HK1139076B - Lyophilized preparation comprising influenza vaccine, and method for preparation thereof - Google Patents

Abstract

Disclosed is a lyophilized preparation improved in the stability of an influenza vaccine contained therein. A lyophilized preparation remarkably improved in the stability of an influenza vaccine contained therein can be prepared by lyophilizing an aqueous solution satisfying the following requirements (A) to (C): (A) the aqueous solution contains (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and an acid addition salt thereof; (B) the component (iii) is contained in an amount of 20 to 85% by weight relative to the total amount of the lyophilized preparation; and (C) the pH value of the aqueous solution aqueous solution is adjusted to pH 8 to 10 by controlling the ratio between arginine and the acid addition salt thereof which constitute the component(iii).

Description

Freeze-dried preparation containing influenza vaccine and preparation method thereof

Technical Field

The invention relates to a freeze-dried preparation containing an influenza vaccine. More particularly, the present invention relates to a lyophilized formulation for improving the stability of an influenza vaccine. Furthermore, the invention relates to a preparation method of the freeze-dried preparation.

Background

Influenza is a disease caused by infection of the respiratory system by influenza virus. Generally, after a latency period of about 1 to 2 days from the start of infection with influenza virus, fever of 38 ℃ or higher occurs, followed by general symptoms such as headache, general malaise, arthralgia, and myalgia, and respiratory symptoms such as cough and sputum, and these symptoms usually recover within 1 week. However, if elderly persons, infants, pregnant women, patients with chronic diseases of the respiratory system or circulatory system, patients with diabetes, patients with chronic renal insufficiency, or the like are infected with influenza, the influenza may cause serious complications such as pneumonia, bronchitis, or the like, and may even cause death in some cases. In addition, influenza causes serious health damage, has strong infectivity, and intensively infects many people in a short time, thereby causing huge social and economic losses.

In order to prevent health damage caused by influenza infection and reduce social and economic losses, administration of influenza vaccines is the most effective method. Liquid preparations for injections and frozen preparations for nasal drops are known as influenza vaccine preparations at present, but no dry preparations having excellent stability have been marketed (see, for example, non-patent document 1).

When influenza vaccines are distributed as liquid preparations, in order to prevent inactivation of influenza vaccines, they must be kept at a low temperature during distribution and storage, i.e., a refrigeration step is indispensable. When the influenza vaccine is distributed as a frozen preparation, it is also required to keep the vaccine in a frozen state during distribution and storage in order to stably maintain the preparation. Since influenza vaccines formulated as liquid preparations or frozen preparations as described above require high temperature management during transportation and storage (see non-patent document 2), it is difficult to distribute influenza vaccines in areas with insufficient power supply or areas without low-temperature transportation means while maintaining their activity.

In order to overcome the above-mentioned disadvantages of the liquid preparation or the frozen preparation, it is effective to circulate the influenza vaccine in the form of a dry preparation. Therefore, in recent years, techniques for formulating lyophilized preparations of influenza vaccines by adding lactose or trehalose have been disclosed, but the stability thereof has not been shown (see non-patent document 3).

Like other vaccines, influenza vaccines are highly sensitive to heat, and are known to be destroyed in activity at high temperatures or below freezing point (see non-patent document 2). Even if only the drying treatment or the lyophilization treatment is performed, there is a problem that the activity of the influenza vaccine decreases with time at the time of preparation or storage. No technique for overcoming the above problems has been found, and no example of successful application of a dry preparation of an influenza vaccine has been reported so far.

Non-patent document 1: deborah A. Buonagreo et al., Vaccine, Vol.24(2006), 2151-.

Non-patent document 2: stephen E.Zweig, Vaccine, Vol.24(2006), 5977-.

Non-patent document 3: robert j. garmeise et al, AAPS PharmSciTech 2006; 7(1) Article 19, E1-E7.

Disclosure of Invention

The present invention has been made in view of the above-mentioned background art, and an object of the present invention is to provide a lyophilized preparation having improved stability of an influenza vaccine.

As a result of intensive studies to solve the above problems, the present inventors have found that a lyophilized preparation having significantly improved stability of an influenza vaccine can be obtained by lyophilizing an aqueous solution satisfying the following conditions (a) to (C) when preparing a lyophilized preparation containing an influenza vaccine:

(A) comprising (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and acid addition salts thereof;

(B) (iv) blending the component (iii) in an amount of 20 to 85 wt% based on the total amount of the obtained lyophilized preparation; and

(C) adjusting the pH to 8-10 by controlling the ratio of arginine and its acid addition salt constituting the above component (iii).

Based on the above findings, further, the present invention has been completed by further repeating improvements.

Namely, the present invention provides a lyophilized preparation containing an influenza vaccine as described below.

Item 1, a lyophilized formulation comprising an influenza vaccine, characterized in that,

the lyophilized preparation is obtained by lyophilizing an aqueous solution containing (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and an acid addition salt thereof,

(iii) the ratio of arginine and an acid addition salt thereof is 20 to 85 wt% relative to the total amount of the lyophilized preparation, and the ratio of arginine and an acid addition salt thereof is within a range such that the liquid property of the aqueous solution is pH8 to 10.

The lyophilized formulation of item 2 or 1, wherein (i) the influenza vaccine is a desalted vaccine.

The lyophilized formulation of item 3 or 1, wherein (ii) the hydrophobic amino acid is phenylalanine, or a combination of phenylalanine and at least one of valine, leucine, and isoleucine.

The lyophilized preparation according to item 4 or 1, wherein the hydrophobic amino acid (ii) is contained in a proportion of 14 to 75 wt% with respect to the total amount of the lyophilized preparation.

The lyophilized preparation of item 5 or 1, wherein (iii) arginine and an acid addition salt thereof is arginine and a hydrochloride thereof.

The lyophilized preparation of item 6 or 1, wherein an acid addition salt of arginine is contained in an amount of 1 to 20 parts by weight based on 1 part by weight of arginine.

The lyophilized preparation according to item 7 or 1, wherein the total amount of the components (i) to (iii) is 80 to 100 wt% based on the total amount of the lyophilized preparation.

The lyophilized preparation according to item 8 or 1, which is an injection that is soluble in water.

The lyophilized preparation according to item 9 or 1, which is a pharmaceutical preparation for transpulmonary administration.

The lyophilized preparation according to item 10 or 1, which is a pharmaceutical preparation for nasal administration.

Further, the present invention provides a method for producing a lyophilized preparation containing an influenza vaccine as described below.

Item 11, a method of preparing a lyophilized formulation comprising an influenza vaccine, the method comprising the steps of:

step 1: preparing an aqueous solution containing (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and an acid addition salt thereof, wherein the content of the (i) influenza vaccine in the aqueous solution is an amount corresponding to 20 to 85% by weight relative to the total amount of the prepared lyophilized preparation, and the pH of the aqueous solution is 8 to 10; and

step 2: and (4) freeze-drying the aqueous solution prepared in the step.

The production method according to item 12 or 11, wherein the (i) influenza vaccine is a desalted vaccine.

The production method of item 13 or 11, wherein (ii) the hydrophobic amino acid is phenylalanine, or a combination of phenylalanine and at least one of valine, leucine, and isoleucine.

The production method according to item 14 or 11, wherein the content of the (ii) hydrophobic amino acid in the aqueous solution used in the step 1 is an amount corresponding to 14 to 75 wt% based on the total amount of the prepared lyophilized preparation.

The process according to item 15 or 11, wherein the arginine and its acid addition salt (iii) are arginine and its hydrochloride.

The production method according to item 16 or 11, wherein the aqueous solution used in the step 1 contains an acid addition salt of arginine in a proportion of 1 to 20 parts by weight relative to 1 part by weight of arginine.

The production method according to item 17 or 11, wherein the total content of the components (i) to (iii) in the aqueous solution used in the step 1 is an amount corresponding to 80 to 100% by weight based on the total amount of the prepared lyophilized preparation.

Since the lyophilized preparation of the present invention can stably maintain the activity of an influenza vaccine during storage, it can be distributed and stored more easily than conventional preparations.

In addition, the lyophilized preparation of the present invention can be used as an injection by dissolving in water for injection at the time of use, or can be used as a pulmonary preparation or nasal drops directly, and therefore, the lyophilized preparation has an advantage that it can be applied to various administration forms.

Detailed Description

In the present specification, the pH is a value measured at 25 ℃.

The lyophilized preparation of the present invention is characterized by containing (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and an acid addition salt thereof, and further having a specific blending ratio of the component (iii), and the ratio of arginine and an acid addition salt thereof constituting the component (iii) satisfying a specific range.

The influenza vaccine (hereinafter, also referred to as component (i)) to be incorporated into the lyophilized preparation of the present invention is preferably an influenza HA (hemagglutinin) vaccine, which is one of subunit vaccines and split vaccines prepared by purifying virus particles that have proliferated in developing eggs. However, it may be an influenza vaccine derived from tissue culture. Further, the vaccine may be a live vaccine for attenuating influenza virus, a component vaccine (component vaccine) detoxified while retaining immunogenicity, or a whole particle vaccine (whole viral particles vaccine) for inactivating all virus particles. The influenza vaccine used in the present invention may be prepared from influenza viruses of either or both of the a-type strain and the B-type strain, and preferably prepared from influenza viruses of both the a-type strain and the B-type strain.

The influenza vaccine used in the present invention may be a vaccine prepared by a known preparation method, or a commercially available vaccine.

When the prepared or commercially available influenza vaccine is in the form of a solution or powder and contains salts such as a buffer and a preservative, it is preferable to desalt the influenza vaccine by desalting the influenza vaccine in advance. By using for the desalting treatment as described above, the stability of the influenza vaccine in the lyophilized preparation of the present invention can be more effectively improved. The method for desalting the influenza vaccine is not particularly limited, and examples thereof include ultrafiltration, precipitation, ion exchange, and dialysis. In addition, from the viewpoint of suppressing the reduction in influenza vaccine activity during desalting treatment, it is preferable to desalt using an aqueous solution adjusted to a pH of 8 to 10, preferably 8 to 9, with an alkaline substance such as sodium hydroxide, potassium hydroxide, or arginine. Particularly preferred is a solution having the same composition as that used for lyophilization in the preparation of the lyophilized preparation of the present invention, except that it does not contain an influenza vaccine.

The blending ratio of the component (i) in the lyophilized preparation of the present invention is appropriately set according to the method of application of the lyophilized preparation, and for example, the blending ratio of the component (i) is in the range of 0.3 to 60% by weight, preferably 1 to 50% by weight, more preferably 2 to 40% by weight, further preferably 3 to 30% by weight, and particularly preferably 3 to 20% by weight with respect to the total amount of the lyophilized preparation.

Specific examples of the hydrophobic amino acid (hereinafter, sometimes referred to simply as component (ii)) contained in the lyophilized preparation of the present invention include amino acids constituting proteins such as valine, leucine, isoleucine, and phenylalanine. In the present invention, the hydrophobic amino acid may be used singly or in combination of two or more. In the present invention, as the hydrophobic amino acid, it is preferable to use phenylalanine alone or a combination of phenylalanine and at least one of valine, leucine, and isoleucine.

The blending ratio of the component (ii) in the lyophilized preparation of the present invention may be appropriately set according to the kind of the component (ii), the method of applying the lyophilized preparation, and the like, and for example, the blending ratio of the component (ii) is in the range of 14 to 75% by weight, preferably 20 to 75% by weight, more preferably 20 to 70% by weight, still more preferably 25 to 65% by weight, and particularly preferably 30 to 60% by weight with respect to the total amount of the lyophilized preparation.

The lyophilized preparation of the present invention further contains arginine and an acid addition salt thereof (hereinafter, sometimes referred to as component (iii)). That is, arginine and its acid addition salt must be combined and compounded in the lyophilized preparation of the present invention.

The acid addition salt of arginine used in the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include inorganic acid addition salts such as hydrochloride, nitrate, and sulfate; and organic acid addition salts such as acetate. In the present invention, arginine hydrochloride is preferably used as an acid addition salt of arginine. In the present invention, the acid addition salt of arginine may be used singly or in combination of two or more.

In the lyophilized preparation of the present invention, the component (iii) is contained in a blending ratio of 20 to 85 wt% based on the total amount of the component (iii) (i.e., the total amount of arginine and its acid addition salt). By containing the component (iii) in the above blending ratio, a decrease in activity of the influenza vaccine during storage can be suppressed, and a preparation having excellent stability can be provided. The amount of the component (iii) is preferably 20 to 80 wt%, more preferably 20 to 75 wt%, still more preferably 25 to 70 wt%, and particularly preferably 30 to 65 wt% based on the total amount of the lyophilized preparation.

The component (iii) satisfies the above blending ratio, and the ratio of arginine and its acid addition salt in the component (iii) is set so that the pH of the aqueous solution used for lyophilization is in the range of 8 to 10. That is, since arginine is basic and the acid addition salt of arginine is acidic, the liquid properties of the aqueous solution for lyophilization are adjusted to be within the above range by adjusting the ratio of arginine to the acid addition salt of arginine within the range satisfying the above mixing ratio of the component (iii). The use of arginine and its acid addition salt as described above adjusts the liquid properties of the aqueous solution for lyophilization to the above-described range, so that the storage stability of the influenza vaccine can be improved. The ratio of arginine and the acid addition salt thereof is preferably set so that the pH of the aqueous solution used for lyophilization is in the range of 8 to 9. Here, the aqueous solution used for lyophilization refers to an aqueous solution used for lyophilization at the time of preparing a lyophilized preparation, that is, an aqueous solution to which all the components contained in the lyophilized preparation of the present invention are added to purified water. The present invention is not particularly limited, and for example, the aqueous solution used for lyophilization is set to contain 0.2 to 20mg, preferably 0.4 to 20mg, more preferably 0.4 to 15mg, still more preferably 0.6 to 12.5mg, and particularly preferably 1 to 10mg of the total weight (total amount of all components) of the lyophilized preparation per 1mL of the aqueous solution.

The ratio of arginine and its acid addition salt in the component (iii) is not particularly limited as long as the liquid property of the aqueous solution used for lyophilization is within the above range, and may be appropriately set according to the kind of the acid addition salt, the kind of the component (ii) to be blended, and the like. Specifically, the ratio of the acid addition salt of arginine to 1 part by weight of arginine is 1 to 20 parts by weight, preferably 1 to 15 parts by weight, more preferably 1.25 to 12.5 parts by weight, still more preferably 1.5 to 12.5 parts by weight, and particularly preferably 2 to 10 parts by weight.

The lyophilized preparation of the present invention may further contain a hydrophilic amino acid other than arginine (hereinafter, sometimes referred to as component (iv)) within a range not affecting the effect of the present invention by adding the above components (i) to (iii). The hydrophilic amino acid may be any amino acid as long as it has a hydrophilic side chain, and may be any amino acid, regardless of whether it is an amino acid constituting a protein. Specific examples of the hydrophilic amino acid include basic amino acids such as lysine and histidine; neutral hydroxy amino acids such as serine and threonine; acidic amino acids such as aspartic acid and glutamic acid; amide amino acids such as asparagine and glutamine; other amino acids such as glycine, alanine, cysteine, and tyrosine. Among the hydrophilic amino acids, preferred are alanine and glycine. The hydrophilic amino acids may be used singly or in combination of two or more.

The proportion of the hydrophilic amino acid to be blended in the lyophilized preparation of the present invention is not particularly limited, but for example, the proportion of the hydrophilic amino acid to be blended is 5 to 50% by weight, preferably 5 to 40% by weight, more preferably 5 to 30% by weight, still more preferably 5 to 25% by weight, and particularly preferably 10 to 25% by weight, based on the total amount of the lyophilized preparation.

In the lyophilized preparation of the present invention, in order to make the stabilization effect on influenza vaccines more remarkable, the total amount of the components (i) to (iii) in the case where the component (iv) is not contained or the total amount of the components (i) to (iv) in the case where the component (iv) is contained is preferably 80 to 100% by weight, more preferably 85 to 100% by weight, even more preferably 90 to 100% by weight, still more preferably 95 to 100% by weight, and particularly preferably 100% by weight, based on the total amount of the lyophilized preparation.

The following components may be contained in the lyophilized preparation of the present invention as long as the effects of the present invention are not affected: monosaccharides such as glucose; disaccharides such as sucrose, maltose, lactose, and trehalose; sugar alcohols such as mannitol; oligosaccharides such as cyclodextrin; polysaccharides such as dextran 40 and pullulan; polyhydric alcohols such as polyethylene glycol; fatty acid salts such as sodium caprate; human serum albumin; an inorganic salt; gelatin; a surfactant; buffers, and the like. Among the surfactants, any surfactant may be used as long as it is generally used in medicines, and any of anionic surfactants, cationic surfactants and nonionic surfactants may be used. The lyophilized preparation of the present invention may further contain an adjuvant if necessary.

The lyophilized preparation of the present invention is prepared by adding predetermined amounts of components (i) to (iii) to purified water, and further adding component (iv) and other additional components as necessary, and subjecting the mixture to lyophilization. Specifically, the lyophilized preparation of the present invention can be prepared by the following steps 1 and 2,

step 1: preparing an aqueous solution comprising (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and acid addition salts thereof; (iii) arginine and an acid addition salt thereof are contained in the aqueous solution in an amount corresponding to 20 to 85% by weight relative to the total amount of the prepared lyophilized preparation, and the pH of the aqueous solution is 8 to 10.

Step 2: the aqueous solution prepared in the above step 1 is lyophilized, thereby obtaining a lyophilized preparation.

The aqueous solution prepared in the above step 1 is used for lyophilization treatment, whereby a lyophilized preparation as a target product can be obtained. Therefore, the aqueous solution for lyophilization is prepared in the above-described step 1 such that the ratio of each component other than the component removed by lyophilization among the components contained in the aqueous solution is the same as the ratio of each component contained in the lyophilized preparation as a preparation object. In addition, the liquid properties of the aqueous solution prepared in step 1 above were adjusted by adjusting the ratio of arginine to arginine acid addition salt, as described above.

The lyophilized preparation of the present invention may be a dried solid (lyophilized cake) obtained by lyophilization, or may be a product obtained by powdering the dried solid.

The mode of administration of the lyophilized preparation of the present invention is not particularly limited. For example, the drug can be subcutaneously administered as an injection by dissolving the drug in a diluent (solution for injection) at the time of use. The lyophilized preparation of the present invention may be dissolved in a diluent to prepare a liquid preparation, and administered as a pharmaceutical preparation for transpulmonary administration or nasal drip administration via the lung or in the nasal cavity. Further, when the lyophilized preparation of the present invention is made into a fine powder by applying air impact to a pharmaceutical preparation for transpulmonary administration or nasal drop administration, the lyophilized preparation may be directly administered transpulmonary or nasally by using a dry powder inhaler capable of applying air impact.

The method of administering the lyophilized preparation of the present invention may be appropriately set according to the age of the subject patient, and the lyophilized preparation may be administered, for example, 1 time or 2 times at about 1 to 4 weeks in an amount corresponding to 3 to 300 μ g of influenza vaccine.

When the lyophilized preparation of the present invention is directly administered, not dissolved in a diluent at the time of use, it is preferably contained in a container in a single dose (1 dose) from the viewpoint of ease of use. In addition, when the lyophilized preparation of the present invention is dissolved in a diluent at the time of administration, it may be contained in a container in a single dose, or may be contained in a single container in combination of doses for a plurality of times.

The lyophilized preparation of the present invention has an advantage that the amount of the lyophilized preparation contained in one container is small because the amount of the excipient to be blended is reduced as compared with a preparation containing an influenza vaccine used as a conventional injection. In the present invention, the amount of the lyophilized preparation contained in one container is not particularly limited, but for example, the amount is 0.1 to 10mg, preferably 0.15 to 8mg, more preferably 0.2 to 6mg, still more preferably 0.25 to 5mg, and particularly preferably 0.3 to 5 mg.

Examples

The present invention will be described more specifically with reference to examples and the like, but the present invention is not limited to these examples.

Reference example 1 discusses desalting conditions of influenza vaccine

Conditions for desalting treatment of influenza HA vaccine solution were investigated. Specifically, as an influenza HA vaccine solution, an influenza HA vaccine "Bio-research" (trade name, manufactured by Denka Ltd., Lot No.308-A) was used, and an appropriate amount of the above solution was added to 4mL of Vivasspinoconcenter (manufactured by Sartorius) and centrifuged at 3000rpm for 30 minutes. Next, the solutions having the compositions shown in Table 1 were added to Vivaspin concentrators, respectively, and centrifuged at 3000rpm for 30 minutes. The above operation was repeated 3 times in total.

The activity (HA valency) of the desalted influenza HA vaccine was determined as follows.

< Activity assay of influenza HA vaccine >

1. Preparation of 5 vol% Chicken erythrocyte suspension

The preserved chicken blood (manufactured by Biotest research, Japan) was added to a test tube, centrifuged at 900g for 5 minutes, and then the supernatant and the leukocyte layer were removed. Then, a diluent (1/200mol/L phosphate-buffered sodium chloride solution (pH7.2)) having the following composition was added to the red blood cells in the test tube, and after stirring, centrifugation was performed to remove the supernatant. The above operation was repeated 3 times. The erythrocytes in the test tube were aspirated by a pipette, and mixed in a container containing a diluent to prepare a 5 vol% suspension of chicken erythrocytes.

Composition of the dilution liquid

NaCl 8.5g

Na₂HPO₄·12H₂O 1.425g

KH₂PO₄ 0.135g

1000mL of refined water

2. Determination of HA valency

50 μ L of influenza HA vaccine solutions of various concentrations were applied to a microplate, and then 50 μ L of a 0.5 vol% chicken erythrocyte suspension (two-stage dilution method) was added thereto, and the microplate was sufficiently mixed and left to stand at room temperature for 1 hour, and then the final dilution factor of the influenza HA vaccine in which erythrocytes were completely aggregated was taken as the HA valence.

The results are shown in Table 1. From the results, it was confirmed that the influenza HA vaccine was reduced in activity when desalted in a solution having a pH of 7 or less, but stably maintained in activity when desalted in a solution having a pH of 8 to 9.

[ Table 1]

	Composition and solution Properties of the solution	Residual activity
			Condition 1	0.5mg of Phe was added to 0.5mL of purified water; adjusting the pH to 3.0 with NaOH	12.5％
Condition 2	0.5mg of Phe was added to 0.5mL of purified water; pH was adjusted to 4.0 with NaOH	6.3％
			Condition 3	0.5mg of Phe was added to 0.5mL of purified water; pH was adjusted to 5.7 with NaOH	12.5％
Condition 4	0.5mg of Phe was added to 0.5mL of purified water; adjusting the pH to 7.0 with NaOH	50％
			Condition 5	Phe was not added to 0.5mL of purified water; adjusting the pH to 7.0 with NaOH	50％
Condition 6	0.5mgPhe was added to 0.5mL of purified water; adjusting the pH to 8.0 with NaOH	100％
			Condition 7	Phe was not added to 0.5mL of purified water; adjusting the pH to 8.0 with NaOH	100％
Condition 8	0.5mg of Phe was added to 0.5mL of purified water; adjusting the pH to 9.0 with NaOH	100％

In table # the residual activity is the ratio (%) of the HA valence after desalting treatment to the HA valence before desalting treatment.

Test example 1 evaluation of stability of influenza vaccine

As an influenza HA vaccine solution, 1mL (equivalent to 90. mu.g of influenza HA vaccine) of the influenza HA vaccine "Bio-research" (trade name, developed by Denka, Lot No.308-A) was added to 4mL of Vivaspin concentrator (manufactured by Sartorius), and centrifuged at 3000rpm for 30 minutes. Then, the solutions having the compositions shown in Table 2 were added to Vivasperincentators, respectively, and centrifuged at 3000rpm for 30 minutes. The above operation was repeated 3 times in total. The final solution to be concentrated and desalted (buffer exchanged) was a solution having the composition shown in Table 2, and the volume was adjusted to the initial volume (1 mL). 0.5mL of the resulting solution was filled in a glass vial and lyophilized to obtain a lyophilized product.

[ Table 2]

	Composition and liquid Properties of solutions used in examples and comparative examples
		Example 1	0.5mg Phe, 0.5mg Arg-HCl, and 0.075mg Arg were added to 0.5mL purified water; pH8
Example 2	0.5mg of Phe, 0.3mg of Arg-HCl and 0.050mg of Arg were added to 0.5mL of purified water; pH8
		Example 3	0.5mg of Phe, 0.2mg of Ile, 0.3mg of Arg-HCl and 0.057mg of Arg were added to 0.5mL of purified water; pH8
Example 4	0.5mg of Phe, 0.2mg of Val, 0.3mg of Arg-HCl and 0.055mg of Arg were added to 0.5mL of purified water; pH8
		Example 5	0.5mg of Phe, 0.2mg of Leu, 0.3mg of Arg-HCl and 0.052mg of Arg were added to 0.5mL of purified water; pH8
Example 6	0.5mg of Phe, 0.2mg of Ala, 0.3mg of Arg-HCl and 0.052mg of Arg were added to 0.5mL of purified water; pH8
		Example 7	0.5mg of Phe, 0.2mg of Gly, 0.3mg of Arg-HCl and 0.052mg of Arg were added to 0.5mL of purified water; pH8
Comparative example 1	0.5mg Phe and a proper amount of NaOH are added into 0.5mL of refined water; pH8
		Comparative example 2	0.5mg of Phe and 0.060mg of Arg were added to 0.5mL of purified water; pH8
Comparative example 3	0.5mg of Arg-HCl and 0.030mg of Arg were added to 0.5mL of purified water; pH8
		Comparative example 4	0.5mg of Phe, 0.5mg of His and 0.030mg of Arg were added to 0.5mL of purified water; pH8

# the solutions used in examples 1 to 7 and comparative examples 2 to 4 were liquid-adjusted to pH8 by adding predetermined amounts of the respective compounding ingredients without using any other pH adjusting agent. After a predetermined amount of the compounding ingredients was added to the solution used in comparative example 1, NaOH was added to make the solution liquid-like pH 8.

The resulting lyophilized preparation was sealed in a glass vial (visual), and stored in this state at 25 ℃/60% RH for 4 weeks in the dark. The HA value of the influenza HA vaccine in the lyophilized preparation was measured in the same manner as in reference example 1 immediately after the preparation and after 1, 2, 3 and 4 weeks of storage.

The results are shown in Table 3. Even when a phenylalanine-containing solution adjusted to pH8 with only sodium hydroxide or arginine alone was used, the reduction in activity of the influenza HA vaccine upon storage could not be suppressed (comparative examples 1 and 2). In addition, even when a solution in which arginine and arginine hydrochloride were added to adjust the pH to 8 without adding phenylalanine as a hydrophobic amino acid was used, the activity reduction of the influenza HA vaccine could not be suppressed (comparative example 3). Furthermore, even when a solution in which phenylalanine, histidine as a basic amino acid, and arginine are combined and the pH is adjusted to 8 is used, the activity reduction of the influenza HA vaccine cannot be suppressed.

On the other hand, it was confirmed that the activity of the influenza HA vaccine in the finally prepared lyophilized preparation could be stably maintained by satisfying the following conditions (1) to (3) for the solution used to prepare the lyophilized preparation.

(1) Complexing hydrophobic amino acids, arginine and arginine acid addition salts; (2) setting the ratio of arginine to arginine acid addition salt such that the solution has a pH of 8; and (3) setting the total amount of arginine and arginine acid addition salt to be a predetermined concentration or more relative to the lyophilized preparation.

[ Table 3]

The residual activity in table # is the ratio (%) of the HA value of the lyophilized preparation after storage to the HA value of the lyophilized preparation immediately after preparation.

The unit% in the composition column of the lyophilized preparation in the table is weight%.

In the table, "-" indicates no measurement.

Claims (17)

1. A lyophilized preparation comprising an influenza vaccine,

the lyophilized preparation is obtained by lyophilizing an aqueous solution containing (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and an acid addition salt thereof,

wherein (ii) the hydrophobic amino acid is at least one selected from phenylalanine, valine, leucine and isoleucine, (iii) the arginine and the acid addition salt thereof are arginine and at least one arginine addition salt selected from hydrochloride, nitrate, sulfate and acetate thereof,

(iii) the blending ratio of arginine and an acid addition salt thereof is 20 to 85 wt% relative to the total amount of the lyophilized preparation, and the ratio of arginine and the acid addition salt thereof is within a range that the liquid property of the aqueous solution is pH8 to 10.

2. The lyophilized formulation of claim 1, wherein (i) influenza vaccine is a desalted vaccine.

3. The lyophilized formulation of claim 1, wherein (ii) the hydrophobic amino acid is phenylalanine, or a combination of phenylalanine and at least one of valine, leucine, and isoleucine.

4. The lyophilized formulation according to claim 1, wherein the (ii) hydrophobic amino acid is contained in a proportion of 14 to 75 wt% with respect to the total amount of the lyophilized formulation.

5. The lyophilized formulation of claim 1, wherein (iii) arginine and its acid addition salt is arginine and its hydrochloride salt.

6. The lyophilized formulation according to claim 1, wherein the acid addition salt of arginine is contained in a ratio of 1 to 20 parts by weight relative to 1 part by weight of arginine.

7. The lyophilized formulation according to claim 1, wherein the total amount of the components (i) to (iii) is 80 to 100 wt% with respect to the total amount of the lyophilized formulation.

8. The lyophilized formulation of claim 1, which is an injection in a dissolution type.

9. The lyophilized preparation according to claim 1, which is a pharmaceutical preparation for transpulmonary administration.

10. The lyophilized formulation of claim 1, which is a pharmaceutical formulation for nasal administration.

11. A method of preparing a lyophilized formulation comprising an influenza vaccine, the method comprising the steps of:

step 1: formulating an aqueous solution comprising (i) an influenza vaccine, (ii) a hydrophobic amino acid, and (iii) arginine and acid addition salts thereof,

wherein (ii) the hydrophobic amino acid is at least one selected from phenylalanine, valine, leucine and isoleucine, (iii) the arginine and the acid addition salt thereof are arginine and at least one arginine addition salt selected from hydrochloride, nitrate, sulfate and acetate thereof,

(iii) the content of arginine and an acid addition salt thereof in the aqueous solution is an amount equivalent to 20 to 85% by weight relative to the total amount of the prepared lyophilized preparation, and the pH of the aqueous solution is 8 to 10; and

step 2: the aqueous solution prepared in the above step was lyophilized.

12. The production method according to claim 11, wherein the (i) influenza vaccine is a desalted vaccine.

13. The production method according to claim 11, wherein (ii) the hydrophobic amino acid is phenylalanine, or a combination of phenylalanine and at least one of valine, leucine, and isoleucine.

14. The preparation method according to claim 11, wherein the content of the (ii) hydrophobic amino acid in the aqueous solution used in the 1 st step is an amount corresponding to 14 to 75% by weight with respect to the total amount of the prepared lyophilized preparation.

15. The process according to claim 11, wherein (iii) arginine and its acid addition salts are arginine and its hydrochloride.

16. The method according to claim 11, wherein the aqueous solution used in step 1 contains 1 to 20 parts by weight of an acid addition salt of arginine per 1 part by weight of arginine.

17. The production method according to claim 11, wherein the total content of the components (i) to (iii) in the aqueous solution used in the 1 st step is an amount corresponding to 80 to 100% by weight relative to the total amount of the prepared lyophilized preparation.