JPH02247128A - Globulin preparation having high titer - Google Patents

Abstract

PURPOSE:To obtain a safe globulin preparation having uniform qualities and high titer free from problems of antigenicity, comprising a monoclonal antibody and an immunoglobulin derived from plasma of the same kind of animal as that of the antibody. CONSTITUTION:(A) An immunoglobulin derived from plasma, such as immunoglobulin prepared by removing aggregate from gamma-globulin for intramuscular injection already used as a drug by acidic treatment is blended with (B) a monoclonal antibody derived from the same kind of animal as that of immunoglobulin of the component A, namely a monoclonal antibody having an antibodyforming cell derived from the same kind of animal as that of immunoglobulin among the antibody forming cell and a multiplicative cell in the case of preparation by cell fusion method in the blending ratio of the component A:B of (10:1)-(10,000:1) to give a high-titer globulin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application] The present invention relates to a monoclonal antibody and a high titer (high antibody activity) immunoglobulin preparation comprising an immunoglobulin derived from the plasma of an animal of the same species as the monoclonal antibody.

[Prior art]

High-potency globulin preparations generally refer to globulin preparations that have high antibody activity against specific antigens, for example,
Various viruses (cytomegalovirus, herpesvirus, HIV, influenza, etc.), virus-derived proteins (
Antibiotics against hepatitis B virus surface antigen, tetanus toxoid, etc.) or tumor-related antigens (carcinoembryonic antigen, α-fetoprotein, etc.) are known, and are used to prevent and treat infections caused by each virus. It is being considered for use in the treatment of tumors.

However, conventional high-titer globulin preparations are prepared using human-derived positive plasma, and the titer is not high enough, and it is difficult to obtain human-derived positive plasma. . Problems such as the difficulty in obtaining products of uniform quality have also been pointed out.

On the other hand, techniques using monoclonal antibodies are being established as a method for preparing antibodies against specific antigens, and some are being considered for clinical application as globulin preparations.

However, because monoclonal antibodies are a homogeneous molecular population, it has been reported that when administered to conspecific animals, they induce the production of anti-idiotype antibodies, which was not observed with plasma-derived globulin preparations (Immuno
logy, 55.197-204.1985).

The anti-idiotype antibody produced binds to the antigen-binding site of the administered monoclonal antibody, neutralizes the antibody, and reduces its ability to protect against infection.

That is, administration of a monoclonal antibody triggers an immune response against the administered antibody even if it is of the same species.

[Problem that the invention seeks to solve]

Therefore, the present inventors conducted various studies in view of the above circumstances, and found that by administering a mixture of monoclonal antibodies and the same kind of plasma-derived immunoglobulin, the immune reaction against monoclonal antibodies could be avoided.
Thus, the present invention was completed by discovering that it is possible to prepare a safe globulin preparation with high titer and no problems such as antigenicity.

[Means for solving problems]

(i) Plasma-derived immunoglobulin Plasma-derived immunoglobulin is derived from the same species as the monoclonal antibody (11). The form of immunoglobulin is not particularly limited, and those prepared by known means can be used.

For example, non-chemically modified types (including PEG-treated types), chemically modified types (alkylated types, sulfonated types), ion exchange resin-treated types, pH4-treated types, enzyme-treated types (plasmin, pepsin, etc.) are used. Specific examples include non-chemically modified T-globulin.

The non-chemically modified γ-globulin of the present invention is defined as: 1) It is in its natural state and has not undergone any modification or change, and is therefore a fragment of γ-globulin, Fab 5F (ab'>2
, does not contain Fc, etc.; ■ No decrease in antibody titer and at the same time, no decrease in antibody spectrum; ■ Anti-complement action (complement fixation) is 20 units (20 units), which is considered safe according to Japanese biological product standards. CH2O value).

It refers to something that has the following properties.

The non-chemically modified r-globulin used in the present invention is
If it is in its natural state and has a low anti-complement value,
γ that can be obtained by any method, but can be manufactured using existing equipment and is already used as a medicine for intramuscular injection.
- The most efficient method is to use globulin and separate its aggregates by acidic treatment. However, if manufacturing complexity and quantity reduction are not an issue, nonionic surfactants can be used to remove γ-globulin aggregates that cause anti-complement action, and γ-globulin with low anti-complement value can be removed. It is preferable to use globulin.

The preparation method may be performed by a known method, for example,
The method described in JP-A-53-47515, JP-A-57-32228, JP-A-63-183539, etc. may be followed.

In addition, it is preferable to perform heat treatment. For example, as liquid heating, JP-A-61-191622, JP-A-63-1
No. 46832, as for dry heating, JP-A-6L-7EB
No. 30, No. 62-228024, No. 62-28393
No. 3, No. 61-289523, etc. may be followed.

The origin of plasma-derived immunoglobulin is not particularly limited. Specific examples include humans, mice, rats, etc., but humans are preferred.

(11) Monoclonal antibody The monoclonal antibody is not particularly limited as long as it is derived from an animal of the same species as the immunoglobulin (i).

The origin of a monoclonal antibody means the origin from the antibody-producing cell side. Therefore, when preparing monoclonal antibodies by the cell fusion method, antibody-producing cells and proliferative cells are fused to prepare hybridomas and monoclonal antibodies are produced. Among the antibody-producing cells and proliferative cells, The antibody-producing cell capsule may be derived from an animal of the same species as the immunoglobulin (i).

In addition, in the case of the transformation method, antibody-producing cells are transformed with a virus, transformants are prepared, and monoclonal antibodies are produced.
) immunoglobulin derived from the same species as the immunoglobulin.

These monoclonal antibodies are prepared by methods known per se.

This will be specifically explained using the cell fusion method.

As described above, the cell fusion method is performed by fusing antibody-producing cells and proliferative cells to prepare hybridomas.

Antibody-producing cells contain various viruses (cytomegalovirus,
herpesvirus, HIv1 influenza, etc.) themselves, virus-derived proteins (hepatitis B virus surface antigen,
tetanus toxin), tumor-associated antigens (carcinoembryonic antigen, α-
These are cells that produce antibodies against fetoprotein, etc.).

Antibody-producing cells are not particularly limited as long as they are derived from an animal of the same species as the immunoglobulin (1).

Specific examples include those derived from humans, mice, rats, etc., but those derived from humans are preferably used.

Antibody-producing cells include lung cells, lymph node vesicles, 892
An example is two balls.

Examples of proliferative eye color include myeloma cells.

As myeloma cells, those derived from mice, rats, humans, etc. are used, for example. Examples include mouse myeloma P3U1, X63-Ag8-.6.5.3, and the like. Preferably, the antibody-producing cells and myeloma cells are derived from the same species of animal.

Cell fusion can be achieved, for example, by Gee, Galfer (G.

Ga1fra) C Neage+ -(Nature) 2
66, 550 (1977)) or a method analogous thereto. At this time, the reaction is carried out using polyethylene glycol (average molecular weight LOOO~4.000>).

Monoclonal antibodies may be obtained by genetic engineering techniques. Alternatively, a chimeric antibody may be obtained by a chimerization technique, consisting of a C region derived from an animal homologous to the plasma-derived immunoglobulin (i>) and a V region derived from an animal other than that.

Furthermore, the monoclonal antibody may be fragmented by known means, such as pab or Fab'.

The monoclonal antibodies obtained in this way can be used for various viruses (cytomegalovirus, herpesvirus, H.
IV, influenza virus, etc.), virus-derived proteins (hepatitis B virus surface antigen, tetanus toxin, etc.), tumor-associated antigens (carcinoembryonic antigen,
Examples include those against α-fetoprotein, etc.).

(iii) Preparation of V agent The mixing ratio of the plasma-derived immunoglobulin in (1) and the monoclonal antibody in (11) is preferably 10:1 to 10,000:1.

After all steps are completed, known methods such as dialysis, sterilization filtration,
It can be made into a liquid preparation by operations such as dispensing. Furthermore, it can also be made into a dry preparation by operations such as freeze-drying.

In the case of liquid preparations, the globulin concentration is 1 to 1511
It is about I/v% (preferably 5 to LOW/V%).

Further, it is preferable to add a stabilizer.

Examples of the stabilizer include sugar, sugar alcohol, and the like.

The amount of stabilizer added is globulin 1 to 1511/
per V%, about 1 to 10 W/V%, preferably 51
An example is about 11/V%.

Further, it is exemplified that the pH of the liquid preparation is about 5.3 to 5.7, preferably about 5.5.

The lower the conductivity, the better, preferably 1 mmho.
Hereinafter, it is particularly exemplified that the temperature is set to 0.6 mmho or less (both converted to 8° C.).

Even in the case of a dry preparation, it is preferable to add a stabilizer. Examples of the stabilizer include sugar, sugar alconopearbumin, and inorganic salts.

The amount of stabilizer to be added is about 1 to 10 parts by weight (preferably 2 parts by weight) of sugar or sugar alcohol, and 0.5 to 5 parts by weight (preferably 1 part by weight) of albumin, per 1 to 15 parts by weight of glob. 0.1 to 1 part by weight (preferably 0.5 part by weight) of the inorganic salt.

In the preparation obtained according to the present invention, immunoglobulin is hardly inactivated, and furthermore, it does not contain contaminants such as anti-human blood group substance antibodies, and since it is heat-treated, contaminant viruses are inactivated and anti-human blood group substances are inactivated. It has properties such as sufficiently low complement activity, and is a safe preparation that complies with the Japanese biological product standards (hereinafter referred to as biological standards) issued in 1985.

Moreover, in the case of a dry preparation, solubility is also good.

When used, the preparation obtained according to the present invention can be used as is in the case of a liquid preparation, or diluted with an appropriate solvent (e.g., distilled water for injection, physiological saline, glucose solution, etc.), or in the case of a dry preparation. is dissolved in an appropriate solvent (e.g., distilled water for injection) and administered intravenously or by infusion to treat specific diseases (
It is used to prevent or treat infections caused by various viruses, hepatitis B, tetanus, tumors, etc.).

〔effect〕

The preparation of the present invention has higher antibody activity (antibody titer) against a specific antigen than ordinary plasma-derived immunoglobulin.1.
This is a so-called high-potency preparation. Especially when administered to humans,
The preparation of the present invention, which consists of human plasma-derived immunoglobulin and human-derived monoclonal antibodies, is more sensitive to specific antigens than conventionally commercially available human plasma-derived immunoglobulin preparations (e.g., Enoglobulin, manufactured by Green Juji). Antibody activity (antibody titer) against

In addition, it is a safe preparation without problems such as antigenicity, is easy to manufacture industrially, and can be prepared with uniform quality.

Therefore, the preparation of the present invention is expected to have extremely high pharmaceutical utility.

〔Example〕

EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited to these in any way.

Example I Immunoglobulin was prepared from BALB/c mouse serum according to Japanese Patent Application Laid-Open No. 5147515. Mouse monoclonal antibody against hepatitis B surface antigen (HBS 8g) and immunoglobulin prepared from mouse serum in a weight ratio of 1:1.
00 to prepare a liquid preparation with an immunoglobulin concentration of 5%.

The antibody titer per 1 ng of mouse monoclonal antibody is 2X10' when measured with a Hebsgen cell, and the above 5%
The anti-HBsAg antibody titer of the liquid preparation was 1 x 106.

0.1 ml of this liquid preparation was administered via the tail vein to female BALB/c mice for 8 weeks, once a week for 6 times. Immediately after the final administration, blood was collected from the orbital venous plexus every day for two weeks to obtain serum. Similarly, anti-11 was diluted to 500 μg and 7 ml with physiological saline.
B5 mouse monoclonal antibody 0.1ml. BAI,
It was administered to B/c mice 6 times to obtain serum.

The anti-tlBsAg antibody level in the obtained serum was measured using a Hebsgen cell, and the half-life was found to be 10 days when the liquid preparation was administered, but 2 days when only the monoclonal antibody was administered. It was suggested that neutralizing antibodies were present in the blood.

Example 2 100 parts by weight of human plasma-derived immunoglobulin and 1 part by weight of human-derived anti-cytomegalovirus monoclonal antibody were mixed to form a liquid with an immunoglobulin concentration of 5% (1!I/V). A formulation was prepared.

Claims (1)

[Claims] A high-potency globulin preparation comprising a monoclonal antibody and an immunoglobulin derived from the plasma of an animal of the same species as the monoclonal antibody, and having a composition of 1:10 to 1:10,000.