JPS6150925A - Purification of antigen or antibody - Google Patents

Abstract

PURPOSE:To recover an antigen or antibody in purified state, by contacting a solution containing an antigen or antibody with an insoluble carrier to effect the adsorption of the antigen or antibody, and separating with an acid having a specific ionic strength. CONSTITUTION:A solution containing an antigen or antibody (e.g. interferon, constituent protein of serum lipoprotein, serum albumin, etc. as the antigen) is made to contact with an insoluble carrier having the antibody or antigen of the above antigen or antibody. The adsorbed antigen or antibody is separated in high yield and purity with an acid solution having anionic strength of <=0.05, preferably <=0.02 and a pH of 1.5-3.0, preferably 2.0-2.5 (e.g. hydrochloric acid, sulfuric acid, formic acid, etc.). EFFECT:The objective product can be transferred immediately to the stable pH range because of the weak buffering action of the recovering agent. The recovered substance can be analyzed easily by various means.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying antigens and antibodies by affinity chromatography using antibodies or antigen-bound plasters, and particularly relates to a method suitable for purifying interferon.

[Conventional technology]

Interferon, which cells produce when stimulated by viruses, has multifaceted biological activities such as antiviral and antitie effects, and is used as a therapeutic or preventive drug for viral diseases such as hepatitis B, Misaki Health, and influenza. It is also expected to be used as an anti-cancer agent for brain tumors, osteosarcoma, leukemia, and other diseases.

There are three types of interferons: α, β, and γ.For example, human interferon α is typified by interferon produced by human white blood cells.
Human interferon β is typified by interferon (human fibroplast interferon) produced by human diploid fibroblasts.

Human interferon γ is also an interferon produced by T lymphocyte cells.

The affinity clost method is used to purify these interferons, but the antibody chromatography method, which uses an insoluble carrier to which antibodies are bound (hereinafter referred to as antibody-bound carrier), is particularly effective in terms of its specificity and usefulness. This is a particularly excellent method of operation.

However, interferon is highly hydrophobic compared to general proteins, and during antibody column chromatography, it tends to cause nonspecific adsorption due to interactions with antibodies in the column or insoluble carriers due to hydrophobic bonds, so it cannot be used in normal antibody columns. Chromatography is often difficult.

In addition, in recent years, with the development of genetic recombination technology, it has become possible to introduce interferon genes into microorganisms such as Escherichia coli and yeast, or into mammalian cells, and to produce genetically recombinant interferon using a production method different from that of natural interferon. This interferon is considered to have higher hydrophobicity than natural interferon due to the deletion of the vj chain.

Therefore, it can be said that non-specific adsorption is more likely to occur during antibody column operation.

As described above, in order to purify a highly hydrophobic protein using an antibody-bound carrier, the elution method must be carefully considered, and the recovery rate can be increased by at least reducing interactions due to hydrophobic bonds.

An effective elution method for interferon purification using an antibody-bound carrier is the acetic acid eluent (Biochemistry, 3830
(1980)), and the citrate buffer eluent by Novick et al. (J, gen, Virol, 69
．． 905 (1983)) and the like are known.

However, none of these methods necessarily yield stable interferon in high yield, and cannot be said to be an efficient purification method.

In addition, affinity chromatography using antibody-binding carriers
In an example of interferon purification using a simple acidic buffer, a high yield could not be obtained using only a simple acidic buffer, and a high concentration of sodium chloride (Maeyer GuigA, -A#, d Nature 27L 622 (1978)) was used.
), polyhydric alcohols such as ethylene glycol and glycerin (Secleer and Burke, Na
ture 285.446 (1980), Yoneh
ara eL al, J, Biol, Ch.
, 256. 3770 (1981)), or nonionic surfactants such as polyoxyethylene paraoctylphenyl ether (Triton X-100) (St
Attempts have been made to add aehelin et al., J. Biol., 9750 (1981)), but all of these recovery agents are undesirable because they destabilize interferon.

[Problem that the invention seeks to solve]

An object of the present invention is to obtain highly purified and highly stable antigens and antibodies (particularly interferon) in high yield.

Contact with an insoluble carrier bound to an antibody or antigen to adsorb the antigen or antibody, and then recover the antigen or antibody using an acid with an ionic strength of 0.05 or less and a pH of 1.5 to 3.0. The present invention provides a method for purifying an antigen or antibody characterized by the following.

The method of the present invention will be described below, focusing on embodiments using interferon as the antigen, but the present invention is not limited thereto.

The specific procedure for purifying interferon is carried out as follows.

That is, a crude interferon solution is brought into contact with an anti-interferon binding carrier to adsorb the interferon, and then unadsorbed proteins are thoroughly washed away.

After that, ionic strength 0.05 or less, ptl 1.5-3
．． A 0.0 acid solution is passed through the tube, and the interferon is recovered as a dissolved substance.

As the crude interferon solution applied to the method of the present invention, a solution containing interferon α and β is preferable because it maintains a stable structure without losing its antiviral activity even under acidic conditions.

For example, in the case of human interferon β, it refers to a human interferon β-containing solution produced in a culture solution after induction treatment with C or the like.

Human interferon that can be applied to the method of the present invention includes natural human interferon produced by culturing the above-mentioned human fibroblast In addition to microorganisms such as yeast and yeast, it also includes recombinant human interferons produced by cells capable of genetic recombination, including mammalian cells.

The anti-interferon antibody used in the method of the present invention is not particularly limited as long as it is an antibody capable of binding interferon, but it may be a monoclonal antibody, such as a mouse monoclonal antibody produced by cell fusion technology, or a monoclonal antibody produced by immunizing an animal with a conventional method. Antibodies and the like obtained by chromatography are not preferred from the viewpoint of purification efficiency.

An anti-interferon antibody-bound carrier is one in which an anti-interferon antibody is bound to an insoluble carrier such as cellulose, agarose, cross-linked dextran, polyacrylamide, or porous glass. This is due to the reaction between the carboxyl group and the activated substituent group inserted into the carrier.

The main substituents of the carrier include a cyanide bromine active group (e.g., "CNBγ-activated Sepharose 4B"; Pharmacia), an N-hydroxysuccinimide ester group (e.g., "activated CH-Sepharose 4B"; Pharmacia, "Affigel"). 1O"; Bioland), hydrazide derivatives (hydrazide polyacryl agaro-5; Miles), oxirane group ("Eupergit C'; Rohm Pharma), anhydride group ("Eupergit A"),
”; ROHM Pharma Co., Ltd.).

The amount of antibody bound to the insoluble carrier is usually 0.00 mg/mg gel.
The amount is 1 to 10 mg.

Contacting the antibody-conjugated carrier with the crude interferon solution includes:
There are batch methods and column methods, both of which are valid.

The interferon solution that is brought into contact with the antibody-bound carrier usually needs to have a pH in the range of 4 to 10, preferably 5 to 9, and if insoluble matter is present, remove the insoluble matter by means such as centrifugation or filtration in advance. It is recommended that you remove the . Further, this contact is preferably carried out until the adsorption of interferon to the antibody-binding carrier is substantially completed.

In the case of the column method, the flow rate per unit volume (S/V) is 0.
5-55. It is desirable to pass the liquid at p"-, preferably 1 to 2, and in the case of a batch method, the contact is carried out for 1 to 100 hours.

After the contact is completed, most of the impurities other than interferon can be removed by washing the antibody-bound carrier with a phosphate buffer containing 1 molar sodium chloride and 30% or less ethylene glycol, an acetate buffer, etc. .

In order to recover interferon adsorbed to this antibody-bound carrier, the ionic strength is 0.05 or less, preferably 0.02.
and the pH is 1.5 to 3.0, preferably 2.0 to
A 2.5 acid-containing solution is used as a recovery agent.

The acid of the present invention is not particularly limited, and may be one type or a mixture of two or more types as long as it satisfies the above range, but specific examples include hydrochloric acid, sulfuric acid, formic acid, and citric acid, such as
Standard hydrochloric acid has an ionic strength of 0.01 and al12.

Although the amount of the recovery agent is not particularly limited, the amount required to substantially recover the interferon that has been adsorbed is used.

The recovery rate is always as high as 90% or more with 0.01 N hydrochloric acid, for example.

Note that the present invention is applicable not only to interferon but also to highly hydrophobic antigens.

For example, Apo-A-ISA-11, B, C-1, C-
What are the constituent proteins of plasma riboproteins such as It and C-■?
Examples include wells for I albumin.

In addition, various specific antibodies can also be purified using the medium recovery agent with low ionic strength of the present invention, contrary to the purification of the target antigen.

In this case, an antigen-binding carrier is prepared by binding an antigen corresponding to the antibody to be purified to an insoluble carrier, and the carrier is brought into contact with a solution containing the antibody to be purified, and the specific antibody of interest is recovered and purified using an anti-absorbing agent. do.

At this time, by adding a concentrated neutral buffer solution to the recovered buffalo chloride solution, a stable neutral solution state can be created.

In all of these methods, the target substance can be immediately transferred to a stable pH due to the weak buffering effect of the recovery agent.

(Effects of the Invention) The interferon purification method using the antibody-bound carrier of the present invention not only provides a high yield and high purity, but also has the following advantages: 1) stability of recovered interferon; , 2) Ease of changing the solvent and pH of the recovered solution 3) Also, in the case of volatile solvents (HCN, formic acid), various analysis operations of the recovered substances are also possible.

To explain in more detail, the recovery agent of the present invention has much better stability of interferon than the recovery agents known up to now.

For example, with 0.01N hydrochloric acid, in the refrigerator (4-8℃)
Even after storage for one month, no deactivation of interferon was observed.

Furthermore, since the interferon solution recovered using the recovery agent of the present invention has a low ionic strength and a weak buffering effect, the composition of the solution can be changed to a desired one by adding an appropriate substance.

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

Combine them to create an antibody-bound carrier.

Using 2 ml of this carrier Q, a solution containing 4 x 10' units of the E. coli-produced gene &lI#A type human interferon β is brought into contact with the carrier Q in a punching manner, and vIL is attached.

After washing with a predetermined washing solution, the adsorbed interferon β was brought into contact with several types of recovery agents in a batch manner, and the recovery rate and purification ratio were compared.

The results are shown in Table 1.

Table 1 Example 2 Solution stability of interferon recovered from antibody column wh
A column of the same antibody-binding carrier as in Example 1 was filled with 2 m Il gel, and recombinant interferon β produced by Escherichia coli (specific activity 3 x 10'ji' L/mg) was added to the column.
contact and adsorb.

After thoroughly washing with the specified washing solution, add 10mM hydrochloric acid (pH2
, 2. Interferon β from the antibody column due to ionic strength 0.01)? I let it come out.

Recovery rate 92%, purification magnification 50 times, specific activity 1.5 x 101
It was in units/mg.

The recovered interferon β was kept at 37° C. for 10 days, and changes in interferon titer over time were monitored.

The results are shown in Table 2.

Table 2 Example 3 About 7 mg of anti-human interferon β mouse monoclonal antibody is bound to an insoluble carrier (Affigel 10) per ml of gel to prepare an antibody-bound carrier, and 1 liter of this antibody is loaded into a column.

A solution (pH 7,
4) Pass the liquid through and completely absorb it.

After cleaning with a specified cleaning solution, 0. O1 normal hydrochloric acid (pH2
, 2, when interferon β adsorbed to the carrier in the column is eluted with an ionic strength of 0.01), 1.70xlQ
11+ units of interferon beta were recovered, yield 97
%, the purification ratio was approximately 6 times, and the specific activity was 8 x 10' units/I1g.

In this way, interferon purification using large columns is also possible.

Example 4 A column was filled with 2 ml of the same antibody-binding carrier as in Example 3, and 2.2 XIO'IO' Chinese hamster cultured cell-produced recombinant interferon-β
(Specific activity 6.I XIO' units/units/m solution is passed through,
It is brought into contact with the antibody in the column and adsorbed.

After thorough washing with the specified washing solution, o, ois molar hydrochloric acid (
pH 2.0, ionic strength 0.015) was passed through the antibody?
, 3r.

The purification magnification was 107 units/B, which was as high as possible.

Example 5 A column was filled with 10 mJ of the same antibody-bound carrier as in Example 3, and a natural interferon-β produced by human fibroblasts (specific activity: 2.5 XIO' units/unit/m solution) was passed through the column at a low flow rate. Drain and absorb.

After thoroughly washing with the specified washing liquid, add 0.01M hydrochloric acid (p
When interferon-β was eluted from the column using H2,2, ionic strength 0.01), the recovery rate was 90%, the specific activity was 1X10'' units/mg, and the purification ratio was 400 times.

Example 6 Purification of anti-interferon antibodies using an interferon-conjugated carrier column.

Purified recombinant human interferon β was added to an insoluble carrier (Affigel 10) at 2.4 g/mJ of gel.
mg to form an interferon-conjugated carrier.

This t ■ body I Qmj! is packed in a column, and an immunoglobulin fraction collected from anti-human interferon β anti-rabbit serum by flow fractionation is passed through the column.

After washing the column with the specified washing solution, 15mM salt Fjl
When the anti-interphenylon β-specific antibody was eluted at (pH 12,0, ionic strength 0.015), 96% of the antibody neutralizing ability was recovered, and the degree of purification was more than 500 times higher.

Example 7 Purification of anti-Escherichia coli protein antibodies.

E. coli cells are disrupted by the Mantoni-Gorlin method, insoluble components are removed by centrifugation, and then nucleic acids are removed using polyethyleneimine.

Thereafter, the soluble protein component is concentrated using a 70% saturated flow to obtain the desired soluble E. coli protein.

This E. coli protein is bound to an insoluble carrier (Affigel 10) at t/img per ml of gel to prepare an E. coli protein-bound carrier.

Qml of this carrier I is packed into a column, and an immunoglobulin fraction (PBS solution) collected from anti-Escherichia coli protein anti-rabbit serum by flow fractionation is passed through.
1 After that, after thoroughly washing the column with PBS, 151 hydrochloric acid (pH 2,
When the anti-colon protein-specific antibody is eluted at an ionic strength of 0.0 and ionic strength of 0.015), about 80% of the antibody neutralization value initially applied to the column is recovered, and the degree of purification is calculated to be about 10 times higher, which is generally said to be Approximately equal to the specific antibody content present in the immunoglobulin fraction containing F [patent applicant Toshi Co., Ltd.

Claims (1)

[Claims] (1) A solution containing an antigen or antibody is brought into contact with an insoluble carrier to which the antibody or antigen is bound, and after the antigen or antibody is adsorbed, the ionic strength is 0.05 or less.
A method for purifying an antigen or antibody, which comprises recovering the antigen or antibody using an acid having a pH of 1.5 to 3.0.