JPH08194192A - Production of freeze-dried material containing proteolytic enzyme - Google Patents

Abstract

PURPOSE: To obtain a method for producing a freeze-dried material contg. proteolytic enzyme with the deactivation suppressed in freeze drying by freeze- drying an aq. composition contg. the enzyme in the presence of a surfactant. CONSTITUTION: An aq. composition contg. proteolytic enzyme is freeze-dried in the presence of a surfactant, especially at least one kind selected from anionic surfactant, amphoteric surfactant and nonionic surfactant. In this case, the deactivation of the enzyme is suppressed by conducting the freeze drying at -3 to -40 deg.C, and the meltability when in use is improved since the grain of the freeze-dried material is refined. Further, as the drying after freeze drying is conducted at <=40 deg.C, the deactivation of the enzyme is effectively suppressed, Accordingly, the enzyme is stably present because the enzyme-contg. material is freeze-dried.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a proteolytic enzyme-containing product in a freeze-dried form. The proteolytic enzyme-containing product is useful as, for example, a contact lens agent.

[0002]

2. Description of the Related Art Contact lenses are roughly classified into hard contact lenses and soft contact lenses. Hard contact lenses are said to be non-hydrophilic and have low oxygen permeability. Has been developed. However, since these lenses are easily contaminated with proteins and the like, it is necessary to perform daily cleaning, sterilization and storage in order to maintain oxygen permeability like soft contact lenses.

By the way, there is a method of decomposing and removing protein stains adhering to the surface of a contact lens with a proteolytic enzyme, and various cleaning agents have been proposed and used. Instability is a major problem. The present inventors have found that, when a proteolytic enzyme-containing aqueous composition is freeze-dried, the water content can be sufficiently removed, and thus the proteolytic enzyme can be stabilized more reliably. It was found that it has excellent solubility in time. Under such circumstances, the present inventors have focused on a freeze-dried product containing a proteolytic enzyme, particularly a freeze-dried contact lens agent.
There is no conventional contact lens formulation in a freeze-dried state, and it is a novel formulation.

However, the activity of proteolytic enzymes decreases when they are freeze-dried. Further, at the time of freeze-drying, there is a problem that the proteolytic enzyme is inactivated even at the time of preparation of the proteolytic enzyme-containing aqueous composition to be subjected to freeze-drying treatment. Have difficulty. For example, at the time of production of a freeze-dried contact lens agent, which is a kind of freeze-dried product, a proteolytic enzyme-containing aqueous composition usually filled in a vial or the like is subjected to freeze-drying treatment. It takes a considerable time to fill the composition into a vial and the like, and to freeze-dry after filling, during which the proteolytic enzyme is inactivated.

[0005]

The present invention has been made in view of the above circumstances, and a problem to be solved by the present invention is to provide a protein in which a decrease in the activity of a protease is suppressed during freeze-drying treatment. It is a method for producing a lyophilized product containing a degrading enzyme.

[0006]

[Means for Solving the Problems] In order to solve the above problems,
The inventors of the present invention have conducted various studies and found that inactivation of proteolytic enzyme is significantly suppressed by freeze-drying the proteolytic enzyme-containing aqueous composition in the presence of a surfactant. Further, at that time, freeze-drying is -3 ° C to-
By being carried out under the temperature condition of 40 ° C, it is possible to suppress the activity decrease of the proteolytic enzyme and to make the freeze-dried product particles fine so that the solubility at the time of use is excellent, and further drying after freeze-drying is performed. It was found that the decrease in the activity of the proteolytic enzyme can be suppressed more effectively by carrying out the treatment under the temperature condition of 40 ° C. or lower.

In the present invention, an aqueous composition containing a proteolytic enzyme is freeze-dried in the presence of at least one selected from surfactants, particularly anionic surfactants, amphoteric surfactants and nonionic surfactants. The present invention relates to a method for producing a lyophilized product containing a proteolytic enzyme, which comprises:

Nonionic surfactants, anionic surfactants and zwitterionic surfactants are effective as the surfactants used in the present invention. In addition, these surfactants can be advantageously used in an appropriate combination. By the way, it is known that these surfactants generally have an antibacterial action at the same time as the washing action.

Examples of anionic surfactants include sodium lauroyl sarcosine, triethanolamine lauroyl-L-glutamate, and sodium myristyl sarcosine, and examples of zwitterionic surfactants include lauryl dimethylamino acetic acid betaine and
2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, alkyldiaminoglycine hydrochloride and the like can be mentioned. Examples of the nonionic surfactant include polysorbate 80, polyoxyethylene hydrogenated castor oil 60, and polyoxyl stearate. 40, polyoxyethylene lauryl ether and the like. Further, as the combination of surfactants, for example, a combination of lauroyl-L-glutamic acid triethanolamine as an anionic surfactant and polysorbate 80 as a nonionic surfactant, and lauroyl-L-glutamic acid triethanolate as an anionic surfactant. The combination of ethanolamine and the amphoteric surfactant lauryldimethylaminoacetic acid betaine is effective.

The concentration of the surfactant in the aqueous protease-containing composition to be lyophilized according to the present invention is 0.01-10 (W / V)%, preferably 0.1-3.
(W / V)%, more preferably 0.5-2.5 (W / V
V)%. Surfactant concentration is 0.01 (W / V)%
If it is less than, the stabilizing effect tends to be insufficient,
If it exceeds 10 (W / V)%, the viscosity of the aqueous composition tends to be high, and it tends to be difficult to freeze. Further, it is preferable to leave a surfactant in the freeze-dried product produced by the method of the present invention. At that time, if the surfactant is present in a high concentration, there is a tendency that a problem that the surfactant is adsorbed to the contact lens occurs.

Examples of the proteolytic enzyme used in the present invention include plant-derived papain, animal-derived trypsin, chymotrypsin, microbial Bacillus-derived protease, and the like. Can be used. Among them, a proteolytic enzyme derived from a microorganism belonging to the genus Bacillus is desirable because of the stability of the enzyme in the liquid, and, for example, bioprase (trade name, manufactured by Nagase Seikagaku Corporation) is preferably used.

In the present invention, for example, when the freeze-dried product is a contact lens agent, the amount of the proteolytic enzyme to be incorporated should be appropriately determined based on the effective amount depending on the cleaning effect to be obtained. However, preferably 10
About 5000 units / ml, more preferably 50-100
The ratio is determined to be about 0 unit / ml. This is because if the compounding amount is too small, the cleaning effect becomes insufficient, and if the enzyme concentration is too high, there is a risk of causing skin damage during cleaning.

In the production of a freeze-dried product, the proteolytic enzyme is inactivated even during preparation of the proteolytic enzyme-containing aqueous composition for freeze-drying treatment. The composition is also preferably prepared in the presence of a surfactant.

The freeze-dried product is produced, for example, as follows. The proteolytic enzyme and the surfactant are dissolved or dispersed in water (preferably purified water). At that time, in order to suppress foaming caused by the surfactant, it is preferable to carry out the dissolution or dispersion operation at a low stirring speed.

Freeze-drying is usually -3 ° C to -40 ° C, preferably -8 to -30 ° C, more preferably -10 to -20.
It is carried out under a temperature condition of ° C. If the temperature is lower than -40 ° C, the freeze-drying time becomes long, so that the industrial property tends to be poor and the enzyme activity tends to decrease. Solubility tends to deteriorate. Freeze-drying is preferably performed under reduced pressure. The drying temperature condition after freeze-drying is 40 ° C or lower, preferably 35 ° C or lower, more preferably 30 ° C or lower, and the lower limit thereof is 5 ° C or higher, preferably 10 ° C or higher. If the drying after freeze-drying is performed at a temperature higher than 40 ° C., the enzyme activity tends to decrease. Further, if the temperature after the freeze-drying is less than 5 ° C, the drying tends to be insufficient. Drying after freeze-drying is performed under reduced pressure.

As the freeze-dried product, a freeze-dried contact lens agent is particularly preferable, and the freeze-dried contact lens agent will be described below. The lyophilized agent for contact lenses is stably stored in a lyophilized state until use, and water (preferably purified water, distilled water for injection, etc.) is used.
It is used by dissolving in. Needless to say, in that case, the stability of the product dissolved in water at the time of use is improved by the presence of the surfactant. When dissolved in water,
Usually, it is desirable that the pH is adjusted to 4-8.

As long as the stability of the freeze-dried contact lens preparation or the contact lens preparation when dissolved in water is not impaired, a commonly used preservative (for example, benzalkonium chloride, chlorhexidine gluconate, parabens, Alkyldiaminoethylglycine hydrochloride, chlorobutanol, sorbic acid, etc.), pH adjusters (eg, hydrochloric acid, phosphoric acid, sulfuric acid, sodium hydroxide, ammonia, etc.), buffers (eg boric acid and its salts, citrate, There is no problem even if excipients such as phosphates, triethanolamine, etc.), chelating agents, disintegrating agents, binders and the like and other enzyme agents such as various additives such as lipolytic enzymes are further added.

The use of the freeze-dried contact lens preparation is as follows.
It is usually carried out by dissolving in water at the time of use, putting a contact lens detached from the eye into this, and immersing for 1 to 12 hours. Thereby, at the same time as the washing is carried out naturally, the surfactant usually has a bactericidal action, so that the sterilization is carried out. By incorporating a preservative, the bactericidal action can be more reliably exhibited. After the immersion, the contact lens is rinsed with tap water and then put on again. It was found that there was no problem even if it was used daily in the same solution for 1 week continuously. In the present invention, the contact lens agent is preferably a hard contact lens agent.

The following are some examples of the present invention,
The present invention will be clarified more specifically, but they are for explaining the present invention and not for limiting the scope of the present invention. Example 1 Bioprase 24000 units Lauroyl-L-glutamic acid triethanolamine 0.5 g Polysorbate 80 0.5 g Disodium hydrogen phosphate dodecahydrate 0.2 g Sodium chloride 0.9 g Phosphoric acid qs Sodium hydroxide qs Purified water qs 100 ml pH 7.0 The solution of the formulation of Example 1 was pre-frozen at -40 ° C under atmospheric pressure, lyophilized at -10 ° C under reduced pressure, and further dried at 35 ° C under reduced pressure to produce a freeze-dried preparation. .
Immediately before the start of freeze-drying and after 8 hours and after 24 hours of freeze-drying, the residual ratio (%) of enzyme activity was calculated. The results are shown in Table 1.

The enzyme activity residual rate (%) was determined as follows.

[0021]

[Equation 1]

[0022]

[Table 1]

Examples 2 to 6 The compositions of the formulations of Example 1 were freeze-dried under the conditions shown in Table 2 to prepare freeze-dried products. The pressure at that time was the same as in Example 1.
Is the same as Immediately before the start of freeze-drying and freeze-drying 8
The enzyme activity residual rate (%) after time was calculated. Table 2 shows the results
Shown in Table 2 shows the results of visually observing the appearance of the crystals.

[0024]

[Table 2]

Example 7 1200 units of bioprase Lauroyl sarcosine sodium 0.03 g Betaine lauryldimethylaminoacetate (35%) 0.05 g Triethanolamine lauroyl-L-glutamate (30%) 0.1 g Boric acid 0.03 g Borax 0 0.018 g Sodium edetate 6 mg Chlorhexidine gluconate 0.3 mg Sodium thiosulfate 0.015 mg Purified water Appropriate amount 100 ml pH 7.0 After pre-freezing the solution of the above formulation at -40 ° C under atmospheric pressure, under reduced pressure -10 Freeze-dry at ℃, and further under reduced pressure 3
It was dried at 5 ° C to produce a freeze-dried preparation. When 6 ml of purified water was added to this freeze-dried preparation and the mixture was allowed to stand for 5 seconds, it was dissolved in a clear colorless solution very well. After leaving the obtained sample solution at room temperature for 7 days, the residual rate of enzyme activity (%) was measured. As a result, the residual rate of enzyme activity was 93.9%, and it showed good detergency in the detergency test for lipid and protein stains performed at the same time. In addition, there was virtually no occurrence of bacteria.

Example 8 1200 units of bioprase Lauroyl sarcosine sodium 0.03 g Betaine lauryl dimethylaminoacetate (35%) 0.05 g Triethanolamine lauroyl-L-glutamate (30%) 0.1 g Boric acid 0.03 g Borax 0 0.018 g Sodium edetate 6 mg Methyl paraoxybenzoate 6 mg Sodium thiosulfate 0.015 mg Purified water Suitable amount Total 100 ml pH 7.0 After pre-freezing the solution of the above formulation at -40 ° C under atmospheric pressure, -10 ° C under reduced pressure. Freeze-drying is carried out and further under reduced pressure 3
It was dried at 5 ° C to produce a freeze-dried preparation. When 6 ml of purified water was added to the freeze-dried preparation of the above composition and left for 5 seconds, it was dissolved in a clear colorless solution very well. After leaving the obtained sample solution at room temperature for 7 days, the residual rate of enzyme activity (%) was measured. As a result, the enzyme activity residual rate was 9
It was 5.1%, and also showed good detergency in the detergency test for lipid and protein stains conducted at the same time. In addition, there was virtually no occurrence of bacteria.

Example 9 Biopulase 1000 units Polyoxyl stearate 40 0.03 g Lauroyl-L-glutamate triethanolamine (30%) 0.1 g Alkyldiaminoglycine hydrochloride 0.6 mg Boric acid 0.03 g Borax 3 mg Sodium edetate 0 0.6 mg Purified water Appropriate amount Total amount 100 ml pH 7.0 The solution of the above formulation was pre-frozen at -40 ° C under atmospheric pressure, and then freeze-dried under reduced pressure at -10 ° C, and further under reduced pressure 3
It was dried at 5 ° C to produce a freeze-dried preparation. The above composition was dissolved in 6 ml of purified water to prepare a proteolytic enzyme-containing contact lens agent. After leaving the obtained sample solution at room temperature for 7 days, the residual rate of enzyme activity (%) was measured. As a result, the residual rate of enzyme activity was 94.5%, and good detergency was exhibited in the washing test for lipid and protein stains performed at the same time. In addition, there was virtually no occurrence of bacteria.

Example 10 The aqueous solution of the formulation of Example 1 was pre-frozen at -40 ° C under atmospheric pressure, lyophilized at -10 ° C under reduced pressure, and further dried at 30 ° C and 41 ° C for 3 days. Then, the residual rate of enzyme activity (%) was measured. As a result, the residual enzyme activity (%) was 82.5% at 30 ° C, and the residual enzymatic activity (%) was 3.9% at 41 ° C. From the above results, it is understood that the drying after freeze-drying is preferably performed at a temperature of 40 ° C. or lower.

[0029]

INDUSTRIAL APPLICABILITY In the method for producing a freeze-dried product containing a proteolytic enzyme in a freeze-dried form of the present invention, the proteolytic enzyme is stabilized during preparation of the freeze-dried preparation, so that the proteolytic enzyme activity is excellent. A proteolytic enzyme-containing freeze-dried product, particularly a proteolytic enzyme-containing contact lens agent can be provided. The lyophilized proteolytic enzyme-containing product thus obtained is in a lyophilized state, so that the proteolytic enzyme is stable and has excellent solubility at the time of use, and when it is stored as an aqueous solution. In addition, since it contains a surfactant, the stability of the proteolytic enzyme can be maintained for a long period of time.

Claims (7)

[Claims] 1. A method for producing a freeze-dried product containing a proteolytic enzyme in a freeze-dried form, which comprises subjecting the proteolytic enzyme-containing aqueous composition to a freeze-drying treatment in the presence of a surfactant. 2. The method according to claim 1, wherein the freeze-drying is carried out under a temperature condition of −3 ° C. to −40 ° C. 3. The production method according to claim 1, wherein the freeze-drying is performed under a temperature condition of −8 ° C. to −30 ° C. 4. The production method according to claim 1, wherein the drying after freeze-drying is performed under a temperature condition of 40 ° C. or lower. 5. The production method according to claim 1, wherein the drying after freeze-drying is performed under a temperature condition of 35 ° C. or lower. 6. The production method according to claim 1, wherein the surfactant is at least one selected from the group consisting of anionic surfactants, zwitterionic surfactants and nonionic surfactants. . 7. The method according to any one of claims 1 to 6, wherein the surfactant concentration in the protease-containing aqueous composition is 0.01 to 10 (W / V)%.