JPS58201722A - Recovery of antiurokinase monoclonal antibody - Google Patents

Abstract

PURPOSE:When other contaminant antibodies are removed from a solution containing antiurokinase monoclonal antibody, anion-exchange chromatography is applied to recover the titled antibody of high purity. CONSTITUTION:A solution containing antiurokinase monoclonal antibody obtained by culturing fused cells producing the same in a vessel for cell cultivation or in mouse abdominal cavity is subjected to anion-exchange chromatography to remove antibodies other than antiurokinase monoclonal antibody, thus producing the same of high purity. As an anion-exchange resin, are cited diethylaminoethyl (DEAE)-Sephadex (trade name registered by Pharmacia Co. Ltd.) or the like, preferably having DEAE groups as functional groups. The electroconductivity of the above solution should be kept in a range from 0 to 15mv/cm on adsorption and rinsing and the eluent should be kept more than 16mv/cm in electroconductivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for removing impure antibodies from a solution containing anti-urokinase single antibodies and recovering highly pure anti-urokinase single antibodies.

Anti-urokinase single antibody is an antibody obtained from the culture medium obtained by culturing anti-urokinase single antibody-producing cells created by cell fusion method, and is used for analysis of urokinase,
Used for refining, etc. In particular, application to affinity chromatography that utilizes antigen-antibody specific binding will dramatically improve the urokinase purification step, so it will be used extensively in the future.

Conventionally, the only method for obtaining large amounts of antibodies has been to purify the desired antibodies from antiserum obtained from immunized animals. Chromatography, affinity chromatography, etc. are well known and commonly used. However, this antiserum method inevitably produces a huge variety of antibodies, and from the antiserum containing many kinds of antibodies,
Even using conventional methods or combinations, it has been extremely difficult to obtain a single target antibody with high purity. The inventors,
In order to improve this point, we used cell fusion method to create anti-urokinase single antibody-producing cells 1. By culturing these cells, we obtained a large amount of anti-urokinase single antibody and used this to recover urokinase. He invented a method to do this and filed an application (Japanese Patent Application No. 3290/1983). However, even in methods using single antibody-producing cells, there is contamination of young impure antibodies, and if a single antibody is to be used as a pure reagent in various fields, it is desirable to remove this impure antibody.

For example, when attempting to use an antibody column as a ligator/dead for purification, the purity of the single antibody used will affect the adsorption capacity, degree of purification, etc. of the prepared antibody column.

The present inventors have found that it is possible to improve these problems by carrying out ion-exchange chromatography in the process of collecting anti-urokinase single antibodies. Based on this knowledge, the present invention was completed.In the present invention, the term "impure antibody" will be used hereinafter to collectively refer to antibodies other than single anti-urokinase antibody. Ru.

That is, the present invention provides a solution containing a single anti-urokinase antibody, which is characterized by removing impure antibodies by performing anion exchange chromatography in the step of recovering the single anti-urokinase antibody. The present invention relates to a method for collecting a urokinase single antibody.

The anti-urokinase single antibody-producing cells used in the present invention are fused cells created by fusion of mouse B cells and myeloma cells. This single antibody-producing fused cell is Mi
lstai'n et al. (Nature, vol. 256.

'495-49' p. 1, 1975), and was first reported by, for example, Clinical Immunology (Aiguchi et al., 12 (1975)).
41: 284-289 negative.

It can be obtained by the method described in 1980.

That is, mouse B was previously immunized with urokinase obtained from human urine or human kidney cell culture fluid as an antigen.
B cells from the spleen of ALB/c♂ (Urokinase used here does not necessarily have to be of high purity (for example, immunity can be achieved if it is 1% or more pure).
and myeloma cells (e.g., P3UIX63Ag8) from the bone marrow abscess of the same mouse were fused in the presence of polyethylene glycol at a cell number lO:1 ratio, and prepared so that only the fused cells could selectively survive. 1
The cells were suspended in HAT culture medium supplemented with 0% beef tallow serum and plated into a 96-well plate. After about a week, most of the cells other than the fused cells of myeloma cells and B cells have died, and a colony of fused cells has been formed.Next, the fused cells are coated with the target antibody against urokinase. In order to investigate whether production is occurring or not, the culture supernatant is used to conduct a 1-double immunoassay” (Eiji Ishikawa et al., Igaku Shoin, 1978).
Check the antibody production ability using a microtiter plate using the enzyme immunoassay method described in 2007). Antibody production (for colonies in the culture supernatant, one colony is one
Cloned to present in one culture hole, 7-10
After culturing for one day, antibody production is checked again using enzyme immunoassay.

Here, too, the antibody productivity (published) is a colony of fused cells that produce the same antibody against the target urokinase, and it is possible to obtain fused cells injected with a single anti-urokinase antibody. The fused cells thus obtained are subcultured into fisheyes and continue to produce single anti-urokinase antibodies.

Next, a solution containing an anti-urokinase single antibody can be obtained by culturing the anti-urokinase single antibody-producing fused cells obtained as described above in a cell culture container or intraperitoneally in a mouse. can. In addition, various conditions related to the culture method using the cell culture container are the conditions described in "Conditions 1, For example, 1 Tissue Culture" used for general cell culture (Junyuki Nakai, Sukerinshu, Asakura Shoten, 1976). be able to. That is, in order to culture cells, a basic culture consisting of a carbon source, a nitrogen source, inorganic salts, etc. is usually required. - This basic medium is also necessary in the present invention, and one example is Minimum Ea
Sen-ti-al Medium) *199 medium, Dulbecco's modified Eagle Mizoia A (Dulbeac
o's Modified Eagle Medium)
, Ham's F-10 medium, Ham's F-1
2 medium, PRMI 1640 medium, etc., and any of them may be used as the basic medium of the present invention.

Furthermore, in addition to these basic media, serum is usually used at 10 to 20%
% (V/V), but the present inventors also made improvements in this respect and invented a method to greatly reduce the amount of serum added by replacing it with other additives, and filed an application earlier. . (Japanese Patent Application No. 56-132,619) Next, the pH and culture temperature of the medium should be adjusted to the conditions normally used for the desired cell growth.
Generally, the pH is 6 to 8 and the culture temperature is 25 to 40°C. Any culture container used for general cell culture, such as a normal culture dish or culture flask, can be applied to the method of the present invention.

Next, the anion exchanger used in the present invention is, for example, DEAE (diethylamine ethyl)-Sephadex ()7 A/W-7, which is used in ordinary anion chromatography.
WBJIA), DEAE-Cellulose.

DEAE-Sepharose (Pharmacia registered trademark 1fs
).

QAE (diethyl-(2-hydroxypropyl)amine ethyltosephadex (registered trademark of Pharmacia), TEAE()!J ethylaminoethyl)-
Examples include cellulose, but a porous ion exchanger having a DEAE group as a functional group is preferred from the viewpoint of binding strength between the ligand and the anti-urokinase single antibody.

According to the method of the present invention, for example, by contacting the anion exchanger packed in a column with a solution obtained by ammonium sulfate precipitation and dialysis from a solution containing the above-mentioned anti-urokinase single antibody, the anti-urokinase single antibody is produced. The antibody is immobilized on the anion exchanger and retained in the column, and unadsorbed impure antibody is eluted and removed. Here, the conductivity during adsorption and washing of a solution containing an anti-urokinase single antibody is θ ~ 15 mJ
/(M), and under this condition most of the impure antibodies can be washed out and removed.Next, the anti-urokinase single antibody adsorbed on the gap ion exchanger has a conductivity of 16 m07.
The ion exchanger can be eluted by using an eluent maintained at a temperature of at least 30 cm, preferably at least 30 cm. When carrying out this method, the washing solution/eluent may be any of those used in ordinary ion exchange chromatography (e.g., alkylamine, aminoethyl alcohol, ammonium, ethylenediamine, imidazole, tris, Phosphoric acid, etc.

Further, it is preferable that all of these solutions be used after adjusting the pH to 6 to 9. Furthermore, as a method for maintaining the conductivity of the washing solution and the eluent, for example, NaH, PO4 + Na
1HPO4, NaCl, KCl, KH,PO4,
Another method is to add one or more inorganic salts such as HPO4* MgCIt +CaC1.

According to the method of the present invention, impure antibodies contained in a solution containing an anti-urokinase single antibody can be easily separated and removed, and a highly pure anti-urokinase single antibody can be obtained. Additionally, since elution can be performed under milder conditions than with affinity chromatography, denaturation of antibodies can be prevented. Furthermore, the anion exchanger used in the present invention is a commercially available product, and has many advantages such as being available at a low price, and is very useful industrially.

Next, according to the example, the failure f! Explain A in more detail (9
) do.

Example: Cells injected with anti-Turokinase single antibody were cultured intraperitoneally in mice, and ammonium sulfate was added to 45% saturation of the obtained ascites.
,0) Dissolve in 5mj and incubate at 4℃ with 200 times the volume of the same buffer.
,-Night dialysis. After the dialysis, the solution obtained by centrifugation was added to Sephacryl 5-aoo (Pharmacia registered trademark),
Charge a 16φX, 74 column and add physiological phosphoric acid a.
By developing with S solution (pH 7) and performing gel filtration,
Antibody fraction from which albumin fraction etc. have been removed (usyy+1)
I got it. This fraction was reduced to 5 mt, and the electrical conductivity was 1.2.
m07cm of 0.025M) lithium-hydrochloric acid buffer (p
Dialysis was performed against H8,0)IJ at 4° C. overnight.

After completion of dialysis, the antibody solution obtained by centrifugation was thoroughly washed with the same buffer solution, and DEAE-cellulose (Wandman jL) was used.
ljDE52) Collar A (16φ, 20ml).

The unadsorbed fraction was converted to a conductivity of 1.2 river/cIIL, o25M.
) Anti-urokinase single antibody '1Nac adsorbed and retained on the column after washing and removing with Lys-HCl-grade surgical solution (pH 8,0)
Elution was carried out using the same buffer solution in which the conductivity (10) degree was gradually increased (1 to 501 RJ/cm2). A2 of the eluted fraction. . The relationship between the antigen (urokinase) binding ability (measured by the enzyme immunoassay described above) and the electrical conductivity is shown in Figure 1. As shown in Fig. 1, most of the antigen binding ability was observed in the recovered fraction. Furthermore, this fraction was found to consist of a single band by polyacrylamide disk electrophoresis.

Example 2 An antibody fraction (257?1 lJ) was obtained from the culture solution 11 obtained by culturing anti-urokinase single antibody-producing cells in a cell culture container by ammonium sulfate precipitation and gel filtration in the same manner as in Example 1. . This fraction was concentrated to 5 ml and transferred to a conductivity of 2.5 rivers/(0.067 M in m) in lithium-phosphate buffer (pH s
Dialysis was performed overnight at 4°C. After dialysis,
Thoroughly wash the antibody solution obtained by centrifugation with the same buffer.
The mixture was passed through a column (16φ, 20 mj) of DEAE-Sepharose CL-6B (Pharmacia S).

The unadsorbed fraction has a conductivity of 2.5/am (0.067M)
! Anti-urokinase single antibody 'f N'a C
Elution was carried out with the same buffer solution whose conductivity was adjusted to 3011 RJ/Cm. The relationship between the eluted fraction A, 8° and the antigen binding ability is shown in Figure 2. 1 and 2, similar to Example 1, most of the antigen binding ability was observed in the recovered fraction, and the electrophoretic I
It was confirmed that C Moho was a single band.

Example 3 Antibody fraction (20w) obtained by the same method as in Example
rl k Reduced by 1ml to 5F)Ij, conductivity 0.5
>n (37 cm of 0.01 M phosphate buffer (pH 8)
,0)ll at 4° C. overnight. After dialysis, the antibody solution obtained by centrifugation was thoroughly washed with the same buffer solution.
AE-Sephadex (registered trademark of Pharmacia) column (lS-.

20ml).

The unadsorbed fraction has an electrical conductivity of 0.5 nrTJ/cyL of 0.0
After washing and removing with 1M phosphate buffer (pH 8,0), by bathing with the same buffer whose conductivity was adjusted to 30 town/cIIL with NaCl, an anti-urokinase single antibody adsorbed on the column. Almost the same results as in Example 2 were obtained.

[Brief explanation of drawings]

(12) Figure 3.1 is a graph showing the relationship between Atao, antigen binding ability, and t conductivity of the bathed fraction showing the DEAE cellulose chromatography pattern in Example 1, and Figure 72 is a graph showing the relationship between Atao, antigen binding ability, and t conductivity of the DEAE cellulose chromatography pattern in Example 2. - It is a graph showing the relationship between A280, antigen binding ability, and electrical conductivity of commonly used fractions showing the Sepharose CL-6B chromatographic pattern. Patent applicant Asahi Kasei Industries, Ltd. (13)

Claims (3)

[Claims] (1) A method for producing an anti-urokinase single antibody characterized by removing impure antibodies by performing anion exchange chromatography in the step of recovering the anti-urokinase single antibody from a solution containing the anti-urokinase single antibody. Collection method (2) DEA in anion exchange chromatography
A method for recovering an anti-urokinase single antibody according to claim 1-1, characterized in that an anion exchanger having 2 E groups is used. (3) In anion exchange chromatography, the conductivity of the adsorbing liquid and the washing liquid is θ~15 rrlJ/ctn, and the eluent is 16 rrlcJ/ctn or more. Method for recovering the anti-urokinase single antibody described