JPS6151000A - Method for purifying human interferon beta - Google Patents

Abstract

PURPOSE:To obtain industrially and advantageously the titled compound having antitumor activity, etc., by bringing a solution of human interferon beta into contact with a specific support for insolubilizing a specific antibody, eluting adsorbed substance, bringing the resultant eluate into contact with hydrophobic group-bonded high-speed chromatographic support, and eluting the adsorbed substance. CONSTITUTION:A solution of human interferon beta is brought into contact with a support containing insolubilized antihuman interferon beta antibody and adsorbed. The resultant adsorbed substance is then eluted with an eluent to give an eluate, which is then passed through a column filled with a hydrophobic group-bonded high-speed chromatographic support, brought into contact with the support and adsorbed thereon. The human interferon beta adsorbed on the high-speed liquid chromatographic support is then eluted with an eluent. The eluent for the substance adsorbed on the support insolubilizing the antihuman interferon beta antibody to be used is preferably acidic buffer solution having <=0.05 ionic strength and 1.5-3.0pH.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to human interferon [1β (hereinafter referred to as IFN-β)].
(abbreviated as β).

[Prior Art] IFN-β is a glycoprotein that is produced by human cells when stimulated by viruses, synthetic nucleic acids, etc. and has antiviral activity and anti-Scancer activity. IFN-β can be produced by stimulating cultured human cells with interferon inducers such as polyI:zltC, and by inserting the 1FN-β gene into microorganisms such as E. mB bacteria and yeast, or into animal cells. There is a genetically recombinant method for producing □ containing □, and both are currently under development. In order to use this IFN-β as a medicine, the crude IFN-β produced by cells and microorganisms must be purified to a purity that is recognized as safe.

Therefore, it is important to know how to efficiently produce IFN-β with high purity.
- Purification and separation at 4°C is an important technique when producing IFN-β as a pharmaceutical product. In particular, when purifying 1q of IFN-β using genetic recombination methods, microorganism-derived proteins, 551 weighed animal-derived proteins, and 7
Since they are contained in large amounts in the target liquid for 1'i production, it is necessary to completely remove them, which requires a sophisticated purification method.

Various chromatography methods have been developed to purify IFN-β, and particularly so-called affinity chromatography, which uses a carrier bound to a substance with affinity for IFN-β, has achieved good results. These affinity chromatographies utilize the affinity of IFN-β to sugar chains and constituent amino acid residues, and are able to detect IFN-β by combining two- or three-stage chromatography.
Although the beta outfit is being carried out, 1. There is still room for improvement in terms of yield and purity.

The most specific type of affinity chromatography is antibody chromatography using an anti-rFN-β antibody as a ligand, and several attempts have been reported (Okan+ura et al., Biochcm
istry, 1C, 3831 (1980)
, 1-10Chkcpl) e I et al., Natu
re, 2 g1, 500 (1981
), Novick et al., J., g.c.
n, V 1ro1. , 6 4.

90.9 (1983)). However, despite its extremely high specificity, it is still difficult to completely purify it with a single step of antibody chromatography, and practically it is necessary to connect it with some other purification method.

[Problems to be Solved by the Invention] Although antibody chromatography has extremely high specificity in the purification of IFN-β, several problems remain.

First, although highly specific, this method does not allow IFN-β
Complete purification of is difficult. In principle, anti-IFN-
β antibody only adsorbs IFN-β, but actually anti-IF
The carrier in which the N-β antibody is insolubilized contains other than IFN-β.
A small amount of lE protein is also adsorbed and may be mixed into the eluate. In addition, the ligand anti-IFN-
There is a possibility that a small amount of β-antibody may be released into the eluate.

In order to completely purify IFN-β, it is necessary to remove these impurities, and it is necessary to connect some purification means after antibody chromatography.

Next, ■FN-β used in antibody chromatography
The elution conditions are mostly acidic mild tI with pH 2.0 to 3.0.
It is a neutral or monoalkaline buffer solution containing high-temperature chaotropic salts and surfactants. These solution systems may interfere with the adsorption of IFN-β when using normal ionic bonds, aqueous bonds, or O-madography, so when connecting to other types of chroma 1-systems, H:! In many cases, operations such as preparation, dilution, dialysis, and desalting are not required. This operation is for IFN-β. Physiologically active proteins such as - may have adverse effects such as loss of activity and protein precipitation, and in the case of treatment of major diseases, it is preferable to avoid them as much as possible due to technical difficulties.

The present invention solves the above-mentioned problems of antibody chromatography, and produces IFN-β with high purity and high yield without limiting the high specificity of antibody chromatography in the IFN-β purification method. The purpose is to refine it.

[Means for Solving the Problems] The present invention comprises a step of bringing an IFN-β solution into contact with a carrier in which an anti-IFN-β antibody has been insolubilized, a step of eluting the adsorbed substance to the carrier with an eluent, and a step of eluting the eluted material with an eluent. a step of contacting a high performance liquid chromatography carrier to which a hydrophobic group is bonded;
This is a method for purifying IFN-β, which consists of a step of eluting β with an eluent.

IFN-β referred to here refers to IFN-β produced by stimulating cultured human cells with viruses or synthetic nucleic acids, and IFN-β produced using genetic recombination technology.
It refers to IFN-β produced by microorganisms such as Bacterium spp. and yeast, or animal cells such as hamsters and ranals, which have incorporated the β-seizogenic gene. The anti-IFN-β antibody used as the ligand in the first stage of purification is not particularly limited as long as it is an antibody capable of binding IFN-β.
Refers to monoclonal antibodies produced by □ cell fusion technology or monoclonal antibodies produced by cell fusion technology.1. Insolubilization of these antibodies requires
Carriers such as cellulose, 77-galose, cross-linked gextran, polyacrylamide, porous glass, etc. are used, and the insolubilization reaction is carried out by bonding the amino group or carboxyl group in the peptide with the active device (Article 8). The main activated substituents include a cyanide bromine active group (e.g., °“CN B l' 21! i-based hyphalose 4B”: Pharmacia), an N-hydroxysuccinimide ester group (e.g., “activated Cl- 1-
Sepharose 4B”:) iSylmacia, ゛°af, C
Gelu 10"; courtesy of BioRad), Hydrazide KM"
J body (for example, Euperkit C11, Rohm)?
- Masha), Ambidrid! ! (For example, "Eupergit A"; Rohm Farm Co., Ltd.).

A carrier in which anti-IFN-β antibody is insolubilized (hereinafter referred to as anti-[FN-β antibody)
The actual operation of antibody chromatography using β antibody insolubilized carrier (abbreviated as β antibody insolubilized carrier) is performed as follows. That is, first, crude IFN-β such as human cell culture supernatant, genetically recombinant Escherichia coli disrupted centrifugation supernatant, or recombinant animal cell culture supernatant.
The solution is contacted with an anti-[FN-β antibody insolubilized carrier and adsorbed.

The adsorption method is a batch method or a column method, but it is dust-free.
Column method has higher adsorption efficiency. Adsorption conditions are pH 5-9
The pH is preferably 6 to 8, and the presence of about 1M inorganic salt does not affect adsorption.

After adsorption, the carrier was thoroughly washed with an appropriate neutral buffer to remove contaminant proteins, and then adsorbed with an acidic buffer of PL-12 to 3 or a neutral or alkaline buffer containing chaotropic salts or surfactants. l Elute FN-β. Many acidic buffers are used, and their compositions are, for example, O., 1M citric acid, 0.1-0.2M glycine hydrochloride, etc., and optionally unmodified salts (for example, 0.5-1M chloride, J chloride, etc.). It is also advisable to add a water-soluble organic solvent (e.g. glycerol or ethylene glycol), a surfactant (e.g. Triton X-100), etc. What composition of acidic buffer should be selected? , the desired elution effect of [FN-β, IFN-β in its buffer]
It is necessary to consider the stability of β, the effect on the antibody that is the ligand, and the adaptability to the next (chroma) system. Considering these points, [FN-β elution When the ionic strength is 0.05 or less, 1) l-11,5 to 3.
An acidic m solution of 0 is preferably used. Such acidity! Examples of the buffer include hydrochloric acid, sulfuric acid, formic acid, and citric acid. These acidic buffers are effective for elution of highly hydrophobic IFN-β, and the yield is quantitative;
The stability was also affected, with no loss of activity occurring in the same solution for at least 1 month at 4°C. In addition, since it does not contain water-soluble organic solvents or surfactants, it has little denaturing effect on the antibody, which is the ligand, and has low ionic strength! Due to its weak chromatographic effect, neutralization, dialysis, and
In many cases, operations such as desalting are unnecessary.

The present inventors have conducted extensive research into hydrophobic chromatography to incorporate it into the second purification step that can effectively remove the slightest remaining contaminant proteins while taking advantage of the above advantages. It has been found that chromatography using high performance liquid chroma 1 to Graphee with attached groups satisfies these requirements.

In order for the Begichroma+'- system used in the second stage of the purification step to meet these conditions, it is necessary to have high protein separation and high yield. Ion-exchange chromatography (for example, ion-exchange chromatography based on sulfopropyl groups), which is often used for protein purification, can remove IFN-β and contaminant proteins71.
The performance was poor and the yield was low, making it unsuitable. Furthermore, even in Affinite r-chromatography, which has been reported as a purification method for IFN-β, chromatography using funcanavalin A as a ligand is not suitable for recombinant IFN-β, which does not have sugar chains.

The so-called high-performance liquid chromatography (hereinafter abbreviated as HPLC) applied in the present invention is a method used for the purification and analysis of proteins and peptides, and is a method used for the purification and analysis of proteins and peptides. Also5tein et al. (p
roc, N aN, A (j+1+1, SC
j.

USA, 77.5'>716 (1980)), but it is applied after blue chromatography and does not predict the present invention, which uses highly specific antibody chromatography.

1-IPLC is a liquid chromatography that is performed under high pressure and high flow rate using 10 high-performance columns, and ion exchange groups and hydrophobic groups are introduced into the carrier. The I-I PLC carrier used in the present invention is not particularly limited as long as it has a hydrophobic group bonded to it and has a 1FN-β adsorption port, but preferably an alkyl group such as C6,01a, a cyanopropyl group, or a phenyl group. Examples include carriers into which groups or the like are introduced. Commercially available products are μm5onda+)ack 018
”,”μm Bondapack CN”,”μm
m3ondapack Phenyl” (J:/
% above, Zorl) ax C8
","Zorl)ax QN"(hereinafter referred to as i'Yupon Co., Ltd.),"Li Chrosorb RP-
8”, “li Chrosorb RP-18”
(Merck & Co., Ltd.), “L Month 1isil C8”,”
IJnisil C18°', ゛() old s
il CN ”, ” Ll n1s
il p Hu,” (Jn1sil C
P” (hereinafter referred to as Gascro Industries).

The actual l-IP L Clfi creation is performed as follows.

That is, Wa obtained in antibody chromatography, C-
(FN-β solution with hydrophobic groups)
Pour into the LC column. Subsequently, elution is performed with a water-soluble organic solvent containing a mild solution. Buffers used include trifluoroacetic acid, formic acid-pyridine, and phosphoric acid! Examples of organic solvents for elution include acetonitrile, 2-propatol, 1-propatol, ethanol, and methanol. When selecting these, a solvent system with good stability of IFN-β and good protein separation ability should be selected, preferably 0.1
% fluoroacetic acid-acetonitrile system is used. This system has good stability of IFN-β at pH 2, and also has excellent separation ability. Furthermore, since there is no ultraviolet absorption,
Protein elution can be monitored by ultraviolet absorption at 280 nm or 210 nm. Moreover, since it is volatile, it is possible to concentrate the eluted solution without JJQ jW. Elution is carried out using, for example, an 8 degree gradient (1%/1ml) of 0→70% ahi-1 to nitrile, but there are no particular limitations as long as the conditions are sufficient to separate proteins.

[Effect of the invention I IFN-β is currently used as an anti-l! Due to its potential as an anti-inflammatory and antiviral agent, there is a social demand for large quantities of it to be supplied for clinical evaluation. The causes of the quantitative limitations of IFN-β to date are that the amount produced by cells itself is small and that it has been difficult to develop a purification method that can achieve high yield and high purity. However, now that genetic engineering technology has made it possible to supply Okawa in terms of production, developing a high-performance purification method has been a major challenge.

IFN-β purified by the method of the present invention has extremely high purity, and no contamination with contaminant proteins or immunoglobulins was observed by bk immunoassay (RIA) or enzyme immunoassay (E fA). That is, it has been found that it is possible to highly purify the product using antibody-based O mudography, and then remove the slight amount of remaining contaminant protein using HPLC.

Moreover, at this time, the anti-[FN-β antibody insolubilized carrier was
By eluting N-β with an acidic solution of low ionic strength, [FN-β can be stabilized]. Log Direct 1-IP
It has been found that this method can be applied to LC. The present invention is a method for easily obtaining purified IFN-β of high quality by directly connecting two types of 7-infinity chromatography with high specificity and separation ability without any special operations.

As stated above, the present invention is the essence of IF, N-β! It provides new technology in the J method.

Among the many types of affinity chromatography, antibody chromatography has attracted attention due to its high specificity, but in practical use, there are problems such as insufficient lees vA, fear of antibody leakage, and high ionic strength elution with low 1)H. Problems such as post-treatment of the liquid remained.

The present invention combines antibody chromatography and HPLC to solve these problems all at once.

Therefore, the purification method according to the present invention makes full use of the high specificity of antibody chromatography, and it is possible to purify IFN-β with a high degree of purification and high yield. The standard product is high quality and highly safe. Furthermore, the chromatographic operation is simple, the reproducibility is high, and the purification method is suitable for large M treatment.

Hereinafter, the present invention will be explained in more detail with reference to Examples. The IFN-β activity in the examples was determined by human amniote F.
L4 [using 1 cell and vesicular inflammation virus (VSV) @
Measured by the Cytopathic Effect (CPE) inhibition method, using standard IFN
−β is calculated by IIA to international units (IU).

Example 1 After sufficiently culturing and proliferating Escherichia coli into which the 1FN-β structural link gene had been incorporated, the bacterial cells were disrupted by high pressure.

Furthermore, polyethyleneimine is added to remove nucleic acids/;
: 11. Salt out the protein by adding 60% saturated ammonium sulfate and add IOIIIM phosphorA containing 0.5M sodium chloride.
Buffer! The resuspension in (1) 87.4) was used as the purified stock solution. This bacterial cell extract contains 5.0X108IU/++
It contained zl protein.

10mα of this bacterial cell extract was mixed with an anti-IFN-β mouse monoclonal antibody (patent application, July 1982-1933) produced by a fusion cell of mouse tile 21111 cells sensitized with IFN-β and mouse milioma cells. Insolubilized agarose column 1mQ (10φX1311111)
℃, flow rate was ``lad/hr.'' The flow-through solution contained 99% of the added protein, but IFN-β
No activity was observed, so ICO was followed by 50% antibody column.
1511M vinegar slim solution containing % ethylene glycol (
1) After washing with l-14,0>20mm, 15mM hydrochloric acid ionic strength 0.015, EIH2,0) 10Ilα
IFN-β was eluted. ■ Recovery rate of FN-β activity is 9
5%, specific activity increased 150 times.

The eluate from the antibody column containing IFN-β activity was roughly applied to an 8φ×100 mm “μm 8ondapack C18” column (Waters) at 1 mQ/mir+ at room temperature. Next, 0.1% trifluoroacetic acid (pH2,
0) containing acetonitrile from O → 70% (1%/m1n
) with a concentration gradient of -1280ni+ and 21Onl
Fractionation was carried out while observing the absorbance of 1 liter.

When we collected both samples with IFN-β activity, we found that IFN-β
The recovery rate was 87% and the specific activity was further increased by 4 times. As a result, the l! of IFN-β relative to the purified stock solution! The yield of liI was 83%, and the specific activity was increased approximately 600 times.

This purified IFN-β sample appeared as a uniform protein band in sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis, and contaminant proteins derived from E. coli were analyzed by radioimmunoassay (RfA) at the detection limit (content rate 0.001%). It was below. Furthermore, the ligand, mouse immunoglobulin, was not detected by enzyme-linked immunosorbent assay (EIA) analysis.

Example 2 Hamster cells enriched with IFN-β (I11-producing gene) were incubated with α'alpha (-) containing 10% fetal bovine serum.
)" (Company 01800) culture medium, and the culture medium containing IFN-β activity was used as a purified stock solution. This culture medium contained 2.,2 x l 0 'I LJ/ml of IFN-β activity.
It contained β activity and 3.6 mg/ml protein.

This cultured slide 1α was applied to a 1 ml agarose column in which the mouse monoclonal antibody used in Example 1 was insolubilized (
It flowed at 10φXb 0m1/hr. Protein I added to the pass-through liquid
It contained 99% of the amount of R, but no IFN-β activity was observed. Next, apply the antibody column to a 15mM acetic acid mild WJ solution containing 50% ethylene glycol (1)H4,0)
20mlt' After washing, add hydrochloric acid (ionic strength 0)
．． 015, pH 2,0) tFN-β was eluted in 10 ml. The recovery rate of IFN-β activity was 98%, and the specific activity was 8.
The price has increased 300 times.

The eluate from the antibody column containing IFN-β activity was further divided into 8φ x 100mm “μm8ondapackC18”
A flow rate of 117'1n was applied to the column (Waters) at room temperature.
It was washed away. Next, 0.1% trifluoro vinegar II (1
) 82.0) containing acetonitrile from O → 70% (1%
/1n) with a m degree gradient of 280nm and 210n
Fractionation was carried out while observing the absorbance of m.

When both parts with IFN-β activity were examined, IFN-β was detected.
The recovery rate was 85%, and the specific activity was roughly 6 times higher. As a result, the recovery rate of IFN-β relative to the purified stock solution was 81%.
%, and the specific activity is about 50,000 F.
.

This Tt5i IFN-β sample appeared as a uniform protein f:1 band in sodium dodecyl sulfate 3-right polyacrylamide gel electrophoresis, and no mouse immunoglobulin was detected in enzyme-linked immunosorbent assay (EIA) analysis.

Comparative Example 1 The eluate of Example 2 eluted from the antibody column was further passed through 2 ml of "Sulfopropyl Sephadex C25" (Pharmacia) (10 φ After washing with buffer solution (1) l-l 7.4> 20ml, 40ml of 50mM phosphate buffer containing 0.5-1 sodium chloride (pl-1a, O) was flowed, and 280 nm
Fractionation was carried out while observing the absorbance of , both fractions with IFN-β activity were collected, and the IFN-β recovery rate was 20%.
The specific activity was further increased by 1.6 times. As a result, the If-N-β recovery rate with respect to the purified stock solution was 19%, and the specific activity was increased by about 13.000 times.

Claims (3)

[Claims] (1) A step of bringing the human interferon β solution into contact with a carrier in which anti-human interferon β antibody has been insolubilized,
A step of eluting the adsorbed substance on the carrier with an eluent, a step of bringing the eluate into contact with a high performance liquid chromatography carrier to which a hydrophobic group is bonded, and a step of removing human interferon β adsorbed to the high performance liquid chromatography carrier. A method for purifying human interferon β, which comprises a step of elution with an eluent. (2) The purification method according to claim (1), wherein the human interferon β is human interferon β produced by genetic recombination technology. (3) Claims in which the eluent for the adsorbed substance on the carrier in which the anti-human interferon β antibody is insolubilized is an acidic buffer with an ionic strength of 0.05 or less and a pH of 1.5 to 3.0. The purification method described in paragraph (1).