JPH01247096A - Recovery of monoclonal antibody - Google Patents

Abstract

PURPOSE:To inexpensively recover the monoclonal antibody of high purity from the culture mixture by adjusting the culture mixture containing the monoclonal antibody to 6.5-8.5pH and treating the culture mixture with a cation exchange resin. CONSTITUTION:A culture mixture containing a monoclonal antibody such as CLN-IgG produced by a human-human hybridoma CLN/SUZ H11 strains is adjusted in pH to 6.5-8.5. The culture mixture is treated with a cation-exchange resin having sulfonic or carboxylic acid groups to adsorb the monoclonal antibody. Then, a NaCl solution of a prescribed concentration is used as an eluent to elute the monoclonal antibody. The recovered monoclonal antibody is of high purity and can be used as a medicine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for recovering monoclonal antibodies.

[Problems to be solved by conventional techniques and inventions 1197
Since 6Hler and Milstein established the hybridoma method in 1995, monoclonal antibodies with various antigen specificities have been produced such as mouse x mouse hybridomas and human x human hybridomas. In addition, human lymphocytes were infected with EBV (Epstein-Barr).
Monoclonal antibodies have also been produced using cells transformed by a virus.

Possible uses for monoclonal antibodies include medicines and diagnostic agents, and many are currently being developed.

High purity is required for use as a medicine.

To prepare large quantities of monoclonal antibodies for use in these applications, hybridomas, etc. are cultured in a culture device such as a spinner flask, or ascites is cultured in the peritoneal cavity of a mouse, etc., and monoclonal antibodies are produced in these culture fluids. This is done by purifying and collecting from the culture solution.
To recover monoclonal antibodies from the culture solution, hybridomas and other solids in the culture solution are removed, adensation and desalting are performed, followed by affinity chromatography or ion exchange chromatography using protein A, anti-IgG antibodies, etc. as ligands. This is done by graphing. After that, gel filtration or the like may be performed to improve the purity or desalt the monoclonal antibody.

When using affinity chromatography to recover monoclonal antibodies from culture fluid, there are the following problems. When performing chromatography using protein A or anti-IgG antibody, etc., as a ligand, which specifically binds to monoclonal antibodies, even if the affinity gel is thoroughly washed after adsorbing the monoclonal antibody to affinity gel, there will be no residue in the culture medium. It is difficult to avoid contamination of the eluent with proteins that coexist with the monoclonal antibody, making it difficult to obtain a highly pure monoclonal antibody. These coexisting proteins may be those added to the medium in advance or those originally contained in ascites, such as albumin and transferrin. In addition, the ligands protein A and anti-I
gG antibodies and the like are biologically derived and expensive, resulting in high costs. Furthermore, since the elution conditions for monoclonal antibodies are usually low pH, there is a risk that the activity of the monoclonal antibody may decrease.

On the other hand, when monoclonal antibodies are recovered using ion exchange chromatography, there are the following problems. Currently, many methods are used for recovering monoclonal antibodies by anion exchange chromatography. Also.

There are few examples of the use of cation exchange chromatography.
Both were conducted under conditions of pH 6.3 or lower (A,
Frej et al.: B10/TECH, S
ep, 777-781.1984. M, Carl
sson et al.: J. Immunol.

Vol, 79.89-98.19851. The recovery method using ion-exchange chromatography involves adsorbing monoclonal antibodies to an ion-exchange resin along with coexisting proteins such as albumin and transferrin in the culture medium, and then increasing the salt concentration gradually or stepwise to increase the coexistence with the monoclonal antibodies. Ion-exchange chromatography, which separates proteins based on the difference in elution time, generally results in insufficient separation, and monoclonal antibodies may be contaminated with coexisting proteins in the culture solution as impurities. mixed in as Moreover, after adsorbing most of the coexisting proteins together with monoclonal antibodies.

In order to perform separation, an amount of ion exchange resin is required that corresponds to the total amount of monoclonal antibodies and coexisting proteins, and the resin cost is relatively high.

Therefore, the present inventors have conducted intensive research on a method for recovering monoclonal antibodies in a culture solution with high purity and at low cost, and as a result, have completed the present invention.

[Means for Solving the Problems] The present invention provides a method for converting a culture solution containing a monoclonal antibody to pi(e
, 5 to 8.5, and treatment with a cation exchange resin.

The monoclonal antibodies applied to the present invention are not limited to specific animal types, including human and mouse types.

In addition, monoclonal antibodies produced from hybridomas and other EBV-transformed cells, such as human x human hybridoma CLN/5UZH11 strain (JP-A-59
-137497: ATCCAB 8307) produced human monoclonal antibody (CLN-IgG
It is named. ) also applies. CLN/5UZH
The 11 strains were created using B cells from cervical cancer patients, and CLN-IgG is of the IgG1 type and has high cancer tissue binding specificity.

Monoclonal antibodies can be prepared by culturing cells transformed with hybridomas or EBV, producing monoclonal antibodies in the culture solution, purifying and recovering from the culture solution.

In vitro culture is carried out by static culture using a culture flask or the like, or agitation culture using a spinner flask or the like. The medium used is a synthetic medium containing sugars, amino acids, hormones, inorganic salts, etc., to which bovine serum or supplements are added. Bovine serum contains proteins such as albumin and transferrin, as well as trace amounts of active ingredients.A medium containing supplements instead of bovine serum is called a serum-free medium.Supplements contain proteins such as albumin and transferrin, trace amounts of hormones, Consists of inorganic salts, etc. For example, the human x human hybridoma strain CL N/S U ZH11, which produces the human monoclonal antibody CLN-IgG, is cultured in RDF medium (Hiroki Murakami et al., Journal of the Japanese Society of Agricultural Chemistry, Vol.
, 58.575-583.19841 supplemented with bovine serum albumin, transferrin, insulin, 2-mercaptoethanol, monoethanolamine, and sodium selenite. Antibiotics may also be added to prevent bacterial contamination. On the other hand, 1 in vivo culture is carried out by transplanting a hybridoma or the like into the peritoneal cavity of a nude mouse or nude rat, and growing the hybridoma in the ascites.

Monoclonal antibodies produced by proliferating hybridomas accumulate in ascites fluid. In addition to monoclonal antibodies, the ascites that has been cultured contains proteins such as albumin and transferrin derived from nude mice or nude rats, hormones, and inorganic salts.

In the present invention, monoclonal antibodies are purified from culture fluid,
A cation exchange resin is used for recovery. In general, both monoclonal antibodies and coexisting proteins in the culture solution other than the monoclonal antibodies are water-soluble and dissociated in an aqueous solution. The pH of the cation exchange resin is adjusted by each protein.
Since the adsorption and elution conditions such as salt concentration are different, by optimizing the conditions, it can be effectively used for the purification of monoclonal antibodies.The cation exchange resin used in the present invention has sulfopropyl groups, carboxymethyl groups, etc. Some have exchange groups. The method for using the cation exchange resin may be continuous chromatography or batchwise operation.

As a result of various studies in order to solve the problems of conventional methods using cation exchange resins, the present inventors have discovered that, in a method for recovering certain monoclonal antibodies from a culture solution,
We have found conditions that allow monoclonal antibodies to be adsorbed onto a cation exchange resin without adsorbing most of the proteins other than the monoclonal antibodies, and then eluted. This condition can be achieved at a pH of 6.5 or higher. Monoclonal antibodies applicable to the present invention have conditions that allow them to be adsorbed to a cation exchange resin at a pH of 6.5 or higher. For example, human monoclonal antibody CLN-IgG is adsorbed to a cation exchange resin at a pH of 8.5 or lower, and most albumin and transferrin in the culture solution are not adsorbed at a pH of 6.5 or higher.

Therefore, the human monoclonal antibody CLN-IgG is adsorbed to the cation exchange resin in the pH range of 6.5 to 8.5, but most of the albumin and transferrin coexisting in the culture solution are not adsorbed. effect is achieved. Further, elution of CLN-IgG from the cation exchange resin is performed by increasing the concentration of NaCf2. Purification using a cation exchange resin under physiological conditions, preferably at pH 7.4, is effective in maintaining the activity of monoclonal antibodies.

The monoclonal antibodies recovered according to the present invention have high purity and are useful, for example, as pharmaceuticals. Furthermore, while the ligands and gels used in affinity chromatography are expensive, the cation exchange resin of the present invention is inexpensive, which is advantageous in terms of cost. Compared to ion exchange chromatography under conditions other than those of the present invention, the present invention is much more advantageous in terms of cost since the amount of cation exchange resin can be determined by taking into consideration approximately the amount of monoclonal antibody adsorbed.

Furthermore, the cation exchange chromatography of the present invention can be used not only as the main step of antibody recovery in the purification of monoclonal antibodies from a culture solution, but also in a repurification step to improve the purity of monoclonal antibodies that have been purified and recovered. Can be done.

[Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples do not limit the scope of the present invention in any way.

Example: Into a sterilized 15I 2-volume spinner flask, sterile-filtered culture medium at IO°C was added, and human x human hybridoma CL was added.
A seed culture of N/SUZ 811 strain was inoculated with 1X10' cells and cultured at 37° C. under 5% CO2 aeration conditions. The medium used was RDF medium supplemented with bovine serum albumin Ig/β, transferrin lomg/β, insulin 10 mg/β, and 2-mercaptoethanol 10.
-'M, monoethanolamine 10-'M, sodium selenite 10-9M were added, and the antibiotics penicillin G 50,000 U/I2, ampicillin 50ag/I2, and streptomycin 50 mg/β were added. .

After culturing for 6 days, hybridomas were removed by centrifuging the culture solution at 300 rpm for 20 minutes. Add ammonium sulfate to this culture solution to 70% saturation,
- Left at 4°C overnight. The resulting precipitate was IO,ooorpI
Collected by centrifugation at I+ for 20 minutes and dissolved in 20 ml of pure water, 10I111 was added to 10mM PH buffer (pH
7,4) Dialysis was performed at 212. After 4 hours, dialysis fluid 2J2
The solution was replaced and dialysis was continued at 4°C overnight.

After filtering the dialysate through a 0.2μ filter, Prot.
H equipped with Pak-5P (Waters) column
Purified using a PLC device. 10m at 0.5ml/min
After flowing MPB buffer (pH 7,4) for 1 hour, 20 ml of filtered dialysate was flowed at a rate of 0.5 ml per minute. after that,
lon+MPB buffer (pH 7,4) 0.5ml/min
It was washed away. After 30 minutes, IM NaCJ2-containing 10-containing 1 was added after 40 minutes while maintaining a total of 0.5 ml per minute.
The solution was eluted by passing the solution through a 0-enrichment gradient. The eluent is UV
Absorbance at 280 nm was continuously measured using a monitor. In addition, the eluate was fractionated at 1 minute intervals using a fraction collector. ELI the absorbance peak fraction of the eluent
The SA method was used to determine the CLN-1gGgG fraction, and the ELISA method used sheep anti-human I as the primary antibody on the in-phase side.
Using gG (Fc fraction specificity, Kabbell),
Peroxidase-conjugated sheep anti-human IgG (
Sanderch method using Fab fraction-specific (Cabbell) was performed.

The CLN-IgG-containing fractions were collected and subjected to gel filtration as follows. For gel filtration, 10mM of heptaphryl 5300 (Pharmacia) was placed in the column, 10mM PBS was flowed at a rate of 0.2ml per minute, and after 150 minutes, 1ml of the above fraction was flowed at a rate of 0.2ml per minute for 5 minutes, and 10mM PH3 was added every minute. This was carried out by flowing 0.2 ml per minute for 120 minutes. The absorbance of the effluent at 280 nm was continuously measured using a UV monitor. Further, the effluent was fractionated and collected using a fraction collector at 5 minute intervals. CLN-I by EL I SA method
The gG fraction was determined and collected: 2.2 mg/ml
A CLN-IgG containing solution 2.5I111 was obtained.

Add 0.5 ml of this solution to NAP-5 (Pharmacia)
using 10mM sodium carbonate buffer (p) 1
9.5) The buffer was exchanged. Thereafter, it was filtered through a 0.2μ filter, and the purity was measured using an HPLC device equipped with a ProtPak-DEAE (Waters) column. After flowing 10 mM sodium carbonate buffer (pH 9.5) at a rate of 0.5 ml per minute for 1 hour, 0.5 ml of the above furnace solution was flowed at the same flow rate. 10mM after 1 minute
Sodium carbonate buffer (pH 9.5) was flowed at the same flow rate.

After 30 minutes, maintain a total of 0.5 ml per minute.
After 0 min, N
It was eluted using aCβ concentration gradient. The absorbance of the eluate at 280°C was continuously measured using a UV monitor, and the CLN-IgG content of the absorbance peak fraction was determined by ELISA, and the purity was calculated from the absorbance. As a result, it was found that this absorbance fraction contained CLN-IgG with a purity of 95% or more.

Comparative Example 10ml of the pure solution of ammonium sulfate precipitate obtained in the above example was
Dialyzed against mM PBS 2I2. After 4 hours, the external dialysate solution 2β was replaced and dialysis was continued overnight at 4°C.6 The dialysate was purified to CLN-IgG using a protein A-bound Sepharose (Pharmacia) column. 0 per minute
．． After 2 ml of lomMPBs was passed through a column packed with 5 ml of protein A-conjugated Sepharose for 4 hours, 10 ml of dialysate was passed at a rate of 0.2 ml per minute. Two hours later,
lomMPBs was passed through the column, and then 0.1M glycine hydrochloride solution (pH 3,0) was passed for 5 hours for elution. The eluent was added to 0.5M 1-Lis-HCl buffer (pH 8,0) at 5 minute intervals.
It was collected in a test tube containing 3 ml and fractionated using a fraction collector. CLN-I by EL I SA method
The gG-containing fraction was determined and collected to be 11.7 ml. 1 of this liquid. The oml was subjected to gel filtration in the same manner as in the example. As a result, CLN-Ig of 0.4 mg/ml
2.5 ml of G-containing solution was obtained.

The purity of 0.5 ml of this liquid was measured using ProtPak-DEAE in the same manner as in the example.

As a result, it was found that it contained CLN-IgG with a purity of about 90% at most.

[Effects of the Invention] According to the present invention, monoclonal antibodies in a culture solution can be recovered with high purity and at low cost.

Hand System Completed Author August 18, 1988 Director General of the Patent Office Yoshi 1) Takeshi Moon 1, Indication of IG Patent Application No. 74559, 1988 2, Name of Invention Method for Recovery of Monoclonal Antibodies 3.7+li Relationship with the case of the person making the correction Patent applicant name (805) Mitsubishi Yuka Co., Ltd. 4, Agent 5, Date of amendment order Voluntary action 6, Subject of amendment Column 7 of detailed explanation of the invention in the specification, Amendment rATCCAB-RO9Q described in page 6, line 3 of the description of contents
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Claims (3)

[Claims] (1) Culture solution containing monoclonal antibody at pH 6.5 ~
8.5, and treatment with a cation exchange resin. (2) The method for recovering a monoclonal antibody according to claim 1, wherein the monoclonal antibody is CLN-IgG produced by the human x human hybridoma CLN/SUZH11 strain. (3) The method for recovering monoclonal antibodies according to claim 1, wherein the cation exchange resin has a sulfonic acid group or a carboxyl group as an exchange group.