JPS585188A - Heat treatment of myeloperoxidase - Google Patents

Abstract

PURPOSE:To deactivate a virus which may exist in a myeloperoxidase-containing solution without denaturing myeloperoxidase, by adjusting the myeloperoxidase-containing solution to a proper pH, followed by heating it. CONSTITUTION:A myeloperoxidase-containing solution is adjusted with a buffer solution such as phosphoric acid buffer solution, citric acid buffer solution, etc. to a pH of 4-8 and a salt concentration of 0.01-0.3M, and, if necessary, a heat stabilizer such as albumin, dextran, etc. is added to the solution, which is heated at 50-70 deg.C, preferably 55-65 deg.C for 8-16hr. The prepared myeloperoxidase is filtered so that microorganisms are removed, and, if necessary, it is lyophilized.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of heat-treating a solution of myeloperoxidase in order to inactivate viruses that may be contained therein.

Mini gastric peroxidase was developed in 1941 by ecAg cell sr.
A-cta
Pkyslol! 1caad, ), j, 5up
p1.8 (1941)] is included in large amounts in bone marrow-derived cells, especially neutral polyglots and monocytes, along with lysochyphids. The content per weight of neutrophils reaches a peak. This enzyme has an fk reserve distribution of approximately 120,000 to 1! ! It is a heme protein with a high basicity and contains 2 atoms of iron per protein molecule. The physiological significance of this enzyme is that it produces hypohydrogen ions in the presence of hydrogen peroxide and hydrogen ions, and through its intense oxidation or addition action, it destroys the constituent proteins of bacteria, viruses, etc. Alternatively, it is thought that it irreversibly changes acidity and exhibits pharmacological effects such as bactericidal, virucidal, or virus inactivation.

However, it is known that viruses such as hepatitis and endemic diseases are present in neutral white blood cells and monocytes, and these viruses must be removed or inactivated. In order to avoid such risks, viruses are usually measured in advance using immunoassays, and raw materials containing high concentrations of viruses are excluded, which can be effective in preventing viral infections to some extent. , this method cannot be adopted as an industrial manufacturing method.

The various human serum protein preparations obtained by fractionating plasma have the problem of viral infections, but albumin preparations can be heated at 60°C for 10 hours to remove albumin. It was found that the virus could be inactivated without deterioration, and albumin preparations have since been subjected to this heat treatment and are safely used clinically. Since it was discovered that heat treatment for 60'C11O hours is effective in preventing viral infections, this method has been applied to other Jinmaro Seitsubo white preparations; For application, the thermal stability of the substance itself must be considered.

The inventors researched the processing conditions for maintaining the thermostability of mini-level oxidase, found effective processing conditions for maintaining the thermostability, and eliminated viruses that are at risk of being infected without altering myeloperoxidase. It was successfully revitalized.

In the present invention, a solution containing myeloperoxidase is adjusted to pH 4 to 8 using a buffer solution, and then heated to inactivate the virus.

Myeloperoxidase subjected to heat treatment is recovered from neutral polynuclear leukocytes and monocytes, which are cells of bone marrow origin, according to a known method. The degree of purification is not particularly limited, but the specific activity is 50 units (U)/m or more, preferably 100 U/d or more. The amount of myeloperoxidase contained in the solution as protein is 0.03
~5% WΔ, preferably 0.1 to 1.0 WΔ,
Appropriate! Adjust the pH of the 1IIIi solution to 4-8. Phosphate buffer, citrate buffer, Plissin-Tovinson buffer, etc. are used as buffer solutions, and the salt concentration is adjusted to 8.01 to 8.01.
Adjust to 0.3M. The heating temperature is 50 to 70°C, preferably 55 to 65°C, and the heating time is 8 to 16 hours.

It is preferable to add a heat stabilizer such as albumin, dexFlan, or an amino acid during the heat treatment.

Myeloperoxidase obtained in this way is #coa
After passing through RP, it is freeze-dried and formulated into a formulation. At this time, a halogen salt is added, and a pharmaceutical carrier, excipient, stabilizer, activator, and preservative are added to the composition.

r This mini-gastric peroxidase preparation is used as a treatment for tuberculosis, etc., and is usually packaged in ampoules or dispensing containers per unit dose for injection or topical use. Approximately 0. Adjust to ~10% W/V solution. Administration is carried out parenterally or orally, and local administration is preferred, but topical administration is also possible. Specifically, the drug is administered by, for example, intravenous administration (in the case of systemic diseases such as sepsis or miliary tuberculosis), intramuscular administration, or spraying into the respiratory tract. In addition, in the case of targeted administration, mini-level oxidase is inactivated by gastric juice, so
Enteric coating prevents contact with gastric fluids. The dosage of this drug to living bodies varies depending on the route of administration, the target disease, its symptoms, etc. For example, in the case of intravenous injection or local administration, the amount of myeloperoxidase administered per day for adults is 100 (1y, 0). .1φυ/
Immediately.゛Experiment 1゛The ratio was adjusted to 8.0, 7.0, 8.0, and 9.0, and the same heat treatment was applied to examine the residual activity rate. The active unit of myeloperoxidase is B, Chan
Methods in Enzymology' (M
ethodin Enzymology), II, 7
64 (1955).

The residual activity rate at each pH value is - as shown in the table below, 79% at pH 3.0 and 68% at pH 9.0rLj.
However, it was 95% or more in the pH range of 4.0 to 8.0, and it was found that this range was effective for thermal stability.

Thermal stabilization effect experiment 3 at various pH levels This experiment was conducted on urokinase samples containing A.
Add cold virus, measles virus, vesicular stomatitis virus, chikung air virus, six encephalitis virus, rubella virus, polio virus, coxalaki virus, and echo virus, heat treatment at 60°C for 10 hours,
The remaining virus infectivity was measured over time, and it was found that the virus had completely lost its infectivity after 10 hours. This result also applies to viruses other than the one used at 60°C.
This suggests that if heat treatment is applied for 0 hours, the virus infectivity will disappear.

At Honkamei, we adjust the pH of the Miewah peroxidase solution to ~8 with a buffer solution, and then heat it to inactivate the virus, thereby eliminating the risk of Kurairi peroxidase without altering the Miewah peroxidase. Since the virus can be inactivated and only the pH needs to be adjusted, it is easy to incorporate into the production process of Φbunkoda peroxidase, and the myeloperoxidase, which is useful as a therapeutic agent for cancer, can be produced in high yield. It has a possible effect.

Example Electron knee peroxidase extracted from human leukocytes was CM
- Highly purified using Sephadex C-5O column WV Todatefy, the 100 υ/d solution was adjusted to pH 7.0 using Pritton-Robinson buffer, and then heated at 60°C for 10 hours. . The activity after heat treatment was 99·U/Wd, and the activity residual rate was 99 arc.

Applicant: Kinsha Co., Ltd. Setorijuji

Claims (1)

[Claims] PR solution containing mini gastric peroxidase with buffer
A method for heat treatment of Mini Cedar Seven, which is characterized by adjusting the temperature to I to 8 and then subjecting it to heat treatment to inactivate the virus.