KR100260634B1 - Process for the preparation of hapatitis b vaccine - Google Patents

Abstract

PURPOSE: A manufacturing process for hepatitis B vaccine is provided, which produces hepatitis B vaccine keeping high antigenicity for a long time. CONSTITUTION: The manufacturing process for hepatitis B vaccine comprises the steps of: diluting recombinant hepatitis B antigen with phosphate buffer(pH 7.5); adding hydroxy propyl cellulose(HPC), and dissolving; controlling concentration to contain fixed amount of protein, and adding a stabilizer(such as natural protein, amino acid, and polysaccharide to prevent deformation of protein), an aluminum gel(such as hydroxy aluminum gel and aluminum phosphate gel to increase immunity), and/or a salt to make the solution to be isotonic; spray-drying by a spray-dryer(the velocity of sprayed nitrogen is 600L/minute, the temperature of influx air is 70deg.C) to get spray-dried product; and dissolving the spray-dried product in the same buffer completely, and measuring antigenic value using test kit.

Description

Manufacturing method of hepatitis B vaccine

FIG. 1 is a diagram showing the particle size and distribution of the spray-dried hepatitis B preparation in a buffer solution and the liquid formulation before spray drying by light scattering particle size analyzer (Brookhaven BI-200SM).

The present invention relates to a method for preparing a hepatitis B vaccine formulation. More specifically, the present invention relates to a method for preparing a hepatitis B vaccine preparation characterized by spray-drying a liquid hepatitis B antigen and a hepatitis B vaccine preparation prepared by the above method.

Hepatitis B caused by the hepatitis B virus poses a serious problem, both immunologically and clinically, but preventive methods have been studied mainly because no effective treatment has been found. It is well known that it is the best way. A suitable prophylaxis is to administer the hepatitis B vaccine to a person who may be infected by the hepatitis B virus. Indeed, this vaccine prevented infant infection rates in Japan from 0.26% in 1985 to 0.04% in 92 (Viral Hapatitis and Liver Disease, 1994, 530-532).

Hepatitis carriers are spread around the world (Centers for Disease Control, 1991), and 23% of the total population, especially in South Korea, and more than 10% of the total population in Southeast Asia and Africa are known to be hepatitis carriers. (Southeast Asean J. Trop. Med. Public Health 23 (1), 17-21 (1992)). Therefore, the hepatitis B vaccine needs to be used worldwide.

Hepatitis B vaccine currently on the market is a liquid formulation, and as the weight of the antigen gradually decreases during the storage period, low-temperature storage is essential, and the antigenicity and immunity of the vaccine during long-distance transportation are a problem. The modification of proteins, including vaccines, is caused by the adsorption of other substances on the surface of the protein in storage, or by the aggregation or precipitation of proteins (Pharmaceutical Research 6 (7), 903-917). This drawback is very problematic in underdeveloped countries where there is a shortage of medical staff and facilities. Given that hepatitis B is particularly prevalent in underdeveloped countries, a dry formulation form that is stable for long term storage and unaffected by temperature changes is preferred.

In Korean Patent Publication No. 93-2263, the liquid formulation is dried and solidified to maintain the titer of the antigen. That is, a dried formulation was prepared by lyophilizing the hepatitis B vaccine with aluminum gel and a stabilizer. However, a total of more than 42 hours is required for the freeze-drying time, and there is a problem that not only multi-step temperature control is necessary to maintain the antigen value of the hepatitis B vaccine during lyophilization, but that the addition of a stabilizer is necessary and inadequate for large-scale scale. .

Compared with lyophilization, spray drying is a method of drying the liquid to obtain the form of powder. It is possible to speed up the process by continuous liquid supply and has a low cost, and has a cooling effect on the surface of the liquid by evaporation heat during evaporation. It is known to be an efficient drying method (Phamaceutical research 11 (1), 12-20 (1994)). Using this spraying method, hormones in the form of desiccant (Macro Mumenthaler, Pharmaceutical Research 11 (1), 12-20 (1994)) or enzymes (J. Broadhead, J. Pharm, Pharmacol. 46, 485-467 (1994) ) To date, the vaccine has been in the form of a desiccant made by lyophilization of an antigen, a stabilizer, and an additive, and no vaccine has been reported in the form of a desiccant by spray-drying a liquid antigen. Therefore, there is a need for a method that can maintain the antigen value of the vaccine while taking advantage of spray drying.

The present inventors have made efforts to develop a method for producing a hepatitis B vaccine by spray drying, which has high productivity, simplicity of process, and at the same time maintains the antigenic value of the vaccine, focusing on the advantages of the spray drying method. It became.

An object of the present invention is to provide a spray drying method of the hepatitis B vaccine.

Another object of the present invention is to provide a dry preparation of the hepatitis B vaccine prepared by the above method.

The method for producing a hepatitis B vaccine of the present invention for achieving the above object is characterized by spray-drying the hepatitis B vaccine.

Hepatitis B vaccine of the present invention for achieving the above another object is characterized in that prepared by spray drying.

According to the present invention as described above, the method of spray drying of hepatitis B vaccine having high productivity, simplicity of process and at the same time can maintain the antigenic value of the vaccine, and the antigenicity of the vaccine prepared using the same, type B is maintained for a long time Desiccant preparations of the hepatitis vaccine are provided.

Hereinafter, the present invention will be described in more detail.

Hepatitis B vaccine desiccant of the present invention has a very stable property that the antigenicity of the vaccine is maintained for a long time, it can be prepared by spray drying the liquid formulation.

In the present invention, before entering the spray drying process, the hepatitis B antigen is adjusted to include protein components in the range of 100 µg / ml to 10 µg / ml, and then stabilizers, aluminum gels and salts are added thereto depending on the conditions.

Stabilizers prevent the transformation of proteins by heat during the hot spray process. Available as stabilizers are natural proteins, amino acids or polysaccharides. Suitable proteins include gelatin, albumin, dextran and the like, and are preferably used in amounts of 0.01-2% (W / V). As amino acids, glycine, alanine, glutamic acid, arginine, lysine or salts thereof can be used, and suitable sugars include glucose, xylose, galactose, fructose, lactose, maltose, saccharose, hydroxy propyl cellulose, and hydroxy. Oxy methyl cellulose, carboxy methyl cellulose, mannitol, or sorbitol. The amino acids and sugars are preferably used at a concentration of 0.1-10% ((W / V)).

Aluminum gel is an adjuvant for increasing the immunity of hepatitis vaccine, and may be mixed after spray drying in addition to the pre-spray vaccine solution, and aluminum hydroxide gel or aluminum phosphate gel may be used.

Salts are those which are added to make the formulation solution physiologically isotonic in the use of spray dried vaccine formulations, which may be used after spray drying or spray drying, such salts include sodium chloride, sodium phosphate, or potassium phosphate, Preference is given to adding in concentrations of 0.1-10 mM.

The vaccine solution mixed with the above additives is put into a container, and then prepared by spray drying into particles having a diameter of 0.1-10 μm. Spray drying is usually performed under the following conditions. That is, the hepatitis B antigen mixture in solution is supplied to the spray dryer at a rate of 1-10 ml / min and sprayed at an air flow rate of 400-1000 l / min, and the temperature of the inlet air is 50 ° C to 150 ° C. Most preferred optimum conditions are that the feed flow rate of the solution is 3 ml / min, the air flow rate is 600 l / min and the temperature of the inlet air is 70 ° C. The process time for spray drying under the above conditions is 5 hours per liter of solution. At this time, the surface temperature of the spray-dried sample is very lower than the actual temperature because of the cooling effect of the solvent evaporation. Therefore, since the antigenicity of the hepatitis B antigen can be maintained without loss, the dependency on the stabilizer is lowered.

When using a spray-dried preparation, it is dissolved in distilled water for injection or physiological saline for injection, and the hepatitis B antigen protein concentration is adjusted to 5 µg / ml-20 µg / ml, and the salt is not added before spray drying. It is desirable to add salt to the solution in which the spray dried formulation is dissolved to bring the formulation into physiological isotonic state. The salts that can be used are the same as those exemplified for the addition before spray drying, and the concentration of addition is preferably 0.1-10 mM. If not mixed with aluminum gel prior to spray drying, mix 0.5 μg / ml aluminum gel with 10 μg / ml hepatitis B antigen protein concentration.

When the spray-dried hepatitis B vaccine preparation of the present invention is dissolved in a buffer solution, hepatitis B antigen forms an aggregated phase. For example, when the spray-dried preparation without aluminum gel is dissolved in distilled water, the size of the hepatitis B antigen is diameter. 50-200nm, which is larger than 20-50nm, the size of hepatitis B antigen in the liquid state before spray drying, which is thought to be due to the increase in hydrophobic bonds due to the strong cohesion that occurs during spray drying. Such aggregated antigens have the advantage of causing a large increase in immunity (see Korean Patent Publication No. 85-7272; P. N. Lelie, Viral Hepatitis and Liver Disease, 1009-1013 (1988)). However, in the above case, the antigenicity is lost by high temperature heat treatment, and the effect of the aggregated antigen is applied only to the antigen extracted from plasma (PN Lelie, Viral Hepatitis and Liver Disease, 1009-1013 (1988)). . In fact, the use of the above method is very limited given that most of the hepatitis B vaccines currently used in the world are made by genetic recombination technology. In contrast, the aggregated antigens of the present patent may similarly increase the immunogenicity, but there is no decrease in antigenicity or molecular modification by temperature. This is because it is a method of utilizing physical cohesion at low temperature, rather than coagulation by chemical modification as before. Therefore, this method can be applied to recombinant vaccines as well as plasma vaccines.

Hereinafter, the present invention will be described in more detail with reference to the following Examples, which are not intended to limit the present invention.

In the following examples, the antibody titer was measured using an AUSAB test kit (available from Abbott, USA), and the antigen titer was measured using an Auzyme test kit (available from Abbott, USA), and the protein was lowry. It was quantified by the method. Genetic recombinant hepatitis B antigens used in Examples and Comparative Examples were Saccharomyces cerevisiae strains transformed by the expression vector pYSAG101 containing a surface antigen gene (Korean Patent Publication No. 90-5959). Obtained by culturing).

Example 1

The recombinant hepatitis B antigen was diluted to a concentration of 300 µg / ml in 10 mM phosphate buffer (pH 7.5) and then supplied to a spray dryer (Buchi 190) at a flow rate of 3 ml / min to obtain a spray-dried preparation. At this time, the flow rate of nitrogen to be injected is 600ℓ / min and the temperature of the incoming air is 70 degrees Celsius. After completely dissolving the spray-dried preparation in the same buffer solution, the antigenic value of hepatitis B antigen was measured using an Ozyme test kit, and this value was compared with that of the spray-dried liquid hepatitis B antigen in cold storage. Relative antigenicity is shown in Table 1 below. At this time, there was no decrease in the antigen value during the spraying process.

Example 2

After diluting the recombinant hepatitis B antigen to a concentration of 300 µg / ml in 10 mM phosphate buffer (pH 7.5), the hydroxy propyl cellulose (HPC) was dissolved therein to a concentration of 3 mg / ml. Example 1 Spray dried formulations were obtained in the same manner. After completely dissolving the spray-dried preparation in the same buffer solution, the antigen value of hepatitis B antigen was measured using an Ozyme test kit, and the value was compared with that of the spray-dried liquid hepatitis B antigen in cold storage. Relative antigenicity is shown in Table 1 below. At this time, there was no decrease in antigen value during the spraying process.

Example 3

The recombinant hepatitis B antigen was diluted to a concentration of 300 µg / ml in 10 mM phosphate buffer (pH 7.5), and then gelatin was dissolved therein to a concentration of 1.5 mg / ml, followed by spray drying in the same manner as in Example 1. Obtained formulations. After completely dissolving the spray-dried preparation in the same buffer solution, the antigen value of hepatitis B antigen was measured using an Ozyme test kit, and the value was compared with that of the spray-dried liquid hepatitis B antigen in cold storage. Relative antigenicity is shown in Table 1 below.

TABLE 1

Example 4

The preservation stability of the spray dried vaccine was evaluated by storing the spray dried vaccine at a constant temperature for a certain period of time, and then adding the buffer solution to the vaccine sample to completely dissolve the antigenicity. That is, a small amount of the spray-dried preparation prepared in Example 2 in a vial and stored at a constant temperature for a period of time as shown in Table 2, and then added to the 10mM phosphate buffer (pH7.5) to dissolve the vaccine and Ozyme kit Antigenicity was measured. The relative antigen value for the standard antigen was calculated using the hepatitis B antigen stored at low temperature as a standard antigen, and the results are shown in Table 2 below.

TABLE 2

As can be seen in Table 2, the spray-dried preparation of the present invention was stable for 6 months at 37 ℃ while maintaining the antigen value unlike the liquid vaccine.

Comparative Example 1

The preservation safety of the liquid formulation was investigated in the same manner as used in Example 4 by dividing the liquid formulation in which the concentration of the hepatitis B vaccine was set to 50 µg / ml by vial method. The measurement method was the same as in Example 4, and the results measured at 37 ° C are shown in Table 3 below in comparison with the results in Example 4.

TABLE 3

From the results in Table 3, it can be seen that the long-term storage of the dry preparation of the present invention maintains a higher antigen value for longer than the liquid preparation.

Comparative Example 2

The average particle size and distribution of the solution prepared in Example 1 dissolved in 10 Mm phosphate buffer solution (pH 7.5) to 100 μg / ml and the liquid preparation before spray drying were measured with a light scattering particle size analyzer (Brookhaven BI-200SM). The measurement results are shown in FIG. In this case, the average particle size was 180 nm and the liquid formulation was 35 nm in size.

Example 5

The preparation prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.5) to 20 μg / ml, and the solution was mixed with 1.0 μg / ml aluminum hydroxide gel at a ratio of 1: 1. The vaccine thus prepared was injected subcutaneously with 1.0 mL of 11 guinea pigs of 300 to 305 g each, and blood was collected one month later, and antibody production rate was measured by using OUSAB. The results are shown in Table 4 below.

Example 6

The same experiment as in Example 5 was carried out using the formulation prepared in Example 2, and the results are shown in Table 4 below.

Comparative Example 3

The same experiment as in Example 5 was carried out using a liquid hepatitis B antigen, and the results are shown in Table 4 below for Examples 5 and 6.

TABLE 4

* GMT: geometric mean titer = (nl.n2 ... nN) ^{1 / N}

From the above results, it can be seen that the hepatitis B vaccine desiccant of the present invention maintains higher antigenicity than the liquid formulation.

Claims (10)

A method of preparing a hepatitis B vaccine preparation, characterized by spray drying the hepatitis B antigen. The method of claim 1, wherein the hepatitis B antigen comprises a protein component in the range of 100 μg / ml to 10 μg / ml in the spray dried solution. The method of claim 1, wherein at least one of an aluminum gel, a stabilizer, and a salt is added to the hepatitis B antigen prior to the spray drying. The method of claim 3, wherein the aluminum gel is at least one of an aluminum hydroxide gel and an aluminum phosphate gel. 4. The method of claim 3, wherein said stabilizer is at least one of natural protein, amino acids and polysaccharides. The method of claim 5 wherein said natural protein is at least one of gelatin, albumin and dextran and is added to the solution prior to spray drying at a concentration of 0.01-2% (W / V). The method of claim 5, wherein the amino acid is at least one of glycine, alanine, glutamic acid, arginine, lysine and salts thereof, and added to the spray dried solution at a concentration of 0.1-10% (W / V). 6. The method of claim 5, wherein said polysaccharide is at least one of glucose, xylose, galactose, fructose, lactose, maltose, saccharose, hydroxy propyl cellulose, hydroxy methyl cellulose, carboxy methyl cellulose, mannitol and sorbitol , Adding to a spray dried solution at a concentration of 0.1-10% (W / V). 4. The method of claim 3 wherein said salt is added to a solution that is at least one of sodium chloride, sodium phosphate and potassium phosphate and is spray dried or to a solution in which the vaccine formulation is dissolved after drying. The method of claim 1, wherein the solution of the spray-dried hepatitis B antigen is introduced at a rate of 1-10ml / min, and spray-dried by introducing 50 ℃ -150 ℃ air at a rate of 400-1000L / min Way.