JPH03133935A - Natural-type human interleukin 6 composition - Google Patents

Abstract

PURPOSE:To provide the subject composition consisting of a mixture of interleukins having 3 kinds of N-terminal amino acid sequences, simultaneously produced from human fibroblast by a super induction method for the production of human IFN-beta and separated and purified from the product. CONSTITUTION:The objective natural-type human interleukin 6 composition contains 3 kinds of human interleukin 6 (IL-6) originated from human fibroblast and having the N-terminal amino acid sequences of formula I, formula II and formula m at ratios of 3:5:2. A cultured liquid of fibroblast produced by super-induction method is treated with an insoluble carrier bonded with a blue dye to adsorb the most part of IFN-beta and obtain a non-specific adsorbed fraction containing >=90% of IL-6. Said fraction is made to contact with a silica adsorbent to effect the adsorption of IL-6 and eluted with an acidic solution. The obtained solution is subjected to salting-out to precipitate IFN-beta and other contaminant proteins and obtain a supernatant containing IL-6. The objective purified composition is produced by hydrophobic chromatographic treatment of the supernatant.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the production of human interferon β from human fibroblasts.
This invention relates to natural human interleukin 6 co-produced by superinduction method to produce . Human natural interleukin-6 according to the present invention is a useful substance that can be widely used as a therapeutic agent for immunodeficiency, bone marrow suppression after bone marrow transplantation, thrombocytopenia, and the like. It is also useful as a standard product for measuring interleukin-6 concentration in blood, etc.

[Prior art] Antigen-stimulated B! It is known that differentiation-inducing factors derived from T cells are necessary for differentiation of JA cells into antibody-producing cells (Nature N, Biol, 237
゜15 (1972)). The general term for factors that differentiate B cells into antibody-producing cells is B CD F (B cell di
It is called ferentiation factor). This BCDF is obtained by separating lymphocytes from human peripheral blood, tonsils, etc. using density gradient centrifugation, etc., and then using Epstein.
It is known that it can be obtained by transforming human B cells using -narr Virus, establishing a human BCDF-producing B cell line, and culturing this.
-169424). In addition, by culturing lymphocyte mixed reaction cells, mitogen-stimulated T cells, T cell hybridomas, etc., we detected BMJ cell differentiation captives (TRTi'1) that have sugar chains in the active site and B cell factors that do not have sugar chains. (T.R.
F2) is known to be obtained (JP-A-60-2
37022). Furthermore, a B cell differentiation factor produced by large cells transformed by human T cell leukemia virus was proposed as a B cell differentiation prisoner protein product (Japanese Patent Application Laid-Open No. 1983-1998).
-115025). Also human T lym
B isolated from the culture supernatant of a human T cell line transformed with phototropicvirLls type I
A 121 kd protein with CDF activity is BSF-
2 (T, l1irano et al., Proc.
, Natl, Acad, Sci, USA, 82.540
0 (1985)). As mentioned above, B cell differentiation factors produced by lymphoid cells such as B cells and T cells have been reported.

Subsequently, with rapid progress in genetic engineering technology, the DNA sequence of the gene encoding B cell differentiation factor 2 (BSF-2) and the amino acid sequence of the protein were determined (Japanese Patent Application No. 184858/1983, i3-1579
90). Furthermore, using this gene, prokaryotic cells transformed with recombinant DNA consisting of vector DNA capable of replicating in prokaryotic cells are cultured in a medium,
It has also become possible to collect the produced human BCDF (Japanese Patent Application Laid-open No. 157996/1983). And this human BC
It has been discovered that an immunotherapeutic agent containing DF as an active ingredient is effective against cancer, primary and secondary immunodeficiency diseases, and various infectious diseases arising from these diseases (Japanese Patent Laid-Open No. 64-63524).
).

On the other hand, HS is a protein that induces acute phase proteins in hepatocytes.
F (hepatocyte stimulation)
ng factor) was known (Ann, N,
Y. Acad. Sci. 408.90 (1983)).

As a result of research, 20H8F has also been found to be functionally BCDF (BS
F-2) was found to be the same as (Proc, N
atl, Acad, Sci, USA, 84.7251 (
1987)).

In addition, ■FN- which is said to have interferon activity
β2 (European Publication Patent No. 022057
4, Proc, Natl, Acad, Sci, USA,
77.7152 (1980)) and 26kd prot
ein (Proc, Natl, Acad, Sci,
tlSA, 72.2708 (1982)) and HPG, a growth factor for hybridomas/plasmacytomas.
It turns out that F is also the same substance.
e, 324.415 (1986), EMBO, J.
6.1219 (1987), J, Exp, Med, 1
65,914. (1987)).

It was proposed that these proteins isolated with different activities are actually the same substance and should be called interleukin 6 (
Nature, 324. 73 (1986), EN
IIO, J.

6.1219 (1987)).

It is known that interleukin 6 is produced by various cells. In addition to the lymphocytes mentioned above, human fibroblasts were
It has been reported that o1y(I), Po1y(C) and cycloheximide are produced by stimulating them (Eur,
J, iochem, 159,625. (1986)
). There are a wide variety of inducers, including IL-1, TNF, and PD.
GF.

IFN-β, LPS, viruses, etc. are also known. It has also been reported that human vascular endothelial cells, macrophages, human glioblastoma, etc. produce it. (Immunol, 142, 144. (1989
), J.

Immunol, 141, 1529. (1988),
JP-A-83-296688)).

By the way, human BCDF (
IL-6) became possible to produce (Unexamined Japanese Patent Publication No. 1983-1)
57!396) Although a lot of biological knowledge has been obtained regarding its biological activity and effects on other cells, it does not have sugar chains, so it is actually different from the natural interleukin 6 produced by human cells. Questions remained as to whether they were equivalent and what about other biological activities. One of the reasons for this is that genetically modified proteins using prokaryotes such as Escherichia coli have very high productivity of specific proteins, whereas human cells have poor productivity, and human natural protein It has been impossible to obtain Leukine 6 in large quantities with constant quality. This is also one of the reasons why there is little physical and chemical knowledge about the amino acid sequence and sugar chain structure of human natural interleukin-6.

The N-terminal amino acid of human BCDF produced using prokaryotic cells transformed with a gene encoding BCDF is Pro-Val-Pr.

-Pro-Gly=... and Ala-Pro-Val-
Pro-Pro-... etc. are known (Japanese Patent Application Laid-Open No.
3-157990). Regarding the natural type, the N-terminal amino acid of interleukin 6 derived from fibroblasts subjected to interleukin l'''C induction is Ala-Pro-Val-
Pro-Pro-Gly-Glu-Asp-Ser-L
ys-Asp-Val ... and Val-Pro-Pr
It has been reported that it is a mixture of o---... (J,
Exp, Med, 165, 914 (1987)). Similarly, the N-terminal amino acid of natural interleukin 6 derived from leukocytes is Ala-Pro-Val-Pro-Pr.
o ... and Val-(Pro)-Pro-Gly-G
lu...(D mixture (J, Im
Munol, 140.1534 (1988)).

In addition, F-DIF (fibroblast-
derived differentiation i
The N-terminal amino acid of
It has been reported that al-Pro-Pro-Gly.
Highly homologous to interleukin 6 (The bi
ology of the 1nterferon S
system 1988. p395). As mentioned above, various reports have been made, suggesting that the physicochemical properties such as the structure of natural interleukin-6 change depending on the type of producing cells and the type of inducer. The same can be said of molecular weight, isoelectric point, sugar chain structure, etc. For example, HGF (hybridoma groin) produced from fibroblasts using interleukin 1
The molecular weight of wth factor) is 23kd and 24k
d (J, Exp, Med,, 165,91
4 (1987)), the molecular weight of human interleukin 6 produced by monocytes is 21.5, 23.5.24, 26, 28.
kd and (FEBS LETTER5, 247, 323 (
1989)) Various. The molecular weight diversity of human interleukin-6 is predicted to be due to the sugar chain structure (O-glycoside type and N-glycoside type) and phosphorylation groups (
FEBS LETTER3, 247, 323 (1989
), Iloichem, Biophys, Res,
Commun, 152.1144 (1988)).
As described above, there are still many aspects of human natural interleukin 6 that are not understood, and its activity, structure, etc. due to differences in cell types and inducers are also unknown.

There is no example of complete purification of human natural interleukin 6 produced by human fibroblasts using the superinduction method, and there are no reports regarding the N-terminal amino acid or sugar chain structure.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to purify natural human interleukin-6 produced from human fibroblasts by the superinduction method and provide interleukin-6 as a substance.

[Means for Solving the Problems] As a result of extensive research to solve the above problems, the present inventors have found that human interferon β can be extracted from human fibroblasts.
By establishing a method for purifying human interleukin-6 from the culture medium produced by the superinduction method used to produce human interleukin-6 and clarifying the structure of the interleukin-6 preparation thus obtained, The present invention has been completed by providing a human interloyl 26 composition derived from fibroblasts, which is different from that of the present invention. The present invention will be explained in detail below.

Now, the present inventors have focused on a method of producing IL-6 by treating human fibroblasts with Borie:BoriC, a synthetic nucleic acid, and cycloheximide, a protein synthesis inhibitor. This magazine is similar to the superinduction method, which is a method for producing human natural interferon-β, and by this method, human fibroblasts are able to produce a large amount of engineered FN-β2 (26kd), which is said to have antiviral activity.
pr.

tein, IL-6) (Proc, Natl, Acad, Sci,
, 79. 2768. (1982)).

This production method is exactly the same as that of the already known human natural interferon β (Proc, Natl, Ac
ad, sci, 67, 464 (1970),
J. Gen. Virol, 56.78 (1970)
) method is fine.

The present inventors have demonstrated that human natural interleukin 6 and human natural interferon β are contained in the fibroblast culture medium obtained using the superinduction method.
This was confirmed by ISA method and HGF activity measurement. Useful substance IFN-β produced by this super induction
can be purified using a blue dye-binding insoluble carrier (blue carrier) such as ``Plu-Sepharose'' and a metal chelate carrier (Japanese Patent Publication No. 12700/1989. USP-4).
541952).

Conveniently, human IFN-β2 (IL 6) is known to be poorly adsorbed to the blue carrier (ENllo
, J., 5.2529 (1986)), but the previous report treats IL-6 as an antiviral active substance, so it does not show the behavior of IL-6 that does not have antiviral activity as described in the present invention. do not have. Since the amount of blue carrier used here is sufficient to substantially bind interferon β, usually about 1 to 5% of human natural interferon β remains unadsorbed on the blue carrier. Under these conditions, more than 90% of human natural interleukin 6
was found to remain in the non-specifically adsorbed fraction. Next, in order to recover human natural interleukin 6 from the supernatant that has passed through the blue carrier, a solution containing crude human natural interleukin 6 is brought into contact with a silica-based adsorbent (silica carrier) and adsorbed using a known method. can do. Silica carriers include, for example, CPG (controlled
pore glass) and silica gel. The concentration method using a silica carrier is CPG (c.

A method of batch adsorption using trolled pare glass and recovery using acid is known (J, Exp,
Med, 165, 914 (1987)). In addition, fibroblast-derived differentiation-inducing factor (F-DI) has the same amino acid sequence as interleukin-6 and is presumed to be the same substance.
A batch adsorption method using microbead silica gel is known for the purification of F) (The Iliology
of the 1nterferan System
1988. p. 395). In the present invention, either a batch adsorption method in which a silica carrier is added to a crude human natural interleukin 6 solution, or a column adsorption method in which a silica carrier is packed in a column and a crude human natural interleukin 6 solution is flowed into the column. But that's fine.

The pore diameter of the silica carrier used is preferably 7 to 1100 nm, preferably 10 to 50 nm. Adsorption conditions are near neutrality,
Preferably the pH is 6.5-8.

When performing the column adsorption method, it is necessary to remove insolubilized proteins and nucleic acids remaining in the crude human natural interleukin 6 solution, as well as water-soluble polymers (such as methylcellulose) that are input for the protective effect on cells. A precolumn may be installed before the silica carrier column. The precolumn may be any insolubilizing carrier, such as a small amount of silica carrier or a blue carrier that does not bind L-6. Before eluting human natural interleukin 6 from the silica carrier, it is desirable to wash it with a solution (neutral or weakly acidic buffer) that does not elute IL-6.

This makes it possible to remove some of the M protein adsorbed onto the carrier. After washing, human natural interleukin 6 is recovered using a removable recovery liquid. The recovery liquid in this case is preferably a known acidic solution. Specifically, glycine-hydrochloric acid buffer (pI (2) or hydrochloric acid with pH 1 to 3,
Mineral or organic acids such as sulfuric acid are used. Moreover, ethylene glycol, glycerin, etc. can be added to these recovery agents as appropriate.

The silica carrier also has an affinity for human natural interferon β, and contaminant proteins and human natural interferon β are also concentrated in the moat recovered with acid. As a method for separating and purifying interleukin-6 from a liquid containing interleukin-6 and interferon β recovered from a silica-based carrier under acidic conditions, neutralization/anti-crude interleukin-6
A method combining voriclonal antibody column/gel filtration/strong acid ion exchange chromatography/reversed phase chromatography (Eur, J., Illiochem,
168, 541 (1987)), it has been reported that strong acid ion exchange chromatography can be used for separation, but the operation is complicated and the degree of separation is not clear.

The present inventors have made extensive studies to solve these problems, and have discovered that combining a salting-out method and hydrophobic chromatography brings about an extremely high effect. Let's be specific.

First, we discovered that human natural interleukin-6 can be separated from contaminant proteins by salting out under acidic conditions (pH 1 to 6), where it is stable. In other words, this is a method in which human natural interleukin 6 remains in the liquid phase and other contaminant proteins are precipitated. As the salt used for salting out, ammonium sulfate, sodium sulfate, monosodium phosphate, sodium chloride, potassium chloride, etc. are used, but ammonium sulfate or sodium sulfate is preferable. For example, it has been found that by adding ammonium sulfate as 10% to 60% saturated ammonium sulfate and removing the precipitate using a centrifuge, it is possible to dramatically increase the purity of human natural interleukin 6 in the supernatant.

The supernatant containing naturally occurring human interleukin 6 in salt contact contains a large amount of salts. On the other hand, hydrophobic chromatography uses the hydrophobicity of proteins to perform adsorption and desorption.
It is used as an effective method for purification (Bioc
hem, Biophys, Res, Common, ,
49.383 (1972)). Particularly in a state where the concentration of salts is high, by contacting the protein as it is, the protein can be subjected to hydrophobic chromatography as it is adsorbed or partially diluted, and human natural interleukin 6 can be efficiently adsorbed. The adsorption method may be a column method or a batch method. In addition, in the above salting out, ammonium sulfate was further added to the supernatant containing interleukin 6 to give a concentration of 65% to 70%.
It is also possible to dissolve the precipitate in saturated ammonium sulfate in a 40% or less saturated ammonium sulfate solution and to adsorb and purify it by the hydrophobic chromatography described above under hydrophobic conditions. The hydrophobic chromatography used here may be any one in which an alkyl group (C+ to Cl8), phenyl group, etc. is chemically bonded to a carrier skeleton. As the skeletal carrier, insoluble carriers such as polysaccharide based materials such as cellulose and agarose, and synthetic polymer based carriers can be used. Under naturally hydrophobic conditions, most of the interleukin-6 and interferon-β in the salting-out supernatant are adsorbed onto the hydrophobic carrier. The target protein could be eluted from the adsorbed carrier by passing a solution with a gradient or stepwise decrease in salt concentration according to a conventional method. In addition to changing the salt concentration, pH and temperature may also be changed. According to this hydrophobic chromatography, for example, when a solution with a reduced salt concentration of p I (5 to 9) is run, human natural interleukin 6 is surprisingly efficiently eluted, and further purified interleukin 6 is eluted efficiently. However, on the other hand, human natural interferon β remained adsorbed to the carrier and was not eluted, making it possible to completely separate interferon β.
Purification was achieved using reverse phase chromatography. To give an example, OD S (C
+e) - By separating the peak of natural human interleukin 6 using a gradient of water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid using a column high-performance liquid chromatography method, the purity is increased and the product is substantially purified. It is possible to obtain a standard IL-6 preparation. As the carrier for reverse phase chromatography, a carrier to which an alkyl group or a phenyl group is bonded is preferably used as in hydrophobic chromatography.

The quantification of natural human interleukin-6 according to the present invention can be carried out by enzyme immunoassay using a monoclonal antibody (p1128237) that strongly neutralizes the biological activity of human interleukin-6, or by using a method that produces engineered gM in response to human BCDF. Human B cell line CL4 (Proc, N
atl, Acad, Sci, , 82.5490. (1
985)) etc.

As a result of structural analysis of the purified human natural interleukin-6 according to the present invention, it was found that more than 90% of the molecular weight was found to be in the range of 21 to 25 kd using the 5DS-PAGE method, and that minor components were present in the range of 27 to 30 kd. Ta. It was found by electrophoresis that 80% of the isoelectric points were between 5.0 and 6.5. These physical properties were consistent with known human interleukin-6. In addition, N-terminal amino acid analysis revealed that the N-terminus was a mixture of A 1 a, P r o, and V a 1, respectively. That is, it is a mixture of the following three types of human interleukin 6.

■ Ala-Pro-val-Pro-Pr.

-Gly-Glu-Asp-8er- ■ Pro-Val-Pro-Pro-Gly-Glu
-Asp-Sep-Lys- ■ Val-Pro-Pro-Gly-Glu-As
p-Ser-Lys-Asp- Also, the mixing ratio of the above ■, ■, ■ is approximately 37 5: 2
It is. N of interleukin 6 derived from human fibroblasts
As terminal amino acids, a peptide mixture of these three N-terminal amino acids was obtained for the first time by the present invention. In addition, analysis of the sugar composition of this human natural interleukin-6 revealed that most of it (80% or more) is O-glycoside type sugar chains (mucin type), and less than one-tenth of it also has N-glycoside type sugar chains. It became clear that it was.

In addition, human natural interleukin 6 obtained in the present invention
For example, human serum albumin can be added as a stabilizer, and the solution can be replaced with a solution suitable for injection by dialysis or Rja Shiokawa chromatography according to a conventional method. Furthermore, it can be formulated into a formulation by appropriately diluting and dispensing it, and if necessary, freeze-drying it.

As described above, fi lf was produced from human fibroblasts by the superinduction method for the production of IFN-β.
The human interleukin 6 having the J structure is the human natural interleukin 6 according to the present invention.

[Example] The present invention will be specifically explained below according to Examples.

Example 1 Human fibroblasts were cultured with beads in 21 glass culture vessels in 11 Eagle's MEM medium containing 5% NC5 to a cell number of 106/ml (beads: Cytodex 1). (Pharmacia), 37°C). Thereafter, the medium was replaced with serum-free Eagle's MEM medium 11 containing a small amount of carboxymethyl cellulose, and 100,000 units/1 human natural interferon β was added as priming. The next day, poly■: BoriC 30mg/l
.

Cycloheximide was added at 10 mg/1. Four hours later, actinomycin D was administered at a dose of 4 mg/1.
After 1 hour, the production medium was replaced with Eagle's MEM medium containing a small amount of methylcellulose, and superinduction treatment was performed.

Thereafter, the culture was continued for 2 days (37°C).

was transferred to another container with a stirrer. Sterilized "Blue Sepharose CL-6B F F" (Pharmacia) was added to this product solution, and batch adsorption was carried out at 15°C for 4 days with stirring. After stopping stirring, the blue carrier was allowed to settle and the supernatant was transferred to another container. The silica carrier is sodium phosphate! After high-pressure steam sterilization (121"C, 130 minutes) in a buffer solution of
It was flowed at 0 m 1 /hr. The two columns were purified separately when the entire volume was run. After flowing 25 ml of sodium phosphate buffer and then flowing 20 mM hydrochloric acid, 10 ml of interleukin 6-containing fractions were collected. Add ammonium sulfate to this hydrochloric acid recovery solution to make it 1.33M? The mixture was stirred gently at 4°C overnight. The precipitate was removed by centrifugation at 3000 rpm for 30 minutes (4°C). The separated supernatant was poured into a column packed with 1 ml of Butyl Toyopearl 650M (Tosoh Corporation), a hydrophobic chromatography carrier, and adsorbed. This column was washed with 20mM hydrochloric acid containing 1.33M ammonium sulfate.
50mM sodium phosphate containing 1°33M ammonium sulfate! After washing with 1 buffer, the cells were recovered with 50 mM sodium phosphate buffer. After that, ODS column (C+s) (YMC-Pac
k ODS A-312S-5120A, high performance liquid chromatography (Shima?jt) equipped with
Using LC-4A), the human natural interleukin 6 peak was fractionated by gradient elution of water containing O01% twafluoro#acid and acetonitrile containing 0.1% trifluoroacetic acid. The human natural interleukin 6 obtained in this way was added to ``Sephadex G-25''.
(Pharmacia) using 5mM formic acid as a solvent to obtain an interleukin 6 solution containing no acetonitrile.

In addition, the measurement of the concentration of human natural interleukin 6 is as follows:
An ELISA method using a 96-well plate was used. That is, the anti-interleukin 6 antibody lG61 (monoclonal antibody) was coated on the plate at a concentration of 1 μg/ml. After blocking with sodium phosphate buffer (pH 7.0, wash buffer) containing bovine serum albumin (BSA), 10 Mg of another anti-interleukin 6 antibody (■C67) labeled with biotin was used as a secondary antibody.
First, 1.50 μl of /ml was placed on a plate, and 50 μl of each of Interleukin 1'-'Fn6 standard product of known concentration and Interleukin 6 solution of unknown concentration were added and reacted for 1 hour with shaking. After washing three times with washing buffer, 100 μm of ``streptavidin-HRP conjugate'' (BRL) diluted 2000 times with washing buffer was added and reacted for 30 minutes. After washing three times with washing buffer, 100% citric acid buffer (pH 5,0) containing orthophenylenediamine and hydrogen peroxide was added.
μm was added to cause a color reaction. The reaction was stopped with 5 N sulfuric acid for 30 minutes, and the amount of color developed in each well was measured at 492-690 nm using a microplate photometer (MultiScan CM, manufactured by Flow Laboratory).

Table 1 shows the purification yield and purity of each purification.

(Left below) Purification yield of 2 is 48% and purification purity by 5DS-polyacrylamide gel electrophoresis (SDS-PAGE) is 90%.
% or more.

The molecular weight was 23,000 ± 2,000 Daltons (90%
above) and minor components of 27,000±1,000 daltons.

Isoelectric focusing p■ determined from isoelectric focusing electrophoresis 5.0-6.
There were several in the 5 range.

Example 2 To determine the N-terminal amino acid sequence of human natural interleukin 6, 20 μg of purified human natural interleukin 6 obtained in Example 1 was first subjected to 5DS under non-reducing conditions.
-Polyacrylamide gel (4-20%, manufactured by Tefco)
Electrophoresis was performed.

This was then plotted on a PVDF membrane, Coomassie stained, the main band of 22-24 kd was highlighted, and an amino acid sequencer (Applied D
iosystems model 477A), and the recovered PTH-amino acid was subjected to IIP using one-third of the amount of ffl.
LC (Applied Biosystems 1
20A, PTH analyzer).

The amounts of amino acids identified for each cycle are shown in Table 2. Three or two amino acids were identified in the same cycle. Also Val, Pro, Pro, Gly, G 1 u,
Human natural interleukin 6 was detected by detecting the A sp and S e r amino acids three consecutive cycles each, and by comparing with the amino acid sequence deduced from the cDNA for interleukin 6.
It was found that the N-terminal amino acid sequence of Also, based on the N-terminal amino acid analysis value, the ratio is approximately 3
:5:2.

■ Ala-Pro-Val-Pro-Pr.

-Gly-, Glu-Asp-8er -■ Pro-
Val-Pro-Pro-Gly-Glu-Asp-8
er-Lys -■ Val-Pro-Pro-Gly
-Glu-Asp-8er-Lys-Asp - (blank below) Example 3 The constituent sugars of human natural interleukin-6 were measured by high performance liquid chromatography (HPLC).

■ Add 1 ml of 2N trifluoroacetic acid to the neutral sugar analysis sample,
Hydrolysis was carried out at 0°C for 16 hours. 500 ng of rhamnose was added to the internal standard substance, dried under reduced pressure, and dissolved in distilled water. The samples thus prepared were analyzed by HPLC. The column is”
TSK-gel Sugar AXG" (Tosoh Corporation), 05M potassium borate pH 8.1 was used as the mobile phase. 1% arginine/3% boric acid was used as the reaction reagent for Bost column labeling, and the detection wavelength was EX: 320n
m.

EM: 430 nm.

(2) Amino sugar analysis 1 ml of 4N hydrochloric acid was added to the sample and hydrolyzed at 100°C for 16 hours. Mannosamine 500n as internal standard substance
g was added, dried under reduced pressure, and dissolved in distilled water. The samples thus prepared were analyzed by HPLC. The column is “TSK-
gel SCX” (Tosoh Corporation), 0.16 for the mobile phase
M sodium borate buffer pH 7.6 was used. 1% arginine/3% boric acid was used as the reaction reagent for Bost column labeling, and the detection wavelength was EX: 320 nm, EM: 43
It was set to 0 nm.

■Add 0.2ml of 0.05N sulfuric acid to the sialic acid analysis sample, and
Hydrolyzed for 11 hours. In addition to this, N-7 is used as an internal standard substance.
1 μg of cetylmannosamine was added. The samples thus prepared were analyzed by HPLC. The column is °' Ge1pa
k C-620-10" (H+ type) (Hitachi Kasei Co., Ltd.), 0.3% phosphoric acid was used as the mobile phase. 0.1% malononitrile was used as the reaction reagent for Bost column labeling, and the detection wavelength was EX : 360 nm, EM: 430 nm.

Table 3 shows the compositional analysis results of each constituent sugar.

(Margins below) From these results, it is estimated that most of the sugar composition of human natural interleukin-6 has a formula ( It is estimated that it has an N-glycoside structure.

-0-G a 1 Ac ・ N-G a l-N eu ・ Ac (■) (Left below margin) uc Ac Man-[GlcNAc-Gal-(Neu),]cN
Ac-Gal-(Neu), 1 Ac y = Q rl n = 1 r2 (II) [Effects of the Invention] The applications of the human natural interleukin-6 of the present invention are very multifaceted. First, an immunoassay system using human natural interleukin 6 and anti-interleukin 6 antibodies can be used for immunological analysis of pathological conditions. Second
Cl1n, Immunol, 21, 1225 (1
It can also be applied to the treatment of various diseases by utilizing the diversity of physiological activities shown in 989). Thirdly, by adding it to the growth medium of hybridomas, the proliferation thereof can be enhanced.

Thus, human natural interleukin-6 is a very widely effective substance.

Furthermore, the present invention has made it possible to provide high quality human natural interleukin 6 in large quantities.

Claims (1)

[Claims] (1) A human natural interleukin 6 composition comprising a mixture of human interleukin 6 derived from human fibroblasts and having the following three types of N-terminal amino acid sequences. [1]Ala-Pro-Val-Pro-Pro-Gl
y-Glu-Asp-Ser- [2] Pro-Val-Pro-Pro-Gly-Gl
u-Asp-Ser-Lys- [3] Val-Pro-Pro-Gly-Glu-As
p-Ser-Lys-Asp-