Virus-inactivated HGI-glycoprotein capable of stimulating proliferation and differentiation of human granulocyte, process for preparing same and leukopenia curative containing same

• US 4,230,697 A
• Filed: 05/09/1979
• Issued: 10/28/1980
• Est. Priority Date: 07/03/1978
• Status: Expired due to Term

• Current Assignee: Green Cross Corporation (Green Cross Holdings Corporation), Morinaga Milk Industry Company Limited
• Original Assignee: Green Cross Corporation (Green Cross Holdings Corporation), Morinaga Milk Industry Company Limited
• Inventors: Yamada, Muneo, Yanai, Nobuya, Nishida, Masayuki, Kuboyama, Morio, Funakoshi, Satoshi, Ogasa, Katsuhiro
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/037,515
• Time in Patent Office: 538 Days
• Field of Search: 424/177, 260/112.5 R
• US Class Current: 424/85.1
• CPC Class Codes: A61K 35/22 Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: Y10S 530/83 Plasma; serum
• CPC Class Codes: Y10S 530/834 Urine; urinary system
• CPC Class Codes: Y10S 530/851 Placenta; amniotic fluid

Abstract

A colony-stimulating factor having definite physical and chemical properties and a function of stimulating activity on human bone marrow cells to proliferate and differentiate, thereby forming granulocyte colonies, is obtained from human urine by concentrating the urine with respect to proteins contained therein by adsorption chromatography with silica gel, salting out with ammonium sulfate and other means, then removing impurities by adsorption on cation exchanger, and further purifying by ion exchanging chromatography on anion exchanger, gel filtrating chromatography with highly crosslinked gels, affinity chromatography with sugar affinitive adsorbents and electrophoresis. This substance is stable in the presence of a stabilizer such as albumin or urinary proteins, against heat-treatment of virus-inactivation and can be used as a leukopenia curative which is precluded from fear of virus-infection.

Claims

1. A virus-inactivated glycoprotein from human urine which stimulates human bone marrow cells to form colonies of granulocytes and which has the following physical and chemical properties:

(a) molecular weight;

75,000 to 90,000 dalton as determined by gel filtration;

(b) solubility;

soluble in water, slightly soluble in chloroform, and insoluble in ethyl alcohol and acetone;

(c) specific optical rotation;

[α

]_D²⁰ =0±

40 (0.25% aqueous solution);

(d) pH;

5.0-6.0 (1% by weight aqueous solution);

(e) isoelectric point;

pH 4.7±

0.2;

(f) thermostability;

on being heated at 60°

±

0.5°

C. for 30 minutes in 1% aqueous solution, the stimulating function on the proliferation and differentation of the human granulocyte is completely lost;

(g) electrophoresis;

the relative mobility is 0.25 in the electrophoresis using sodium dodecyl sulfate-polyacrylamide gel;

(h) infrared absorption;

characteristic absorption at the following wave numbers (cm^-1);

3600-3200 (strong absorption), 1700-1600 (strong absorption), 1550 (medium absorption), 1430-1380 (medium absorption), and 1150-1000 (broad band);

(i) color reaction;

colors characteristic of saccharides are produced by the α

-naphthol-sulfuric acid reaction, indole-sulfuric acid reaction, anthrone-sulfuric acid reaction and phenol-sulfuric acid reaction;

colors characteristic of polypeptide linkage and amino acids are produced by the Lowry-Folin'"'"'s reaction and by the ninhydrin reaction after hydrolysis with hydrochloric acid;

(j) constituent amino acids of the protein moiety;

proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, triptophan and arginine;

(k) color and shape;

substantially white and amorphous;

(l) sugar composition of the polysaccharide moiety;

10.0-13.0% by weight in terms of glucose of neutral sugars, 3.0-7.0% by weight of sialic acids and 1% by weight of other amino sugars;

(m) weight ratio of protein to polysaccharide;

75-85;

13.0-20.0; and

(n) elementary analysis;

42.3-47.3% of carbon, 5.7-7.8% of hydrogen, 9.6-14.3% of nitrogen, 34.4-39.4% of oxygen and 0.2% or less of sulfur.

2. A therapeutic agent for leukopenia containing as active material a glycoprotein according to claim 1.

3. A therapeutic agent for leukopenia containing as active material a glycoprotein according to claim 1 having a specific biological activity of 35,000 units/mg or more.

21. A process for stimulating human bone marrow cells to form colonies of granulocytes comprising supplying to the bone marrow an amount of the glycoprotein of claim 1 effective to increase the colonies of granulocytes.

4. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating urine with of normal humans respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material collecting fractions of a relative effluent of 1.11 to 1.60, adjusting the aqueous solution containing the collected fractions to pH 5-9, and heating the resulting aqueous solution at 50°

-70°

C. for 8-30 hours in the presence of albumin of the human origin to inactivate viruses.

5. A process according to claim 4, wherein the concentration of albumin in the aqueous solution is 2.0% (W/V) or more.

6. A process according to claim 4, wherein the albumin originates from human serum or human placenta.

7. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline silution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to adsorb the effective ingredient, eluting the adsorbed active material with a 20-100 mM saccharide solution, adjusting the aqueous solution containing the collected fractions to pH 5-9, and heating the resulting aqueous solution at 50°

-70°

C. for 8-30 hours in the presence of albumin of the human origin to inactivate viruses.

8. A process according to claim 7, wherein the concentration of albumin in the aqueous solution is 2.0% (W/V) or more.

9. A process according to claim 7, wherein the albumin originates from human serum or human placenta.

10. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to absorb the active material, eluting the adsorbed active material with a 20-100 mM saccharide solution, subjecting the eluate to preparative zone electrophoresis, eluting the active material with a salt solution, adjusting the eluate to pH 5-9, and heating the resulting eluate at 50°

-70°

C. for 8-30 hours in the presence of albumin of the human origin to inactivate viruses.

11. A process according to claim 10, wherein the concentration of albumin in the aqueous solution is 2.0% (W/V) or more.

12. A process according to claim 10, wherein the albumin originates from human serum or human placenta.

13. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to absorb the active material, eluting the adsorbed active material with a 20-100 mM saccharide solution, subjecting the eluate to preparative zone electrophoresis, eluating the active material with saline solution, adjusting the concentration of active material in the eluate to at least 70 mg/ml and the pH to 5-9, and heating the resulting eluate at 50°

-70°

C. for 8-30 hours to inactivate viruses.

14. A process according to claim 13, wherein the concentration of active material in the final heat treatment step is 100-200 mg/ml.

15. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, and collecting fractions of a relative effluent of 1.11 to 1.60;

the active material containing fraction including the concentrated human urine with respect to urinary proteins in any of the above steps being subjected to virus inactivating treatment by heating the fraction in the form of aqueous solution at 50°

-70°

C. for 8-30 hours under such conditions that the protein content of the aqueous solution has been adjusted to at least 70 mg/ml and the pH to 5-9.

16. A process according to claim 15, wherein the protein content of the aqueous solution is 100-150 mg/ml.

17. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to absorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to adsorb the active material, and eluting the adsorbed active material with a 20-100 mM saccharide solution, the active material containing fraction including the concentrated human urine with respect to urinary protein in any of the above steps being subjected to virus inactivating treatment by heating the fraction in the form of aqueous solution at 50°

-70°

C. for 8-30 hours under such conditions that the protein content of the aqueous solution has been adjusted to at least 70 mg/ml and the pH 5-9.

18. A process according to claim 17, wherein the protein content of the aqueous solution is 100-150 mg/ml.

19. A process for producing a virus-inactivated glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which comprises concentrating human urine with respect to proteins contained therein, contacting the slurry proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the active material, eluting the active material with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to adsorb the active material, eluting the adsorbed active material with a 20-100 mM saccharide solution, subjecting the eluate to preparative zone electrophoresis, and eluating the active material with a saline solution to recover the active material in pure form;

the active material containing fraction including the concentrated human urine with respect to urinary protein in any of the above steps being subjected to virus inactivating treatment by heating the fraction in the form of aqueous solution at 50°

-70°

C. for 8-30 hours under such conditions that the protein content of the aqueous solution has been adjusted to at least 70 mg/ml and the pH to 5-9.

20. A process according to claim 19, wherein the protein content of the aqueous solution is 100-150 mg/ml.