Novel csf and method for obtaining the same

Claim 2

Granulocyte colony stimulating factor (G-CSF) that has the ability to promote the differentiation and proliferation of human bone marrow cells to granulocytes having the following physicochemical properties: i) Molecular weight: 19,000±1,000 as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis; ii) Isoelectric point: Having at least one of the three isoelectric points, A, B and C, shown in Table 1: iii) UV absorption: Maximum absorption at 280 nm and minimum absorption at 250 nm; iv) The N-terminal 21 amino acids are (5) (10) (17) (20) H 2 N-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser-Phe-Leu-Leu-Lys-X-Leu-Glu-X-Val-, wherein X represents a naturally occurring unidentified amino acid residue.

Claim 3

The G-CSF according to Claim 1 wherein the isoelectric point pl is A.

Claim 4

The G-CSF according to Claim 1 wherein the isoelectric point pl is B.

Claim 5

The G-CSF according to Claim 1 wherein the isoelectric point pl is C.

Claim 6

The G-CSF according to Claim 1 wherein the molecule is glycosylated.

Claim 7

The G-CSF according to Claim 1 having a specific activity of 3.94xlO l U/mg or higher.

Claim 8

A method for preparing the G-CSF according to Claim 1, said method comprising culturing a cell line having a human G-CSF producing ability in a serum-free culture solution, subjecting the supernatant of the culture to steps (1) to (3) indicated below, and optionally subjecting the resulting fractions to either step (4) or (5): step (1) subjecting the supernatant of the culture to gel filtration using a gel having an effective fraction range of 5,000-70,000 daltons, and recovering fractions having the neutrophile-dominant colony stimulating activity (CSA); step (2) adsorbing the recovered fractions onto a carrier for reverse-phase high-performance liquid chromatography and performing elution by the density gradient technique with a mixture of water and an organic solvent so as to recover fractions having the neutrophile-dominant CSA: step (3) subjecting the so recovered fractions to high-performance molecular sieve chromatography so as to recover fractions having the neutrophile-dominant CSA; step (4) subjecting the so recovered fractions to isoelectric point electrophoresis so as to recover fractions having the neutrophile-dominant CSA; or step (5) subjecting the fractions recovered in step (3) to the step of removing sialic acid so as to recover fractions having the neutrophile-dominant CSA.

Claim 9

A method according to Claim 7 wherein the cell line having the human G-CSF producing ability is CHU-1 (C.N.C.M., Deposit-No. 1-315).

Claim 10

Cell line CHU-1 (C.N.C.M., Deposit-No. 1-315).

Claim 11

Cell line CHU-1 (C.N.C.M., Deposit-No. 1-315).

Claim 12

Pharmaceutical composition comprising the G-CSF according to Claims 1 to 5 and a pharmacologically acceptable carrier.

Claim 13

Pharmaceutical composition for stimulating the differentiation and proliferation of bone marrow cells to granulocytes comprising an effective amount of the G-CSF according to Claims 1 to 5 and a pharmacologically acceptable carrier.

Claim 14

Pharmaceutical composition for assisting in curing infectious diseases comprising an effective amount of the G-CSF according to Claims 1 to 5 and a pharmacologically acceptable carrier.