JPS5920225A - Method for stabilizing tumor necrotic factor (tnf) - Google Patents

Abstract

PURPOSE:To stabilize the titled factor useful as a carcinostatic agent to keep the activity thereof for a long term even after the separation and purification or freeze-drying step, by adding globulin or protamine to the tumor necrotic factor (TNF). CONSTITUTION:A tumor necrotic factor (hereinafter abbreviated to TNF) is stabilized by adding globulin, e.g. bovin blood serumal globulin, or protamine, e.g. protamine of a salmon, or a salt thereof thereto. For using the TNF induced in an animal or a tissual culture system as a carcinostatic agent, the separation and purification are important, and the storage in a solution or a frozen state for a long term is essential. However, the activity is remarkably reduced by the operation. The addition of the globulin and/or protamine inhibits the reduction in the activity. The concentration of the added globulin and/or protamine is 1mug or more, optimally 100mug or more, based on 1ml solution containing the TNF. Preferably, the separation and purification are carried out at 0-30 deg.C, and the freeze-drying is carried out at 0 deg.C or below.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing cancer necrosis factor (hereinafter abbreviated as TNF), and more particularly, to a method for stabilizing TNF during storage, separation and purification, freeze-drying, and other operations. It is something.

TNF in the present invention is defined as ``one or more substances that have a reticuloendothelial system activating effect: by injecting a membrane into a mammal, and then injecting endotoxin derived from Durham-negative bacteria; A physiologically active substance that is induced by adding endotoxin derived from Durham-negative bacteria to a tissue culture system containing activated macrophages derived from mammals, and is a factor that causes necrosis of certain cancers when inoculated into tumor-bearing animals. It is a substance defined as In addition to causing necrosis in certain cancers, TNF is known to have non-species-specific effects. For example, ``rNF obtained from rabbits can cause tumor necrosis in mice.
It is known that in vitro it has almost no harmful effect on normal cells and has the ability to kill certain cancer cells (for example, LM cells derived from mouse cancer). As described above, TNF has an anticancer effect, is seed valve specific, and does not act on normal cells, so it is expected to be an anticancer agent.

Traditionally, TN induced in animals or tissue culture systems
It is known that the amount of FO is extremely small. TN
In order to widely and safely use Fi as an anticancer drug, it is important to separate and purify it, and long-term storage in a solution or frozen state and freeze-drying are important methods for the industrial pressure production of TNF. This is an essential operation. However, the present inventors have discovered that when a highly purified TNF solution is stored, frozen, or lyophilized, its activity is significantly reduced. There are no reports examining the stability of shredded, high-purity TNF. Under these circumstances, despite its anticancer effect, it is impossible to efficiently and stably provide highly purified TNFi on an industrial scale.

As a result of extensive research into stabilizing TNF, the present inventors have found that the addition of globulin or procumin or its salts can stabilize TNF even after long-term storage, and even after separation and purification, freeze-drying, and other operations. It was found that the activity of TNF was maintained. The present invention is based on this knowledge.

The present invention relates to a method for stabilizing TNF, which is characterized by adding globulin''!, protamine, or a salt thereof to TNF.

The TNF raw material used in the present invention is produced by a known method. As such, for example, Mattbe
ws et al., Br, J., Cancer,
42 (1980) 416, Green et al., J. Natl. Cancer In5t1.5
9 (1977) 1519. The method will be explained below.

i.e. mammals (e.g. mice, woolly
A substance that activates the reticuloendothelial system in guinea pigs, etc.
Intravenous injection of one or more seeds! , inject intraperitoneally. Durum-positive bacteria, protozoa, or yeast are usually used as substances that have a reticuloendothelial system activating effect, and the trc
is absorbed into the membrane as a bacterial cell extract component.

Here, examples of Durham-positive bacteria include, for example, Propio
nibacterium acnes (Coryne
bacterium parvu+n), Pro
pionibacterium granulosu
rn(Corynebacterium granule
osum ) like Propionibacteri
a, 13acillus Calmette −
Guerin (+3CG), Mycol) acterl
Mycobacterium like um smegmatis
ria, Nocardia erythropo
lis.

Nor like Nocardih gThrdneri
Examples include a rd i as. Examples of protozoa include malaria parasites and toxoplasma gondii. In the case of yeast, usually 5 acc++ aromyces c
Zymosan extracted from E. erevisiae etc. is used. 1. Synthetic polymeric compounds such as pyran copolymers can also be used.

Endotoxin obtained from Durham-negative bacteria 7 to 14 days after administration of a substance that has a reticuloendothelial system activation effect, for example,
E. coli, Pseudomonas aeruginosa, and Salmonella typhoid-derived Noriboboli satucharide are injected intravenously into the mammal. 1.5 to 2 hours after injection, the mammal's body fluids (e.g., ascites, lymph, etc.) and/or serum or plasma are obtained, or organs such as the liver, spleen, etc. of the animal are homogenized, and physiological Extract with saline to obtain TNF raw material.

The method for producing the TNF raw material used in the present invention is not limited to the above. That is, T
NF raw material production method can also be adopted.

The TNF raw material produced in this manner is separated and purified using a combination of conventional biochemical separation and purification methods. Examples of such methods include salting out using ammonium sulfate, ion exchange chromatography using anion exchange resin, gel filtration, and electrophoresis. When these methods are combined and the separation and purification steps are carried out to increase the degree of purification, TNF gradually becomes unstable. For example, a TNF sample purified to a specific activity of 500,000 units/mg (the activity of TNF contained in 1 mg of total protein; units of activity will be described later) is extremely unstable, as shown in the Examples. TNF samples with lower specific activity can also be degraded to varying degrees by storage, freezing, lyophilization, etc.
Its activity decreases. The object of the present invention is such highly purified TNF which has become unstable, and may be in the form of either a solution or a powder, but in particular TNF
, (containing solution.) The pH of the TNFi-containing solution used in the present invention is 5-5.
It is preferably maintained at 10, and more preferably adjusted with an appropriate buffer.

Such buffers include, for example, phosphate buffers,
tri/([: tris (Jlydroxymet
byl) aminometbane)-hydrochloric acid buffer and the like.

Depending on the purpose, salt may be added. Salts used include sodium chloride, potassium chloride, etc.
Its concentration is determined by the purpose.

For example, when used for injection, sodium chloride in 15MK is added to make an isotonic solution.

Globulins used in the present invention include serum globulins derived from animals, such as cows, horses, sheep, goats, humans, etc., and there is no significant difference in stabilizing effect among them. When used as a stabilizer for injectable preparations, human gamma globulin purified according to known methods is most preferred.

Examples of the protamine used in the present invention include protamines such as salmon, herring, and shark A, and salts thereof (eg, sulfates), and there is no significant difference in stabilizing effect among them. When used as an injectable preparation, protamine or a salt thereof purified for medical use by a known method is preferred.

The concentration of globulin and protamine or their salts is 921 μm or more, more preferably 10 μm or more, and even more preferably 10 μm/rnt of TNFf/containing solution.
It is 0 μ7 or more. The upper limit of the concentration added is determined from common sense and economical viewpoints, and is 50 rng per ml of the same solution. The amounts of globulin and protamine or their salts to be added to the TNF-containing powder are selected so that the above-mentioned solution concentration is achieved when the powder is dissolved.

The method of addition is not particularly limited, but for example, powders of globulin and protamine or their salts are directly added to TNF.
Examples include a method of adding the powder to a containing solution, a method of adding the same powder by dissolving it in gold water or an appropriate buffer solution, and a method of adding the same powder by mixing it with a TNF-containing powder. The addition time may be during the separation and purification process or during the formulation process.

Also included in the method of the invention is a method of adding globulin and protamine or a salt thereof together.

In that case, the total amount of both substances is equal to the added amount nL mentioned above.
and the amount should be adjusted.

In this way, the solution containing 'l' NF to which globulin or iL/ and protamine or its salt has been added is kept at a temperature of 0 to 30°C, more preferably 0 to 30°C, while in solution state.
It is preferable to store the solution at 10°C or perform separation, purification, and formulation operations.Furthermore, when the same solution is stored in a frozen state, it is desirable to keep it at 0°C or lower, more preferably -20°C or lower. In the solution containing TNF to which globulin and/or protamine or a salt thereof is added in the present invention, the TNF activity is maintained even during storage in a solution state or frozen state, or during separation/purification and formulation operations.

The method of the invention is also effective for freeze-drying operations. That is, when a solution containing TNFf is freeze-dried by a conventional method (especially in the case of high purity), its activity decreases, but when globulin'-primary and/or protamine or its base is added to the same solution, by tnf
activity does not decrease.

Furthermore, globulin or/and protamine or a salt thereof may be added after lyophilization. Preferably, the powder is stored at room temperature or below room temperature.

There are two ways to measure the activity of TNF: one is to measure the tumor necrosis effect in vivo, and the other is to determine the total cancer cell killing effect in vitro.

In vivo methods include, for example, Carswel
The method of l et al., Proc, Nat, Acad, Sc
i, USA, 72 (1975) 3666. This method uses a transplanted MethA sarcom
This is to measure the effect of JPnaJ't 'I' NF on necrosis by a. That is, (BALI3/
c×C57BIJ/6) FI mouse intradermally 2
X 10S Co MeH+ A sarcoma Jll
transplant the cyst. After 7 days, the size of the transplanted tumor was 7 days later.
Select a mouse that has reached ~8 nnn and has good blood circulation without hemorrhagic necrosis, and inject a full sample from the tail vein. t#:
0.51 nl of TNF sample diluted with saline is injected, and after 24 hours the following criteria are used for evaluation.

H: Changed U →: Faint hemorrhagic necrosis (廿): Moderate hemorrhagic necrosis (necrosis extending from the center of the transplanted cancer surface to more than 50%) (uY+: b1′: Significant J (+1 case of necrosis) (Severe necrosis in the middle part of the transplanted cancer, with only a small amount of surrounding cancer tissue remaining) Measurement of TNF activity using an in vitro method is described, for example, by Ruff et al.
ts Vol, 11. ed, by E.

Pick, Academic Press, N, Y
, (1980) 235) or Kull et al.
Immunol, 126 (1981) 1279)
Methods include:

The method used by the present inventors is an improvement on these, and measures the effect of TNF on killing LM cells (American Quiz Culture Collection, CCL1, 2) (i7). , 0.1 - of the TNF sample diluted in the medium and 0.1 - of the medium suspension of LM cells at a concentration of 10 cells/me were placed in a 96-well tissue culture microplate (Flow Laboratory). The medium is Eagle's Minimum Essential Medium containing 10 v/v% fetal bovine serum (the composition of which can be found, for example, in "Tissue Culture", edited by Junnosuke Nakai et al., Breakfast Shoten, 1).
(described in 967). The microplate is incubated at 37° C. for 48 hours in air containing 5% carbon dioxide. After culturing, add 20 μt of glutaraldehyde.
Add to fix the cells. After fixation, the microplate was washed, dried, and treated with 0.0"5 thymethylene blue solution'f:
Add 0.1 me and stain the surviving cells.

After washing away excess methylene blue and drying, extract with the remaining methylene blue f:S% hydrochloric acid solution, and extract the 665
The absorbance at nm is measured using Quitartic Multiscan (Flow Laboratory). This absorbance is proportional to the number of surviving cells. The dilution rate of the TNF sample corresponding to the absorbance value of 50% of the control without the addition of the TNF sample is determined using a graph or η4, and the dilution rate is defined as the unit (U)/me. Hereinafter, all in vitro activities of TNF in the present invention are expressed in this unit.

According to the method of the present invention, TNF, which is expected to be an anticancer agent, can be melted, frozen, freeze-dried, and stored in a dry state for 11 minutes. +1. Its activity is also confirmed during purification and formulation operations.
Since it is retained, it becomes possible to efficiently and stably provide high-purity TNF on an industrial scale. When human globulin or protamine or its salts are used as a stabilizer, it is safe even when applied to the human body, and TNF'
It is extremely advantageous when used as an anticancer drug.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 T from rabbit with a specific activity of 500,000 U/my
TN with an activity of 1200 U/me using NF
F solution (0.1M phosphate buffer containing 0.15M sodium chloride 1) H3o) 5 was prepared. Various concentrations of human gamma globulin were added to the TNF solution, stored at 4°C, and evaluated over time (days 2, 7, and 30) using the in vitro and in vivo evaluation methods described herein. Residual activity was measured. In the case of the in vitro method, the residual activity rate (H) was totally calculated from the obtained measurement values.

The results are shown in Table 1 together with the results of other Examples. The figure also shows the residual activity of TNF after 7 days of storage at various concentrations of human gamma globulin.

In addition, for 1 in vivo evaluation, the treated solution was concentrated 20 times using an ultra-superconcentrator, mini module NM-3 (manufactured by Asahi Kasei Kogyo, sold by Funakoshi Yakuhin), and 0.5 ml per animal was injected into the tail vein using membrane force. The degree of oral necrosis was evaluated for 24 hours after membrane tension.
Observed in groups.

Example 2 A TNF solution having the same active concentration as in Example 1 was prepared, human gamma globulin at various concentrations was added to the TNF solution, and the mixture was repeatedly frozen (-70°C) and thawed once, 3 times.
(times), and its residual activity was measured by an in vitro evaluation method. In addition, after freezing the solution-fI knee at 70°C, freeze-drying was performed using a freeze dryer, and then the freeze-dried product was
After leaving it at room temperature for a week, add sterile distilled water to dissolve f'/F l.
, , remaining activity rate in vitro.

Obtained using an evaluation method. The freeze-dried product was subjected to K in vivo evaluation in the same manner as in Example 1, and the anticancer effect was also confirmed. The results are shown in Table 1.

Example 3 In the same manner as in Example 1, salmon sulfate protamine was used instead of human gamma globulin to examine its stabilizing effect. The results are shown in Table 1.

Example 4 In the same manner as in Example 2, salmon protamine sulfate was used instead of human gamma globulin to determine its stabilizing effectff.
i FA solid. The results are shown in Table 1.

Comparative Example To a TNF solution having the same active concentration as in Example 1, each amino acid, metal salt, and chelating agent, which are well known as usual solution stabilizers for physiologically active substances, were added.
TNF residual activity rate f in after storage at ℃ for 7 days
Table 2 shows the results obtained by in vitro evaluation.

Table 2 Comparison Example and above examples,
As is clear from the comparative examples, according to the stabilization method of the present invention, it is possible to stably maintain the activity of TNF during operations such as storage in a solution state, freezing, thawing, and freeze-drying. Not only can it be applied to the purification process and formulation process during TNF production, but it also guarantees product stability when commercialized.

[Brief explanation of drawings]

Figure 1 shows the added concentration of human gamma globulin.
It is a graph showing the relationship with the TN F in vitro residual activity rate after storage for 7 days at °C.

Claims (1)

[Claims] 1. A method for stabilizing cancer necrosis factor, which comprises adding globulin or protamine or a salt thereof to cancer necrosis factor.