JPS58198292A - Thermal stabilization of leucine aminopeptidase originating from human placenta - Google Patents

Abstract

PURPOSE:When human placenta-originating leucine aminopeptidase is heat-treated in order to inactivate viruses included therein, specific aminoacids are added to increase its thermal stability. CONSTITUTION:When a solution containing leucine aminopeptidase originating from human placenta is heat-treated so as to inactivate viruses which might be included in the solution, at least one selected from a group consisting of leucine, methionine, alanine and epsilon-aminocaproic acid is added by 0.1-20w/v%, when only one aminoacid is used, or 1-20w/v% in combination.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a solution containing human placental leucine aminopebutida, which is heat-treated to inactivate viruses that may be contained in the solution. The present invention relates to a method for increasing the thermostability of leucine aminopeptidase.

Human placental leucine aminopeptidase (hereinafter abbreviated as LAP) is a type of angiotensinase that exists in human placental tissue and has the ability to degrade angiotensin.
It functions as a blood pressure regulating factor in vivo. In other words, when the renin-angiotensin system is abnormally activated, blood pressure rises and hypertonic pressure develops, but LAP decomposes the pressor substances angiotensin-1 and 1-2 and inhibits their physiological activity. This maintains blood pressure homeostasis. Furthermore, even without the light path of the renin-angiotensin system, when LAP is reduced, the function of the angiotensin decomposition and metabolic system is also reduced, and angiotensin is stored in the body, resulting in hypertension. Administering LAP to such hypertensive patients has the advantage of being safer than antihypertensive drugs.

On the other hand, there is a possibility that viruses such as hepatitis and endemic diseases exist in human placenta tissue, and if LAP made from this is administered for serious medical purposes without virus removal or inactivation treatment, viral infections may occur. There is a risk of exposure to In order to avoid such risks, viruses are usually measured in advance using an immunoassay method, and raw materials containing high concentrations of viruses are excluded, thereby achieving a certain degree of effectiveness in preventing viral infections. However, this method is not suitable for medical use.
It cannot be applied to APO large-scale manufacturing methods.

The problem of viral infections is also included in individual human serum protein preparations obtained by fractionating plasma. However, it has been found that virus infectivity can be inhibited without altering albumin by subjecting albumin preparations to heat treatment at 60°C for 10 hours. , and has been safely used clinically. Since it was found that preparations heat-treated at 60° C. for 10 hours were effective in preventing post-administration viral infections, this method has been applied to other human serum protein preparations. A material to which a heat treatment method of 60° C. for 10 hours can be applied must be stable against this treatment. Therefore, various stabilizers have been researched to enable this heat treatment, and in the presence of stabilizers, 60
This heat treatment is applied to substances that can withstand heat treatment at ℃ for 10 hours, and amino acids, sugars, etc. are used as stabilizers for human serum proteins at physiologically isotonic or lower concentrations.

When LAP is also subjected to this heat treatment at 60℃ for 10 hours,
At the same time as the virus was inactivated, LAP activity also decreased. The present inventors conducted research on stabilizers to improve the heat stability of LAP, and found that among the amino acids, sugars, salt, albumin, etc. that have been conventionally used as stabilizers for protein cakes, leucine, It was discovered that only amino acids such as methionine, alanine, and ε-aminocaproic acid have a remarkable heat stabilizing effect on LAP, and the present invention was completed based on this new knowledge.

The present invention involves heating a solution containing LAP in the presence of at least one type of amino acid selected from the amino acid group consisting of leucine, methionine, alanine, and direct-aminocaproic acid to inactivate the virus. It is processed.

The placental angiotensin-degrading enzyme purified according to the separation and purification method (Japanese Patent Application No. 55-151234) is used. The amount of LAP as protein contained in the heat treatment solution is 0.01 to 10% W/V, preferably 0.1 to 10% W/V.
2%V. The pH of the heat-treated solution is preferably adjusted to 5 to 7 with an appropriate buffer, and the salt concentration is preferably adjusted to 0.01 to 0.3 M.

Leucine, methionine, alanine, and 6-aminocaproic acid are purified for medical use, and when each of these amino acids is used alone, the total addition amount is 0.1 to 20 OΔ, and when used in combination, the total amount added is It is 1 to 20% W/V. The upper limit of the amount added is selected based on the mixing ratio in the final product. The temperature change in the heat treatment is 50 to 70°C, more preferably 55 to 65°C, and the heating time is 8 to 12 hours. The thus heat-treated LAP is sterilized and then lyophilized to form a formulation. If an excess of amino acids is added, it is reduced to 1% or less using known methods such as dialysis and dilution.

In order to examine the heating effect of the present invention, we investigated the infectivity of each Kusunoki virus that may be included in LAP preparations.
Experiments were conducted to determine the heating effect with the addition of a stabilizer and the heating effect without the addition of a stabilizer. In this experiment, LAP samples were infected with smallpox virus, mumps virus, measles virus, vesicular stomatitis virus, titangonia virus, Japanese encephalitis virus, hepatitis B virus, rubella virus, poliovirus, coxalaki virus, and echovirus. In addition,
A heat treatment was performed at 60° C. for 10 hours, and the remaining virus infectivity was measured over time. After 10 hours, the infectivity was completely lost regardless of whether a stabilizer was added or not. This result suggests that the infectivity of viruses other than the virus used can be inactivated if the heat treatment of the present invention is applied.

Since the present invention uses amino acids such as leucine, methionine, alanine, and C-aminocaproic acid to stabilize LAP under heat, there is no possibility that antigenic substances may be mixed into the preparation.
L
It is easy to incorporate into the manufacturing process of AP, and virus inactivation treatment Biochen+, Biopl+ys, Ac
ja, + 19L 66 & (1969).

Example 1 p) 16.3 of 0.1M) Lisumarein #! Angiotensin-degrading activity dissolved in the solution: 250μy/br/
mq of LAP aqueous solution 201 (5011t/br/ln
l), add 200 v of leucine dissolved in the same buffer volume.

After thoroughly stirring this, it is heated at 60° C. for 10 hours. Thereafter, the mixture was rapidly cooled with ice water, albumin was added to a concentration of 1%, and after sterilization filtration, the mixture was divided into 2 portions and freeze-dried to obtain a virus-inactivated LAP preparation.

Example 2 To a 10/(32 μf/hr/rd) aqueous LAP solution with angiotensin-degrading activity of 315 μf/br/mQ dissolved in 0.1 M phosphate buffer at pH 5.5, 100 μf of methionine dissolved in the same buffer was added. After stirring well, the mixture was heated at 60°C for 10 hours. After that, it was rapidly cooled with ice water, and then albumin was added to 1% a degree and the liter was removed.
! ! After filtration, the LAPIj! virus was inactivated by dispensing 2/2 aliquots and freeze-drying. A li agent was obtained.

Example 3 Angiotensin degrading activity of pna, s dissolved in 0.05M phosphate buffer 218 pt S'/hr r/mt
i LAP aqueous solution 5 / (100+l/hr r/d
) was added with 150 fI of alanine dissolved in the same buffer solution, stirred well, and heated at 60°C for 10 hours.

Thereafter, the mixture was rapidly cooled with ice water, albumin was added to a concentration of 1%, and after removal of 1ilIv5, the mixture was dispensed at 2m/each and freeze-dried to obtain a virus-inactivated LAP preparation.

Example 4 In 0.2M) lis-malein buffer solution with pH 6.0
'Dissolved angiotensin degrading activity 150μy/b
r/■ LAP aqueous solution 5/(100μy/hr r/
s/), Leucine-16P and C dissolved in the same buffer.
-Aminocarbric acid 84y- is added, stirred well, and then heated at 60°C for 10 hours. Then rapidly cooled with ice water,
Next, albumin was added to a concentration of 1%, and after sterile filtration, 2 m
A LAP preparation in which the virus was inactivated was obtained by dispensing the solution in 1/2 portions and freeze-drying it.

EXPERIMENTAL EXAMPLE In Example 1, various stabilizers were used for heat treatment at 60'C for 110 hours, and their thermal stabilization effects were compared and studied. The activity of the sample before heating was set as 100%, and after the heat treatment the stabilizer was removed by dialysis and the active percentage was examined. The results are shown in Table 1.

The viruses in the table are the same as those listed on page 6.

Margin below Table 1 3. Person who corrects Relationship to the incident: Applicant Kinsha Midori Juji Co., Ltd. 4 agents 6. Subject of Supplement 11- Detailed description of the invention in the specification 7. Contents of correction

Claims (1)

[Claims] Heating a solution containing human placental leucine aminopeptidase to inactivate viruses in the presence of at least one type of amino acid selected from the amino acid group consisting of leucine, methionine, alanine, and ε-aminocarboxylic acid. A method for heat stabilizing human placental leucine aminopeptidase, which comprises subjecting it to a treatment.