JPH02152995A - Separation and purification of human granulocyte-macrophagecolony-stimulation factor - Google Patents

Abstract

PURPOSE:To separate and purify the subject factors produced by animal cell and having different sugar-chain structures without damaging the sugar-chain structure according to the content of the sugar chain by using two specific kinds of column chromatographic means in combination. CONSTITUTION:A human granulocyte-macrophage colony stimulation factor (hGM-CSF) produced by an animal cell is treated by immobilized wheat embryo lectin affinity chromatography column to separate into an adsorbed fraction mainly containing 2N-mer hGM-CSF and a non-adsorbed fraction. The adsorbed fraction is treated by a hydrophobic chromatography to separate the 2N-mer hGN-CSF from said fraction. If necessary, the non-adsorbed fraction of the affinity chromatography column is treated by the hydrophobic column chromatography to separate ON-mer or 1N-mer hGM-CSF from the fraction.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention provides human granulocyte/macrophage colony stimulating factor (
human granulocyte-macropH
agecolonystimulaLing fact
The present invention relates to a method for separating and purifying hGM-C3F (hereinafter abbreviated as hGM-C3F).

<Prior Art and Problems> GM-C3F is a type of cytotoxic cell that acts on white blood cell stem cells present in the bone marrow and promotes their differentiation and proliferation into granulocytes and macrophages. It is thought to be beneficial in the treatment of leukopenia caused by administration of anticancer drugs, etc.

Various human cells produce hGM-C3F, and these are called natural hGM-C3F.As a result of immunological analysis using anti-CM-C3F antibodies, natural hGM-C3F was produced.
The molecular weight of CSF was found to be heterogeneous from 16 to -35, and the molecular weight of its main component was found to be 2530 (R, ε, Donahue et al. ColdSpri
ng 1larbor 5yIllposia
on QuantitaLiveBiology 5
1'J 685i 1000), while hcM-C
The 3F gene has already been cloned (GWong
5science, vol. 228, p. 810, 1985+
F-Lee et al. Proc, Nap, Acad, S.
ci, vol. 82, p. 4360, 1985), hcM-C3F has been produced using Escherichia coli, yeast, or animal cells as hosts using genetic engineering techniques;
tJ for M-C3F! Due to the existence of a, natural h
Recombinant hGM-C with a structure as close as possible to GM-CSF
For the production of 3F, it is desirable to use animal cells as a host. Examples of host animal cells include CO3 derived from monkey kidneys.
CellsChinese hamster ovary cells (CHO cells)
Human lymphoblastoid Namalva cells are used, but genetically recombinant hGM-C derived from these animal cells
Heterogeneity in the sugar chain structure is also known in 5F.

hGM-C3F produced in animal cells includes Mi recombinant type,
Regardless of the natural type, 0-2 N-glycosidic bonds and multiple O-glycosidic bonds tJh per molecule
chains exist, and the molecular weight heterogeneity is mainly 11G M
This is due to the number of N-glycoside-linked sugar chains added to CSF. (K, ni aushansky rari ocJ+emi
sLry, vol. 26, p. 4861, 1987),
When hGMC3F is divided according to the number of N-glycosidic linkage sugar chains added, the one with both added positions is the 2N form, the one with only one place is the IN form, and the one with only the O-glycoside linkage added is the 0N form. Their molecular weights are approximately 23-35 for the 2N-isomer and 18-23 for the 1N-isomer.
and 0N body: 16-18.

However, these molecular weights may vary depending on the producing cells, culture conditions, detection method, etc. hGM-
IJ at C3F! Although the significance of the chain is largely unknown, it is predicted that it is involved not only in increasing the efficiency of secretion from producing cells, but also in in vivo stability, mW distribution, and signal transmission.

On the other hand, when using hGM-C8F for therapeutic purposes, it goes without saying that it is desirable to develop hGM-C3F that has a sugar chain structure with the highest therapeutic effect, and for this purpose, it is necessary to separate hGM-CSF based on the sugar chain structure. However, 1. IT
At present, separation techniques based on differences in chain structure have not been established. That is, hGM-C3 from animal cell culture
Reverse-phase chromatography is often used as a purification method for F, but in reverse-phase chromatography, an acid such as trifluoroacetic acid is often included in the moving phase to obtain high separation power, and the sugar chain of bGM-C3F is There is a risk that the sialic acid present at the end of the hGM-C3F may be eliminated by acid hydrolysis, and it has been difficult to fractionate hGM-C3F produced in the culture solution and having a non-uniform molecular weight based on its sugar content.

On the other hand, concanavalin A (
Affinity chromatography in which lectins such as Con A) and wheat germ lectin (WGA) are immobilized on an insoluble carrier is commonly used, and even in the case of hGM-C3F, Con A (A, J, Luis et al.
od vol. 57, p. 13, 1981) and W.G.A.
(P. Moonen et al. Proc. Nati. Ac
ad, Sci, vol. 84, p. 4428, 1
There is a report using 987). However, these reports involve the process of partially purifying hGM-C5F by removing contaminant proteins from the hGM-C3F-containing culture solution, and the partially purified hGM-C3F is of low purity and still contains many contaminant proteins. However, the behavior in columns of 0N, 1N, and 2N bodies is unknown. Therefore, clearly different from the method of the present invention, hC;MC3F is converted into N7'J
Cosidic bond 1! It was difficult to separate and purify it due to the number of t1 additions.

Therefore, in purifying hGM-C3F produced by animal cells, it is important to establish a separation and purification method that can fractionate according to the sugar chain content without damaging its sugar chain structure.
This is extremely meaningful in developing hGM-C3F as a therapeutic drug.

<Means for Solving the Problem> As a result of intensive research, the present inventors have found that by combining 21% m column chromatography, hGMCSF produced in animal cells with different sugar chain contents can be separated according to their sugar chain content. They found a method for purification and completed the present invention.

That is, among various protein purification methods such as gel i11 perchromatography, ion exchange chromatography, affinity chromatography, hydroxyapatite chromatography, hydrophobic chromatography, and reverse phase chromatography, wheat germ lectin (
We have found that by combining immobilized lectin affinity chromatography using (WGA) and hydrophobic chromatography, it is possible to purify hGM-CSF with different sugar chain contents by a number of minutes depending on the sugar chain content.

The present invention subjects bGM-C5F produced by animal cells to wheat germ lectin affinity chromatography, and then uses hydrophobic chromatography to determine the number of N-glycoside-linked sugar chains bonded to the molecule.
A method for separating and purifying 3F is provided. hcM-C3F separated and purified by this method contains I or a plurality of O-glycoside-linked sugar chains, but the number of the bonds is not uniformly determined.

The animal cells used in the present invention include human-derived cell lines such as monocytic cell line U-937 and leukemia cell Mo,
Various human cells and tissues, such as T cells, monocytes, macrophages, fibroblasts, endothelial cells, placental cells, lungs, bladder, etc., or genetically engineered cells and tissues transformed to produce hGM-C3F, e.g. Examples include animal cells such as CO5 cells, Cl-1 cells, and Namalva cells. Industrially, animal cells transformed by genetic engineering are preferred. hGM-C produced by these cells naturally or under some stimulus, such as plant lectins, phorbol esters, calcium ionophores, etc.
Any hGM-C3F that has been purified or partially purified using the general protein purification methods described above or a combination thereof from a culture solution containing 3F can be used as a starting material in the present invention. When separating and purifying the 2N form, a sample solution in which the purity of hGM-C3F is about 10% (W/W) or higher (i.e., containing hGM-C3F at about 10% (W/W) or more) is used. is available, but also 0
When separating and purifying N-isomer and 1N-isomer, hGM
A sample solution purified so that the purity of -C3F is about 70% (W/W) or more, more preferably 90% (W/W) or more is preferable. hcM-C3F sample solution is protein M
It can be prepared using common purification means or a combination thereof, and for example, it can be prepared as follows. h
c; M-C3F producing cells are cultured for several days in an appropriate animal cell culture medium such as RPMI-1640 medium containing fetal calf serum (FCS) or dish-free medium. After culturing, cells are removed by centrifugation, filtration, etc. to obtain a culture supernatant.

This culture super-soak is concentrated using appropriate means such as ultrafiltration and ammonium sulfate precipitation. 'a condensate with anti-hcM-C3
The column is added to a F monoclonal antibody adsorption column, gel filtration column, anion exchange column, etc., and after washing the column, 7 volumes of 1+ GM-CSF are discharged using an appropriate eluent.

The obtained purified or partially purified hGM-CS
The F-containing sample solution was converted into a tris-salt M mild IIi solution containing sodium chloride,
Salt-containing HEPES (N-(2-hydroxyethyl)
piperazine-N″-2-ethanesulfonic acid) buffer,
Usually used 11 fJ such as salt-containing phosphoric acid 11 solution
Add to a WGA immobilized affinity column equilibrated with i't&. As a carrier, carriers used in ordinary column chromatography such as agarose and cellulose can be used, but because of their high processing speed and reproducibility, carriers such as superhardened agarose, synthetic polymers, or silica gel are usually used in high performance liquid chromatography (HPLC). and agarose are preferable. Examples of carriers bound with WGA include WGA-agarose (Seikagaku Corporation) and WGA-Sepharose (Pharmacia Corporation), preferably WGA-agarose (Seikagaku Corporation). be able to. The hGM-C3F-containing sample solution has, for example, p114 to about 9, and several g/mf to several mg/
Although it is preferable that about mN of bGM-CSF be contained, the present invention is not particularly limited thereto. sample? 8
The buffer used for the solution is the usual mild i! I-liquid can be used. The sample is preferably added in an amount of, for example, IO μg to 10 mg/+nem, but is not particularly limited thereto.

The WGA immobilized affinity column contains 2N hG
Adsorbs MC3F, but 0N form and 1NNO2-C3
Most of F is not adsorbed.

After washing the column with the buffer used for equilibration, the adsorbed 2N hGM-C5F is eluted. It is important to use a buffer containing N-acetylglucosamine or N-acetylneuraminic acid as an eluent. The sugar concentration is not particularly limited, but is generally preferably about 1mM or more, more preferably 0.2M or more. The buffer solution contains, for example, about 1"OO to 200mM of sodium chloride, preferably about 9.5 to 100mM of p114-9.5 to 100mM Tris-HCl solution, HE r'ES buffer, phosphate buffer, etc. In addition, those used in ordinary column chromatography can be used as appropriate.The salt concentration is not limited to the above concentration as long as non-specific adsorption is avoided. The concentration is not limited to the range listed in L as long as it has a slow fJi effect.The column size is, for example, a column size of approximately 5 mm to 5 cm in diameter and 5 cm to 1 m in length, which is used in ordinary affinity chromatography. Although the flow rate varies depending on the size of the column, it is preferable that sample addition, washing, and elution are performed at, for example, a linear flow rate of about 1 to 1000 nff/cm''/hr. At that time, about 5 to 50 times the column volume,
Preferably, a washing solution of about 5 to 10 times the volume and an eluent of about several times the column volume, preferably about 2 to 5 times the volume of the column, can be used. Are these conditions also normal? This can be done within the scope of affinity chromatography. The column temperature is, for example, 50°C or less, preferably 4
"C to room temperature, but there are no particular restrictions as long as the temperature range does not deactivate the sample."Next, using hydrophobic chromatography, the 2N hGM-C3F-containing Wi8 obtained above was Separate the 0N body and 1NNO2-C3F that are still slightly mixed in the liquid, and also separate the 0N body GM-C3F and 1NNO2 contained in the non-absorbed fraction.
- Separate from CSF.

The 2N hGM-C3F-containing elution fraction or the non-adsorbed fraction was added with the buffer described later, and the obtained 2N hGM-
The C3F-containing solution is added to a hydrophobic column chromatograph, such as phenyl superose, equilibrated with the buffer. After washing the column with the buffer used for equilibration, as the salt concentration contained in the buffer was lowered, based on the difference in hydrophobicity of hGM-C3F due to the difference in sugar chain content,
From bGM-C3F with large molecular weight to l1ii1? 8 is served.

As a result, 0N, 1N and 2N GM-C5F can be separated and purified.

Ligands used in hydrophobic chromatography include:
For example, a hydrophobic functional group having a carbon number of C0 to C4, such as a phenyl group, a neopentyl group, a butyl group, an ethyl group, etc. can be used, and a phenyl group is more preferably used. As the bound insoluble lu form, carriers used in ordinary column chromatography can be used, as in the above-mentioned affinity chromatography.
A carrier commonly used in high performance liquid chromatography (HPLC) is preferred. Examples of such carriers bound include Phenyl Superrose (Pharmacia) and T S K gel Phenyl 5PW (Tosoh).
It is important that it is greater than M, preferably about 1.5 to 2M. Examples of buffer solutions include sodium sulfate,
Contains sodium citrate, ammonium sulfate, etc.
Preferred examples include, but are not limited to, buffers containing ammonium sulfate, preferably around 10 to 100 mM phosphate buffers, with pH around 4 to 9.

Lower the salt concentration by increasing the proportion of salt-free buffer. For example, apply a gradient of 15 to 60 m1n, preferably about 30 ain, and hGM-C3
I can get 8 F's. Although it differs depending on the column size, sample addition, washing, etc.
mj! /cm”/hr.

At this time, it is preferable to use a washing solution of about 5 to 50 times the strength, and to add the sample in an amount of, for example, IO μg to 100 mg/rnf carrier, but the amount is not particularly determined. The eluent volume is appropriately determined based on column size, flow rate, and gradient time.

The column temperature is set within a temperature range that does not deactivate v: and t:+. The column size is, for example, about 5ffi in diameter.
A normal one with an I11 to 5 cm and a length of about 5 cm to 1 m can be used. These conditions can be carried out within the range of conditions for conventional hydrophobic chromatography, preferably hydrophobic chromatography using HPLC.

2N body, 1N body, 0N body, bC obtained by the above method
; M-C3F is present in the above buffer, but the buffer can be easily replaced with another desired buffer by a commonly used method, such as dialysis or gel filtration. .

The 2N, 1N, and 0N hGM-C3F separated and purified by hydrophobic chromatography each exhibited a single band in polyacrylamide electrophoresis, where 0 to 2
From hGM-C3F, which is a mixture of N-isomers, 2N-isomer and 1N-isomer
It is confirmed that 0N and 0N (*h GM-CSF are separated and purified, respectively).

Next, the present invention will be explained in more detail with reference to Examples.

However, the present invention is not limited thereto and may be modified as commonly practiced in the technical field of the present invention.

Example In this example, hGM-C3+ was quantified using the following two methods.

(]) Colony hamster femurs were removed aseptically and treated with 2% live fetal blood.
Contains a MEM culture! ? &(Stanners, c,
p-++rL, a+, Nature New Biology, 230:52 (1972) (manufactured by Flow)) is injected, the bone marrow cells are washed out, the cells are broken up by pipetting, and the cells are allowed to stand for 3 minutes. cell float
Il! After taking the solution and washing with the same culture solution, the cells were adjusted to a cell concentration of 1×10/ml with α-MEM culture solution containing 40% tallow serum. This 11/lung cells) γ fluid and 2%
Human GM-C5F, which was serially diluted with α-MEM culture medium containing live fetal serum, was added to each well at 100 μP, and 37
C., 5% carbon oxide. After 42 hours of culture, 25 μ2 of tritium-labeled thymidine prepared at 40 pci/fIdl with α-MEM culture medium is added to each well, and after a further 6 hours, the radioactivity incorporated into the cells is measured.

The specific activity of GM-C5F was determined by defining CM-C3FII, which induces tritium 1 m thymidine uptake of (CV/CC)/2, as 50 units/d. However, CM represents the maximum amount of tritium-labeled thymidine uptake, and CC represents the amount of tritium-labeled thymidine included in the CM-CSF absence count.

(2) Sandwich radioimmunoassay using anti-hGM-C5F monoclonal antibody (Immun).

One polystyrene ball (first antibody immobilized ball) on which an anti-hGM-C5F monoclonal antibody was immobilized (Radiometric As5ay: IRMA) was prepared.
C; M-C3F-containing sample LOOul.

0.25% 5% run n 7 /I/ 7゛mi 7 (BSA
) and 0.02% NaN, containing 0.03 MIJ 7
Add 400 ul of acid buffer (Pf17.4) mixed solution and incubate at 37°C for 24 hours. After that, 2 ml of 0.85% NaCl! Wash the ball 3 times with It', (l)pill the second antibody solution at 1100μffi.
and 0.25% B S A ・0.02% NaN 3
Contains 0.03M phosphate buffer (P117.4) 400μ
Add the mixture of E and incubate at 4°C for 18 hours and then at 37°C.
Incubate for 5 hours at 1.

Thereafter, the ball is washed three times with 0.85% NaCI2mR, and the radioactivity of the It51-second antibody bound to the first antibody-immobilized ball is determined using a T-counter. 4
m! A calibration curve was prepared using hGM-C3F of known concentration as a quasi-substance, and hcM-cs in the sample was determined from this test IvA.
Calculate F concentration.

The amount of protein was measured using Protein Analyzer manufactured by Biola Ndo Co., Ltd. using bovine serum albumin as a standard substance, and electrophoresis was performed using the method of Laesmi et al. (Nature, 227) using a 3% concentration gel and a 12.5% separation gel.
S680 p. 1970).

Example 1 h 2N isomer, 1N isomer and 0 from GM-CSF
Isolation of N-body GM-C8F Main production process l〉 Technique M Futami Σ
029555) in RP old-1640 medium containing 10% fetal bovine serum (Fe2) in the presence of 5% carbon dioxide.
Culture at 7°C for several days, centrifuge after completion of culture, and Iv
A culture supernatant from which cells were removed was obtained by filtration. In this section, the supernatant was concentrated by ultrafiltration or ammonium sulfate precipitation, and equilibrated with phosphate buffered saline (PBS).
°C) anti-hGM-C3F Monochrome I:J-Nal antibody adsorption column (see patent application No. 63-87554) (2°2C)
mx12.5cm) to 60m1! It was added at a flow rate of /hr.

After washing the column with 480 ae PBS, hGM-C3F was eluted with 0.1 M sodium citrate (ph 2.8). The eluate was fractionated into tall portions and immediately neutralized by the addition of 2M Tris-HCl (P) 1B,0).

hGM purified using an antibody adsorption column in this way
-C3F has a purity of 90% (5DS-polyacrylamide gel electrophoresis (SDS-PAGE)).
-8) or above and a 2N form with a molecular weight of 16-35 KDa, 1
It was a mixture of N-form and ON-form. (Fig. 1) Step 2> WGA-Agarose Calm Chroma It CM-CS, which is the O-2N mixture obtained in Step 1
F(3,82B) in 20% solution containing 100mM NaCl
+sM) WGA-agarose column (3.2 cm) equilibrated (4'C) with Lis-HCl buffer (pl+7.2)
x6.2cm) and 20m1! It was added at a flow rate of /hr.

After washing the column with 400 d of the buffer used for equilibration, 0.
2M N-acetylglucosamine and 100mM N
Tris-hydrochloric acid I1 containing aC1 lf! (pH 7,2)
And hGM-C3F? I rolled an 8. As a result, most of the hGM-C3F contained in the elution fraction was 2N, and the flow-through fraction and the washed/? The non-adsorbed fraction combined with liquid I contains 2N GM-
C3F was hardly detected.

Column adsorption fraction (2N body) and non-adsorption fraction (0N, 1N
The hGM-C3F recovered in the body) was 2.151I
g ([!Monthly yield 56%), 1.39 mg (recovery rate 3
7%).

Step 3> Phenyl Superrose Calm Chromatography 1" horizontal (1) 2N hGM-C3F obtained in purification step 2 of 2N hGM-C3F (1° 19 mg
) was dissolved in 100mM phosphoric acid containing 3°0M ammonium sulfate;
After diluting 2 times with an acid solution (PI+7.0), a phenyl superose column (0
, 5 cm + x 5 cm) at a flow rate of 0.5 affi/win (Fast Protein Liquid
ChroIllatographHy (FPLC Famarnoa)), buffer L & 5 t* used for equilibration
In step 1, apply a targeted gradient directly to the column with fcfp solution so that the ammonium sulfate concentration becomes OM for 30 minutes hC; M-C3
F was eluted. By separating the peak eluting at ammonium sulfate concentrations of 1.0-0.83M, 0N and 1N
2N body hGM-C3F0.
99 mg (recovery rate 83%) was obtained. Furthermore, the buffer solution was replaced with PBS by gel filtration using Sephadex G25 (Furu9fu Co., Ltd.) equilibrated with PBS.

The molecular weight of the 2N hGM-CSF thus purified was 23-35 KDa as a result of 5DS-PAGE (Fig. 1). Furthermore, the specific activity of 2N body bC;M-C3F measured using hamster bone marrow cells was approximately 4X10'
unit/IIIg protein.

(The WCA-agarose non-adsorbed fraction obtained in separation and purification step 2 of 2J 0N body and 1NNO2-C3F was
1 by ultrafiltration using O membrane (Amicon).
After concentrating to 5 ml, it was added to a phenyl superose column under the conditions shown in section (1).

Bimodal peaks were observed at the elution positions at ammonium sulfate concentrations of 0.77M and 0.65M, with ammonium sulfate concentrations of 0.82-0.
The peak eluting at 70M was 1NN GM-C3F, 0.
The peak eluting at 680.50M is 0NN GM-C3.
It was collected as F.

1N body and 0NN body C;M separated and purified in this way
-The molecular weight of C3F is 18-2 respectively on 5DS-PAGE.
3K (1N form) and 16-18K (0N form) (Fig. 1) Example 2 Purification of 2N form hGM-CSF from hGM-C3F <Step 1>1'' 2C3I hGM-C3F-producing Namalva cells (2) cultured in a medium containing Saidasa serum-free medium 2 Neetrichyte 11 (Boehringer)
After ultrafiltration and ammonium sulfate precipitation at 80% saturation, the culture supernatant of Japanese Patent Application No. 63-029555 was treated with 2 containing 50mM NaCl.
Ultrogel AcA34 column (2.6 cm
95 cm, IBF) at a flow rate of 30 id/hr, and gel filtration chromatography was performed. hGM-
The C3F-containing eluate was transferred to a MonoQ column (0.5 cm x 5 am,
2111197) at a flow rate of 1I11/sin, and anion exchange chromatography was performed. NaC
The protein was released by linearly increasing 111 degrees to 400a+M.

hC; hGM contained in M-C3F-containing 18i' eluate
C3FI is 115μ8. The protein amount was 680j7g, the purity was about 1Ox (W/W), and the specific activity was about 57 times higher than that of the culture supernatant.

<Step 2> Partially purified hGM-C3F obtained in Step 1 (115 pg
) was applied at 4.4 d /
It was added at a flow rate of hr.

After washing the column with the buffer solution 25 used for normalization, 0°2M N-acetylglucosamine and 150mM N
The protein adsorbed on the column was eluted with a 5011M Tris-HCl buffer containing aC1. Most of the proteins contained in the column elution fraction were 2N hGM-C5F, but some contaminant proteins were also contained. Also, anti-hGM-C
It was confirmed by Western blotting using SF antibody that the column-unadsorbed fraction did not contain 2N GM-C3F.

hGMC3F recovered from WGA-agarose column
was 36 Mg (recovery rate 31%), and the specific activity increased about 7 times during this process.

Next, WGA containing this hGM-C3F (24Mg)
- Transfer the agarose column elution fraction to l containing 3.4M ammonium sulfate.
After diluting 2 times with oosM phosphate buffer (ρ117.0), 501IM phosphate buffer containing 1.7M ammonium sulfate (pl+7
．． Phenyl Superrose column (0) equilibrated with
0.5cm
hGM-C3F adsorbed on the column was eluted by applying a linear ammonium sulfate gradient under the conditions shown in Step 3 (I) of Example 1. Collect a single peak eluting at ammonium sulfate concentration and collect 2N ttc;
19 μg (recovery rate 79%) of M-CSF was obtained.

2 thus purified from partially purified hGM-C5F.
When N-isomer hGM-C3F was subjected to 5DS-PAGE, a single band was detected at Oa with a molecular weight of 25-35.

[Brief explanation of the drawing]

Figure 1 shows the S of ttGM-C3F purified according to the present invention.
Lane 1 shows the results of DS polyacrylamide gel electrophoresis. Lane 1 is a mixture of 0N to 2N hG before separation and purification.
M-C3F, lane 2 is 2N hGM C3F. Lane 3 is 1N hGM SF. Lane 4 is the electrophoresis result of 0N hGM-C3F. Figure (KDa) 14.7

Claims (5)

[Claims] (1) A method for separating and purifying human granulocyte-macrophage colony stimulating factor produced by animal cells, which consists of the following steps (A) and (B), or (A), (
A method for separating and purifying human granulocyte-macrophage colony-stimulating factor characterized by comprising B) and (C) Step (A): human granulocyte-macrophage colony-stimulating factor produced by animal cells is added to immobilized wheat germ lectin. Using an affinity chromatography column, step (B) is used to fractionate into an adsorbed fraction mainly containing 2N human granulocyte-macrophage colony-stimulating factor and a non-adsorbed fraction; using hydrophobic chromatography, step (B) Step (C) of separating and purifying the 2N human granulocyte-macrophage colony stimulating factor from the adsorbed fraction of A); using hydrophobic chromatography, the 0N or 1N human granulocyte-macrophage colony stimulating factor is isolated from the non-adsorbed fraction of step (A); Separating and purifying at least one human granulocyte-macrophage colony stimulating factor (2) The method according to claim 1, wherein the ligand for hydrophobic chromatography is selected from a phenyl group or a hydrophobic functional group having a carbon number of C_1 to C_1_0. (3) The method according to claim 1 or 2, wherein the carrier for affinity chromatography is selected from agarose, superhardened agarose, synthetic polymer, or silica gel. (4) Claims 1, 2 or 2, characterized in that the carrier for hydrophobic chromatography is selected from agarose, superhardened agarose, synthetic polymers, or silica gel. Method described in Section 3 (5) The animal cell is a cell transformed with DNA encoding human granulocyte/macrophage colony stimulating factor to produce human granulocyte/macrophage colony stimulating factor. Section 1,
The method described in paragraph 2, paragraph 3 or paragraph 4