4th through 6th are 9th is Lys, and from the llth through 13th are and (iv) Isoelectric point: 7.2 ± 0.2 (as determined by isoelectric focusing).

Claim 15

A method of producing human IL-2A, B or a mixture thereof which comprises cultivating human cells capable of producing IL-2 in the presence of an inducer and thereby causing formation and accumulation of human IL-2A and B in the culture broth and subjecting the culture fluid to a series of steps, namely the steps of: (a) applying the culture fluid to a sulfopropyl-crosslinked dextran column, for adsorption, and performing elution with a buffer, (b) applying the eluate to a column packed with an anion exchange chromatographic carrier, for adsorption, and conducting elution with a salt solution, (c) subjecting the eluate to gel filtration on a polyacrylamide-agarose column, (d) applying the eluate to a DEAE-cellulose column, for adsorption, and carrying out elution of activity peaks with salt solutions, the'activity peak being eluted at a lower salt concentration to give IL-2A-containing solution and at a higher salt concentration to give IL-2B-containing solution, and then, when the IL-2A-containing solution is subjected to the following steps, IL-2A is obtained; when the IL-2B-containing solution is subjected to the following steps, IL-2B is obtained; and when both the IL-2A-containing solution and the IL-2-containing solution are subjected to the following steps, a mixture of IL-2A and B is obtained: (e) subjecting the eluate to the second gel filtration on a polyacrylamide-agarose column, and (f) subjecting the eluate to high performance liquid chromatography and performing elution with a solvent.

Claim 16

A method of producing human IL-2A, B or a mixture thereof which comprises cultivating human cells capable of producing IL-2 in the presence of an inducer and thereby causing formation and accumulation of human IL-2A and B in the culture broth and subjecting the culture fluid to a series of steps, namely the steps of: (a) applying the culture fluid to a sulfopropyl-crosslinked dextran column, for adsorption, and performing elution with a buffer, (b) applying the eluate to a column packed with an anion exchange chromatographic carrier, for adsorption, and conducting elution with a salt solution, (c) subjecting the eluate to gel filtration on a polyacrylamide-agarose column, (d) applying the eluate to a DEAE-cellulose column, for adsorption, and carrying out elution of activity peaks with salt solutions, the'activity peak being eluted at a lower salt concentration to give IL-2A-containing solution and at a higher salt concentration to give IL-2B-containing solution, and then, when the IL-2A-containing solution is subjected to the following steps, IL-2A is obtained; when the IL-2B-containing solution is subjected to the following steps, IL-2B is obtained; and when both the IL-2A-containing solution and the IL-2-containing solution are subjected to the following steps, a mixture of IL-2A and B is obtained: (e) subjecting the eluate to the second gel filtration on a polyacrylamide-agarose column, and (f) subjecting the eluate to high performance liquid chromatography and performing elution with a solvent.

Claim 22

A method of producing a mixture of human IL-2A and B, which comprises cultivating human cells capable of producing IL-2 in the presence of an inducer and thereby causing formation and accumulation of human IL-2A and B in the culture broth and subjecting the culture fluid to a series of steps, namely the steps of: (a) applying the culture fluid to a sulfopropyl-crosslinked dextran column, for adsorption, and performing elution with a buffer, (b) applying the eluate to a column packed with an anion exchange chromatographic carrier, for adsorption, and conducting elution with a salt solution, (c) subjecting the eluate to gel filtration on a polyacrylamide-agarose column, and (d) sujecting the eluate to high performance liquid chromatography and performing elution with a solvent.

Claim 23

A method of producing a mixture of human IL-2A and B, which comprises cultivating human cells capable of producing IL-2 in the presence of an inducer and thereby causing formation and accumulation of human IL-2A and B in the culture broth and subjecting the culture fluid to a series of steps, namely the steps of: (a) applying the culture fluid to a sulfopropyl-crosslinked dextran column, for adsorption, and performing elution with a buffer, (b) applying the eluate to a column packed with an anion exchange chromatographic carrier, for adsorption, and conducting elution with a salt solution, (c) subjecting the eluate to gel filtration on a polyacrylamide-agarose column, and (d) sujecting the eluate to high performance liquid chromatography and performing elution with a solvent.