JPH06211897A - Immunoglobulin purification method - Google Patents

Abstract

(57) [Summary] [Object] To provide a method for efficiently purifying an immunoglobulin having divalent reactivity in a large amount at a time to a high purity efficiently by a simple operation without pretreatment. [Structure] Immunoglobulin having divalent reactivity
It is subjected to HPLC (manufactured by Tosoh Corporation) using gel Ether-5PW (manufactured by Tosoh Corporation), and separated and eluted using a solution containing ammonium sulfate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying immunoglobulins having two different types of antigen specificity (hereinafter referred to as divalent immunoglobulins). More specifically, it relates to a method for efficiently and simply purifying the substance by subjecting a sample containing an immunoglobulin having divalent reactivity to chromatography utilizing a hydrophobic action in a short time and with high purity.

Immunoglobulins having divalent reactivity are currently being studied as clinical test reagents or therapeutic agents,
It is a protein that is highly expected in the future. As described above, a highly purified method for immunoglobulin having bivalent reactivity, which can be clinically applied in many fields, is particularly desired.

[0003]

2. Description of the Related Art There are several known methods for purifying immunoglobulins having bivalent reactivity. Among them, a typical example is a gel filtration method in which the difference in the molecular weight of proteins in a sample solution is used for separation. From this principle, this method can very easily separate proteins having greatly different molecular weights, but it is impossible to separate them with proteins having an equivalent molecular weight. The immunoglobulin having F (ab ′) ₂ type bivalent reactivity has a molecular weight of about 110.
The gel filtration method can separate the F (ab ′) ₂ type having monovalent reactivity.

Further, a hybrid hybridoma (quadrommer obtained by further fusing two types of hybridomas)
IgG type divalent immunoglobulins secreted from Ma) range from 10 types of combinations of H chains and L chains, and among them, the immunoglobulin having the desired divalent reactivity is selectively purified. It is very difficult to separate.

In addition, a purification method utilizing ion exchange chromatography is known. Specifically, a sample solution containing an immunoglobulin having divalent reactivity is dialyzed against a buffer solution having a low salt concentration, and a cation exchange resin (sulfopropyl (S
P), a resin having a carboxymethyl (CM) group), or an anion exchange resin (diethylaminoethyl (DEA)
E), a resin having a quat-tanary aminoethyl (QAE) group) is adsorbed on the column to gradually increase the salt concentration or to raise the salt concentration all at once, thereby adsorbing the bivalently reactive immunoglobulin. It is a method of selectively eluting and separating and purifying.

According to this method, many F (ab ′) ₂ type immunoglobulins are eluted with the initiation buffer, and F (ab ′) ₂ type immunoglobulins having a divalent reactivity are selectively obtained. It is difficult to avoid, and the yield is unavoidable. Further, the operation of dialyzing against the starting buffer solution as a pretreatment for applying to the column is complicated, and there is a disadvantage that it has to be carried out for a long time.

Recently, using hydroxyapatite gel,
A method for selectively purifying a bivalent reactive immunoglobulin secreted from a hybrid hybridoma has been reported (Tada, H. et al., Hybr.
Idoma, 8, 73-83, 1989). However, the hydroxyapatite column is expensive, and is not suitable for scale-up because of problems such as pressure resistance and easy deterioration.

On the other hand, a purification method (affinity chromatography) that makes good use of the antigen specificity of a protein having an affinity for an immunoglobulin having a bivalent reactivity or an immunoglobulin having a bivalent reactivity can be considered.
Antibodies (polyclonal antibodies, monoclonal antibodies) that have specificity for the constant region of immunoglobulins that have bivalent reactivity and immunoglobulins that have bivalent reactivity by immobilizing the antigen itself on an insoluble support And then reversibly adsorbed, an organic solvent such as dioxane (10%), an acidic solution such as propionic acid (pH = 2.5, 1M), hydrochloric acid (pH = 2.5), ethylene glycol ( 50%), chaotropic ions (I ⁻ , CF ₃ COO ⁻ , SCN ⁻ , CC
l ₃ COO ⁻ , pH = 7.0 to 8.0, 3M) and the like, or by decreasing the interaction between the bivalently reactive immunoglobulin and the antibody or antigen therefor, or urea (pH = 7. 0.8M), guanidine hydrochloride (pH =
(3, 6M) and the like are used to release and elute the bivalently reactive immunoglobulin with a protein denaturant. This 2
It is possible to obtain the immunoglobulin having the desired bivalent reactivity by using a column on which each type of antigen is immobilized.

However, in general, a decrease in antibody activity and yield is inevitable. Further, the carrier deteriorates quickly, which is disadvantageous in terms of reproducibility and cost. Naturally, there is a disadvantage in stepping up to large-scale purification. Furthermore, at present, the relatively inexpensive recombinant protein A and protein G, which are widely used for the purification of antibodies (immunoglobulin G class), hardly adsorb immunoglobulins having F (ab ′) ₂ type bivalent reactivity. So it is not available.

When the latter purification method is used for obtaining a large amount of highly pure immunoglobulin having a high degree of reactivity, the cost problem is large as described above. For this, the former non-specific purification method using ion exchange chromatography is used.

However, when the former purification method using ion exchange chromatography is performed, it is necessary to perform a pretreatment of dialysis against a starting buffer solution having a low salt concentration and desalting. Thus, the protein sample is exposed to a dilute solution, which is very unstable condition for the protein. Some samples may irreversibly precipitate, which may lead to the inconvenience of having to start the chromatography starting buffer with a high salt concentration.

It is also difficult to selectively separate and purify immunoglobulin having divalent reactivity, and it is not suitable for effective purification in one step. In other words, ion exchange chromatography and affinity chromatography,
The purification method is a combination of hydroxyapatite chromatography.

Concentration operation is difficult with culture medium in which hybrid hybridomas of immunoglobulin-producing strains having divalent reactivity are cultured. Particularly, it is difficult to remove the pH indicator such as phenol red added, and it cannot be sufficiently removed by a simple and effective salting out method using ammonium sulfate or an ultrafiltration method. This greatly influences the separation and purification of bivalently reactive immunoglobulins to the end. It is also accepted that the pH indicator cannot be eluted easily once it is adsorbed on the filler, and often accelerates the deterioration of the filler.

[0014] In the near future, a large-scale culture method of human-type bivalent immunoglobulin has been established, and the bivalent-reactive immunoglobulin is used as a drug or a carrier for a drug particularly against neoplasms such as cancer and virus-infected cells. However, it is expected to be applied therapeutically, but at that time, it is clear that the purification method also occupies a very important weight in the manufacturing process, and the development of the method is indispensable.

[0015]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for purifying an immunoglobulin having divalent reactivity, which is significantly more useful than the above purification method.

[0016]

The present inventor has arrived at the present invention as a result of extensive studies on the above problems. That is, the present invention is a method for purifying immunoglobulin, which comprises subjecting a sample containing an immunoglobulin having bivalent reactivity to hydrophobic interaction chromatography to selectively separate and elute the immunoglobulin of interest. Is. Hereinafter, the present invention will be described in more detail.

In the present invention, there is no particular limitation on the method for producing the purified bivalent-reactive immunoglobulin,
Examples thereof include those obtained by chemical synthesis, those obtained by fusing two or more kinds of hybridomas, those obtained by genetically engineering, and the like. Such divalent immunoglobulins include various impurities such as normal monovalent immunoglobulins. In the present invention, such impurities have a hydrophobic interaction. It is removed by using chromatography utilizing.

Chromatography utilizing the hydrophobic interaction used here is not particularly limited, but preferably the carrier comprises a non-porous resin, sepharose, a hydrophilic polymer resin, and a functional group. Preferred are those having an oligoethylene glycol group, a butyl group, a phenyl group, an octyl group, a neobenzyl group and the like. Specifically, TSKgel Ether-5P
W, Phenyl-5PW, Butyl-NPR, etc. are listed.

A buffer containing salt or a salt is added as it is to a sample containing immunoglobulin having bivalent reactivity,
Purification is carried out by adsorbing to a column in a salt solution, gradually or at once decreasing the salt concentration, and selectively separating and eluting the divalent-reactive immunoglobulin from the packing material. The salt used here may be one generally used, but preferably ammonium sulfate, sodium sulfate,
Examples thereof include potassium phosphate and sodium phosphate.

[0020]

As is apparent from the above description, according to the present invention, compared to the conventional method, a simple operation can be performed in a short time, without pretreatment, with high purity efficiently, and in a large amount at a time. It is possible to purify reactive immunoglobulins.

[0021]

EXAMPLES Hereinafter, the conditions will be described in detail with reference to examples. However, the present invention is not limited to these examples.

Example 1 (1) Chemical Synthesis of Immunoglobulin Having Bivalent Reactivity from Two Mouse Monoclonal Antibodies A monoclonal antibody against human thyroid stimulating hormone (TSH) (hereinafter referred to as TS07, IgG1) was used. 0.
Thoroughly dialyzing against 1 M citrate buffer (pH 3.7),
Pigs prepared with 1M citrate buffer (pH 3.7)
Pepsin was added to TS07 at 1% (w / w). After limited hydrolysis at 37 ° C. for 2 hours, the F (ab ′) ₂ component of TS07 was obtained. Similarly, a monoclonal antibody against alkaline phosphatase (hereinafter abbreviated as AP05, IgG1) is subjected to limited hydrolysis with porcine pepsin,
AP05 F (ab ') ₂ was obtained.

AP05 F (ab ') ₂ was added to 10 mM acetate buffer (pH 5.5) containing 100 mM sodium chloride.
Was adjusted to 1 mg / ml. 10 mM β in the solution
-H of F (ab ') ₂ by adding mercaptoethanol
The interchain disulfide bonds were reduced. Target AP0
Fab ′ of 5 was obtained by separating it from the reducing agent, unreduced F (ab ′) ₂ by gel filtration chromatography. Fab '
Is a monovalent antibody having one antigen binding site.
Immediately, 5,5′-dithiobis (2-nitrobenzenic acid) (hereinafter abbreviated as DTNB) was added to the solution so as to have a concentration of 50 mM, and TNB was chemically bonded to the sulfide group of Fab ′. Excess DTNB was removed by gel filtration chromatography to prepare Fab′-TNB.

On the other hand, F (ab ′) _{2 of} TS07 was also reduced with β-mercaptoethanol in the disulfide bond between H chains. Excess β-mercaptoethanol was separated by gel filtration chromatography, and after removing the excess reducing agent, equimolar AP ′ Fab′-TNB was immediately added. 1% of tris (hydroxymethyl) aminomethane (pH 8.0) (hereinafter referred to as Tris) prepared to 1M
/ 20 volume was added and the reaction was carried out at 4 ° C. overnight.

(2) Purification of immunoglobulin having divalent reactivity Powdered ammonium sulfate was added to the solution reacted in (1) so as to have a concentration of 1.5 M, and well stirred to dissolve. The reaction solution was TSKgel Ether-5PW (7.
5 mmI. D. × 75 mm, manufactured by Tosoh Corporation was applied to HPLC (manufactured by Tosoh Corporation). Fifty
The column was washed with ml of PBS containing 1.5 M ammonium sulfate to elute non-adsorbed components.

Immediately after washing, the ammonium sulfate concentration was lowered with a linear gradient from 1.5 M to 0 M PBS over 30 minutes. The results are shown in Fig. 1.
In the figure, the horizontal axis represents the elution time and the vertical axis represents the absorbance at 280 nm. From the chromatograph in FIG. 1, three main peaks were observed. Peak 1, 2, 3 from the peak with the shortest elution time
And The flow rate at this time was 1.0 ml / min, and the detection wavelength was 280 nm. The eluate is 0.5 ml / tu
Fractionated by a fraction collector at be.

(3) Examination of the presence or absence of antigen specificity for alkaline phosphatase In each well of an untreated microtiter plate (96-well Nunc plate, manufactured by Intermed), (2)
Each sample fractionated in step 10 was multiplied 10 times with PBS and added with 50 μl of each, and incubated at 4 ° C. overnight. Remove the solution from each well and add 0.4% tween to PBS.
en) -20, a solution containing PBS (hereinafter PBS-T) was washed three times, and then 0.1% bovine serum albumin (BSA) was used.
300 μl of PBS solution in which was added to each well,
Blocking treatment was performed at 4 ° C. and the sample was stored as it was.

The microtiter plate was returned to room temperature, washed with a PBS-T solution, and then 5 μg / m in PBS.
50 μl of a solution of alkaline phosphatase (hereinafter abbreviated as ALP), which was designated as 1, was added. 90 at 37 ℃ in this state
Let stand for a minute. 50 mM carbonate buffer (pH 9.8) -1
100 μl of para-nitrophenyl phosphate (PNPP) adjusted to 3 mg / ml with 0 mM magnesium chloride was added to each well. After color reaction for 30 minutes at room temperature, add 1N sodium hydroxide solution to 1
The enzyme reaction was stopped by adding 00 μl.

For each well of the above microtiter plate, the absorption intensity at a wavelength of 405 nm and a control wavelength of 492 nm was measured by an automatic microtiter plate reader (East Sotho).
The results measured by MPR-A4 (trade name, manufactured by Co., Ltd.) are shown by black circles in FIG. In the figure, the left axis is the color reaction 405 nm.
The absorbance of the fractions was plotted on the horizontal axis, and the antibody titer of each fraction was shown. Fractions having alkaline phosphatase activity were found in peaks 1 and 2.

(4) Examination of the presence or absence of antigen specificity for thyroid stimulating hormone (TSH) 0.1 M was added to each well of an untreated microtiter plate (96 well Nunc plate, manufactured by Intermed).
100 dissolved in sodium carbonate buffer (pH 9.6)
50 μl of a solution of TSH of μIU / ml was added and incubated overnight at 4 ° C. Remove the solution from each well and add PB
After washing three times with a solution containing 0.4% tween-20 in S (hereinafter PBS-T), PBS solution 3 in which 0.1% bovine serum albumin (BSA) was dissolved
00 μl was added to each well, blocked at 4 ° C., and stored as it was.

After returning the microtiter plate to room temperature and washing with PBS-T solution, each sample fractionated in (2) was multiplied by 10 with PBS and added to each well in an amount of 50 μl. In this state, it was left at 37 ° C. for 90 minutes. Next, goat anti-mouse IgG labeled with peroxidase
(F (ab ′) ₂ specific; manufactured by Tago) antibody was PBS-T
Dilute the solution 5000 times and add 50μ to each well.
I was added in increments of 1. Incubate at room temperature for 90 minutes
The solution was removed and washed with a PBS-T solution three times. In addition, 0.3 mg / ml of 2,2-azinodi-
Each of the substrate solutions consisting of (3-ethylbenzthiazoline sulfate) -diammonium salt (ABTS) and 0.1 M citrate buffer (pH 4.1) containing 0.01% hydrogen peroxide (H ₂ O ₂ ). Add 100 μl to the well and incubate at room temperature for 30
After color reaction for 10 minutes, add 200 mM oxalic acid solution to 10
The enzyme reaction was stopped by adding 0 μl.

For each well of the above microtiter plate, the absorption intensity at a wavelength of 415 nm and a control wavelength of 492 nm was measured by an automatic microtiter plate reader (East Sotho).
The result of measurement by MPR-A4 (trade name, manufactured by Co., Ltd.) is shown by a white circle in FIG. In the figure, the right axis shows the color reaction of 415 nm.
The absorbance of the fractions was plotted on the horizontal axis, and the antibody titer of each fraction was shown. The fractions in which the antibody titer to thyroid stimulating hormone was recognized were peak 2 and peak 3.

(5) Examination of the presence or absence of antigen specificity (bivalent reactivity) for both TSH and ALP Untreated microtiter plate (96 well Nunc plate, interface)
100 μIU / ml TS dissolved in 0.1 M sodium carbonate buffer (pH 9.6) in each well of Med Co., Ltd.
50 μl of H solution was added and incubated at 4 ° C. overnight. Remove the solution from each well and add 0.4% Tween to PBS.
Solution containing tween-20 (hereinafter PBS-
After washing 3 times with T), 300 μl of PBS solution in which 0.1% bovine serum albumin (hereinafter BSA) was dissolved was added to each well, blocked at 4 ° C. and stored as it was.

After returning the microtiter plate to room temperature and washing it with a PBS-T solution, the sample of each peak fractionated in (2) was multiplied by 10 with PBS and 50 μl was added to each well. It was In this state, it was left at 37 ° C. for 90 minutes. Next, 50 μg / ml A dissolved in PBS
100 μl of LP solution was added and incubated at 37 ° C. for 90 minutes. After washing with PBS, 50mM carbonate buffer (pH 9.8) -10mM magnesium chloride for 3m
Paranitrophenyl phosphate (PNPP) adjusted to g / ml was added to each well in an amount of 100 μl. After color reaction for 30 minutes at room temperature, 100 μl of 1N sodium hydroxide solution was added to stop the enzymatic reaction.

For each well of the above microtiter plate, the absorption intensity at a wavelength of 405 nm and a control wavelength of 492 nm was measured by an automatic microtiter plate reader (East So-
The results of measurement by MPR-A4 (trade name, manufactured by Co., Ltd.) are shown in FIG. In the figure, the absorbance at 405 nm in the color reaction is plotted on the vertical axis and the fraction number is plotted on the horizontal axis, showing the antibody titer of each fraction. The fraction showing alkaline phosphatase activity is only peak 2, that is, TSH.
It was shown that this peak is a divalent immunoglobulin because it has antigen specificity to both ALP and ALP. From the above, it was shown that by using hydrophobic interaction HPLC, an immunoglobulin having bivalent reactivity can be effectively purified in one step.

[Brief description of drawings]

FIG. 1 is a diagram showing an elution pattern of a hydrophobic interaction chromatography in (2) of Example 1.

FIG. 2 is a graph showing antibody titers against alkaline phosphatase and thyroid stimulating hormone in (3) and (4) of Example 1.

FIG. 3 is a diagram showing the bivalent antigen specificity to alkaline phosphatase and thyroid stimulating hormone in (5) of Example 1.

Claims (4)

[Claims] 1. An immunoglobulin characterized by subjecting a sample containing immunoglobulins having two different antigen specificities to hydrophobic interaction chromatography to selectively separate and elute the immunoglobulin of interest. Purification method. 2. The method according to claim 1, wherein the packing material for chromatography uses a non-porous resin, sepharose or a hydrophilic polymer resin as a carrier. 3. The method according to claim 1 or 2, wherein
Immunoglobulins with two different antigen specificities
A method which is either one obtained by chemical synthesis, one obtained by fusing two or more kinds of hybridomas, or one obtained by genetic engineering. 4. The method according to any one of claims 1 to 3, wherein the salt used is ammonium sulfate, sodium sulfate, potassium phosphate, or sodium phosphate.