JPS58176547A - Carrier for immunoaffinity-chromatography - Google Patents

Abstract

PURPOSE:To obtain a carrier for immunoaffinity-chromatography having no nonunique absorptivity for impurities, by activating a water-insoluble gel having OH groups with a bifunctional compd. having an epoxide group, and combining it with a specified polyethyleneglycol deriv. CONSTITUTION:Water-insoluble gel having OH groups, such as agalose or poval beads, is activated with a bifunctional compd., such as epihalohydrin or bisoxirane, and combined with one of polyethylene glycol derivs. represented by formulae I and II (n=2-3, m=1-10) to obtain an immunoaffinity chromatographic carrier. This carrier is especially suitable for separation and purification of biological active substances, and, for example, the carrier combined with an antibody combines with an antigen in an organism fluid antiquely without being affected other components, therefore, the purified antigen can be obtd. with high recovery yield by eluting the adsorbed antigen.

Description

Detailed Description of the Invention The present invention relates to a carrier for immunoaffinity chromatography that binds a useful biologically active substance, particularly an antibody, as a ligand. Immunoaffinity, which improves antibody recovery efficiency, prevents nonspecific adsorption, can withstand repeated use, and can obtain an adsorbent for immunoaffinity chromatography with high immobilization efficiency, by introducing a polyethylene glycol derivative. O Regarding carriers for chromatography In recent years, technological innovations in biotechnology have been remarkable, and genetic recombination and cell fusion technologies have been used to produce useful substances such as hormones and enzymes, especially inter-7-ron, somatostatin, and incilin, by mass culturing microorganisms or cells. This made it possible to produce trace amounts of useful substances such as Furthermore, the production of lymphokines by cell fusion of human lymphocytes is also beginning to attract attention as an antitumor effect and immunostimulant. (Proc, Natj
, Acaa Sci.

U, S, A, Engineering 8 7717 ('81)) Many of these biologically active and useful substances are expected to be used as therapeutic agents, and the quality of their purification process will determine the success or failure of industrialization. be.

Currently, separation and purification methods include differences in solubility, differences in charge,
Methods that utilize differences in molecular size, shape, chemical or physical affinity, etc. are widely used, but the separation principles of these methods are based on differences in the physical or chemical properties of substances. Therefore, the specificity of separation is generally low, and several methods must be used in combination to increase purification purity, which has the disadvantage of low purification efficiency.

On the other hand, the biological affinity of bioaccompanying polymers is extremely specific due to their unique higher-order structure, and affinity chromatography, which utilizes this biological affinity for separation and purification, has high purification efficiency. It is an excellent means of purification, and is particularly suitable for separating and purifying trace amounts of active ingredients that have biological activity. Immunoaffinity chromatography uses a particularly small amount of highly biologically active and highly specific antibodies as ligands, so it has extremely high purification efficiency and can be used to purify all substances with antigenicity. (・It has a range of applications.

In the purification of interferon, the activity could be increased 5000 times with one round of immunoaffinity chromatography. (Nature.

In immunoaffinity chromatography, antibodies used as ligands have conventionally been purified from the serum or ascites of immunized animals using a cold ethanol method or the like. These are a mixture of antibodies with different specificities, and it has been difficult to produce antibodies with constant antigen specificity and antibody titer.

However, in 1975, 3hler and Milstein
Since the establishment of a cell fusion method for lymphocytes (Na
ture 256 495. ('75)), it became possible to prepare monoclonal antibodies with high binding specificity in large quantities and homogeneously, and the possibility of using immunoaffinity chromatography on an industrial scale was realized.

In immunoaffinity chromatography, which requires high-purity purification, it is necessary to avoid contamination with impurities other than the target substance as much as possible, and suppressing nonspecific adsorption of impurities is an important issue.

Furthermore, in practical terms, since antibodies are expensive, repeated use is an essential condition, and the stability of the prepared adsorbent is also important.

Ligands to water-insoluble gels generally have hydroxyl groups.

Covalent bonding is an excellent method for bonding, and the activation of water-insoluble gels with hydroxyl groups using cyanogen halides has been frequently used, but this method introduces a charged moiety into the binding moiety, resulting in non-specific There were problems such as adsorption and ligand leakage.

An example of an activation method that does not introduce a charged moiety is the epoxide activation method. According to this method, chemically stable alkylamine bonds and ether bonds are formed at the bridge between the carrier and the ligand, resulting in electrostatic interaction. There is no non-specific adsorption due to oxidation, making it suitable for use on an industrial scale. (J, Ch.
romatography.

135.427 ('77)) Special public service 1977-2358
Publication No. 8 discloses a method of activating a water-insoluble gel using an epoxide activation method and binding an enzyme or an enzyme inhibitor thereto.

The present inventors attempted to obtain an adsorbent for immunoaffinity chromatography using this method, but the amount of antibody bound was low, the reaction required a long time under high conditions, and the antibody activity decreased. It was not possible to obtain the desired adsorbent. Therefore, an attempt was made to further introduce a spacer, and a commonly used spacer having a -(C1h)n- structure was used, but non-specific adsorption of impurities could not be avoided.

The present inventors have found that the immobilization efficiency is high, there is no non-specific adsorption,
In order to obtain a carrier that has a high antigen recovery rate and is suitable for an adsorbent for immunoaffinity chromatography that can withstand repeated use, we conducted intensive research and developed a water-insoluble gel with hydroxyl groups that was activated with epoxide and used as a spacer.
The present invention was completed by introducing a polyethylene glycol derivative having a -(CH2-Cl-42-0-)m- structure.

That is, since the spacer according to the present invention has hydrophilicity, there is no non-specific adsorption of impurities, and when used in combination with an epoxy activation method, an excellent carrier for immunoaffinity chromatography can be obtained.

The present invention will be explained in detail below.

Water-insoluble gels are used as carriers for immunoaffinity chromatography, and examples of natural products include agarose (Sepharose), dextran, cellulose,
Examples of porous cellulose beads and synthetic polymers include polyacrylamide, porous glass, etc. Among them, agarose is excellent from the viewpoint of binding capacity for the antibody as a ligand, elimination of non-specific adsorption, etc. Furthermore, a water-insoluble gel obtained by crosslinking polyvinyl alcohol (hereinafter referred to as Botchi Beads) is an excellent material that has almost the same effect as agarose.

In activating a water-insoluble gel having a hydroxyl group, a bifunctional compound having an epoxide group, such as shrimp halohydrin and bisoxirane, is used. The former is preferably epichlorohydrin, and includes epoxides.

These compounds react under relatively mild conditions and can easily introduce epoxy groups.

The spacer is necessary to prevent a decrease in the antigen recovery rate due to steric hindrance, and the spacer is necessary for the present invention. CH20
)m-(CH2)n-NH2...(1) or NH2-NH・Co-(Q-b)n-0-(CX-1
2.CH20) m-(Q(2).

-CO11NH@NHz (2) is used.

In the formula, m is 1 to 10, and m is 10 to prevent steric hindrance.
The amount below is sufficient, and even if it is more than that, not only the effect will not improve but the antigen recovery rate will decrease. In the formula, n is 2
~3 is appropriate. The compounds (1) and (2) above may be produced by any method, for example, a method of introducing an amino group or a hydrazide group into polyethylene glycol by a conventional method ('l"etrahedron Letters
N131. P, 2839 ('78)), and also commercially available intermediates such as 1,2-bis(2-cyanoethoxy)
There is a method of introducing an amine group or hydrazide group by reduction or hydration reaction of ethane (manufactured by Tokyo Kasei Co., Ltd.) (Chemistry and Industry of Cyanide Compounds, Okada et al., Saiwai Shobo), and it is also possible to purchase products sold by RLJ. can. For example, floor chemistry,
α,ω-bis(3-aminopropyl)polyethylene glycol ether natocal, manufactured by Co., Ltd.

Attachment of polyethylene glycol derivatives to epoxide-activated water-insoluble gels is achieved by reacting with epoxy groups under strongly alkaline conditions. The amount of polyethylene glycol derivative used is 10 to 10 of the epoxy groups introduced into the water-insoluble gel.
0x morteal.

Usually pH 9-j3. React at 25-75°C for 3-20 hours.

Methods for binding the antibody to the carrier of the present invention include a binding method using geltaraldehyde, a method for binding by periodic acid oxidation of the glycol moiety of the sugar chain portion of the antibody, and a method for binding using N-hydroxyconotate. The method of binding by imidonidelization is also a binding method that does not cause side reactions, but the operation is somewhat complicated, □□□thodeEnzymal, 34.7
7 ('74)) and other known methods of attachment can be used.

In the binding method using glutaraldehyde, the carrier is reacted with a glutaraldehyde solution at pH 5 to 9 for 15 to 60 minutes and thoroughly washed. Glutaraldehyde is used in an amount of 10 to 100 times the molar amount of the amino group or hydrazide group bonded to the carrier. The carrier was then mixed with the antibody solution pi (6,5~9, temperature 4~
React for 3 to 24 hours at 25°C, and combine the aldehyde group bound to the carrier with the amino group in the antibody molecule using a Schiff base reaction.In addition, the sugar chain portion of the antibody is oxidized with periodate. The method first oxidizes the antibody using periodic acid or a salt thereof under acidic conditions. At this time, oxidation occurs between adjacent hydroxyl groups of the glycol, and at the same time 1 mole of 104- is reduced to IO3-, this site is cleaved and 2 moles of aldehyde groups are generated.

The generated aldehyde group is brought into contact with the amine group or hydrazide group of the carrier of the present invention and bonded by a Schiff base reaction.

Schiff bases are unstable under acidic or alkaline conditions, and in immunoaffinity chromatography, when elution of adsorbed tannochlorite is carried out under acidic or alkaline conditions, it is desirable to stabilize the Schiff base, and sodium borohydride or cyanohydride is preferred. It can be easily stabilized by reducing with sodium boron or other reducing agent at a temperature of 4 to 25° C. for several tens of minutes to several tens of hours.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

In addition, in the following examples, the antigen recovery rate was measured by the following method.

■) Antigen radioactive iodine (1251 identification Chlorami 7T method (Nature, 194 "495 (
'62)) and purified by gel filtration.

2) Measurement of the amount of antigen binding of soluble antibodies Add an equal amount of diluted labeled antigen solution to a certain amount of antibody solution. Stir vigorously, leave for a certain period of time, centrifuge, and radiate the supernatant or precipitate. The activity was measured and the maximum amount of antigen that could be bound was determined.

(The antigen concentration, antibody concentration, and standing time were determined through preliminary experiments.) 3) Measurement of the amount of antigen binding of the adsorbent (immobilized antibody) The adsorbent was packed into a column, and the antigen labeled with 125I was supplied to increase the affinity. Chromatography was performed and the radioactivity of the adsorbed antigen was measured to determine the amount of antigen bound to the adsorbent.

4) Calculation of antigen recovery rate It was calculated from the ratio of the maximum amount of antigen adsorbed in the actual column reaction (3) to the amount of antigen that can be bound by the soluble antibody used to prepare the adsorbent.

That is, antigen recovery rate = y x 1o o %.

Wash well on a glass filter and suction-filter Sepharose 4B (manufactured by Pharmaci 71J) 20Ji'\( (wet weight) into a round-bottomed flask, add 30ml of distilled water and 20ml of distilled water.
M, Na01-113 ml, x Pichlorhydry
3 ml were added one after another and shaken in a constant temperature bath at 40'C for 2 hours.

Next, the activated gel was obtained by washing thoroughly with distilled water on a glass filter. Approximately 500 μmole/9 (solid content) of epoxy groups were introduced. The determination of epoxy groups was performed using a titration method using hydrochloric acid (J, Chromatogr, 90°87
('74)). (The same applies to the following Examples and Comparative Examples) 20 g (wet weight) of epichlorohydrin-activated Sepharose 4B prepared in Example 1-1 was placed in a round bottom glass: 7K.
Add 100 ml of distilled water, 2M NaOH10
0me1 α,ω-bis(3-aminopropyl)-diethylene glycol ether (manufactured by Fusugaku Kagaku Co., Ltd.) 2゜y was added, and the mixture was shaken overnight in a constant temperature bath at 40°C.

Approximately 450 μmole/'J (solid content) α, ω-
Bis(3-aminopropyl) diethylene glycol ether was attached. The amount of α,ω-bis(3-aminopropyl)diethylene glycol ether bonded was determined by the nitrogen content analysis method using the Kjeldahl method. (Organic Quantitative Analysis, Basic Analytical Chemistry Course 11, Kyoritsu Publishing) (The same applies to the following Examples and Comparative Examples).

20 g of poval with a degree of saponification of 99.5 mol% and an average molecular weight of 6000 was dissolved in 300 ml of toluene, and 1 mol% of tolylene diisocyanate was added to the poval.
After reacting at a temperature of 5 hours with vigorous stirring, the mixture was extracted with acetone and washed with distilled water to prepare crosslinked poval peas.

The poval beads obtained in Example 2-1 were used in place of Sepharose 4B, and the poval beads were activated in the same manner as in Example 1-1, except that the amount of epichlorohydrin used was 6 ml. Approximately 300 μmoleZg (solid content) of epoxy groups were introduced.

A carrier of the present invention was obtained by bonding a polyethylene glycol ether derivative in the same manner as in Example 1-2, except that the ehyrylohydrin activated ossified poval beads obtained in Example 2-2 were used. Approximately 200μmo! α, ω of e/g (solid content)
-bis(3-aminopropyl)diethylene glycol ether was attached.

Example 3-1 Binding example 1 of anti-bovine albumin antibody
5 g (wet weight) of each of the carriers of the present invention prepared in Example 2-2 (using Sepharose) and Example 2-3 (using Poval beads) was treated with 5 ome of 5% glutaraldehyde for 30 minutes.
01M phosphate buffer (hereinafter referred to as P 13 S) (pi
-18,0) was thoroughly washed.

Separately, an anti-bovine albumin antibody was prepared from rabbit antiserum (manufactured by Fuji Kinki Co., Ltd.) by ammonium sulfate precipitation and DEAE cellulose column chromatography. 0.01 g of this anti-bovine albumin antibody 75m9 in PBS (pH 8,0) 50
ml and reacted with each phase activated with glutaraldehyde overnight at 4°C. Further, unreacted aldehyde groups were blocked by treatment with 0.2 M ethanolamine (p+(s,s) for 2 hours at room temperature.Next, Na
B8450■ was reacted at a temperature of 4° C. for 6 hours to reduce the Schiff base and obtain an adsorbent for immunoaffinity chromatography. The amount of antigen binding in this case is shown in Table 1.

Anti-bovine albumin antibody-conjugated Sepharose 4B prepared in Example 3-1 from Table 1 and 51 IIt of Povalpeas were each packed into an ICInφ column, and bovine serum 0.2°me
is supplied from the top of the column, and 0.02M boric acid buffer f
-(p118.0. o, t 5M, Na (containing J)
was allowed to flow down until the 01)280 nm of the eluate became 0.005 or less. Next, 0.2M glycine-hydrochloric acid buffer (PH2,5) was passed through the column to elute the adsorbed bovine albumin. Antigen recovery rate and purification purity are shown in Table 2. Purification purity is determined by subjecting the glycine-hydrochloric acid buffer elution fraction to SDS polyacrylamide electrophoresis, Coomassie Priliant Blue staining, and Shimadzu dual-wavelength chromatography scanner C8-9to (
(manufactured by Shimadzu Corporation). (The same applies to the following Examples and Comparative Examples.) The flow rate in the column reaction was 5 ml/2.h.

Example 3-3 Purification of albumin by repeated use When the same column operation as in Example 3-2 was repeated, the adsorbent for immunoaffinity chromatography obtained in Example 3-1 was obtained using anti-bovine albumin-bound Sepharose. No decrease in antigen recovery rate or purification purity was observed for both 4B and Povalpeas after repeated use 30 times. The results are shown in FIG.

γ without antigen binding ability in place of purified anti-bovine albumin antibody
- An adsorbent was obtained in the same manner as in Example 3-1 except that globulin (Bovine, manufactured by Miles) was used. 1 lIn9 of γ-globulin was bound per carrier (11 wet weight).

Example 4-2 Measurement example of non-specific adsorption amount 1-1
5 ml of the adsorbent prepared in above was packed into a 1 crrLφ column, 5 m9 of bovine serum albumin (BSA) was supplied from the second part of the column, and 002 M boric acid buffer (pl (8,0
, 0.15M, containing NaC7) was allowed to flow down. The same Banoff 7 solution was passed through the column until the OD2so nm of the eluate from the column became 0.005 or less, and then 1.0.2 M glycine-hydrochloric acid buffer (pH 2.5) was passed through the column. The BSA recovery rate at this time is shown in Table 3. Table 3 shows that according to the present invention, non-specific adsorption is almost eliminated.

Table 3 Note that the protein was quantified by the method of Lowry.

(J, Biol, Chem, 193.256(+
51)). In addition, the flow rate in the column reaction is 5 m/! /
It was CrrL2·h.

Purified anti-bovine albumin antibody was prepared from rabbit antiserum (Fuji Organ Co., Ltd.) by ammonium sulfate precipitation.

This purified anti-bovine albumin antibody 40m9 was dissolved in 10 ml of 0.1 M acetate buffer (pH 3,7), and NaI
041m9 was added and reacted for 30 minutes at room temperature. Next, ethylene glycol was added to give a final concentration of 0.1M, and the mixture was reacted at 4°C for 4 hours. Furthermore, this reaction solution was dialyzed against o1M acetate buffer (pH 3,7) 11 at 4°C for 18 hours.

5 g (wet weight) of the carrier for immunoaffinity chromatography prepared in Example 1-2 was added to the aldehyde group-introduced anti-bovine albumin antibody prepared in Example 5-1, and 1M sodium carbonate buffer (pH 9) was added. ) was adjusted to 6. After reacting overnight at 4°C, NaBH410m9 was added to the formed Schiff base and treated at 4°C for 5 hours at pH 85 to reduce and stabilize. An adsorbent for graphics was obtained.The amount of antibody bound in this case is shown in Table 1.

Albumin in bovine serum was purified in the same manner as in Example 3-2, except that the adsorbent for immunoaffinity chromatography prepared in Example 5-2 was used. The antigen recovery rate and purification purity are shown in Table 2. Further, no decrease in antigen recovery rate or purification purity was observed even after repeated use 30 times.

Comparative Example 1) An epoxy group was introduced in the same manner as in Example 1-1.
r loin 4839 (wet weight) was mixed with purified anti-bovine albumin antibody (Example 3-1) solution 3 oml (1.5 me)/
me) and reacted at room temperature with gentle shaking. The antibody binding amount m9/g (wet weight) of the obtained adsorbent is shown in Table 4 and Table 1. The antigen recovery rate and purification purity are shown in Table 2.

Comparative Example 2 CNBr-activated Sepharose 4B (manufactured by Pharmacia)
1 g (dry weight) was immersed in ll'l 1 g old C1C15o for 15 minutes, and coupled with coupling buffer (0.5M, NaC6
0.2 M, NaHCO3 containing 7-r-pt (
'8.5). γ-globulin without antigen-binding ability (Bovine, -, Iy,,, < manufactured by Co., Ltd.) 1
s omg wo power y fu +) 7 'Gubatufu t50m
CNBr-activated Sepharose 113 was dispersed in the solution and reacted overnight at 4'C with gentle shaking. 6 m per carrier (wet weight)! 7' γ-globulin was bound. Further, active groups were blocked by treatment with 0.2°N1 ethanolamine (pH 8.5) at room temperature for 2 hours. Using the adsorbent thus obtained, a bovine serum albumin (BSA) elution test was conducted in the same manner as in Example 4-2. Table 3 shows the recovery rates of the boric acid buffer I-eluted fraction and the glycine hydrochloride buffer eluted fraction.

Comparative Example 3 Purified anti-tunalpmin antibody was purified using the same method as Comparative Example 2.
It was bound to Nl3r-activated Sepharose 4B ()' manufactured by Almacia. 5 #i of antibodies were bound per gram (wet weight). These values are also listed in Table 1.

Further, albumin in bovine serum was purified in the same manner as in Example 3-2, and the antigen recovery rate and purification purity are shown in Table 2. Furthermore, repeated tests using the same column showed a significant decrease in the amount of bovine albumin adsorbed after 7 uses. The results are shown in FIG.

Comparative Example 4 485 g (wet weight) of Sepharose activated in Example 1-1 was dispersed in 30 ml of aqueous ammonia (25%).
Amination was carried out by reacting at 0°C for 2 hours.

When the amino group was quantified by the Kermer method, 4
80 μmole/9 (solid content) noamino groups were introduced.

This aminated Sepharose 4 B 5 ,! When purified anti-sialbumin antibody was bound to i' (wet weight 1i) in the same manner as in Example 3-1, the bound amount was i (wet weight).
It was 10m9 per area. (Also listed in Table 1) Albumin in bovine serum was also purified in the same manner as in Example 3-2,
The antigen recovery rate and purification purity are shown in Table 2.

Comparative Example 5 5 g (wet weight) of Sepharose 4B activated in Example 1-1 was added to 25 ml of distilled water 1.2 M, Na in a round bottom flask.
01125 m11 hexamethylene t/7 noamine 1,5I
400μ/g (solid content)
A mole of hexamethylene diamine was introduced.

This hexamethylenediamine-conjugated sepharose 4B
When purified anti-bovine albumin antibody was bound to s ji (wet weight) in the same manner as in Example 3-1, the bound amount was 11 m9 per 1 g (wet weight).

(Also listed in Table 1) Albumin in bovine serum was purified in the same manner as in Example 3-2, and the antigen recovery rate and purification purity are shown in Table 2.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the amount of bovine albumin adsorbed and the number of repetitions in column operation. Patent Applicant Denki Kagaku Kogyo Co., Ltd. Agent Patent Attorney Sada Suzuki Letter of Amendment to Repeated Number of Procedures August 27, 1980 Mr. Kazuo Wakasugi, Commissioner of the Patent Office ■, Minor Case Notification 1980, Patent Application No. 59627 2, Name of the invention: carrier for immunoaffinity chromatography 3,
Person making the amendment Relationship to the case Patent applicant 4, Agent 〒1505, Date of amendment order Voluntary 6, Number of inventions increased by the amendment, 1)
"Target substance" on page 4, line 16 of the specification is corrected to "Target substance." (2) “Me t
hod (EnzymaIJ [1methods En
Corrected by LymolJ. (3) In the same page, page 13, line 13, "manufactured by Budsugaku Kagakusha" is corrected to "manufactured by Tokotsuna Kagakusha." (4) Insert "Contains 0.15M NaCl" after "...processed for 30 minutes" on page 15, line 10. (5) Same, page 16, line 3, ``1 antigen binding amount'' is corrected to ``1 antibody binding lid''. (that's all)

Claims (1)

[Claims] A compound of the following formula in a water-insoluble gel having a hydroxyl group activated with a difunctional compound having an epoxide group. N112-(CI-12),-0-(CH2-CH
20) m-(CH2)--Mklz...(1) or Nl2-N)(-Co-(CH2)n-o-(C
H2-CH20)--(CH2)rl-CO-Nl-1
・N1(2...(2) (where n = 2 to 3 in the formula)
, m = 1 to 10).