JPH02111726A - Stabilization of human interleukin 1 - Google Patents

Abstract

PURPOSE:To improve stability of human interleukin 1 in preservation or lyophilization by adding chondroitin sulfuric acid, a metallic salt, polyvinyl alcohol and/or glycogen to human interleukin 1. CONSTITUTION:Human interleukin 1 (human IL-1), preferably alpha type is blended with one or more substances selected from chondroitin sulfuric acid, a metallic salt thereof (preferably alkali or alkaline earth metallic salt), polyvinyl alcohol and glycogen as a stabilizer in such a way that the total amount of the stabilizer is 2-20mg, preferably 10-20mg, especially 10mg based on 1ml solution containing human IL-1 to prevent reduction in activity of human IL-1 when human IL-1 is subjected to pharmaceutical manufacturing operation such as lyophilization or spray drying and preserved in a solution or dry state.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing human interleukin 1, and more particularly, to a method for stabilizing human interleukin 1 during storage or during operations such as freeze drying and spray drying.

Gerry et al. discovered a substance in the culture supernatant of human macrophages that promotes mitogen-induced mouse thymocyte division, and they combined this with lymphocyte activating factor (Iympho).
However, since 1979, the name interleukin 1 (hereinafter abbreviated as rlL-IJ) has been used.

When high-purity IL-1 is subjected to formulation operations or stored in a solution or dry state, its activity is significantly reduced.

The present inventors conducted intensive research on the stabilization of human IL-1 and found that by adding certain polysaccharides or vinyl polymers to human IL-1, operations such as freeze-drying or spray-drying were performed. Even if it is stored for a considerable period of time,
It was discovered that the activity was retained, and the present invention was completed.

The present invention uses chondroitin sulfate for human IL-1.

The present invention relates to a method for stabilizing human TL-1, which comprises adding at least one substance selected from chondroitin sulfate metal salt, polyvinyl alcohol, and glycogen.

Human IL-1 used in the present invention includes human α type or β type, with α type being preferred.

This human IL-1 may be in either a solution or a dry state, but a dry product containing human IL-1 is particularly preferred.

When the dried product containing human IL-1 is made into a solution, it is preferable that the pH is maintained at 3 to 9, and it is more preferable to adjust the pH with an appropriate buffer. , for example, phosphate buffer, Tris (iri
s(hydroxymethylLaminoeth
ane) hydrochloric acid buffer, etc.

Depending on the purpose, salt may be added, and the salts used include sodium chloride, potassium chloride, etc., and the concentration is determined depending on the purpose. For example, when used for injection, dissolve sodium chloride in distilled water. After, 0
．． Add to 15M and make a freeze-dried product.

Among substances that stabilize Hill-IL-1 (hereinafter referred to as "stabilizers"), chondroitin sulfate may be of any type A to E, but it is necessary to make the solution alkaline when adding it. For convenience of use, alkali metal salts of chondroitin sulfate, such as sodium salts and potassium salts, and alkaline earth metal salts thereof, such as calcium salts and magnesium salts, which do not require such a process, are preferred.

The stabilizers can be used alone or in appropriate combinations of two or more.

The concentration of stabilizer added was as follows: solution 1 containing human IL-1
2B~20mg per 1, preferably 1011g~20
+g, and 10s+g is most preferable from a common sense and economic point of view.The amount of stabilizer added to the dried product containing human IL-1 is as follows when the resulting product is dissolved. The solution concentration is chosen to be .

The method of addition is not particularly limited, but examples include adding the stabilizer powder directly to the IL-1-containing solution, or, if necessary, drying it by freeze-drying, spray-drying, or other methods. It will be done. The addition time may be during the separation and purification process or during the formulation process.

When two or more types of stabilizers are added, the total amount thereof may be adjusted to the above-mentioned concentration and amount.

The dried product containing human IL-1 to which a stabilizer has been added is preferably stored or formulated at a temperature below 30°C, preferably below 10°C. The dried product containing IL-1 retains IL-1 activity during the formulation procedure.

Examples are given below to further explain the present invention in detail, but the present invention is not limited thereto.

(Left below) 8.5m prepared in Example 2 of JP-A-61-271222
g/ml human interleukin 1α (hereinafter rrH
ulL, -1α), 134 μg/ml
rHu IL-1α solution (0.1M phosphate buffer containing 0.15M sodium chloride, pH 5.1)
) was prepared. Sodium chondroitin sulfate A was added to this rHu IL-1α solution at a concentration of 4 mg/ml, and after freeze-drying, it was stored at 40°C, and after 30 days, EIA
The residual activity was determined by the method. The results are shown in Table 1.

Residual activity was measured in the same manner as in Example 1 except that glycogen was used as the stabilizing agent instead of sodium chondroitin sulfate. The results are shown in Table 1.

11EL Residual activity was measured in the same manner as in Example 1 except that polyvinyl alcohol was used as a stabilizer instead of sodium chondroitin sulfate.

The results are shown in Table 1.

The residual activity was measured in the same manner as in Example 1, except that the substances listed in Table 1 were used as the coarse additives. The results are shown in Table 1.

As can be seen from Table 1, the stabilizer used in the present invention has extremely superior effects compared to other additives and no additive.

rHu I with the same active concentration as used in Example 1
A L-1α solution was prepared, and sodium chondroitin sulfate A was added to it at a concentration of 0.1 w/ν% and 0.2 w/νχ.

0.4 w/νχ, 1. Ow/vχ and 2. Ow/v
χ and stored at 40°C after freeze-drying.
After 30 days, rHu ILlα residual activity was determined in vitro.
It was measured by the o evaluation method, and the residual activity rate was calculated based on the value.

The results are shown in Figure 1.

Remaining amount after storage at 40″C for 30 days (χ) when the initial value is 100 Table 1 Effect of additives on stability of rHu IL-1α lyophilized product Table 1 (continued) Remaining amount after storage at 40℃ for 30 days (1) when the value is lOO

[Brief explanation of drawings]

Figure 1 shows that I
It shows the change in L-1 residual activity.

Claims (3)

[Claims] (1) Human interleukin 1, chondroitin sulfate, chondroitin sulfate metal salts, polyvinyl alcohol,
A method for stabilizing human interleukin 1, which comprises adding at least one substance selected from glycogen. (2) The stabilization method according to claim 1, wherein human interleukin 1 is α type. (3) The stabilization method according to claim 2, wherein the human interleukin 1 has the following amino acid sequence. [There is a gene sequence]