JPS5925364A - Stabilization of interferon-beta having no saccharic chain - Google Patents

Abstract

PURPOSE:To stabilize the titled compound useful asa drug, by adding an inorganic salt to interferon-beta having no saccharic chain. CONSTITUTION:An inorganic salt(e.g., salt, potassium chloride, ammonium sulfate, etc.) is added to interferon-beta (interferon-beta produced from a microorganism by recombination DNA technique), so that interferon having no saccharic chain is stabilized. The concentration of the inorganic salt is preferably >=0.3M, especially preferably 0.5-1.0M. The interferon-beta is deactivated at a temperature by about 15 deg.C lower than interferon-beta produced by human fibroblast and unstable, however, this method improves the unstability.

Description

[Detailed description of the invention] The present invention relates to a method for stabilizing interferon-β.

In particular, it relates to a method for stabilizing interferon β molecules that do not have sugar chains.

Interferon (IFN) is a virus or double-stranded RNA
It is a certain type of protein that has a wide range of biological activities, such as suppressing the proliferation of viruses produced by animal cells and anticancer effects when stimulated by animals, and its effects are known to be species-specific.

Currently, two types of human interferon are known to exist. That is, there are the α type produced by white blood cells, the β type produced by fibroblasts, and the γ type produced by lymphocytes, and it has been proven that they differ in chemical structure and biological activity.

Since interferon, which is effective for humans, is produced only by human cells, it has been very difficult to obtain large amounts of interferon. Recent advances in recombinant DNA technology have made it possible to produce IFN-1, IFN-β, and IFN-γ using microorganisms such as Escherichia coli and yeast, and the prospect of producing large amounts of interferon has become possible. (Goeddol + D, ■.

et aL Nucleic Ac1d Re5ea
rch, IL 4057 (198D), Natur
e, 287, 411 (1980), Nature 2
95 +' 503 (19B2)) Interferon β (hereinafter referred to as F-IFN) produced by human fibroblasts
-β) is a glycoprotein with a molecular weight of approximately 23,000±2,000, and the molecular weight of the protein portion is approximately 20,000.
, is a substance in which sugar with a molecular weight of around 6000 is bound.

On the other hand, interferon-β (hereinafter referred to as G-I
FN-β) has a molecular weight of approximately 200 without binding sugars.
00 simple protein, and in this respect the natural IFN-
It is a different substance from β, and therefore there are some differences between the two in terms of chemical and biological properties.

Although interferon β itself is a fairly unstable substance, it has become clear that G-IFN-β is also, in some respects, an even more unstable substance and easily deactivated. That is, when comparing the thermal stability, which is a typical index of stability, as shown in Figure 1, G-IFN-β is higher than F-I.
It is inactivated at about 15° lower temperature than FN-β. ()-IF
The instability of N-β is considered to be a major drawback when it is used clinically as a drug.

The present inventors continued intensive studies to improve the instability of G-IFN-β, and as a result, they arrived at the present invention.

That is, the present invention provides a method for stabilizing interferon β, which is characterized by adding a polyol to interferon β, which does not have a sugar chain.

The interferon β without sugar chains of the present invention is IF
I produced by microorganisms such as E. coli that have been recombined with the N-β gene.
FN-β (G-IFN-β), with a molecular weight of approximately 20.0
00 simple protein.

The ()-IFN-β of the present invention can be obtained, for example, by the method of Noguguchi et al.
Proc, Natl, Acad, 5ci-, 77,
5230 (1980)), it can be manufactured as follows.

m from human fibroblasts that produce interferon-β
- Isolate the RNA mixture and the corresponding e-DN
A is prepared and ligated to E. coli plasmid pBR322 to obtain E. coli having the structural gene of interferon-β. At this time, in order to express the interferon structural gene in E. coli and produce interferon protein, a promoter sequence for transcribing DNA into m-RNA is linked upstream of the structural gene, and the resulting m-
RNA needs to have an SD sequence that allows it to bind to liposomes and be translated into protein. To this end, the interferon β structural gene is linked to the E. coli lactose operon promoter and downstream of the SD sequence.

Using the plasmid (+) L0117R) containing the promoter region and the structural gene of IFN-β obtained here, E. coli HB
Rearrange 101.

The thus obtained recombinant was precultured in L and B medium, and then inoculated into new LB medium in approximately 2 cm.
After culturing at 0° for several hours, the bacteria are collected, washed, treated with lysozyme to dissolve the cell wall, frozen and thawed, and centrifuged. G-IFN-β produced and accumulated within the E. coli cells is solubilized. An extract exhibiting interferon activity is obtained.

Examples of the inorganic salt of the present invention include, but are not limited to, common salt, potassium chloride, ammonium sulfate, and the like. As the inorganic salt, common salt is particularly preferable.

The concentration of the inorganic salt is preferably 0.3M or more, particularly 0.5 to 1.0M.

The present invention will be specifically explained below with reference to Examples.

Reference example H) Plasmid pL0117 containing the IFN-β gene
E. coli HBI DI (T.

Taniguch et al. + Proc. N
atl, Acad, Sci, +775230 (
(1980)) was precultured in LB medium, and then 1 t of L
Approximately 2% of the cells were inoculated into B medium.

The cells were cultured with shaking at 30° and collected at 60 points when the bacterial count OD reached 0.6, washed with 50 mM Tris buffer pH 8.0 containing 25% sucrose, and collected by centrifugation.

The bacterial cells were mixed with 20-mM EDTA-50mM Tr.
The suspension was suspended in IS buffer pH 8.0, 2.0 µ/-gluzozyme was added, and after standing for 30 minutes, the cells were lysed by repeating freezing and thawing twice. The lysate was centrifuged at 15,000 rpm for 60 minutes, and the supernatant was used as the stock solution of G-IFN-β.
It was used as a material for stability experiments. F-IFN-β was partially purified from a stock solution produced by human fibroblasts. (S,
Kobayashi et.

al,,The Cl1nical Potentia
l of Interferois”Japan Me
dical Research Foundation
Pukli-cation N1115+ p6
0 (1980) University of Tok
yo Press). ()-IFN-β stock solution is 110
0U/mg, F-IFN-β stock solution is 35 CI U/
The activity of ml was confirmed by CPE-50 method (FL-cell-VSV system).

Figure 1 shows the inactivation curves when both stock solutions were heated in a water bath at various temperatures for 5 minutes. C+-I FM-β is F-IFN-
It was more likely to undergo heat inactivation than β, and the temperatures at which 50% inactivation occurred were 41°C and 55°C, respectively, a difference of about 14°.

Example 1 As shown in the reference example, G-IFN-β has a thermal stability of F-
IFN-β is also bad. The difference is particularly noticeable between 400 and 50°C. Therefore, 1 volume of saline solution was added to each of the filtration volumes of both stock solutions, and the final salt concentration was adjusted to 0 to 1. OM was deactivated by heating at 43° C. for 5 minutes, and the residual activity was measured to obtain Figure 2. As can be seen from Figure 2, 4 of G-IFN-β
Inactivation (70%) due to heating at 6° C. for 5 minutes was prevented by the addition of common salt, and almost 100% activity remained in the presence of about 0.4 M common salt. When the salt concentration increases to 1M, 1
．． In some cases, about 5 times the activity was observed.

[Brief explanation of the drawing]

Figure 1 shows F -IFN- after heating at various temperatures for 5 minutes.
Figure 2 shows the residual activity of β and G-IFN-β.
- shows the residual activity rate of β. Patent applicant Toshi Co., Ltd. Training temperature (5 minutes) Figure 1 0 0.2 0.4 0.6 0.
8 10 N1NaCl 9 degrees Figure 2

Claims (4)

[Claims] (1) A method for stabilizing interferon-β without sugar chains, which comprises adding an inorganic salt to interferon-β without sugar chains. (2) Interferon-β without sugar chains is recombinant D
The method according to claim 1, which is interferon-β produced from microorganisms by NA technology. (3) The method according to claim 1, wherein the inorganic salt is common salt. (4) The method according to claim 1, wherein the concentration of the inorganic salt is 0.3M or more.