JPS5925333A - Stabilization of interferon-beta free from glycose chain - Google Patents

Abstract

PURPOSE:To stabilize the titled substance, by adding a polyol to an interferon-beta free from glycose chain. CONSTITUTION:An interferon-beta free from glycose chain is stabilized by adding a polyol (e.g. glycerol, ethylene glycol, or sugars such as sucrose) to the interferon in an amount of >=5%, especially 10-30%. The interferon-beta free from glycose chain is G-IFN-beta which is a simple protein having a molecular weight of about 20,000 and produced by microorganisms such as Escherichia coli recombined with IFN-beta gene.

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 (IPN) 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 S. nigra, and its effects are known to be species-specific.

Six types of human interferon are currently 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. With the recent development of recombinant DNA technology, it has become possible to produce IFN-a, 1FN-β, and IFN-r using microorganisms such as Escherichia coli and yeast, and the prospect of producing large amounts of interferon has become possible. (Goeddol, D.

V, ct al+ Nucleic Ac1d Re
5earch+ 8.4057 (1980), Natu
re, 287.411 (1980).

Nature 295,. 503 (19B 2
) ). Interferon-β (hereinafter abbreviated as F-IPN-β) produced by human fiber cells is a glycoprotein with a molecular weight of approximately 23,000 ± 2,000, with the protein portion having a molecular weight of approximately 20,000 and a molecular weight of 3,000.
It is a substance in which sugars before and after are bound together. On the other hand, interferon-β (hereinafter abbreviated as G-FN-β) produced by microorganisms such as Escherichia coli that have been recombined with the interferon-β gene is a simple protein with a molecular weight of approximately 20,000 that does not have sugars attached to it, and in this respect it is a natural protein. It is a different substance from IFN-β, and therefore there are some differences between the two in terms of chemical and biological properties.

Interferon-β itself is a highly unstable substance°
However, it has become clear that G-IFN-β is also an even more unstable substance in some respects and is easily deactivated. That is, when comparing the thermal stability, which is a typical index of stability, as shown in Figure 1, G-IFN-β has P
- Inactivates at about 15° lower temperature than IPN-β. G-
The instability of IFN-β is considered to be a major drawback when it is used clinically as a pharmaceutical.

The present inventors continued intensive studies to improve the instability of G-11''N-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-IPN-β), molecular weight approximately 20.0
00 simple protein. G-IFN-β of the present invention
For example, Taniguchi et al.'s method (Proc, Natl,
Acad, Sci, 77 + 5250 (198
0)) can be manufactured as follows.

In this study, an m-RNA mixture was isolated from human fibroblasts producing terferon-β, and the corresponding bio-DNA
Create E. coli plasmid 1) 101.322
and obtain E. coli containing the structural gene of interferon-β. At this time, in order for the structural gene of interferon to be expressed in E. coli and to produce interferon protein, DNA m-E (
It is necessary to bind a promoter sequence for transcription to NA, and to have an SD sequence or the like that allows the resulting m-4NA to bind to ribosomes and be translated into protein. for that,
The structural gene of interferon β is linked to the downstream of the lactose oper/p1 motor and SD sequence of E. coli. A plasmid (pL0117R) containing the promoter region obtained here and the structural gene of IFN-β is used to infect E. coli T.
Reassemble lB101.

The recombinant obtained in this way was precultured in LB medium, and this was inoculated into about 2 sections of new LB medium.
After culturing for several hours, the cells were harvested, washed, treated with lysozyme to lyse the cell wall, frozen and thawed, and centrifuged to solubilize the 0,-IFN-β that had been produced and accumulated within the E. coli cells. An extract exhibiting interferon activity is obtained.

Polyols of the invention include, for example, glycerin, ethylene glycol or saccharides. Among sugars, sugars other than polysaccharides, ie, monosaccharides and oligosaccharides are preferred. Particularly preferred polyols are glycerin and sucrose. The concentration of the polyol is preferably 5 or more, especially 10 to 30.

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

Reference example Plasmid pL0117 containing the human 1FN-β gene
E. coli HB 101 (T.

Taniguch et al. + Proc. N
atl, Acad, Sci, 775230 (1
980)) was precultured in LB medium, and this was added to 1 t of LB medium.
Approximately 2 inoculated cells were added to the medium. Culture with shaking at 3C0, bacterial count 0D6
When 6O reached 0.6, collect the bacteria and add 50mM Tris buffer containing 25% sucrose to warm pi(s,
The cells were washed with o and collected by centrifugation. 12 bacterial cells
0 ml of 20mM EDTA-50mM Tri
Suspended in s buffer pH 80, 2.0 mq/m
1 Norizozyme was added, and after standing at 0° for 30 minutes, freeze-thaw was repeated twice to achieve constant bacterial lysis. 15,000 ml of lysate
Centrifuge at 0 rpm for 60 minutes and transfer the supernatant to G-IF.
A stock solution of N-β was used as a material for -stability experiments. F-
IFN-β was partially purified from a stock solution produced by human 8-line fibroblasts. (S.Kobaya
shi et, at,, ”The koC
l1nical Potential of Inte
rferon! ”Japan as Medical
Re5search Foundation Pukl
cation 'Nα15+ P2O(198
0) University of Tokyo
(Press). ()-IFN-β stock solution is 1100
U/ml! , F- and IFN-β stock solution is 350 shots/-
The activity of OPD activity-50 method (FL-cell-vs.
It was approved and implemented in V series).

The heat inactivation was also more likely to occur, and the degree of 50% inactivation was 41°C and 55°C, respectively, a difference of about 4°.

Example 1 As shown in the reference example, G-IFN-β has a thermal stability of F-I.
Worse than FN-β. The difference is particularly noticeable between 40° and 50°C. Therefore, GIFN-β stock solution and F-IFN
-β stock solution and 0% glycerin solution were mixed to give a glycerin content of 5 to 60%. (The IFN activity is the same).

The second result was obtained by heating the solution at 43° C. for 5 minutes to determine the remaining activity. From Figure 2, 46℃ of G-FN-β,
Inactivation (80%) by heating for 5 minutes is completely protected in the presence of more than 10% glycerin. In the presence of 30%, a slight enhancement of 1 L was observed.

Example 2 The heat-inactivated protective effects of 20% glycerin and 20% sucrose on G-FN-β were investigated. The results are shown in Table 1. In the case of no additive, about 55% of the activity was inactivated by heating at 4° C. for 5 minutes, but the inactivation was almost completely protected by the addition of glycerin or sucrose.

Table 10-IPN-β at 43°C. Possible heat deactivation for 5 minutes Protective effect of Riolu

[Brief explanation of the drawing]

Figure 1 shows F-IFN-β after heating at various temperatures for 5 minutes.
Figure 2 shows the residual activity of G-IFN-β when glycerin was added and heated at 43°C for 5 minutes.
The residual activity rate of FN-β is shown. Patent applicant Toshi Co., Ltd. Chi10 [Incorrect heating temperature (5 minutes) Figure 1

Claims (5)

[Claims] (1) A method for stabilizing interferon-β that does not have sugar chains, which comprises adding a polyol to interferon-β that does not have sugar chains. (2) Interferon-β without sugar chains is recombinant 1
1. The method according to claim 1, wherein the method is inter7eron-β produced from microorganisms by 1NA-technology. (3) The method according to claim 1, wherein the polyol is glycerin. (4) Claim 1 in which the polyol is sucrose
The method described in section. (5) The method according to claim 1, wherein the concentration of the polyol is 5% or more.