SE451844B - GLYCOPROTEIN, PROCEDURE FOR PREPARING THEREOF AND THERAPEUTIC AGENTS INCLUDING THE GLYCOPROTEIN - Google Patents

Description

"451 844 2 10 20 25 30 35 in recent years have tried to develop therapeutic substances for the treatment of leukopenia which are more effective and have less side effects. It was known that a colony stimulating factor (hereinafter referred to as CSF) stimulates proliferation. and differentiation of bone marrow cells.CSF acts on the bone marrow cells and stimulates proliferation and differentiation to form granulocytes or macrophages.It is an essential property of marrow cells, when grown in vitro, that they form granulocyte cells rophag cell aggregate (hereinafter referred to as granulocyte cell.macrophage colony) by simultaneous proliferation and differentiation [ïchikawa, Y., Proceedings of the National Academy of Science, Vol. 56, p. H88 (l966); Metcalf, D., Experimental Hematology, Vol. 1, p. 185 (l97§]. Since CSF induces granulocyte and macrophage colonies from bone marrow cells, some researchers have suggested that CSF should be considered as separate factors, ie a granulocyte indu factor and Hlm fl kf fi f fi gí fl du fi ßf flfi dflfi factor EStan- ley, E.R.et al, Journal of Experimental Medicine, Vol. 1H3, p 631 (1976¶. However, these factors are usually analyzed together as CSF in in vitro analysis of mouse bone marrow cells.

Many factors that stimulate the formation of in vitro colonies of mouse bone marrow cells have been isolated from various sources, such as serum, urine, various organ extracts, media conditioned by various tissues and cell lines, body fluids such as serum and urine; conditioned media of cells such as leukocytes, and tissues [Éherldan, J.w., Journal of cell Physiology, vol. 78, p H51 (l97l].

CSFs that have an effect on human bone marrow cells have been isolated from human sources, ie various organ extracts, serums, media conditioned by tissues [Metcalf, D. and Moare, MAS, "Cíba Foundation Symposium 13, Haemopoietic Stem Cells", p. 157 However, each CSF obtained from different organs, different cells and conditioning media is therefore not a single substance common to all sources. For example, the molecular weight of CSF obtained from media is conditioned by human placental cells. 30,000 daltons [Eur- gess, AW et al, Blood, Vol. H9, p. 575 (l97Z], while CSF from human serum is Ä5 000 daltons Kšhan, SH et al, British Journal of Haematology, Volume 20, p 329 (l97l fl. Two types of CSF with a molecular weight of 35,000 and less than 1,300, respectively, were isolated from media conditioned by human leukocytes lšrice, GB- et al, B100d, V01- 32, P-331 (l97} Ã] Furthermore, each CSF Uï lJ \ 11 20 25 30 \) J UI 3,451,844 has different shares white, some acting on the respective type of cells that grow and differentiate into granulocyte or macrophage, others on both types of cells. CSF isolated from different sources are therefore considered to be substances that differ from each other: hetcalf and Moore, loc. cit., (l97} j.

It is also known that in human urine there is a type of CSF that can stimulate bone marrow cells from mice to form colonies of granulocytes and macrophages in vitro 'Stanley, al., Federation Proceedings, Vol. 34, pp. 2272 (l§75); Stanly, E.R.

Medical Science, Vol. H7, p. H67 (1969 ?. Here it was reported that ER et and Netcalf, D., Journal of Experimental Biology and this CSF have a molecular weight of 45.00OJ and stimulate proliferation and differentiation of bone marrow cells from mice to form a macrophage dominant colony. In contrast to this stimulating effect on mouse bone marrow cells, it hardly stimulates the formation of granulocyte or macrophage colonies of human bone marrow cells but consistently stimulates the formation of clusters. In this description, "colony" and "collection" refer to human bone marrow cells, cell aggregates that contain H0 or more cells and 3 to less than H0 cells, respectively, according to Metcalf's defini l57 (l97 ") - When examining substances with CSF activity in urine (Metcalf, D. , Experimental Hematology, Vol. 2, p. From man, a new HGI glycoprotein has now been found and isolated in its pure state, which, unlike previously known CSFs, has a molecular weight of about 85,000 and with an effect on bone marrow cells from both humans and mice in the formation of pure granulocyte colonies in vitro. Furthermore, pure HGI glycoprotein isolated from human urine has been succeeded in surprisingly affecting human bone marrow cells and stimulating proliferation and differentiation of pure granulocyte colonies (hereinafter referred to as biological activity). Furthermore, this HGI glycoprotein was identified, a preparative method with good reproducibility was developed and uses were found, which led to the present invention.

An object of the present invention is to provide a new CSF.

Another object of the invention is to provide a process for producing this new CSF.

A further object of the invention is to provide a therapeutic agent for leukopenia which contains the novel CSF. According to the present invention there is provided a glycoprotein characterized in that it stimulates human bone marrow cells to form cones of granulocytes and which glycrotein has the following physical and chemical properties: (a) soluble in water, slightly soluble in chloroform and insoluble in ethyl alcohol and acetone: (b) specific optical rotation: / ÛWÉO = 0 f 000 (0.25 *% aqueous solution); (c) pH; 5.0-6.0 (1% by weight aqueous solution); (d) isoelectric point: pH 4.7 f 0.2; (e) thermostability: when heated at 60+: 0.5 ° C for 30 minutes in 1% aqueous solution, the stimulating effect on proliferation and differentiation of humanogranulocytes is completely lost; (f) electrophoresis: the relative mobility is 0.25 in electrophoresis using sodium dodecyl sulfate-polyacrylamide gel; (g) infrared absorption: characteristic absorption at the following wavelength (cm_1): 3600-3200 (strong absorption), 1700-1600 (strong absorption), 1550 (medium absorption), 1430-1380 (medium absorption) and 1150 -1000 (wide band); (h) color reaction: colors characteristic of saccharides are obtained in the (X-naphthol-sulfuric acid reaction, the indole-sulfuric acid reaction, the anthron-sulfuric acid reaction and the phenol-sulfuric acid reaction; colors characteristic of polypeptide bonding and amino acid retention; and in the ninhydrin reaction after hydrolysis with hydrochloric acid: (i) amino acids contained in the protein moiety, proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan and arginine; (J) color and shape: essentially white and amorphous; (k) sugar composition of the polysaccharide moiety: 10.0-13.0% by weight expressed as neutral sugar glucose, 3.0-7.0 wt. percent sialic acids and 1 weight percent other amino sugars: (1) weight ratio of protein to polysaccharide: 75-B5: 13.0-20.0, (m) elemental analysis 42.3-47.3% carbon, 5.7-7, 8% hydrogen, 9.6-14.3% nitrogen, 34.4-39.4% oxygen and 0.2% or less sulfur; Molecular weight: 75 ÛÛÛ to 90,000 daltons, determined by gel filtration, whereby the glycoprotein is obtained from urine of healthy human, which urine is first concentrated with respect to proteins therein, which are then brought into contact with a cation exchanger and then with an anion exchanger, active material after elution, the eluate was subjected to gel filtration chromatography, fractions collected therefrom were subjected to sugar affinity chromatography and the adsorbed active material was eluted, the eluate was subjected to preparative zone electrophoresis and electrophoresis.

The HGI glycoprotein of the invention is prepared by concentrating proteins found in human urine, bringing these urinary proteins into contact with cation exchangers. removal of impurities by adsorption on the ion exchanger, bringing the flow into contact with an anion exchanger to adsorb the active material, eluting the active material with a saline solution through a linear concentration gradient elution, exposing the eluate to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material, up to a relative effluent of 1.11 to 1.60, affinity chromatography of the collected fractions with an adsorbent having an affinity for sugar to adsorb the active material, eluting of the adsorbed active material with a 20-10 mM saccharide solution, subjecting the eluate to preparative zone electrophoresis, eluting the active material with collection of saline solution fractions and recovering the active material in pure form.

The invention is described in detail below.

A typical process for the production of HGI glycoprotein according to the invention is carried out in the following manner. Fresh urine obtained from normal humans is adjusted to pH 6-9, preferably 7-8, with dilute acidic or alkaline solutions and then centrifuged to remove insoluble matter present in the urine. The supernatant is contacted with a siliceous adsorbent such as silica gel, silica gel-magnesium silicate, diatomaceous earth, quartz glass or bentonite and the adsorbed constituents were eluted with an alkaline solution having a pH of suitably 9 or higher. The alkaline solution used for the elution is not specific, but may suitably consist of an aqueous solution of ammonium hydroxide, sodium hydroxide or the like in a concentration of 0.5 - 1.5 M. The eluate thus obtained is adjusted to pH 7-8 with Acid solution and is added with a neutral salt, such as ammonium sulfate, to a saturation of 70 Z to salt out the active substance, thereby obtaining a crude protein fraction containing the HGI glycoprotein.

The above crude protein fraction is redissolved in a small amount of an alkaline solution, freed from low molecular weight substances by ultrafiltration, diluted with a saline buffer solution and contacted with a cation exchanger (eg carboxymethyldextran, carboxymethylcellulose or phosphorus). contained in this solution. The above contact is performed at neutral pH and the crude fraction of HGI glycoprotein and cation exchanger has been adjusted to pH 6-8 preferably with 0.01 - 0.15 M saline buffer solutions before contact. Most of the HGI glycoprotein I passes through the cation exchanger without adsorption after concentration, the concentrated stream is equilibrated with a dilute buffer solution of pH 6-8 and subjected to ion exchange chromatography with an anion exchanger, eg DEAE cellulose, as equilibrators. , wherein the HGI glycoprotein is adsorbed on the anion exchanger. Thereafter, the adsorbed HGI glycoprotein is eluted by so-called linear concentrations - 0.3 M saline solutions such as sodium chloride. The HGI glycoprotein elutes at a salt concentration of 0.1 M or higher but perfect separation is difficult. The flow fractions at a salt concentration of 0.1 - 0.3 M ion gradient elution using 0.1 are collected and, if necessary, subjected to desalination and concentration.

It is also possible to use stepwise elution with 0.1 - 0.5 M saline solution to elute the HGI glycoprotein from the ion exchanger.

For further purification, the combined fraction obtained above is subjected to gel filtration chromatography on a highly crosslinked polymer gel with a water recovery value of 10 - 20 ml / g such as Sephadex ® G-150 or Biogel ® P-100 and the active substances are developed with a .05 - 0.1 M saline buffer solution. Fractions with a relative flow volume of 1,11 to 1,60, preferably 1,11 to 1,5 H5, are collected, desalted and concentrated or lyophilized.

The semi-purified substances thus obtained which contain HGI glycoprotein can be used as pharmaceuticals.

The relative flow volume mentioned here is a volume that can be expressed using the ratio Ve / Vo (where Ve denotes the volume of solvent required to elute the substance contained in the column and Vo denotes the void volume). in the column).

For further purification, the semi-purified substances obtained above are dissolved in a dilute saline buffer solution of 1.0 - 2.0 M, such as a phosphate buffer solution of pH 6.0 - 8.0, preferably 6.0 - 7.0, containing 1 0 - 2.0 M NaCl and subjected to affinity chromatography with absorbents with affinity for sugar, such as concanavalin A-Sepharose 4 B (from Fine Chemical Laboratory), equilibrated with the same buffer solution. The HGI glycoprotein adsorbed on the affinity column is eluted with a 1.0 - 2.0 M saline in dilute buffer containing 20 ~ 100 mm fl saccharides in dilute buffer solution containing 1.0 - 2.0 M salt at pH 6.0 8.0, for example the saccharide is α-methyl-D-glucoside or the like at pH 6.0 - 8.0, preferably 6.0 - 7.0. The fractions containing HGI glycoprotein are collected and desalted and concentrated or lyophilized, if necessary.

For further purification of the HGI glycoprotein by electrophoresis, the pooled fraction obtained from the affinity chromatography is subjected to a preparative zone electrophoresis using an acrylamide gel or agarose gel as vehicle, at pH 7.0-9.0 and the The purified HGI glycoprotein is recovered from the support with a dilute saline solution under cooling, desalted and concentrated or lyophilized.

According to the present invention, it becomes possible to recover urokinase, kailikrein and lysozyme from humans during the production of the HGI glycoprotein.

The HGI glycoprotein thus obtained is a powder of white or light brown color, which is tasteless, odorless and somewhat hygroscopic and which has the physical and chemical properties described below.

Fig. 1 shows the infrared absorption spectrum of the HGI glycoprotein; Fig. 2 shows the relationship between the relative mobility of electrophoresis and the molecular weight; Fig. 3 shows the ultraviolet absorption spectrum of the HGI glycoprotein; and Fig. 4 shows the relationship between the added amount of HGI glycoprotein and the number of colonies that develop during in vitro analysis.

The physical and chemical properties were determined on sample No. 6 in Example 1 (described below). (1) Molecular Weight The molecular weight of the HGI glycoprotein of the invention was found to be about 85,000 daltons measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 75,000 to 90,000 daltons measured by gel filtration using Sephadex ® G 150. Consequently, the most likely molecular weight range appears to be from 75,000 to 90,000 daltons. (2) Solubility The solubility of the HGI glycoprotein in different solvents is given in Table 1.

Table 1 Solvents Solubility Water Soluble Ethyl alcohol Insoluble Acetone Insoluble Chloroform Slightly soluble 1 M sodium chloride solution Soluble io 'zaacano solution Soluble It is also readily soluble in a dilute saline solution, such as a dilute phosphate diluent solution or a dilute phosphate solution. It is also soluble in a dilute saline solution in the pH range from 1 to 12. (3) The pH The pH of a one percent aqueous solution of the HGI glycoprotein is 5.0 to 6.0, i.e. in the acidic range. (4) Specific optical rotation The optical rotation was measured on a 0.25% aqueous solution of HGI glycoprotein at 20 ° C. The specific optical rotation angle λ 23 was found to be in the range 0 in 40. (5) IR absorption spectrum The IR absorption spectrum of the HGI glycoprotein measured according to the method with KBr pellets is shown in Fig. 1. The characteristic absorption bands are given in table 2. w l- '\ J | 20 R) k !! 50 35 451 844 9 Absorption Degree of Comment Wave number (cm) absorption 3600 - 3200 Strong The broad absorption band appears to originate from left-OH groups that form different degrees of hydrogen bonds. 1700 - 1600 Strong The broad absorption band appears to be derived from 1590 Medium from Ä -co -CNH bonds in protein fragments lü} O - 1580 Medium 1150 - 1000 Medium The broad absorption band appears to be derived from -CCC bonds in polysaccharide fragments. (6) Isoelectric point 7 The isoelectric point of the HGI glycoprotein is pH (7) Color reaction 5,7 1 0,2, measured by isoelectric focusing on polyacrylamide gel.

Different color reactions were examined on HGI glycoprotein dissolved water. Results obtained are given in Table 3.

Table 3 Color reaction Evolved Comment 'color Lowry-Folín's reaction Blue Peptide bonds Ninhydrin reaction (hyd-Violet α-amino acids rolyserag with 6N HCl at 110 C for 22 hours) u-Naphthol-sulfuric acid reaction- Violet Saccharides reaction (Molischß s reaction) -sulfuric acid reaction Brown "(Dische's reaction) Antron-sulfuric acid reaction Dark green" Phenol-sulfuric acid reaction Brown "451 844 (8) Thermostability 1D After heating a 1% aqueous solution of HGI glycoprotein at 60 1 0.5 OC for 30 min (9) Amino acid composition of the protein fragment HGI-glycoprotein was hydrolyzed with 1N hydrochloric acid at 110 ° C and the amino acid composition of the protein fragment was determined by means of an auto-analyzer for amino acids, the results given in Table U were obtained. 13 Table M Amino Acid Weight% Mole (13) Praline _ 3.2 0.392 -; Aspartic Acid 3.3 1,038 "" Treonin 0 331 Serine 11,9 l: 596 Glutamic Acid 13,8 1,322 Glycine ll 0 2 066 Aiinin âis 11155 Va in '0, 771 Methionine 2: 5 0.256 Iscleucine 2.5 0.269 Leucine 7.0 0.753 Tyrosine 5.8 0, ü5l Phenylalanine 12.8 1,050 Lysine 2 2 0.212 Histiain 1: 0 0.091 Tryptophan trace - 2; Arginine "-" Ammonium 0.5 Table U shows that the protein fragment of the HGI glycoprotein is composed of 17 amino acids, of which acidic and neutral amino acids predominate, while basic amino acids constitute constituents in smaller amounts. is also one of the characteristics that over 70% of the total amino acids are linear amino acids comprising aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine and leucine.35 (10) Electrophoresis - According to Laemuli's method Ehature, volume 227, p 680 (1970i and using a sodium dodecyl sulfate-polyacrylamide gel, the HGI glycoprotein showing a single band in a position of relative mobility of 0.25, trypsin inhibitor (molecular weight 21,500), ovalbumin (molecular weight 43,000), Serum albumin monomer (molecular weight 65,000) and human serum albumin dimer (molecular weight 150 000) by means of the movement of the substances with known molecular weights and the same for HGI -g lycoprotein, the molecular weight of the latter was found to be about 85,000 (Figs. 2). In Fig. 2, a, D, c and Ö denote trypsin inhibitor, ovalbumin, human serum albumin monomer and human serum albumin dimer, respectively, and the arrow denotes the HGI glycoprotein. (II) UV absorption spectrum The UV absorption spectrum of the HGI glycoprotein, measured on a 0.1 5 g aqueous solution in a 1 cm silica cell, is shown in Fig. 5. It shows maximum absorption at 280 nm and terminal absorption in the 1% wavelength range shorter than 250 nm. The optical density E ICF at 280 nm is 5.8. (12) Sugar composition of the polysaccharide fragment Neutral sugars were determined by the phenol-sulfuric acid reaction, sialic acids by the warren's thiobarbital method Journal of Bio- iogieai cnemistry, vo1 .__ 234, p. 1971 (1959LÉ, the amino sugar by means of the Elson V The results were as follows: neutral sugars: 10.0 - 15.0%; sialic acids: 5.0 - 7.0%; amino sugars less than 1.0%; total sugars: 13.0 - 20 (15) Protein to polysaccharide ratio The protein content of the HGI glycoprotein is 75 - 85 2, determined according to Kieldahl's semi-micro method. The total sugar content is 15.0 - 20.0%, as stated above. (14) Elemental analysis Results of elemental analysis for HGI glycoprotein are as follows: C, 42.5 - 47.5%; H, 5.7 - 7.8%; N, 9.6 - 14.5%; 59.4%; S less than 0.2%.

The HGI glycoprotein with stated physical and chemical properties is capable of stimulating proliferation and differentiation of both human and mouse granulocytes as shown in Experiment 1 (described later) and shows no acute toxicity, as shown in Experiment 4 (described later). As can be seen from the results of Experiment 5 (described later), it can be further used as leukopenia chemotherapeutics.

HGI glycoprotein produced from human urine according to the above procedure is aseptically lyophilized in glass vessels and hermetically sealed. It is also possible that before lyophilia: Biochemical Journal, Vol.27, p. 1 ZA (1933). ' Provide the HGI glycoprotein with an aqueous solution containing human serum albumin as a stabilizer and an amino acid or saccharin as a solubilizing aid; the resulting solution is sterilized by membrane filtration and then lyophilized aseptically. Prior to use, the seal on the glass vessel is broken and the HGI glycoprotein is dissolved by adding sterilized physiological saline solution, sterile water or a sterile isotonic solution. The resulting solution is administered to patients with leukopenia by intravenous, intramuscular or subcutaneous injection.

The results of experiments 1 and 2 (described later) show that the effective dose is 0.75 mg or more, preferably 0.75 - 2.2U mg, per day per kg body weight ”. Semi-purified products, produced on a larger scale, with a specific biological activity of 35,000 units / mg or more such as those containing HGI glycoprotein corresponding to samples N and N 5 of Example 1 (described later) can also be used as pharmaceuticals .

The effect of HGI glycoprotein on the proliferation and differentiation of granulocytes is described in detail below.

Experiment with stimulating effect on proliferation and differentiation of mouse and human granulocytes in vitro.

In each 35 mm diameter plastic petri dish was placed 1 ml of McCoy's 5A medium containing 0.05, 5.1, 0.15 or 0.2 μg of HGI glycoprotein (Sample No. 6 in Example 1), 20% fetal calf serum, 0.3% agar and 7.5 x 10 6 bone marrow cells from mice or 25 x 10 bone marrow cells from normal individuals or patients with iron deficiency anemia. The medium in the petri dish was incubated in a humidified atmosphere with 5% CO 2 at 37 ° C for 7-9 days. The difference in the number of cells supplied between mice and humans was due to a larger number of stem cells in mice. After incubation, the number of discrete colonies containing more than 50 cells for mice and more than H0 cells for humans with an inverted microscope was counted. For morphological analysis of colonies, some of these were picked up with microhematocrit tubes and stained with 0.6% orceini H0% acetic acid. The results obtained are shown in Fig. U. Fig. H shows the relationship between the dose of HGI glycoprotein and the number of colonies formed in vitro. In Fig. H, -0-0- refers to mouse bone marrow cells and -0-ë- human bone marrow cells. As shown in Fig. U, HGI glycoprotein stimulates proliferation. | .- | UI 20 25 30 * 55 1; 451 844 fermentation and differentiation of bone marrow cells from mice and humans, which form colonies and there is a dose-response relationship between HGI glycoprotein and the number of colonies formed.

In morphological analysis of the cells that form the colonies, it was observed that these cells were all mature granulocytes.

As stated above, HGI glycoprotein has an effect on bone marrow cells from both humans and mice in the formation of colonies of granulocytes, the number of colonies being proportional to the dose of HGI glycoprotein, and there is a definite ratio. the formation of colonies of bone marrow cells between mouse and human.

Therefore, in all the following experiments, only bone marrow cells were used: ß robbery mouse.

Simulating effect on proliferation and differentiation of granulocytes in vivo.

Sixty CSYBL male mice (average body weight 20 g) were randomly divided into six groups of ten mice each. To a control group, subcutaneous 0.0U mg / mouse human serum albumin dissolved in 0.2 ml of sterile normal saline solution was administered once daily for three consecutive days. The remaining five experimental groups, i.e. groups 1, 2, 3, 4 and 5, were added subcutaneously with 0.005, 0.01, 0.02, 0.03 and 0.04 mg / mouse HGI glycoprotein, respectively (Sample No. 6 in Example 1 as described later) each dissolved in 0.2 ml of sterile normal saline solution, once a day for three consecutive days.

Blood samples were taken from vena.coccygea. in each mouse before administration and 2, 4, 6, 8 and 10 days after administration.

The leukocytes in each blood sample were stained with 1% gentian violet solution and the leukocyte count was counted with a Bürker-Türk counting chamber.

Furthermore, each blood sample was smeared on a slide, stained with Wright-Giemsal solution, after which the amount of granulocytes in leukocytes was measured under a microscope.

The number of granulocytes was calculated according to the following formula:) (number of leukocytes in 1 mm x (amount of granulocytes in leukocytes) = number of granulocytes in 1 mmš.

The results obtained are shown in Table 5. \ I 1 IJ \ J'1 20 30 35 451 844 11 Table 5 rupp Kon- 1 2 3 4 5 Ä ^ troll - 0 OS (m8) 0 0.005 0.01 0.02 0, 03 0.011 Days 0 1150 1152 1155 1150 11118 1125 2 380 1120 550 600 775 B00 11 372 590 800 1100 11100 11120 6 380 595 1100 1800 2200 2100 s 1110 1100 620 750 950 971 10 1130 1110 1170 1160 1165 # 50 “* Each numeric value denotes the average number 3 for ten mice. granulocytes per mm As shown in Table 5, the number of granulocytes in the experimental groups administered with 0.01 - 0.0H mg / mouse of the HGI glycoprotein began to increase after two days of administration and amounted to 5 - 6 times the value of the control group after 6 days. - gar.

The number of granulccytes decreased and returned to normal after ten days. When the daily dose was increased to above 0.02 mg, there was no significant increase in granulocytes corresponding to the increasing dose of HGI glycoprotein.

These results suggest that granulocytosis can be achieved in a sufficient amount by daily injection of 0.01 mg or more, preferably 0.01 - 0.03 mg, of HGI glycoprotein into a mouse (average body weight about 20 U). However, since the stimulatory effect of HGI glycoprotein on bone marrow cells from mice in vitro is on average about 1.5 times higher than the effect on human bone marrow cells (Experiment 1), the effective dose for humans is likely to be 1.5 times higher. than the same for mice determined in vivo in the compound (equivalent to 1 15 451 844 search 2. The effective daily dose per kg of human body weight is accordingly estimated at 0.75 mg or more preferably 0.75 - 2.2ü mg.

Experiment 3 Protective effect of HGI glycoprotein in leukopenia caused by carcinostatic substances. '' 30 C57 BL male mice aged H-5 weeks, were randomly divided into three groups of ten mice. To the control group was administered by intraperitoneal injection 30 mg / kg body weight lo LD 50) Qytosin-D-arabinoside dissolved in 0.2 ml of sterile normal saline solution, once a day under consecutive days. In addition, 0.2 ml / mouse of a sterile normal saline solution was administered subcutaneously once a day for consecutive days. To another group (HGI leukoprotein administered group), Qytosin-D-arabinoside was administered in the same manner as in the control group. In addition, 0.03 mg / mouse HGI glycoprotein (Sample No. 6 in Example 1 described later) was administered subcutaneously once a day for 11 consecutive days. To the remaining group (Cepharantine-administered group) cytosine D-arabino was administered side in the same way as in the control group and in addition, 0.5 mg / mouse Cefarantine (Kakan Pharmaceuticals'Co ;; conventionally used against leukopenia) dissolved in 0.2 ml of sterile normal saline solution was administered subcutaneously, once a day for lay on each other for consecutive days.

Blood samples were collected from the coccygeal vein of each mouse before administration and 2, U, 6, 8, 10, 12 and lü days after administration. The number of leukocytes was measured: æm in Experiment 2 and the percentage reduction in the number of leukocytes after administration was obtained by setting the number before administration to 100.

The results are given in Table 6. 16 451 844 .QWCE O fi æ LÛQ ®Ü§W> H @ UÜEämm LW ®UÄm> H®M% fi w m fi LÜ> n. Fl @ mm mm ~ OHH @. @ = Q »fifi 0.0» wo fi @, @ ~ m flfl n.mm ow NH @. @@: OH c. @ æ QNH mm @ mm GH M. fi> Q fifi o. @@ ONH m ~ n> OHH w O. @ æ QNH cßww øn fi O . @ æ QNH @ @. @ w CWH fi. mæ ßw fl m.mw mm fi = n.nw mw fi o.ow ow fi @. : www nxwcwï ncmpæcoxzmq nxwcwz acmuæooxsmq uxwcwz xuæooxswq nmmßm aELU ;; $ fl: @ EÉä ußäwhæw fi C fl EUd HHOhnCO ¥ QQSLU c: ~ @: c% æ] o | Compared to the control group, the HGE glycoprotein-administered group showed a marked preventive effect on the reduction of leukocyte counts after ten days after the start of HGI glycoprotein administration, which effect is comparable or superior to the effect of Cepharantine. By the fourteenth day after the start of administration, the number of leukocytes in the control group had decreased to H6.6%, while for the HGI glycoprotein-administered group it was 73.3 ä, a smaller decrease than for the Cepharantine-administered group. It therefore seems likely that the HGI glycoprotein is effective: in the therapy of human leukopenia.

It was also confirmed that the HGI glycoprotein is effective when other carcinostatic substances, such as 5-fluorouracil and daunomycin, were administered, which are known to cause a reduction in the number of leukocytes in a manner similar to cytosine D-arabinoside. No preventive effect on the reduction in leukocyte count could be observed when human serum albumin was examined in the same manner as described above.

Experiment H Acute toxicity of the HGI glycoprotein.

The acute toxicity of HGI glycoprotein prepared in Example 1 (Samples Nos. H and No. 6) was examined in male male Mice according to the method described by Lichied and Wilcoxon in the Journal of Pharmacology and Experimental Therapeutics, Vol. 99: P 99 (19H9).

No fatal outcome was found after administration of H 000 mg / kg body weight or intravenous administration of 2,000 mg / kg body weight. Consequently, it was practically impossible to estimate LD50; LD50 Vië Subcutaneous in; intraperitoneal injection was above U 000 mg / kg body weight and LD50 at intravenous injection was above 000 mg / kg body weight.

Four hundred liters of fresh urine obtained from normal humans were adjusted to pH 8 with 10% sodium hydroxide and centrifuged by continuous centrifugation at 15,000 G at 0 ° C to remove insoluble matter. The supernatant was adjusted to pH 7 with 10% hydrochloric acid and passed through a silica gel column (10 X 80 cm). The substances adsorbed on the silica gel were eluted with H0 liters of 5% ammonium solution. The eluted solution was adjusted to pH 7.5 with 1 N sulfuric acid and added with ammonium sulfate to 70% saturation, after which it was allowed to stand at 0 ° C overnight. The precipitate was collected by filtration, dissolved in 2 liters of 5 μg of ammonium solution, placed in cellophane tubes (Visking Co.) and dialyzed against a 0.05 M phosphate buffer solution (pH 6.5). The dialyzed solution was given a volume of 10 liters by adding the same buffer solution and passed through a CM Sefadex C-50 ® ion exchange column (4.0 x H0 cm) equilibrated with 0.05 M phosphate buffer solution (pH 6.5), for adsorption of impurities on the ion exchange resin. Ten liters of the effluent were concentrated using a DIAFLO ® hollow fiber ultrafiltration apparatus (Amicon DC-30, USA, suspended molecular weight approx. 10 G90). The concentrated solution was dialyzed against 0.1 M Tris -HO 1 buffer (pH 7.0) at 5 ° C overnight. The dialyzed solution was given a volume of 1 liter with the same buffer solution (the resulting solution is referred to as Sample No. 1).

The above solution was passed through the DEAE cellulose column (H, 0 x H0 cm) equilibrated with 0.1 M Tris-HCl buffer (pH 7.0) and the column was washed with a sufficient volume of 0.1 M Tris-HO buffer (pH 7.0). The charged column was eluted stepwise with 0.1 M Tris-HCl buffer solution (pH 7.0) containing 0.5 M sodium chloride. The fractions capable of effecting proliferation and differentiation of granulocytes, which were tested in the same manner as in Experiment 1, were collected and dialyzed against 0.1 M Tris? -HCl buffer (pH 7.0) (this solution is referred to as sample No. 2). The dialyzed solution was again passed through the DEAE cellulose column (1.0 x H0 cm) equilibrated with 0.1 M trisf HCl buffer (pH 7.0) and the charged column was subjected to a linear concentration gradient eluting with sodium chloride (0 to 0.5 M). The active fractions were collected and added with ammonium sulphate to a saturation of 70%. The precipitates were collected by centrifugation and dissolved in a small volume of 0.1 M Tris-HO1 buffer (pH 7.0) and dialyzed against the same buffer solution (this dialyzed solution is called Sample No. 3).

Twenty ml of the dialyzed solution was applied to a Sefadex ® G-150 column (,, 0 x 60-cm) which was equilibrated with 0.1 M Tris-HCl buffer (pH 7.0) and the fractions obtained at a ratio of Ve / Vo of l, ll - 1, H5 was collected. The combined fraction was carefully dialyzed against distilled water at 5 ° C and the dialyzed solution was lyophilized to produce about 500 mg of a powder (this semi-purified HGI glycoprotein is called Sample No. U).

KM | ..4 CJ | _. | Two hundred mg of the semi-purified HGI glycoprotein was dissolved in 0.02 M phosphate buffer (pH 7.0) containing 1.0 M sodium chloride and passed through 100 ml of concanavalin A-Sepharose ÄB affinity column equilibrated . with the same buffer. After thorough washing of the column with the same buffer, the HGI glucoprotein was eluted with 0.02 M phosphate buffer (pH 7.0) containing 50 mM α-methyl-D-glucoside and 1.0 M sodium chloride. The fractions capable of producing proliferation and differentiation of granulocytes in vitro were collected and dialyzed against distilled water. The dialyzed solution was lyophilized (this is referred to as a sample. About 30 mg of the lyophilized powder indicated on the otovan was dissolved in ml of 0.125 M Tris -glycine buffer (pH 6.8) containing 10% glycerol. The resulting solution was electrophoresed at 10 mA while cooling with using a preparative electrophoresis apparatus (type Fuji Kabára ll from Fuji Riken Co., Japan) using 5% acrylamide gel (pH 8.9; 20 mm x 25 mm) The fracture with a relative mobility of 0, H6 was recovered with 0.025 M tris-glycine buffer (pH 8.3) and dialyzed against distilled water The dialyzed solution was lyophilized to give about 10 mg of the HGI-, g glycoprotein (designated Sample No. 6).

Samples No. 1 to No. 6 obtained at various stages of preparation were tested for the ability to effect proliferation and differentiation of bone marrow cells from both humans and mice in a manner similar to that in Experiment 1. The ñGI glycoprotein or a fraction containing this was added to the medium in an amount necessary to form 200 colonies per dish.

The specific activity was calculated using the following formula, where one unit corresponds to a formed colonization; specific activity (units / mg) = number of formed colonies (units) protein content (mg) in analyzed sample The results are given in Table T. 20 451 844 m. ~ wH = Q.m ~ HH æqæ fl w .tå @ .m fi. ømnwmwcwcøm ooo oww ooo oæ oæ oo ooo mn com m cnvm m oo fl 4._-@&&YLwuwcccV = o; fi> «Jxm x fi u fi omaw rzugzzwš: æ ;; ; a «He; æ:; mE: @n> m w> Hm: <iaf.

H. @ = Wn>. ~ Ow Nnm fi w m. ~ = Ømnwwwc fi cmm _. | «||. |! | .L ||. ||||||| wsš cmpu Lm fifi wumwnmëcmn> m wæHmc <OQO OOO H OQO OHN ooo Nm OQO HH com z Dum ^ mE \ Lm @ m: cwv @@@ fi> fi »xw x fi g fl vwaw w Lz m Lz: Lz M Lm N H Hz Example 2 To 100 mg of HGI glycoprotein powder (Sample No. 6) obtained in the same manner as in Example 1 was added 100 ml of an aqueous solution containing 5% human serum albumin (Sigma Co., USA) and 1 ml. % glycine (Wako Pure Chemicals Co., Japan). The resulting solution is sterilized in a Millipore filtration system (Millipore Co., USA) equipped with a membrane filter with a pore size of 0.1 H5 μm. The sterilized solution was aseptically filled in ml portions into glass vessels sterilized by heating at 170 ° C for two hours.

After lyophilization, the glass vessels were hermetically sealed. In this way, 95 vials of a therapeutic agent were obtained: leukopenia, each vial containing 1 mg of HGI glycoprotein. Example 3 In the same manner as in Example 1, one liter of a concentrated solution containing HGI glycoprotein, analogous to sample No. 1, was prepared. , of 1,000 l of fresh urine obtained from normal human beings. This concentrated solution was diluted with 10 L of 0.1 M Tris-HCl buffer (pH 7.0). After thorough stirring, the diluted solution was reconcentrated to about 1/10 of the original volume using a hollow fiber DIAFLO ® ultrafiltration apparatus. The concentrated solution was added to 5 L of 0.1 M Tris -HCl (pH 7.0) and 5 L of DEAE cellulose suspension containing 300 g of DEAE cellulose based on the dry weight, which was equilibrated with 0.1 M Tris -HCl. buffer (pH 7.0). The mixture was stirred for 30 minutes, allowed to stand for 10 minutes and filtered under vacuum on a Büchner funnel to collect the DEAE cellulose. The collected DEAE cellulose was saturated with 10 L of 0.1 M Tris -HCl buffer (pH 7.0) and collected by filtration in the same manner as above. DEÅE'0® 1 'lulosant was further washed with 10 L of 0.1 M Tris -HCl buffer (pH 7.0) containing 0.05 M sodium chloride and collected in the same manner as above. To the DEAE cellulose thus treated was added 10 L of 0.1 M Tris -HCl buffer (pH 7.0) containing 0.5 M sodium chloride, and the whole was stirred to release the HGI glycoprotein from the DEAE cellulose. The mixture was filtered in the same manner as above and the filtered solution was collected. The filtered solution was desalted by DIAFLO ® fiber ultrafiltration. The desalted solution was lyophilized to give about 15 g of a powder. The powder was dissolved in 150 ml of distilled water and applied to a Sefadex ® G-150 column (6.0 x 80 cm) equilibrated with 0.1 M Tris-HCl buffer (pH 7.0). The fractions corresponding to Ve / Vo ratios of 1.11 - a \ l1 p: UI EG f \) UI SEK 50 '\ H 451 844 22 1.60 were collected. The combined fraction was thoroughly dialyzed against distilled water. The dialyzed solution was concentrated using a DIAFLO ® hollow fiber concentrator (type DC-2) to a concentrate of about 100 ml containing about 3 g of crude SGI glycoprotein. To the concentrated solution were added 1 g of glycine (Wako Pure Chemicals Co.) and 5 g of serum albumin (Sigma Co.). The resulting solution was sterilized by filtration in the same manner as in Example 1 and aseptically filled into bottles in 2.5 ml portions. After aseptic lyophilization, the bottles were hermetically sealed. Thereby, H0 vials with a therapeutic agent against leukopenia were obtained, each vial containing about 3.8 mg of the HGI glycoprotein.

Due to the clinical use of the HGI glycoprotein according to the invention, it is very important that pathogenic viruses present in urine and which can cause hepatitis and endemic diseases be removed from a drug prepared from the HGI glycoprotein as well as other drug additives with human blood elements. When a drug made from human urine is administered to patients without prior treatment who remove or inactivate the virus, there is always a risk of viral infections. This also applies to the HGI glycoprotein according to the invention. To avoid the risk of viral infections caused by viral contamination, a first examination must be made of the urine used as a starting material so that virus-contaminated urine can be excluded. Although this has proven to be effective to some extent in preventing viral infection, it is impossible to use such a practice in the industry where one simultaneously treats urine obtained from tens of thousands of people.

Serum proteins produced from human plasma for medical purposes are also prone to problems with viral infection. Since it was found that the viral infection caused by serum albumin agents can be prevented by heat treatment at 60 ° C 10 hours, without denaturation, all serum albumin agents have been subjected to a similar heat treatment and become safe for clinical use. These heat treatments are therefore also used in the production of other human serum protein agents.

In order to be able to use a waste treatment at 60 ° C for 10 hours, the treated drug constituents must be stable under these conditions. Various stabilizers have been found for this purpose. Among the substances which can not withstand such a heat treatment, some can be made heat-resistant in the presence of a stabilizer. To stabilize human serum proteins against heat treatment, certain types of amino acids or saccharides have been used 23 at the physiologically isotonic concentration or lower. Although it is desirable to inactivate viruses by heat treatment of the HGI glycoprotein, the biological activity of the HGI glycoprotein is lost by heat treatment at 60 ° C for 10 hours.

It has now been found that the HGI glycoprotein in aqueous solution has an improved thermal stability and becomes resistant to the above-mentioned heat treatment without losing its characteristic properties under the following conditions. The HGI glycoprotein is stable in heat treatment at pH 5 - 9, when albumin is present in the solution in an amount of over 2 2 or when the concentration of HGI glycoprotein in the solution is as high as 70 mg / ml or higher, or proteins from human urine is present in a concentration of 70 mg / ml or higher.

According to the present invention, HGI glycoprotein is obtained which has been subjected to an inactivation treatment for viruses which contaminate the EGI glycoprotein. The inactivation treatment comprises heating an aqueous solution of the HGI glycoprotein, which has been stabilized W me: heat in the following manner, at 50 ° C, preferably 70 ° C, preferably 55 ° C: iii 65 ° C for 8 to 50 hours, preferably 8 ° C. - 12 hours, one via one: pa of 5 to 9.

When albumin is used as a stabilizer, the degree of purification of the HGI glycoprotein present in the aqueous solution is not decisive, but HGI glycoprotein fractions with a degree of purity corresponding to the same for samples Nos. 4 to 6 in Example 1 are preferred. The concentration of the HGI glycoprotein in the aqueous solution to be heat treated is 0.1% (w / v, ie the number of units by weight in 100 units of volume; this applies in the following to all aqueous solutions) or higher, preferably 0.5 to 15%. The aqueous solution is adjusted = pH 5 - 9, preferably 6 - 8, by adding an acid or alkali solution, preferably a buffer solution. The albumin preparation used according to the invention may be derived from human serum or human placenta, both of which are suitably purified for medical use and have a purity of 80% or higher, as evidenced by electrophoresis. The amount of albumin to be added to the HGI glycoprotein-containing aqueous solution is 2.0% or more, preferably 10-20%.

The upper limit of the amount of albumin to be added is not fixed but the appropriate amount depends on the allowable amount of albumin in the final product. The use of albumin of human origin for stabilizing the HGI glycoprotein against heat eliminates the risk of contamination of the final product with antigenic substances and enables effective stabilization of the HGI glycoprotein against heating. Since albumin makes glycoprotein preparations stable during storage, it is not necessary to remove the albumin after the heat treatment as long as UT is used as an appropriate amount in the heat treatment. The heat treatment according to the invention is therefore an effective step which is to be included in the process for the production of the HGI glycoprotein and certainly forms part of the industrial production process which comprises a step for inactivating viruses.

Experiment 5 The effect of heat treatment in the presence of albumin on virus 5.4 .mu.g In infectivity was examined using various viruses capable of contaminating EGI glycoprotein preparations.

HGI glycoprotein prepared in the same manner as Sample No. U in Example 1 was dissolved in water to prepare a solution of 100 mg / ml. This solution was inoculated with 0.2 ml of a virus suspension (with a virus infection dose of 1x 105 - lx 106 / 0.2 ml measured according to a method described below) of chickenpox virus, mumps virus, measles virus, herpes virus, chikungunyavirus, Japanese encephalitis virus, rubella virus, poliovirus, coxsackievirus or echovirus followed by 0, 20, 100 or 200 mg / ml human serum albumin (Sigma Chemical Co.). The resulting solution was adjusted to pH 7.0 with 0.1 M Tris-HCl buffer, then heat treated at 60 ° C for 10 hours, then immediately cooled in ice-cold water, desalted, sterilized by membrane filtration and lyophilized to give a powder. was obtained. The biological activity and the viral infection were analyzed by the following methods and compared with them before the heat treatment.

The analysis of biological activity was performed by observing as a parameter the colony formation of bone marrow cells from mice in vitro. In each 35 mm diameter plastic petri dish was placed 1.0 ml of supplemented McCoy's 5A medium containing 20% fetal calf serum, 10% (0.1 ml) sample, 0.3% agar, 7.5 x 10 6 bone marrow cells. from mouse and 10% (0.1 ml) sample, making the concentration of each sample equal. After 7 days of incubation in a humidified atmosphere with 5% CO 2, discrete colonies containing more than 50 cells were counted with an inverted microscope. The ratio (in%) ... i | ..: UI 29 25 451 844 between the number of colonies formed after the heat treatment and that formed before the heat treatment was called recycled biological activity.

The dose of virus infection was determined as follows. Chickenpox virus was successively transplanted into HeLa cells and the plaque-forming unit virolegy, vel. 36, pp. 171: - 179 (1968f was determined for the same cell. Successive transplants of bovine virus, measles virus and Japanese encephalitis virus were performed with VERO cells and of police virus, coxsackievirus and echovirus with HeLa cells.

The infectivity of each virus was analyzed by determining the infectious dose for 50% of the tissue culture -National Institution of Health, Japan (ed.) "Experimental Virology", Maruzen Co., Tokyo, 196; whereby the cytopathic effect was used as an indicator. The observation period was ten days.

Herpesviruses and chikungunyaviruses were successively transplanted into FL cells and VERO cells, respectively, and the plaque-forming unit was determined ("Experimental Virology", loc. Cit.). Red dog viruses were successively transplanted into BHK-21 cells and the plaque-forming unit was determined with VERO cells.

The result obtained is given in Table 8. a 451 844 26 5 o 7.2 o S. SH So fi coš o Ûo fl ... oz og E, 9: oom HE fc HE fi no: ETÉC Two co fi oxæoc fi man .Hmåc: o fl oxwu: mm Lm fi:: aëzn fi m »Uo fl š fl ozm lmzk; lo fl oz .Hmorå | c.nw:.:> | o fi ox Hmomš. . xw fl wo fi cïo. . Lš fi wm ocmës:> w cwcczïomaæ ac fi coowcaa fi. nmvrš wc fi cuuwcma: wuwz co fi omnucmocox w H fi wndå \ JI 25 30 451 844 27 As shown in Table 8, the virus infectivity had been completely lost in each sample after the heat treatment at 60 ° C for ten hours, whether or not albumin was added. This fact indicates that other viruses can also be inactivated by the heat treatment according to the invention. It was found that the biological activity of the HGI glycoprotein could be preserved in the samples containing albumin, which confirmed sufficiently effective. that an addition of albumin is the result of a heat treatment under other conditions similar to those stated above. In the next experiment, the value of the aqueous solution of HGI glycoprotein containing albumin was varied for an examination of the change in biological activity after heat treatment.

Experiment 6 The same HGI glycoprotein used in Experiment 5 was dissolved in the water to prepare an aqueous solution of 20 mg / ml. To the solution was added 10 mg / ml human serum albumin (Sigma Co.). The solutions covering the pH range 2 to 10 were prepared in intervals on one unit as follows; pH 2 - 6, 0.1 M citric acid-sodium phosphate buffer, pH 7 ~ 10, 0.1 M tris-HCl buffer. Each sample was heated at 60 ÛC for ten hours, the remaining biological activity could be analyzed in the same way as in Experiment 5. The ratio (in%) between the number of colonies formed after the heat treatment and that formed before the heat treatment was designated â- recovered biological activity. The results obtained are given in Table 9. The biological activity of the HGI glycoprotein was remarkably preserved in the pH range 5 to 9, which shows that an adjustment of the pH of the HGI glycoprotein solution in this range is necessary before the heat treatment.

UI 451 844 28 Table 9. Number of colonies Recovered bio-ph per 0.1 ml logical activity Before heat treatment 86 100.0 2 0 0 BO 0 H 0 G 5 82 95.3 6 95 111.6 7 116 137.2 8 100 116.5 In another experiment not described later, an aqueous solution containing at least 70 mg / ml of HGI glycoprotein was adjusted to pH 2 - 10 with an acid or alkali solution and the recovered biological activity after heat treatment was examined. . It was found that the recovery of biological activity was about H0 - 50 at a pH in the range 5 - 9, while the biological activity of an aqueous solution of the HGI glycoprotein solution at a pH lower than 5 or higher than 9 completely disappeared after the heat treatment.

Experiment 7 The dependence of the effect of the heat treatment on the concentration of the HGI glycoprotein in aqueous solution was investigated.

A purified HG1 glycoprotein analogous to Sample No. 6 in Example 1 was dissolved in water and adjusted to pH 7.0 with 0.1 M Tris-H01 buffer. The solution was further adjusted to a final concentration of 10, 50, 60, 70, 80, 100, 150 and 200 mg / ml, respectively. Each solution was heated to 60 ° C for ten hours, then immediately cooled in ice water, sterilized by filtration and lyophilized.

The biological activity of each sample was analyzed in the same manner as in Experiment 5. Results obtained are given in Table 10. Table 10 Concentration of Number of colonies Recovered biological HGI glycoprotein per 0.1 ml activity (mg / ml) 0 50 31 25, H 60 35 28.2 70 MM 55.5 80 50 U0.3 100 5 41.1 150 55 42.7 200 56 55.2 Before heating bellows 100.0 document As shown in table 10, , when the concentration of HGI glycoprotein is 50 mg / ml, the recovered biological activity after heat treatment 25, U%. It was found that in order to recover a biological activity of at least 35% after heat treatment, one must have a concentration of HGI glycoprotein which is at least 70 mg / ml, preferably 100 - 200 mg / ml.

Experiment 8 The same HGI glycoprotein used in Experiment 5 was dissolved in water, adjusted to different pH values in the range 2 - 10 by adding 0.01 M citric acid / sodium phosphate buffer for the pH range 2 - 6 and 0.1 M Tris ~ HCl buffer for pH range 7 - 10 and each solution was adjusted to a final concentration of 100 mg / ml with distilled water, heated at 60 ° C for 10 hours, then immediately cooled in ice water and examined, as in experiments 5, with respect to biological activity after heat treatment. Some of the above solution (pH U, 5) was used before adjusting the pH.

The results obtained are given in Table ll. k: \ H f \ _1 CD f \ 7 \ 31 451 844 30 Table ll Number of colonies Biological pH recovered per 0.1 ml activity (%) Before heat treatment 136 lCC action 2 Ü CB 0 CCO 5 51 3?, 5 6 511 39,? 7 59 Ä3,3 8 63 146.5 9 * 9 35, Û 10 OC pH eg set GC As shown in table ll, when the pH of the HGI glycoprotein solution was in the range from 5 to 9, the recovery of biological activity 36, 0 - H6.3%. If the pH was below 5 or above 9, the biological activity was completely lost. The results of experiments 7 and 8 showed that in heat treatment of the HGI glycoprotein solution the concentration of HGI glycoprotein should be at least 7% mg / ml and the pH should be in the range from 5 to 9.

Experiment 9 The procedure of Experiment 5 was repeated except that the HGI glycoprotein was dissolved in water at a concentration of 230 mg / ml of water; serum albumin was not added and the solution was adjusted to pH 7.0.

After the heat treatment, only the dose of virus infection was determined as in Experiment 5. In the test, the virus activity was found to have disappeared completely. The results indicate that viruses other than those used can also be inactivated by the heat treatment according to the invention.

The above virus inactivation method by heating with a stabilizer should be applied to an HGI glycoprotein with a certain degree of purity. Attempts have therefore now been made to achieve a practical Uï | ... | (J | .. | UI 20 25 30 _55 451 844 31 process which can be used for the production of purified HGI glycoprotein. It has been found that an HGI glycoprotein solution containing at least 70 mg / ml of a protein derived from urine used as a starting material is a heat-resistant solution suitable for the purpose.This new process for stabilizing the HGI glycoprotein solution against heating is described in detail below.

One liter of fresh urine obtained from normal humans generally contains 30-50 mg of protein. Such proteins were concentrated in a known manner by ultrafiltration, ion exchange chromatography, adsorption on silica gel, salting out or a combination of these methods. Urine proteins including the HGI glycoprotein were further concentrated in vacuo to adjust the protein content to at least 70 mg / ml, preferably 100 to 150 mg / ml. If the protein content of the HGI glycoprotein-containing fraction is below 70 mg / ml, the loss of biological activity of the HGI glycoprotein upon netting becomes undesirably large. Although any crude fraction containing 70 mg / ml or more of protein from urine is suitable for the purpose, a crude fraction obtained in the above process for the production of HGI glycoprotein according to the invention is preferable in the new stabilization process. . The crude fraction is adjusted to a protein content of urine of at least 70 mg / ml and to pH 5 to 9, preferably pH 6 to 8.

The heat-stabilized HGI glycoprotein solution thus obtained or, if necessary, after purification according to the procedure described above, is heat-treated under the above conditions to give a fraction ready for use as a drug or a product having the above-mentioned physical properties. and chemical properties.

Experiment 10 The procedure of Example 1 was repeated to prepare an HGI glycoprotein-containing fraction analogous to Sample No. 1, except for the following modification: The precipitate formed by salting out with ammonium sulfate to 70% saturation was divided into eight parts. Each portion was dissolved in water and adjusted to pH 7.0 with 0.1 M Tris-ICl buffer.

The 8 solutions were set to a final protein content of 10, 50, 60, 70, 80, 100, 150 and 200 mg / ml, respectively. Each solution was further divided into two groups. One group was heat treated at 60 ° C for 10 hours and the other was not heated. All solution samples were further purified, as described in Example 1, to the level of Sample No. H in Example 1. This gave two series of samples with different proteinaceous activity in Table 32 (unprocessed). were examined with respect to biologist 451 844 content, of which one series (experimental sample) was heat treated and the other (control sample Each sample un in the same way as in experiment 5. The results obtained are stated m. ~ 0H Hsa cow wa @ .w @ H “wa ewa me n.NCH ANH oø fi z fifi ä 9: å H. m _o ~ o. E m.mm mo om fifi> oLmmxnwL®m m.Oæ om om OH.

AIN: m: OH m 1 ßn fi ccm w 1: NH ewa N 1 æ flfi co fi e 1 .f fi. cæ m -: OH os w | m fifl om> ohQHHonu: oz | Hae om m | wc fi ca H ez |: §1i = ~ wl.; @ »~» wwwm ||| ===. @ <Ls fi zc fi cx ~ m @: <Q fi mzc fi oaoam> oLL _: -, -i! I: 1 || l: i: | 5 |: i au. | . : Ii-s I.

NH * Massa _ \ T1 | ._ | Example M The procedure of Example 1 was repeated several times to give 100 mg of HGI glycoprotein with a purity corresponding to sample No. 6. To 100 mg of the thus obtained purified HGI glycoprotein was added. 100 ml of 10% human serum protein (Green Cross Co., Japan) as a stabilizer. The resulting solution was adjusted to pH 6.8 with 10% sodium hydroxide solution, heated at 60 ° C for 10 hours, then immediately cooled in ice water and sterilized with Millipore filtration system (Millipore Co.) with membrane filter with a pore size of 0.1 H5 μm. Each 1 ml of sterilized solution was aseptically filled into 1 ml portions in glass vessels sterilized by heating at 180 ° C for 2 hours. After lyophilization under aseptic conditions, the glass vessels were hermetically sealed to give 97 glass vessels each containing 1 mg of a preparation containing heat-treated HGI glycoprotein.

The preparations obtained above were tested for biological activity and viral infectivity in the same manner as in Experiment 5.

The biological activity was comparable to that of an untreated sample and no viral infectious activity could be detected.

Example 5 One thousand liters of fresh urine obtained from normal humans was treated in the same manner as in Example 1. The flow from the CM Sephadex C-50 ion exchange column was concentrated to 1 liter of a crude HGI glycoprotein solution. To the crude concentrate was added 10 liters of 0.1 M Tris 101 buffer (pH 7.0), the whole was stirred thoroughly and then concentrated to about one tenth using a hollow fiber DIAFLO ultrafiltration apparatus. To the concentrate were added 5 liters of 0.1 M: rie-Hcl buffer (pH 7.0) and 5 liters of DEAE-cellulose suspension (300 g DEAE cellulose, dry weight) equilibrated with 0.1 M trisz-H01 buffer ( pH 7.0). The mixture was stirred for 50 minutes and filtered after being allowed to stand under vacuum to collect DEAE cellulose. Thus, DBAE cellulose was washed with the addition of ten liters of 0.1 M Tris -H01 buffer (DH 7.0).

The DEAE cellulose was collected by filtration under vacuum, washed again with 10 liters of 0.1 M Tris-HO1 buffer containing 0.05 M sodium chloride and filtered under vacuum to collect the DEAE cellulose. To the DEAE cellulose was added 10 liters of 0.1 M Tris-HCl buffer (pH 7.0) containing 0.3 M sodium chloride and the whole was stirred to elute the HGI glycoprotein-containing fraction from Lu lJ kn h) CD 25 30 35 451 844 3, DEAE-cellulcsan. The eluate was desalted using a DIAFLO® hollow fiber ultrafiltration apparatus (type DC-30). The desalted fraction was lyophilized to give about 15 g of a powder. The powder was dissolved in 150 ml of distilled water and applied to a Sephadex® G-150 column (6.0 X 80 cm), which was equilibrated with 0.1 M Tris-HCl buffer (pH 7.0). The fractions corresponding to Ve / Vo ratios of 1.11 - 1.60 were collected. The combined fraction was thoroughly dialyzed against distilled water and it. _ Ü. The dialysis solution is concentrated by using a DiArso ß P hollow fiber ultralilting apparatus (type DC-2), yielding 100 ml of a concentrate containing about 3 g of crude HG2 glycoprotein. . 10 g of powdered albumin from human placenta with a purity of 98%, determined by electrophoresis, was dissolved in the concentrate obtained above. The resulting solution was adjusted to pH 6.5 with a 10% aqueous solution of sodium hydroxide, sterilized by trituration as in Example H, aseptically filled into 2.5 ml portions in glass bottles, lyophilized aseptically and hermetically sealed to give HO Vials each containing approximately 5.8 mg of heat-treated HGI glycoprotein.

The above preparations were tested for biological activity and viral infectivity in the same way as in Experiment 5. The biological activity was comparable to that of a preparation which was not heat treated and no viral infectivity could be detected.

Example 6 Four hundred liters of fresh urine collected from normal humans were adjusted to pH 8 with 10% sodium hydroxide solution and centrifuged under cooling at 10 ° C using a continuous centrifuge at 15,000 G to remove insoluble matter.

The supernatant was adjusted to pH 7 with 10 2 g of hydrochloric acid and passed through a silica gel column (10 x 30 cm). The substances adsorbed on the silica gel were eluted with NO liters of 5% ammonium solution. The eluate was adjusted to pH 7.5 with 1 N sulfuric acid and added with ammonium sulfate to a saturation of 70%, allowed to stand overnight at 0 ° C and the precipitate formed was collected by filtration. The precipitate was dissolved in 2 liters of 5% ammonium solution, placed in cellophane tubes (Vishking Co.) and carefully dialyzed against 0.05 M phosphate buffer (pH 6.5). The dialyzed solution was given a volume of 10 liters with the same buffer and passed through the 10 H UI 20 301 451 844 35 CM Sephadex® C-50 ion exchange column (H0 x HO cm), which was equilibrated with 0.05 M phosphate buffer (pH 6, 5), for the removal of the impurities by adsorption. Ten liters of the stream were concentrated using a DIAFLO® hollow fiber ultrafiltration apparatus (Amicon Co., type DC-30) and the concentrate was dialyzed against 0.1 M Tris-HCl buffer (pH 7.0) at 5 ° C overnight on similar method described above. The dialyzed solution was given a volume of one liter with the same buffer and passed through the DEAE cellulose column (4.0 x MO cm) equilibrated with the same buffer. The column: was thoroughly wetted with 0.1 M Tris-HCl buffer (pH 7.0) and then eluted with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.3 U sodium chloride. The fractions that can stimulate the proliferation and differentiation of mouse bone marrow cells in vitro, which were analyzed in the same way as in Experiment 5, were collected. The combined fraction was dialyzed against 0.1 M Tris-HCl buffer (pH 7.0). The dialyzed solution was again passed through the DEAE-cellulose sac clone (ε, 0 x M0 cm) equilibrated with the same buffer. The column was eluted by a linear concentration gradient elution with sodium chloride (C to 0.3 M). The fractions having a stimulating effect on the proliferation and differentiation of granulocytes were collected and mixed with ammonium sulfate to a saturation of 70%. The precipitate formed was collected, dissolved in a small amount of 0.1 M Tris -HO1 buffer (pH 7.0) and dialyzed against the same buffer.

Twenty ml of the dialyzed solution was applied to a Sephadex® G-150 column (H, 0 X 60 cm) equilibrated with 0.1 M Tris - HCl buffer (pH 7.0) and the flow fractions obtained at Ve / Vo ratios of 1.1, 1.45 were collected. The combined fraction was thoroughly dislylined against distilled water and the dialyzed solution was lyophilized to give about 500 mg of a powder.

Two hundred mg of the above powder was dissolved in 0.02 N phosphate buffer (pH 7.0) containing 1.0 M sodium chloride and passed through a column containing 100 ml of concanavalin A-Sefaros ® UB (Fine Chemical Laboratory). equilibrated with the same buffer.

The column was washed thoroughly with 0.02 M phosphate buffer (pH 7.0) containing 1.0 M sodium chloride and then eluted with a 0.02 M phosphate buffer (pH 7.0) containing 50 mN u-methyl-D-glycoside and 1.0 M sodium chloride. The fractions having a stimulating effect on the proliferation and differentiation of granulocytes, which were analyzed according to the method described in Experiment 5, were collected and dialyzed against distilled water. The dialyzed solution was lyophilized.

About 50 mg of the lyophilized powder obtained above was dissolved in 1 ml of 0.125 M tris-glycine buffer (pH 6.8) containing 10% glycerol and subjected to electrophoresis at 10 mA while cooling.

Using a preparative electrophoresis apparatus (type Fuji Kabara II from Fuji Riken Co., Japan) using 8% acrylamide gel (pH 8.9; 20 mm x 25 mm). The fraction with a relative mobility of 0.66 was recovered with 0.025 M tris-glycine buffer (at 8.5), then oylated against distilled water and the dialyzed solution was lyophilized to give about 10 mg of HGI glycoprotein.

The above procedure was repeated a number of times and about 1 g of HGI glycoprotein was obtained. One gram of the EGE glycoprotein thus obtained was completely dissolved in ten ml of water and added to 6.8 with a 10% aqueous solution of sodium hydroxide. The resulting solution was heated at 60 ° C for ten hours, then cooled in ice water, diluted 10-fold with sterile water and sterilized by filtration with a Millipore filtration system (Millipore Co.) equipped with a membrane filter with a pore size of 0 , ü5 um. Each 1 ml of the sterilized solution was aseptically filled into glass bottles sterilized by heating at 180 ° C for 2 hours. After aseptic lyophilization, the bottles were hermetically sealed. This gave about 97 glass bottles, each containing 10 mg of a heat-treated HGI glycoprotein preparation.

The above preparations were tested for biological activity and viral infectivity in the same manner as in Experiments 5 and 7. The biological activity was about H0% of the same for the preparation before the heat treatment and no viral activity could be detected.

Example 7 One thousand liters of fresh urine obtained from normal humans was treated in a similar manner to Example 6 and 2.5 liters of an aqueous solution containing the crude HGI glycoprotein obtained from the effluent of a CM Sefadex ® C-50 column . To the solution was added 25 liters of 0.1 M Tris-HCl buffer (pH 7.0). After thorough stirring, the solution was concentrated to about 1/25 of the original U1 | __ | CJ | .. | KJ! 50 .35 451 844 37 original volume using a DIAFLO® hollow fiber ultrafiltration apparatus. To the concentrate were added 5 liters of 0.1 M Tris-HCl buffer and 5 liters of DBAE cellulose suspension (300 g of DEAE cellulose in the dry state) equilibrated with 0.1 H Tris-HCl buffer (pH 7.0). The mixture was stirred for 50 minutes, allowed to stand and filtered under vacuum to collect the DEAE cellulose. The collected DEAE cellulose was washed by adding ten liters of 0.1 M Tris -HO 1 buffer (pH 7.0), filtered. under vacuum, washed with C.1 M Tris -HO1 buffer (7.0) containing 0.05 U sodium chloride and filtered in vacuo. The thus treated IEAE cellulose was stirred in ten liters of 0.1 M Tris-HCl buffer (at 7.6) containing 0.5 H sodium chloride to elute the EGI glycoprotein-containing fraction from the DEAE cellulose. The eluate was removed by using a DIAFLO® hollow fiber ultrafiltration apparatus (type DC-30). After lyophilization, about 15 g of a powder was obtained.

The lyophilized powder obtained above was dissolved in 153 ml of distilled water and applied to a Sephadex® G-150 column (6.0 x 80 cm) equilibrated with 0.1 M Tris-HCl buffer (at 7.0).

The HGI glycoprotein-containing fractions obtained at Ve / Vo ratios of 1.11 - 1.60 were collected and thoroughly dialyzed against distilled water.

The dialyzed solution was concentrated using a DIAFLO® hollow fiber ultrafiltration apparatus (type DC-2) to give 30 ml of a concentrate containing about 3 g of crude HGI glycoprotein. The concentrate was adjusted to pH 6.8 with sodium hydroxide and heated under the same conditions as in Example 6. The heat-treated concentrate was sterilized by filtration and aseptically filled into 1.0 ml portions in glass bottles.

After aseptic lyophilization, the glass bottles were hermetically sealed. There were thus obtained 30 bottles each containing 5.2 mg of the heat-treated HGI glycoprotein preparation.

The above preparations were examined for biological activity and viral infectivity according to the same method as in Experiments 5 and 7. The biological activity was about HO% of the same before the heat treatment and no viral activity could be detected.

Example 8 The procedure of Example 1 was repeated to give Ca-500 may (about 26 mg of active substance) HGI glycoprotein powder corresponding to sample No. H, except that the following steps were performed before the dialysis of the 5% aqueous ammonia solution of the precipitate obtained at 70% saturation of ammonium sulfate.

About 20 g of the precipitate that formed was dissolved in water to give a volume of 200 ml (100 mg / ml protein concentration). The resulting solution was heated at 60 ° C for 10 hours and then cooled in ice water to prepare a fraction containing heat-treated HGI glycoprotein. This fraction was dialyzed in the same manner as in Example 1. The biological activity of the obtained powdered HGI glycoprotein was comparable to that of the fraction before the heat treatment and no viral activity could be detected.

Example 9 About H50 mg of a powder corresponding to Sample No. H of Example 1 and containing 23.5 mg of HGI glycoprotein was obtained by repeating the procedure of Example 1, except for the following modification. _ ._ U _ U _ _. Ten liters of the effluent water solution: wax Ch Sepnaoex The ion exchange column was concentrated using a DIAPLO® hollow fiber ultrafiltration apparatus to a protein concentration of 70 mg / ml. The concentrated aqueous solution was heated at 60 ° C for ten hours and then cooled in ice water. The insoluble material formed was removed and the aqueous solution was placed in cellophane tubes (Visking Co.) and dialyzed against 0.1 M Tris -HO1 buffer (pH 7.0) at 5 ° C. The dialyzed solution was given a volume of 1 liter with the same buffer, whereby a solution corresponding to sample No. 1 in Example 1 was obtained.

The biological activity of the obtained HGI glycoprotein powder was substantially comparable to that of the powder obtained without heat treatment. No viral activity could be detected.

Example 10 About 500 mg of a powder corresponding to Sample No. U in Example 1 and containing about 26 mg of HGI glycoprotein was obtained by repeating the procedure of Example 1, except for the following modification.

The precipitate obtained by salting out the flow from the DEAE cellulose column with ammonium sulfate was dissolved in 0.1 M Tris -HCl buffer to a protein content of 150 mg / ml. The resulting solution was heated at 60 ° C for 10 hours and cooled in ice water. After the precipitate formed was removed by filtration, \ I1 | .. | The aqueous solution against 0.1 M Tris -HCl buffer to prepare a dialyzed solution was used instead of Sample No. 3 in Example 1.

The biological activity of the HGI glycoprotein-containing powder finally obtained was substantially comparable to that of the preparation obtained without heat treatment. No viral activity could be detected. EXAMPLE 11 Fifty liters of fresh urine obtained from normal humans were neutralized with a 10% sodium hydroxide solution and centrifuged by means of a continuous centrifuge with a coolant running at 10,000 g. The supernatant was concentrated. . It is immediately centrifuged ten times with the aid of an EIAFUO high-fiber ultrafiltration apparatus (Aminon Co., type DC-30, after equipped with a molecular weight separator of 1000,000 membranes). After adding 50 liters of water, the solution was again concentrated to prepare 3 liters of concentrate. The concentrated urine was further concentrated by means of a rotary vacuum evaporator to give about 50 ml of a concentrate with a protein content of 70 mg / ml. The concentrate was heated at 60 L 0.5 ° C for tic hours and cooled rapidly to prepare a heat treated HGI glycoprotein-containing fraction. After the formation of insoluble matter was removed, the concentrate was given a volume of 5 liters with 0.1 M Tris-HCl buffer (pH 7.0) and passed through the DEAE-cellulose column (1.0 x H0 cm), which equilibrated with 0.1 M tris-HCl buffer so that the HGI glycoprotein was adsorbed on the DEAE cellulose. The column was washed thoroughly with 0.1 M Tris-HCl buffer (pH 7.0) and eluted with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.3 M sodium chloride. Thereafter, the procedure of Example 1 was repeated, except that the eluate obtained above was used instead of the 5% aqueous ammonium solution of the precipitate obtained at 70% saturation with ammonium sulfate. In this way, about 200 g of HGI glycoprotein powder corresponding to sample No. H were obtained by repeating the procedure of Example 1.

The biological activity of the finally obtained HGI glycoprotein powder was substantially comparable to that of the preparation obtained without heat treatment. No viral activity could be detected.

Example 12 The procedure of Example 1 was repeated to collect it at 451 844 40 fraction corresponding to sample No. 3 in Example 1. The fraction was concentrated to a protein content of 70 mg / ml and heated at 60 ° C for 10 hours. Thereafter, the fraction was cooled in ice water, freed from the precipitate formed and treated in the same manner as in Example 1 to give about 10 mg of an HGI glycoprotein powder corresponding to sample No. š in Example 1. The biological activity of the finally obtained HGI glycol the copretein powder was essentially comparable to that of the preparation ezan heat treatment.

Claims (5)

451 844 f-ü ~, _ Glycoprotein, characterized in that it stimulates human bone marrow cells to form colonies of granulocytes and which glycoprotein has the following physical and chemical properties: (a) solubility: soluble in water, slightly soluble in chloroform and insoluble in ethyl alcohol and acetone; (b) specific optical rotation: Zi fi åo = 0 f 400 (0.25% aqueous solution>; (c) pH; 5.0-6.0 (1% by weight aqueous solution); (d) isoelectric point: pH 4.7 In 0.2; (e) thermostability: when heated at 601 0.5 ° C for 30 minutes in 1% aqueous solution, the stimulating effect on human proliferation and differentiation of humanogranulocytes is completely lost; (f) electrophoresis: the relative mobility is 0.25 in electrophoresis using sodium dodecyl sulphate-polyacrylamide gel; (g) infrared absorption: characteristic absorption at the following weight (cm "1): 3600-3200 (strong absorption), 1700-1600 (strong absorption ), 1550 (medium absorption), 1430-1360 (medium absorption) and 1150-1000 (broad band); (h) color reaction: colors characteristic of saccharides are obtained by the W-naphthol-sulfuric acid reaction, the indo1-sulfuric acid reaction , the anthron-sulfuric acid reaction and the phenol-sulfuric acid reaction; dyes characteristic of polypeptide binding and amino acids are maintained in the Lowry-Folin reaction and in the ninhydrin reaction after hydrolysis with hydrochloric acid; (i) amino acids contained in the protein moiety, proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan and arginine; (j) color and shape: substantially white and amorphous; (k) sugar composition of the polysaccharide moiety: 10.0-13.0% by weight expressed as glucose of neutral sugars, 3.0-7.0% by weight of sialic acids and 1% by weight of other amino sugars: (1) weight ratio between protein and polysaccharide: 75-85: 13.0-20.0; (m) Elemental analysis: 42.3-47.3% carbon, 5.7 ~ 7, BX hydrogen, 9.6-14.3 l nitrogen, 34.4-39.4% oxygen and 0.2% or less sulfur; and 451,844 4% (n) molecular weight: 75,000 to 90,000 daltons, determined by gel filtration, the glycoprotein being obtained from urine from a healthy human, which urine is first concentrated with respect to proteins contained therein, which are then contacted with a cation exchanger and then with an anion exchanger, active material after elution, the eluate was subjected to gel filtration chromatography, from there collected fractions were subjected to sugar affinity chromatography and the adsorbed active material was eluted, the eluate was subjected to preparative zone electrophoresis and the active material. A therapeutic agent for leukopenia, characterized in that it comprises at least 500,000 units of glycoprotein as active ingredient and excipient, which glycoprotein stimulates the human bone marrow cell to form colonies of granulocytes and which glycoprotein has the following physical and chemical properties: (a) soluble in water, slightly soluble in chloroform and insoluble in ethyl alcohol and acetone; (b) specific optical rotation: ÅQYÉÛ = Ü 2 400 (Ü, 25% aqueous solution); (c) pH; 5.0-6.0 (1% by weight aqueous solution); (d) isoelectric point: pH 4.7 I 0.2; (e) thermostability: when heated at 50: 0.5 ° C for 30 minutes in 1% aqueous solution, the stimulating effect on proliferation and differentiation of human granulocytes is completely lost; (f) electrophoresis: the relative mobility is 0.25 in electrophoresis using sodium dodecyl sulfate-polyacrylamide gel; (g) infrared absorption: characteristic absorption at the following weight (cm'1): 3600-3200 (strong absorption), 1700-1600 (strong absorption), 1550 (medium absorption), 1430-1380 (medium absorption) and 1150-1000 (wide band); (h) dye reaction: dyes characteristic of saccharides are obtained in the N-naphthol-sulfuric acid reaction, the indole-sulfuric acid reaction, the anthron-sulfuric acid reaction and the phenol-sulfuric acid reaction; dyes characteristic of polypeptide binding and amino acids are obtained in the Lowry-Folin reaction and in the ninhydrin reaction after hydrolysis with hydrochloric acid; (i) amino acids contained in the protein moiety, proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, (q 451 844 G5 methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan and arginine (j) color and shape: substantially white and amorphous; (k) sugar composition of the polysaccharide moiety: 10.0-13.0% by weight expressed as neutral sugar glucose, 3.0-7.0% by weight of aialic acids and 1% by weight of other amino sugars: (1) weight ratio of protein to polysaccharide: 75-85: 13.0-20.0; (m) elemental analysis: 42.3-47.3 X carbon, 5.7-7.8 X hydrogen, 9.6 14.3 X nitrogen, 34.4-39.4 X oxygen and 0.2% or less sulfur, and in) molecular weight: 75,000 to 90,000 daltons, determined by gel filtration, the glycoprotein being obtained from urine from a healthy human, which urine was first concentrated with respect to proteins present therein, which were then contacted with a cation exchanger and then with an anion exchanger, active material then eluted, the eluate subjected to gel filtration chromatography, from which fractions collected were subjected to sugar affinity chromatography and the adsorbed active material was eluted, the eluate was subjected to preparative zone electrophoresis and the active material was eluted. Therapeutic agent for leukopenia according to claim 2, characterized in that the viral infectivity is eliminated. The therapeutic agent for leukopenia according to claim 2, characterized in that the glycoprotein has a specific biological activity of at least 35,000 units / mg. A process for the production of a glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which protein has the following physical and chemical properties: (a) soluble in water, slightly soluble in chloroform and insoluble in ethyl alcohol and acetone; specific optical rotation: Zoíšß = 0 3 40 ° c (0.25% aqueous solution); (c) pH; 5.0-6.0 (1% by weight aqueous solution); (d) isoelectric point: pH 4.7 1 0.2; (e) thermostability when heated at 60: 0.5 ° C for 30 minutes in 1% aqueous solution, the stimulating effect on proliferation and differentiation of human granulocytes is completely lost; If) electrophoresis: the relative mobility is 0.25 in electrophoresis using sodium dodecyl sulphate-polyacrylamide gel; (g) infrared absorption: characteristic absorption at the following weight (cm'1): 3600-3200 (strong absorption), 1700-1600 (strong absorption), 1550 (medium absorption), 1430-1380 (medium absorption) and 1150-1000 (wide band); (h) dye reaction: dyes characteristic of saccharides are obtained in the drnaphthol-sulfuric acid reaction, the indole-sulfuric acid reaction, the anthron-sulfuric acid reaction and the phenol-sulfuric acid reaction; dyes characteristic of polypeptide binding and amino acids are obtained in the reaction of Lowry-FoLin and in the reaction of ninhydrin after hydrolysis with hydrochloric acid; (i) amino acids contained in the protein moiety, proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan and arginine; (j) color and shape: substantially white and amorphous; (k) sugar composition of the polysaccharide moiety: 10.0-13.0% by weight expressed as glucose of neutral sugars, 3.0-7.0% by weight of sialic acids and 1% by weight of other amino sugars: (1) weight ratio between protein and polysaccharide: 75-85: 13.0-20.0; (m) Elemental analysis: 42.3-47.3% carbon, 5.7-7.6 X hydrogen, 9.6-14.3 X nitrogen, 34.4-39.4 X oxygen and 0.2 X or less sulfur; and (n) molecular weight: 75,000 to 90,000 daltons, determined by gel filtration, characterized in that healthy human urine is concentrated with respect to proteins contained therein, these proteins being contacted with a cation exchanger to remove contaminants by adsorption on the ion exchanger, the flow is contacted with an anion exchanger for adsorption of the active material, active material is eluted with a saline solution according to linear concentration gradient elution, the eluate is subjected to gel filtration chromatography on a highly crosslinked polymer gel to develop the active material with fractions. 1.11 to 1.60 are collected, the collected fractions are subjected to affinity chromatography with an adsorbent having an affinity for sugar to adsorb the active material, the adsorbed active material is eluted with a 20-100 mM saccharide solution, the eluate is subjected to preparative zone electrophoresis, the active u,