JPWO2008068885A1 - Protective agent for lyophilization and method for producing physiologically active substance - Google Patents

Abstract

An object of the present invention is to provide a protective agent for preventing the physiologically active substance from being altered when freeze-drying a mixture composed of the physiologically active substance and water.
The present invention relates to a protective agent for preventing alteration of a physiologically active substance when a mixture composed of the physiologically active substance and water is freeze-dried, and is an amino acid ester in which the amino group at the α-position is modified. A lyophilization protective agent comprising an amino acid amide having a modified amino group at the α-position. The modified amino group is preferably an acylamino group represented by the formula (1) or an imino group represented by the formula (2).
-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)
R ¹ is a hydrocarbon group having 1 to 21 carbon atoms which may contain a hydrogen atom or a hetero atom, R ² and R ³ are each independently a hydrocarbon group having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom. Represents.

Description

   The present invention relates to a freeze-drying protective agent and a method for producing a physiologically active substance.

Physiologically active substances {enzymes, recombinant proteins, antibodies, peptides, amino acids, nucleic acids, sugars, antibiotics, vitamins, etc.} are widely used as various research and development, diagnostic / testing drugs, pharmaceuticals, and the like. It is important that the physiological activity (titer) of the physiologically active substance is not impaired during the manufacturing process or during the storage period.
For example, a freeze-dried pharmacological preparation of G-CSF {granulocyte colony stimulating factor} containing saccharides {mantose, raffinose, sucrose, trehalose or amino sugar is known (Patent Document 1).

JP-T 8-504784 {WO94 / 14465 pamphlet}

However, even if a conventional freeze-dried pharmacological preparation is used, there is a problem that the physiological activity of the physiologically active substance is impaired during the storage period, and the physiological activity (titer) is maintained only about 10%.
An object of the present invention is to provide a protective agent for preventing deterioration of a physiologically active substance and maintaining the activity for a long period of time when a mixture composed of the physiologically active substance and water is freeze-dried. .

The inventors of the present invention have reached the present invention as a result of studies to achieve the above object.
That is, the feature of the lyophilizing protective agent of the present invention is that the physiologically active substance is prevented from being altered when freeze-dried a mixture composed of the physiologically active substance and water, and is protected for maintaining the activity for a long period of time. An agent,
The gist is that it contains an amino acid ester in which the amino group at the α-position is modified or an amino acid amide in which the amino group at the α-position is modified.

The method for producing a physiologically active substance of the present invention comprises an amino acid ester modified with an amino group at the α-position or an amino acid amide modified with an amino group at the α-position in the presence of a lyophilization protective agent.
The gist is that a bioactive substance is produced by freeze-drying a mixture composed of the bioactive substance and water.

The lyophilization protective agent of the present invention can prevent the physiologically active substance from being altered when the mixture composed of the physiologically active substance and water is lyophilized. And even if the bioactive substance obtained is preserved for a long time, the bioactivity (titer) hardly decreases.
Therefore, the protective agent of the present invention is a field using physiologically active substances {enzymes, recombinant proteins, antibodies, peptides, amino acids, nucleic acids, sugars, antibiotics, vitamins, etc.} {various research and development, diagnostic / inspection drugs, pharmaceuticals, etc.} Is extremely useful.
The physiological activity (titer) means the purity of the physiologically active substance.

  Examples of physiologically active substances include, but are not limited to, enzymes, recombinant proteins, antibodies, peptides, amino acids, nucleic acids, sugars, antibiotics and vitamins.

  Enzymes include oxidoreductases {cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc.}, hydrolases {lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc.}, isomerase {glucose isomerase, etc. Etc.}, transferase {acyltransferase, sulfotransferase, etc.}, synthase {fatty acid synthase, phosphate synthase, citrate synthase, etc.} and elimination enzyme {pectin lyase etc.}.

  Recombinant proteins include protein preparations {interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), sodium And diuretic peptides, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like} and vaccines {hepatitis A vaccine, hepatitis B vaccine and hepatitis C vaccine} and the like.

  Examples of antibodies include monoclonal antibodies and polyclonal antibodies.

  The peptide is not particularly limited in amino acid composition, and examples thereof include dipeptides and tripeptides.

  Examples of amino acids include glutamic acid, tryptophan, and alanine.

  Examples of the nucleic acid include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

  Antibiotics include streptomycin and vancomycin.

  Examples of the saccharide include hyaluronic acid, albumin, ceramide, erythritol, trehalose, lipopolysaccharide, and cyclodextrin.

  Examples of vitamins include vitamin A, vitamin B6, vitamin B12, vitamin C, and derivatives and salts thereof.

  Of these physiologically active substances, enzymes and recombinant proteins are preferred, enzymes are more preferred, oxidoreductases and hydrolases are particularly preferred, and oxidoreductases are most preferred.

The mixture composed of the physiologically active substance and water may contain an aqueous solvent.
Examples of aqueous solvents include buffer solutions {phosphate buffer solution, acetate buffer solution, citrate buffer solution and borate buffer solution} and water miscible organic solvents {methanol, ethanol, 2-propanol, butanol, ethylene glycol, glycerin, dimethyl ester. Sulfoxide, dimethylformamide, etc.}.

  As the modified amino group of the amino acid ester or amino acid amide, the type of the modified amino group is not limited as long as the reactivity of the amino group is suppressed. However, the modified amino group is preferably an acylamino group represented by the formula (1) or an imino group represented by the formula (2).

-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)

R ¹ is a hydrocarbon group having 1 to 21 carbon atoms which may contain a hydrogen atom or a hetero atom, R ² and R ³ are each independently a hydrocarbon group having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom. Represents.

Examples of R ¹ include a hydrogen atom, methyl, ethyl, propyl, pentyl, cyclohexyl, heptyl, benzyl, heptadecenyl and henicosyl. Of these, methyl and ethyl are preferable, and methyl is more preferable.

Examples of R ² and R ³ include a hydrogen atom, methyl, ethyl, propyl, pentyl, cyclohexyl, heptyl, benzyl and heptadecenyl. Of these, methyl, ethyl and propyl are preferable, methyl and ethyl are more preferable, and methyl is particularly preferable.

  As amino acids constituting the amino acid ester or amino acid amide, glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, cysteine, methionine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, aspartic acid And glutamic acid. Of these, arginine, histidine, lysine, asparagine and glutamine are preferable from the viewpoint of retention of physiological activity (titer), more preferably arginine and histidine, and particularly preferably arginine.

  When the amino acid is an aminodicarboxylic acid {aspartic acid and glutamic acid, etc.}, the amino acid ester or amino acid amide may not necessarily be a diester or diamide, and the carboxyl group at the ω position is a free carboxyl group (—COOH) or a carboxylate group. (-COOM) or a mixture thereof may be used.

  M includes alkali metals {lithium, sodium, potassium, etc.}, alkaline earth metals {magnesium, calcium, etc.}, alkanolamines {monoethanolamine, diethanolamine, triethanolamine, etc.} and alkylamines having 1 to 4 carbon atoms { Methylamine, ethylamine, propylamine, butylamine and the like} and the like.

  As amino acid esters, alkyl {methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, cyclohexyl, octyl, nonyl, decyl, lauryl, palmityl, stearyl, oleyl and behenyl etc.} esters, aryl {phenyl, And monoesters with methoxyphenyl, benzyl and methylphenyl} esters and polyhydric alcohols {glycerin, glucose, sucrose, sorbitol, mannitol, trehalose, etc.}. Of these, alkyl esters are preferable, methyl esters and ethyl esters are more preferable, and ethyl esters are particularly preferable.

As an amino acid amide, in addition to a free amide (—CONH ₂ ), N-alkyl {methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, cyclohexyl, octyl, nonyl, decyl, lauryl, palmityl, Stearyl, oleyl and behenyl etc.} amide, N-aryl {phenyl, methoxyphenyl, benzyl and methylphenyl} amide and N-hydroxyalkyl {hydroxyethyl, hydroxypropyl, 2,3-dihydroxypropyl, glucosyl etc.} amide etc. It is done. Of these, free amides and N-alkylamides are preferred, and free amides, N-methylamides and N-ethylamides are more preferred.

  An amino acid ester in which the α-position amino group is modified and an amino acid amide in which the α-position amino group is modified are known methods {methods of modifying amino groups, methods of esterifying carboxylic acids, or methods of amidation}. Etc. can be easily obtained. It is preferable to esterify or amidate after modifying the amino group.

The protective agent for lyophilization of the present invention may contain water and / or an aqueous solvent.
When water and / or an aqueous solvent is contained, the content (% by weight) of the amino acid ester in which the α-position amino group is modified or the amino acid amide in which the α-position amino group is modified is modified in that the α-position amino group is modified. Based on the total weight of the amino acid ester or amino acid amide in which the amino group at the α-position is modified and water and / or an aqueous solvent, 0.01 to 20 is preferable, and 0.1 to 10 is more preferable.

  The protective agent for lyophilization of the present invention includes known protective agents such as sugars {glucose, lactose, mannose, raffinose, sucrose, trehalose, etc.}; or amino acids {arginine, glycine, histidine, etc.} and / or peptides {silk Protein etc.} may be included.

  When a saccharide is included, the content (% by weight) of the amino acid ester in which the α-position amino group is modified or the amino acid amide in which the α-position amino group is modified is the amino acid ester in which the α-position amino group is modified or α Based on the total weight of the amino acid amide in which the amino group at the position is modified and the saccharide, 0.01 to 20 is preferable, and 0.1 to 10 is more preferable.

  When the amino acid and / or peptide is contained, the content of the amino acid ester in which the α-position amino group is modified or the amino acid amide in which the α-position amino group is modified is the same as that in the case of the saccharide.

  When the lyophilization protective agent of the present invention contains water, an aqueous solvent, a saccharide, an amino acid, and / or a peptide, the lyophilization protective agent of the present invention is an amino acid ester in which the amino group at the α-position is modified or It can be easily obtained by uniformly mixing an amino acid amide in which the amino group at the α-position is modified with water, an aqueous solvent, a saccharide, an amino acid and / or a peptide.

  In the method for producing a physiologically active substance of the present invention, the use amount (% by weight) of the lyophilizing protective agent is preferably 1 to 1000, more preferably 10 to 500, and next preferably based on the weight of the physiologically active substance. Is 30 to 300, particularly preferably 60 to 100, and most preferably 64 to 81. Within this range, the retention rate of physiological activity (titer) is further improved.

  The protective agent of the present invention and the mixture composed of the physiologically active substance and water may be mixed uniformly immediately before lyophilization or may be mixed uniformly in the previous step.

  There are no restrictions on the freeze-drying conditions {temperature, degree of vacuum, etc.}, but rapid freezing is preferred.

  The following examples further illustrate the invention, but the invention is not limited thereto. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.

<Example 1>
N-α-acetylarginine {Arginine acetamide, MP Bio Japan Co., Ltd.} 12.6 parts (0.05 mole part), 1 part of methanesulfonic acid and 92 parts (2 mole parts) of ethanol were mixed uniformly at 80 ° C. The mixture is heated and stirred for 5 hours, concentrated with an evaporator, recrystallized from water, dried under reduced pressure {60 ° C., 20 Pa}, and the lyophilized protective agent (1) {N-α-acetylarginine ethyl ester} of the present invention is added. Obtained.

<Example 2>
N-benzoyl-arginine ethyl ester {MP Bio Japan Co., Ltd.} was used as it was as the lyophilization protective agent (2) of the present invention.

<Example 3>
After 50 mg of lysozyme {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg} was dissolved in 10 g of ion-exchanged water, 32.3 mg of the lyophilization protective agent (1) obtained in Example 1 ( 0.13 mmol) was added and uniformly mixed to prepare an aqueous solution for lyophilization. This aqueous solution was transferred to a glass bottle with a cap of 20 mL, and the glass bottle was immersed in liquid nitrogen for 1 minute to freeze, and then the glass bottle was transferred to a freeze dryer {DF-01H manufactured by ULVAC, Inc.] under reduced pressure (13 .3 Pa) and lyophilized at −40 ° C. for 40 hours to obtain a dried product. Subsequently, the dried product was stored for 3 days in a desiccator containing phosphorus pentoxide to obtain freeze-dried lysozyme (R1).

<Example 4>
Example 3 and Example 3 except that “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” was changed to “protective agent for lyophilization (2) 40.5 mg (0.13 mmol)” Similarly, freeze-dried lysozyme (R2) was obtained.

<Example 5>
Lyophilized cholesterol oxidase (R3) was obtained in the same manner as in Example 3 except that “lysozyme” was changed to “cholesterol oxidase {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg}”.

<Comparative Example 1>
Trehalose {Wako Pure Chemical Industries, Ltd.} was used as a comparative freeze-drying protective agent (H1).

<Comparative example 2>
Glucose {Wako Pure Chemical Industries, Ltd.} was used as a comparative freeze-drying protective agent (H2).

<Comparative Example 3>
Except for changing “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” to “protective agent for lyophilization (H1) 50 mg (0.13 mmol)”, in the same manner as in Example 3. A freeze-dried lysozyme (HR1) was obtained.

<Comparative example 4>
The same as Example 3 except that “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” was changed to “protective agent for lyophilization (H2) 23.8 mg (0.13 mmol)” Thus, lyophilized lysozyme (HR2) was obtained.

<Comparative Example 5>
“Lysozyme” was changed to “cholesterol oxidase {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg}”, and “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” A freeze-dried cholesterol oxidase (HR3) was obtained in the same manner as in Example 3, except that was changed to “Freeze-drying protective agent (H1) 50 mg (0.13 mmol)”.

  Freeze-dried lysozyme (R1), (R2), (HR1) and (HR2) obtained in Examples and Comparative Examples and freeze-dried cholesterol oxidase (R3) and (HR3) obtained in Examples and Comparative Examples were frozen. The enzyme activity (titer) immediately after drying and the enzyme activity (titer) when stored for 6 months were measured and summarized in Table 1.

<Measurement of enzyme activity (titer) of lysozyme>
After dissolving 16 mg of a measurement sample (lyophilized lysozyme) in 5 g of ion-exchanged water, 10 μL of this aqueous solution was added to 30 ° C. 0.4% Bacillus subtilis suspension (1 ml of 20% Bacillus subtilis suspension, ion-exchanged water). (46.5 mL and 1 mol / L potassium phosphate aqueous solution 2.5 mL uniformly mixed) 3 mL) was added to obtain a measurement solution. Immediately, the absorbance (A0) at 450 nm was measured at 30 ° C. with a spectrophotometer (Shimadzu Corporation, UV-2550), and the measurement solution was allowed to stand at 30 ° C. for 5 minutes. A5) was measured, and the difference (A5-A0) (ΔA) was calculated.

  On the other hand, the difference in absorbance (A5−A0) (A5−A0) in the same manner as above except that “measurement sample (lyophilized lysozyme)” was changed to “lysozyme {Wako Pure Chemical Industries, Ltd., titer 20000 units / mg}”. ΔAb) was calculated, and “retention rate of enzyme activity (titer)” was calculated from the following equation.

Retention rate of enzyme activity (%) = (ΔA / ΔAb) × 100

  The freeze-dried lysozyme (R1), (R2), (HR1) and (HR2) ”obtained in Examples and Comparative Examples was stored at 25 ° C. in a sealed state for 6 months, and the absorbance was measured in the same manner as described above. The difference in absorbance (A5-A0) (ΔA ′) was calculated, and “retention rate of enzyme activity (titer) after 6 months storage” was calculated from the following formula.

Retention rate of enzyme activity after 6 months storage (%) = (ΔA ′ / ΔAb) × 100

<Measurement of enzyme activity (titer) of cholesterol oxidase>
An enzyme aqueous solution was prepared by dissolving 16 mg of a measurement sample (lyophilized cholesterol oxidase) in 5 g of ion-exchanged water.
On the other hand, sodium cholate 78 mg (0.18 mmol, 60 mmol / L), nonylphenol ethylene oxide adduct {Union Carbide and Plastics, trade name: Triton X-100} 9 mg (0.3 wt%), aminoantipyrine 0. 85 mg (0.004 mmol, 1.4 mmol / L), phenol 5.9 mg (0.063 mmol, 21 mmol / L), peroxidase {Toyobo Co., Ltd.} 15 units (5 units / ml), cholesterol 1.0 mg (0 .0027 mmol, 0.9 mmol / L) and 3 mL of 50 mmol / L phosphate buffer (pH 7.0) were uniformly mixed to prepare a substrate solution. In addition, the density | concentration described in the parenthesis represents the density | concentration with respect to a phosphate buffer.

  Subsequently, 10 μL of the enzyme aqueous solution was added to 3 mL of the substrate solution to obtain a measurement solution. Immediately, the absorbance (B0) at 500 nm was measured at 30 ° C. with a spectrophotometer {Shimadzu Corporation, UV-2550} at 30 ° C., and after standing at 30 ° C. for 5 minutes, the absorbance at 30 ° C. again ( B5) was measured, and the difference (B5-B0) (ΔB) was calculated.

  On the other hand, the difference in absorbance (B5-B0) was similar to the above except that “measurement sample (lyophilized cholesterol oxidase)” was changed to “cholesterol oxidase {Wako Pure Chemical Industries, Ltd., titer 20000 units / mg}”. ) (ΔBb) was calculated, and “retention rate of enzyme activity (titer)” was calculated from the following equation.

Retention rate of enzyme activity (%) = (ΔB / ΔBb) × 100

  The freeze-dried cholesterol oxidase (R3) and (HR3) obtained in Examples and Comparative Examples were stored for 6 months in a sealed state at 25 ° C., and then the absorbance was measured in the same manner as described above, and the difference in absorbance (B5-B0 ) (ΔB ′) was calculated, and “retention rate of enzyme activity (titer) after 6 months storage” was calculated from the following formula.

Retention rate of enzyme activity after 6 months storage (%) = (ΔB ′ / ΔBb) × 100

When the freeze-drying protective agent of the present invention was used, the enzyme activity (titer) retention rate was remarkably high. Furthermore, even when sealed and stored at 25 ° C. for 6 months, a high retention rate was maintained.
As a main cause of a decrease in the titer of an enzyme or the like, a three-dimensional structure of the enzyme or the like may be distorted by freeze-drying. However, when freeze-dried using the freeze-drying protective agent of the present invention, it is considered that this distortion could be greatly suppressed.

The inventors of the present invention have reached the present invention as a result of studies to achieve the above object.
That is, the feature of the lyophilizing protective agent of the present invention is that the physiologically active substance is prevented from being altered when freeze-dried a mixture composed of the physiologically active substance and water, and is protected for maintaining the activity for a long period of time. An agent,
an amino acid ester in which the amino group at the α-position is modified or an amino acid amide in which the amino group at the α-position is modified , wherein the modified amino group is an acylamino group represented by the formula (1) or the formula (2) And the amino acid constituting the amino acid ester or amino acid amide is arginine, histidine, lysine, asparagine, or glutamine .

-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)

R ¹ is a hydrogen atom, methyl, ethyl, propyl, pentyl, cyclohexyl, heptyl, benzyl, heptadecenyl or henicosyl, R ² and R ³ are each independently a carbon atom having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom Represents a hydrogen group.

The production method of the physiologically active substance of the present invention is characterized by containing an amino acid ester in which the amino group at the α-position is modified or an amino acid amide in which the amino group at the α-position is modified , wherein the modified amino group has the formula (1 ) Or an imino group represented by formula (2), and the amino acid constituting the amino acid ester or amino acid amide is arginine, histidine, lysine, asparagine, or glutamine . In the presence,
The gist is that a bioactive substance is produced by freeze-drying a mixture composed of the bioactive substance and water.

-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)

R ¹ is a hydrogen atom, methyl, ethyl, propyl, pentyl, cyclohexyl, heptyl, benzyl, heptadecenyl or henicosyl, R ² and R ³ are each independently a carbon atom having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom Represents a hydrogen group.

< Reference Example 1 >
N-benzoyl-arginine ethyl ester {MP Bio Japan Co., Ltd.} was used as it was as a reference freeze-protecting agent (2).

<Example 2 >
After 50 mg of lysozyme {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg} was dissolved in 10 g of ion-exchanged water, 32.3 mg of the lyophilization protective agent (1) obtained in Example 1 ( 0.13 mmol) was added and uniformly mixed to prepare an aqueous solution for lyophilization. This aqueous solution was transferred to a glass bottle with a cap of 20 mL, and the glass bottle was immersed in liquid nitrogen for 1 minute to freeze, and then the glass bottle was transferred to a freeze dryer {DF-01H manufactured by ULVAC, Inc.] under reduced pressure (13 .3 Pa) and lyophilized at −40 ° C. for 40 hours to obtain a dried product. Subsequently, the dried product was stored for 3 days in a desiccator containing phosphorus pentoxide to obtain freeze-dried lysozyme (R1).

< Reference Example 2 >
Example 2 with the exception that “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” was changed to “protective agent for lyophilization (2) 40.5 mg (0.13 mmol)” Similarly, freeze-dried lysozyme (R2) was obtained.

<Example 3 >
Lyophilized cholesterol oxidase (R3) was obtained in the same manner as in Example 2 except that “lysozyme” was changed to “cholesterol oxidase {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg}”.

<Comparative Example 3>
The same procedure as in Example 2 was followed, except that “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” was changed to “protective agent for lyophilization (H1) 50 mg (0.13 mmol)”. A freeze-dried lysozyme (HR1) was obtained.

<Comparative example 4>
The same as Example 2 except that “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” was changed to “protective agent for lyophilization (H2) 23.8 mg (0.13 mmol)” Thus, lyophilized lysozyme (HR2) was obtained.

<Comparative Example 5>
“Lysozyme” was changed to “cholesterol oxidase {Wako Pure Chemical Industries, Ltd., titer 20,000 units / mg}”, and “protective agent for lyophilization (1) 32.3 mg (0.13 mmol)” Was changed to “50 mg (0.13 mmol) of the lyophilizing protective agent (H1)” in the same manner as in Example 2 to obtain lyophilized cholesterol oxidase (HR3).

Example lyophilized lysozyme obtained in Reference Example and Comparative Example (R1), (R2), (HR1) and (HR2), and Example, freeze-dried cholesterol oxidase obtained in Reference Examples and Comparative Examples (R3) and For (HR3), the enzyme activity (titer) immediately after lyophilization and the enzyme activity (titer) when stored for 6 months were measured and summarized in Table 1.

The freeze-dried lysozyme (R1), (R2), (HR1) and (HR2) ”obtained in Examples , Reference Examples and Comparative Examples was stored at 25 ° C. in a sealed state for 6 months, and the absorbance was as described above. Was measured, and the difference in absorbance (A5-A0) (ΔA ′) was calculated. From the following formula, “retention rate of enzyme activity (titer) after 6 months storage” was calculated.

The inventors of the present invention have reached the present invention as a result of studies to achieve the above object.
That is, the feature of the lyophilizing protective agent of the present invention is that the physiologically active substance is prevented from being altered when freeze-dried a mixture composed of the physiologically active substance and water, and is protected for maintaining the activity for a long period of time. An agent,
The physiologically active substance is an enzyme, a recombinant protein, an antibody, a peptide or an amino acid,
containing α-position amino acid ester in which an amino group has been modified, the modified amino group, an acylamino group represented by the formula (1), the amino acids constituting the amino acid ester is a gist point is arginine To do.

-NHCOR ¹ (1)

R ¹ represents methyl .

Features of the method of manufacturing the physiologically active substance of the present invention contains an amino acid ester wherein the amino group of the α-position is modified, the modified amino group, an acylamino group represented by the formula (1), the amino acid ester In the presence of a lyophilization protective agent, wherein the amino acid comprising
This is a production method for producing a physiologically active substance by freeze-drying a mixture composed of a physiologically active substance and water , and is summarized in that the physiologically active substance is an enzyme, a recombinant protein, an antibody, a peptide or an amino acid .

-NHCOR ¹ (1)

R ¹ represents methyl .

Claims (8)

A protective agent for preventing deterioration of a physiologically active substance when freeze-drying a mixture composed of the physiologically active substance and water,
A protective agent for lyophilization, comprising an amino acid ester in which the α-position amino group is modified or an amino acid amide in which the α-position amino group is modified. The protective agent according to claim 1, wherein the physiologically active substance is at least one selected from the group consisting of enzymes, recombinant proteins, antibodies, peptides, amino acids, nucleic acids, sugars, antibiotics and vitamins. The protective agent according to claim 1 or 2, wherein the modified amino group is an acylamino group represented by formula (1) or an imino group represented by formula (2).

-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)

R ¹ is a hydrocarbon group having 1 to 21 carbon atoms which may contain a hydrogen atom or a hetero atom, R ² and R ³ are each independently a hydrocarbon group having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom. Represents. The protective agent according to any one of claims 1 to 3, wherein the amino acid constituting the amino acid ester or amino acid amide is arginine, histidine, lysine, asparagine or glutamine. In the presence of a lyophilization protective agent containing an amino acid ester in which the amino group at the α-position is modified or an amino acid amide in which the amino group at the α-position is modified,
A method for producing a physiologically active substance, which comprises freeze-drying a mixture composed of a physiologically active substance and water to produce a physiologically active substance. The production method according to claim 5, wherein the physiologically active substance is at least one selected from the group consisting of enzymes, recombinant proteins, antibodies, peptides, amino acids, nucleic acids, sugars, antibiotics and vitamins. The production method according to claim 5 or 6, wherein the modified amino group is an acylamino group represented by the formula (1) or an imino group represented by the formula (2).

-NHCOR ¹ (1)
^{-N = C (R 2) R} 3 (2)

R ¹ is a hydrocarbon group having 1 to 21 carbon atoms which may contain a hydrogen atom or a hetero atom, R ² and R ³ are each independently a hydrocarbon group having 1 to 17 carbon atoms which may contain a hydrogen atom or a hetero atom. Represents. The production method according to any one of claims 5 to 7, wherein the amino acid constituting the amino acid ester or amino acid amide is arginine, histidine, lysine, asparagine or glutamine.