JPS61191699A - Method of purifying mouse interferon - Google Patents

Abstract

PURPOSE:To purify simply a large amount of mouse interferon, by bringing a solution of mouse interferon into contact with a silica adsorbent, treating the adsorbent with an eluant, bringing the eluate into contact with a carrier having copper by chelate bond, and treating the carrier with an eluant. CONSTITUTION:A solution of mouse interferon is brought into contact with a silica adsorbent. Then, the adsorbed material of the adsorbent is eluted with an eluant (containing preferably neutral salt, ethylene glycol, or surface active agent such as sodium cholate, etc., buffer solution at 4-6pH). The eluate is brought into contact with a carrier having copper by chelate bond, the adsorbed material of the carrier is eluted with an eluant, so mouse interferon is purified.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for purifying mouse interferon.

[Prior Art] Interferon is a protein with multipurpose effects including antiviral and anticancer effects, and its development as a pharmaceutical has progressed rapidly in recent years. However, there are still many unknown points regarding the in vivo behavior and medicinal efficacy of interferon, as well as its physical properties, and many questions remain to be clarified. When such problems are evaluated non-clinically using animals, human interferon cannot be used due to the species specificity of interferon, and interferon from the animal is required. Even in the case of model experiments using such animals, it is necessary to use a large amount of interferon from that animal species that has been purified to the point that it does not contain any impurities in order to make a normal evaluation.

Animals used in non-clinical experiments include mice, rats, rabbits, dogs, and monkeys, but mice are the most widely and commonly used animals, and are no exception in the field of interferon research. Therefore, in order to develop interferon as a pharmaceutical product, it is important to properly evaluate mouse-mouse interferon through allogeneic animal experiments, and high-quality mouse interferon is needed.

Conventionally, the purification method for mouse interferon was
ra et al. (J, Biol, Chem, 253.5074
(1978) ), De ) layerer-Guig
nard et al. (Nature, 271.622 (197
2)) Many other methods have been reported, among which the method using an antibody against mouse interferon is an excellent method that allows a highly pure sample to be obtained through one-step purification.

However, highly specific antibodies are difficult to prepare;
Since it is difficult to obtain in large quantities, it is difficult to apply it to large-scale purification. Therefore, there is a need to establish a method for purifying murine interferon with high purity and in large quantities using carriers that are inexpensive and available in large quantities in place of the antibody method.

[Problems to be Solved by the Invention] As mentioned above, the present invention aims to establish a high purity purification method for mouse interferon in order to supply high-quality purified mouse interferon necessary for non-clinical research. do.

[Means for Solving the Problems] The present invention includes a step of bringing a mouse interferon solution into contact with a silica-based adsorbent, a step of eluting the adsorbed substance to the adsorbent with an eluent, and a step of chelating copper with this eluate. This is a method for purifying mouse interferon, which comprises the steps of bringing it into contact with a carrier, and eluting the substance adsorbed onto the carrier with an eluent.

The mouse interferon referred to herein may be any of the α-type, β-type, and γ-type, and includes, for example, mouse interferon produced by mouse cells such as mouse L-929 cells and mouse C-243 cells, and mouse interferon produced using genetic recombination technology. Examples include mouse interferon that can be produced by microorganisms such as Escherichia coli and yeast into which a mouse interferon structural gene has been incorporated, and animal cells such as hamsters and monkeys.

The adsorbent used in the present invention can be any adsorbent containing silica, but for example, "control pore glass" (EIeC1rOnuCIeOniC3
(manufactured by Fuji Davison) and "Microbead Silica" (manufactured by Fuji Davison).

Purification of mouse interferon using a silica-based adsorbent used in the first stage of purification is performed as follows. That is, first, a solution containing mouse interferon is contacted and adsorbed onto a silica-based adsorbent. Adsorption can be carried out by either a batch method or a column method, but the column method has higher adsorption efficiency. Mouse interferon is adsorbed at pH 6-9.

Elution is determined by the pH value, ionic strength, and hydrophobicity of the eluent. For example, mouse interferon elutes in an acidic region (pH 2 to 5). Further, under high ionic strength, elution becomes possible in a wide pH range (pH 2 to 9). This ionic strength can be increased by increasing the concentration of buffer salts such as phosphoric acid, acetic acid, citric acid, boric acid, etc., or by adding neutral salts such as sodium chloride or potassium chloride (0.2-1.0M). can. If the eluent contains a solvent that weakens hydrophobic interactions such as ethylene glycol or propylene glycol, or contains a surfactant such as "Triton X-100" or sodium cholate (0.1-1%)
In some cases, the elution power becomes even stronger.

The composition, concentration, and volume of the eluent are not particularly limited, and should be the concentration necessary to maintain an effective pH for removing contaminant proteins contained in the crude mouse interferon used as the material, and the amount of adsorbed material. The amount necessary for practical recovery of mouse interferon is used.

Among them, highly pure mouse interferon can be obtained by selectively eluating only mouse interferon without eluating most of the contaminant proteins, and pH 4 to 6, preferably pH 4.5
A solution containing 0 to 15% (V/V) of an organic solvent such as ethylene glycol or propylene glycol that weakens hydrophobic interactions in proteins with an acidic buffer of ~5.5% is particularly preferably used. As the buffer, acetate buffer, citrate buffer, etc. are used. Such an eluent has stable and mild conditions for mouse interferon, and can be directly applied to the second stage chromatography.

Furthermore, the present inventors investigated chromatography to be introduced into the second purification step in order to effectively remove a small amount of contaminant protein remaining in the purified mouse interferon eluate obtained by the above chromatography. As a result of extensive research on various chromatography methods, we have found that chromatography using a carrier in which copper ions are chelated (hereinafter abbreviated as copper chelate carrier) satisfies this requirement.

As a copper chelate carrier, a carrier in which an exchange group having a chelating ability such as a biscarboxymethylimino group [-N(CH2COOH)2] is bound to agarose, cellulose, polyacrylamide gel, etc. can be used as a carrier with a copper salt such as copper sulfate. Examples include solution-treated carriers. Preferably,"
Chelating Self 10-S” (Pharmacia
A carrier made by chelating copper to an insoluble polysaccharide carrier (manufactured by Co., Ltd.) is used.

Purification of mouse interferon using a copper chelate carrier is performed as follows. The mouse interferon solution eluted from the silica-based adsorbent is preferably adsorbed under conditions of pH 5 or higher. Adsorption can be done by either a batch method or a column method, but the column method has higher adsorption efficiency.

Elution is performed with an acidic buffer such as phosphoric acid, acetic acid, citric acid, etc., and D)-15 or less is preferable. However, under high ionic strength, elution at even higher pH becomes possible. The ionic strength can be increased by increasing the concentration of buffer salts such as phosphoric acid, acetic acid, citric acid, etc., or by adding neutral salts such as sodium chloride, potassium chloride (0.2-1°0M). Elution The composition and liquid amount of the agent are not particularly limited,
Optimal elution conditions are appropriately determined depending on the amount of contaminant proteins present, mouse interferon, column dimensions, etc.

In this way, mouse interferon is adsorbed and eluted on a silica-based adsorbent, and this eluate is adsorbed and eluted on a copper chelate carrier, thereby removing contaminant proteins mixed in the crude mouse interferon and selectively Mouse interferon can be purified and isolated.

[Effect of the invention] Although mouse interferon is needed for non-clinical studies of interferon using mice and for protein chemical research on interferon, a high-purity purification method that can be easily produced in large quantities has not yet been reported. Not yet.

The present invention is a purification method that is simple, allows large-scale processing, and obtains highly pure mouse interferon. Mouse interferon obtained from water droplets is different from that before purification.
Polyacrylamide electrophoresis (SDS-PAGE) with specific activity several tens of times higher and in the presence of sodium dodecyl sulfate
It was purified to a purity of 99% in the assay conducted by the company, and is a purified sample sufficient for use in non-clinical tests. The silica-based adsorbent used in the first stage is hard, chemically stable, and easy to handle, so it is particularly suitable for processing a large amount of crude liquid in the first stage of purification. Chromatography using a copper chelate carrier, which is roughly the second purification step, has high specificity for mouse interferon, and this makes it possible to remove some remaining contaminant proteins. As described above, the present invention has made it possible to directly connect two types of affinity chromatography with high specificity and separation ability without any special operations, and to easily obtain a large amount of purified mouse interferon with high purity.

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

Mouse interferon activity in the examples is mouse LY
Cells and vesicular stomatitis virus (■SV) were measured by the cytopathic effect (CPE) inhibition method and converted to international units (■-U) using standard mouse interferon.

Example 1 After culturing Escherichia coli (Kaikokenki No. 7662) into which a mouse interferon-β structural gene had been incorporated, the cells were crushed to obtain a crude mouse interferon-β solution as a centrifuged supernatant. This crude mouse interferon solution contains '1.6X1
Contains 0.7 IU/ml of mouse interferon activity;
It contained 19 mq/ml of protein. 2,500 ml of this solution was added to "Control Pore Class" 30
0m1 (44φx200mm> 4°C1 flow rate 150m
It flowed at 1/hr. This column was then injected into a 20mM phosphate buffer (pH 7, O
) After washing with 3,300 ml of 0.5M sodium chloride and 10% ethylene glycol, elution was carried out with 50mM acetate buffer (pH 5.0>).Mouse interferon-
β was purified to a purity of 87% with an activity recovery rate of 70% and a 17-fold increase in specific activity.

Next, “Chelating Self 10-S 6B IF
Column 100ml (26φX200mm>50mM
A copper chelate carrier was prepared by pouring 500 ml of copper sulfate solution made into a 1% solution in acetate buffer (pH 5.0>).Next, 3200 ml of the above eluate containing mouse interferon-β activity was directly poured into the above copper chelate carrier. 4° to chelate carrier
The C1 flow rate was 100 ml/hr. Subsequently, this column was washed with 3400 ml of 20mM acetate buffer (pH 5, 6) containing 0.5M sodium chloride, and then 20mM acetate buffer (p)-14 containing 0.5M sodium chloride,
8), mouse interferon-β was eluted. Mouse interferon-β has an activity recovery rate of 80% and a purity of 99%.
% purified. As a result, the recovery rate of mouse interferon-β from the purified stock solution was 56%, and the specific activity was approximately 2.
0 times, and a purified sample with a purity of 99% was obtained.

Example 2 After culturing Escherichia coli (Kaikokenki No. 7662) into which the mouse interferon-β structural gene had been incorporated, the bacterial cells were crushed,
A crude mouse interferon-β solution was obtained as a centrifugation supernatant. This crude mouse interferon solution contains 1.8x1
Contains 0.7 IU/ml of mouse interferon activity.
It contained 3 mq/ml of protein. Add 10α of this solution to “Microbead Silica” 3180φX60
0 mm) at 4°C and at a flow rate of 1.5α/hr. This column was then added to a 20ml column containing 50% ethylene glycol.
After washing with M phosphate buffer (pH 7, 0>2i.
Elution was performed with 50 mM acetate buffer (pH 5°O) containing 5M sodium chloride and 10% ethylene glycol. Mouse interferon-β has an activity recovery rate of 72% and a specific activity of 2.
The product was raised to 1x J and purified to a purity of 90%.

The above eluate 4 containing mouse interneuron-β activity
600ml was directly transferred to copper chelate carrier 1Q (80φ x 200mm) prepared in the same manner as in Example 1 at 4°C for 50 minutes.
The flow rate was 0 m1/hr. Tsutsui lever column 0.5M
20mM acetate buffer (pH 5, 6) containing sodium chloride
) After washing with 26ff, mouse interferon-β was eluted with 20mM acetate buffer (pH 4,8) containing 065M sodium chloride. Mouse interferon-β
was purified to a purity of 99% with an activity recovery rate of 95%. As a result, mouse interferon-β
The activity recovery rate was 68%, the specific activity was increased 20 times, and a purified sample with a purity of 99% was obtained.

Comparative Example 1 After culturing Escherichia coli (Kaikokenki No. 7662) into which the mouse interferon-β structural gene had been incorporated, the bacterial cells were crushed,
A crude mouse interferon-B solution was obtained as a centrifugation supernatant. This crude mouse interferon solution contains 7.7x1
Contains mouse interferon activity of 0.5 TU/ml;
It contained 8mC1/m+ proteins. Add 320ml of this solution to “Control Pore Class” 10
0m 1 (26φ x 188mm), flow rate 100m
It flowed at l/hr. Next, this column was added to a 0.1M boric acid buffer solution containing 0.5M sodium chloride (pH 9,0).
The cells were washed with >2000 ml and eluted with 0.1 M acetate buffer (pH 5.0) containing 0.5 M sodium chloride and 50% ethylene glycol.

Mouse interferon-β had an activity recovery rate of 60% and a 7-fold increase in specific activity.

Continuing, “’Chelating Cephas 6B t
Each metal chelate column was prepared by flowing 15 ml of each metal salt solution of zinc chloride, cobalt chloride, and nickel chloride in a 50 mM acetate buffer (1% solution at pH 5,0>) into a 3 ml o column (10φ x 33 mm). 40 m+ of the above solution containing interferon-β activity was applied to each of the above metal chelate carriers at 4°C and at a flow rate of 3 ml/hr.
When the carrier was flushed with water, all of the mouse interferon-β passed through without adsorption to the carrier.

Claims (2)

[Claims] (1) A step of bringing the mouse interferon solution into contact with a silica-based adsorbent, a step of eluting the adsorbed substance to the adsorbent with an eluent, a step of bringing the eluate into contact with a carrier to which copper is chelated, A method for purifying mouse interferon, which comprises a step of eluting the adsorbed substance on a carrier with an eluent. (2) The purification method according to claim (1), wherein the eluent for eluting the adsorbed substance on the silica-based adsorbent is a buffer solution with a pH of 4 to 6.